Carlberg C. Molecular Immunology. How Science Works 2022

Carsten Carlberg
Eunike Velleuer
Molecular
Immunology
How Science Works
Molecular Immunology
Carsten Carlberg · Eunike Velleuer
Molecular Immunology
How Science Works
Carsten Carlberg Eunike Velleuer
Institute of Biomedicine Department of Cytopathology
University of Eastern Finland Heinrich Heine University Düsseldorf
Kuopio, Finland Düsseldorf, Germany
Institute of Animal Reproduction and Food Centre for Child and Adolescent Health
Research Helios Clinic
Polish Academy of Sciences Krefeld, Germany
Olsztyn, Poland
ISBN 978-3-031-04024-5 ISBN 978-3-031-04025-2 (eBook)

https://doi.org/10.1007/978-3-031-04025-2
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Preface
The recent experience of the COVID-19 (coronavirus disease 2019) pandemic, the
discussion about the origin of SARS-CoV-2 (severe acute respiratory syndrome coro-
navirus 2) as well as effective vaccination against it remind all of us that we should
know more about the system that keeps us alive: immunity. Our immune system is
composed of biological structures like the lymphatic system and bone marrow, cell
types like leukocytes (cellular immunity) and proteins like antibodies and comple-
ment proteins (humoral immunity). The perfect balance of these components protects
us against infectious diseases and cancer. Molecular immunology aims to understand
the collective and coordinated response of these cells and proteins to substances
that are foreign to our body. The main purpose of this immune response is the
fight against microbes, such as viruses, bacteria, fungi and parasites. However,
the example of allergic reactions, which nowadays are getting continuously more
common, demonstrates that also non-microbial molecules can induce a strong reac-
tion of our immune system. Moreover, incorrect reactions of the immune system can
lead to autoimmune diseases, such as diabetes type I and multiple sclerosis. Thus,
immune responses can cause tissue injuries that may harm our body more
than the effects of pathogenic microbes. These collateral damages may make us
severely ill or even kill us, such as in case of bacterial sepsis or strong responses to
SARS-CoV-2 infections.
Therefore, the main purpose of this book is to provide an essential background in
molecular immunology. This includes the basic principles and underlying processes
of immunity against bacteria and viruses, immune reactions in case of cell and organ
transplantation, the overboarding immune system in context of allergies and autoim-
mune reactions as well as the way how a proper functioning immune system protects
us against cancer. Understanding these mechanisms will highlight that a fight against
viruses uses the same mechanisms as the battle against thousands of transformed
cancer cells arising every day in each of us.
The content of the book is linked to the lecture course in “Molecular Immunol-
ogy”, which is part of a series with courses in “Molecular Medicine and Genetics”,
“Cancer Biology” and “Nutrigenomics”, that is given by one of us (Carsten Carlberg)
v
vi Preface
in different forms since 2005 at the University of Eastern Finland in Kuopio. Accord-
ingly, this book relates to the textbooks Mechanisms of Gene Regulation: How
Science Works (ISBN 978-3-030-52321-3), Human Epigenetics: How Science Works
(ISBN 978-3-030-22907-8), Cancer Biology: How Science Works (ISBN 978-3-030-
75699-4) and Nutrigenomics: How Science Works (ISBN 978-3-030-36948-4), the
studying of which may be interesting to readers who like to get more detailed infor-
mation. The clinical impact of the book is based on personal experience by one of
us (Eunike Velleuer).
Chapters 1–3 of this book focus on the cellular basis of immunology, Chaps. 4–6
will discuss the key molecules mediating the effector functions of B and T cells and
Chaps. 7–11 will provide a link of molecular immunology to infections by bacteria
and viruses (including influenza and SARS-CoV-2), organ transplantation, allergy
and autoimmunity as well as different types of cancers. A glossary in the Appendix
will explain the major specialist’s terms.
We hope that readers will enjoy this rather visual book and get as enthusiastic
as the authors about life and its protection reflected in the fine-tuned molecular
immunology.
Kuopio, Finland Carsten Carlberg

Düsseldorf, Germany Eunike Velleuer
January 2022
Contents
1 Cells and Tissues of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Global Burden of Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Innate and Adaptive Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Hematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4 Primary and Secondary Structures of the Immune System . . . . . . 13
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2 Innate Immunity and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.1 Pathogen- and Danger-Associated Molecular Patterns
and Their Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.2 Myeloid Cells of Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3 Mechanisms of Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.4 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.5 Lymphoid Cells of Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . 36
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3 Adaptive Immunity and Antigen Receptor Diversity . . . . . . . . . . . . . . 41
3.1 Classes and Responses of Adaptive Immune Cells . . . . . . . . . . . . 41
3.2 Lymphocyte Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.3 Mechanisms of Antigen Receptor Diversity . . . . . . . . . . . . . . . . . . 50
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4 B Cell Immunity: BCRs, Antibodies and Their Effector
Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.1 Structure and Diversity of Antibodies and BCRs . . . . . . . . . . . . . . 59
4.2 BCR Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.3 Humoral Adaptive Immune Response . . . . . . . . . . . . . . . . . . . . . . . 67
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
vii
viii Contents
5 Antigen-Presenting Cells and the Major Histocompatibility

Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.1 Antigen-Presenting Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.2 MHC Proteins and the HLA Locus . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.3 MHC Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
6 T Cell Immunity: T Cell Receptors and Their Effector
Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
6.1 TCR Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
6.2 T Cell Effector Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.3 T Helper Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
6.4 Cytotoxic T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
7 Immunity to Bacterial Pathogens and the Microbiome . . . . . . . . . . . . 109
7.1 Principles of Immune Responses to Infections . . . . . . . . . . . . . . . . 109
7.2 Immune Responses to Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
7.3 Emerging Microbial Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
7.4 Immunity to the Microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
8 Immunity to Viral Pathogens and the Virome . . . . . . . . . . . . . . . . . . . . 135
8.1 Principles of Immune Responses to Viruses . . . . . . . . . . . . . . . . . . 135
8.2 Chronic Virus Infections and Emerging Viral Pathogens . . . . . . . 139
8.3 Influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
8.4 COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
9 Tolerance and Transplantation Immunology . . . . . . . . . . . . . . . . . . . . . 155
9.1 Central and Peripheral Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
9.2 Graft Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
9.3 Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
10 Immunological Hypersensitivities: Allergy and Autoimmunity . . . . . 171
10.1 Classification of Hypersensitivities . . . . . . . . . . . . . . . . . . . . . . . . . . 171
10.2 Immunity of Allergies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
10.3 Type 2 and 3 Hypersensitivities . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
10.4 T Cell-Mediated Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
10.5 The Equilibrium Model of Immunity . . . . . . . . . . . . . . . . . . . . . . . . 193
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Contents ix
11 Cancer Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

11.1 Outline of Cancer Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
11.2 Recognition of Cancer Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
11.3 Monoclonal Antibodies in Cancer Immunotherapy . . . . . . . . . . . . 206
11.4 Immune Cell Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Abbreviations
3D 3-dimensional
A Adenine
ACE2 Angiotensin-converting enzyme 2
ADCC Antibody-dependent cellular cytotoxicity
ADCP Antibody-dependent cellular phagocytosis
AID Activation-induced deaminase
AIDS Acquired immune deficiency syndrome
AIRE Autoimmune regulator
AKT Akt murine thymoma viral oncogene homolog
AMPK AMP-activated protein kinase
AP1 Activation protein 1
APS1 Autoimmune polyglandular syndrome type 1
ASC Apoptosis-associated speck-like protein containing a CARD
ATP Adenosine triphosphate
BAK BCL2 antagonist/killer 1
BAX BCL2-associated X, apoptosis regulator
BCL2 BCL2 apoptosis regulator
BCL2L1 BCL2 like
BCR B cell receptor
BID BH3-interacting domain death agonist
BLK BLK proto-oncogene, Src family tyrosine kinase
BLNK B cell linker
bp Base pair
BTK Bruton tyrosine kinase
C Cytosine
CAMP Cathelicidin
CAR Chimeric antigen receptor
CASP Caspase
CBIF Cobalamin-binding intrinsic factor
CCL C-C chemokine ligand
CCR C-C chemokine receptor
xi
xii Abbreviations
CD Cluster of differentiation
CD40LG CD40 ligand
CDC Complement-dependent cytotoxicity
CDR Complementarity determining region
CDS Cytosolic DNA sensor
CEBPA CCAAT enhancer-binding protein alpha
CFB Complement factor B
CLEC12A C-type lectin domain family 12 member A
CLP Common lymphoid progenitor
CMP Common myeloid progenitor
CMV Cytomegalovirus
CNS Central nervous system
COVID-19 Coronavirus disease 2019
CpG Cytosine–guanine dinucleotide
CR Complement receptor
CRP C-reactive protein
CSF Colony-stimulating factor
CTLA4 Cytotoxic T-lymphocyte-associated protein 4
CXCL Chemokine (C-X-C motif) ligand
CXCR Chemokine (C-X-C motif) receptor
D Diversity
DAG Diacylglycerol
DAMP Damage-associated molecular pattern
DEFB4 Defensin, beta 4A
DKK1 Dickkopf WNT signaling pathway inhibitor 1
EBV Epstein–Barr virus
EGF Epidermal growth factor
EMA European Medicines Agency
EOMES Eomesodermin
ER Endoplasmatic reticulum
FACS Fluorescence-activated cell sorting
FAS Fas cell surface death receptor
FASLG Fas ligand
Fc Fragment crystallizable
FcεRI Fc epsilon receptor type I
FDA US Food and Drug Administration
FKBP FK506-binding protein
FOXP3 Forkhead box P3
FPR1 Formyl peptide receptor 1
FYN FYN proto-oncogene, Src family tyrosine kinase
G Guanine
GATA GATA-binding protein
GR Glucocorticoid receptor
GRB2 Growth factor receptor-bound protein 2
GvHD Graft-versus-host disease
Abbreviations xiii
HBV Hepatitis B virus

HIV-1 Human immunodeficiency virus 1
HLA Human leukocyte antigen
HPV Human papilloma virus
HSC Hematopoietic stem cell
HSV Herpes simplex virus
IBD Inflammatory bowel disease
ICAM Intercellular adhesion molecule
ID2 Inhibitor of DNA binding 2
Ig Immunoglobulin
IGF Insulin-like growth factor
IL Interleukin
IL1RN IL1 receptor antagonist
IL2RA Interleukin 2 receptor subunit alpha
ILC Innate lymphoid cell
INF Interferon
INPP5D Inositol polyphosphate-5-phosphatase D
INS Insulin
IP3 Inositol trisphosphate
IRF3 Interferon response factor 3
ITAM Immunoreceptor tyrosine-based activation motif
ITGB2 Integrin subunit beta 2
ITIM Immunoreceptor tyrosine-based inhibition motif
J Joining
KIR Killer cell immunoglobulin-like receptor
KITLG KIT ligand
LCK LCK proto-oncogene, SRC family tyrosine kinase
LPS Lipopolysaccharide
LTA Lymphotoxin alpha
LTB Lymphotoxin beta
LTC4 Leukotriene C4
LYN LYN proto-oncogene, Src family tyrosine kinase
MAIT Mucosa-associated invariant T
MAP2K1 Mitogen-activated protein kinase kinase 1
MAPK Mitogen-activated protein kinase
Mb Mega base pairs (1,000,000 bp)
MBP Major basic protein
MDSC Myeloid-derived suppressor cell
MERS-CoV Middle East respiratory syndrome coronavirus
MHC Major histocompatibility complex
MICB MHC class I polypeptide-related sequence B
MIS-C Multisystem inflammatory syndrome in children
MPP Multipotent progenitor
MTOR Mechanistic target of rapamycin kinase
NAFLD Non-alcoholic fatty liver disease
xiv Abbreviations
NASH Non-alcoholic steatohepatitis

NET Neutrophil extracellular trap
NFAT Nuclear factor-activated T cells
NFκB Nuclear factor κB
NK Natural killer
NKT Natural killer T
NLR NOD-like receptor
NO Nitric oxide
NOS Nitric oxide synthase
PAF Platelet-activating factor
PAMP Pathogen-associated molecular pattern
PCR Polymerase chain reaction
PDCD1 Programmed cell death 1, also called PD1
PDCD1LG2 Programmed cell death 1 ligand 2, also called PDL2
PGD2 Prostaglandin D2
PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase
PLA2 Phospholipase A2
PLCG Phospholipase C gamma
PPIA Peptidylprolyl isomerase A
PRKC Protein kinase C
PTPN6 Protein tyrosine phosphatase non-receptor type 6
PU.1 Purine-rich box 1
RAC RAC family small GTPase 1
RAG Recombination-activating gene
RAS Rat sarcoma
RLR RIG-like receptor
RORC RAR-related orphan receptor gamma
ROS Reactive oxygen species
SAP Serum amyloid P-component
SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
SCFA Short-chain fatty acid
scFv Single-chain variable fragment
SCID Severe combined immunodeficiency disease
SEL Selectin
SLAMF7 SLAM family member 7
SLE Systemic lupus erythematosus
SNARE Soluble NSF attachment protein receptor
SOS1 SOS RAS/RAC guanine nucleotide exchange factor 1
SRC SRC proto-oncogene, non-receptor tyrosine kinase
STAT Signal transducer and activator of transcription
SYK Spleen-associated tyrosine kinase
T Thymine
TAM Tumor-associated macrophage
TAP Transporter, ATP-binding cassette subfamily B member
TAPBP TAP-binding protein, also called tapasin
Abbreviations xv
TBX21 T-box transcription factor 21

TCR T cell receptor
TdT Terminal deoxynucleotidyl transferase
TET Ten-eleven translocation
TGFβ Transforming growth factor beta
TH T helper
THBD Thrombomodulin
TLR Toll-like receptor
TMPRSS2 Transmembrane serine protease 2
TNF Tumor necrosis factor
TNFRSF TNF receptor superfamily member
Treg T regulatory
TSH Thyroid-stimulating hormone
TSLP Thymic stromal lymphopoietin
U Uracil
UV Ultraviolet
V Variable
VDR Vitamin D receptor
VEGF Vascular endothelial growth factor
VZV Varicella-zoster virus
X-SCID X-linked severe combined immunodeficiency disease
ZAP70 Zeta chain of T cell receptor-associated protein kinase 70
Chapter 1
Cells and Tissues of the Immune System
Abstract This chapter will provide a first overview on the cells and tissues forming
the immune system. First, we will discuss the general role of immunity in detecting
and neutralizing pathogens, in order to reduce the global burden of infectious
diseases. In human history of the last thousands of years pathogenic microbes have
caused numerous severe pandemics, of which that of SARS-CoV-2 is the most recent
one. We will distinguish the features and cell types of innate and adaptive immunity
and describe their response over time after contact with an antigen. Basically, all cell
types of the immune system are created by the process of hematopoiesis, perturbation
of which can lead to significant disruption of immune cell production. Finally, we
will describe the primary and secondary structures of the immune system, such as
bone marrow, thymus, lymph nodes and spleen, and the migration of immune cells
between them.
Keywords Pathogenic microbes · Infectious diseases · Pandemics · Innate

immunity · Adaptive immunity · Specificity · Memory · Hematopoiesis · Bone
marrow · Lymph nodes · T cell migration
1.1 Global Burden of Infectious Diseases
The immune system is meant to protect us against pathogenic microbes, such as

extra- and intracellular bacteria, viruses, fungi and parasites like protozoa and worms
(helminths) (Table 1.1). The effector mechanisms of immunity should eliminate the
pathogens and create a memory of their encounter (Sect. 7.1). In this way, individuals
become “immune”, i.e., they are protected against future infections with the
microbe. Immune cells and individuals that have not been in contact with a given
microbe are considered as “naïve”.
The immune system should protect us from a large variety of life-threatening
infectious diseases. Nevertheless, human history is full of devasting pandemics (Table
1.2), the first of which were caused by bacteria, such as Yersinia pestis and Mycobac-
terium tuberculosis (Sect. 7.3), and the more recent by viruses like influenza A virus
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 1

C. Carlberg and E. Velleuer, Molecular Immunology,
https://doi.org/10.1007/978-3-031-04025-2_1
2 1 Cells and Tissues of the Immune System
Table 1.1 The immune

Type of pathogen Examples Diseases
system protects against
pathogens Bacteria Extracellular Salmonella Food poisoning
enteritides Pneumonia
Streptococcus Tetanus
pneumoniae
Clostridium
tetani
Intracellular Mycobacterium Tuberculosis
tuberculosis Leprosy
Mycobacterium
leprae
Viruses HIV-1 AIDS
Influenza Influenza
SARS-CoV2 COVID-19
Fungi Candida Systemic
albicans candidiasis
Parasites Protozoa Plasmodium Malaria
falciparum Sleeping
Trypanosoma sickness
brucei
Helminths Schistosoma Schistosomiasis
mansoni
List of examples of four types of pathogenic microbes (and diseases
caused by them), against which the immune system is protecting
us
(Sect. 8.3), Ebola virus, human immunodeficiency virus 1 (HIV-1) and SARS-CoV-
2 (Sect. 8.4), which originated from animals. Thus, in the past infectious disease
were worldwide the most common cause of death.
Advanced knowledge in hygiene and medicine within the recent 150 years, such
as the identification of microbes, disinfection, vaccination and the use of antibiotics,
significantly reduced the burden of infectious diseases. Nevertheless, in the year 2017
worldwide still 7.3 million people (i.e., 12.8% of all) died from infectious diseases,
such as tuberculosis, acquired immune deficiency syndrome (AIDS), malaria, menin-
gitis or hepatitis, as well as from general microbe-related disorders like lower respi-
ratory infections (including influenza) and diarrheal diseases (Fig. 1.1, top). For
comparison, more than 73% of deaths worldwide relate to non-communicable disor-
ders, such as cardiovascular diseases and cancer. During the past decades the under-
standing of immunology significantly advanced by describing additional distinct
roles of the immune system controlling the homeostasis of our body, such as tissue
development and maintenance. This implies that mal-functions of the immune
system have a large impact also for non-communicable diseases, such as cancer,
metabolic and neonatal disorders.
In industrialized countries, such as in Finland, in 2017 less than 1000 of 53,722
deaths (i.e., less than 2%) were due to an infectious disease, but there was a similar
1.2 Innate and Adaptive Immunity 3
Table 1.2 Major pandemics in human history

Year Name Deaths Comments
430 BC “Plague of Athens” 100,000 First documented pandemic
541 Justinian plague 30–50 million Killed half of world population
(Yersinia pestis)
Around “Black death” 50 million Killed a quarter of world
1340 (Yersinia pestis) population
Around Tuberculosis Many millions Since 60,000 years with humans;
1500 (Mycobacterium tuberculosis) pandemic in middle ages
Around “Hueyzahuati” 3.5 million Pandemic in Central America
1520 (Variola major) brought by Europeans
1918 “Spanish flu” 50 million Pandemic in the whole world,
(Influenza virus) killed 2% of population
1976–2000 Ebola (Ebolavirus) >15,000 Many regional epidemics in
Africa
Since 1981 AIDS (HIV-1) 37 millions Ongoing pandemic with most
victims in Africa
Since 2019 COVID-19 (SARS-CoV2) >4 million Ongoing pandemic with victims
all over the world
Newly emerging infectious diseases were threatening humans since hunter-gatherers settled into
villages and domesticated animals. The number of victims before 1900 are estimates
percentage of deaths related to diseases associated with problems of the immune

system (Fig. 1.1, bottom). However, in the year 2021 the COVID-19 pandemic
(Sect. 8.4), i.e., a disease that in 2017 did not exist, had 3.02 million victims world-
wide and 1077 in Finland. The numbers for 2021 are even larger than for 2020
(1.81 million/561), although the largest ever worldwide vaccination campaign had
been started. Thus, understanding and appreciating the function of the immune
system is more important than ever.
1.2 Innate and Adaptive Immunity
The immune system of humans and other mammals uses a remarkably effective set
of tools for fighting against pathogenic microbes. The core of these mechanisms
is the detection of a wide variety of molecules, known as antigens. The exact site
of a complex antigen, such as a protein, that is recognized by antigen receptors is
called determinant or epitope. Epitopes are often found on the surface of microbes
or malignant cells and one major role of the immune system is to distinguish them
from epitopes of own healthy tissues (self-antigen).
The immune system is subdivided into innate and adaptive immunity. The innate
immune system is evolutionary older (Box 1.1) and provides a first line defense
against invading pathogens, since it responds within a few hours to the presence of
Cardiovascular diseases 17.79 million

Cancers 9.56 million
Respiratory diseases 3.91 million
Lower respiratory infections 2.56 million
Dementia 2.51 million
Digestive diseases 2.38 million For comparsion:
Neonatal disorders 1.78 million
Diarrheal diseases 1.57 million in 2021 3.02 million deaths
Diabetes 1.37 million
Liver diseases 1.32 million due to COVID-19
Road injuries 1.24 million
Kidney disease 1.23 million
Tuberculosis 1.18 million
HIV/AIDS 954,492
Suicide 793,823
Malaria 619,827
Homicide 405,346
Parkinson disease 340,639
Drowning 295,210
Meningitis 288,021
269,997
Protein-energy malnutrition 231,771
Maternal disorders 193,639
Alcohol use disorders 184,934
Drug use disorders 166,613
129,720
Hepatitis 126,391
Fire 120,632
Poisonings 72,371
Heat (hot and cold exposure)
Terrorism
53,350
26,445 World: Number of deaths
Natural disasters 9603
Cardiovascular diseases 21,359

Cancers 13,089
Dementia 8546
Digestive diseases 2416
Respiratory diseases 1784
Liver diseases 1178
Suicide 868 For comparsion:
Lower respiratory infections 713
Parkinson disease 682
598
in 2021 1077 deaths
Alcohol use disorders
Kidney disease
Diabetes
569
390
due to COVID-19
Road injuries 289
Drug use disorders 289
Drowning 116
Heat (hot and cold exposure) 104
Homicide 87
Fire 73
Tuberculosis 57
Diarrheal diseases 48
Neonatal disorders 46
Meningitis 19
Poisonings 18
12
HIV/AIDS 6
Protein-energy malnutrition 4
Hepatitis 2
Maternal disorders 2
Terrorism 2
Malaria
Natural disasters
0
0
Finland: Number of deaths
0
Fig. 1.1 Leading causes of death worldwide and in Finland. In the year 2017 about 7.3 million
(12.8%) of 57 million annual deaths worldwide (top) are the direct result of infectious diseases
(red), while at least another 15 million deaths (26.7%) are related to a mal-functional immune
system (orange). The percentages for infectious diseases are in an industrialized country, such as
Finland (bottom), with less than 2% significantly lower, while the percentage of diseases associated
with immune system dysfunctions (27.7%) are even slightly higher. For comparison, the victims of
COVID-19 of the year 2021 are indicated. Data are based on https://ourworldindata.org/causes-of-
death
antigens or injured cells (Chap. 2). Innate immune cells, such as macrophages, are
stimulated when a limited set of pattern-recognition receptor recognize molecules
common to groups of microbes or expressed by damaged host cells. In contrast, the
adaptive immune system reaches its full activity just a week after first contact with an
Table 1.3 Comparing innate and adaptive immunity

Features Innate immunity Adaptive immunity
Specificity For molecules recognized by B and T cell clones, each with a
pattern recognition receptors BCR or TCR specific for a different
antigen
Diversity Limited by the number of different Very large, since there are millions
pattern recognition receptors of B and T cell clones
(approximately 100)
Memory Limited to trained immunity Yes, based on memory B and T cells
Barrier functions Skin, mucosal epithelia, secreted Lymphocytes in epithelia,
anti-microbial peptides antibodies secreted in epithelia
Humoral component Complement proteins Antibodies
Cellular component Phagocytes (neutrophils and B and T cells
macrophages), dendritic cells, NK
cells, mast cells, ICLs
antigen (Chap. 3). The adaptive immune system has far higher specificity and diver-
sity in antigen recognition than the innate immune system (Table 1.3). However,
immune functions are not unique to hematopoietic cells, since also a number of other
cell types forming the epithelium, endothelium and connective tissue display barrier
functions and are therefore contribute to the basic mechanisms of pathogen defense
(Type 4 immune response, Sect. 10.5).
Box 1.1: Evolution of the immune system Mechanisms for defending the
host against microbes are present in all multicellular organisms. The phylo-
genetically oldest mechanisms of host defense are those of innate immunity,
which are present even in plants and insects. The innate immune system co-
evolved with microbes and recognizes microbial molecules that are essential
for their survival. For example, Toll-like receptors (TLRs), which are already
found in insects, are highly evolutionary conserved membrane receptors that
start a signal transduction cascade ending up with the activation of the tran-
scription factor NFκB (nuclear factor κB). In fact, most of the mechanisms of
innate immunity appeared when the first multicellular organisms evolved some
750 million years ago. Approximately 500 million years ago, jawless fish, such
as lampreys and hagfish, developed an immune system containing lymphocyte-
like cells that may function like lymphocytes in more advanced species and even
respond to immunization. The antigen receptors on these cells were proteins
with limited variability that were capable of recognizing many antigens but
were distinct from the highly variable antibodies and TCRs that appeared
later in evolution. The more specialized defense mechanisms that constitute
adaptive immunity are found in vertebrates only. Most of the components
of the adaptive immune system, including lymphocytes with highly diverse
antigen receptors, antibodies and specialized lymphoid tissues, evolved coor-

dinately within a short time in jawed vertebrates (e.g., sharks) approximately
360 million years ago. In humans (and other mammals) the immune system
has shaped to primarily respond efficiently to acute infections in young people,
in order to assure their survival during the reproductive and child-caring age.
This includes fighting against microbes but also the control of tissue repair,
wound healing, elimination of dead and cancer cells and the formation of the
healthy gut microbiota. However, beyond the reproductive age there is lack
of major evolutionary pressure and the genetic traits selected to ensure
fitness in early-life may lead to immunophenotypes with a high rate of
chronic inflammation (Box 8.6). Accordingly, advanced longevity is a recent
phenomenon occurring under optimized conditions of civilization.
The innate immune system is composed of (Fig. 1.2, left):

• physical and chemical barriers, such as epithelia cells and the anti-microbial
molecules that they secrete
• phagocytic cells, such as neutrophils and macrophages, monocytes dendritic cells,
mast cells, natural killer (NK) cells and innate lymphoid cells (ILCs) (Chap. 2)
• proteins of the blood, such as proteins of the complement system and inflammatory
molecules.
Some innate immune cells, such as macrophages, dendritic and mast cells, are
tissue resident and perform surveillance for possible invading microbes. In contrast,
other cells of innate immunity, such as monocytes, NK cells and ILCs, are recruited
from the bloodstream to those tissues that send out signals of disturbance, such as
during infections, damage and emerging inflammation (Chap. 2).
Lymphocytes are the cellular component of the adaptive immune system and are
distinguished into B and T cells (Fig. 1.2, right). Both types of cells express on
their surface highly specific antigen receptors, B cell receptors (BCRs) and T cell
receptors (TCRs), respectively. In contrast to innate immune cells that use pattern-
recognition receptors with limited specificity for pathogen-associated molecular
patterns (PAMPs), i.e., microbe-associated molecules, such as lipopolysaccharide
(LPS) (Sect. 2.1), the antigen recognition domains of BCRs and TCRs can adapt
millions of different structures that are able to bind basically any molecule existing
in nature. However, T cells respond via their TCR only to small peptides presented
on major histocompatibility complex (MHC) proteins on the surface of antigen-
presenting cells, i.e., the T cell response is restricted to proteins. In contrast, B
cells are more flexible and bind to any antigen molecule in solution or being presented
by other cells. Thus, not only proteins, but also polysaccharides, lipids and small
molecules are able to induce antibody responses. The total population of lympho-
cytes of an individual is composed of different clones of B and T cells, each of which
have a different BCR or TCR on the surface (Sect. 3.3). In this way, the immune
system of an individual can recognize 10–1000 million distinct structures that may
Innate immunity Adaptive immunity
Microbe Epithelial
barriers
Antibodies
Naïve B cell
Phagocytes
(macrophages +
neutrophils) Dendritic
cells
Mast cells
NK cells
and ILCs
Complement Naïve T cell

proteins
0 6 12 1 4 7
Hours Days
Time after infection
Fig. 1.2 Innate and adaptive immunity. Protective immunity against microbes is mediated by
the early reactions of the innate immune system as well as the later responses of the adaptive
immune system. Innate immunity is used for the initial defense against infections, while responses
of adaptive immunity need up to 7 days to develop to full potency. Key cell types are shown. More
details are provided in the text
act as antigenic determinants, i.e., the adaptive immune system has a very high diver-
sity. This is essential for the defense of an individual against the large number of
potential pathogens that he or she is exposed to during lifespan. When the large sets
of some 10 million different naïve B and T cells are exposed to a previously unknown
antigen, this molecule is bound effectively only by a very few lymphocytes. These
cells are then activated and proliferate, in order to generate thousands of identical
progenies with the same specificity, i.e., a cell clone. This process is referred to as
clonal expansion. B cell effector cells are plasma cells that secrete their BCR in form
of antibodies, i.e., antibodies are a soluble form of BCRs with identical specificity
(Sect. 4.1). Antibodies are also often referred to as immunoglobulins (Igs), since
they represent the immunity-conferring portion of the globulin fraction of the serum.
Antibodies are the mediators of humoral immunity of the adaptive immune system
(Sect. 3.1). Their main function is to neutralize microbes by covering their surface
and to promote their elimination by cellular (phagocytes) and humoral (complement

system) responses of the innate immune system (Sect. 4.3).
The adaptive immune system has a memory function for antigen contact and is
capable to respond stronger and faster to repeated exposures with an identical antigen
(Fig. 1.3). This second boosted immune response is based on long-lived memory
B cells and represents the mechanistic basis of vaccinations. Similar principles
apply also to T cells. Cells of the innate immune system can also be trained by
epigenetic changes. The latter prepares macrophages for a stronger response to a
possible re-exposure with PAMPs but does not represent traditional immunologic
memory (Table 1.3).
Antigen X Antigen X
+ antigen Y
Plasma cells
Secondary
Memory
Serum antibody titer
anti-X
B cells
response
Plasma cells
Plasma cells
Memory
Primary B cells Primary
Memory
anti-X anti-Y
B cells
response response
Naïve B cells
Weeks 2 4 6 8 10
Fig. 1.3 Specificity, memory and self-limitation of B cells. Schematic illustration of the speci-
ficity of B cells producing different antibodies to antigens X and Y. Immunologic memory implies
that a second exposure to antigen X results in a faster and stronger response in terms of secreted
antibodies specific to antigen X than the primary response. Self-limitation means that a few weeks
after each boost of response both the titer of specific antibodies as well as the amount of specific
plasma B cells decreases. However, after each response the number of long-lived memory B cells
increases. The same principles apply to the response of T cells
1.3 Hematopoiesis 9
1.3 Hematopoiesis
Cells of the immune system have a rapid turnover and are therefore able to show
a maximal adaptive response to environmental changes. In an adult, all circulating
blood cells, including immune cells, are produced in the bone marrow by a process
termed hematopoiesis (Fig. 1.4). Exceptions are tissue-resident macrophages, which
origin from fetal liver and colonized the organs during embryogenesis. Examples for
such tissue-resident macrophages are microglial cells in the CNS (central nervous
system), Kupffer cells in the liver or alveolar macrophages in the lungs.
Hematopoiesis is the process of the lifelong regeneration of our blood
cells. Long-lived, self-renewing hematopoietic stem cells (HSCs) divide either into
daughter stem cells or into progenitor cells for the more than 100 phenotypically
distinct cell types in 11 major lineages, most of which belong to the immune system.
HSCs differentiate into immature progenitor cells, such as multipotent progenitors
(MPPs), which then give rise to the progenitors of the myeloid or lymphoid lineages,
called common myeloid progenitors (CMPs) and common lymphoid progenitors
(CLPs), respectively. Thus, the first distinction of the hematopoietic cascade is the
differentiation into either the myeloid line or the lymphoid line. In further differ-
entiation steps of the myeloid line there is progressive commitment to erythrocytes
(red blood cells essential for oxygen transport), megakaryocytes creating platelets
(needed for blood coagulation after injuries), mast cells, basophils, eosinophils,
neutrophils and monocytes (which can further differentiate into dendritic cells or
macrophages). With the exception of erythrocytes and megakaryocytes these myeloid
cells belong to the innate immune system. Differentiation of the lymphoid line results
in B and T cells of the adaptive immune system as well as NK cells and ILCs of the
innate immune system.
Due to the turnover of cells and the persistent hematopoiesis most cells of the
immune system are replaced every few days to weeks. Every day HSCs give rise
to some 3 × 1011 cells (most of which are erythrocytes and neutrophils); i.e., over
our lifespan the bone marrow produces far more cells than any other tissues of our
body. Most of the different end-products of hematopoiesis leave the bone marrow
as mature cells, while T cells (Sect. 3.2) and mast cells (Sect. 10.2) terminate their
differentiation in the thymus and in epithelial tissue, respectively.
The decisions for the differentiation in different lineages are driven by epige-
netic changes, such as in chromatin accessibility (Box 1.2), and by the expression of
lineage-determining master transcription factors (Box 1.3). A number of extrinsic and
intrinsic factors, such as growth factor-stimulated signal transduction cascades, tran-
scription factors and chromatin modifying enzymes (chromatin modifiers), regulate
the equilibrium between self-renewal and differentiation of HSCs. Thus, chromatin
modifiers and transcription factors work together in creating appropriate epigenetic
profiles on the level of DNA methylation and histone modifications, which determine
the respective functions of the different cell types of the hematopoietic system. Epige-
netic regulation is fundamental for the differentiation of immune cells but also for
T cell
pro-T cell pre-T cell
B cell
pro-B cell pre-B cell
common innate lympho-
lymphoid cyte cells
progenitor
pro-ILC
NK cell
pro-NK cell
Dendritic
cell
CFU-M Monoblast Monocyte Macrophage

HSC multi-
potent
progenitor
Eosinophil
CFU-eo Eo myelocyte
Neutrophil
CFU-G Neutro myelocyte
Basophil
early
progenitor CFU-b Baso myelocyte
with myeloid
potential
Mast cell
CFU-Mc
Erythrocyte
CFU-E Erythroblast
Platelets
CFU-Mg
Megakaryoblast
Fig. 1.4 The hierarchy of hematopoiesis. The scheme illustrates the hematopoietic tree of the
development of the major lineages of blood and immune cells
1.3 Hematopoiesis 11
their adaptive response to several environmental challenges, such as microbe infec-

tions. These epigenetic processes are also the basis for trained immunity representing
a memory function of the innate immune system. In general, epigenetic profiling of
immune cells is an important tool for a molecular description of health and diseases
being as different as allergy (Sect. 10.2) and cancer (Chap. 11).
Box 1.2: The impact of epigenetics in hematopoiesis During hematopoiesis

the genome-wide DNA methylation pattern of differentiating blood cells
changes dynamically and is very locus specific, i.e., at some genomic regions
there is a rise in DNA methylation, while it decreases at other regions.
Hematopoietic cells can be easily segregated based on their DNA methylation
profile, i.e., their DNA methylome. In general, the commitment to a specific cell
lineage increases the level of DNA methylation, since a larger set of genes is
not anymore needed in these terminally differentiated cells. Interestingly, the
myeloid lineage is the default outcome of hematopoiesis, since its differ-
entiation requires less correction by increased DNA methylation than that of
the lymphoid lineage. Parallel to alterations in the DNA methylome, also key
chromatin modifiers are changing their expression during hematopoiesis and
lead to characteristic histone modification patterns. Both DNA methylation and
histone modification patterns determine accessibility of genomic DNA for tran-
scription factors and polymerases. Accordingly, a mis-regulation of the genes
encoding for chromatin modifiers can result in hematopoietic failure, such as
cell cycle arrest of HSCs, pre-mature differentiation termination, apoptosis and
defective self-renewal and finally leads to hematological malignancies, such as
leukemia, myelodysplastic syndrome or even complete bone marrow failure.
Box 1.3: The impact of master transcription factors in hematopoiesis In

addition to chromatin modifiers, some master transcription factors, such as
CEBPα (CCAAT/enhancer binding protein α), PU.1 (purine-rich box 1) and
GATA2 (GATA binding protein 2), have a key role in hematopoiesis. They act as
pioneer factors that directly bind nucleosomal DNA to prime enhancers for acti-
vation. These transcription factors then recruit chromatin remodeling and modi-
fier complexes, which in turn facilitate removal and post-translational modifica-
tion of nucleosomes at these genomic regions. For example, in myeloid progen-
itors the sustained expression of CEBPα generates macrophages, whereas
under sustained expression of GATA2 mast cells are created. However, when
initially CEBPα and afterward GATA2 are expressed, eosinophils are produced,
while the reversed order leads to basophils. Furthermore, CEBPα interacts with
the chromatin modifier TET2 (ten-eleven translocation, a DNA demethylating
enzyme), so that its target genes get demethylated during hematopoiesis. The
activity of TET2 may be the key mechanism, why myeloid cells are epigenet-
ically closer to HSCs than lymphoid cells. This fits with the observation that
TET2 is mutated in several myeloid malignancies.
In homeostasis, the highly differentiated cell types representing the end points
of the hematopoietic tree are produced in proportion to the needs, i.e., the turnover
of the cell types. In contrast, a disruption or mis-regulation of this hematopoietic
homeostasis can lead to hematological disorders, such as leukemia, lymphoma and
myeloma. In these diseases the excessive production of leukocytes in the bone marrow
may lead to a significant raise in their levels in blood circulation. In the opposite
situation, bone marrow failure leads to a decline of all blood counts. Moreover,
because all hematopoietic cells have the same origin, i.e., HSCs, a mis-regulation in
one lineage has also impact on the other ones. Therefore, a disruption in any stage
of hematopoiesis affects the production and function of every blood cell and
may have severe consequences, such as the inability to fight against infections
or the risk of uncontrolled bleeding.
When the body is perturbed by low oxygen levels (e.g., at high altitudes), an
infection or the onset of an inflammation-related common disease, the bone marrow
responds by increased production of erythrocytes, monocytes or neutrophils, respec-
tively, in order to achieve an adapted state of homeostasis. In turn, deviations from
the average blood counts for different types of leukocytes (Table 1.4) are regularly
used in clinical practice as disease biomarkers.
Table 1.4 Normal blood cell counts

Cell type Mean count per μl Normal range
Erythrocytes (red blood cells) 4,000,000 3,500,000–4,500,000
Leukocytes (white blood cells) 7400 4500–11,000
Neutrophils 4400 40–60% of leukocytes
Lymphocytes 2500 20–40% of leukocytes
Monocytes 300 2–8% of leukocytes
Eosinophils 200 1–4% of leukocytes
Basophils 40 <1% of leukocytes
Humans have in average 5 L of blood, i.e., there are approximately 35 billion (109 ) leukocytes only
in the blood (more are found in the lymphatic system)
1.4 Primary and Secondary Structures of the Immune System 13
1.4 Primary and Secondary Structures of the Immune

System
After birth the bone marrow is the key tissue for the generation of nearly all immune
cells (Sect. 1.3). Most immune cells mature in the bone marrow before they go into
circulation in the blood or lymphatic system (Box 1.4), but pro-T cells migrate for
maturation to the thymus (Box 1.5) (Fig. 1.5). Primary lymphoid organs are those
sites, where B and T cells first express antigen receptors (BCRs and TCRs)
and obtain phenotypic as well as functional maturity. These sites provide growth
factors and other signals required for B and T cell maturation and present self-
antigens for selecting non-self-reacting cells. When mature B and T cells emerge
from the bone marrow and thymus, respectively, they are functionally quiescent
and called “naïve” (i.e., immunologically inexperienced), since they have not yet
been in contact with any specific antigen. Naïve and memory B and T cells are also
called “resting” lymphocytes, as they are not proliferating or performing effector
functions. However, after antigen binding to their BCR or TCR, naïve lymphocytes
Generative Blood, Peripheral

lymphoid organs lymph lymphoid organs
Bone marrow Common Lymph
lymphoid nodes
precursor
Recirculation
Pro-B cell
Spleen
HSC
Mature B
cell
Immature
Pro-T cell B cell
Thymus
Recirculation
Mature
T cell
lymphoid tissues
Fig. 1.5 Maturation of B and T cells. The precursors of B and T cells evolve out of the
hematopoiesis process (Sect. 1.3) in the bone marrow. Pro-B cells finish their maturation in the
bone marrow (Sect. 3.3), while pro-T cells migrate to the thymus and mature there. Mature B and T
cells circulate in the blood and lymph system to secondary lymphoid organs like lymph nodes and
spleen as well as to epithelial tissues all over the body. There they wait for possible contact with
antigens or recirculate to other secondary lymphoid organs
activate signal transduction cascades that lead to their proliferation, referred to as

clonal expansion (Chaps. 4 and 6). In this way, the number of antigen-specific B cells
increases by a factor of up to 5000 and that of cytotoxic T cells even by a factor of
50,000. In parallel, B and T cells differentiate into effector cells that are involved in
different processes eliminating microbes carrying the antigen for which the activated
lymphocytes are specific for (Sect. 3.1). A subset of these cells differentiates into
long-lived memory cells.
Box 1.4: The lymphatic system The main functions of the lymphatic system
are the removal of interstitial fluid from tissues, carrying lipids from the
digestive system, transporting lymphocytes from and to the lymph nodes and
moving antigen-presenting dendritic cells (Sect. 5.1) from infected tissues to
lymph nodes (Fig. 1.6), in order to get there in contact with B and T cells.
The lymphatic system is composed of a large network of lymphatic vessels
and lymph nodes (Box 1.6). The lymphatic vessels transport a clear fluid
called lymph and are arranged parallel to blood vessels. The lymph capil-
laries absorb interstitial fluid from tissues, lymph vessels transport it towards
larger collecting ducts, such as the right and left (thoracic) lymphatic duct,
from where the fluid returns to the bloodstream. Every day 8–12 L of lymph
are transported through a human body. The unidirectional flow of the lymph
is controlled by large number of intraluminal valves. In contrast to the cardio-
vascular system, the lymphatic system is not a closed system and has no active
pump. Nevertheless, like in blood vessels there is active movement of immune
cells along endothelial cells, which, however, is far slower than the movement
of erythrocytes in the blood. Lymph nodes connect the lymphatic system
with the blood system and are the sites where lymphocytes “jump” from one
system to the other.
Box 1.5: The thymus The thymus is a primary lymphoid organ that is located
beneath the sternum in the upper front part of the chest, stretching upwards
towards the neck. Due to postnatal antigen stimulation, the thymus increases in
size of up to 50 g until puberty. At that rather early age the organ already starts
to become atrophic and regresses due to the replacement of thymic stroma
with adipose tissue. The main function of the thymus is to provide an inductive
environment for the development of T cells from pro-T cells that derive from the
bone marrow. Moreover, stromal cells of the thymus are used for the selection
of functional and self-tolerant T cells, i.e., the thymus has a major role in central
tolerance (Sect. 9.1).
Lymph node Naïve

without antigen T cell
High endothelial
venule Activated
T cell
Peripheral tissue
site of infection/
Microbes
vessel
Blood vessel
Lymph node lymphatic

with antigen vessel
vessel
Left brachio-
cephalic vein Thoracic
to heart duct
Fig. 1.6 T cell recirculation. Mature, naïve T cells leave the bloodstream and enter lymph nodes via
high endothelial venules, while antigen-presenting dendritic cells enter lymph nodes via lymphatic
vessels. Activation of a T cell is initiated after the specific interaction of its TCR with an antigen.
After activation, the T cell proliferates via clonal expansion, i.e., the proliferation of the exact same
T cell to up to 50,000 identical copies that differentiate into effector cells. These cells return to the
circulation via efferent lymph vessels and the thoracic duct. At sites of inflammation, effector and
memory T cells leave the bloodstream and enter the respective peripheral tissues through venules
Naïve as well as memory B and T cells cannot be distinguished morphologically

in a microscope, since they are small (diameter 8–10 μm), have a large nucleus
with dense heterochromatin and only a thin rim of cytosol. In contrast, activated
lymphocytes are larger (diameter 10–12 μm), have more cytosol and organelles
and are called lymphoblasts. The half-life of naïve B and T cells is 1–3 months. In
homeostasis, the pool of naïve B and T cells is constant, i.e., the number of daily
produced lymphocytes equals those that are dying.
Secondary lymphoid organs are the sites of lymphocyte activation, since they
provide an optimized environment for the interaction of B and T cell interaction with
antigens. Secondary lymphoid organs are lymph nodes (Box 1.6) and the spleen
(Box 1.7) but also pharyngeal nasopharyngeal tonsils, the appendix, Peyer’s patches
in the gut and mucosal-associated lymphoid tissues. At these sites antigen-presenting
cells and lymphocytes meet and antigens are concentrated (Chaps. 4–6). Moreover,
secondary lymphoid organs filter body fluids, such as blood lymph and mucosal
contents, to capture and display antigens, in particular to B cells. In order to increase
the chance for antigen contact ever further, naïve lymphocytes often circulate from
one secondary lymphoid organ to another. The latter applies in particular to T cells
(Fig. 1.6), i.e., they are more mobile than B cells. This is an example of “leukocyte
homing”. This mechanism is very efficient and leads to a daily net flux of some 25
billion lymphocytes through the about 500 lymph nodes of a human body. Other
examples of leukocyte homing are the recruitment of activated effector lymphocytes
(as well as monocyte and neutrophils) to tissue of the origin of the antigen, for which
they carry a specific antigen receptor. Moreover, memory B and T cells migrate into
mucosal tissues, skin and other tissues. The homing and recruitment of leukocyte is
based on the interaction of the immune cells and the endothelial cells lining blood
vessels at the site of injury or infection via adhesion molecules on the surface. The
latter are activated by cytokines and chemokines produced by inflammatory reactions
of macrophages, neutrophils and dendritic cells (Sect. 2.3).
Box 1.6: Lymph nodes Humans have approximately 500 lymph nodes, which
are part of the lymphatic system (Box 1.4). Via several afferent lymph vessels
lymph nodes allow the lymph (and cells contained in it) to pass back to the
blood (Fig. 1.7). Lymph nodes clusters at the proximal ends of legs and arms
and in the neck, where lymph is collected from regions of the body likely to
sustain pathogen contamination from injuries. The inner portion of a lymph
node is the medulla being surrounded by the cortex on all sides except the
bottom part known as hilum, where efferent lymph vessel as well as arteries
and veins are connected with the node. This gives the otherwise spherical lymph
node a bean-shaped structure. Lymphocytes enter a lymph node via specialized
high endothelial venules in the paracortex. Germinal centers of lymph nodes
(Sect. 4.3) contain mature naïve B cells, while the medulla contains mature
naïve T cells. Dense collections of B and T cells form follicles, which change in
Dendritic cell
lymphatic vessel
Naïve
B cell
T cell zone
High endothelial B cell zone
venule
ch
T cell
B cell
Artery
Cortex Medulla
Hilum
T cell
lymphatic vessel
Fig. 1.7 The lymph node: A site where B cells and T cells meet. Naïve B and T cells leave the
blood stream by moving across the wall of the high endothelial venule of an artery passing the
lymph node. Both cell types then migrate to different zones of the lymph node following a gradient
of specific chemokines. Dendritic cells enter the lymph node via an afferent lymphatic vessel and
migrate to the T cell-rich areas, where they present the antigen that they have captured at a site of
infection
number, size and configuration depending on the functional state of the lymph
node. Thus, when lymph nodes encounter an antigen, they can significantly
expand and represent an easily detectable sign of an ongoing infection.
Box 1.7: The spleen The spleen is a highly vascularized bean-shaped organ
(12 × 7 × 5 cm) located in the left upper side of the abdomen, which can
expand in case of an infection. It acts as a filter of the blood and removes
particulate matter, aged red blood cells and platelets. Moreover, the white pulp
of the spleen is the major single site of B cell accumulation and the place where
B cells finish their maturation, such as isotype switching (Sect. 4.1). At this
secondary lymphoid organ blood-borne antigens are most likely recognized
by specific BCRs, which then induces the expansion of the respective B cell
clone and its differentiation to antibody-producing plasma cells. In this way,
the spleen is the main source of antibodies. Thus, the spleen can be considered
as a gigantic lymph node. Moreover, the spleen stores erythrocytes, in order
to replace them in the blood in case of an emergency.
Abnormal (ectopic) lymph node-like structures that form in peripheral tissues at

sites of chronic inflammation are considered as tertiary lymphoid organs. They often
form at sites of chronic infection (Sect. 8.2), graft rejection of transplanted organs
(Sect. 9.2), autoimmune diseases (Sect. 10.4) and cancer (Chap. 11).
Further Reading
1. Flajnik MF (2018) A cold-blooded view of adaptive immunity. Nat Rev Immunol 18:438–453
2. Morens DM, Fauci AS (2020) Emerging pandemic diseases: how we got to COVID-19. Cell
182:1077–1092
3. Simon AK, Hollander GA, McMichael A (2015) Evolution of the immune system in humans
from infancy to old age. Proc Biol Sci 282:20143085
Chapter 2
Innate Immunity and Inflammation
Abstract The innate immune system has effector functions that allow a very early
response to invading pathogens as well as to non-microbial danger signals. In this
chapter, we will discuss the properties and functions of myeloid cells of innate immu-
nity, such as monocytes, dendritic cells, macrophages and neutrophils. The latter two
cell types perform phagocytosis, i.e., a process of ingesting extracellular substrates,
such as microbes, followed by intracellular destruction. The recognition of PAMPs
via pattern-recognition receptors starts a signal transduction cascade that involves
the activation of the inflammasome and the secretion of cytokines, such as inter-
leukin (IL) 1β. Also, non-microbial danger-associated molecular patterns (DAMPs)
can induce comparable inflammatory responses. Inflammation is mostly local but
can also be systemically and depending on cytokine levels it may be protective or
pathologic, such as in sepsis. We will finish this chapter by describing the functions
of lymphoid cells of innate immunity, such ILCs and NK cells.
Keywords Innate immunity · Pattern-recognition receptors · PAMPs · DAMPs ·

Myeloid cells · Macrophages · Neutrophils · Phagocytosis · Chemokines ·
Cytokines · Inflammation · Inflammasome · Innate lymphoid cells
2.1 Pathogen- and Danger-Associated Molecular Patterns

and Their Receptors
Microbes contain uniquely (or at least at far higher concentrations than host cells)
different types of PAMPs (Table 2.1). PAMPs are relatively invariant structures shared
by large groups of microbes, such as nucleic acids like double-stranded RNA in
viruses and unmethylated DNA sequences in bacteria, proteins containing the unusual
amino acid N-formylmethionine, lipids and carbohydrates like LPS, lipoteichoic acid
and terminal mannose residues, which are recognized by one or more members of
the pattern-recognition receptor family found in cells of the innate immune system.
Pattern-recognition receptors are located in the plasma membrane, the membrane of
phagocytic vesicles like endosomes and lysosomes and in the cytosol of different
cell types of the innate immune system (Fig. 2.1). Importantly, some of these
PAMPs are indispensable for the survival of the microbes, i.e., they cannot easily

https://doi.org/10.1007/978-3-031-04025-2_2
20 2 Innate Immunity and Inflammation
Table 2.1 Key PAMPs and DAMPs as well as the receptors recognizing them
PAMPs Microbe type Pattern-recognition
receptor
Nucleic acid Single-stranded RNA Virus, parasite TLR7, TLR8, RLRs
Double-stranded RNA Virus TLR3, NRLs
Cytosolic DNA Virus, bacteria CDS
Non-methylated CpG Virus, bacteria, TLR9
parasite
Ribosomal RNA Bacteria TLR13
Protein Peptidoglycans Bacteria TLR2, NRLs
Flagellin Bacteria TLR5
Profilin Parasite TLR11, TLR12
Lipoprotein Bacteria, parasite TLR2, TLR6
Lipids LPS Gram-negative TLR4
bacteria
Lipoteichoic acid Gram-positive TLR2, ficolins
bacteria
Carbohydrates Mannan Fungi, bacteria C-type lectins
Glucans Fungi Dectins
DAMP
Stress-induced Heat shock proteins TLR2, TLR4
proteins
Crystals Monosodium urate Inflammasome
Proteolytically Proteoglycan peptides TLR4
cleaved extracellular
matrix
Mitochondria and Formylated peptides and FPR1
mitochondrial ATP
components
Nuclear proteins High-mobility group TLR2, TLR4
box 1
Single- and double-stranded RNAs, which make up the genomes of some viruses and are generated
during the life cycle of most viruses, are primarily distinguished from respective RNAs of the host
cell via their location in endosomes, which reflects their origin by phagocytosis of microbes. Flag-
ellin is a protein subunit component of the flagella of motile bacteria. ATP = adenosine triphosphate;
CpG = cytosine-guanine dinucleotide; FPR1 = formyl peptide receptor 1
change by mutations. In this way, the innate immune system has evolutionary
adapted to key molecules of microbes and ensures that microbes cannot evade
innate immunity. In contrast, microbes use many ways to evade recognition by the
adaptive immune system via loss or mutation of antigens recognized by BCRs or
TCRs (Chaps. 7 and 8). DAMPs are recognized by the same mechanisms as PAMPs,
i.e., by the same set of pattern-recognition receptors as well as soluble molecules in
2.1 Pathogen- and Danger-Associated Molecular … 21
Extracellular
Fungal polysaccharide
Bacterial cell TLR
wall lipid Lectin
Plasma membrane
RLR Endosomal membrane
viral DNA
Endosomal
CDS
Microbial DNA, RNA
Microbial DNA
TLR
NLR
Bacterial peptidoglycan
Cytosolic
Fig. 2.1 Pattern-recognition receptors and their location. In innate immune cells pattern-
recognition receptors are located either in the plasma membrane (TLR1, 2, 4, 5 and 6, C-type
lectin receptors, as well as FPR1 and dectins (not shown)) recognizing extracellular PAMPs, in the
membrane of endosomes (TLR3, 7, 8 and 9) binding to microbial nucleic acids and in the cytosol
(NLRs, RLRs and CDS) interacting with cytosolic PAMPs and DAMPs
the blood and mucosal secretions. DAMPs are often altered endogenous molecules
that are the result of cell damage or death caused by infections or trauma, toxins,
burns or loss of blood supply, i.e., in many cases microbes are not involved in the
activation of innate immunity.
The cell types of innate immunity, in particular phagocytes and dendritic cells,
recognize sites of infections through PAMPs as well as tissue damage through
non-microbial endogenous DAMPs. In humans, the PAMP and DAMP recognition
process is based on only approximately 60 different pattern-recognition receptors,
such as TLRs (Table 2.1). This is a limited set of receptors compared with the
millions of different BCRs and TCRs mediating adaptive immunity. In this way,
the innate immune system is recognizing only some 1000 different molecules
deriving from microbes or damaged cells. This explains the reduced specificity
of the innate immune system in recognizing only general patterns mostly shared
by a large number of antigens. However, most cells of the innate immune system
are already effector cells before or very shortly after antigen exposure, while
naïve B and T cells need a week to become effector cells and then start their
fight against microbes. When membrane-bound pattern-recognition receptors like
TLRs as well as cytosolic receptors like CDS (cytosolic DNA sensor), NLR (NOD-
like receptor) and RLR (RIG-like receptor) are stimulated by PAMPs or DAMPs,
they initiate signal transduction cascade via activating the transcription factors NFkB
(nuclear factor κB), AP1 (activation protein 1), IRF3 (interferon response factor 3)
and IRF7. NFkB and AP1 regulate the expression of genes involved in inflammatory
responses, such as the inflammatory cytokines TNF (tumor necrosis factor) and IL1β,
the chemokines CCL2 (C–C chemokine ligand 2) and CXCL8 (chemokine C-X-C
motif ligand 8) as well as endothelial adhesion molecules. IRF3 and IRF7 stimulate
the expression of different types of interferons (IFNs) being important cytokines of
innate immunity against viruses (Sect. 8.1). Cytosolic pattern-recognition receptors
are the key sensors for infections with microbes, since part of their life cycle happens
in the cytosol of host cells. Moreover, some microbes are able to escape the phago-
cytotic process in endosomes and lysosomes and enter the cytosol (Sect. 2.3). In
addition, there are pattern-recognition receptors like pentraxins, C-lectin receptors,
ficolins and members of the complement system (Sect. 7.2) that recognize PAMPs in
the blood and extracellular fluids. These proteins facilitate the clearance of microbes
from the latter fluids by enhancing their recognition by immune cells like phagocytes
(Sect. 2.3) and NK cells (Sect. 2.5). All pattern-recognition receptors of the innate
immune system are encoded by individual genes, while BCRs and TCRs of the
adaptive immune system use somatic recombination of gene segments of only a few
genes for reaching their vast diversity (Sect. 3.4).
2.2 Myeloid Cells of Innate Immunity
The myeloid compartment of innate immunity comprises circulating monocytes,

neutrophils, basophils and eosinophils as well as macrophages, dendritic cells and
mast cells in tissues (Fig. 2.2). Monocytes have a poly-shaped nucleus and fine gran-
ules as well as phagocytic vacuoles in the cytosol. There are different subsets of mono-
cytes, but the majority of them produce inflammatory mediators, are phagocytic and
are rapidly recruited to sites of infection or tissue injury. Depending on the cocktail of
additional signals, such as cytokines (Box 2.1) and chemokines (Box 2.2), to which
monocytes are exposed within a tissue, they differentiate into dendritic cells and
macrophages. Thus, a central role of monocytes is to refill the pool of macrophages
and dendritic cells in response to inflammation and other stimuli. The differen-
tiation of monocytes is based on epigenome changes in response to contacts with
antigens, such as infectious microbes. These epigenetic changes create a memory of
the microbe encounter. Dendritic cells are the most efficient antigen-presenting cells
2.2 Myeloid Cells of Innate Immunity 23
Innate immunity
Monocyte Macrophage NK cell
Band neutrophil Mature neutrohil Aged neutrophil

Erythrocytes
Granules
Nucleus
Cytoplasm
Eosinophil Basophil
Adaptive immunity
Lymphocyte Active lymphocyte Plasma cell
Fig. 2.2 Microscopic pictures of cells of innate and adaptive immune system. Above, cells of
innate immunity are show. Monocytes have some vacuoles, are quite round shaped but display a poly-
shaped nucleus. Macrophages display numerous vacuoles and have a more flexible cytosol, both
representing activation. NK cells are rather large cells with a high cytosol-nucleus ratio. Within the
cytosol many small granules can be found. All neutrophils found in the peripheral blood have effector
function. Band neutrophils represent young neutrophils and are a sign for an acute infection (mostly
bacterial infection) whereas in steady-state mostly mature neutrophils with a dense, polymorphic
nucleus, which is typically subdivided into segments, are found. Therefore, aged neutrophils display
several segments. Eosinophils are densely packed with granules containing their effector molecules.
These red stained granules gave this granulocyte subtype its name. Likewise, basophils display dark
blue granules. Below, cells of adaptive immunity are shown. Lymphocytes cannot be distinguished
between T and B cells as well as ICLs morphologically. But the ratio of cytosol and nucleus represent
their activity state. Plasma cells display a blue cytosol. Of note, the Golgi complex displays as a
bright area in close contact to the nucleus. Microscopical pictures were taken by E. Velleuer after
preparation of smears from peripheral blood samples and stained according to Pappenheim or with
Diff-Quick
and instruct T cells of the adaptive immune system. The latter happens at lymph
nodes, for which the dendritic cells migrate from infected or damaged peripheral
tissues (Sect. 5.1).
Box 2.1: Cytokines Most cytokines are rather small proteins that are secreted
by immune cells (as well as by many other cell types) and influence the behavior
of other cells. For example, they positively or negatively regulate the intensity
and duration of the immune responses, such cellular proliferation, survival,
migration or differentiation. ILs are a large family of cytokines systematically
named IL1 until IL36 (in human). Similarly, there are 33 human IFNs with
major subgroups alpha, beta and gamma. In contrast, other cytokines, such as
TNF, still have individual names that often rely to the history of their discovery.
Cytokines can act autocrine on the cells that release them or paracrine on
cells in their vicinity depending on that the target cells express the respective
cytokine receptor. However, the systemic release of cytokines can lead to a
life-threatening cytokine storm or septic shock. Therefore, cytokine produc-
tion is tightly regulated on the level of gene expression as well as on mRNA
and protein stability.
Box 2.2: Chemokines Most chemokines are small polypeptides (8–10 kDa)
that are secreted by leukocytes and platelets but also by endothelial cells,
epithelial cells and fibroblasts. Some chemokines are secreted in response to
external signals, such as TNF stimulation, and are involved in inflammatory
reactions, while others are produced constitutively and maintain the distribu-
tion of immune cells, such as B and T cells in lymph nodes (Box 1.6). In
inflammation, chemokines recruit circulating leukocytes, such as monocytes
and neutrophils, from the blood to infected or damaged tissues. In humans there
are 47 different chemokines in four families (CC, CXC, C and CX3C) defined
via cysteine residues forming internal disulfide loops. The nomenclature of the
two major families is CCL or CXCL that bind to a signal through the respec-
tive receptors CCLR or CXCLR of the superfamily of seven-transmembrane G
protein-coupled receptors. When these receptors are activated by their specific
ligands, they stimulate signal transduction cascades that result in morphological
changes, such as cytoskeletal changes and increased cell motility. The migra-
tion of leukocytes is often stimulated by a chemokine concentration gradient,
referred to as chemotaxis. Thus, leukocytes migrate towards infected and
damaged cells in tissues, where chemokines are produced. Similarly, the
migration of dendritic cells from sites of infection into draining lymph nodes
is directed by chemokines. For example, when dendritic cells encounter a
2.2 Myeloid Cells of Innate Immunity 25
microbe, they express the chemokine receptor CCR7, which is stimulated by

chemokines produced in the lymphatic system (Sect. 5.1). Since naïve T cells
also express CCR7, they are able to find the dendritic cells, once they have
reached the lymph node.
Macrophages are together with neutrophils the key phagocytic cells of our body
(Fig. 2.3). The blood counts of neutrophils (Table 1.4) are more than 10-times higher
Pathogen
Basophils and
Dendritic cells Macrophages Neutrophils
mast cells
· Antigen presen- · Phagocytosis · NETs

tation · Cytokines · Cytokines
· Phagocytosis · Anti-microbial · ROS
· Cytokines peptides · Degranulation
· IFNγ · IFNγ · Cytokines
· ROS
Cytokine and Adhesion Co-stimulatory

FcγR
chemokine molecule receptor
receptor Cytokines
Complement
MHC-II TLR FcεR
receptor
Fig. 2.3 Functional profile of myeloid cells of the innate immune system. Dendritic cells,
macrophages, mast cells, basophils and neutrophils are different types of effector cells of the innate
immune system and all belong to the myeloid line. The cell types express partially overlapping
groups of membrane receptors, such as TLRs, cytokine and chemokine receptor, MHC proteins,
FcRs, adhesion molecules and receptors for co-stimulatory molecules and complement proteins.
This is the basis of the specificity of the responses listed below
than that of monocytes but the cells survive only for hours to days. Therefore, an
adult has to produce every day some 1011 neutrophils, i.e., the vast majority of the
daily produced leukocytes, in order to replace the loss. Rapidly after the entry of
microbes, neutrophils migrate to sites of infection, where they have a number of
effector functions for the fight against microbes (Sect. 2.3).
Basophils in the blood and mast cells in tissues are the key cell types performing
the battle against parasitic infections. Since the latter became in the past 100 years
significantly more seldom, at least in industrialized countries, the cells are nowa-
days more often recognized in context of allergic reactions (Sect. 10.2). Mast cells
are found in skin, mucosal epithelia and connective tissues adjacent to small blood
vessels and nerves. They contain numerous granules being filled with preformed
inflammatory mediators like histamine and acidic proteoglycans, which are released
within minutes after the stimulation of the cells, e.g., in the fight against larger
microbes like helminths (worms). Moreover, in a second wave mast cells secrete
cytokines like TNF and other inflammatory mediators. Eosinophils in the blood are
also involved in the fight against parasites via the release of enzymes that are harmful
to the cell walls of parasites but also act against bacteria and viruses.
All myeloid cells of innate immunity express cytokine and chemokine receptors
and most of them TLRs (Fig. 2.3). Dendritic cells are specialized using MHC-II
proteins for antigen presentation (Sect. 5.1). Basophils and mast cells present the
antibody receptor FcεRI (fragment crystallizable (Fc) epsilon receptor type I) on
their surface specifically binding IgE (Sect. 10.1). The direct or indirect contact of
myeloid cells with PAMPs and DAMPs results in effects on gene expression that
are often stronger than in any other tissue or cell type of our body. The strong
reaction is necessary, since bacteria proliferate far faster than human cells and may
represent immediate danger to our body. However, the different myeloid cells of
innate immunity differ in their ability to detect danger signals via different types of
membrane receptors and to activate specific effector functions as a result of signal
transduction cascades activated by these proteins. The strength and specificity of
the response of the immune cells, such as different populations of macrophages,
depends on their epigenomic profile before encountering microbes. This implies
that the proper epigenomic programing of our immune cells before contact with
antigen is essential for an optimal response. Thus, proper epigenomic programing
of our immune cells during hematopoiesis and antigen encounter is critical for
a well-functioning innate immune system.
Neutrophils, basophils and eosinophils are referred to as granulocytes, since they
develop from the same progenitor and have granules in their cytosol. Granules
are vesicles filled with aggressive molecules like histamine that can be secreted,
Neutrophiles contain two types of granules, of which one is filled with enzymes
like lysozyme, collagenase and elastase and the other with anti-microbial peptides
like defensins and cathelicidins (CAMPs). Defensins form a pore in the membrane
of microbes causing its permeabilization and cell death, while CAMPs destroys the
lipoprotein membranes of microbes. Moreover, granulocytes are characterized by
a dense, polymorphic nucleus, which is typically subdivided into several segments
(Fig. 2.2). In contrast, other innate immune cells, such as monocytes, dendritic cells,
2.3 Mechanisms of Phagocytosis 27
NK cells, ILCs as well as lymphocytes, are mononuclear cells. Neutrophils, basophils

and eosinophils circulate in the blood and have a half-life spanning from only a few
hours to days. In contrast, when eosinophils enter tissues, they can survive like mature
mast cells for weeks to months. Thus, although basophils, eosinophils and mast
cells share a number of effector functions, their location significantly influences
their lifespan.
2.3 Mechanisms of Phagocytosis
Neutrophils and monocytes circulate via the blood throughout the body and perform
surveillance for possible sites of infection or tissue damage. Although the cells are
able to detect and destroy bacteria in the blood, their main site of effector func-
tions are extravascular sites, where monocytes differentiate into macrophages and
dendritic cells. The relative amounts of recruited neutrophils and monocytes depends
on their relative expression of adhesion molecules and chemokine receptors. For
example, neutrophils express the chemokine receptors CXCR1 and CXCR2, which
bind the chemokine CXCL8 produced by tissue-resident macrophages (Box 2.3).
In contrast, when these macrophages rather secrete the chemokine CCL2, primarily
monocytes are recruited that carry the chemokine receptor CCR2 on their surface.
Neutrophils are faster than monocytes (differentiating to macrophages) concerning
entering infected or damaged tissues, but their half-life in inflamed tissues is with
1–2 days significantly shorter. This explains why pus does not only contain dead
pathogens and cellular debris but also large amounts of dead neutrophils. Neutrophils
are terminally differentiated cells that largely stopped gene expression, i.e., in their
short life they mainly rely on the action of preformed proteins including enzymes.
In contrast, the effector function of macrophages like the production of cytokines
(Fig. 2.4) is primarily based on induced gene expression. Therefore, at later stages
of the immune response, i.e., several days after onset of infection, macrophages
are the dominant effector cells.
Box 2.3: Tissue-resident macrophages In addition to macrophages that

derive from differentiated monocytes, there are tissue-resident macrophages
at strategic points throughout the body, where there is a high likelihood of
microbial invasion or accumulation of foreign particles. Therefore, populations
of tissue-resident macrophages are found in most tissues and organs, such as
Kupffer cells in the liver, Langerhans cells in the skin, alveolar macrophages
in pulmonary alveoli, microglia in the CNS and osteoclasts in bone. Under
resting conditions most tissues contain only a few resident macrophages,
while at acute inflammation (Sect. 2.4) the number of macrophages drasti-
cally increases based on monocytes entering the tissue and differentiation to
Classically activated Alternatively activated

(M1-type) Mircobial (M2-type)
TLR ligands, LPS
IFNγ
Monocyte
IL13, IL4,
TGFβ
ROS, NO, lyso- IL1, IL12, IL23 IL10, Proline poly-
somal enzymes chemokines TGFβ amines, TGFβ
Anti-microbial Anti- Wound

actions: mation repair,
phagocytosis
and killing of
many bacteria
and fungi
Fig. 2.4 Activation of macrophages. In tissues monocytes differentiate into M1-type macrophages
(classically activated macrophages), when they are stimulated by LPS or the cytokine IFNγ, or into
M2-type macrophages (alternatively activated macrophages), when they are exposed to the cytokines
IL4, IL13 and TGFβ1. M1- and M2-type macrophages represent the extremes of a continuous
spectrum. More details are provided in the text
macrophages. Tissue-resident macrophages have a wide variety of homeo-

static and immune surveillance functions ranging from the clearance of cellular
debris, the response to infections and the resolution of inflammation. However,
they are not derived from monocytes but from yolk sac and the fetal liver, i.e.,
they were positioned already before birth in the respective tissues. Tissue-
resident macrophages are maintained also during adult life independently of
monocytes by self-renewing and are not exchanged during a bone marrow
transplant, where the rest of the immune system is replaced by that of another
person (Sect. 9.2).
Depending on the signals the monocytes are exposed to during their differentiation
in tissues, they develop into a wide spectrum of functionally different macrophages
(Fig. 2.4). For simplicity only the endpoints of this spectrum, M1-type (classically
activated) and M2-type (alternatively activated) macrophages, are discussed. M1-
type macrophages derive from monocytes that in tissues get in contact with PAMPs,
2.3 Mechanisms of Phagocytosis 29
such as LPS (Table 2.1), and cytokines confirming the presence of bacteria. The
macrophages then secrete further pro-inflammatory molecules, in order to sustain
the inflammatory reaction. This provokes the adaptive immune system to respond
through the proliferation of T helper (TH ) cells type 1 (TH 1) (Sect. 6.2). TH cells
are effector T cells that are characterized by the expression of the surface molecule
CD4 (Box 2.4) (Sect. 5.3). In contrast, M2-type macrophages are formed, when the
monocytes are exposed to the cytokines IL4 and IL13. These types of macrophages
exert an almost opposite immuno-phenotype (Box 8.6) than M1-type macrophages.
They do not produce ROS (reactive oxygen species) or NO (nitric oxide) required for
killing of microbes but provoke immunotolerance and TH 2-type immune responses.
M2-type macrophages produce anti-inflammatory molecules, such as TGFβ1 (trans-
forming growth factor beta 1, Sect. 9.1), IL10 or IL1RN (IL1 receptor antagonist),
and inhibit the secretion of pro-inflammatory cytokines. In the following, we will
discuss primarily the function of M1-type macrophages in phagocytosis (this section)
and inflammation (Sect. 2.4) and come back later to the anti-inflammatory actions of
M2-type macrophages in the context of tissue repair, allergies and cancer (Sects. 6.3,
10.1 and 11.1).
Box 2.4: Cluster of differentiation Immune cells are regularly phenotyped

(Box 8.6), e.g., by the flow cytometry method fluorescence-activated cell
sorting (FACS), by a set of more than 370 surface proteins, referred to as the
cluster of differentiation (CD). CD molecules often act as receptors or ligands
initiating a signal transduction cascade (Sect. 4.2), but some of them also func-
tion as adhesion proteins. A criterium for being member of the list of CDs
is that two independent monoclonal antibodies recognize the surface protein.
Immune cells are usually defined by the CD molecules that they are expressing
(“+”) or sometimes also by the molecules that they are not expressing (“−”).
For example, “CD3+ , CD4+ , CD8− ” cells represent TH cells, while “CD3+ ,
CD4− , CD8+ ” cells are cytotoxic T cells. CD3 is associated with the TCR,
i.e., its expression is a marker for a T cell, while CD4 interacts with MHC-II
proteins and CD8 with MHC-I proteins (Sect. 5.3). In addition, sometimes the
index hi, mid or low is used, in order to indicate the expression level of a given
CD molecule.
Phagocytosis is an active, energy-dependent multistep process that aims on a

destruction of microbes (and other large particles with a diameter of more than
0.5 μm) in phagocytes like macrophages and neutrophils (Fig. 2.5). The first step
is the binding of the microbe by receptors on the surface of phagocytes. When
the microbe contains a PAMP that is bound by a pattern-recognition receptor, it is
bound directly. However, also indirect binding is possible via receptors that recog-
nize molecules, such as complement proteins (Sect. 7.2) or antibodies (Sect. 4.1), that
cover (opsonize) the microbe. The latter option is an example of an efficient inter-
action of the adaptive immune system (providing with the antibody a high-affinity
Microbes bind to Lectin receptor

pattern-recognition
receptors
Phagocyte membrane Lysosome with

zips up arround microbe enzymes
Fusion of
phagosome
Activation of phagocyte with lysosome
Arginine
NOS2 Citrulline
Killing of microbes by ROS, NO

NO and proteolytic enzymes
in phagolysosomes
Phagolysosome ROS
O
Phagocyte
oxidase
Fig. 2.5 Phagocytosis is a multistep process. Phagocytes like macrophages and neutrophils use
pattern-recognition receptors to bind directly to microbes or to microbes opsonized by complement
proteins. This allows the internalization of the microbes, so that they end up inside of phagosome
vesicles in the cytosol. The phagosomes fuse with lysosomes, in which the microbes get in contact
with aggressive chemicals like ROS and NO and proteolytic enzymes. This will kill und lyse the
microbes
2.4 Inflammation 31
sensor for the microbe) and the phagocyte of the innate immune system taking over
the elimination of the microbe. In a second step the microbe will be internalized
by an endocytosis process, so that the microbes are engulfed by an endosome-like
membrane vesicle referred to as phagosome. Phagosomes then fuse with a lysosome,
which is an organelle that contains reactive chemicals like ROS and NO produced by
the enzymes phagocyte oxidase and inducible NOS (NO synthase), respectively, and
proteolytic enzymes like the serine proteases elastase and cathepsin G. The contact
with these aggressive chemicals and proteins kills the microbes and lead to their
lysis. In a similar way, macrophages not only remove microbes but also senescent
or necrotic host cells including dying neutrophils. Macrophages engulf apoptotic
cells before they release their contents and may induce inflammation. This clearing
process is important for returning to homeostasis after an infection or an injury and
is performed by M2-type macrophages. In this way, macrophages promote the
repair of damaged tissues, including stimulating the growth of new blood vessel
(angiogenesis) and the synthesis of collagen-rich extracellular matrix (fibrosis).
Some microbes, such as Candida albicans fungi, produce large hyphae and fila-
ments, which are too large for be phagocytosed. In this case neutrophils release
NETs (neutrophil extracellular traps) (Fig. 2.3), which are an extracellular struc-
ture consisting of genomic DNA and histones to which anti-microbial effector
molecules like lysozyme, elastase and defensins are bound that trap and kill microbes.
However, the extrusion of nuclear contents leads also to the death of the respective
neutrophil. After clearing the infection, neutrophils secrete resolvin proteins that
support recovery of the tissue.
2.4 Inflammation
Throughout our body immune cells are found that can mediate pro-
inflammatory as well as anti-inflammatory responses via the secretion of
cytokines, hormones and neuropeptides. These cells are in strategic positions to
sense, process and communicate signals on the perturbation of our body. Eradicating
pathogens is probably the most important result of these inflammatory responses
(Sect. 10.5) that are directed against:
• intracellular bacteria and viruses (type 1 immune response)
• helminths (type 2 immune response)
• extracellular bacteria and fungi (type 3 immune response).
Clinically, inflammation is characterized by:
• erythema and hyperthermia caused by increased blood flow to inflammatory
lesions
• swelling due to fluids and cells entering the tissue
• pain based on nerves sensing the swelling
• collateral tissue damage.
On the cellular level, phagocytes like neutrophils and macrophages secrete a

number of signaling molecules in response to their activation by PAMPs derived
from infection or injury. These cytokines, chemokines and growth factors affect the
migration and activity of other immune cells. Moreover, important plasma proteins,
such as complement proteins, antibodies and acute-phase proteins, enter the site of
infection or injury. The resulting accumulation of leukocytes, proteins and fluids
derived from blood in extravascular tissue is called acute inflammation. Due to the
short half-life of neutrophils, most inflammatory lesions are dominated by monocyte-
derived macrophages. They perform their effector functions like killing microbes
(Sect. 2.3). The combined action of innate and adaptive immune cells (Chaps. 4 and
6) eradicates infectious microbes but also results in collateral tissue damage, such
as cytotoxicity due to ROS production and the degradation of extracellular matrix
via proteases. After the clearance of pathogens and the removal of dead neutrophils
by macrophages, there is recruitment or phenotypic switching of macrophages into
the M2-type, i.e., into a pro-resolving phenotype. Therefore, inflammation usually
resolves within a few days to weeks and the host returns to homeostasis.
Inflammation can also derive from DAMPs that are based in changes in the
concentration of nutrients and metabolites, which occurs in many lifestyle-related
diseases, such as obesity, insulin (INS) resistance, type 2 diabetes and atheroscle-
rosis. Moreover, also neurodegenerative diseases, such as Alzheimer’s disease, most
types of cancer, allergy and autoimmune diseases, have an inflammatory component.
In these cases, the immune system often cannot eliminate the primary stimulus, so
that chronic inflammation develops. Sites of chronic inflammation often undergo
tissue remodeling including angiogenesis and fibrosis. While acute inflammation is
dominated by the innate immune system, in chronic inflammation components of
the adaptive immune system, such as cytokines derived from T cells or antibodies
produced by B cells, take a more prominent role.
Mechanistically inflammation is explained by cytosolic multiprotein complexes,
referred to as inflammasomes. The core protein of the inflammasome is a cytosolic
pattern-recognition receptor, which mostly origins from the NLR family (Fig. 2.6).
The main function of the inflammasome is to generate active forms of the pro-
inflammatory cytokines IL1β and IL18 within minutes after stimulation of the cell
with cytosolic PAMPs and DAMPs. Both cytokines are stored in the cell in an inac-
tive form that requires proteolytic activation. The latter is performed by the cysteine
protease caspase (CASP) 1, which is in its inactive conformation part of the inflam-
masome complex. The adaptor protein ASC (apoptosis-associated speck-like protein
containing a CARD) connects the CASP with the pattern-recognition receptor, so
that the activation of the latter by a PAMP or DAMP results in the activation of the
CASP. Since this signal transduction process is based on protein–protein interactions
and enzymatic actions but not on new gene transcription and protein translation, the
cells release within minutes active IL1β and IL18.
The inflammasome assembles only, when its sensory component detects a change
in the levels of cytosolic molecules like an increase in microbial products (PAMPs)
or in crystals derived from environmental or endogenous compounds (DAMPs) or
2.4 Inflammation 33
Pathogenic bacteria
Extracellular ATP K+
Plasma membrane
K+
NLRP3 Bacterial products
(sensor) + Crystals
ASC K+
(adaptor) + ROS
CASP1
(inactive enzyme)
innate signals
(e.g., via TLRs)
NLRP3
pro-IL1β
CASP1
(active)
IL1B gene
transcription
secreted IL1β
Nucleus
IL1β
Fig. 2.6 Function of the inflammasome. At the example of the NLRP3 inflammasome the molec-
ular mechanism of acute inflammation is illustrated. The inflammasome is a cytosolic protein
complex that is composed by multiple copies of a sensor for PAMPs and DAMPs, such as the
pattern-recognition receptor NLRP3, of the adaptor protein ASC and of the inactive form of the
protease CASP1. Stimulation of the inflammasome by a PAMP or DAMP leads to the activation of
the CASP, which processes inactive pro-IL1β into active IL1β (as well as pro-IL18 into IL18). In
this way, within minutes after a stimulus the cell secretes the pro-inflammatory cytokines IL1β and
IL18 and the process of acute inflammation starts
reduction in K+ ion concentration. Realizing that crystalline substances potently acti-

vate the inflammasome provides a mechanistic understanding to some inflammatory
diseases. For example, the painful inflammatory condition of joints, named gout, is
associated with deposition of monosodium urate crystals in joints. Accordingly, IL1
antagonists are nowadays used for the therapy of severe forms of the disease. Simi-
larly, excessive amounts of endogenous substances, such as cholesterol crystals
within macrophages, free fatty acids and lipids in adipose tissue and β-amyloid
in neurons, were found to act as DAMPs inducing the inflammasome in the
context of atherosclerosis, the metabolic syndrome and Alzheimer’s disease,
respectively. Furthermore, there are a few autoinflammatory syndromes, such as
familial Mediterranean fever, where the inflammasome is inappropriately triggered
by an autosomal gain-of-function mutations in one of its components. Therefore, in
these cases IL1 antagonists are an effective therapy.
The cytosolic protein gasdermin D is another target of CASP1, which then
oligomerizes and forms a pore in the plasma membrane leading to ionic imbal-
ance and osmotic lysis. This form of programmed cell death is called pyroptosis
and happens in macrophages and dendritic cells but not in neutrophils and most
other cell types. The process is characterized by swelling of cells, loss of plasma
membrane integrity and the release of the pro-inflammatory cytokines TNF, IL6 and
IL8. Pyroptosis amplifies the inflammatory response and enhances the clearance of
bacteria but may also lead to a septic shock (Sect. 7.2).
The main cytokines of innate immunity are TNF, IL1 and IL6, which are primarily
produced by tissue macrophages and dendritic cells. At local inflammatory lesions
these cytokines act in a paracrine fashion on cells in their vicinity (Fig. 2.7, left). This
enhances the production of adhesion molecules by endothelial cells and increases
permeability of blood vessels. Moreover, in combination with chemokines (Sect. 2.2)
the cytokines attract monocytes and neutrophils to the site of infection. All these
actions increase the local number of cells that can fight infections and repair tissues.
In cases of more severe infection the production of cytokines is elevated so much
that significant amounts of them enter the circulation, which causes systemic effects.
The protective effects of systemic cytokines are (Fig. 2.7, center):
• enhancing the production of neutrophils from progenitor cells in the bone marrow
• production and secretion of acute-phase proteins like the soluble pattern-
recognition factors CRP (C-reactive protein) and SAP (serum amyloid P-
component) of the pentraxin family (Sect. 2.1). CRP levels in the blood are an
important clinical biomarker diagnosing infections or inflammatory diseases
• inducing TNF and IL1 via prostaglandin production in the hypothalamus an
increase in body temperature, i.e., causing fever.
Fever is often considered as an unwanted side effect of the fight of the immune
system against infections, so that often prostaglandin synthesis inhibitors, such as
aspirin and ibuprofen, are used as therapy. However, elevated body temperature
relates to the enhanced metabolic functions of immune cells, decreases the
metabolism of microbes and reminds the patient to rest.
2.4 Inflammation 35
Local Systemic Systemic
Endothelial cells Brain Heart
TNF, IL1 TNF IL1, chemokines TNF, TNF

IL1, Low
IL6 output
Fever
Liver Endothelial cells/
blood vessel
Adhesion IL1, TNF
Endothelial cell
molecule IL6
Increased
permeability
Acute phase Increased
Leukocytes proteins Thrombus permeability
IL1, IL6 Bone marrow Multiple tissues
TNF, chemokines
IL1 TNF, TNF
IL1, IL6 Skeletal
muscle Insulin
resistance
Leukocyte production
Fig. 2.7 Local and systemic actions of pro-inflammatory cytokines. TNF, IL1 and IL6 are the
main pro-inflammatory cytokines of the innate immune system. They have local (left) and systemic
effects that are either protective (center) or pathologic (right). More details are provided in the text
In contrast, the pathologic effects of systemic cytokines, in particular of TNF, are

(Fig. 2.7, right):
• inhibition of myocardial contractility and vascular smooth muscle tone leading to
a marked decrease in blood pressure
• causing intravascular thrombosis via impairment of the normal anti-coagulant
properties of the endothelium as well as leakage of the capillaries
• wasting of muscle and fat cells (cachexia) due to appetite suppression and reduced
synthesis of lipoprotein lipase as well as increased glucose level as a result of a
transient insulin resistancy.
The systemic complication of severe bacterial infection (mostly in the blood) is
sepsis (Sect. 7.2), which is a syndrome characterized by fever, low blood pressure,
fast heart and respiratory rates, elevated leukocyte count, metabolic abnormalities
like INS resistance and mental disturbances. A septic shock is the most severe form
of sepsis and involves vascular collapse and disseminated intravascular coagulation.
In this way, the overreacting innate immune response can be deadly.
Nutrient deprivation and infection by pathogens are the most challenging events
for the survival of an organism. Thus, there was a co-evolution for the responses
to food via the endocrine regulation of metabolism and to infectious diseases
via the innate immune system. This suggests that there is an interface between
metabolism and immunity, which is largely mediated by macrophages. Moreover,
the basal inflammatory response increases with age, which is often referred to as
“inflammaging”, and leads to low-grade chronic inflammation that is maladaptive
and further promotes the aging process. This may be due to:
• the accumulation of senescent cells that secrete pro-inflammatory cytokines
• an increased likelihood that a failure of the immune system does not effectively
clear pathogens and dysfunctional host cells
• overactivity of the transcription factor NFκB
• a defective autophagy response ultimately leading to increased ROS production.
In all these cases, not microbes but the excess of endogenous molecules, such
as lipoproteins, saturated fatty acids or protein aggregates initiate the inflammatory
response. Metabolic dysregulation associated with chronic inflammation accompa-
nies not only aging itself but also most common age-related diseases. Thus, sterile
(i.e., non-microbial) induction of low-grade chronic inflammation is a critical
characteristic of aging as well as of metabolic diseases.
2.5 Lymphoid Cells of Innate Immunity
The lymphoid line of hematopoiesis (Sect. 1.3) produces not only cells of the adap-
tive immune system, but with ILCs and NK cells also members of the innate immune
system. ILCs and NK cells have lymphocyte-like morphology, resemble function-
ally different subtypes of T cells but have the important difference that they do not
express a TCR. Thus, they have to rely in their function and specificity of germline-
encoded receptors on their surface, such as pattern-recognition receptors (Sect. 2.1)
and cytokine receptors. The functional profile of ILCs resembles that of CD4+ TH
cell subtypes (Sect. 6.3): ILC1 act similar to TH 1 cells, ILC2 resemble TH 2 cells and
ILC3 are comparable to TH 17 cells (Fig. 2.8). Accordingly, ILC1s and TH 1 cells both
respond to intracellular pathogens like viruses and transformed cells (type 1 immune
response, Sect. 10.5), ILC2s and TH 2 cells fight together against large extracellular
parasites like helminths and respond to allergens (type 2 immune response), while
ILC3s together with TH 17 cells react on extracellular microbes like bacteria and
fungi (type 3 immune response). However, in contrast to T cells ILCs are able to
react promptly to signals from infected or injured cells like secreted cytokines and
overexpressed surface proteins. About a week after the onset of immune reaction, T
cells have undergone clonal expansion and both types of cells are active and cross-
regulate each other. For example, ILCs can express MHC-II proteins and present
antigens to T cells, while T cells secrete IL2, in order to stimulate ILCs. In addition
to these positive feedback loops, ILCs and T cells can also compete for resources
like cytokines and survival factors. Thus, ILCs and TH cells mirror each other
only partly and an orchestration of their activity over time is essential for the
proper coordination between innate and adaptive immune response.
2.5 Lymphoid Cells of Innate Immunity 37
Common lymphoid
progenitor
ID2 ILC precursor

Development IL15, IL7 IL7
of ILCs IL7
RORG
ILC1 ILC2 ILC3

IL25
IL12 IL33 IL1
IL18 TSLP IL23
Activation
of ILCs
IFNγ IL25, IL33, TSLP IL17, IL22

Intestinal barrier
Defense against
functions Defense against function;
helminths;
of ILCs viruses lymphoid
organogenesis
Type 1 reaction Type 2 reaction Type 3 reaction
Fig. 2.8 ILC subsets. The three subsets of ILCs all develop in the bone marrow from a common
precursor identified by the transcription factor ID2 (inhibitor of DNA binding 2). Under the influence
of the cytokines IL7 and IL15 and the specific transcription factors TBX21, GATA3 and RORC the
precursor cells differentiate into ILC1, ILC2 and ILC3, respectively. Each ILC subset produces a
different set of cytokines and has the indicated effector functions. In this way, ILCs are the innate
counterpart of TH cell subsets of adaptive immunity
The development of ILC1s depends on the transcription factor TBX21 (T-box tran-
scription factor 21) and under stimulation of IL12 and IL18 they primarily produce
the cytokine INFγ (Box 2.5). For ILC2s GATA3 is the key transcription factor and
when exposed to IL25, IL33 and TSLP (thymic stromal lymphopoietin) they secrete
IL5 and IL13. The transcription factor RORC (RAR-related orphan receptor gamma)
is most critical for ILC3s and IL1 and IL23 induce in them the production of IL17 and
IL22. ILCs are resident in epithelial barrier tissues and are involved in the organiza-
tion of these tissues, e.g., during fetal development. Moreover, their prompt response
to microbe encounter makes ILCs important for tissue homeostasis. For example, at
mucosal barriers in the intestine, ILC3s critically interact with the microbiome and
distinguish these commensal bacteria from pathogenic microbes (Sect. 7.4).
Box 2.5: IFNγ Although this cytokine shares its name with type I IFNs
(Sect. 8.1), it has no significant anti-viral functions on its own. In contrast,
it acts primarily as activator of effector cells of the innate (ILC1 cells, NK cells
and macrophages) and adaptive (TH 1 cells, Sect. 6.3) immune system, i.e., it is
an important player of cell-mediated immunity against intracellular microbes.
IFNγ activates macrophages to ingested microbes and kill them by phago-
cytosis. Moreover, IFNγ-activated phagocytes take up fragments of apoptotic
cells after they had been specifically detected by cytotoxic T cells (Sect. 6.4).
Moreover, the cytokine stimulates the differentiation of CD4+ T cells into TH 1
cells and inhibits the production of TH 2 and TH 17 cells. In addition, in B cells
IFNγ induces isotype switching to IgGs (Sect. 4.3). In cells expressing IFNγ
receptors signal transduction is induced that leads to the enhanced expression
of genes involved in antigen presentation and T cell activation.
NK cells are cytotoxic cells that circulate in the bloodstream, where they represent
5–15% of the mononuclear cells. Morphologically they are comparable to large
lymphocytes and contain a number of granules in their cytosol. They can kill virus-
infected normal and tumor cells (Fig. 2.9a) via the secretion of the protein perforin.
Like the complement protein C9, perforin creates membrane holes (Sect. 7.2) through
which other granular enzymes, granzymes, get into the cytosol of target cells and
start there the process of apoptosis (Sect. 6.4). Thus, NK cells have properties
comparable to cytotoxic T cells, which are T cells that are characterized by the
expression of the surface protein CD8. Through the elimination of virus-infected
and transformed cells, NK cells and cytotoxic T cells eliminate virus reservoirs and
prevent tumorigenesis in particular of hematopoietic cells. Moreover, NK cells share
a number of features with ILC1s, such as the sensitivity to IL12, the production of
INFγ (Fig. 2.9b) and the interaction with infected and transformed cells. However,
ILC1s are tissue-resident, while NK cells are circulating. A major role of IFNγ being
produced by NK cells is the stimulation of macrophages to finish their phagocytosis
process.
Since NK cells express no TCR, they have to use regular surface receptors for
distinguishing infected and stressed cells from healthy cells. These receptors are
either activating, such as KIRs (killer cell immunoglobulin like receptors), CD16
(FcγRIIIA) or C-type lectin receptors (Sect. 2.1), or inhibitory and are connected
with respective signal transduction cascades. Activating receptors have ITAMs
(immunoreceptor tyrosine-based activation motifs) on the cytosolic sides, while
inhibitory receptors are characterized by ITIMs (immunoreceptor tyrosine-based
inhibition motifs). These motifs are found also at other immune receptors that will
Further Reading 39
a
transformed cell
Killing of
cancer cell
NK cell
Killing of
infected cell
Infected cell
Killing of
NK cell
IFNγ phagocytosed
IL12
microbes
Macrophage with
phagocytosed microbes
Fig. 2.9 Functions of NK cells. NK cells detect molecules on the surface of transformed or infected
cells and kill them (a). IL12 produced by macrophages, which have phagocytosed microbes, activate
NK cells that in turn secrete INFγ, which activates the macrophages to terminate the phagocytosis
process (b)
be discussed in Sects. 4.4 and 6.1. The balance between the ligands of these recep-
tors determines the functional outcome of the NK cells, i.e., a majority for activating
receptors represents an infected cells that subsequently will be destructed, while
healthy cells carry ligands for inhibitory receptors, such as MHC-I proteins. Thus,
when cells repress the expression of MHC-I proteins they are likely be destructed
by NK cells.
Further Reading
1. Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling.

Nat Rev Immunol 16:407–420
2. Cerwenka A, Lanier LL (2016) Natural killer cell memory in infection, inflammation and cancer.

3. Ebbo M, Crinier A, Vely F, Vivier E (2017) Innate lymphoid cells: major players in inflammatory
diseases. Nat Rev Immunol 17:665–678
4. Fitzgerald KA, Kagan JC (2020) Toll-like receptors and the control of immunity. Cell 180:1044–
1066
5. Gong T, Liu L, Jiang W, Zhou R (2020) DAMP-sensing receptors in sterile inflammation and
inflammatory diseases. Nat Rev Immunol 20:95–112
6. Huber-Lang M, Lambris JD, Ward PA (2018) Innate immune responses to trauma. Nat Immunol
19:327–341
7. Monticelli S, Natoli G (2017) Transcriptional determination and functional specificity of myeloid
cells: making sense of diversity. Nat Rev Immunol 17:595–607
8. Nemeth T, Sperandio M, Mocsai A (2020) Neutrophils as emerging therapeutic targets. Nat Rev
Drug Discov 19:253–275
9. Vivier E, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, Koyasu S, Locksley RM,
McKenzie ANJ, Mebius RE et al (2018) Innate lymphoid cells: 10 years on. Cell 174:1054–1066
Chapter 3
Adaptive Immunity and Antigen
Receptor Diversity
Abstract This chapter will provide an overview on the roles of B and T cells in the
response of the adaptive immune system to microbe invasion. In this context, we will
discuss the impact of memory cells over human lifespan. Adaptive immunity is able
to respond more specifically than the innate immune system (Chap. 2), because it uses
with TCRs and BCRs (that develop into antibodies) very specific antigen receptors.
For an understanding how the diversity of these antigen receptors is created based
on very few genes, we will discuss lymphocyte maturation, clonal selection and the
mechanisms of somatic recombination.
Keywords Adaptive immunity · Humoral response · Cell-mediated response · B

cells · TH cells · Cytotoxic T cells · Memory cells · Lymphocyte maturation ·
Negative and positive selection · Somatic recombination · Antigen receptor
diversity
3.1 Classes and Responses of Adaptive Immune Cells
Like the innate immune system, also the adaptive immune system functions via
a humoral and a cell-mediated component. The actions of antibodies represent
the humoral part of adaptive immunity (Fig. 3.1, left). Antibodies neutralize the
infectivity of microbes and label them for more efficient elimination (Sect. 4.2)
through effector mechanisms of the innate immune system (Chap. 2). They are very
effective in the fight against extracellular microbes and the toxins that these microbes
produce but cannot reach any intracellular microbes (Sect. 7.2).
The main function of B cells is the specific recognition of antigens via their clonal
BCR followed by proliferation as well as differentiation into antibody-producing
plasma cells, i.e., B cells are not directly involved in the elimination of microbes
(Fig. 3.2a). When microbes have entered host cells, such as intracellular bacteria
that resist phagocytosis (Sect. 7.3) or viruses replicating in cells (Sect. 8.1), effector
functions of cell-mediated adaptive immunity are needed (Fig. 3.1, center and right).
One arm of the latter are TH cells, which do not themselves eradicate microbes but
instruct phagocytes to do so (Sect. 6.3). TH cells coordinate the action of other
immune cells both by cell–cell contacts as well as by cytokine secretion (Fig. 3.2b).

https://doi.org/10.1007/978-3-031-04025-2_3
42 3 Adaptive Immunity and Antigen Receptor Diversity
Humoral immunity Cell-mediated immunity

Extracellular
microbes
Microbe
Phagocytosed Intracellular microbes

microbes that can live (e.g., viruses) replicating
Extracellular microbes within macrophages within infected cell
Responding
lymphocytes
B cell Helper T cell Cytotoxic T cell
Activated macrophage
MHC I
mechanism MHC II
Secreted antibodies Neutrophil Killed infected cell
Block infections Activated Kill infected

and eliminate phagocytes cells and eliminate
Functions extracellular reservoirs of
kill microbes
microbes infection
Fig. 3.1 Humoral and cell-mediated adaptive immunity. The adaptive immune system can be
distinguished into a humoral part, which is mediated by antibodies secreted by B cells (left), and
a component represented by TH cells coordinating the action of other immune cells (center) and
cytotoxic T cells destructing infected and transformed cells (right). More details are provided in
the text
Cytotoxic T cells are the other arm of cell-mediated adaptive immunity (Fig. 3.1,
right). They directly detect and destruct infected cells of the host (Fig. 3.2c), i.e., their
main effector function is to prevent further spreading of microbes (Sect. 6.4).
The different sizes of microbes, ranging from small viruses to very large
helminths require different mechanisms of the adaptive immune system to fight
against them. Plasma cells (Fig. 2.2, bottom) are effector B cells that have enlarged
dramatically primarily due to drastically increased ER (endoplasmic reticulum). The
latter is the production site of antibodies (Sect. 4.1). These antibodies are able to
3.1 Classes and Responses of Adaptive Immune Cells 43
Antigen recognition
a B cell
Neutralization
+ of microbe,
Microbe phagocytosis,
Antibody complement
activation
b Cytokines
Helper T cell Activation of
macrophages
Activation
Microbial antigen
(proliferation and
presented by antigen-
presenting cell via MHC II
of T and B cells
c Cytotoxic T cell
Killing of
infected cell
Infected cell expressing

mirobial antigen via MHC I
d Regulatory T cell
Suppression of
lymphocytes
Regulatory T cell
Activated T cell
Fig. 3.2 Effector functions of B and T cells. B cells recognize via their BCR many different types
of antigens and develop into antibody-secreting plasma cells (a). TH cells detect via their TCR
antigens displayed by MHC-II proteins on the surface of antigen-presenting cells and subsequently
secrete cytokines that stimulate a variety of innate and adaptive immune cells (b). Cytotoxic T
cells contact via their TCR antigens presented by MHC-I proteins, which are specific for infected
or transformed host cells, and kill the respective cells (c). Regulatory T cells suppress immune
responses (d)
Table 3.1 Different types of vaccines

Type of vaccine Vaccines in use First used
Live attenuated Measles, mumps, rubella, yellow 1796 (smallpox)
fever, influenza, polio, typhoid,
rotavirus
Killed microbe Pertussis, hepatitis A, rabies 1896 (typhoid)
Toxoid Diphtheria, tetanus 1923 (diphtheria)
Protein (protein, recombinant Hepatitis B, influenza, typhoid 1970 (anthrax)
protein) or polysaccharide
Virus-like particle Papillomavirus 1986 (hepatitis B)
Outer membrane vesicle Group B meningococcal 1987 (Group B
meningococcal)
Viral vectored Ebola 2019 (Ebola)
Nucleic acid vaccine SARS-CoV-2 2020 (SARS-CoV-2)
bind to microbes and prevent them from infecting cells due to steric hindrance
of binding to their cellular receptors (Fig. 3.2a). This neutralization mechanism
of antibodies is very effective, since it stops an infection before it is established.
Therefore, vaccination primarily aims to stimulate the production of potent
antibodies (Box 3.1). Moreover, antibodies coat microbes and make them in this
way an easy target of phagocytes, such as neutrophils and macrophages (Sect. 2.3).
In addition, antibody-bound microbes are more easily recognized by complement
proteins leading to microbe destruction (Sect. 7.2). Some antibody-secreting plasma
cells migrate to the bone marrow or mucosal tissues and survive there for years as
memory cells that continue to produce antibodies at lower levels. These antibodies
provide immediate protection, when the individual gets infected again by the same
microbe.
Box 3.1: Principles of vaccination In general, prophylactic immunization

against infectious diseases is a powerful tool and have led to the complete (e.g.,
smallpox) or nearly complete (e.g., polio) eradication of many of these disor-
ders. In vaccination, a killed (i.e., a non-live vaccine) or attenuated (i.e., a live
vaccine) form of a microbe is administrated. This elicits an immune response
and the production of microbe-specific B and T memory cells and should
provide protection against infection with the respective microbe. Vaccines are
most effective, if the microbe does:
• not establish latency (Sect. 8.2)
• not interfere with the host immune response
• not undergo antigenic variation
• not have an animal reservoir (Sect. 8.2).
For example, a vaccine against HIV-1 is difficult to develop, since the virus
is latent and lyses TH cells. Most of today’s vaccines induce humoral immu-
nity, i.e., constant production of high-affinity antibodies long-lived plasma
B cells. In addition to live and non-live vaccines, recently also vaccines have
been developed that are based on viral vectors, virus-like particles, DNA and
RNA (Sect. 8.4). (Table 3.1). All these vaccinations are so called active vacci-
nations because they cause an active response of the immune system. Passive
vaccinations are discussed in Box 4.1.
In contrast to B cells, T cells do not have a secreted form of their antigen

receptor, i.e., also as effector cells they function via a membrane-bound TCRs
(Sect. 6.1). Therefore, also as effector cells T cells need to get into physical contact
with antigens. These antigens are exclusively peptides, e.g., fragments of proteins
derived from microbes (Sect. 5.3). T cells are primarily distinguished into TH cells
(CD4+ ) that coordinate the action of other immune cells (Fig. 3.2b) and cytotoxic
T cells (CD8+ ) that detect and destroy virus-infected cells (Fig. 3.2c). Naïve CD4+
T cells that specifically interact with peptides displayed by MHC-II proteins on the
surface of an antigen-presenting cell, such as a dendritic cell, get activated, undergo
clonal expansion and turn into different types of effector TH cells (Sect. 6.3). These
effector T cells produce different sets of cytokines that either activate macrophages
to:
• continue with the destruction of phagocytosed microbes (Sect. 2.3)
• stimulate neutrophiles to promote the inflammation process (Sect. 2.4)
• induce the clonal expansion and differentiation of B cells (Sect. 4.1).
In contrast, naïve CD8+ T cells get exposed to peptides presented on MHC-I
proteins that derive from cytosolic proteins of virus-infected or transformed cells.
When these peptides significantly differ from the normal set of self-peptides that are
regularly presented by healthy body cells (Sect. 9.1), the T cells get activated, prolif-
erate and differentiate into cytotoxic effector T cells. Like NK cells (Sect. 2.5) these
cytotoxic T cells kill virus-infected body cells, in order to destroy virus reservoirs
and prevent their further spreading. Furthermore, there is a subtype of CD4+ effector
T cells, regulatory T (Treg ) cells, that act as suppressors to the actions of other T cells
(Fig. 3.2d). Treg cells prevent overboarding activities of both TH and cytotoxic T
cells and will be discussed in more detail in the context of immunological tolerance
(Sect. 9.1).
The typical response of the adaptive immune system to the encounter of an antigen,
such as a microbe causing an infectious disease, has multiple steps (Fig. 3.3). The first
step is the antigen recognition phase, in which either a naïve B cell clone specifically
binds via its BCR a soluble antigen or an antigen-presenting cell is exposing a peptide
originating from a microbial protein to the TCR of a naïve T cell clone (Sect. 5.1).
The following activation phase takes approximately 7 days, in which the respective
lymphocytes proliferate (clonal expansion) and differentiate into effector cells. In this
Antigen Lymphocyte Antigen Contraction

Memory
recognition activation elimination (homeostasis)
Antibody pro-
Antibodies
ducing cell
T cells
Magnitude of response
T cells
Clonal
expansion Surviving
Antigen-
memory
presenting Apoptosis
cell cell
Naïve T cells
Naïve B cells
days 7 14 21
Time after antigen exposure
Fig. 3.3 Time course of responses of adaptive immunity. The adaptive immune response happens
in multiple steps. The first step is antigen recognition at timepoint 0 leading to the activation of
only those B and/or T clones that specifically bind the antigen. The effector phase of the following
14 days is composed of lymphocyte activation step and the antigen elimination step. When the
antigen has disappeared, the contraction phase of immune response starts, most B and T cells
die by apoptosis and only a lower number of long-lived cells survive and provide memory of the
antigen encounter
status the lymphocytes are able to eliminate microbes via different effector functions,
such as:
• antibody production by plasma cells
• coordination the response of other immune cells via cytokine secretion by TH
cells
• killing of virus-infected cells by cytotoxic T cells.
This antigen elimination phase takes another 7–10 days and ideally ends with
the complete disappearance of the microbe. In the subsequent contraction phase,
most of the effector B and T cells die by apoptosis, because signals for lymphocyte
activation disappeared together with the microbes. Keeping the effector cell alive
would require a substantial amount of energy from the host and increases the risk
of unwanted overreactions of the immune system like autoimmune diseases. In this

way, the body returns to homeostasis and the patient will feel again healthy. However,
a few effector cells survive as long-lived memory cells for years or even the whole
remaining lifespan of the individual. This is the memory phase concerning the
specific antigen. Thus, at older age, in contrast to naïve cells, the number of memory
cells with a BCR or TCR specific for a given antigen is significantly higher. Memory
cells respond already after 1–2 days to a repeated exposure with the same antigen.
Moreover, the response of memory cells is stronger and often qualitatively different
to that of naïve cells (Sect. 4.3). Such repeated antigen exposures may even not
be recognized by the individual, i.e., the person is considered immune and does
not get infected after contact with the respective microbe.
The immune system of newborns shows many deficiencies, which is in part due
to the fact that the fetus needed to be tolerant to cells of the mother (Box 3.2).
However, adaptive immunity is a lifelong, never-ending learning experience.
Every exposure of the adaptive immune system with a previously unknown antigen
leads to the production of additional long-lived memory B and T cells. Therefore, the
number of circulating memory T cells increases over lifespan from less than 5% at
birth to up to 80% of all T cells in the elderly (Fig. 3.4). In parallel, the involution of
the thymus after puberty (Box 1.5) reduces during lifespan the percentage of naïve T
cells in circulation from nearly 100% at birth to 20% in older people. Accordingly, the
Thymic output
100
Naïve T cells
80
% Blood T cells
Memory T cells
60
40
20
0
0 20 40 60 80
Age (years)
Fig. 3.4 Proportions of naïve and memory T cells change with age. Due to the regression of the
thymus after puberty the daily production of short-lived naïve T cells declines over age. In contrast,
the livelong exposure with foreign antigen increases the number of long-lived memory T cells, so
that the proportion between both types of T cells significantly changes during lifespan
daily output of the thymus of 16 million cells in young adults reduces to less than one
million cells in elderly. The huge collection of memory cells in older people seems
to be able to compensate the loss of naïve T cells. However, in some aspects the
immune system of older people resembles that of the newborn, such as reduced
anti-microbial activity by neutrophils and macrophages, reduced antigen presentation
by dendritic cells and decreased activity of NK cells.
Box 3.2: The neonate immune system At birth the innate immune system
of the neonate is muted, since the fetus had to tolerate non-shared maternal
antigens and to ignore the stress and remodeling during development. This
makes the newborn, and particularly a premature baby, very susceptible to
infections. Neonates have all components of the cellular immune response, but
the signaling of B and T cells is immature, i.e., the innate immune cells show
reduced activity and therefore the generation of antibodies is delayed. Thus,
in the first months of life the immune response is hindered, which explains
the large proportion of infant deaths from infectious diseases. Interestingly,
the neonatal immune system is not as uniform as assumed for long time
but there are variable immune profiles among newborns, which are based
on differences in in utero conditioning, genetics, gestational age, modes
of delivery, transferred maternal antibodies by breastfeeding and envi-
ronmental factors before and after birth. This can also explain in part the
variability in the immunophenotype (Box 8.6) of individuals when they are
adults. However, across all infants the immune response is immature and has
difficulties to protect the young children effectively from infectious diseases.
Events during the first weeks and months of life, such as infections, maternal
antibodies and microbial colonization, have not only an immediate impact
on the immunity of newborns but also long-term consequences on disease
susceptibility.
3.2 Lymphocyte Maturation
During hematopoiesis (Sect. 1.3) HSCs in the bone marrow give rise to CLPs, which
then further differentiate into pro-B and pro-T cells (in contrast to pre-lymphocytes
pro-B and pro-T cells do not express any antigen receptors). The pro-B cells mature
into follicular and marginal zone B cells, while pro-T cells develop into T cells
carrying a “classical” TCR formed by an α and β chain. In contrast, pro-B and pro-T
cells that derive before birth from fetal liver differentiate into B-1 cells and T cells
carrying a TCR formed by γ and δ chains.
The principles of the maturation of B and T cells are very comparable (Fig. 3.5).
The molecular processes happening during the production of millions of different
3.2 Lymphocyte Maturation 49
Pre-B antigen Antigen Positive and

Proliferation receptor Proliferation receptor negative
expression expression selection
mature
Immature B cell
pre-B cell:
B cell:
expresses
expresses
pro-B cell one chain of
complete
antigen
antigen
receptor
receptor
weak
antigen Positive
recognition selection
strong
antigen
recognition
Negative
selection:
cell death
Failure to express
pre-antigen receptor;
cell death Failure to express
antigen receptor;
cell death
Fig. 3.5 Maturation of B and T cells. Pro-B cells in the bone marrow and pro-T cells in the
thymus have to learn expressing their BCR or TCR, respectively. In this multistep process first the
heavy and then the light chain of the BCR (or first the β and then the α chain of TCR) are expressed
and functionally tested. Cells failing the checkpoints of this lymphocyte maturation process are
negatively selected and die by apoptosis. Positive selection is used for perfectly expressed antigen
receptors with weak affinity for test antigens. More details are provided in the text
B and T cell clones, such as somatic recombination and the creation of junctional
diversity (Sect. 3.3), are unique for lymphocytes and require careful control. For
example, due to specific requirements concerning positive and negative selection for
mature lymphocytes expressing either a perfect BCR or TCR, these developmental
processes happen in separate primary lymphoid organs. Thus, B maturation takes
places in the bone marrow, while pro-T cells migrate for their maturation to the
thymus.
Lymphocyte maturation is a multistep process, in which at each checkpoint the
developing cells are tested (Fig. 3.5). Maturation continues only when the preceding
step was successfully completed. In the alternative case, a system of negative and
positive selection drives the failing cells into apoptosis. One checkpoint with negative
selection is the expression of the first of the two chains of the antigen receptor, which
is the heavy chain of the BCR or the β chain of the TCR (Sect. 3.3). This test is
possible through the expression of a surrogate second chain replacing the missing
polypeptide in the respective pre-antigen receptor. Another checkpoint with negative
selection assures that both chains, heavy and light chain in case of BCR or β and
α chain for TCR, form a functional antigen receptor. Both tests are essential, since
for each of the two antigen chains somatic recombination determines their specific
assembly, which with a change of 2 of 3 creates a frameshift in protein translation
causing a non-functional protein (Sect. 3.3). Another checkpoint selects in a positive
way for cells that carry antigen receptors with reasonable but not too strong affinity for
antigens. For example, for T cells these are TCRs that recognize self-MHC proteins.
In contrast, cells with a BCR or TCR that strongly reacts to a host self-antigen are
eliminated by negative selection. In this way, only those lymphocytes survive that
have a perfectly formed antigen receptor showing tolerance to self-antigens.
This is called central tolerance and will be further discussed in Sect. 9.1.
3.3 Mechanisms of Antigen Receptor Diversity
Antigen receptors are formed by two different polypeptides, which are the heavy and
light chain for BCR (two copies each, Sect. 4.1) (Fig. 3.6a) as well as the α and β
chain for TCR (Fig. 3.6b). Each of the four polypeptides can be distinguished into a
variable and constant domain. In addition, the heavy chain, α chain and β chain have
in addition a transmembrane domain and a short cytosolic domain. Sections 4.1 and
6.1 will present the variable domain of all four polypeptides and the constant domains
of light chain, α chain and β chain as an Ig domain formed by 110 amino acids. In
contrast, the constant domain of the heavy chain is longer and composed of four
Ig domains. Via disulfide bridges between the constant domains of heavy and light
chain as well as of α and β chain, respectively, BCR and TCR are stabilized. However,
functionally more important are the variable domains, which serve as antigen binding
sites via three complementary determining regions (CDRs) (Fig. 3.6). The term
“variable” already suggests that each B and T cell clone differs in the exact amino
acid sequence and 3-dimensional (3D) structure of the latter domains of their BCR
or TCR, respectively. In this way, each lymphocyte clone recognizes a different
antigen.
There is one gene encoding for the heavy chain and two (κ and λ) for the light
chain (Fig. 3.6a) as well as one gene each for β and γ chain and a combined gene
locus for the α and δ chain (Fig. 3.6b). These in total six antigen receptor gene loci
have the special property that they are subdivided into three or four cluster of up to 50
similar but not identical segments that encode for parts of the respective polypeptide
chains. For example, for the heavy chain there are about 45 V segments (their number
varies between individuals), 23 D segments and 6 J segments (Fig. 3.6a). In contrast
to exons of regular genes that are combined in a linear fashion, for each antigen
receptor gene there is an apparent random selection of exactly one segment of each
type, which are combined and form the codon for the respective polypeptide chain.
This unique system provides insight how the huge diversity of B and T cell clones
can be created based on only six different genes.
3.3 Mechanisms of Antigen Receptor Diversity 51
V region C region
a Immunoglobulins Transmembrane
DH region
VH CH1
Ig heavy (μ) chain (membrane form) N C
JH
Ig light chain N CH2 CH3 CH4
VL CL
CDR1
CDR2
CDR3
H chain locus (1250 kb; chromosome 14)

VH (n = ~ 45)
DH (n = 23) JH
VH1 VH2 V Hn Cμ Cδ Cγ3 Cγ1 Cα1 Cγ2 Cγ4 Cε1 Cα2
5’ 3’
L L L enh enh
κ chain locus (1820 kb; chromosome 2)
Vκ (n = ~ 35)
Jκ
Vκ1 Vκ2 Vκn Cκ
5’ 3’
L L L enh enh
λ chain locus (1050 kb; chromosome 22)
Vλ (n = ~ 30)
Vκ1 Vκ2 Vκn Jλ1 Cλ1 Jλ2 Cλ2 Jλ3 Cλ3 Jλ7 Cλ7
5’ 3’
L L L enh
V region C region
b T cell receptor
Dβ
Vβ Cβ
TCR β chain N C
Jβ
TCR α chain N C
Vα Cα Trans-
CDR1
membrane
CDR2
CDR3 region
Human TCR β chain locus (620 kb; chromosome 7)

Vβ (n = ~ 50)
Vβ1Vβ2 Vβn Dβ1 Jβ1 Cβ1 Dβ2 Jβ2
Cβ2
5’ 3’
L L L enh
Human TCR α/δ chain locus (1000 kb; chromosome 14)

Dδ1
L Vδ2 Dδ2 Dδ3 Jδ enh Cδ L Vδ3
Vα (n = ~ 45) 3’
Vα1 Vα2 Vαn
5’ 3’
L L L Jα (n = ~ 55) Cα enh
Human TCR γ chain locus (200 kb; chromosome 7)
Vγ (n = ~ 5)
V γ 1 Vγ 2 V γn Jγ1 Cγ1 Jλ2 Cγ2
5’ 3’
L L L enh
Fig. 3.6 Protein domain and gene organization of antigen receptors. Both types of antigen
receptors are formed by two different polypeptides, i.e., for BCR (a) and TCR (b) each two gene
loci are used. The color coding of protein (sub)domains corresponds to the gene fragments encoding
for them
The variable region of the heavy, β and δ chain is formed by one variable (V),
one diversity (D) and one joining (J) gene segment, while for the variable region of
the κ and λ light chain as well as of the α and γ chain only V and J gene segments
are used. The mechanism behind this gene segment fusion is somatic recombination
(Box 3.3), which happens for a given B or T cell once for V-J joining and even
twice for V-D-J joining (first D-J, then V-D). Recombination is a genetic mechanism
that normally occurs only in germ cells (spermatocytes and oocytes) during meiosis,
where homologous segments of the genome copy deriving from the father of an
individual is exchanged with that of the mother. V(D)J joining is an exception that
occurs only during maturation of B and T cells. The cells eliminate the genomic
DNA between the joining DNA elements, in order to ensure that the respective cell
clone sticks with the decision, which elements are combined. T cells with a γδTCR
represent less than 10% of all T cells in the thymus. Since the gene segments of the
δ chain are located within the α chain gene locus, the δ locus is deleted when the α
chain gene segments are rearranged, so that T cells cannot express both an αβTCR
and a γδTCR.
Box 3.3: Mechanism of somatic recombination Somatic recombination is a

multistep process (Fig. 3.7) that involves:
• epigenetic processes open chromatin at the recombination sites, in order
to give access to specific enzymes. These enzymes are either specific to
lymphocytes, such as RAG (recombination activating gene) 1 and 2, or are
ubiquitous DNA double-stranded break repair enzymes
• arranging the two DNA segments that need to be combined next to each
other following the rule that at the break points one of the segments is
flanked by a 12 bp (base pair) spacer and the other by a 23 bp spacer
• introducing double-strand breaks by RAG1, adding nucleotides (referred to
as P nucleotides) via the lymphoid-specific enzyme TdT (terminal deoxynu-
cleotidyl transferase) or removing them by exonucleases and ligating the
DNA segments by using enzymes of the ubiquitous nonhomologous end
joining DNA repair process.
The epigenetic status, such as histone modification, at the recombination
site is important, in order to restrict gene rearrangement to a specific phase
during lymphocyte maturation and to attract RAG2 via histone 3, lysine 4
hypermethylation. Mutations in the genes RAG1 or RAG2 cause SCID (severe
combined immunodeficiency disease), in which patients lack B and T cells. In
most cases the genomic DNA between the joint segments is deleted, but in some
cases it is only inverted, in order to join the segments in correct orientation.
The spacer sequences are unique to antigen receptor genes, so that only those
and no other genes in B and T cells get rearranged. The random addition of up
a
Stage of Pro-B Large Pre-B Small Pre-B Immature B Mature B
Stem cell
maturation
Proliferation
RAG expression
TdT expression
alternative
VH to DJ re- Recombined splicing of
Germline DH to JH combination, H chain gene μ and κ/λ VDJ-C RNA to
Ig DNA, RNA DNA (VDJ), V to Jκ RNA
μ mRNA form Cμ and
recombination
Cδ mRNA
Membrane
None None Cytosolic μ IgM (μ + κ Membrane
Ig expression pre-BCR
or light chain) IgM and IgD
Anatomic site Bone marrow Periphery
Negative sel-
Response to ection (del-
None None None
antigen etion), re-
ceptor editing
b
Stage of Double Single positive
Stem cell Pro-T Pre-T Mature T
maturation positive (immature T)
Proliferation
RAG expression
TdT expression
Recombined
uncombined β chain gene
TCR DNA, RNA (germline) [V(D)J-C]; Recombined β and α chain genes [V(D)J-C];
DNA β chain β and α chain mRNA
mRNA
pre-TCR
TCR expression None None (β chain/ Membrane αβ TCR
pre-Tα)
Anatomic site Bone Thymus Periphery
marrow
Positive and Activation
Response to (proliferation
None None None negative
antigen and dif-
selection
ferentation)
Fig. 3.7 Molecular steps of B and T cell development. The molecular events corresponding to
the steps of the maturation of B cells (a) and T cells (b) are indicated. More details are provided in
the text
to 20 nucleotides (referred to as N nucleotides) via TdT is another mechanism

of junctional diversity.
The V(D)J recombination at the genomic regions of the BCR and TCR genes each
creates a single exon that encodes for the complete variable domain of the respective
antigen receptor chains. CDR1 and CDR2 are encoded exclusively by the V segment,
while CDR3 is encoded by a combination of V, D (only in heavy, β and δ chain) and J
sequences and shows even higher variation than the first two. Accordingly, the CDR3
regions of BCR and TCR contribute most to the specificity of antigen binding
(Sects. 4.1 and 6.1). The constant (C) region(s) of the antigen chain genes are located
downstream of the J segments and are separated by an intron (Fig. 3.6). The λ light
chain locus has four C segments, while the κ light chain locus has only one. The
heavy chain gene locus has even nine C segments encoding for the constant regions
of different antibody types (IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgE)
(Sects. 4.3). For comparison, the β and γ chain gene loci have two C segments each
and that of the α and δ chain only one. Thus, isotype switching is a special feature
of antibodies but not of the TCR.
After two (heavy and β chain) or one (light and α chain) recombination events,
including adding P and N nucleotides, the rearranged genes are transcribed to form
a primary mRNA transcript (Fig. 3.8). Then RNA splicing joins the V(D)J exon
with the C region exons and forms an mRNA that is translated at the membrane of
the ER to the respective antigen receptor polypeptide chain. After processing and
glycosylation, the polypeptides get mature and combine to BCRs or TCRs, which
are exposed on the surface of the respective B or T cell. The addition of P and
N nucleotides at the recombination sites may introduce frameshifts (with a change
of 2 in 3) that likely result in non-functional antigen receptors. The respective B
and T cell clones are eliminated by negative selection during lymphocyte maturation
(Fig. 3.5). This inefficient antigen receptor production is the payoff of generating
junctional diversity.
The molecular counterpart to the steps of lymphocyte maturation discussed in
Sect. 3.2 are the stages of B and T cell development (Fig. 3.7):
• HSCs and CLPs proliferate in the bone marrow and give rise to pro-B and pro-T
cells
• Pro-B cells stay in the bone marrow and pro-T cells migrate into the thymus,
where both express RAGs and TdT, in order to start recombination for first for
D-J joining and then for V-D joining including the creation of junctional diversity
of the gene loci of the heavy chain or β chain, respectively
• Pre-B and pre-T cells express the heavy chain or β chain polypeptide together
with surrogate light chain or α chain, respectively. In case a functional receptor
is produced, the respective lymphocyte clones escape negative selection and
proliferate.
• The re-expression of RAGs starts the recombination of the light chain or α chain
in pre-B and pre-T cells, respectively
• Immature B and T cells express a complete BCR or TCR, respectively, which is

functionally tested by mechanism of negative and positive selection
• Lymphocytes that pass all checkpoints are mature B and T cells. They leave the
primary lymphoid organs and move to the periphery.
a μ heavy chain b κ light chain
Vμ1 Vμ2 Vμn Dμ 1-23 Jμ1-6 Cμ Vκ1 Vκ2 Vκn Jκ1-5 Cκ

Germline
5’ 3’ 5’ 3’
DNA L L L L L L
D-J joining
Vμ1 Vμ2 Vμn D1 D2 Jμ1-6 Cμ
5’ 3’ V-J joining
L L L
Rearranged V(D)J joining
DNA
Vμ1 D2 Jμ1-6 Cμ Vκ2 Jκ1-5 Cκ
5’ 3’ 5’ 3’
Transcription Transcription
Vμ1 D2 Jμ1-6 Cμ Vκ2 Jκ1-5 Cκ
Primary RNA 3’
5’ 5’ 3’
transcript
RNA processing RNA processing
Jμ1 Jκ1
Messenger AAA AAA
RNA (mRNA)
Translation Translation
Nascent L V C L V C
polypeptide
Processing, Processing of protein
glycosylation of protein
Mature V C V C
polypeptide
Fig. 3.8 Recombination and expression of antigen receptor genes. Recombination, transcrip-
tion, splicing and protein processing of the genes μ heavy chain (a), λ light chain (b), β chain
(c) and α chain (d). More details are provided in the text
c TCR β chain d TCR α chain

Vβ (n = ~ 50) Jβ1 Jβ2
Vβ1Vβ2 Vβn Dβ1 Cβ1 Dβ2 Cβ2 Vα1 Vα2 Vαn Jα (n = ~ 55) Cα
Germline
5’ 3’ 5’ 3’
DNA L L L enh L L L enh
D-J joining
Vβ1Vβ2 Vβn Dβ1 Jβ Cβ1 Dβ2 Cβ2
5’ 3’ V-J joining
L L L enh
Rearranged V(D)J joining

DNA
Vβ1Dβ1 Jβ Cβ1 Dβ2 Cβ2 Vα1 Jα Cα
5’ 3’ 5’ 3’
enh enh
Transcription Transcription
Primary RNA Vβ1Dβ1 Jβ Cβ1 Dβ2 Cβ2 Vα1 Jα Cα

5’ 3’ 5’ 3’
transcript
RNA processing RNA processing
Messenger AAA AAA

RNA (mRNA)
Translation Translation
Nascent L Vβ Cβ L Vα Cα
polypeptide
Processing, Processing of protein
glycosylation of protein
Mature Vβ Cβ Vα Cα
polypeptide
Fig. 3.8 (continued)
Thymocytes are developing T cells in the thymus, in the cortex of which they
express αβTCRs or γδTCRs. Medullary thymic epithelial cells present self-peptides
on MHC proteins for negative selection. In fact, up to 95% of all produced T cells
die by apoptosis. Surviving T cells with αβTCR mature either into CD4+ TH cells or
CD8+ cytotoxic T cells and exit the thymus towards circulation.
The different combinations of V, D and J gene segments as well as the addition and
removal of nucleotides at the junctions are the basis of the tremendous diversity of
Further Reading 57
Table 3.2 Diversity in BCR and TCR genes

BCR αβTCR γδTCR
Mechanism Heavy chain κ light chain λ light chain α chain β chain γ chain δ chain
V segments 45 35 30 45 50 5 2
D segments 23 0 0 0 2 0 3
J segments 6 5 4 55 12 5 4
Total 1011 1016 1018
potential
(including
junctional
diversity)
antigen receptors (Table 3.2). Since each lymphocyte has its individual choice of V, D
and J gene segments for both chains of its antigen receptor (combinatorial diversity)
and random junctional diversity, it represents a clone that expresses one version of a
BCR or TCR with unique specificity for an antigen. Although the theoretical number
of different antigen receptors is 1011 to 1018 , at a given time only approximately
107 different B and T cell clones are in circulation.
The potential number of antigen receptors generated via junctional diversity is
larger than that generated by combinations of V, D and J gene segments for both
chains of the respective antigen receptor.
Further Reading
1. Gascoigne NR, Rybakin V, Acuto O, Brzostek J (2016) TCR signal strength and T cell
development. Annu Rev Cell Dev Biol 32:327–348
2. Müller V, de Boer RJ, Bonhoeffer S, Szathmary E (2018) An evolutionary perspective on the
systems of adaptive immunity. Biol Rev Camb Philos Soc 93:505–528
3. Pollard AJ, Bijker EM (2021) A guide to vaccinology: from basic principles to new
developments. Nat Rev Immunol 21:83–100
Chapter 4
B Cell Immunity: BCRs, Antibodies
and Their Effector Functions
Abstract B cells respond to antigen stimulation of their BCR via the production of
antibodies. This is a key feature of the adaptive immune system and a major aim of
vaccination. In this chapter, we will discuss the structure and function of BCRs and
antibodies, in particular of their variable regions, and their adaption to antigens, such
as affinity maturation due to somatic hypermutations and isotype switching based on
somatic recombination. In this context, we will present the different phases of the
humoral response of B cells and their communication with T cells, in particular in
germinal centers of lymph nodes. The effector functions of B cells will be discussed
on the cellular level as well as in the context of BCR signaling.
Keywords BCR · Antibody · CDR · Antigen binding · Affinity maturation ·

Isotype switching · Somatic hypermutation · Humoral immune response · Primary
and secondary response · T cell dependency · Germinal center · Signal transduction
4.1 Structure and Diversity of Antibodies and BCRs
The recognition of antigens by BCRs being expressed on the surface on naïve B

cells activates them to proliferate and differentiate into plasma cells (Fig. 2.2) that
secrete antibodies of the same specificity as the BCR. Thus, antibodies are the
secreted form of BCRs, i.e., they are encoded by the same genes, which are exclu-
sively expressed in B cells (Sect. 4.3). Antibodies are the mediators of the humoral
response of B cells against all types of microbes and other antigenic molecules.
Thus, antibodies are effector molecules, while BCRs are sensors for antigens.
Antibodies and BCRs are together with TCRs and MHC proteins those proteins that
bind antigens with high affinity and specificity (Table 4.1). The main function of
BCRs and TCRs is to respond to antigen binding via the induction of a signal trans-
duction cascade within the respective B and T cells (Sects. 4.2 and 6.1). In contrast,
the function of MHC proteins is to display antigens to TCRs, i.e., they do not induce
signaling in cells carrying them. Antigens bound by MHC proteins are peptides in a
size range of 10–30 amino acids (Sect. 5.2) and TCRs are specialized to recognize
MHC protein-peptide complexes (Sect. 6.1). In contrast, antibodies and BCRs are
more flexible and bind a wide variety of molecular structures ranging from proteins

https://doi.org/10.1007/978-3-031-04025-2_4
60 4 B Cell Immunity: BCRs, Antibodies …
Table 4.1 Proteins recognizing antigens with high affinity and specificity
Feature Antigen-binding receptor
Antibody/BCR TCR MHC-I/MHC-II
Cells carrying None/B cell T cell Any cell
receptor (MHC-I)/antigen-presenting
cell (MHC-II)
Antigen-binding 2 × 3 CDRs in V 2 × 3 CDRs in V Peptide-binding cleft
site domain of heavy domain of α and β
and light chain chain
Type of antigen Proteins, lipids, Peptide-MHC Peptides
carbohydrates, complexes
nucleic acids
Antigenic Linear and Linear Linear
determinants conformational
Affinity (KD in 0.01–100 100–10,000 1000
nM)
via carbohydrates and lipids to nucleic acids. Moreover, antibodies and BCRs recog-
nize linear as well as conformational antigens, while TCRs and MHC proteins bind
only a few amino acids of a linear peptide. Therefore, antibodies and BCRs have
with a KD in the order of 0.01–100 nM in most cases a clearly higher affinity for
their specific antigen than TCRs and MHC proteins.
Antibody molecules are composed of four polypeptides, which are two identical
heavy chains and two identical light chains (Fig. 4.1, left). The heavy chain is formed
by 4–5 Ig domains, while the light chain is composed of only two Ig domains. The
globular Ig domains are formed of 110 amino acids in two layers of 3–5 β sheets that
are held together by a disulfide bridge (Fig. 4.2, left). The Ig domain is very stable
and is used as building block for many other immune molecules (and even for
proteins that have no relation to the immune system), which all together form the
Ig superfamily. The amino-terminal Ig domains of both heavy and light chains are
variable, in particular via their three CDR motifs, which are formed by protruding
loops between anti-parallel β sheets. Due to processes explained in Sect. 3.3, these
CDR motifs are extremely variable in the sequence of the 9–11 amino acids forming
them (Fig. 4.2, right). Accordingly, the 3D structure of the CDRs is very flexible. The
in total six CDRs of the variable domain of the heavy and light chain are positioned
opposite to each other and form the antigen-binding surface. The CDRs act like
“fingers” that can take basically any position in space being complementary to the
antigen structure, i.e., any possible antigen can be recognized.
The four polypeptide chains forming an antibody molecule are covalently linked
by disulfide bonds (Fig. 4.1). Moreover, non-covalent interactions between the vari-
able domains of the heavy and light chain as well as between the respective constant
domains further stabilize the antibody molecule. In this way, every single antibody
molecule has two identical antigen-binding sites. Due to the hinge region between
4.1 Structure and Diversity of Antibodies and BCRs 61
Secreted IgG Membrane IgM / BCR

Antigen-
Framework binding site
Antigen- regions
N N
Light Heavy binding site N N
chain chain VH
N N VL
N N CH1
Rs
VH
CD
Hinge CL
CH1 VL
Hinge CL
Fab CH2
FcR/comple- region
ment protein CH 2 CH3
binding sites
Fc region
CH3 CH4
Plasma mem-
Tail piece brane of B cells
C C
C C
Ig domain
Fig. 4.1 Structure of antibodies and BCRs. Antibodies have a Y-like structure and are composed
of two heavy and two light chains composed of 4–5 and 2 Ig domains, respectively. Here the structure
of an IgG molecule is shown (left). The N-terminal domains of both chains a variable and involved in
antigen binding, while the remaining domains are constant and do not contribute to antigen contact
but interact with complement proteins and FcRs. BCRs are membrane-bound antibodies that carry
a transmembrane domain at their C-terminus that allows anchoring to the plasma membrane of B
cells (right)
the second and third Ig domain of the heavy chain, the distance of two antigen-binding
sites can be flexibly adapted to the size of the antigen-carrying microbe.
There are five different isotypes of antibodies, IgA, IgD, IgE, IgG and IgM, that
differ in the type of constant region of their heavy chain (Table 4.2). In humans
there are two subtypes of IgA, IgA1 and IgA2, and even four forms of IgG, IgG1,
IgG2, IgG3 and IgG4. These all together nine antibody types relate to the nine
segments for the constant region within the human gene locus of the heavy chain,
which are arranged in the order Cμ, Cδ, Cγ3, Cγ1, Cα1, Cγ2, Cγ4, Cε1 and Cα2
(Fig. 3.6). Since the Cμ and Cδ segments are first in line, mature B cell clones
express BCRs that are membrane-bound forms of IgM and IgD with identical antigen
specificity. Therefore, the primary response of B cells to antigen exposure is the
secretion of IgMs (Sect. 4.3), while IgD is found only in traces. Most of the about
96 100
N
CDR3 CDR1 CDR2 CDR3
42 26 80
47
Variability
CDR1
60
53
70 40
82 CDR2 20
61
112 20 40 60 80 100 110

15
Amino acid residue #
Fig. 4.2 Hypervariable regions of variable Ig domains. 3D structure of the variable Ig domain
(left). Hypervariable CDR1 (green), CDR2 (blue) and CDR3 (red) are highlighted as loops
connecting anti-parallel β sheets. A Kabat plot indicates the extent of variability of each of the
110 amino acids of the Ig domain (right). Amino acids composing the CDRs show the highest
variability and are indicated using the same color code
Table 4.2 Antibody isotypes

Isotype Subtypes Plasma Half-life (days) Secreted form Functions
(Heavy con-centration
chain) (mg/ml)
IgA IgA1, IgA2 3.5 6 Mainly dimer Immunity of
mucosa
IgD None Trace 3 Monomer BCR
IgE None 0.05 2 Monomer Defense against
helminths;
allergy
IgG IgG1, IgG2, 13.5 23 Monomer Classical
IgG3, IgG4 antibody
IgM None 1.5 5 Pentamer Native antibody
3 g of antibodies that an adult is producing every day are IgAs, which are primarily
secreted from mucosal epithelia, in order to neutralize microbes in the lumen of
mucosal tissues. In this way, inhaled and ingested microbes should be blocked from
infecting the respiratory and gastrointestinal tracts of the host. In addition, IgGs of the
mother are actively transported across the placenta and protect the neonate until the
baby’s immune system becomes mature (Box 3.2). Moreover, IgAs are secreted into
maternal milk and provide newborns with additional passive immunity (Box 4.1).
IgEs are found only at very low levels in the blood or extracellular fluids, since they
are bound with high affinity by FcεRIs on the surface of mast cells, basophils and
4.1 Structure and Diversity of Antibodies and BCRs 63
activated eosinophils (Sect. 10.2). IgGs are the most stable antibodies with a half-
life of approximately 3 weeks (Table 4.2), whereas the other antibody classes are
stable for only a few days. The constant region of the heavy chain of IgEs and IgMs
is composed by four tandem repeats of Ig domains, while that of IgAs, IgDs and
IgGs contains only three Ig domains. Moreover, IgAs form covalently connected
dimers and IgMs even pentamers. These antibody complexes have 4 or even 10
antigen-binding sites of the same specificity. Thus, by multimerization IgA and
IgM bind their antigens with higher avidity, which expresses the overall strength
of the binding between antibody and antigen. Importantly, the presence of microbe-
specific IgMs in the serum of a patient is a sign for a recent infection, while respective
IgGs represent past infections.
Box 4.1: Passive immunity The transfer of specific antibodies, e.g., against a
snake toxin, can be life-saving. In addition, some microbes, such as Clostridium
tetani causing tetanus (Sect. 7.1), produce poisonous exotoxins that can be
neutralized with specific antibodies. Moreover, the transfer of antibodies by
breast-feeding from mother to child provides protective immunity. However,
this passive immunization is short-lived, since it is limited by the half-life of
the administrated antibodies in circulation. Thus, passive immunization does
not induce memory, i.e., no memory B cells are produced.
The constant Ig domains of the heavy and light chain are not involved in antigen
recognition. However, the constant part of the heavy chain interacts with proteins
in solution or bound to the surface of cells, such as complement proteins and FcRs.
These receptors specifically bind different constant regions of the heavy chain and
are expressed primarily on the surface of innate immune cells, such as macrophages,
NK cells and mast cells. The different types of interactions of the constant region
mediate the effector functions of antibodies (Sect. 7.2).
The default structure of the heavy chain of the BCR contains at its carboxy-
terminal end is a hydrophobic α-helical transmembrane domain and a short (3 amino
acids for IgM and IgD and up to 30 amino acids for IgG and IgE) cytosolic domain
(Fig. 4.1, right). This extended heavy chain is used for the binding of the BCR to the
plasma membrane of B cells. These approximately 50 extra amino acids represent
the only difference between antibodies and BCRs. When a B cell clone gets activated
by an antigen, it differentiates in most cases to an antibody-secreting plasma cell. In
this differentiation step, the gene encoding for heavy chain is expressed at far higher
levels but due to alternative processing of its mRNA, such as RNA cleavage and the
choice of polyadenylation sites, without the transmembrane domain. In this way, the
BCR loses the contact with the plasma membrane of the B cell and enters as an
antibody the circulation (Fig. 4.3a).
In the process of isotype switching (Fig. 4.3b, more details in Sect. 4.3) anti-
bodies change after repeated exposure to the same antigen the constant part of their
Changes in antibody structure Antigen

a recognition functions
Change from Change from

membrane to B cell receptor
No change
secreted form function to
b
IgE
IgA
Each isotype
Isotype serves a
No change
switching
IgG
maturation
(somatic Increased No change
mutations in
variable region)
Fig. 4.3 Changes in antibody structure. There are three main ways how BCRs or antibodies
can change their structure during humoral responses. Basically, all activated B cells shift their
expression of BCRs to secreted antibodies either before or after affinity maturation (a). This does
not change antigen affinity but causes a shift to effector function. The change of the gene segment
for the constant part of the heavy chain leads to isotype switching, which does not affect antigen
affinity but changes effector function (b). The antigen-binding site of the BCR may undergo affinity
maturation due to somatic mutations in the gene segments encoding for the variable region, which
increases the affinity for antigen but does not change the way of effector function (c)
heavy chain without losing their antigen specificity. However, the different anti-
body isotypes are involved in different effector functions, most of which are
mediated by different types of FcRs. Affinity maturation (Fig. 4.3c, more details in
Sect. 4.3) is a mechanism, where somatic hypermutations in B cells are allowed, so
that amino acids within the variable domain of both the heavy and light chain are
exchanged and in an evolutional process antibodies with an even high affinity for
their antigen are generated. Thus, the type of effector function is not changed by
affinity maturation but the efficiency of antigen recognition.
4.2 BCR Signaling 65
4.2 BCR Signaling
The binding of an antigen to at least two BCR molecules on the surface of a B cell
induces within the cell a signal transduction cascade (Box 4.2, Fig. 4.4). The simul-
taneous binding of two or more BCRs is more efficient in the case of multivalent
antigens, such as polysaccharides, than with proteins, which contain each epitope
only once per molecule. BCR has only a very short cytosolic tails (Fig. 4.1, right),
but it is associated with the molecules Igα and Igβ, which have longer cytosolic tails
containing ITAMs. The ITAMs are phosphorylated by SRC (SRC proto-oncogene,
non-receptor tyrosine kinase) kinases like LYN (LYN proto-oncogene, SRC family
tyrosine kinase), FYN (FYN proto-oncogene, SRC family tyrosine kinase) or BLK
Cross-linking
of BCRs by
antigen
Cellular membrane
Cytoplasma FYN FYN P

BCR P
P P GRB P P Tyrosine
2
Igα/β
P P phosphorylation
SYK
events
PLC
S
SO
G2
GTP/GDP exchange PLCG2 activation Biochemical

on RAS, RAC
intermediates
RAS•GTP, RAC•GTP Increased cytosolic Ca2+ DAG
Active
ERK, JNK Ca2+ dependent enzymes PRKC enzymes
Transcription
Nucleus
FOS factors
Target genes Target genes p50 p65
Target genes
JUN
AP1 NFAT NFκB
Fig. 4.4 BCR signaling. Antigen-binding to BCRs on the surface of B cells leads in the cytosol to
the activation of tyrosine kinases of the SRC family, which phosphorylate ITAMs at the cytosolic
tails of the Igα and Igβ molecules. This allows docking of SYK, tyrosine phosphorylation of further
adaptor proteins and the induction of canonical signal transduction cascades of PLCG2 and RAS,
which end with the activation of the transcription factors NFAT, NFκB and AP1. More details are
provided in the text
(BLK proto-oncogene, SRC family tyrosine kinase), which are linked by lipid
anchors to the inside of the plasma membrane. The phosphorylated ITAMs are bound
by the kinase SYK (spleen associated tyrosine kinase), which phosphorylates adaptor
proteins, such as BLNK (B cell linker) and then attracts other adaptor proteins like
GRB2 (growth factor receptor bound protein 2) and SOS1 (SOS RAS/RAC guanine
nucleotide exchange factor 1) as well as the kinase BTK (bruton tyrosine kinase).
This stimulates PLCG2 (phospholipase C gamma 2) as well as the small G-proteins
RAS (rat sarcoma) and RAC (RAC family small GTPase 1). PLCG2 activates via
increased Ca2+ levels in the cytosol Ca2+ -dependent enzymes, such as calcineurin,
the transcription factor NFAT (nuclear factor activated T cells) and via the second
messenger DAG (diacylglycerol) and protein kinase C (PRKC) the transcription
factor NFκB. Furthermore, RAS and RAC stimulate a cascade of mitogen-activated
protein kinases (MAPKs), which finally results in the activation of the transcription
factor AP1. All three transcription factors belong to the most potent gene regulatory
proteins not only in B cells but also in T cells (Sect. 6.1) and in cells of the innate
immune system (Sects. 2.1 and 10.2). Thus, the activation of BCR by an antigen
results in a strong response on the level of gene regulation. Within the list of BCR
target genes are those encoding for costimulatory molecules like CD80 (also called
B7-1) and CD86 (also called B7-2), adhesion molecules like ICAM1 (intercellular
adhesion molecule 1), migration receptors, pro-survival molecules and cell cycle-
related protein. This is important for stimulating proliferation and differentiation of
B cells.
Box 4.2: Signal transduction cascades The cascades of signal transduction

pathways have a common structure and typically start with an extracellular
ligand binding to its cognate membrane receptor, which then at its cytosolic
part interacts with adaptor proteins and activates them. This activation status
is mostly transferred to a cascade of protein kinases that eventually either stim-
ulate a transcription factor or a chromatin modifying enzyme in the nucleus
or other key enzymes located in the cytosol. This often orchestrates gene expres-
sion changes that result in functional alterations of the concerned cell. In this
way, extracellular (and in part also intracellular) signals are perceived by dozens
of signal transduction pathways. The latter form networks of cellular circuits
that represent key lines of intracellular communication, i.e., mechanisms by
which a cell integrates and transduces intra- and extracellular signals from
their source, e.g., the extracellular environment, into an action of the cell,
such as to move, excite granules, grow, differentiate or keep homeostasis, i.e.,
of its fate. Some of these pathways do not involve gene expression changes.
Since proteins encoded by genes regulating cell fate, cell survival and genome
maintenance often interact with each other, their respective pathways overlap,
i.e., their classification may not be as distinct as indicated.
4.3 Humoral Adaptive Immune Response 67
In case the antigen is a protein, the B cells internalize the BCR-bound antigen to
an endosome, which fuses with a lysosome. Peptides derived from the antigen are
presented on the cell surface via MHC-II proteins. Through the communication with
TH cells (Sect. 4.3) the activated B cell gets a second confirmatory signal, which is
essential for the stimulation of proliferation and differentiation. Alternative second
signals are the activation of the CR2 (complement receptor 2, also called CD21) by
complement proteins like C3b (Sect. 7.2). Activated CR2 acts as a complex with
the membrane proteins CD81 and CD19, the cytosolic tails of which become tyro-
sine phosphorylated and stimulate via PI3K (phosphatidylinositol-4,5-bisphosphate
3-kinase) BTK and PLCG2. Thus, the CR2 co-receptor activation significantly
enhances BCR signaling. In this way, also non-protein antigens can induce T cells
independently an efficient B cell response (Sect. 4.3). Furthermore, B cells express
also inhibitory, ITIM domain-carrying co-receptors, such as FcγRIIb, which bind
IgGs being produced by the same B cell. This stimulates the negative regulatory phos-
phatases INPP5D (inositol polyphosphate-5-phosphatase D) and PTPN6 (protein
tyrosine phosphatase non-receptor type 6) that dampen BCR signaling. This anti-
body feedback control down-regulates the B cell response a few weeks after first
antigen encounter.
4.3 Humoral Adaptive Immune Response
The humoral form of the response of the adaptive immune system is a multi-
step process that ends up with the production of antibodies (Fig. 4.5). These anti-
bodies have effector functions primarily against extracellular pathogens but they are
also effective against intercellular pathogens and neutralize them before they infect
the host cell. Humoral response is in particular important against pathogens that
are covered by polysaccharides and lipids, such as Streptococcus pneumoniae and
Neisseria meningitidis, which are difficult to detect by T cells being restricted in
recognizing protein antigens.
First a naïve B cell clone is activated through the specific binding of an antigen
to its BCR (Sect. 4.2). In case the antigen is a protein, the activation is enhanced by
signals from TH cells or physical contacts with them. However, multivalent antigens
with repeating determinants, such as polysaccharides, can activate B cells without the
support of TH cells, i.e., such antigens are T cell independent. The latter responses are
rapid but are mostly mediated only by low-affinity IgMs. In contrast, T cell-dependent
responses are slower but become more potent. The activated B cell performs clonal
expansion, i.e., within a week it may proliferate into a pool of up to 5000 cells
with identical BCRs. In case these B cells are not exposed to any additional signal,
they differentiate into plasma cells that secrete IgM-type antibodies, which have the
same antigen specificity as the BCR that originally detected the antigen. Thus, when
the antigen was a polysaccharide, lipid or nucleic acid, the B cells will not be
further stimulated by TH cells and stick with their decision to produce IgMs.
In contrast, protein antigens enable TH cell support, which often induces isotype
Naïve IgM+, Antigen

IgD+ B cell Antigen recognition
TH1 cell,
other stimuli
B cell proliferation
Proliferation
IgG-expressing Ig-expressing B cell

B cell
Plasma
cell
IgM
IgG Memory B cell
IgG
Antibody secretion
Isotype switching
Fig. 4.5 Phases of B cell responses. A naïve B cell clone is activated via the binding of its specific
antigen to a BCR on its surface. Stimuli through cytokines produced by TH cells or other cells induce
the proliferation of the B cell (clonal expansion). Then the B cell either (i) directly differentiates
into a plasma cell secreting IgM-type antibodies, (ii) first performs isotype switching (in response to
respective signals) and then differentiates into a plasma cell secreting either IgGs (shown here), IgAs
or IgEs or (iii) first allows affinity maturation and then differentiates into a plasma cell secreting
antibodies with higher affinity or into a long-lived memory B cell
switching into IgAs, IgEs and IgGs, in order to adapt the effector function better to
the antigen. Moreover, the contact with TH cells also induces affinity maturation, in
order to enhance the affinity of the antibody for the antigen. B cells with optimized
IgAs, IgEs or IgGs can also develop into long-lived memory cells, which are able to
fight effectively against the antigen-carrying microbe, when the host gets infected a
second time. Thus, depending on the nature of the antigen the humoral response
of the adaptive immune system leads to the secretion of IgMs, IgAs, IgEs or
IgGs into the blood, lymph and interstitial fluids. The specific antibodies mediate
their effector function by:
• neutralizing microbes or their toxic products, i.e., preventing their entry and spread
• agglutinating two or more pathogens with the same antigen into an aggregate,
which is then more efficiently eliminated
• activation of the system of complement proteins (Sect. 7.2)
• opsonization (i.e., tagging) the surface of microbes for enhanced phagocytosis by
macrophages and neutrophils
• antibody-dependent cellular cytotoxicity (ADCC) (Sect. 11.2), i.e., targeting
microbe-infected cells for lysis by cells of the innate immune system, such as
NK cells carrying FcRs and other leukocytes
• activation of mast cell for expelling parasitic worms.
About half of all mRNA produced by a plasma cell is encoding for antibodies,
so that a single cell can secrete thousands of antibody molecules per second. Thus,
the 5000 plasma cells of a B cell clone originating from a single naïve B cell
may produce together every day some 1012 antibody molecules. This extreme
expansion is essential for competing with rapidly dividing microbes.
While the first infection with a microbe results primarily in the production of
IgMs by previously naïve B cells, the second response after a re-exposure with the
same microbe is mediated by memory B cells (Box 4.3) that often have adapted their
secreted antibody for more effective effector function and higher antigen affinity by
isotype switching and affinity maturation, respectively (Fig. 4.6). The magnitude of
the secondary response is larger than the primary response, i.e., a larger quantity of
antibodies is produced. Plasmablasts are circulating plasma cells that derive from
memory B cells. At steady state, their number in the blood is low but significantly
increases within a week after repeated infection. Some of these plasmablasts migrate
to the bone marrow and develop into long-lived plasma cells. The secondary response
is mediated by IgAs, IgEs or IgGs with higher average affinity as the IgMs within
the first response, but is effective only against protein antigens. Interestingly, the
immune response to many bacteria and viruses in the blood is primarily mediated by
IgGs, while in mucosal tissues like intestine and airways the same microbes initiate
the production of IgAs. The effector function of IgAs is primarily the neutralization
of their antigens, i.e., their complete coverage, since this antibody isotypes often
operates at areas of the body that are not reached by phagocytes. Since IgGs are
more stable than other antibody types, a switch to IgGs improves the effectiveness of
the immune response. In this way, the secondary response is often so efficient in
Primary antibody response Secondary antibody response
First infection IgG Repeat infection

Plasma
cells
IgM
Amount of antibody
Short-lived plasma
cells in lymphoid Low-level
organs antibody
production
Activated Long-lived
B cells plasma cells
in bone
marrow Memory
Naïve Long-lived B cells
B cells plasma cells
in bone Memory
marrow B cells
0 7 >30 0 3 10 >30
Days after antigen exposure
Feature Primary antibody response Secondary antibody response

Magnitude Smaller Larger
Antibody Relative increase in IgG and, at
Usually IgM > IgG
isotype certain situations, in IgA or IgE
Antibody
variable
Induced by All immunogenes Only protein antigens
Fig. 4.6 Primary and secondary immune responses of B cells. The primary and secondary
response of B cell differs in strength, main antibody isotype and antigen affinity. Most secondary
responses are T cell-dependent, so that the antigen needs to be a protein. More details are provided
in the text
the elimination of the microbe that the host even does not feel the re-exposure
to the microbe.
Box 4.3: Memory B cells and vaccination Memory B cells are generated
mainly within germinal centers of lymph nodes, i.e., they preferentially derive
from secondary humoral responses and are T cell-dependent. Thus, antibodies

produced by memory B cells recognize exclusively protein antigens. The
memory B cells can survive for decades, since the express high levels of
anti-apoptotic proteins. Most memory B cells typically express high-affinity
BCRs and produce IgG-type antibodies, i.e., they underwent affinity matu-
ration and isotype switching. Thus, the production of large quantities of
isotype-switched, high-affinity antibodies after repeated exposure to an
antigen is based on the activation of memory B cells. Accordingly, the main
goal in the development of an effective vaccine against a microbe or microbial
toxin is the induction of both affinity maturation and memory B cell formation.
Therefore, vaccines need to activate also TH cells. In case, the targeted antigen
is a polysaccharide, which, e.g., encapsulates a bacterium, such as Pneumo-
coccus, Meningococcus or Haemophilus, it needs to be covalently linked to a
foreign protein, in order to create a hapten-carrier conjugate. The respective
vaccines are called conjugate vaccines and are particularly effective in infants
and young children, who less likely make strong T cell-independent responses
to polysaccharides than adults. Interestingly, a polysaccharide vaccine like
against Pneumococcus can induce long-term protective immunity.
FcRs and complement proteins mediate the effector function of antibodies only,
when they bind to adjacent molecules. Thus, a microbe can stimulate a humoral
adaptive immune response, only when it is recognized by at least two anti-
body molecules. This is an important condition, since it prevents that circulating
free antibodies trigger any effector responses, which would be inappropriate and
dangerous.
Humoral responses of the adaptive immune system are initiated primarily in
secondary lymphoid organs, such as lymph nodes and the spleen. Antigens reach
the lymph nodes from tissues via the lymph, while the spleen is exposed to antigens
circulating in the blood. B cells recognize an antigen in its intact native conforma-
tion, while for the contact with T cells a linear peptide derived from it needs to be
presented on MHC-II proteins on antigen-presenting cells, such as dendritic cells
and macrophages (Fig. 4.7). Importantly, B cells are also able to internalize protein
antigens and can present them in form of a peptide on their MHC-II proteins to TH
cells. Follicular B cells and TH cells are located in different zones of lymph nodes,
i.e., they get independently activated by the same antigen. Please note that the likeli-
hood that two given B or T cell clones are specific for the same antigen is 1:100,000
to 1:1,000,000. B and T cells activated in their respective zones follow a chemokine
gradient and migrate towards each other. They meet at the edges of the follicles and
test via the contact of the MHC-II protein on the B cell and the TCR on the T cell,
whether they share specificity for the same antigen. In fact, it is likely that the confor-
mational epitope of the antigen that is recognized by the BCR is not identical to the
linear peptide, for which the TCR is specific. The B cell may even present multiple
linear peptides to different T cells. Nevertheless, this T cell-dependent extrafollicular
Protein antigen
T cell
B cell zone epitope
(primary follicle)
B cell
T cell zone
epitope
Dendritic
cells
B cell
T cell
Initial T-B interaction
Extrafollicular focus
Short-lived
Germinal center
plasma cells
Follicular
TH cell
Follicular
Extrafollicular dendritic cell
TH cell
Fig. 4.7 T cell-dependent immune responses of B cells. In different zones of the lymph node
naïve B cells and TH cells are activated by the same protein antigen (but maybe by a different
epitope). Then both cell types move towards each other and have physical contact at the interface
of the B and T cell zones. The B cells proliferate, may perform isotype switching and differentiate
to short-lived plasma cells. Some T cells develop into follicular TH cells and form together with
activated B cells a germinal center, in which affinity maturation, further isotype switching and the
generation or memory B cells and long-lived plasma cells happens
primary antibody response will result in the production of antibody with specificity
to the conformational epitope. In contrast, marginal zone B cells in the spleen and
B-1 cells in mucosal tissues preferentially recognize multivalent antigens and start
a T cell-independent response. Both types of responses result in plasma cells that
produce IgMs with relatively low antigen-binding affinity. Moreover, the produced
plasma cells of the primary response are rather short-lived and do not move to distant
sites, such as the bone marrow, i.e., they stay in the secondary lymphoid tissues.
The interaction between activated B and TH cells takes place not only at the inter-
face of two follicles, but also at germinal centers of follicles, where some of the acti-
vated B and TH cells (referred to as follicular TH cells) migrate (Fig. 4.7). Germinal
centers are substructures within follicles that form 4–7 days after the initiation a
T cell-dependent B cell activation. Importantly, the cells of a germinal center derive
from one or only a few B cell clones. Germinal centers are distinguished into light and
dark zones. Light zones contain follicular TH cells and follicular dendritic cells, which
are absent from dark zones. Follicular dendritic cells are specialized stromal cells
that are highly efficient at capturing and displaying antibody- and/or complement-
coated antigens. For this purpose, the dendritic cells have cytosolic processes that
form a meshwork around which a germinal center is formed. Dark zones of germinal
centers are areas of densely packed B cells that proliferate with an extremely high
rate of every 6–12 h and undergo somatic hypermutation (Box 4.4). The progenies
of the proliferating B cells move to the light zones, where they are selected for
increased antigen affinity by contacts with follicular dendritic cells. Thus, follicular
TH cells and follicular dendritic cells stimulate the genetic diversification of B
cells via the expression of surface receptors, such as FcRs and CRs. Due to effective
elimination of antigen, its concentration in the germinal centers decreases over time
and the BCRs need to increase their antigen affinity, in order to assure the survival
of their B cell clone. Therefore, multiple rounds of mutations in the dark zone
and selection in the light zone result in affinity maturation. Positively selected
B cells differentiate into long-lived plasma cells or memory B cells, both of which
leave the germinal center. Most of these cells migrate to the bone marrow, which
2–3 weeks after the antigen encounter becomes the major antibody producing site.
These long-lived plasma cells may continue to secrete antibodies for decades, even
if the antigen is no longer present. This provides immediate protection in case the
antigen is encountered later. Thus, about half of the antibodies circulating in the
blood of an adult represent antigen encounters of the past.
Box 4.4: Somatic hypermutation and affinity maturation The repeated or

prolonged exposure of B cells with the antigen that they are specific for is a sign
that the B cell response needs to be improved. This is achieved by the expression
of the enzyme AID that converts at the tetranucleotide AGCT (which is found
preferentially within the rearranged V(D)J exon) C into U. After replication
the U is either converted to T or excised by DNA repair mechanisms. Thus,
this genomic region experiences a specific mutation rate of 1 in 1000 bp

per cell division, which is far higher as for any other gene. This somatic
hypermutation results in B cell clones whose BCRs show a wide variety in
affinity for the antigen that initiated the B cell response. In the light zone of
germinal centers these B cell clones get in contact with follicular dendritic cells
and follicular TH cells presenting the antigen. A strong interaction indicates a
high affinity BCR and leads to survival of the B cells, while a weak interaction
initiates the elimination of the respective B cells. In this way, B cell clones with
high antigen-binding affinity of their BCRs become dominant. This selection
procedure, referred to as affinity maturation, may be repeated multiple times
until it results in a B cell with a significantly improved antigen affinity. While
in primary immune responses the KD value of the BCR-antigen interaction is
in the range of 1–100 nM, it increases in secondary responses to 0.01 nM.
Even when the pathogenic microbe is eliminated, long-lived plasma cells and
memory B cells survive for years. When the host is re-infected after years
memory T cells activate the memory B cells, which then rapidly multiply and
produce (after differentiation to plasma cells) antibodies with improved antigen
binding affinity.
Activated TH cells, such as follicular TH cells in the light zone of the germinal
center, express the surface protein CD40LG (CD40 ligand, also called CD154), the
receptor of which, CD40, is found on the surface of B cells (Fig. 4.8). The CD40LG-
CD40 interaction stimulates within the B cells a signal transduction cascade, which
results in the activation of the transcription factors NFκB and AP1 (Sect. 4.2). This
stimulates the proliferation of the B cells and increases the production and secre-
tion of antibodies. Moreover, this activates the enzyme activation-induced cytidine
deaminase (AID), which is required for affinity maturation and isotype switching.
In T cell-dependent B cell responses, some of the B cells undergo heavy chain
isotype switching from μ/δ to γ, α or ε. In principle, isotype switching can already
happen during the primary response of B cells, i.e., at extrafollicular sites, but it occurs
most efficiently during the secondary response within germinal centers. Isotype
switching is regulated by cytokines that are produced by TH cells (Fig. 4.8). The
choice to which isotype the heavy chain is changed depends on the class of microbe,
to which the T cells had been exposed to as well as on the tissue-specific needs.
The encounter of helminthic parasites leads to the production of IL4 and stimulates
the secretion IgEs (Sect. 10.2), while in mucosal tissues under the influence of the
cytokine TGFβ1 IgAs are released. Isotype switching requires somatic recombination
between the V(D)J exon of the heavy gene locus, which had been formed during B
cell maturation (Sect. 3.3), and the respective C region of the desired isotype. Without
recombination B cells produce IgMs, once they have differentiated into plasma cells,
i.e., they stick with the expression of the constant Ig domains of the μ type. Nucleotide
sequences within introns between the J and C regions allow the recombination, in the
way of which the intervening DNA segments for unused constant regions are deleted.
Follicular TH cell
IgM+ B cell Activated B cell
CD40LG CD40
? IL4 Mucosal tissues;

cytokines (e.g., TGFβ)
Isotype
switching
IgG subclasses
IgM IgE, IgG4 IgA
(IgG1, IgG3)
Complement Opsonization and Immunity against Mucosal immunity

activation phagocytosis; helminths; (transport of IgA
Principal complement mast cell through epithelia)
activation; neonatal degranulation
functions immunity (immediate
(placental transfer) hypersensitivity)
Fig. 4.8 Mechanisms of heavy chain isotype switching. After triggering activated B cells by TH
cells they either produce (i) IgM when they are not exposed to any other signal, (ii) IgG1 and IgG3
after presently unknow signal contact, (iii) IgE and IgG4 in response to IL4 exposure or (iv) IgA
after stimulation with a wide range of cytokines including TGFβ1
Isotype switching demonstrates the remarkable plasticity of the response of B

cells, which are able to generate antibodies with distinct effector functions, so that
they can fight most effectively against various types of microbes (Chaps. 7 and 8).
Both somatic hypermutation as well as the somatic recombination during isotype
switching bear the risk of DNA breaks that allow the chromosomal translocation of
oncogenes into the active locus of the heavy chain gene. Therefore, B cell tumors,
referred to as lymphomas, often develop from germinal centers. Moreover, there is
the risk that the reactions within a germinal center creates self-reactive B cell clones
that may cause antibody-mediated autoimmune diseases (Sect. 10.3).
Further Reading
1. Cyster JG, Allen CDC (2019) B cell responses: cell interaction dynamics and decisions. Cell
177:524–540
2. Laidlaw BJ, Cyster JG (2021) Transcriptional regulation of memory B cell differentiation. Nat
Rev Immunol 21:209–220
Chapter 5
Antigen-Presenting Cells and the Major
Histocompatibility Complex
Abstract In this chapter, we will discuss the principles of antigen presentation. We

will learn that the major purpose of antigen-presenting cells, such as dendritic cells,
macrophages and B cells, is to display peptide antigens to T cells. These peptides
are derived from extracellular microbes and are presented via MHC-II proteins to
CD4+ T cells (TH cells), which are found with highest concentration at secondary
lymphoid organs, such as lymph nodes. In contrast, antigen peptides that origin from
intracellular microbes or damaged cellular proteins are displayed by all kind of body
cells on MHC-I proteins to CD8+ T cells (cytotoxic T cells). Thus, we will understand
that MHC proteins are central in segregating antigens that are produced inside of
regular cells, such as viral proteins, from those that internalized from outside, such
as proteins from extracellular bacteria.
Keywords Antigen-presenting cells · Dendritic cells · T cells · MHC-I · MHC-II ·

Antigen peptides · CD4 · CD8
5.1 Antigen-Presenting Cells
Since naïve B and T cells are concentrated in secondary lymphoid organs, such as
lymph nodes, where the chance that an antigen is specifically recognized is signifi-
cantly higher than the probability to be found by a lymphocyte in circulation (please
remind that the chance that a B or T cell clone is specific for a given antigen is
1:100,000–1:1,000,000). Thus, keeping the lymphocytes at a limited number of
sites and organizing the transport of antigen to these locations is more efficient
than the patrol of B and T cells throughout the body. Antigens that are recognized
by the BCR of B cells are often free circulating in the body and reach either lymph
nodes via the lymph or the spleen via the blood stream (Sect. 4.1). The latter antigens
reach the spleen either directly from tissues or via the thoracic duct from the lymph
and blood.
T cells are specialized to MHC protein-bound peptide antigens, since only these
can be recognized by their TCR. Accordingly, the major function of antigen-
presenting cells is to capture antigens from their site of entry, which are often
epithelium-lined surfaces like the skin and to transport them to lymph nodes for

https://doi.org/10.1007/978-3-031-04025-2_5
78 5 Antigen-Presenting Cells and the Major Histocompatibility Complex
presentation to naïve CD4+ T cells. In addition, antigens are also concentrated in

other secondary lymphoid organs, such as Peyer’s patches and tonsils, which are
located in mucosal tissues of the ileum (the final and longest segment of the small
intestine) and the pharynx (the throat behind the mouth and nasal cavity), respectively.
The term “antigen-presenting cell” is specifically applied for those cells that
express MHC-II proteins, display foreign antigens and bind to CD4+ T cells, such
as dendritic cells (Box 5.1), macrophages and B cells (Fig. 5.1). In addition, endothe-
lial and some epithelial cells are able to express MHC-II proteins and may present
antigens to T cells. For example, in the thymus epithelial cells present peptides via
Antigen Antigen
Response
uptake presentation
Dendritic cell Costimulator Naïve T cell

(e.g., CD80) activation:
CD28
clonal expansion
T cells
Naïve
T cell T cells
Macrophage
activation:
activation of
macrophages
(cell-mediated
T cell immunity)
Killed
microbe
B cell
activation:
B cell activation
and antibody
production
(humoral
T cell Antibody immunity)
Fig. 5.1 Antigen-presenting cells. CD4+ T cells recognize antigen-carrying MHC-II proteins on
the surface of dendritic cells (top), macrophages (center) and B cells (bottom). This results either
in clonal expansion and differentiation of T cells, stimulation of the macrophages to finish the
phagocytosis process or the differentiation of B cell into antibody-producing plasma cells. More
5.1 Antigen-Presenting Cells 79
MHC-I and MHC-II proteins as a central part of the selection process of maturing T
cells (Sect. 3.3). In contrast, all nucleated cells (i.e., all body cells besides erythro-
cytes) express MHC-I proteins, display self-antigens and are recognized by CD8+ T
cells but are not considered as classical antigen-presenting cells. Naïve CD4+ T cells
are activated most effectively by dendritic cells (Fig. 5.1, top), while MHC-II protein
expressing macrophages (Fig. 5.1, center) and B cells (Fig. 5.1, bottom) communi-
cate preferentially with effector CD4+ T cells, i.e., with different types of TH cells.
The antigen presentation results either in:
• the clonal expansion and differentiation of the activated T cells into effector cells
(Sect. 6.1)
• the activation of the macrophages, in order to terminate the phagocytosis process
(Sect. 2.3)
• the stimulation of the B cells to differentiate into antibody-producing plasma cells
(Sect. 4.3).
Box 5.1: Dendritic cells Dendritic cells derive from the myeloid lineage of
hematopoiesis (Sect. 1.3), i.e., they belong to the innate immune system. In
tissues, monocytes can differentiate into dendritic cells, which then mediate
together with neutrophiles and macrophages the inflammatory response
(Sect. 2.4). Moreover, in utero (i.e., before birth), dendritic cells develop from
embryonic precursors in the yolk sac or fetal liver and take up residence in the
epithelial layer of the skin. Immature dendritic cells phagocytose microbes and
turn into mature antigen-presenting cells using MHC proteins. These mature
cells activate both naïve CD4+ and CD8+ T cells as well as memory CD4+ T
cells. Dendritic cells are the most efficient antigen-presenting cells for initiating
a response of naïve CD4+ T cells, because they:
• are positioned strategically at common sites of microbe entry as well as in
tissues that are often colonized by microbes
• have due to long membranous projections (looking like dendrites on
neurons) a very large surface-to-volume ratio allowing them to efficiently
sampling pathogens, such as viruses and bacteria, from their surrounding
• express membrane receptors capturing microbes and responding to them
• migrate via the lymphatics from the sites of microbe capture into the T cell
zones of lymph nodes
• express after maturation increased amounts of MHC proteins, co-
stimulators and cytokines.
In addition to the display of peptide antigens to T cells, antigen-presenting cells

contribute further stimuli inducing the full responses of T cells. These second signals
are provided either by membrane-bound proteins (Sect. 6.1) or cytokines that are
secreted by antigen-presenting cells. These co-stimulators are in particular impor-

tant for the activation of naïve T cells. Dendritic cells and macrophages express
pattern-recognition receptors, such as TLRs, which detect the presence of microbes.
Therefore, microbes but not non-microbial antigens are able to enhance the function
of antigen-presenting cells by stimulating the expression of MHC-II proteins and
co-stimulatory proteins improve the antigen presentation. Moreover, the production
of cytokines is boosted, which increases the efficiency of the interaction with T cells.
Typical entry sites of foreign antigens, such as microbes, are the skin as well as
the epithelia of the respiratory and gastrointestinal tract. Dendritic cells are located
close to these epithelia, in order to optimize their chance in capturing antigens. These
tissue-resident dendritic cells express on their surface a number of different pattern-
recognition receptors, such as C-type lectin receptors, that are able to bind microbes
(Sect. 2.1). Captured microbes are internalized by endocytosis and digested in lyso-
somes. The resulting microbial peptides are loaded on MHC-II proteins (Sect. 5.3). In
parallel, the activated dendritic cells (also called mature dendritic cells) detach from
the epithelial tissues and express the chemokine receptor CCR7 (Box 2.2). The latter
allows the dendritic cells to follow a gradient of the chemokines CCL19 and CCL21
that are produced in lymphatic vessels and lymph nodes. This guides the migration
of dendritic cells towards the T cell zones of the closest lymph node. Numerous
lymphatic capillaries in these epithelia drain lymph towards regional lymph nodes
and transport free antigens as well as dendritic cells with MHC-II protein-bound
antigen peptides to the secondary lymphoid organs, in order to concentrate the anti-
gens there. During their 12–24 h migration activated dendritic cells increase their
MHC-II protein expression, i.e., they differentiate into potent antigen-presenting
cells. Naïve T cells also express CCR7 receptors, which directs them to the T cell
zones of the lymph nodes. This maximizes the chance that they get in contact with
antigen-presenting cells. Moreover, the T cells express CD40LG, which interacts with
its receptor CD40 on the surface of dendritic cells and macrophages (Sect. 4.3). In
addition, T cells secrete cytokines, such as IFNγ, that activate their specific receptors
on the surface of antigen-presenting cells.
5.2 MHC Proteins and the HLA Locus
MHC proteins are surface receptors that are able to bind and present peptides, which
are recognized by TCRs of T cells. While MHC-I proteins are expressed on all nucle-
ated cells, MHC-II proteins are found only on antigen-presenting cells (Sect. 5.1).
MHC-I proteins are formed of a large (44–47 kD) α chain and a small (12 kD) β
chain, which is referred to as β2-microglobulin (Fig. 5.2, left). In contrast, MHC-II
proteins are composed of about equally sized α and β chains (29–34 kD) (Fig. 5.2,
right). Nevertheless, the 3D structure of both types of proteins is comparable, the
most important part of which is the peptide-binding cleft. In MHC-I proteins the
binding cleft is tighter (since it is formed only by the α chain) and can accommo-
date only a smaller peptide (8–11 amino acids) that origins from the degradation of
5.2 MHC Proteins and the HLA Locus 81
MHC-I protein MHC-II protein
Peptide-binding cleft
α2 α1
α1 β1
NN
N N
β2 α3 β2 α2
Transmembrane region
^^
C C C
Ig domain
Fig. 5.2 Structure of MHC-I and MHC-II proteins. MHC proteins are membrane-anchored
proteins that have a peptide-binding cleft. MHC-I proteins are formed by a large polymorphic α
subunit containing the peptide-binding cleft and a small non-polymorphic β2-microglobulin subunit
(left). In contrast, MHC-II proteins are composed by two equally sized polymorphic α and β chains,
both of which contribute to the peptide-binding cleft and are integrated to the membrane (right).
In both types of proteins, the α and β chains are non-covalently linked
globular cytosolic proteins within the proteasome (Sect. 5.3). In contrast, in MHC-
II proteins both the α and β chain contribute to the binding cleft, which is open
to both ends. This leaves space for a larger peptide (10–30 amino acids, but the
optimal size is 12–16 residues) that results from digestion of microbial proteins in
lysosomes. Both types of MHC proteins can bind a large variety of peptides,
i.e., in contrast to TCRs they are not restricted a specific antigen. Peptides are
bound non-covalently to MHC proteins, but they have a very slow off-rate. Half-
lives of peptide-MHC complexes range from hours to many days assuring that the
peptides are displayed long enough for a recognition by TCRs. Moreover, the peptide
contributes to the stable interaction of the α and β chain of MHC proteins. In this way,
only peptide-loaden MHC proteins are stably expressed on the cell surface. Thus,
MHC proteins are trimeric complexes of an α chain, a β chain and a peptide
that are only functional in this constellation.
Both types of MHC proteins contain similar but not identical Ig domains
(Sect. 4.1), which are specifically recognized by the co-receptors CD4 and CD8,
respectively, which are expressed on T cells (Sect. 5.3). The expression of either
CD4 or CD8 proteins is the main distinction between TH cells and cytotoxic T cells.
CD8+ T cells contact MHC-I protein bearing normal body cells, while TH cells
interact only with MHC-II protein carrying antigen-presenting cells. Thus, CD4+ T
cells are MHC-II restricted and CD8+ T cells are MHC-I restricted.
MHC-I and MHC-II proteins are encoded by the HLA (human leukocyte antigen)
gene cluster (Fig. 5.3), which is a genomic region in chromosome 6 that shows
higher interindividual variations than any other region of the human genome. Thus,
Proteasome
TAP2
TAP1 DOB
DP DM DQ DR
TAPBP B2 B1 A1 DOA A B B2 A2 B1 A1 B1-B9 A
Class II
region
0 200 400 600 800 1000
CFB TNF
C4B C4A C2 LTA LTB MICB
Class III
region
1000 1200 1400 1600 1800 2000
MICA HLA-B HLA-C HLA-E

Class I
region
2000 2200 2400 2600 2800 3000
HLA-A HLA-G HLA-F

Class I
region
3000 3200 3400 3600 3800 4000
Fig. 5.3 The HLA gene cluster. Schematic representation of the about 4 Mb of the genomic region
of the cluster of the HLA genes on human chromosome 6. The location of selected genes is indicated,
which encode for MHC-I proteins (class I), MHC-II proteins (class II) and other immune-related
proteins (class III), such as C4A, C4B, CFB, C2, LTB, TNF, LTA and MICB
5.2 MHC Proteins and the HLA Locus 83
MHC proteins are highly polymorphic, i.e., they show a large number of variant
amino acids in particular in adjacent to their peptide-binding cleft. This polymorphic
protein domain is the contact point for the CDRs within the variable domain of TCRs
(Sect. 6.1). At present (January 2022) more than 24,000 alleles of HLA class I genes
(the major genes HLA-A, HLA-B and HLA-C as well as the minor genes HLA-E, HLA-
F and HLA-G) and nearly 8900 alleles of HLA class II genes (HLA-DRA, HLA-DRB1-
9, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DOA, HLA-DOB, HLA-
DPA1, HLA-DPB1, HLA-DPB2, HLA-DMA, HLA-DMB) are known (http://hla.allele
s.org). Furthermore, the HLA gene locus also contains class III genes, which do not
encode for MHC proteins but for other important immune-related proteins, such
as the cytokines TNF, LTA (lymphotoxin alpha) and LTB (lymphotoxin beta), the
complement proteins C2, C4A, C4B and CFB (complement factor B) and MICB
(MHC class I polypeptide-related sequence B).
Since HLA genes are expressed from both copies of the genome, there are a large
number of different MHC proteins available for antigen display. Nevertheless, most
individuals use only eight HLA class II genes. Importantly, TCRs not only contact
the peptide carried by the MHC proteins but also the proteins themselves. Therefore,
T cell recognize the MHC proteins of a different person as foreign and initiate
an immune response. This is the molecular basis of graft rejection after organ
transplantation (Sect. 9.2). Moreover, MHC proteins do not only display foreign
peptides but even a far larger number of self-peptides, i.e., fragments of regular
proteins from the same individual. However, in most cases T cells are carefully
selected by mechanisms of central tolerance (Sect. 9.1) during their maturation in the
thymus (Sect. 3.3) not to recognized these self-peptides. A failure of self-tolerance
can lead to autoimmune diseases (Sect. 10.4).
The variant MHC-II protein subtypes differ in their preference for antigenic
peptides. For example, if an individual shows a strong allergic reaction against a
pollen protein, the person is likely to carry a class II HLA gene allele, the protein
product of which efficiently binds an immunodominant peptide (Box 5.2) derived
from the respective antigenic protein. This HLA allele may be inherited and explains
why children show the same type of allergies as their mother or father. Furthermore,
genetic predisposition for certain antigenic peptides can also be the reason why other
antigens are not recognized at all and a person is non-responsive to a vaccine.
Box 5.2: Immunodominant peptides Complex antigenic proteins need to be

digested into short linear peptides, in order to be presented via MHC proteins
to T cells. In most cases only a few of the large number of possible peptides
turn out to be effective activators of T cells. These immunodominant epitopes
or determinants represent only a minor part of the whole antigen. Knowing the
structural basis of this immunodominance is very important for the prediction
of the most effective peptides. Thus, knowing the amino acid sequence of an
antigenic protein, e.g., a viral spike protein, as well as its 3D structure, is

essential for the design of an effective vaccine.
Cytokines that are secreted by both innate and adaptive immune cells up-regulate
the expression of MHC proteins. A rising number of MHC-I and MHC-II proteins
promotes the display of antigen peptides and increases the response of cytotoxic T
cells and TH cells, respectively, boosting the adaptive immune response. For example,
many viruses induce an early innate immune response leading to the production of
IFNα and IFNβ (Sect. 8.1), which increase HLA class I gene expression. Accordingly,
there is a larger number of MHC-I proteins that present peptides derived from viral
proteins produced and degraded in the cytosol of infected cells. In contrast, the
expression of HLA class II genes in antigen-presenting cells, such as dendritic cells
(Sect. 5.1), is stimulated by IFNγ, which is initially secreted by NK cells and later by
effector T cells. This is one of several mechanisms, how the innate immune system
stimulates the adaptive immune system. A further mechanism is the stimulation of
HLA class II genes in response to signaling of microbe-activated TLRs.
5.3 MHC Pathways
MHC proteins can bind only peptides. There are two different mechanisms how
antigen proteins are digested and the resulting peptides are loaded on MHC
proteins. These antigen processing pathways distinguish primarily between proteins
that are located and lysed within the cytosol, i.e., being of intracellular origin, and
proteins that are taken up by endocytosis, i.e., being of extracellular origin, and
degraded in lysosomes (Box 5.3). The pathways assure that peptides from intracel-
lular proteins are loaded on MHC-I proteins and presented to CD8+ T cells, while
peptides from extracellular proteins end up on MHC-II proteins that are displayed
to CD4+ cells. Since both types of T cells have after clonal expansion and differen-
tiation significantly different effector functions (Sects. 6.3 and 6.4), this distinction
is essential for a proper function of the adaptive immune system (Fig. 5.4).
Box 5.3: Protein degradation All proteins of a cells are synthesized at ribo-
somes that are located in the cytosol or attached to the ER. The synthesis of
proteins and in particular their folding into correct 3D structures has a reason-
able failure rate (approximately 20%). These mal-formed proteins are rapidly
degraded in the proteasome to smaller peptides and single amino acids. In
addition, even correctly folded proteins have a limited half-life and, based
on their ubiquitination status, are degraded through the same process hours,
5.3 MHC Pathways 85
Nucleus
Viral infection Exocytic
vesicle
CD8
ERAP
TAP1/2
Viral protein
translation Peptides
TAPBP
Synthesized β2m
viral Golgi
protein Proteasome
Class I
MHC α chain
Chaperone
Ubiquitination
Normal body cell ER
Antigen presenting cell

Protein antigen
Lysosome CD4
Clip
HLA-DM
Endocytic
vesicle
Endosome
Invari- Exocytic
ant chain vesicle
α
Chaperone
β
Class II Golgi
MHC
ER
Fig. 5.4 The pathways of MHC-I and MHC-II proteins. In the MHC-I pathway (top), cytosolic
proteins are digested by proteasomes and resulting peptides are transported into the ER, where they
bind to MHC-I proteins. In the MHC-II pathway (bottom), proteins from extracellular sources are
taken up by endocytosis and degraded within lysosomes. The resulting peptides stay enclosed by
vesicles, which fuse with vesicles derived from the ER that contain MHC-II proteins. Further details
are provided in the text
days or weeks after their synthesis. Proteins that are degraded by the protea-
some are either regular cellular proteins and result in the production of self-
peptides or they origin from microbes that are able to reach the cytosol, such as
viral proteins (Sect. 8.1) and extracellular bacteria injecting proteins into the
cytosol (Sect. 7.3). Moreover, some cytosolic peptides may represent micro-
bial proteins that were internalized by phagocytosis (Sect. 2.3) but escaped the
phagosomes into the cytosol, such as observed with the bacterium Listeria
monocytogenes. The proteasome is a large multiprotein enzyme complex
located in the cytosol and nucleus. In contrast, proteins that that are taken
up from extracellular sources via endocytosis first locate in endosomes and
traffic then to lysosomes. The latter are specialized organelles, which contain
proteases like cathepsins that degrade the ingested proteins to larger peptides
and single amino acids. Thus, the location of a protein is important for the site of
its degradation. Accordingly, peptides that are lysed by the proteasome repre-
sent cytosolic proteins, while peptides produced in lysosomes serve as markers
of extracellular proteins. Therefore, it is important that cytosolic peptides
are loaded only on MHC-I proteins, while peptides of extracellular origin
bind to MHC-II proteins.
In both antigen processing pathways, the peptides are loaded to respective MHC
proteins before they are expressed on the cell surface. Like all other membrane
bound proteins, both types of MHC proteins are synthesized at the membrane of
the ER. The ER membrane contains the transport proteins TAP1 (transporter 1, ATP
binding cassette subfamily B member) and TAP2 that together pump in an ATP-
dependent process cytosolic peptides into the lumen of the ER (Fig. 5.4, top). In
this way, self-peptides as well as antigenic peptides are available in the ER lumen.
This is important, since heterodimeric MHC protein complexes are only stable in
the presence of a peptide in their binding cleft (Sect. 5.2). Moreover, TAPBP (TAP
binding protein, also called tapasin) links TAP1-TAP2 heterodimers specifically with
freshly assembling MHC-I proteins. Interestingly, the genes encoding for TAP1,
TAP2 and TAPBP are also located within the HLA gene cluster (Fig. 5.3). MHC-I
proteins that are stabilized by peptide binding dissociate from TAPBP, move from
the ER to the Golgi complex and are transported by exocytic vesicles to the cell
surface. There they may be recognized by TCRs of cytotoxic T cells, which via their
co-receptor CD8 verify that they are communicating with MHC-I proteins.
Peptides loaded on MHC-II proteins derive from extracellular proteins that are
taken up by either endocytosis, pinocytosis or phagocytosis or they result from
autophagy of the cell’s own protein located in membranes or vesicles (Fig. 5.4,
bottom). All these proteins are targeted to lysosomes (Box 5.3), where they are
digested. Thus, the resulting peptides derive from antigens that bind to receptors on
the cell surface and are then internalized. After synthesis of MHC-II proteins in the
ER their peptide binding cleft is filled by a subdomain of a protein called invariant
chain. This assures that peptides of cytosolic origin are not loaded to MHC-II proteins.
5.3 MHC Pathways 87
The invariant chain protein directs the MHC-II proteins via the Golgi complex to
late endosomes and lysosomes containing the peptides of extracellular antigenic
origin. Then the invariant chain protein is digested by lysosomal proteases, disso-
ciates from the MHC-II proteins and is replaced by the peptides. This exchange
process is facilitated by the non-polymorphic HLA-DM protein, which in parallel
selects for peptides binding with high affinity. Furthermore, proteases fit the peptides
to the size of the MHC-II protein’s binding cleft by trimming overhanging ends. The
peptide-stabilized MHC-II proteins are then delivered to cell surface. There they can
be recognized by TCRs of T cells, which prove via their co-receptor CD4 the identity
of MHC-II proteins.
Interestingly, some dendritic cells are able to take up via phagocytosis both virus-
infected and tumor cells (Fig. 5.5). As usual, the antigenic proteins of these cells are
digested in lysosomes to peptides, i.e., they follow the MHC-II pathway. However,
some of the antigenic proteins escape the phagosome and are transported to the
cytosol, where they enter the MHC-I pathway, i.e., they are digested by the protea-
some and their resulting peptides are presented via MHC-I proteins to cytotoxic T
cells. Due to this cross-presentation dendritic cells can inform both TH cells and
cytotoxic T cells about the presence of virus infection (Chap. 8) or tumor burden
(Chap. 11). Thus, instead of exclusively relying on the recognition of virus-
infected cells and tumors by cytotoxic T cells, the additional activation of TH
Antigen Cross- T cell

capture presentation response
Infected cells and
TH cell Viral antigen
viral antigens picked
up by host antigen- enters cytosol
presenting cells MHC-II
Virus
infected cell
Viral Dendritic cell

MHC-I antigen Cytotoxic
Co-stimulation T cell
Fig. 5.5 Cross-presentation of antigens to cytotoxic T cells. Virus-infected cells can be phago-
cyted by dendritic cells. Instead of presenting peptides derived from antigenic proteins only via
the MHC-II pathway to TH cells, the dendritic cells allow some antigenic proteins to escape to the
cytosol. There they enter the MHC-I pathway and are presented to cytotoxic T cells
cells stimulates the production of antibodies by B cells and enhances the phago-
cytic capacity of macrophages (Sect. 6.3). In this way, both type of T cells get more
effectively involved in the fight against infections and cancer.
Further Reading
1. Trowsdale J, Knight JC (2013) Major histocompatibility complex genomics and human disease.
Annu Rev Genomics Hum Genet 14:301–323
2. Petersdorf EW, O’HUigin C (2019) The MHC in the era of next-generation sequencing:
Implications for bridging structure with function. Hum Immunol 80:67–78
3. Wieczorek M, Abualrous ET, Sticht J, Alvaro-Benito M, Stolzenberg S, Noe F, Freund C (2017)
Major histocompatibility complex (MHC) class I and MHC class II proteins: conformational
plasticity in antigen presentation. Front Immunol 8:292
Chapter 6
T Cell Immunity: T Cell Receptors
and Their Effector Functions
Abstract T cells constitute up to 30% of all circulating leukocytes and are a major
component of the adaptive immune system. In this chapter, we will describe that T cell
immunity is, like B cell immunity (Chap. 4), highly specific to antigens. The TCR is
the specific antigen receptor of T cells and recognizes exclusively peptides displayed
on MHC proteins (Chap. 5). Modulated by the activity of accessory proteins, antigen-
bound TCR initiates a signal transduction cascade that activates the same set of
transcription factors as BCR signaling. Activation of TCR signaling results in the
expansion of the respective T cell clone and the transformation to effector cells that
contribute to the neutralization of microbes. We will learn that based on different
type of antigen and signal exposure, CD4+ T cells differentiate into TH 1, TH 2 and
TH 17 cells. The different TH cells diverge in their functional profile of coordinating
other cells of the immune system in their fight against microbes. Finally, we will
understand that cytotoxic T cells directly attack viral-infected host cells and can kill
them by inducing apoptosis.
Keywords TCR structure · Accessory proteins · TCR signaling · Clonal

expansion · T cell effector function · TH cell subtypes · Cytotoxic T cells ·
Apoptosis pathways
6.1 TCR Signaling
The TCR is the specific antigen receptor of T cells, which resembles the antigen-
binding fragment of the Y-shaped BCR. Typically, TCR is a heterodimer of two
equally structured and sized α and β chains, each of which is formed of two Ig
domains (Fig. 6.1). However, in more rare γδ T cells the TCR is formed by γ and
δ chains (Box 6.1). In analogy to the variable domain of BCRs (Sect. 4.1) the N-
terminal Ig domain of both chains of the TCR is highly variable. The diversity of the
variable Ig domains is based on the same mechanism of somatic recombination as for
the analogous domains of the heavy and light chain of BCRs (Sect. 3.3, Fig. 3.8b).
Moreover, like in the BCR the variable domain contains three hypervariable CDRs
that like protruding fingers act as the T cell-specific antigen-binding interface of the
TCR (Fig. 6.1). However, in contrast to the BCR, which is able to recognize any

https://doi.org/10.1007/978-3-031-04025-2_6
90 6 T Cell Immunity: T Cell Receptors and Their Effector Functions
N N
Antigen-
binding
site
CDRs
Vβ Vα
bond
Carbohydrate
β chain α chain groups
Ig domain
Cβ Cα
Transmembrane
region
C C
T cell
Fig. 6.1 Schematic structure of the TCR. A typical TCR is formed by an α and a β chain, each of
which is composed of two Ig domains and carries a C-terminal transmembrane domain (bottom).
The N-terminal Ig domains of both chains (Vα and Vβ) are highly variable, in particular the three
protruding hypervariable CDRs forming the peptide-MHC protein binding site
macromolecular structure as a possible antigen, the antigen-binding interface of the

TCR interacts only with peptide-bound MHC proteins (Sect. 5.2), i.e., the TCR is
peptide-restricted.
Box 6.1: γδ T cells and other specialized T cells There are smaller popula-
tions of T cells with distinct features and specialized functions in host defense
that are different from CD4+ and CD8+ T cells, such as γδ T cells (< 5% of
6.1 TCR Signaling 91
all T cells), mucosa-associated invariant T (MAIT) cells and natural killer T

(NKT) cells. γδ T cells are mainly found in epithelial tissues, where they are
involved in the early response to microbes before other cells of the adaptive
immune system getting effective. In contrast, MAIT cells are located prefer-
entially in the liver (> 50% of T cells in this tissue), where they form a barrier
to intestinal bacteria that may have entered the blood. NKT cells resemble NK
cells of the innate immune system (Sect. 2.5). Specialized T cells are involved
in the surveillance against stressed body cells, which are infected or damaged.
In addition, the specialized T cells secrete cytokines that modulate the activity
of other cells of the adaptive immune system. The antigen receptors of γδ T
cells, MAIT cells and NKT cells have limited diversity, so that the cells recog-
nize a lower number of different antigens (despite a high potential of diversity,
Sect. 3.3) that are not displayed by MHC proteins. In contrast to αβ T cells,
some γδ T cells recognize small phosphorylated molecules, alkyl amines or
lipids of microbial origin, i.e., they are not peptide-restricted.
The constant Ig domain of each to the two TCR chains has at its C-terminal end a
short hinge region, which covalently connects the α and β chain by a disulfide bond.
The transmembrane region embeds each TCR chain into the plasma membrane, so
that only 5–12 amino acids face the cytosol. The latter are too few forming an inter-
action domain with cytosolic proteins. However, the transmembrane region contains
positively charged lysine and arginine residues that enable a tight contact with nega-
tively charged aspartic acid residue of the transmembrane region of the proteins
CD3 and ζ. In analogy of the proteins Igα and Igβ in BCR signaling (Sect. 4.2), CD3
and ζ proteins carry each an ITAM domain, which transduces TCR activation to a
signal transduction cascade in the cytosol. The whole TCR complex is formed of the
two TCR chains, four copies of CD3 and a dimeric ζ protein that contacts together
with the co-receptors CD4 or CD8 peptide-bound MHC-II or MHC-I proteins on the
surface of antigen-presenting cells or normal body cells, respectively (Fig. 6.2). This
immune synapse (also referred to as supramolecular activation cluster) connects TH
cells with an antigen-presenting cells as well as cytotoxic T cells with normal body
cells (Sect. 5.3). The co-receptors CD4 and CD8 are critical for the decision with
what types of antigen-carrying cells a T cell is interacting with.
Like in other signaling cascades of innate and adaptive immune cells, such as NK
cells (Sect. 2.5) and B cells (Sect. 4.2), the effects of the core signal receiving receptor,
the TCR, are modulated by activating and inhibiting co-receptors of the CD28 family
(Fig. 6.2). CD28 itself is a typical T cell co-receptor that is stimulated when it interacts
with either CD80 or CD86 proteins on the surface of antigen-presenting cells. CD28
is expressed on T cells when their activation is needed, such as in response to micro-
bial products and innate immune reactions. Moreover, resting antigen-presenting
cells express only low levels of CD80 and CD86 preventing an accidental activa-
tion of naïve T cells. The activation of CD28 boosts TCR signaling via recruiting
the kinase PI3K that activates its downstream kinase AKT (Akt murine thymoma
Receptors of CD4+ Receptors of MHC-II expressing

T helper cell antigen-presenting cells
Signal CD28 CD80/CD86

transduction CD4
Antigen
recognition TCR MHC-II
ITAM
CD3
ζ
Signal
transduction
CTLA4 CD80/CD86
ITIM
PDCD1 CD274/
PDCD1LG2
Adhesion ITGB2 ICAM1
Fig. 6.2 The immune synapse and its accessory proteins. The immune synapse is formed by
the TCR, CD3 and ζ proteins, the co-receptors CD4 or CD8 and peptide-bound MHC-II or MHC-I
proteins. In addition, TH cells and cytotoxic T cells have a large number of common accessory
proteins on their surface, such as CD28, CTLA and PDCD1 binding CD80, CD86 and PDCDLG2
as well as the integrin ITGB2 (integrin subunit beta 2) binding ICAM1
viral oncogene homolog) as well as the Ca2+ signaling trigger PLCG2 (Sect. 4.2).
Moreover, CD28 contributes to the activation of the transcription factor NFκB via
RAC and MAPK. This leads to increased survival, proliferation and differentiation of
antigen-specific T cells. Thus, CD28-CD80/CD86 activation is an essential second
signal for boosting the activation of naïve T cells (Sect. 3.1). In contrast, effector
or memory T cells are less dependent on CD28 co-stimulator activation.
Another CD28 family member, CTLA4 (cytotoxic T lymphocyte associated
protein 4, also called CD152), binds CD80 and CD86 proteins even stronger than
CD28, but this interaction has inhibitory effects on TCR signaling. Thus, the expres-
sion of CTLA4 inhibits the initial activation of T cells in lymph nodes (Sect. 4.3).
Similarly, the binding of PDCD1 (programmed cell death 1, also called PD1) on T
cells with CD274 (also called PDL1) and PDCD1LG2 (programmed cell death 1
ligand 2, also called PDL2) on non-T cells leads to the inhibition of TCR signaling
in effector cells (Sect. 6.2). While the activation of T cells is critical, in order
to fight efficiently against pathogens, in certain situations it is also important to
6.1 TCR Signaling 93
down-regulate the immune response. Thus, CTLA4 and PDCD1 are expressed on
T cells, in order to prevent overboarding responses of the adaptive immune
system. This contributes to immune tolerance (Sec. 9.1). The blocking of CTLA4
and PDCD1 by commercially produced monoclonal antibodies is a promising tool
in the immunotherapy of cancer (Sect. 11.3). However, the therapy compromise
self-tolerance and may cause autoimmune responses of the patients (Sect. 10.4).
Since T and B cells origin from the same cell lineage, they do not only apply
the same mechanisms for obtaining the high diversity of their antigen receptors
(Sect. 3.3), but also the signal transduction cascades downstream of BCR and TCR
(Sect. 4.2) are very comparable (Fig. 6.3). When the co-receptors CD4 and CD8,
Phosphorylation, release Dephosphorylation MAP kinase, SAP

and degradation of IκB of cytoplasmatic NFAT kinase pathway
DAG
P PP GTP RAS
PRKC GTP RAC
P
G2
Ca2+
PLC
IκB
IκB Ca2+
p50 p65 P P
Calmodulin
p50 p65
inactive NFκB inactive
NFAT Calcineurin RAF1
active NFAT
p50 p65 MAP2K1
active NFκB
FOS IL2
p50 p65 JUN
NFκB NFAT AP1
Fig. 6.3 TCR cell signaling. Signaling cascades that origin at the TCR end with the transcription
factors NFκB, NFAT and AP1 converge on the enhancers of genes like IL2. More details are provided
in the text. MAP2K1 = mitogen-activated protein kinase kinase 1
which are at their cytosolic part associated with the SRC kinase LCK (LCK proto-
oncogene, SRC family tyrosine kinase), confirm via a specific contact with MHC-II
or MHC-I proteins, respectively, the correct formation of the immune synapse of TH
cells and cytotoxic T cells, LCK gets in close contact with the in total 10 ITAMs
of CD3 and ζ. Thus, the longer and stronger the TCR is activated by antigen,
the more ITAMs get phosphorylated by LCK. This induces the recruitment and
activation of the tyrosine kinase ZAP70 (zeta chain of T cell receptor associated
protein kinase 70). ZAP70 belongs to the same family of tyrosine kinases as SYK
in BCR signaling (Sect. 4.2) and has analogous function. Therefore, basically all
further downstream signaling steps TCR and BCR signaling are identical. In brief,
with BLNK, GRB2 SOS1 and BTK further adaptor proteins and kinases get activated
that induce via:
• the second messenger DAG and the kinase PRKC the transcription factor NFκB
• PLCG2 and increased cytosolic Ca2+ levels activating through calmodulin the
kinase calcineurin the transcription factor NFAT
• the small G proteins RAS and RAC and the MAPK cascade the transcription
factor AP1.
The three potent transcription factors NFκB, NFAT and AP1 synergistically acti-
vate enhancers regulating key immune genes, such as IL2 (Fig. 6.3) as well as others
encoding for cytokine receptors and T cell effector proteins. Thus, TCR activa-
tion results in the activation of genes critical for the regulation of survival,
proliferation and differentiation of T cells.
6.2 T Cell Effector Functions
After the exposure with antigen, T cells undergo the same principles of clonal expan-
sion as already described for B cells (Sect. 4.3). Accordingly, microbe-specific naïve
CD4+ and CD8+ T cells get activated via the contact of their TCR with peptides
presented on MHC-II and MHC-I proteins, respectively. Millions of naïve T cells
with individual TCRs, which are continuously produced in the thymus (Sect. 4.3),
circulate through our body. They move from one secondary lymphatic organ (mostly
lymph nodes) to another and meet there a large number of dendritic cells presenting
different antigens (Sect. 1.4). Mostly, one dendritic cell is presenting a number of
different peptides originating from the same antigenic protein (Sect. 5.3). When a
resting naïve T cell recognizes a peptide-MHC complex, for which it shows high
specificity, an immune synapse is formed and TCR signaling is initiated (Sect. 6.1).
Please note that the chance that a given T cell is specific for an antigen is 1:100,000–
1:1,000,000. The strength of the interaction and T cell activation largely depends
on co-stimulatory proteins of the CD28 family. The initiated signal transduction
cascade causes significant changes in the epigenome and transcriptome of the T cell.
This results in the consecutive expression of cytokines, cytokine receptors and other
surface molecules, such as IL2, CD69, IL2RA (interleukin 2 receptor subunit alpha,
6.2 T Cell Effector Functions 95
also called CD25) and CD40LG. This initiates clonal expansion of the T cell, i.e., in
a massive proliferation resulting within a week in up to 5000 (CD4+ ) to even 50,000
(CD8+ ) identical copies of T cells showing identical antigen specificity (Fig. 6.4).
Importantly, other T cells that are not specific to the given antigen do not proliferate
at all. Thus, within a week from a small number of antigen-specific T cells large
counts of effector cells with specificity for the same antigen are generated.
In parallel to their clonal expansion, the T cells become larger and differentiate
into different types of effector TH cells when carrying a CD4 co-receptor (Sect. 6.3)
or into cytotoxic T cells when being CD8+ (Sect. 6.4). Both types of effector T cells
mediate cellular immunity. Most of these cells leave the secondary lymphoid organs
and migrating via the blood stream to the site of infection. At this extravascular site
the microbe-specific T cells are retained by adhesive and chemotactic interactions
and contribute to the elimination of the microbes (Sect. 7.2). Furthermore, CD4+ T
cells that differentiate into follicular TH cells stay in the lymph nodes, translocate in
there to lymphoid follicles and stimulate B cells to produce high-affinity antibodies
of different isotypes (Sect. 4.3).
Clonal
expansion
10
Contraction
(homeostasis)
10
10 CD8+ T cells
Memory
Infection CD4+ T cells
days 7 14 200
Fig. 6.4 Dynamics of microbe-specific T cell numbers. Within the first week after onset of an
infection microbe-specific CD4+ and CD8+ T cells undergo clonal expansion and differentiate into
effector TH cells and cytotoxic T cells, respectively. After elimination of the microbe the T cell
numbers contract due to apoptosis of most of the effector T cells and only long-lived microbe-specific
CD4+ and CD8+ memory T cells remain
Humoral immunity only reacts on extracellular microbes that can be recognized

by antibodies, while cellular immunity is able to respond to intracellular microbes. TH
cells of types 1 and 17 (Sect. 6.3) both recognize macrophages presenting peptides
derived from ingested microbes on MHC-II proteins but produce different sets of
cytokines activating either the macrophages in finishing the phagocytosis or stim-
ulating neutrophiles to mediate inflammation, respectively (Fig. 6.5a). TH cells do
not directly eliminate microbes but stimulate other cells to do so. Thus, the major
effector function of TH cells is the production of cytokines, in order to coordinate
the function of other immune cells.
In contrast, cytolytic T cells directly interact with their targets (Sect. 6.4). The
latter are regular body cells that are microbe-infected and present peptides derived
from microbial proteins on MHC-I proteins (Fig. 6.5b). This results in killing of
the infected cells via the induction of apoptosis to them, i.e., the cells are dying by
an orderly process of programmed cell death. Thus, the effector function of both
types of T cells contribute with high efficiency to the elimination of microbes
throughout the whole body. Moreover, T cells can differentiate into Treg cells that are
involved in immune tolerance and inhibit overboarding responses of effector CD4+
and CD8+ T cells, e.g., by the production of IL10 and IL35 (Sect. 9.1). This reduces
a Phagocytes with ingested b Infected cell with microbes

microbes in vesicles or antigens in cytoplasm
CD4+ CD4+ CD8+
T cells (TH1 cells) T cells (TH17 cells) T cells (cytolytic
T cells)
Cytokine secretion
Macrophage
activation =>
killing of ingested killing of Killing of
microbes microbes infected cell
Fig. 6.5 Contribution of T cells to the fight against infections. TH 1 and TH 17 cells respond to
macrophages having ingested intracellular bacteria (a). They produce cytokines stimulating either
macrophages or neutrophiles to eliminate the microbe. Cytolytic T cells recognize virus-infected
body cells and induce apoptosis to them (b)
6.3 T Helper Cells 97
tissue destruction and immunopathology. However, Treg cells are not considered as
effector cells.
The effector phase is followed by a contraction phase, in which the T cell responses
decline after the microbes are eliminated. Most of the effector T cells undergo apop-
tosis after their task is finished, because due to the disappearance of the antigens
they lack a survival stimulus. In this way, the immune system returns to homeostasis.
However, some microbe-specific memory T cells survive for years or even lifetime.
This is based on the increased expression of anti-apoptotic proteins, such as BCL2
(BCL2 apoptosis regulator) and BCL2L1 (BCL2 like 1). Furthermore, memory cells
are able to self-renew, i.e., they are slowly proliferating. With aging the percentage of
memory T cells in the total number of T cells increases (Sect. 3.1). After an infection
the number of antigen-specific memory T cells is 10- to 100-fold higher than that of
the initial naïve T cells. Moreover, the epigenome of memory T cells is at regions
of key immune genes, such as those encoding for cytokines, in a more responsive
“poised” state. Since immunity is systemic, memory cells migrate to basically every
site in the body. Therefore, a second exposure with the same antigen will lead to a
stronger and more rapid response, so that the host often does not get aware of the
recurrent infection. The success of a vaccination is largely based on this memory
effect.
6.3 T Helper Cells
The general function of TH cells is to support (“help”) other cells of the immune
system, such as the maturation of B cells into antibody producing plasma cells
(Sect. 4.3) or the activation of macrophages (Sect. 6.2). TH cells are character-
ized by the expression of the CD4 glycoprotein on their surface, which restricts
their direct communication to antigen-presenting cells, such as dendritic cells, that
display peptide-loaden MHC-II protein on their surface (Sect. 5.3). Depending of
the type of microbe that antigen-presenting cells have encountered, they produce
different types of cytokines, in order to induce the production of a TH subtype that
combats the respective type of microbe most efficiently, i.e., environmental factors
shape the differentiation of the T cells. For example, intracellular bacteria, such
as Listeria and mycobacteria, parasites, such as Leishmania, as well as some viruses
lead to the production of IFNγ (Box 2.5), IL12, IL18 and type I IFNs by cells of
the innate immune system, such as dendritic cells, macrophages and NK cells. All
these signals induce a differentiation into TH 1 cells and a so-called type 1 immune
response (Sect. 10.5). In turn, IL4 is produced after a contact with helminthic para-
sites and the naïve T cells differentiate into TH 2 cells, which is the core of the type 2
immune response.
Signal transduction pathways in response to stimuli from antigen-presenting cells
induce different types of epigenetic programming of the differentiating TH cells,
such as specific epigenetic changes in DNA methylation and histone modification at
enhancer and promoter regions. Moreover, TH subtype-specific transcription factors,
such as TBX21, STAT (signal transducer and activator of transcription) 1 and 4

(TH 1), GATA3 and STAT6 (TH 2) as well as RORC and STAT3 (TH 17), are activated,
which results in specific gene expression patterns. The changes in the epigenome and
transcriptome are inherited in the progeny of proliferating cells, so that all members
of the T cell clone become committed to the same pattern. This guides the differen-
tiation of the T effector cells into TH 1, TH 2 and TH 17 subtypes, i.e., the developing
cells become more and more committed to a specific cytokine expression profile
(Table 6.1). This is the production of.
• INFγ (Box 2.5) and IL2 by TH 1 cells
• IL4 (Box 6.2), IL5 and IL13 by TH 2 cells
• IL17 (Box 6.3) and IL22 by TH 17 cells
• IL21 by follicular TH cells.
In turn, the respective cytokine patterns amplify the TH cell subtype differenti-
ation. For example, IFNγ promotes TH 1 cell differentiation and inhibits the devel-
opment of TH 2 and TH 17 cells. This increases the polarization of the subtypes, so
that different T cell populations accumulate. Most extreme polarization occurs
in the context of chronic infections, which are characterized by prolonged immune
stimulation (Sect. 8.2). However, as already discussed in the context of M1- and M2-
type macrophages (Sect. 2.3), the three types of TH cells should be understood as
extremes of a continuum of multiple intermediate cell states. The multitude of envi-
ronmental signals that affect the activation pattern of these T effector cells and
impose specific epigenetic programming. This results in many different expression
patterns of cytokines and other immune-related genes.
Table 6.1 Major subsets of TH cells. Naive CD4+ T cells differentiate into different subsets of
effector cells, which are distinguished by the major cytokines that they are producing, the types of
cells they are primarily interacting, the immune reactions that they are modulating, their general
role in host defense and their impact in disease
T cell type Major Principal Major immune Role in host Impact in
produced target cells reactions defense disease
cytokines
TH 1 IFNγ, IL2 Macrophages Activation of Intracellular Autoimmune
macrophages microbes diseases,
chronic
inflammation
TH 2 IL4, IL5, Eosinophiles Activation of Helminths Allergy
IL13 eosinophiles
and mast cells
TH 17 IL17, IL22 Neutrophiles Recruitment Extracellular Autoimmune
and activation bacteria and diseases,
of neutrophils fungi inflammation
Follicular TH IL21 B cells Stimulation of Extracellular Autoimmune
cells antibody microbes diseases
production
Box 6.2: IL4 This is the master cytokine of TH 2 cells, since it acts both as
an inducer and effector of this TH cell subset. The latter are the stimulation
of intestinal peristalsis and the recruitment of eosinophiles. IL4 is structurally
and functionally similar to IL13; together both cytokines activate the alternative
pathway of M2-type macrophages and suppress the IFNγ-mediated classical
pathway of M1-type macrophages. In addition, IL4 produced by follicular TH
cells stimulates B cell to perform antibody isotype switching from IgM to IgE
and IgG4 (Sect. 4.3).
Box 6.3: IL17 This cytokine for a class of six IL17 subtypes (A-F) and does
not resemble other cytokines. The main family member is IL17A, which is
secreted by TH 17 cells as well as by some innate immune cells, such as ILC3
cells. IL17 connects cellular adaptive immune responses with acute inflam-
matory actions of neutrophiles, i.e., IL17 is a pro-inflammatory cytokine. The
latter are more severe and prolonged, when TH 17 cells are involved, as with
innate immunity alone.
The cytokine expression patterns explain most of the different functional profiles
of the cell populations, i.e., their impact in health and disease. TH cell-dependent
inflammation evolved as a mechanism of anti-microbial defense. However, the acti-
vation of macrophages and neutrophiles by TH 1 and TH 17 cells, respectively, can
lead to tissue injury. This collateral damage is called delayed-type hypersensitivity
and causes much of the pathology associated with certain types of infection and
chronic immunologic diseases (Chaps. 7, 8 and 10). Therefore, TH 1 and TH 17 cells
are often associated with inflammatory autoimmune diseases (Sect. 10.4), while TH 2
have a key role in allergic reactions mediated by eosinophiles (Sect. 10.2) (Table 6.1).
TH 1, TH 2 and TH 17 cells differ in the expression of chemokine receptors and
adhesion molecules. TH 1 cells have high levels of CXCR3 and CCR5 as well as of
SELE (selectin E) and SELP (selectin P), which attracts them to sites of prominent
inflammation mediated by cells of the innate immune system. In contrast, CCR3,
CCR4 and CCR8 are found preferentially in TH 2 cells and guide them to sites of
infections by helminths in mucosal tissues. Finally, the chemokine CCL20 leads
CCR6-expressing TH 17 cells to sites of bacterial and fungal infections, in particular
in mucosal tissues of the gastrointestinal tract. TH 17 cell development depends on
the gut microbiome and therefore this TH subset is involved in the development of
pathologic intestinal inflammation (Sect. 7.4).
The main function of TH 1 cells is to stimulate macrophages to destroy ingested
microbes (Fig. 6.6, left). TH 1 cells communicate with macrophages both via
CD40LG-CD40 interactions and by IFNγ secretion. This microbe destroying
Bacteria Antigen-
Bacteria presenting cells
Naïve CD4+
T cell
Naïve T cell
Fungi
Proliferation and
Antigen-
presenting cells
TH17 cells
TH1 cell
IL17 IL22
Leukocytes
and tissue cells
Macrophage Tissue cells
IFNγ
Chemokines, TNF,
IL1, IL6, CSFs
Anti-microbial
peptides
Classical macrophage
activation (enhanced Increased
microbial killing) neutrophil response barrier function
Fig. 6.6 Functions of TH 1 and TH 17 cells. When TH 1 cells interact with macrophages presenting
antigenic peptides, for which they are specific, they secrete IFNγ (left). The cytokine stimulates the
classical macrophage pathway, in which the cells increase their phagocytosis and kill the ingested
microbes. In contrast, TH 17 cells produce either IL17, which recruits neutrophils as well as other
leukocytes and increases production of anti-microbial peptides, or IL22, which promotes epithelial
barrier functions (right). More details are provided in the text
pathway is referred to as “classical” and mediated by M1-type macrophages

(Sect. 2.3). In contrast, TH 2 cells activate the “alternative” pathway, which is
performed by M2-type macrophages and results in tissue repair. Activation of CD40
on the surface of macrophages induces a signal transduction cascade that results in
the activation of the transcription factors NFκB and AP1, the induction of their target
genes, such as NOS2, leading to the production of NO, proteolytic enzymes and ROS
within lysosomes. The latter compounds kill ingested microbes when phagosomes
fuse with lysosomes. Furthermore, activated macrophages secrete cytokines like TNF
and IL1, which recruits other leukocytes and initiates inflammation. Furthermore,
TH 1 cells also secrete large quantities of IL2, which enhances the proliferation and
survival of cytotoxic T cells (Sect. 6.4).
TH 17 cells are primarily involved in the recruitment of neutrophils to sites of
infection by extracellular bacteria and fungi (Fig. 6.6, right). Neutrophiles are potent
phagocytes (Sect. 2.3) that destroy the microbes, which is mostly linked to inflam-
mation. Accordingly, pro-inflammatory cytokines produced in response to the pres-
ence of bacteria and fungi, such as IL6, IL1 and IL23, stimulate the differentia-
tion of CD4+ T cells into TH 17 cells. Interestingly, the anti-inflammatory cytokine
TGFβ supports this process. In contrast, the key TH 1 and TH 2 cytokines, IFNγ and
IL4, suppress TH 17 differentiation. IL17 is the main cytokine secreted by TH 17
cells (Box 6.3) and determined the name of this TH subset, which mediates type 3
immune response (Sect. 10.5). In gastrointestinal mucosa, IL17 stimulates leuko-
cytes and tissue cells to produce chemokines and the pro-inflammatory cytokines
TNF, IL1 and IL6. These signaling molecules recruit and activate neutrophiles. IL22
is another cytokine produced by TH 17 cells, the main function of which is to main-
tain the integrity of epithelial tissues, in particular in the skin and the intestine,
by promoting their barrier function stimulating repair reactions and inducing the
production of anti-microbial peptides.
In contrast to other TH cell subsets, TH 2 cells do not mediate their actions via
the recruitment of phagocytes. This is mainly due to the fact that the natural targets
of TH 2 cells, helminthic parasites, are too large to be phagocytosed. Therefore, the
main mediators of the TH 2 response, eosinophiles and mast cells, run a different
strategy in combating helminths (Fig. 6.7). They secrete a number of aggressive
chemicals, ranging from vasoactive amines, such as histamine, to lipid mediators,
such as leukotrienes and prostaglandins, as well as cytokines (Sect. 10.2). The differ-
entiation of TH 2 cells is majorly controlled by the cytokine IL4 (Box 6.2). In turn,
IL4 is together with IL5 and IL13, the major cytokine produced by TH 2 cells. These
three cytokines mediate the effector functions of TH 2 cells, which are:
• stimulating peristalsis in the intestine (IL4 and IL13)
• provoking the alternative pathway of macrophage activation (IL4 and IL13)
• increasing the secretion of mucus from of epithelial cells airways and intestine
(IL13)
• activating mature eosinophils by stimulating their proliferation, differentiation
and recruitment (IL5).
These mechanisms block the entry and mediate the expulsion of microbes from
epithelial surfaces (called barrier immunity), kill helminths and terminate inflamma-
tion combined with the initiation of tissue repair. Importantly, IL4 produced by follic-
ular TH cells stimulates antibody isotype switching in B cell, so that they secrete IgEs
that are specific to helminths. These IgEs bind to FcεRIs on the surface of eosinophiles
and mast cells, so that these cells of the innate immune system recognize helminths
with high specificity and combat them by releasing their granule contents. IgEs use
these mechanisms also in the context of allergic reactions (Sect. 10.2).
Naïve CD4+
Helminths or T cell
protein antigens
Antigen- Proliferation and

presenting cell
B cells follicular TH cells TH2 cells

M2-type
IL4, macrophage
IL13
IL4
Eosinophil
IgG4 IL4, Alternative

IL5 macrophage
IL13
IgE Antibody activation
production (tissue repair)
Helminth
Mast cell Intestinal mucus Eosinophil

degranulation secretion and peristalsis activation
Fig. 6.7 Functions of TH 2 cells. IL4 secreted by follicular TH cells stimulates B cells to produce
IgE-type antibodies that bind to FcεRIs on mast cells. Moreover, IL4 produced by TH 2 cells enhances
together with IL13 immunity at mucosal barriers and induces the alternative macrophage pathway,
which leads to tissue repair. In addition, TH 2 cells secrete IL5 that activates eosinophiles to fight
against helminths. More details are provided in the text
6.4 Cytotoxic T Cells
Cytotoxic T cells are distinguished from TH cells (Sect. 6.3) by the expression of the
co-receptor CD8 instead of CD4. This allows them to bind with their TCR peptide-
loaden MHC-I proteins on the surface of all nucleated body cells. Cytotoxic T cells
recognize most efficiently viral antigens presented by MHC-I proteins on the surface
of specialized dendritic cells expressing the membrane glycoprotein THBD (throm-
bomodulin). The latter cells are not typically infected by viruses themselves, but
6.4 Cytotoxic T Cells 103
via cross-presentation of proteins of virus-infected cells, which they have ingested.

Thus, dendritic cells are able to present antigens from all types of viral proteins
(Sect. 5.3). Moreover, TH cells push the responses of cytotoxic T cells to latent viral
infections (Sect. 8.1), organ transplants (Sect. 9.2) and tumors (Sect. 11.2), all of
which are not well recognized by the innate immune system, via further activation
of antigen-presenting cells.
Many viruses infect body cells, such as liver cells by HBV (hepatitis B virus),
that lack an effective system of phagosomes and lysosomes being able to destroy
the viruses (Sect. 8.2). Moreover, when intracellular microbes, such as the bacteria
Mycobacterium tuberculosis or Listeria monocytogenes (Sect. 7.3), manage to escape
the vesicles, by which they were taken up, and reach the cytosol of their target cells,
there is no other chance than killing the infected cells. Therefore, the main function
of cytotoxic T cells is the induction of apoptosis to microbe-infected target cells.
Thus, the killing of the infected body cells is the only possibility to eliminate
intracellular microbes without spreading them.
After the specific recognition of virus-infected cells, cytotoxic T cells use two
different mechanisms to induce apoptosis in their target cells (Fig. 6.8). In this way,
cytotoxic T cells resemble NK cells of the innate immune system (Sect. 2.5). Similar
principles apply to the surveillance for transformed cells, which are recognized by
cytotoxic T cells via the display of tumor antigens on MHC-I proteins (Sect. 11.2).
However, cytotoxic T cells play also key roles in the acute rejection of transplanted
organs (Sect. 9.2) and in the destruction of host tissues in autoimmune diseases
(Sect. 10.4).
The clonal expansion of CD8+ T cells has the remarkable rate of 50,000 for the
multiplication of antigen-specific cells (Fig. 6.4). This creates a sufficiently large
pool of cells that recognize and destroy the source of the antigen throughout the
whole body. During the approximately 7 days of massive proliferation the cells turn
into effector cytotoxic T cells that have acquired the tools for destroying the virus-
infected cells. The differentiation of cytotoxic T cells is promoted by the cytokines
IL2, IL12 and type I IFNs leading via signal transduction cascades and the activation
of the transcription factors TBX21 and EOMES (eomesodermin) to specific changes
of their epigenome. In this process the cells develop a large number of granules, which
are modified lysosomes containing proteins like perforin and granzymes. Moreover,
cytotoxic T cells express a number of cytokines, the most important of which is IFNγ
(Box 2.5). This cytokine activates macrophages to remove fragments of apoptotic
cells without inducing inflammation.
Cytotoxic T cells kill target cells that express the same MHC-I protein-bound
peptide, which triggered their proliferation and differentiation. This process
needs to be highly specific, in order not to harm neighboring non-infected cells.
Therefore, perforin and granzymes are not freely diffusing to the target cells but are
delivered in granules via the cleft of the immune synapse formed by TCR, CD8
and peptide-loaden MHC-I proteins as their counterpart for specific recognition
as well as by adhesion molecules forming a stabilizing ring around the synapse
(Sect. 6.1). The granules are transported by microtubules towards the synapse and
a Perforin/granzyme mediated cell killing
CD8+
Target T cell
cell
Endosome
Perforin
Perforin induces uptake
Granzymes
of granzymes into
target cell endosomes
their release into the cyto-
sol activating caspases
Cytolytic T cell releases

granule contents
into immune synapse Apoptosis of target cell
b FAS/FASLG-mediated cell killing
CD8+
Target
T cell
cell
FASLG FAS
FASLG on cytolytic T cell

interacts with FAS on target cell
Fig. 6.8 Function of cytotoxic T cells. Granule exocytosis releases complexes of perforin and
granzymes from cytotoxic T cells, which have specifically recognized their target cells (a). Perforin
allows granzymes to enter the cytosol of the target cells, where they induce apoptosis. An alterna-
tive mechanism of inducing apoptosis is the contact between the protein FASLG, which after the
recognition of infected cells is expressed on the surface of cytotoxic T cells, with FAS on target
cells (b). More details are provided in the text
6.4 Cytotoxic T Cells 105
taken up by target cells via endocytosis (Fig. 6.8a). Perforin is homologous to the
complement protein C9 (Sect. 7.2) and makes the endosomal membrane perme-
able. This allows granzymes to reach the cytosol of target cells. Granzyme B, the
most important member of the granzyme family, is a serine protease that cleaves
proteins after aspartate residues and activates in this way CASPs, such as CASP3,
as well as BID (BH3 interacting domain death agonist). The latter protein belongs
to the BCL2 family, members of which are the main regulators of this mitochon-
drial (intrinsic) pathway of apoptosis (Fig. 6.9). Some members of the BCL2
family are pro-apoptotic, while others are anti-apoptotic. They act as sensors of
cellular stress and can be activated by growth factor deprivation, noxious stimuli,
DNA damage or receptor-mediated signal transduction pathways. The main stress
sensor in lymphocytes is BCL2L11, which activates the pro-apoptotic proteins BAX
(BCL2 associated X, apoptosis regulator) and BAK (BCL2 antagonist/killer 1) that
increase the permeability of the outer mitochondrial membrane. This leads to the
leakage of cytochrome C and other mitochondrial proteins into the cytosol and acti-
vates CASP9. In the following further downstream CASPs induce fragmentation of
genomic DNA and apoptotic cell death. When cells undergo apoptosis, fragments
of the nucleus and cytosol form membrane-bound apoptotic bodies that are recog-
nized by receptors on phagocytes. In an efferocytosis called process neutrophiles
and macrophages rapidly engulf and eliminate apoptotic bodies without causing an
inflammatory response.
In case of apoptosis induced by cytotoxic T cells, the delivery of cytotoxic proteins
to the target cells takes only minutes. Thus, the cytotoxic T cells have to be attached
only for a short time, while in the following 2–6 h cell death by apoptosis occurs to
the target cells. This is the principal pathway, how cytotoxic T cells kill their target
cells. In addition, they use a pathway that is independent of the release of gran-
ules (Fig. 6.8b). This extrinsic apoptosis pathway is mediated by the membrane
protein FASLG (Fas ligand) that interacts with FAS (Fas protein) on the surface of
their target cells (Fig. 6.9). FAS belongs to the death receptor family, the activation
of which initiates the activity of a series of proteolytic enzymes, including CASP8.
The directed induction of apoptosis does not harm the cytotoxic T cells themselves,
so that they can move on to their next target cells. Apoptosis of virus-infected cells
ends with the phagocytosis (Sect. 2.3) of their cell fragments, i.e., with the prote-
olytic digestion of all cellular ingredients (Fig. 6.9). In this way, also the viruses are
destroyed and the reservoir of infection is eradicated.
Cell injury:
factors, survival Mitochondrial (intrinsic) pathway

signals
- DNA damage
- protein misfolding
BH3-only (BAX, BAK)

proteins
Cytochrome C and other
pro-apoptotic
Regulators proteins
DNA and nuclear (BCL2, BCL2L11) Initiator caspases:
fragmentation CASP9
Endonuclease Death receptor (extrinsic) pathway

activation FAS FASLG
Initiator
caspases: TNF
Executioner CASP8
TNFR
caspases
Receptor-ligand
interactions:
- FAS-FASLG
- TNFR-TNF
Apoptotic body
Fig. 6.9 Apoptosis pathways. Apoptosis can be induced by the intrinsic pathway, which leads the
release of cytochrome C from mitochondria, and the extrinsic pathway that is initiated by liganded
death receptors on the cell surface. Both pathways lead to the fragmentation of the cells and the
phagocytosis of apoptotic bodies. More details are provided in the text
Further Reading
1. Gaud G, Lesourne R, Love PE (2018) Regulatory mechanisms in T cell receptor signalling. Nat
2. Gieseck RL, Wilson MS, Wynn TA (2018) Type 2 immunity in tissue repair and fibrosis. Nat
Further Reading 107
3. Henning AN, Roychoudhuri R, Restifo NP (2018) Epigenetic control of CD8+ T cell

differentiation. Nat Rev Immunol 18:340–356
4. Mayassi T, Barreiro LB, Rossjohn J, Jabri B (2021) A multilayered immune system through the
lens of unconventional T cells. Nature 595:501–510
5. Spolski R, Li P, Leonard WJ (2018) Biology and regulation of IL-2: from molecular mechanisms
to human therapy. Nat Rev Immunol 18:648–659
Chapter 7
Immunity to Bacterial Pathogens
and the Microbiome
Abstract The major physiological function of the immune system is the protection
of our body against microbe infections. In this chapter, we will first summarize the
principles of immune responses to control infections with all kinds of microbes. Then
we will focus on the effector functions of humoral and cell-mediated innate and adap-
tive immunity against bacterial infections. We will learn that an overreaction of the
immune system to the presence of bacteria can lead to life-threatening sepsis. Next,
we will present emerging bacterial pathogens and discuss at the example of tubercu-
losis a more than 70,000 year-long interaction of Homo sapiens with Mycobacterium
tuberculosis. Finally, we will explain the impact of the interaction of our immune
system with the gut microbiome in the context of health and disease.
Keywords Host–pathogen interactions · Bacterial pathogens · Complement

system · Sepsis · Tuberculosis · Microbiome · Dysbiosis
7.1 Principles of Immune Responses to Infections
Infectious diseases have two major components, the pathogenic microbe and the
immune system of the host, both of which are modulated by environmental conditions
(Fig. 7.1). The nature and outcome of the microbe-host interaction, such as:
• entry of the microbe via mucous membranes of the respiratory, gastrointestinal
and genitourinary tract or the skin (e.g., via wounds)
• invasion and colonization of host tissues by the microbes
• the ability of the microbes to evade host immunity (Box 7.1)
• the amount of injury or impairment caused to host tissue by the microbes
• determines, whether the host stays healthy or gets ill. Thus,
• the host may be either resistant against an infection with a given microbe or
susceptible to it
• the host’s immune system may be either very responsive or deficient
• the host may be either tolerant against the microbe or the latter has high
pathogenicity (Box 7.2)
• the microbe may be either virulent or not (Box 7.2).
https://doi.org/10.1007/978-3-031-04025-2_7
110 7 Immunity to Bacterial Pathogens and the Microbiome
Environment
Host Pathogen
Host response
Replication
and rewiring
Disease Health
Resistance Susceptibility
Acquired immunity
Tolerance Pathogenicity
Non-virulence Virulence
Fig. 7.1 Host and pathogen are interdependent. Host and pathogen have a very complex rela-
tionship that determines, whether the individual stays healthy or gets ill. Moreover, the environment
plays a central role on the effects of the microbe on the host as well as on the resulting effector
functions of the host’s immune system on the pathogen. More details are provided in the text
Box 7.1: Principles of immune evasion by microbes The virulence (Box 7.2)
of a microbe critically depends on its ability to resist mechanisms of innate
immunity. For example, bacteria with polysaccharide-rich capsules are difficult
to eliminate by phagocytosis, since sialic acid residues in the bacterial capsules
inhibit the activation of the alternative complement pathway (Sect. 7.2). A
common instrument how microbes evade adaptive immune responses via
neutralizing antibodies is the variation of their surface antigens due to rapid
changes of their genome. Similarly, also the structure of surface carbohydrates
can be varied due to changes in the expression of glycosidases. Intracellular
microbes use other mechanisms to evade immune destruction, such as the inhi-
bition phagolysosome fusion, an escape to the cytosol or scavenging aggressive
chemicals produced by lysosomes. In general, immune evasive microbes tend
7.1 Principles of Immune Responses to Infections 111
to cause chronic infections, such as tuberculosis (Sect. 7.3) or Herpes viruses

(Sect. 8.2), that are difficult to eliminate and last over years or decades. Such
microbes survive in a quiescent latent state in their host cells, but become active
when the host becomes immune-compromised.
Box 7.2: Pathogenicity and virulence Pathogenicity describes the ability of

a pathogenic microbe to cause a disease within a host, while virulence is the
degree of pathogenicity. The virulence of a microbe is often expressed by lethal
dose 50 (LD50 ), i.e., the number of microbes that are required to kill 50% of
infected hosts, while ID50 is the infectious dose, i.e., the microbe load that
makes 50% of all hosts ill. Both rates are average numbers and there are large
interindividual differences. Nevertheless, the lower LD50 or ID50 of a microbe
are, the more virulent is the pathogen. The likelihood that an infection causes
a disease primarily depends on the microbe count and the resistance of the
host. Many features of microbes determine their virulence and a multitude of
mechanisms contribute to the pathogenesis of infectious diseases. For example,
the susceptibility of the host is affected by gender, fatigue, pre-existing illness,
age, nutritional status, lifestyle, emotional disturbance as well as by weather
and climate.
Microbes aim to optimize the interaction with their host, in order to create a local
environment that allows maximal replication rates either inside or outside of host
cells. For example, pathogenic extracellular bacteria grow in the blood, in connec-
tive tissues and in spaces between tissues. They cause disease either by the induction
of inflammation, i.e., tissue injury at the local site of infection, or by the presenta-
tion or release of toxins, i.e., everywhere in the body. Gram-negative bacteria carry
endotoxins, such as LPS, on their surface that act as PAMPs (Sect. 2.1) and stimu-
late cytokine production by innate and adaptive immune cells. Furthermore, secreted
exotoxins, such as in the bacterial diseases diphtheria, tetanus, cholera and anthrax,
reduce protein synthesis, affect salt-water transport, inhibit neuromuscular trans-
mission or disturb signaling pathways, respectively, in various host tissues. Special
types of toxins are superantigens (Box 7.3) that can cause a systemic inflammatory
response syndrome.
Box 7.3: Superantigens Some bacterial strains, such as Staphylococcus

aureus, produce toxins that are able to act as superantigens. These proteins
activate various different T cell clones (5–20% of all compared to 0.01% in
case of regular antigens), because they are not processed to peptides by antigen-
presenting cells but displayed as intact proteins on the surface of macrophages.
Superantigens are able to bind to the TCR of TH cells as well as to MHC-II
proteins, i.e., they cross-link, e.g., TH 1 cells and macrophages. This leads to
a massive increase in the number of activated T cells and in the levels of the
cytokines produced by them and other immune cells. In particular, high levels
of TNF, IL1 and IL6 cause a cytokine storm that may lead to systemic toxicity,
a sepsis-like systemic inflammatory response syndrome (Sect. 7.2) and even
death. Genetic polymorphisms of HLA genes of class II (Sect. 5.2) contribute
the susceptibility to superantigens.
The presence of pathogenic microbes induces a number of effector mechanisms

of innate and adaptive immune system of the host. This provides many distinct
and specialized ways to respond to microbes and eliminate them (Sect. 7.2). In
contrast, the survival and pathogenicity of microbes is critically dependent on their
ability to evade or resist these different effector mechanisms. Moreover, a too strong
response of the immune system may cause collateral damage to the host, such as tissue
damage and impairment. Ideally, there is a balance between activating signals to elim-
inate the pathogenic microbe and inhibitory signals that avoid tissue pathology, i.e.,
the immune system should respond in an appropriate magnitude. However, illness
is caused often rather by the immune response than by direct actions of the
microbe.
The principles of the responses of the innate and adaptive immune system to
microbe infection have already been described in Sect. 1.2. In a typical infec-
tion (Fig. 7.2), the effector functions of innate immune cells, such as neutrophils,
macrophages, NK cells and ILCs, slow down the growth of pathogenic microbes
but are mostly not able to completely eradicate them. Since the clonal expansion
of microbe-specific B and T cells takes about a week, the innate immune system
has to control the infection for this period until adaptive immunity takes over. Some
pathogenic microbes even developed resistance against effector functions of innate
immunity (Box 7.1), so that survival critically depends on adaptive immunity. The
highly specific effector functions of B and T cells as well as the large numbers of
cells and antibodies then enable the complete elimination of the microbes. When this
task is completed, most of the effector cells undergo apoptosis, but a lower number of
microbe-specific long-lived memory cells survive and provide the body with immu-
nity against a recurrent infection with the same microbe. Although immune or vacci-
nated individuals have the same infection risk as non-vaccinated persons, the invasion
of microbes is mostly asymptomatic in the former and resolves without developing
illness. In these cases, memory B and T cells rapidly respond and within 1–3 days
efficient effector mechanisms are available that eradicate the microbes before they
are able to grow to a substantial number.
7.1 Principles of Immune Responses to Infections 113
Innate immune Adaptive immune

response memory
Infection
established
Number of organisms
Infection
ended
Microbe
entry
Time course of infection
Fig. 7.2 Innate and adaptive immunity together control a typical infection. A plot of microbe
number over time indicates that early responses to pathogenic microbes are mediated by cells of
the innate immune system. These slow down the growth of the microbes but often do not eradicate
them. In case of a first contact with the microbe, it takes approximately one week until microbe-
specific clones of T and B effector cells have grown to sufficient numbers that are able to eliminate
the microbe. Some of the T and B cells survive as long-lived memory cells and enable more rapid
responses in case of a repeated infection with the same microbe
The above described outcome of a typical infection focused on the microbe number
over time. An infected person is considered ill, when he or she has developed symp-
toms, such as fever, in response to the presence of the microbes or diarrhea to the
toxins produced by them. Therefore, illness occurs mostly some days after first
contact with the microbe. In addition, the symptoms of the disease are often primarily
a consequence of the immune response. Therefore, the health status can be reduced
for a while after the microbes have been eliminated. In this time frame the immune
system returns to homeostasis and contributes to the repair of injured tissues.
An alternative representation of microbe infections is a plot of health status over
microbe number (Fig. 7.3). This allows to define not only the effects of pathogenic
microbes in reducing the health status of an individual but also to characterize symbi-
otic interactions of microbes with the host that may improve well-being. In this
scheme a typical acute infection, such as measles or gastritis, is represented by
curve 1: first the microbes grow without affecting the health status, then the disease
symptoms start, i.e., health status decreases, the immune system reduces the microbe
number, while initially health further declines until in the recovery phase the original
health status is fully reconstituted. In contrast, curve 2 describes an infection that
results in a permanent disability, i.e., a reduced heath status, such as damage caused
by meningitis or encephalitis. In case of an unstable disability, such as in context of
8
9
Mutualists
7
Health
Pathogens
2
3
5
6
Microbe number
Fig. 7.3 Comparing the routes of infections with pathogens and mutualists. A plot of health
status over microbe number compares six scenarios of infections with pathogenic microbes with
three schemes of infections with mutualists. More details are provided in the text
the autoimmune diseases rheumatic fever or reactive arthritis, the end point may be
an inflammatory stage that causes further tissue damage (curve 3). Non-resolving,
persistent infections like herpes (Sect. 8.2) or tuberculosis (Sect. 7.3) are represented
by curve 4 indicating a variant status of remaining health. Thus, the pathogenicity of
a microbe critically depends on its ability to evade or resist the effector mecha-
nisms of immunity (Box 7.1). In extreme, the immune system is not able to control
the microbe number sufficiently (curve 5) or not at all (curve 6), so that health
declines until death of the patient. In the latter cases, often the microbes do not kill
the host directly but the overboarding reactions of the host’s immune system cause
so much damage that the person dies. This will be further discussed in context of
sepsis (Sect. 7.2). Moreover, inherited and acquired defects of the immune system,
7.2 Immune Responses to Bacteria 115
such as AIDS following an insufficiently treated HIV-1 infection or the long-term

immunosuppression after organ transplantation (Sect. 9.3), increase the susceptibility
for infections.
Curves 7–9 (Fig. 7.3) describe mutualistic microbe-host interactions that are
symmetric to the curves 1, 2 and 4, respectively. Curve 7 displays the short-term
transient colonization of the intestinal track with beneficial microbes, such as a
probiotic bacterial strain. In contrast, curve 8 reflects the infection with a microbe,
such as a live vaccine, that is eventually cleared but leads via gained immunity to an
improved health status of the host. Finally, curve 9 represents a long-term interaction
with a mutualist, such as a beneficial component of the gut microbiome (Sect. 7.4).
7.2 Immune Responses to Bacteria
Most pathogenic bacteria grow in extracellular spaces of the host (Sect. 7.1), but some
species, such as Mycobacterium tuberculosis, survive inside of macrophages and are
referred to as intracellular bacteria (Sect. 7.3). Both innate and adaptive immunity
have developed distinct mechanisms to fight against extra- and intracellular
pathogenic bacteria. The main instruments of the innate immune system against
extracellular bacteria are:
• the rapid recruitment of neutrophils and macrophages to the site of infection by
cytokines and chemokines secreted by tissue-resident macrophages and dendritic
cells. This is part of the inflammatory response (Sect. 2.4)
• the activation of phagocytosis in neutrophils and macrophages, in order to ingest
and kill the bacteria (Sect. 2.3)
• the activation of the complement system that makes microbes detectable by
phagocytes, supports the inflammatory response and leads to the lysis of microbes
(Box 7.4).
Box 7.4: The complement system The humoral response of the innate
immune system is primarily mediated by some 50 different complement
proteins (primarily produced by the liver) that circulate in the blood, lymph and
extracellular fluids. Once activated, these proteins interact with each other in
a highly regulated fashion, in order to opsonize microbes, promote the recruit-
ment of phagocytes (Sect. 2.3) or directly eliminate microbes (see below). The
three pathways to activate complement proteins are (Fig. 7.4):
• the classical pathway that uses antibodies of adaptive humoral immunity, in
order to specifically recognize microbes. The large multimeric complement
protein C1 binds with its C1q subunit to the Fc regions of antigen-bound
IgG3 and IgM antibodies. This starts proteolysis of complement proteins C2
Initiation of
complement Early steps Late steps
activation functions
Classical
pathway
Membrane attack
C3b is deposited
complex
on microbe
Alternative
pathway
C5a C9
Lectin
pathway C3a
Lectin C3b:
C3a: C5a: C5b-9: Lysis
Opsonization and
of microbe
phagocytosis
Mannose
binding lectin
Fig. 7.4 Principles of complement activation. The complement system can be activated by the
classical, alternative or lectin pathway, all of which lead to the proteolysis of C3 protein into C3a and
C3b. The latter binds to the surface of microbes and initiates the proteolysis of C5 into C5a (causing
inflammation) and C5b (initiating the polymerization of C9 to the membrane attack complex). More
and C4, their fragments C2a and C4b form the serine protease C3 convertase
that splits the most abundant but inactive complement protein C3 into the
active fragments C3a and C3b.
• the alternative pathway that functions without the involvement of anti-
bodies is activated when complement protein C3 is hydrolyzed sponta-
neously on the surface of microbe to C3b. This pathway distinguishes
host cells from microbes, because the latter do not express complement
regulatory proteins like CD35, CD46 CD55 or CD59.
• the lectin pathway that is based on the mannose-binding protein lectin that
specifically identifies mannose on the surface of microbes.
The three pathways are redundant to each other, since they all result in the
activation of the C3 convertase. The C3b fragment binds covalently to microbe
surfaces or to antibodies binding microbes. In this way, within minutes millions
of C3b molecules attach to microbes, so that they become “visible” to phago-
cytes expressing receptors for C3b. In the following, the enzyme C5 convertase
is activated, which cleaves complement protein C5 into C5a and C5b. The frag-
ments C3a and C5a both stimulate inflammation. In parallel, a cascade of the
proteins C5b, C6, C7, C8 and C9 lead to the formation of the membrane attack
complex on the surface of microbes. The complex forms a transmembrane
channel, which causes osmotic lysis in particular to gram-negative bacteria.
Importantly, host cells are not harmed by the complement system, since
they carry on their surface sialic acid residues that inhibit complement
activation. The same strategy is used by some pathogenic microbes, in order
evade the activation of the alternative or lectin pathway (Box 7.1). However,
these bacterial pathogens can be still detected by the classical complement
pathway.
The adaptive immune system reacts to the presence of extracellular bacteria

primarily via its humoral response (Sect. 4.3). Proteins or carbohydrates on the
surface of bacteria as well as secreted exotoxin proteins are recognized by naïve B
cells, which then proliferate and differentiate to plasma cells that produce massive
amounts of highly specific antibodies (Fig. 7.5a). These antibodies either:
• neutralize bacteria or their toxins by completely covering their surface. Most of
the neutralizing antibodies have due to affinity maturation high affinity for their
targets (Sect. 4.3). Neutralizing IgGs are found primarily in the blood, while IgAs
act in the lumen of mucosal organs and represent a major defense line against
recurrent infections: the aggregation of pathogenic microbes with IgAs reduces
their infectivity, traps them in mucus and facilitates their clearance by peristalsis.
• opsonize bacteria, i.e., mark them for destruction by antibody-dependent cellular
phagocytosis (ADCP). This process significantly enhances the efficiency of
microbe destruction by neutrophils and macrophages and depends on the expres-
sion of different types of FcRs on their surface, such as FcγRI (CD64, high affinity
binding of IgG1 and IgG3), FcγRII (CD32, low affinity binding of IgG1 and IgG3)
and FcγRIII (CD16).
• activate complement-dependent cytotoxicity (CDC), i.e., phagocytosis of C3b-
coated bacteria, the induction of inflammation and the lysis of the microbes.
When protein antigens of extracellular bacteria are displayed by MHC-II proteins
of antigen-presenting cells, TH cells are activated to produce cytokines and express
surface proteins (Sect. 6.3). TH 17 cell secrete IL17, TNF and other pro-inflammatory
cytokines and stimulate inflammation by recruiting neutrophils and monocytes.
TH 1 cells produce INFγ and support phagocytic and microbicidal activities of
a
Neutralization
Opsonization and
Antibody FcR-mediated
Bacteria phagocytosis
B cell
Phagocytosis of
C3b-coated
bacteria
T helper cells
(for protein antigens)
Complement activation
Lysis of microbes
b
IL17, TNF,
other
cytokines
TH17
Bacteria
Macrophage
IFNγ
activation =>
phagocytosis and
TH1
bacterial killing
various
cytokines Antibody
follicular TH response
Fig. 7.5 Adaptive immune responses to extracellular bacteria. The adaptive immune system
responds to extracellular bacteria and their toxins via antibody production (a) and the activation of
TH 1, TH 17 and follicular TH cells (b) More details are provided in the text
macrophages and neutrophils, while follicular TH cell stimulate antibody production

(Fig. 7.5b).
Pathogenic intracellular bacteria, such as Mycobacterium tuberculosis (Sect. 7.3)
differ from extracellular bacteria by their property to resist phagocytosis, i.e., they
are ingested but survive and even replicate within macrophages. In this way, intracel-
lular bacteria cannot be recognized by complement proteins and antibodies, i.e., they
are located in a niche that is “invisible” to humoral responses of innate and adap-
tive immunity. Therefore, mechanisms of cell-mediated immunity need to activate
phagocytosis within the infected macrophages via INFγ secretion by NK cells and
ILC1s of the innate immune system (Sect. 2.5). Similarly, TH 1 cells of the adaptive
immune system reach the same effect by a direct CD40LG-CD40 contact and the
production of INFγ (Sect. 6.2). Moreover, other cytokines, such as TNF, are impor-
tant stimuli of efficient phagocytosis. When intracellular bacteria, such as Listeria,
manage to escape the phagosome and reach the cytosol of the macrophages, there is
no other option than inducing their apoptosis by cytotoxic T cells (Sect. 6.4).
During a bacterial infection the host mostly feels weak and/or ill, because the
response of his/her immune systems uses a substantial amount of the daily energy
for immune cell production in the bone marrow and elevating the body temper-
ature. The local inflammatory response, which is primarily the eradication reac-
tion of neutrophils and macrophages, not only reduces the number of microbes but
also causes tissue injury (Sect. 2.4). This is followed by a repair phase, in which
inflammation is resolved and tissues are repaired, so that the immune system can
return to homeostasis (Fig. 7.6, top). However, when pathogenic microbes manage
to evade the effector mechanisms of the immune system, often higher amounts of pro-
inflammatory cytokines like TNF, IL1β, IL12 and IL18 are produced. This induces
the secretion of acute-phase proteins from the liver. In this way, the immune system
fails in returning to homeostasis but develops a whole body (systemic) response
to the infection. Thus, a severe local or systemic infection with bacteria or fungi
(Box 7.5) can lead to sepsis. The latter is a highly heterogenous systemic inflamma-
tory response syndrome, the common characteristic of which is an unbalanced host
response to infection. Early clinical symptoms of sepsis are elevated body temper-
ature (>38 ºC), heart rate (>90 beats/min), respiratory rate (>20 breaths/min) and
leukocyte count (>12,000/μl). In the context of sepsis abnormalities in tissue blood
perfusion, coagulation, metabolism and organ function are found (Fig. 7.6, bottom).
These can lead to a septic shock, which is a collapse of blood circulation combined
with systemic intravascular coagulation. The mortality rate of sepsis was extreme
in the past, e.g., due to surgeries or giving birth under non-sterile conditions,
but is even todays 20%.
Box 7.5: Immune response to infections with fungi Fungi can live outside
of host cell or survive in macrophages as described for extra- and intracel-
lular bacteria, respectively. Accordingly, similar mechanisms of innate and
adaptive immunity apply for the defense against fungi. The innate immune
system primarily uses the complement system, e.g., in the fight against Candida
reaching the blood stream, for opsonization and destruction by phagocytes, in
particular by neutrophiles. TH 17 cells of the adaptive immune system are crit-
ical for the defense against extracellular fungi, since they recruit neutrophils
for phagocytosis. In contrast, TH 1 cells (often in cooperation with cytotoxic T
cells) are used for the fight against intracellular fungi by either inducing effec-
tive phagocytosis or killing the infected macrophages. Some infections with
fungi are endemic and are caused by species that are present in the environment
Immune suppression
CD4+ T cells: CD8+ T cells:
• Apoptosis • Apoptosis
• Exhaustion • Exhaustion
Protective immunity • TH2 cell polarization • Cytotoxic function
Local repair mecha- Neutrophils: Antigen-presenting

nisms: • Apoptosis cells:
matory • Inhibition and resolu- • Immature cells with de- • Reprogramming of
mechanisms creased antimicrobial macrophages to M2
• Tissue repair functions phenotype
• Return to homeostasis • Reduced HLA-DR ex-
Lymph node: pression
• Apoptosis of B cells and
follicular dendritic cells Others:
• Expansion of regulatory
PAMPs
T cell and MDSC po-
pulations
TLRs
RLRs
Homeostasis NLRs
Leukocytes and par- Endothelium:

Protective immunity enchymal cells:
matory mediators
Localized innate im- matory mediators • Adhesive and pro-
mune response: • Cell injury with release coagulant properties
matory of DAMPs • Barrier function
response matory mediators
• Leukocyte recruitment Platelets: Others:
• Complement activation • Coagulation activation
• Coagulation activation matory mediators (microvascular throm-
• Activation of neutrophils bosis)
and the endothelium • Complement activation
• Microvascular thrombi
Fig. 7.6 Normal and excessive responses of the immune system to bacterial infection. In the
normal, balanced response of the innate immune system to infection with extracellular bacteria,
there is a local inflammatory response, such as cytokine and chemokine release, phagocyte recruit-
ment as well as activation of the complement and coagulation system, at the site of infection leads
to the elimination of the pathogens (top). This is followed by a return to homeostasis, where inflam-
mation is resolved and tissues are repaired. In contrast, during some infections the immune response
becomes unbalanced and can develop a life-threatening sepsis (bottom). This is characterized by
excessive inflammation combined with immune suppression. More details are provided in the text
7.3 Emerging Microbial Pathogens 121
and whose spores enter humans. Other fungal infections are opportunistic, i.e.,
in the healthy individual they cause no or only mild disease but break out in
persons with compromised immunity, such as a deficiency in neutrophils or
after taking immunosuppressive drugs (Sect. 9.3).
On the cellular level sepsis is associated with excessive inflammation that system-
ically activates complement proteins as well as the coagulation system. This disrupts
the integrity of the endothelial barrier and causes leakage of intravascular proteins
and plasma into the extravascular space. Moreover, this leads to microvascular throm-
bosis and hemorrhage (i.e., bleeding) due to the consumption of clotting factors and
platelets. Accordingly, the main reason for early mortality in sepsis is a cardiovascular
collapse and multiple organ dysfunction. In contrast, sepsis also leads to immuno-
suppression via apoptosis of T and B cells as well as of dendritic cells, T cell exhaus-
tion, increase of Treg cells and myeloid-derived suppressor cells (MDSCs) popula-
tions and decreased expression of HLA-DR genes in antigen-presenting cells. Thus,
during sepsis the immune system turns into two opposite directions, the relative
extent of which varies between individuals. Furthermore, the immune response is
disturbed by the release of DAMPs that activate pattern-recognition receptors on
innate immune cells (Sect. 2.1), i.e., sepsis is associated with a strong activation of
innate immunity.
7.3 Emerging Microbial Pathogens
Homo sapiens evolved some 200,000 years ago in East Africa and was spreading
first over the whole continent before our species migrated some 80–60,000 years
ago also to Asia, Australia, Europe and the Americas (Fig. 7.7). Some 30,000 years
Malaria Tuberculosis Smallpox Leprosy Cholera AIDS SARS-CoV2
~ 200,000 ~ 100,000 - 50,000 12,000 2200 500

Today
years ago years ago years ago years ago years ago
Modern hu- Migrations Migrations Early agriculture Silk road links European colo-
mans emerge within out (neolithic demo- Africa, Europe nization of Am- Globalization
in Africa Africa of Africa graphic transition and Asia ericas begins
Fig. 7.7 Emerging microbial pathogens during human history. A time line of key events in
the history of Homo sapiens of the past 200,000 years relates to the estimate time period when the
listed major infectious diseases emerged
ago humans begun to domesticate different animal species, such as dogs, cattle,
sheep, pigs and chicken, and some 12,000 years ago they started agriculture. Until
then humans lived in smaller groups, i.e., the risk of getting infected or spreading
an infection was low. However, microbe infections became far more likely, when
humans had close contacts with domesticated animal species as well as with rodents
or wild birds and lived at higher densities in villages and cities. At latest from that time
on pathogenic microbes belong to the strongest evolutionary drivers of our species.
However, evolutionary adaption, e.g., developing resistance against a pathogen based
on the selection of advantageous genetic variations, takes many generations. When
more than 2000 years ago transcontinental trade connected Europe with Asia and
Africa and in particular when 500 years ago the Americans were colonized by Euro-
peans, previously isolated human populations came in contact with each other. For
example, Europeans had the chance to adapt to smallpox (Variola major) infec-
tions, while the bacterial pathogen was completely unknown to the Native American
population. In consequence, millions of the latter died following the rediscovery of
the Americas in 1492 by Columbus. Thus, pathogenic microbes have the highest
risk to kill human hosts, when there is no time for a genetic adaptation. This
had been the case 1918 with influenza (Sect. 8.3) and todays with SARS-CoV-2
(Sect. 8.4). Moreover, high population densities and intensive contact between basi-
cally all human populations, as caused by massive global trade and pleasure travel,
i.e., globalization, further increase the risk. Since viral pathogens are in average far
more contagious and reassort their genomes more rapidly than bacteria, in the past
100 years primarily viral infectious diseases, such as influenza, AIDS and COVID-
19, were the cause of major pandemics (Table 1.2). Pandemics develop in particular
when herd immunity of the population did not yet establish.
Microbes that cause human diseases must have existed in a different environ-
ment or species before they infected us. The oldest known infectious disease of
humans, malaria, is caused by unicellular Plasmodium parasites (Box 7.6) and needs
mosquitoes as an intermediate host, i.e., malaria-infected persons are not infectious
to other humans. In humans, Plasmodium lives preferentially inside of erythrocytes
and hepatocytes. Malaria exists only in regions with a warm climate, such as in trop-
ical Africa, Asia and America, where mosquitoes of the genera Culex and Anopheles
are found. Since both the parasite and its vector are rapidly evolving, there is still no
effective vaccination against malaria. The co-evolution of humans and Plasmodium
parasites made malaria a chronic infectious disease but does not kill adult hosts that
have a potent immune system. Nevertheless, annually hundreds of thousand young
children, who have not finished the development of their immune system (Box 3.1),
die from malaria. Moreover, in adults chronic infectious disease, such as malaria,
tuberculosis and leprosy, as well as parasitic worms (Sect. 10.2) impair fertility,
growth, cognitive development and nutrition.
Box 7.6: Immunity against parasites Parasites are unicellular protozoa,

multicellular worms (helminths), and ectoparasites, such as ticks and mites.
Parasites enter our body mostly via bites from their intermediate hosts, such as
insects or snails. Parasitic infections are often chronic, since the response of the
innate immune system to them is rather weak and the parasites usually evade or
resist effector functions of the adaptive immune system. For example, protozoa
often survive inside macrophages and do not get destroyed by phagocytosis,
while helminths can become resistant against the attack with microbicidal
molecules secreted by eosinophiles. Intracellular protozoa can be eliminated
by cell-mediated immunity via TH 1 cells (type 1 immune response), while
the TH 2 response is the major mechanism in the fight against helminths (type 2
immune response, Sect. 10.2).
The master example for a host–pathogen co-evolution is tuberculosis, which is

caused by Mycobacterium tuberculosis infections. This started some 70,000 years
ago in East Africa and spread around the world by human migration. Only some 10%
of infected individuals develop an active disease, i.e., the intracellular bacterium has
adapted well not to harm its host too much. As we will discuss in more detail in
context of the virome (Sect. 8.2), this is an important pre-condition for a chronic
infectious disease. Nevertheless, due its widespread distribution the yearly death
toll of tuberculosis is more than a million individuals (Fig. 1.1), most of which are
immunocompromised, e.g., by HIV-1 infection, old age or other impairments. Then
tuberculosis causes chronic cough, fever, sustained weight loss, wasting and coughing
up blood or blood-stained mucus. In the past, a typical treatment of tuberculosis was
the exposure with UVB, which increases the endogenous production of vitamin D3
(Box 7.7). It had been estimated that in human history no other pathogen killed
more persons than Mycobacterium tuberculosis (more than a billion individuals).
Moreover, at present some 2 billion people are hosts for the intracellular bacterium,
i.e., more than ever.
Box 7.7: Vitamin D, UV-B and tuberculosis Humans are able to produce
vitamin D3 , when their skin is exposed to ultraviolet (UV) B radiation. A
metabolite of vitamin D3 , 1,25-dihydroxyvitamin D3 , binds and activates
the transcription factor VDR (vitamin D receptor), which is a member of
the nuclear receptor superfamily. In monocytes and macrophages, the VDR
target genes CAMP and DEFB4 (defensin, beta 4A) encode for anti-microbial
peptides that are able to kill Mycobacterium tuberculosis. This explains why
sun or artificial UV-B exposure is efficient in the supportive treatment of tuber-
culosis, vitamin D deficiency is associated with more aggressive tuberculosis,
variations of the VDR gene increase the susceptibility to Mycobacterium tuber-

culosis and humans with dark skin living distant from the equator have an
increased susceptibility to tuberculosis infection.
The success of Mycobacterium tuberculosis in its adaption to the human body is

related to the fact that it lacks classical bacterial virulence factors, such as capsules for
avoiding phagocytosis, exotoxins to poison the host, pili for adherence to host cells
or flagella for motility. Mycobacterium tuberculosis behaves different to commensal
pathogens that may be found in human mucosa. The latter got in contact with humans
far later than Mycobacteria and cause diseases, such as pneumonia or diphtheria,
with questionable benefit for the microbe’s evolutionary survival. Mycobacterium
tuberculosis carries on its surface the masking lipid phthiocerol dimycocerosate that
hides PAMPs, so that the bacterium is not detected by microbicidal macrophages. In
parallel, a recruiting surface lipid, phenolic glycolipid, induces via the chemokine
CCL2 the recruitment of permissive macrophages. Permissive macrophage are cells
that ingest the bacteria but are unable to eliminate them by phagocytosis, because the
fusion of phagosomes with lysosomes is disabled and the bacteria break out into the
cytosol. Mycobacterium tuberculosis does not compete with other pathogens for a
niche in the upper airways but uses permissive macrophages, in order to circumvent
host defenses and to reach the lower airways (Fig. 7.8). In the lower parts of the
lung, the infected macrophages recruit further macrophages that get also infected,
i.e., an inflammatory response is initiated. In this aggregate of macrophages, called
granuloma, the membranes of the infected cells form tight contacts like epithelial
cells. Thus, granulomas are niches, in which Mycobacterium tuberculosis survives
and replicates, as long the adaptive immune response via TH 1 cells secreting IFNγ
(Sect. 6.3) is not effective enough to initiate the completion of phagocytosis within
the infected macrophages.
Granuloma can occur at different sites of the body also with other intracellular
bacteria, such as Mycobacterium leprae causing leprosy. The inflammatory reaction
of the host at the granuloma localizes the bacteria and aims to prevent their spread.
Nevertheless, granuloma often cause severe functional impairment via necrosis and
fibrosis of the concern tissues. Mycobacterium tuberculosis can survive for years
in granuloma and in this latency phase the host is clinically asymptomatic and not
contagious. However, when due to overload with bacteria infected macrophages die
by apoptosis or necrosis, some of the bacteria are liberated and start to replicate while
new macrophages are recruited to the granuloma and get infected. Patients with this
active form of tuberculosis carry a number of granuloma with necrotic cores, around
which cells of the innate and adaptive immune system are aggregated. When these
granuloma break down, they release bacteria into the lungs and the patient coughs out
small aerosol droplets containing only one to three bacteria each. The hydrophobic
surface of the bacteria is responsible for this infection strategy, which is more effective
than a smaller number of larger droplets each carrying thousands of bacteria. When
the small droplets are inhaled by other persons, a new infection cycle starts.
a Macrophage with
engulfed
M. tuberculosis
The b
transmission
cycle
c
Granuloma
Fig. 7.8 Transmission cycle of tuberculosis. An infection with Mycobacterium tuberculosis is

initiated by fine aerosol droplets containing 1–3 bacteria, which are coughed up by an individual
with active disease and inhaled by a new host (a). The bacteria use the strategy to recruit and
enter permissive macrophages, which transport them to the lower airways (b). There a granuloma
is formed through the recruitment of further macrophages and other immune cells to the original
infected macrophages (c). In this way, the bacterium can replicate and spread to additional host cells.
However, the granuloma reduces bacterial growth in response to T cells of the adaptive immune
system. When the infected macrophages in the granuloma undergo necrosis and form a necrotic
core (d), bacterial growth is pushed and the disease is activated. Then the next host may be infected
by a cough, propelling bacteria into the air (a)
Mycobacterium leprae survives in Schwann cells of the peripheral nervous system.

Therefore, leprosy leads to the damage of nerves and the patients do not feel pain,
so that repeated unnoticed wounds cause the loss of parts of their extremities. Until
500 years ago leprosy was endemic in Europe and today still is a major public health
burden in India, China and Southern America. Both Mycobacteria strains originate
from non-pathogenic Mycobacteria of soil dwellers like amoeba that found in humans
a new habitat. In this way, both types of intracellular bacteria clearly differ from most
extracellular pathogenic bacteria, such as Yersinia pestis causing plaque, that do not
use the human body as permanent host. The regular hosts of Yersinia pestis are
rodents and the bacteria enter only by chance those humans that have been bitten
by fleas originating from infected rats. In this zoonotic transfer, the human disease
plague can be considered as an “accident” of nature, since it does not contribute to the
long-term survival of the pathogenic bacteria. Similar principles apply to COVID-19,
since the natural host of SARS-CoV-2 are bats and the zoonosis just happened in
2019 (Sect. 8.4).
7.4 Immunity to the Microbiome
The symbiosis of prokaryotes and multicellular eukaryotes was and still is an impor-
tant driver of evolution. Like any other multicellular species also humans carry a
microbiome (Box 7.8). This is defined as the community of mostly commensals (i.e.,
microbes that use food supplied by the internal or external environment of their host)
and mutualists (Sect. 7.1) occupying niches of our body and interacting with basically
all organs. In contrast to bacterial infections, which represent the transient overgrowth
of one specific pathogenic species, such as Variola major (Sect. 7.3), the microbiome
functions as an ecological system formed by a multitude of interacting microbe
species that permanently occupy our body and generally contribute to its well-
being. The microbiome also comprises small protozoa, archaea, fungi and algae, but
we will focus in this section primarily on bacteria. Moreover, the viral microbiome,
referred to as virome, will be discussed in Sect. 8.2.
Box 7.8: Evolution of the human microbiome Bacterial lineages inhabit the
gut of humans and their ancestors since millions of years. Therefore, regular
gut bacteria are far better adapted to their host then any pathogenic bacteria,
such as Mycobacterium tuberculosis (Sect. 7.3). The rapid changes in diet
and environment that happened since the onset of industrial revolution some
200 years ago provided a pressure on the genome of humans and those of the
bacterial species forming their microbiome. The far shorter generation time of
the microbes gave them an advantage in evolutionary adaption. This may be the
basis for the significant interindividual differences in the gut microbiome that
7.4 Immunity to the Microbiome 127
largely depend on dietary habits, gender, age and disease status. Interestingly,
the stomach of humans is far more acidic (pH 1.5) than that of most other
species. This has an impact on its microbiome as well as on that of the intestine.
Bacteria are found throughout our body but mainly on external and internal
surfaces of the gastrointestinal tract, the skin and the oral cavity. The total number
of bacteria is in the order of 4 × 1013 (Table 7.1) and their total mass is about 200 g.
This is very comparable to the 3 × 1013 cells forming an adult human body, 83%
of which are red blood cells. However, the amount of bacteria in the gastrointestinal
track depends on the fasting and feeding state, i.e., it responds dynamically to our
eating habits and biorhythm. Due to a low pH in the upper part of the gastrointestinal
track, the concentration of bacteria in the stomach, duodenum and jejunum is only
103 –104 /ml, while in the ileum (.i.e., the lower part of the small intestine) and in
particular in the colon the number of microbes is far higher. Thus, by number and
mass most of the human microbiome is localized within the colon.
In healthy individuals, proteobacteria, such as Enterobacteria, Lactobacillales and
Erysipelotrichales, are found in the nutrient-rich small intestine. In contrast, most
nutrients have been resorbed when the diet reaches the colon and only indigestible
fibers remain, which are used by Bacteroidetes and Clostridia as energy source.
All these gut bacteria live in symbiosis with their human host, since they extract
energy from indigestible carbohydrates and produce vitamins, such as vitamin B12
and folate, for the synthesis of which humans lack specialized enzymes.
The gut microbiome majorly influences the epigenetic and transcriptional
programing of innate immune cells, such as ILCs (Fig. 7.9). The communication
of the microbiome and innate immunity works primarily via metabolites, such as
tryptophan derivatives and the SCFAs (short chain fatty acids) butyrate, propionate
Table 7.1 The human microbiome in numbers

Location Concentration of Organ volume or size Total number of bacteria
bacteria
Large intestine 1011 /ml 400 ml 40 × 1012
(colon)
Teeth 1011 /ml <10 ml 1 × 1012
Skin <1011 /m2 1.8 m2 0.18 × 1012
Lower small intestine 108 /ml 400 ml 0.1 × 1012
(ileum)
Saliva 109 /ml <100 ml 0.1 × 1012
Stomach 103 –104 /ml 250–900 ml 0.00000025–0.000009 ×
1012
Upper small intestine 103 –104 /ml 400 ml 0.0000004–0.000004 ×
(duodenum and 1012
jejunum)
Intestinal lumen
Microbiota B. fragilis
polysaccharide A
Mucus layer SCFAs
Intestinal
epithelial
cell
Laminar propria
IL22
Serum
IgA
amyloid A
CD4+ T cell
Dendritic
cell ILC3
IL23 Plasma cell
IL17 Tregcell IL10

TH17 cell
Fig. 7.9 The microbiome modulates both innate and adaptive immune cells. SCFAs produced
by the microbiome mediate the development of Treg cells. In addition, the microbiota stimulate the
production of IgAs by B cells. More details are provided in the text
and acetate, which are bacterial fermentation products of fibers. The microbes stimu-
late via pattern-recognition receptors on the surface of innate immune cells (Sect. 2.1)
the NLRP6-associated inflammasome, which leads to the secretion of anti-microbial
peptides that control the composition of the microbiome. Moreover, healthy micro-
biota collaborate with host epithelial cells to maintain the intestinal wall via up-
regulation of mucus production. In addition, local B cells are stimulated to produce
and secrete IgAs into the lumen of the gut. In addition, macrophages are primed for
possible inflammatory responses by up-regulating IL1B gene expression. In addi-
tion, microbe-stimulated dendritic cells activate via IL23 ILCs, which through IL22
and serum amyloid A proteins induce the differentiation of T cells into TH 17 cells.
Furthermore, the microbes influence the balance between TH 1 and TH 2 cells and
affect in this way the types of immune response (Sect. 10.5). Interestingly, bacteria
species, such as Bacteroides fragilis, can induce via the secretion of SCFAs the devel-
opment of Treg cells (Sect. 9.1). This has major impact on the tolerance of the host
against the presence of a large number of bacteria in the gut and at other sites of the
body. Thus, the microbiome influences both innate and adaptive immune cells
and majorly contributes to the homeostasis of the immune system.
The microbiome plays an important role in the maturation and “education”
of immune cells. For example, during vaginal delivery Lactobacillales and Prevotella
species of the mother are transferred to the newborn and breast feeding is adding
the lineages Bifidobacteria, Staphylococcus and Enterococcus (Fig. 7.10). The intro-
duction of solid food further increases the diversity and functional capacity of the
gut microbiome. This gradual transition to a functionally more mature microbiome
Prenatal Postnatal
First 1000 days
Early-stage
In utero Neonatal period Puberty Adulthood
maturation
“Sterility” hypothesis Vertical transmission First 2-3 years Sexual maturation Microbiome stability
Presence of microbes? • Antibiotic resistance • Introduction to solid • “Core microbiome”
• Semen genes food mones Richness
• Placenta Complexity
Vaginal delivery Microbiome microbial popula- Flexibility vs. resilience
• Umbilical cord blood “Resembles” vaginal richness tions to dietary changes
• Meconium microbiome • Establishment of
• Low diversity Lactobacillus male and female Pregnancy
Proteobacteria Prevotella Placenta development
Proteobacteria
C-section delivery Resistome Actinobacteria
“Resembles” skin • Acquisition of anti-
microbiome biotic resistance
Staphylococcus genes
Corynebacterium trimesters
Propionibacterium
Breast milk on physiology?

Lactobacillus
Staphylococcus
Enterococcus
Formular milk
Alterations in early
colonizers?
Intestinal length
Microbial diversity and number
Aerobes
Anaerobes
Host organ maturation
Fig. 7.10 The gut microbiome changes from neonates to adults. Infants start with a relatively
simply composed microbiome, but due to nutrition, lifestyle, hormonal changes, immunity and a
gut-brain crosstalk the complexity of the microbiome increases by maturation. More details are
provided in the text
happens in the same phase of the life (first 3 years), in which the immune system
shows rapid development (Box 3.1), i.e., the microbiome appears to be involved in the
training of the immune system. Later, sexual maturation during puberty leads to the
development of gender-specific microbiomes. In parallel, the microbiome composi-
tion shifts from aerobic and facultative anaerobic bacteria to a larger number of anaer-
obic microbes, such as Bacteroidetes and Firmicutes. Finally, adults have developed
a stable core microbiome, which, however, shows large interindividual variations in
its composition. Nevertheless, the microbiome of healthy adults is rather similar in its
functionality. This allows flexibility in the response to challenges provided by infec-
tious diseases as well as by non-infectious disorders. Importantly, the maturation of
the microbiome parallels with the development of host organs, such as the intes-
tine but also of the CNS (Box 7.9). During aging the microbiome changes again by
shifting to the genera Bacteroides, Alistipes and Parabacteroides. Moreover, in older
persons the interindividual variability further increases compared to young adults,
which is one aspect of immune senescence (Box 7.10). Thus, the gut microbiome
significantly contributes to the development and homeostasis of our body.
Box 7.9: The gut-brain-microbiome axis The rather large distance between
the gut and the brain does not immediately suggest that both organs are inten-
sively communicating with each other. The vagus nerve, which runs from the
brainstem to the gut controls the digestive process, while peptide hormones
secreted by the gut and sensed in the brain influence perception and behavior.
Moreover, the gut microbiome interacts with the CNS, so that mental health and
neurological development may both shape and be shaped by these bacteria. For
example, in the irritable bowel syndrome physical effects, such as constipation
and diarrhea, often come along with psychiatric problems, such as anxiety and
post-traumatic stress. Digestive discomfort and psychiatric symptoms are far
stronger with a perturbed microbiome than in persons with a healthy micro-
biome containing probiotic bacteria. The microbiome seems to influence the
production of neurotransmitters, such as serotonin, 95% of which is produced
in the gut. Changes in the microbiota impede the function of the intestine and
alter its signaling to the brain leading to an amplification of the stress response,
e.g., against an infection or a food allergen.
Box 7.10: The aging immune system Within the aging process the immune
system is remodeled and often declines. This starts as early as after puberty,
when the thymus regresses and less naïve T cells are produced (Box 1.5).
Moreover, with age the basal inflammatory response increases (inflammaging),
i.e., there is significantly more low-grade chronic inflammation (Sect. 2.4).
This immune senescence increases not only the risk of acute bacterial and
viral infections (Sect. 8.1) but also their mortality rates, such as observed with
influenza (Sect. 8.3) and COVID-19 (Sect. 8.4). In parallel, the efficiency of
vaccines decreases with age, while latent viruses, such as varicella-zoster virus,
are more easily reactivated (Sect. 8.2). Furthermore, a failing immune system
is not possible to keep sufficient self-tolerance (Sect. 9.1), which substantially
increases the risk for autoimmune diseases (Sect. 10.4). In addition, in the aged
immune system the homeostatic equilibrium between the microbiome and the
host is disturbed. The reduced bacterial diversity in the microbiome of the
elderly may lead to overgrowth of Clostridium difficile, which causes diarrhea.
Furthermore, the risk for inflammation-related, non-infectious disorders, such
as cancer (Sect. 11.1), cardiovascular disease, stroke and Alzheimer’s disease,
significantly raises, when the immune system is less functional.
Since commensal and pathogenic bacteria require similar conditions for their
growth in the intestine, they compete with each other for the same niche. For
example, commensal bacteria produce anti-microbial peptides and SCFAs that inhibit
the growth of other bacterial strains including pathogens, i.e., the presence of
regular components of the gut microbiome prevents the colonization with pathogenic
microbes. However, pathogens have also developed strategies to overcome coloniza-
tion resistance of commensals. They counteract to the competition for nutrients by
focusing on alternative carbon sources, such as galactose, hexuronates, mannose and
ribose, or by using common resources, such as iron, more efficiently. In addition,
pathogens often get a growth advantage over commensal bacteria, when through
the expression of endotoxins they induce inflammation to the intestine of the host
(Sect. 7.1).
A treatment of the host with antibiotics, in order to fight against pathogenic
bacteria, does also disrupts the commensal microbial community. For example,
healthy microbiota of the species Bifidobacterium are replaced by the species
Ruminococcus. This dysbiosis reduces the ability of healthy microbiota to prevent
a future colonization by pathogens and may allow the over proportional growth of
indigenous pathobionts, such as Clostridium difficile, i.e., of potentially patholog-
ical microbes that under normal circumstances live as a non-harming symbionts.
This can cause infectious diarrhea, severe intestinal inflammation and in worst case
a septic shock (Sect. 7.2). Dysbiotic microbiota affect the immune system of the
host through modulation of inflammasome signaling via metabolites secreted by
microbes, disturbing TLR signaling and degrading secreted IgAs. Dysbiosis often
gets persistent, i.e., the gut microbiome may not shift to its healthy state. Then it
is associated with inflammatory diseases of the intestine, such as the inflammatory
bowel diseases (IBDs) Crohn’s disease and ulcerative colitis (Sect. 10.4), as well
as of other organs, such as type 2 diabetes, asthma, obesity, autism and rheumatoid
arthritis (Fig. 7.11) (Box 7.9). Thus, a healthy gut microbiome has a large impact
on human health.
Diet / Bowel Infection or Xenobio-

Exercise Hygiene Genetics
Fluid intake movement tics
tion
Dysbiosis Healthy microbiota
Brain Lung Liver Adipose tissue Intestine Systemic disease

• Stress • Asthma • NAFLD • Obesity • IBD • Type 1 diabetes
• Autism • NASH • Metabolic • Celiac • Atherosclerosis
• Multiple syndrome disease • Rheumatoid
sclerosis arthritis
Fig. 7.11 Dysbiosis and the risk for disease development. Dysbiosis is influenced by many
factors, such as inflammation, infection, diet (which in turn has major impact on bowl movement),
amount of exercise, xenobiotics, hygiene and genetic predisposition. Dysbiotic microbiota affect
via their metabolites and toxins a large range or intestinal and non-intestinal diseases. NAFLD =
non-alcoholic fatty liver disease, NASH = non-alcoholic steatohepatitis
Box 7.11: Dysbiosis and diseases susceptibility Many common disorders,

such as different elements of the metabolic syndrome, cancer and Alzheimer’s
disease, are associated with chronic inflammation. The latter is often modu-
lated by the microbiome, in particular when it has developed dysbiosis. For
example, in obesity the low-grade inflammation of adipose tissue is based on
its recruitment of inflammatory cells, which had been primed by signals of
the microbiome. Similarly, macrophages involved in tumor-associated inflam-
mation may have received their first signals from microbial metabolites and
toxins. This affects in particular colon cancer but applies also to cancers in
other organs. Importantly, also diseases of the CNS, such as autism spectrum
disorders, are affected by the microbiome, when it is in dysbiosis. Thus, the
individual’s microbiome in its healthy state as well as in dysbiosis is an
important predictor for diseases susceptibility.
Further Reading 133
Further Reading
1. Cadena AM, Fortune SM, Flynn JL (2017) Heterogeneity in tuberculosis. Nat Rev Immunol
17:691–702
2. Cambier CJ, Falkow S, Ramakrishnan L (2014) Host evasion and exploitation schemes of
Mycobacterium tuberculosis. Cell 159:1497–1509
3. Eckhardt M, Hultquist JF, Kaake RM, Huttenhain R, Krogan NJ (2020) A systems approach to
infectious disease. Nat Rev Genet 21:339–354
4. Hall AB, Tolonen AC, Xavier RJ (2017) Human genetic variation and the gut microbiome in
disease. Nat Rev Genet 18:690–699
5. Kundu P, Blacher E, Elinav E, Pettersson S (2017) Our gut microbiome: the evolving inner self.
Cell 171:1481–1493
6. Levy M, Kolodziejczyk AA, Thaiss CA, Elinav E (2017) Dysbiosis and the immune system.
7. Lynch SV, Pedersen O (2016) The human intestinal microbiome in health and disease. N Engl
J Med 375:2369–2379
8. Schneider DS (2011). Tracing personalized health curves during infections. PLoS Biol
9:e1001158
9. van der Poll T, van de Veerdonk FL, Scicluna BP, Netea MG (2017) The immunopathology of
sepsis and potential therapeutic targets. Nat Rev Immunol 17:407–420
Chapter 8
Immunity to Viral Pathogens
and the Virome
Abstract This chapter will focus on virus infections and how our immune system
is combating them. First, we will discuss the principles of the effector functions of
innate and adaptive immunity against acute virus infections. Then we will shift to
chronic virus infections and understand that each of us carries an individual virome.
The latter is formed by dozens of different virus types, many of which are not harming
us when we are healthy but may get a problem in the context of a multitude of disease.
Next, we will learn how microbes can be transferred from different animals to humans
and will use influenza virus and SARS-CoV-2 as master examples. Finally, we will
elaborate the impact of vaccination against influenza and COVID-19.
Keywords Virus infection · Effector mechanism · Chronic virus infection ·

Virome · Influenza · SARS-CoV-2 · COVID-19 · Vaccination
8.1 Principles of Immune Responses to Viruses
Since more than 100 years the majority of newly emerging and re-emerging
infectious diseases are caused by viruses. More than 200 different virus species are
known to infect humans. For their effective replication, viruses often take control on
protein synthesis within their host cells. This harms the function of the infected cells
and may lead to their lysis when new viral particles are released. In addition, virus
infection can stimulate inflammatory responses that cause tissue damage.
Viruses can be categorized by the presence of absence of a lipid bilayer cover
as enveloped or non-enveloped as well as by the material of their genome as RNA
or DNA viruses. Examples of enveloped RNA viruses are Zika virus (Box 8.1),
influenza A virus (Sect. 8.3) and SARS-CoV-2 (Sect. 8.4), while poliovirus is a non-
enveloped RNA virus. Moreover, vaccinia virus and herpes simplex virus (HSV)
are enveloped DNA viruses, whereas adenovirus is a non-enveloped DNA virus. In
contrast to other microbes, viruses need a host, in order to replicate, i.e., the key point
in the life cycle of every virus is, how it enters a host cell. Viruses typically enter
cells by receptor-mediated endocytosis, for which they have to bind with sufficiently
high affinity to one or more receptors on the surface of their host cell. For example,
HIV-1 binds to CD4 and therefore has a tropism for TH cells, but also needs the
https://doi.org/10.1007/978-3-031-04025-2_8
136 8 Immunity to Viral Pathogens and the Virome
chemokine receptor CCR5 for entering a cell (Box 8.2). Furthermore, influenza A
virus uses sialic acid covered receptors on respiratory epithelial cells (Sect. 8.3),
while SARS-CoV-2 binds to the receptor angiotensin-converting enzyme 2 (ACE2)
on lung alveolar epithelial cells (Sect. 8.4).
Box 8.1: Zika virus Zika is an enveloped RNA virus of the flavivirus family,
which is spread by daytime-active mosquitoes. The virus is known since
decades, but until 2015 it never caused a human epidemic. However, when
a mutation led to a single amino acid change in the external viral glycoprotein,
the virus suddenly spread pandemically around the tropical belt. It infected
millions of individuals and is able to spread from a pregnant woman to her
baby. This can result in microcephaly, severe brain malformations and other
birth defects.
Box 8.2: HIV-1 and AIDS The retrovirus HIV-1 started to spread worldwide
in the human population since the 1980s and has killed more than 37 million
individuals. HIV-1 infects and destroys preferentially CD4+ TH cells immune
cells and leads in this way to a failure of the immune system, referred to as
AIDS, that allows other (chronic) infections, such as tuberculosis, to break
out. Less than 100 years ago HIV-1 originated from zoonotic transmission
of simian immunodeficiency viruses from African primate species, such as
chimps. Interestingly, homozygotic carriers of a 32 bp-deletion in the CCR5
gene cannot be infected by HIV-1.
The infection with a virus means for the immune system the sudden appearance of
a new structure, to which the effector functions of innate and adaptive immunity are
responding. The main mechanisms of the innate immune system are the production of
type I IFNs (Box 8.3) and the induction of apoptosis to virus-infected cells by NK cells
(Fig. 8.1a). INFs are produced by cells that sense via TLR3, TLR7, TLR8 or TLR9
in their endosomes the presence of viral RNA or DNA (Sect. 2.1). In parallel, also
cytosolic pattern-recognition receptors of the RLR family recognize nucleic acids
of viruses that managed to escape into the cytosol (Fig. 2.1). In both cases signal
transduction pathways are initiated and converge in the activation of the transcription
factors IRF1, IRF3 and IRF7, which together regulate 300–1000 genes. Major target
genes encode for type I IFNs, which inhibit the replication of viral genomes via
the activation of proteins of the restriction factor family. Moreover, other target
genes booster NK cells as well as the adaptive immune system. An immune evasive
mechanism of viruses is the down-regulation of MHC-I proteins or the prevention
of peptide antigen presentation on these receptors (Sect. 5.3) (Box 8.4). Therefore,
the cells are not recognized by cytotoxic T cells but preferentially killed by NK cells
8.1 Principles of Immune Responses to Viruses 137
Innate immune Adaptive immune

a
response memory
Type I IFNs B cell Antibodies

Protection
against
infection
Anti-viral state Neutralization
NK cell Cytotoxic T cells Infected cell
Infected cell
Eradication
of
established
infection
Killing of infected cell Killing of infected cell

b
NK
cells cytotoxic T cells
Type I Antibodies
IFNs
Magnitude of response/infection
Virus
titer
2 4 6 8 10 12
Days after viral infection
Fig. 8.1 The response of the innate and adaptive immune system against virus infections.
The innate and adaptive immune system use different mechanisms to prevent and eradicate virus
infections (a). Time course of main effector functions of innate and adaptive immunity after infection
with a virus (b) More details are provided by the text
that act independent of MHC proteins and use patter-recognition receptors, such as
KIRs, FcγRIIIA and C-type lectin receptors (Sect. 2.5).
Box 8.3: Type I INFs The main function of this cytokine family (composed
of 13 variants of IFNα and IFNβ) is to mediate the early responses of the innate
immune system against viral infections. Accordingly, the strongest stimulus
for the production of type I INFs are viral RNA or DNA genomes, which
are detected by pattern-recognition receptors in endosomes and the cytosol of
virus-infected cells. Since the receptor for type I INFs is found on all nucleated
cells, the cytokines are able to inform in a paracrine fashion all neighboring non-
infected cells about the presence of viruses, so that they can switch into an anti-
viral state. Moreover, INFs cause the sequestration of lymphocytes in lymph
nodes, increase the cytotoxicity of NK cells and cytotoxic T cells, promote the
differentiation TH 1 cells and up-regulate MHC-I protein expression.
Box 8.4: Immune evasion by viruses The genome of RNA viruses, such
as influenza viruses, flaviviruses, enteroviruses and coronaviruses, is rapidly
evolving, since the viruses do not use any error correction or repair mecha-
nism during the replication of their genome. This leads to frequent changes
in the amino acid sequence and structure of viral surface proteins. Since the
latter are most commonly serve as antigens, the viruses are no longer targets of
immune responses, in particular by those that are mediated by antibodies. Point
mutations and other minor changes in the viral genome lead to an antigenic
drift, e.g., in rhinoviruses that cause the common cold as well as in HIV-1. In
contrast, reassortments of whole segments of the viral genomes cause an anti-
genic shift, e.g., in the case of the influenza A virus (Sect. 8.3). Another viral
immune evasion mechanism is the inhibition of the production of INF type I
by host cells, as it commonly occurs by all types of coronaviruses (Sect. 8.4).
Furthermore, some viruses inhibit the presentation of peptides from cytosolic
proteins on MHC-I proteins, which reduces the recognition of virus-infected
cells by cytotoxic T cells. In addition, some viruses, such as EBV (Epstein-Barr
virus), produce mimics of cytokines or chemokines, so that their receptors are
antagonistically blocked and respective immune cells are not activated.
The main effector function of the adaptive immune system against viruses are
antibodies that neutralize the virus by binding to its envelop or capsid (Sect. 3.1). In
this way, extracellular viruses are blocked to bind surface receptors and cannot enter
host cells. High affinity antibodies that are the result of affinity maturation of B cells
in germinal centers (Sect. 4.3) are most suited for effective neutralization of viruses.
Moreover, for the protection of viral infections of the mucosa-covered respiratory or
8.2 Chronic Virus Infections and Emerging Viral Pathogens 139
intestinal tract, IgAs are most potent. Thus, vaccinations against viral diseases aim
to induce the production of IgAs. However, when anti-viral antibodies are captured
by FcRs on the surface of macrophages and other cells of the innate immune system,
the entry of the virus may even be facilitated. This antibody-mediated enhancement
can increase lung inflammation, e.g., in the context of an infection with SARS-CoV-2
(Sect. 8.4).
When viruses are intracellular, killing of virus-infected cells by cytotoxic T cells
is the only possibility to eliminate the microbes. Therefore, the main function of
cytotoxic T cells is the surveillance of the body for possible virus-infected cells
(Sect. 6.4). However, virus-infected cells can only be detected and eliminated by
cytotoxic T cells, when peptides originating from viral proteins are presented by
MHC-I proteins.
The typical time course of a viral infection starts with the production of type I
INFs, which peak already 2 days after onset of infection, and is followed by NK cell
proliferation with a maximum at days 3–4 (Fig. 8.1b). However, in most cases the
virus titer does not decline before virus-specific cytotoxic T cells achieve highest
counts after day 7 and virus-specific antibodies reach their maximal titer at days 9–
10. When the virus is eliminated, the immune system returns to homeostasis and only
a few hundred virus-specific memory B and T cells remain. However, sometimes the
immune system does not manage to eliminate all viruses. This leads to a chronic
viral infection, i.e., the persistence of the viral antigens (Sect. 8.2). After the initial
typical response, the immune system may either return to homeostasis “accepting”
the presence of the viral antigen on a constant, rather high level or it stays permanently
alert and the viral titers are fluctuating in waves, e.g., due to an antigenic drift as
observed with influenza virus infections (Sect. 8.3). Another possibility is that the
viral antigen titer increases very slowly, so that there is no or only very weak response
of the immune system. An important parameter in these scenarios is how the
antigen titers change over time. This does not only apply to viral infections, but
also to other immune responses, such as in chronic autoimmune diseases like SLE
(systemic lupus erythematosus) and multiple sclerosis (Sects. 10.3 and 10.4).
8.2 Chronic Virus Infections and Emerging Viral

Pathogens
Viruses that were emerging rather recently in the human population, such as influenza
virus (Sect. 8.3) and SARS-CoV-2 (Sect. 8.4), had no time to adopt to us as a
host. They cause in some individuals severe illness or may even kill them, while
in the majority of the infected persons they are cleared within 1–2 weeks by effector
functions of the immune system. The viruses did not establish a stable relationship
with their new host and can only survive when they manage to jump over to a new
host before the first host either dies or has eliminated them. In contrast, 5–8% of
our genome is composed of retrovirus sequences, i.e., in the past many viruses
managed to persist in our ancestors. Moreover, some viruses, such as the eight human
herpesviruses, have a common ancestor in birds, reptiles and mammals, indicating
joined co-evolution of these viruses and their hosts over more than 100 million years.
When during an acute viral infection, the effector functions of the immune system
are insufficient and/or immune evasive mechanisms of the virus are very potent, the
infection cannot be resolved. In extreme case, this may have devastating conse-
quences and the host may die. However, often the viruses just hide in niches, such
as neurons, hematopoietic cells or stem cells. Then effector functions of the immune
system and the actions of the virus get into an equilibrium, so that the infection
becomes chronic (Fig. 8.2). The continuous presence of viral antigens during chronic
infections sometimes leads to T cell exhaustion and/or anergy (Box 8.4). Thus, in
chronic infections the immune system adjusts to the presence of the virus and
its effects, such as causing a chronic inflammation. In this case, excessive damage of
infected cells and tissues is prevented and viral replication is restricted to an accept-
able level. However, the equilibrium between the actions of the host immune system
and the virus is metastable and can become dangerous but also benign or even symbi-
otic. Chronic viruses form our virome, which is part of the microbiome (Sect. 7.4).
Thus, in a similar way as the gut microbiome trains the immune system, also
the virome interacts with innate and adaptive immunity.
Box 8.5: T cell exhaustion The progressive loss of effector function of cyto-
toxic T cells during chronic viral infections is referred as exhaustion. The term
“exhaustion” implies that first a normal T cell response developed but it then
declined. In contrast, in tolerance anergic T cells fail to develop effector func-
tion (Sect. 9.1). Exhaustion may have developed, in order to limit collateral
tissue damage due to persistent T cell activity. For example, exhaustion of cyto-
toxic T cells is due to the persistence of their antigen and based on changes
in their epigenome and transcriptome. These changes result in the overexpres-
sion of inhibitory receptors, such as PDCD1, altered expression of transcription
factors, changes in signal transduction and down-regulation of key metabolic
genes, so that the T cells are unable to clear their antigen. Blocking PDCD1
by a monoclonal antibody (Sect. 11.3) reverses the exhaustion.
The majority of us is permanently infected by more than 10 types of chronic

viruses. Most of these are DNA viruses, such as HSV, cytomegalovirus (CMV), EBV
and varicella-zoster virus (VZV) (Table 8.1). In addition, anelloviruses and adeno-
associated viruses infect most humans by the end of their childhood and respective
integrated viral sequences are found in the genome of many individuals. In these
infections the viral genome persists in host cells but the productive replicative cycle
is not lytic but latent, i.e., no infectious virus is produced. Latent viruses are most
successful in long-lived cells, such as HSV and VZV in neurons, CMV in HSCs as
well as HIV-1 and EBV in memory lymphocytes. In many cases these latent viruses
do not produce any obvious symptoms. However, the viruses can become activated
Viral strategies Immune strategies
• Rapid replication
Acute infection • Innate immunity
• Immune evasion and • Antigen presentation
subversion • Entry • Cytokines
• Immune privilege • Primary replication • Clonal expansion of
• Tissue damage • Spread lymphocytes
• Viral adaptation • Secondary replication
(genetic, mutation) • Tissue damage mechanisms
• Shedding • Regulatory cell inter-
actions
Decision point
Recovery Chronic infection
• Clearance of damaged cells • Continuous/intermittend antigen

• Elimination of virus • Tissue damage
• Re-establish immune system • Altered immune system
• Re-establish homeostasis • Altered homeostasis
Immune strategies Viral strategies

• Continuous replication • Dampen responses
cells • Latency • Chronic activation
• T cell memory • Immunopathology
• B cell memory • Lymphocyte func-
• Remodeling lymphoid regulation tion/dysfunction
tissues • Mutation • Repertoire contrac-
• Immunoprivilege tion
Fig. 8.2 Acute versus chronic viral infection. An acute infection represents non-equilibrium
phase of competition between the strategies of the virus and the host immune response. When the
host dominates the infection, it is either cleared and the host recovers or it may become chronic. In
the latter case the virus may get latent or a metastable equilibrium between viral and host strategies
is established. More details are provided in the text
Table 8.1 Chronic human virus infections

Virus type Infection rate (%) in Major sites of Chronic disease in
human populations persistence normal individuals
Endogenous 100 All cells Unknown
retroviruses
Anelloviruses 90–100 Many tissues Unknown
Human herpesviruses >90 Lymphocytes Unknown
6 and 7
VZV >90 Sensory ganglia Herpes zoster
neurons, lymphocytes
CMV 80–90 Myelomonocytic cells Rare
EBV 80–90 Pharyngeal epithelial Burkitt’s lymphoma,
cells, B cells non-Hodgkin’s
lymphoma
Polyomaviruses BK 72–98 Kidneys Unknown
and JC
Adeno-associated 60–90 Many tissues Unknown
virus
HSV-1 50–70 Sensory ganglia Cold sores,
neurons encephalitis, keratitis
Adenovirus Up to 80 Adenoids, tonsils, Unknown
lymphocytes
Viruses that affect at least 50% of human populations are listed
and replicate, in particular, when the immune system is weakened, e.g., when an
individual is immunocompromised. The strategy of latent viruses is comparable to
that of Mycobacterium tuberculosis, which at present survives in some 2 billion
individuals, while it causes only in some 10% of them an active disease (Sect. 7.3).
The human metagenome does not only comprise the human genome but also
the genomes of all eukaryotic (parasites), bacterial and viral species that form our
microbiome. The different components of the microbiome influence each other while
they interact with their host in health as well as in disease (Fig. 8.3). As individuals
differ in their gut microbiome (Sect. 7.4), they also have their personal virome.
Therefore, each of us is experiencing an individual training of the immune
system by all components of the metagenome. This extends the one-microbe-one-
disease paradigm to a multimicrobe-based understanding of diseases and explains
the different immunophenotypes (Box 8.6) of individuals. For example, dysbiosis of
the bacterial arm of the microbiome, which is caused by the widespread use of antibi-
otics, significantly increased the rate of autoimmune diseases (Sect. 10.4), while the
presence of helminths provides resistance against them. This observation also relates
to the increase of allergies in developed countries, which have high antibiotic use
and absence of helminths, and is summarized as the hygiene concept (Sect. 10.2).
Thus, the competence to respond to immune challenges, as provided by microbe
infections but also by non-transferable disorders, such as cancer, diabetes and
Latent herpesvirus confers

resistance to bacterial infections
Bacteria Pat Susceptibility Viruses

ho
g microbiome
en
s
Antibiotics Dysbiosis viral infections
reshape linked to
community obesity, asthma
s
on
ti
fe c
l in
Vira
Disease
Symbiosis
Parasitic infections
Helminths confer
Virulence resistance
to autoimmune
diseases
Eukaryotes
Fig. 8.3 The metagenome in health and disease. This scheme displays the complex interaction
of eukaryotic, bacterial and viral components of the microbiome with their human host in health
and disease. More details are provided in the text
Alzheimer’s disease, is largely dependent on the status of our metagenome.

Since chronic CMV infections affect the immune senescence of T cells, even aging
is affected by the individual’s immunophenotype.
Box 8.6: Immunophenotype The immunophenotype of an individual is repre-

sented by the different types and relative number of immune cells in circulation.
Immune cells are traditionally described by more than 370 different proteins
expressed on their surface, which are part of the CD categorization (Box 2.4).
This can be determined by FACS and related techniques on cell suspensions as
well as on fresh or fixed tissue. The immunophenotype is used for describing the
general competence of the immune system as well as for diagnosis and prog-
nosis of immune related diseases, such as autoimmune diseases (Chap. 10)
and cancer (Chap. 11). Individuals vary in their immunophenotype due to vari-
ations in the genome and different environmental exposure. There are thou-
sands of known variants, most of which have only a minor contribution to
the immunophenotype, but together they may explain 50% of the individual’s
immune response and risk for immunological diseases. The other half is due
to age [Box 3.2, see also inflammaging (Sect. 2.4)], gender, environmental
exposure, diet and microbiome (Sect. 7.4).
All species carry their own type of metagenome, to which they are adapted. When
two different species get into close contact, there is the chance that they infect each
other with their viruses or other microbes. A microbe that is well adapted, i.e.,
symptom-less, to one species may cause severe illness in another species that
had not experienced this microbe before. The recent transfer of SARS-CoV-2
from bats to humans at the wet market in Wuhan (China) is a master example of
this concept (Sect. 8.4). In general, zoonotic transmission between other species and
humans can be distinguished in five different stages (Fig. 8.4). Stage 1 represents
probably the majority of all microbes, which are specific to one non-human species
Transmission
Stage
to humans
Stage 5:
Only from
exlusive human
humans
agent
Stage 4: From animals

long outbreak or (many cycles)
humans
Stage 3: From animals

limited or (few cycles)
outbreak humans
Stage 2:
Only from
primary
animals
infection
Stage 1:
exlusive animal None
agent
Rabies virus Ebola virus Dengue virus HIV-1
Fig. 8.4 Stages of zoonotic transmission of animal pathogens to humans. Details are provided
in the text
8.3 Influenza 145
and have not yet been transferred to humans. This stage unfortunately provides a large
repertoire of possibilities for future zoonotic transmissions to humans. Microbes of
stage 2, such as those causing anthrax (Bacillus anthracis) or rabies (rabies virus),
can infect humans that eat meat from infected animals or get bitten by them. However,
the microbes cannot be transferred from one infected individual to another person.
In stage 3 pathogenic microbes from animals, such as Marburg and Ebola virus
(Box 8.7), manage a few cycles of transmissions between humans but do not establish
themselves over longer time in a human population,i.e., they cause only to short
outbreaks. Stage 4 depicts animal diseases, such as avian influenza (Sect. 8.3), yellow
fewer and dengue fever from mosquitos, that often perform zoonotic transmission to
humans and other animal species, such as farm poultry and pigs in case of influenza.
The microbes are stable for many infection cycles between humans and can lead
to larger outbreaks. Finally, stage 5 describes infectious diseases, such as measles,
mumps and syphilis, that spread between humans without the involvement of other
species. However, they still origin from animals but their zoonotic transmission was
long time ago.
Box 8.7: Ebola virus Ebola virus is a member of the filovirus family of
enveloped RNA viruses, to which also Marburg virus belongs. Filoviruses
are occasionally transmitted by zoonosis from fruit bats to humans and other
mammals. Human pathogenic filoviruses appeared so far only in equatorial
Africa, i.e., outbreaks are restricted to local regions in this part of the world. A
clinical hallmark of filovirus infections is a hemorrhagic fever, i.e., bleeding
internally and externally due to decreased blood clotting. In average 50% of the
infected persons die within a few days to weeks after infection, i.e., Ebola is a
very deadly disease. Ebola virus is transmitted via virus-contaminated bodily
secretions, i.e., person touching contaminated fluids or surfaces in nursing care,
burial services etc. are frequently infected.
8.3 Influenza
Every year one billion individuals are infected by seasonal influenza, which results
in 3–5 million cases of severe illness and 300,000–500,000 deaths worldwide. Like
in many other infectious diseases, morbidity and mortality of influenza infection
is significantly increased in old age. Accordingly, 90% of the influenza deaths are
elderly (>65 years of age), probably because they have a drastically decreased thymic
output (Sect. 3.1). The recurrence of the epidemic is based on the efficient transmis-
sion of the virus between humans via respiratory droplets, direct contact and contami-
nated surfaces. Outbreaks of influenza occur in the winter months, when transmission
is favored by low temperatures and humidity. The symptoms of influenza range from
milder effects on the upper respiratory tract, such as sore throat, fever, cough, running
nose, headache, muscle pain and fatigue to lethal pneumonia due to high levels of
viral replication in the lower respiratory tract in combination with a cytokine storm
or secondary bacterial infections. Moreover, there can also be complications of the
heart, CNS and other organs.
Influenza viruses belong to the family of orthomyxoviruses of enveloped RNA
viruses. The genome of influenza viruses A and B is formed by eight segments of
single-stranded RNA, which allows a rapid antigenic drift and shift of the virus
(Box 8.4). For the major glycoproteins on the viral envelop, hemagglutinin and
neuraminidase, 18 and 11 subtypes are known in various species. They are the major
determinants for the different strains of the influenza A virus (Fig. 8.5). For example,
8 genome segments, most

important encoded proteins:
Hemagglutinin (HA)
(18 subtypes, H1 - H18)
Neuramidase (NA)
(11 subtypes, N1 - N11)
Nomenclature:
Animal/place/no/year
NEP
HA NA Examples:
nuclear export protein
A/Fujian/411/92 (H3N2)
M1 matrix protein M2 ion channel A/Hong Kong/156/97 (H5N1)
H1N1
H3N2
? H1N1 H2N2 H1N1
1918 1957 1968 1977 2009
Fig. 8.5 Influenza virus. The genome of influenza A virus is composed of eight ribonucleoprotein
complexes that encode for RNA polymerase subunits, the glycoproteins hemagglutinin (HA) and
neuraminidase (NA), the viral nucleoprotein (NP), the matrix protein M1, the membrane protein
M2, the nonstructural protein NS1 and the nuclear export protein (NEP) (left). The 18 variants of
HA and the 11 variants of NA determine the “flu alphabet” of influenza virus strains (right). A
time line describes the main virus strains causing pandemics and seasonal outbreaks of the disease
(bottom)
8.3 Influenza 147
the “Spanish flu” (Box 8.8) of 1918 was based on the strain H1N1. The following
pandemics of the years 1957, 1968 and 2009 were caused by the strains H2N2,
H3N2 and H1N1, respectively. Importantly, the H1N1 variant caused between 1919
and begin of the 1950s several epidemics until it was replaced by the strain H2N2.
Moreover, highly pathogenic (60% fatality rate) H5N1 infections occurred during
the past decades, but only occasional human-to-human transmissions were observed,
which prevented the outbreak of pandemics. The influenza B strains circulates in
humans since at least 80 years but has no animal reservoir as well as strains C and
D that do not cause major disease in humans.
Box 8.8. Spanish flu The influenza A virus strain H1N1 pandemic of the years
1918 to 1920 was with 50 million deaths (translating to 200 million victims in
the present world population) the largest ever (Table 1.2). Although influenza A
viruses circulate in human populations since at least 2000 years, this pandemic
was particularly lethal even to the young population, because.
• the hemagglutinin protein was unusually cytopathic and immunopathogenic
compared to previous strains
• co-infections with pneumopathogenic bacteria caused fatal bacterial bron-
chopneumonia.
The pandemic had three major waves: the first wave in spring 1918 (fatality
rate 0.5%), autumn 1918 (fatality rate 2.5%) and winter 1918/19 (fatality rate
1%). Of note, the pandemic was called “Spanish flu”, because newspapers
in neutral Spain were the first to report about the disease, while most other
affected countries participated in World War I and did not allow their press to
spread the news. Thus, the pandemic did not originate from Spain but was
probably imported to the US by guest works from China and then spread by
hundreds to thousands of US American soldiers that had been shipped to the
battlefields in Europe.
Influenza viruses enter the human body via oral and nasal cavities and settle on
the epithelial mucosa of the respiratory tract. When the virus successfully passes
the mucosa, it spreads both to epithelial cells, where the virus is effectively repli-
cating, and immune cells, such as macrophages and dendritic cells. The hemagglu-
tinin protein binds to sialic acid on the surface of host cells and induces the fusion
of the viral envelop with the cell membrane. In contrast, the neuraminidase protein
allows new virions to leave infected cells by releasing the hemagglutinin-sialic acid
contact and facilitating movement through the mucus. The innate immune system
detects influenza virus RNA in infected cells via pattern-recognition receptors, such
as TLR8, RLR and NLRP3, and initiates the INF type I response of macrophages,
dendritic cells and cells of the lung. This induces the anti-viral state to neighboring
cells and tissues (Sect. 8.1). However, pro-inflammatory cytokines cause local inflam-
mation in the lung and induce systemic effects, such as fever and anorexia. Moreover,
chemokines recruit neutrophils, monocytes (differentiating to macrophages) and NK

cells to the airways, which leads to clearance of infected cells by phagocytosis and
the induction of apoptosis. Despite these effector functions of innate immune cells,
the virus may spread, so that only the adaptive immune system can lead to virus
clearance. The homotrimeric hemagglutinin protein complex is the major target of
adaptive immune responses, which are mediated by specific antibodies and cytotoxic
T cells. The antibody response to influenza infection is robust and often lasts for the
rest of the life. However, the antigenic drift of the virus, in particular of the head
domain of the hemagglutinin protein, may change the antigen so much that a new
immune response needs to be initiated.
Since the genome of influenza viruses is segmented, an interchange between two
strains, which have infected the same cell, can lead to new assortments, i.e., to an
antigenic shift (Fig. 8.6). For example, pigs that are infected at the same time with
influenza from chicken and humans (e.g., on a farm, where all three species live in
close vicinity), a new strain can be generated that may be more virulent at least for
one of the three species. For example, the influenza pandemic of 2009 was caused
by a new H1N1 strain that reassorted in pigs (“swine flu”). The zoonotic origin of
influenza A viruses has been wild birds, in particular migratory ducks and geese.
The transmission to other species, such as marine mammals, domestic ducks and
further poultry, may be primarily via contaminated water. Also, other species, such
as pigs, horses, dogs, cats and finally humans, can be infected via aerosols as well
as contaminated surfaces and water.
Due to the rapid antigenic shift and drift of influenza viruses, the formulation of
vaccines needs to be adapted every six months. The effectiveness of the vaccines
varies from year to year. At present, the most common vaccine is a mixture of each
two strains of influenza A and B, e.g., the influenza A strains H1N1 and H3N2 as
well as the influenza B strains Victoria and Yamagata. Influenza virus vaccines are
traditionally produced in embryonated chicken eggs, i.e., live viruses are grown and
then chemically inactivated,e.g., by alkylating chemicals, ether or detergents. For
an increased yield in virus production, the currently circulating influenza strains are
assorted with a highly growing strain (A/Puerto Rico). However, current influenza
vaccines do not induce neutralizing antibodies that recognize multiple virus strains,
so that antibody-mediated protection is mostly short-lived. In addition, anti-viral
drugs, such as amantadine and rimantadine targeting the M2 ion channel of the virus
or oseltamivir inhibiting the enzymatic activity of the neuraminidase, are used for
the prevention and treatment of patients, who are at high risk or severely ill.
8.4 COVID-19 149
• Stamping out
• Decontamination
• Water treatment • Movement restrictions • Vaccination
• Biosecurity • Compensation • Market hygiene
• Indoor raising • Quarantine • Live market closure
• Zoning
Marine mammals
Pigs
Wild birds ? Domestic Poultry

ducks
Humans
Bats
Horses
Dogs Cats
Transmission via water Transmission via aerosols, water

and contaminated surfaces and contaminated surfaces
Fig. 8.6 Spreading of influenza A virus between species. Details are provided in the text. Dashed
lines represent transmission that bypasses a domestic duck intermediate
8.4 COVID-19
The coronavirus strains HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoV-HKU1

are endemic in humans since a few hundred years. They typically infect the upper
respiratory tract and cause in most cases only mild symptoms, such as a common
cold. However, since coronaviruses are neurotrophic, in rare cases also encephalitis
is observed. Moreover, different types of coronaviruses are found in bats and are able
to infect camels, birds, cats, horses, mink, pigs, rabbits, pangolins and other animals.
Thus, there is a large variety of species from which zoonotic transmission of
coronaviruses to humans can happen. From this animal reservoir, zoonotic trans-
mission of SARS-CoV happened in 2003 probably from civet cat and Middle East
respiratory syndrome coronavirus (MERS-CoV) in 2012 from the camel. In this line,
SARS-CoV-2 is the youngest member of zoonotic microbes that not only managed
its transmission from bats but immediately learned to spread between humans. All
three recent coronavirus strains replicate also in the lower respiratory track, where
they cause pneumonia that can be fatal. Most likely SARS-CoV-2 will not be the last
example of a zoonotic transmission of coronaviruses, i.e., probably sooner than
later SARS-CoV-3 will be observed in humans. Since the majority of persons that
had been infected with SARS-CoV-2 have no or only mild symptoms, the virus was
quickly spreading to basically all countries of the world, which todays are intensively
connected by global travel and trade. This was the start of the COVID-19 pandemic
that very quickly outnumbered the victims of the SARS-CoV outbreak and the H1N1
swine flu pandemic in 2009 (Sect. 8.3).
Coronaviruses are enveloped, single-stranded RNA viruses. The genome of
SARS-CoV-2 has a size of 30 kb and encodes for a RNA-dependent RNA poly-
merase, proteases and structural proteins forming the nucleocapsid as well as the
trimeric spike glycoprotein, the envelop protein and the matrix glycoprotein that
are all integrated in the envelop. The spike protein binds to ACE2 proteins on the
surface of epithelial cells of the upper and lower respiratory track, such as airway
epithelial cells, alveolar epithelial cells, vascular endothelial cells and macrophages
in the lung. The enzyme TMPRSS2 (transmembrane serine protease 2) on the host
membrane cleaves the spike protein and facilitates the entry of the virus into the cells
(Fig. 8.7, top left). After replication, multiple copies of the virus are released from
infected cells, which then undergo pyroptosis, i.e., an highly inflammatory form of
programmed cell death that often occurs with cytopathic viruses (Sect. 2.4). This
leads to the release of DAMPs, such as ATP and nucleic acids, and triggers in neigh-
boring epithelial cells, endothelial cells and alveolar macrophages the production of
pro-inflammatory cytokines and chemokines, such as IL6, CXCL10, CCL2, CCL3
and CCL4 (Fig. 8.7, top right). Monocytes, macrophages and T cells are attracted to
the site of infection and a pro-inflammatory feedback loop is established. In persons
with an overreactive immune system this attracts further immune cells to the lungs
and leads to the overproduction of pro-inflammatory cytokines, such as IL1, IL6,
IL12 and TNF, which cause damage to the lung infrastructure (Fig. 8.7, bottom
left). The resulting cytokine storm affects other organs and can lead to their damage
and failure. Furthermore, antibody-dependent enhancement by non-neutralizing anti-
bodies intensifies organ damage. Thus, the severity of COVID-19 is rather based
on the strong response of the immune system than on any direct effect of the
virus. In contrast, in persons with a normal responding immune system (i.e., 80–90%
of all infected individuals) the initial inflammation attracts virus-specific cytotoxic T
cells and the infected cells are eliminated before the virus is able to spread (Fig. 8.7,
bottom right). Furthermore, neutralizing antibodies block viral infection and alveolar
macrophages recognize and clear neutralized viruses as well as apoptotic cells by
phagocytosis. This leads to clearance of the virus and only minimal damage to the
lung, i.e., in recovery.
8.4 COVID-19 151
ASC oligomer Host DNA

Airway
IL1
ACE2 Viral RNA
ATP
Epithelial cell
TMPRSS2 Release of virus
Virus maturation
Virus
replication
Viral proteins
Secretes
Secretes
Alveolar
Pyroptosis macrophage
IL6 Positive T cell
CCL3 CCL2 feedback
CCL4 CXCL10 Monocyte

IFNγ Macrophage
Endothelial layer
Leakage caused by vascular permeability
Alveolar macrophages Neutralizing antibody

clear up neutralized binds and inactivates
virus virus
Cytokine storm
No virus release
Alveolar macrophages
recognize and phagocytose
apoptotic cell
CD8+ T cell recognizes

and eliminates infected
cells
Non-neutralizing antibody may cause antibody- CD4+ T cell mediates efficient immune response
dependent enhancement of infection
Dysfunctional immune response: Healthy immune response:

• Infected cells rapidly cleared
phages and T cells • Virus inactivated by neutralizing antibodies
• Systemic cytokine storm
• Pulmonary edema and pneumonia
damage
Fig. 8.7 Chronology of SARS-CoV-2 infection. The first two step are virus infection (top left) and
inflammation caused by lysed cells (top right). In pre-diseased patients (bottom left) inflammation
worsens and leads to a cytokine storm, which causes organ damage and possible failure. In contrast,
in individuals with an healthy immune system (bottom right) the inflammation attracts virus-
specific cytotoxic T cells, which eliminates virus-infected cells before they can spread the virus
SARS-CoV-2 spreads primarily via respiratory droplets and contaminated

surfaces (Fig. 8.8). The incubation period is 4–5 days before symptoms like cough,
fever and fatigue occur. Individuals with only mild symptoms of COVID-19 have a
robust type I INF response (Sect. 8.1) as well as virus-specific TH 1 cells and cytotoxic
T cells, which results in the rapid clearance of the virus. Children in vast majority have
only a mild form of COVID-19, but in rare cases they may present 1–2 months after
the infection the multisystem inflammatory syndrome in children (MIS-C), which
seems to be an autoimmune disease with over boarding immune reactions. In elderly
patients and others that are pre-diseased with cancer, cardiovascular disease or respi-
ratory disorders, a SARS-CoV-2 infection may worsen 5–10 days after symptom
onset and complications occur, such as hypoxia, respiratory failure, acute respi-
ratory distress syndrome and septic shock. In these severe cases of COVID-19,
the initial anti-viral response of the innate immune system is delayed, which is an
Transmission Symptoms
Fever, ~ 90% (44% at time of diagnosis)

Dry cough, ~ 70%
Transmission Fatigue, ~ 40%
Sputum production, ~ 30%
Shortness of breath, ~ 20%
Muscle and joint pain, ~ 15%
Sore throat, ~ 14%
• Distance > 2 m Headache, ~ 14%
Chills, ~ 10%
• Handwashing
Nausea/Vomiting, ~ 5%
• Desinfection depending on age,
Nasal congestion, ~ 5%
• Mask for mouth genomic variations and
Diarrhea, ~ 4%
and nose Anosmia underlying diseases
Incubation Symptomatic Resolution

infection
Disease severity
Severe Critical
Acute respiratory dis-

Asymptomatic Fever, cough tress syndrome, sepsis, Recovery or death
organ failure
Milde -
Moderate
Viral response
Host immune response
Time
Fig. 8.8 Transmission and clinical course of SARS-CoV-2 infections. The virus is spread
primarily via aerosols and contaminated surfaces. Patients exhibit fever and dry cough. In addi-
tion, they may have difficulty in breathing, muscle and/or joint pain, headache/dizziness, diarrhea,
nausea and the coughing up of blood. Severe cases of COVID-19 cases progress to acute respiratory
distress syndrome. More details are provided in the text
8.4 COVID-19 153
immune evasive mechanism of SARS-CoV-2. Then pro-inflammatory cytokines are

secreted, neutrophiles and monocytes move into the lung and initiate there a cytokine
storm. The adaptive immune system lacks its initial priming and the vial burden gets
very high. This leads to increased vascular permeability and causes respiratory
failure, which is the main reason for the fatal cases of COVID-19. In some persons
a chronic form of COVID-19 develops, referred to as “long-COVID-19”, showing a
mild or severe form depending on the severity of the original disease. The symptoms
of long COVID-19 are fatigue, reduced lung capacity and inability to fully exercise
or work.
The global effort in vaccination against SARS-CoV-2 aims to bring the COVID-
19 pandemic under control. However, it can be expected that similar to different
influenza A virus strains (Sect. 8.3) SARS-CoV-2 will stay endemic with humans.
New variants of SARS-CoV-2 (Box 8.9) may emerge both by antigenic drift, i.e., by
point mutations of the viral genome, as well as by antigenic shift due assortment with
coronaviruses from other species. This may make yearly vaccinations necessary of
at least of the endangered part of the population.
Box 8.9: SARS-CoV-2 variants In contrast to stable RNA viruses, such

as measles and polioviruses, that exhibit antigenic stability and unchanging
serotypes over periods of many years, within less than 2 years SARS-CoV-2
showed already a number of variants. They are characterized by their transmis-
sibility, disease severity and ability to evade humoral immunity. For example,
the Alpha and Delta variants of SARS-CoV-2 show increased transmissibility
and greater disease severity, while the Beta, Gamma and in particular the
Omicron variant display increased transmissibility, since they evade humoral
immunity and cause re-infections. For example, the point mutation D614G in
the spike protein increased infectivity. Therefore, it cannot be expected that
vaccination against SARS-CoV-2 will last for the rest of the individual’s
life, as it is the case for vaccines of measles and polio.
The most potent vaccines against SARS-CoV-2 are based on mRNA encoding
for viral proteins. Importantly, mRNA vaccines can be rapidly developed, since
mutations of emerging virus variants can easily be introduced into the RNA template.
Moreover, there is no need to produce and purify larger amounts of protein antigens.
The mRNA is encapsulated in lipid nanoparticles that facilitate uptake by cells, such
as dendritic cells, and also function as adjuvants.
In addition to the protection of the individual one other goal of vaccination is to
generate herd immunity, i.e., a protection of the whole population. When enough
persons in a population are vaccinated and in parallel the vaccination also prevents the
infection, the transmission of the pathogenic microbe can be interrupted. However,
for highly transmissible pathogens, such as measles and COVID-19, the vaccination
rate must be more than 90%.
Further Reading
1. Bartleson JM, Radenkovic D, Covarrubias AJ, Furman D, Winer DA, Verdin E (2021) SARS-
CoV-2, COVID-19 and the aging immune system. Nat Aging 1:769–782
2. Castells MC, Phillips EJ (2021) Maintaining safety with SARS-CoV-2 vaccines. N Engl J Med
384:643–649
3. Fajgenbaum DC, June CH (2020) Cytokine storm. N Engl J Med 383:2255–2273
4. Krammer F, Smith GJD, Fouchier RAM, Peiris M, Kedzierska K, Doherty PC, Palese P, Shaw
ML, Treanor J, Webster RG et al (2018) Influenza. Nat Rev Dis Primers 4:3
5. Lee S, Channappanavar R, Kanneganti TD (2020) Coronaviruses: innate immunity, inflamma-
some activation, inflammatory cell death and cytokines. Trends Immunol 41:1083–1099
6. Morens DM, Fauci AS (2020) Emerging pandemic diseases: how we got to COVID-19. Cell
182:1077–1092
7. Pulendran B, Davis MM (2020) The science and medicine of human immunology. Science 369
8. Schultze JL, Aschenbrenner AC (2021) COVID-19 and the human innate immune system. Cell
184:1671–1692
9. Sette A, Crotty S (2021) Adaptive immunity to SARS-CoV-2 and COVID-19. Cell 184:861–880
10. Virgin HW (2014) The virome in mammalian physiology and disease. Cell 157:142–150
Chapter 9
Tolerance and Transplantation
Immunology
Abstract In this chapter, we will first discuss about the impact and mechanisms of
immunological tolerance, which is differentiated into central and peripheral toler-
ance. Failure of tolerance is a main contributor to autoimmune and allergic disease
(Chap. 10). One major mechanism of peripheral tolerance is the action of Treg cells
that suppress other self-reactive lymphocytes. Then we will discuss the molecular and
cellular basis of transplantation immunology and distinguish between hyperacute,
acute and chronic reactions of the host’s immune system against a graft obtained from
a different person. Finally, we will explain the molecular mechanisms of immuno-
suppressive drugs, which are used for the prevention of graft rejections, and will
discuss their side effects.
Keywords Central tolerance · Peripheral tolerance · Regulatory T cells ·

Transplant rejection · Immunosuppressive drugs · Anti-inflammatory drugs
9.1 Central and Peripheral Tolerance
Adaptive immunity relies on that mature naïve B and T cells that leave their generative
organs, bone marrow and thymus (Sect. 3.2), recognize a wide variety of foreign,
i.e., non-self, antigens (also called immunogens), but do not react with their effector
mechanisms to the own (self) antigens of the individual. Since the generation of
antigen receptors follows random processes of recombination, assembly and editing
of respective gene segments (Sect. 3.3), also BCRs and TCRs are created that show
high affinity for the individual’s own cells. When such self-reactive B and T cells
would reach the periphery, they may cause autoimmune diseases. This is prevented
by mechanisms of immunological tolerance. The non-reactivity to self is referred
to as tolerance and distinguished into central and peripheral tolerance. Central
tolerance (Fig. 9.1, left) happens in the bone marrow and thymus, where immature B
and T cells are exposed to self-antigens (also called tolerogens), in order to test the
functionality and affinity of their BCRs and TCRs, respectively. However, central
tolerance is only effective to 60–70% of self-reactive T cells. Therefore, there is in
addition peripheral tolerance (Fig. 9.1, right) taking place in secondary lymphoid
organs, such as the spleen, lymph nodes and mucosal lymphoid tissues. Importantly,
https://doi.org/10.1007/978-3-031-04025-2_9
156 9 Tolerance and Transplantation Immunology
Central tolerance: Peripheral tolerance:

Generative lymphoid organs (thymus, bone marrow) Peripheral tissues
Recognition of Development of regu- Recognition of Suppression

self antigen latory T cells self antigen
(CD4+ T cells only)
Lymphoid
precursors Change in antigen
receptors
(editing in B cells)
Immature Mature Apoptosis
lymphocytes lymphocytes (deletion)
Apoptosis
Anergy
(deletion)
Fig. 9.1 Central and peripheral tolerance. Central tolerance (left) is a control mechanism taking
place in primary lymphoid organs. Immature B cells in the bone marrow as well as immature T
cells in the thymus are selected not to recognize self-antigens. In case of high affinity antigen
recognition, the respective lymphocytes are either eliminated by apoptosis, the specificity of their
antigen receptor is changed (in case of B cells) or their differentiation to Treg cells is induced (in
case of CD4+ T cells). In peripheral tolerance (right) self-reactive mature lymphocytes that escaped
central tolerance may either sent to anergy, deleted by apoptosis or suppressed by Treg cells
whether an antigen acts as an immunogen or a tolerogen, depends on whether it is

recognized during lymphocyte maturation in the bone marrow and thymus or in the
periphery with or without support of cells of innate immunity (Box 9.1).
Box 9.1: Immunogenicity versus tolerogenicity The immunogenicity or

tolerogenicity of a protein antigen, e.g., for the efficient production of poly-
clonal antibodies after a vaccination, depends on a number of parameters.
Short-lived antigens favor an immune response, while their persistence over
longer time leads to tolerance, as it is the case with the antigens of commensal
bacteria of the microbiome. Furthermore, subcutaneous application of an
antigen like a vaccine is more efficient as its uptake in the mucosa or intra-
venous injection. Vaccines are often applied in combination with an adjuvant,
such as aluminum salts, paraffin oils or dead bacteria, in order to increase
9.1 Central and Peripheral Tolerance 157
the immunogenicity, while in their absence co-stimulators are missing and a

tolerogenic response is favored. Finally, the presentation of an antigen, such
as a self-antigen, via activated dendritic cells carrying a high amount of co-
stimulators is very immunogenic, while inactive antigen-presenting cells have
far lower levels of co-stimulators and rather cause a tolerogenic response.
In the thymus, specialized dendritic cells present via MHC-I and MHC-II proteins
a set of ubiquitous, widely disseminated self-peptides that either are expressed in the
thymus or brought there via the blood to immature CD8+ and CD4+ T cells. In
contrast, immunogens normally do not reach immune generative organs. Moreover,
self-peptides deriving from tissue-specific proteins, such as INS being produced
exclusively by β-cells of the pancreas, are often not presented to immature lympho-
cytes. This increases the risk that B and T cells with high affinity for tissue-restricted
self-antigens escape the control mechanisms of central tolerance. Furthermore,
medullary thymic epithelial cells test the reactivity of immature T cells to MHC
proteins. These cells express the transcription factor AIRE (autoimmune regulator)
that has the unusual property to induce the expression of thousands of proteins that
are normally tissue-restricted, so that peptides derived from these proteins can be
displayed to maturating T cells. Importantly, mutations of the AIRE gene lead to
the multiorgan autoimmune disease APS1 (autoimmune polyglandular syndrome
type 1).
When T cells show via their TCR high affinity for either the presented self-
peptides or MHC proteins, they are eliminated by the induction of the mitochondrial
pathway of apoptosis (Sect. 6.4). This process is referred to as deletion or negative
selection (Fig. 9.1, left). In net effect only 1% of all cells survive the process of T
cell maturation and are allowed to circulate in the periphery. Self-reactive B cells
with strong affinity for self-antigens are also eliminated by apoptosis, while cells
with a BCR of intermediate affinity for self-antigen may go into a state of anergy
(Box 8.4). In anergy the signal transduction within the respective B and T cells is
working suboptimal, e.g., due to:
• a block of antigen receptor signaling
• the lack of active co-stimulatory receptors
• no involvement of innate immune cells
• the presentation of inhibitory receptors, such as CTLA4 and PDCD1 (Sect. 6.1).
Thus, the cells become unresponsive to self-antigens, i.e., they do not develop
effector mechanisms after antigen contact but neither get killed. Alternatively, self-
reactive BCRs may get edited by recombination of the gene encoding for the light
chain (Sect. 3.3) and get tested again for self-reactivity. BCR editing is a common
process and applied to 25–50% of all mature B cells released into the periphery.
Also in peripheral tolerance (Fig. 9.1, right) self-reactive lymphocytes are either
eliminated by apoptosis or anergy is induced to them. This is in particular important
for B and T cells that recognize tissue-specific self-antigens that were not presented
during central tolerance.
In central tolerance, some self-reactive CD4+ T cells differentiate into Treg cells
(Fig. 9.2). This involves the expression of the transcription factor FOXP3 (forkhead
box P3) and anti-apoptotic survival signals. Moreover, the cytokines IL2 and TGFβ
are important for the generation, survival and function of the cells. Treg cells act not
only in lymphoid organs but throughout the whole body. They have a central role
in the prevention of autoimmune diseases (Chap. 10) via the active suppression of
adaptive immune responses either by inhibiting dendritic cells in activating T cells
or through direct suppression of T cell activation. One mechanism of action is the
expression of inhibitory receptors, such as CTLA4 and PDCD1, which due to their
higher affinity to CD80 and CD86 molecules on the surface of antigen-presenting
cells outcompete co-stimulatory receptors, such as CD28, on self-reactive T cells.
While Treg cells inhibit via CTLA4 primarily the initial activation of T cells in
secondary lymphoid organs, they terminate through PDCD1 the response of cytotoxic
T cells in peripheral tissues. Furthermore, Treg cells produce the immunosuppressive
Thymus Lymph node
Regulatory
T cells
FOXP3
FOXP3
Recognition of self Recognition of antigen

antigen in thymus in peripheral tissues
Inhibition of Inhibition of
T cell responses other cells
NK cell
Dendritic cell Naïve T cell B cell
Fig. 9.2 Generation and function of Treg cells. Treg cells primarily develop in the thymus (and
sometimes also in peripheral lymphoid organs) due to the overexpression of the transcription
factor FOXP3. In peripheral tissues, Treg cells suppress the effector functions of other self-reactive
lymphocytes
9.2 Graft Rejection 159
cytokines TGFβ and IL10 (Box 9.2). Moreover, Treg cells suppress B cell activation
and inhibit the proliferation and differentiation of NK cells. Importantly, Treg cells
also prevent strong immune responses to the microbiome (Sect. 7.4) as well as of
pregnant females to their fetus.
Box 9.2: Immunosuppressive cytokines TGFβ and IL10 are important

immunosuppressive cytokines that are produced by many different cell types
including Treg cells. There are three different genes encoding for TGFβ1,
TGFβ2 and TGFβ3, but in immune cells, such as Treg cells and macrophages,
TGFβ1 is dominant. The cytokine suppresses the activation of inflammatory
M1-type macrophages, neutrophiles and endothelial cells, but promotes the
production of tissue regenerative M2-type macrophages (Sect. 2.3). In this
way, TGFβ1 acts anti-inflammatory. Moreover, the cytokine stimulates the
generation of TH 17 cells and Treg cells but inhibits TH 1 and TH 2 development
(Sect. 6.3). Moreover, TGFβ1 is critical for antibody isotype switching to IgAs
(Sect. 4.3). The cytokine IL10 is produced by macrophages, dendritic cells,
Treg cells, TH 1 cells and TH 2 cells and acts as a negative feedback regulator
of activated macrophages and dendritic cells. In these cells, IL10 inhibits the
production of the pro-inflammatory cytokine IL12 and reduces IFNγ secretion
(Box 2.5). The latter has a general suppressive effect on innate and adap-
tive immunity. Moreover, IL10 inhibits in macrophages and dendritic cells the
expression of MHC-II proteins and co-stimulatory molecules. This causes the
inhibition of T cell activation.
9.2 Graft Rejection
During the last 50 years the transplantation of cells, tissues or whole organs (collec-
tively referred to as grafts) from one individual (the donor) to a different person
(the host) became medical routine. This includes also the transfer of blood cells or
plasma, referred to as transfusion. Worldwide millions of people have benefited from
this man-made procedure of replacing blood cells, HSCs (Box 9.3), kidneys, livers,
hearts and other organs. The technical aspects of graft transplantation surgeries are
well under control, but the responses of the host’s immune system against the
graft are still a major issue limiting the success of the transplantation.
Box 9.3: HSC transplantation The transplantation of HSCs, often also

called bone marrow transplantation, is used to treat defects in hematopoi-
etic cell lineages, such as X-SCID (X-linked severe combined immunode-
ficiency disease), beta-thalassemia major and sickle cell disease, as well as
blood cancers like leukemias. Moreover, in the past years, HSC transplanta-
tion became also a very effective treatment option for some inherited metabolic
diseases like mucopolysaccharidosis. This treatment is only an option for severe
cases, where patients totally lack specific enzymes. By replacing the HSC pool
of the patient the amount of enzymes produced in the new blood cells is suffi-
cient to compensate the lacking enzyme activity. During HSC transplantation,
first the host’s own HSCs are destroyed by chemotherapy, immunotherapy or
irradiation and then they are replaced by HSCs from a healthy donor. Thus,
the host obtains a new immune system. A serve complication of HSC trans-
plantation can be GvHD (graft-versus-host disease), which is based on donor’s
mature T cells that recognize alloantigens of the host, such as minor histocom-
patibility antigens. Acute GvHD is characterized by the death of epithelial cells
in skin, liver and gastrointestinal tract, which can be fatal when it occurs exten-
sively in the first 100 days after HSC transplantation. In contrast, chronic GvHD
causes fibrosis and atrophy of one or multiple organs but no cell death and may
lead to complete dysfunction of the affected organ. In order to prevent possible
GvHD, immunosuppressive drugs (Sect. 9.3) are given for prophylaxis.
For ethical and practical reasons, the principles of graft rejection have been studied
primarily in mice. In general, mice, rats, chicken, fruit flies and other animal species
are often used as model organisms in immunology (Box 9.4). Members of inbred
mice strains are genetically identically to each other, i.e., they carry the same genomic
variants also in their HLA gene locus (Sect. 5.2). The principles of transplantation
immunology are demonstrated at the example of acceptance or rejection of skin
grafts in comparison of two different mice strains and their crossed F1 generation:
• a transplantation between two genetically identical [i.e., syngeneic (Box 9.5)]
individuals (inbred mice or monozygotic humans) does not cause any graft
rejection (Fig. 9.3a)
• a transplantation between two genetically different (i.e., allogeneic) individuals
causes after 10–14 days an inflammatory reaction referred to as graft rejection,
i.e., the transplanted skin undergoes necrosis and falls off (Fig. 9.3b)
• a transplantation from a homozygous individual to an individual that is heterozy-
gous (in mice from the parent strain to the F1 generation of a breed of two different
strains) does not cause any graft rejection (Fig. 9.3c)
• a transplantation from an heterozygous individual to a homozygous person (in
mice from F1 generation of a breed of two different strains to the parent strain)
causes a graft rejection after 10–14 days (Fig. 9.3d).
Donor Host Graft rejection
a Skin graft
Strain A (MHC Strain A (MHC

Syngeneic graft is not
rejected
b
Strain B (MHCb) Strain A (MHC

Fully allogeneic graft is
rejected
Strain B (MHCb) Strain A x B (MHC /b) Graft from inbred parental

strain is not rejected by
F1 hybrid
Strain A x B, F1 (MHC /b) Strain A (MHC Graft from F1 hybrid is

rejected by inbred
parental strain
Fig. 9.3 Genetic principles of graft rejection. The inbred mouse strains A and B differ in the
variants of their HLA genes encoding for MHC proteins. Syngeneic grafts are not rejected (a), while
allografts are always rejected (b). Grafts from homozygous A or B mice are not rejected by A/B
heterozygous mice (c), but grafts from A/B heterozygous mice are rejected by homozygous A or B
mice (d)
Box 9.4: Model organisms in immunology Many fundamental findings in

immunology were obtained in model organisms. For example:
• the clonal selection theory was validated in rats
• the role of the thymus in generating T cells was found in mice
• B and T cells as organizing principles of adaptive immunity were discovered

in chickens
• the role of pattern-recognition receptors, such as TLRs, in innate immunity
was described first in mice and fruit flies
• the impact of dendritic cells as central coordinators of immune responses
was observed first in mice.
However, humans diverted from the closest of these model organisms, the
rodents, some 100 million years ago. This questions, whether all observations
made in rodents can be transferred 1:1 to humans. This is of particular impor-
tance for the testing of therapeutics, such as monoclonal antibodies (Sect. 11.3).
Therefore, it is important to verify findings from model organisms in a human
setting. In fact, first experiments in immunology, such as the vaccination trials
by Jenner in 1796, had been done in humans.
Box 9.5: Transplantation vocabulary When a graft is transplanted within

the same individual, e.g., in case of replacement of burned skin, it is referred
to as autologous graft. The transplantation between genetically identical indi-
viduals, such as between monozygotic twins or within inbred strains, is called
syngeneic graft. The term allogeneic graft (or allograft) applies, when donor
and host are genetically different and xenogeneic graft (or xenograft) is used,
when donor and host are from different species. Accordingly, foreign molecules
in grafts are called alloantigens and xenoantigens. Furthermore, lympho-
cytes and antibodies reacting with them are referred to as alloreactive and
xenoreactive.
The onset of a graft rejection takes 10–14 days, which indicates that the process
is mediated by adaptive immunity that needs time for clonal expansion after the first
contact with the foreign tissue. However, the repeated exposure of an individual with
a graft from the same donor leads to a more rapid reaction, i.e., in transplantation
immunology there is a memory effect that is very comparable to repeated infections
with the same type of microbe. In contrast, the reaction to the exposure with a graft
from a different donor again takes 10–14 days, i.e., there is immunological specificity
as observed with infectious diseases (Chaps. 7 and 8). Importantly, rapid response to
a graft can also be obtained, when lymphocytes from a pre-exposed individual were
transferred before transplantation to the naïve host.
The term “major histocompatibility complex” already indicates that MHC proteins
being expressed in grafts are the major alloantigens responsible for rejection reac-
tions. MHC proteins present self-peptides, i.e., alloantigens, of the donor to T cells
of the host (Fig. 9.4, left). Moreover, since the HLA gene locus, which encodes for
the different MHC proteins, is highly polymorphic (Sect. 5.2), MHC proteins of the
a b c
Foreign peptide Donor peptide Donor peptide

Self MHC Donor MHC Donor MHC
Donor
Host
Fig. 9.4 Peptide-laden MHC proteins as alloantigens. Alloantigens of the direct recognition of
MHC-peptide complexes by the TCR of a host’s T cell are either the self-peptide of the donor via
host’s own MHC protein (left), the MHC protein of the donor (center) or both (right)
donor are also recognized as alloantigens (Fig. 9.4, center). Often the host’s T cells
recognize both the self-peptide and the MHC proteins of the donor as alloantigens
(Fig. 9.4, right). Other proteins that differ between donor and host may act as addi-
tional alloantigens, but compared with MHC proteins their contribution is minor.
Therefore, they are called minor histocompatibility antigens.
Allogeneic MHC proteins of the donor are presented either directly or indirectly to
T cells of the host. The direct alloantigen presentation happens within lymph nodes
of the host that are located close to the graft (Fig. 9.5a). Tissue-resident dendritic
cells of the donor, which were transplanted together with the organ, move to these
lymph nodes. There they interact via their MHC-I and MHC-II proteins with TCRs
of host’s CD8+ and CD4+ T cells, which then proliferate and differentiate to TH cells
and cytotoxic T cells, respectively. Thus, antigen-presenting cells of the host are not
involved in this process. The cytotoxic T cells are further stimulated by cytokines,
which were secreted by TH cells, and initiate apoptosis to parenchymal cells of the
graft, i.e., the cells of the graft are killed by a direct mechanism. This reaction is very
strong, since a significant number of the host’s T cells (1–10% of all) recognize the
MHC proteins of the donor. MHC proteins are very immunodominant, because they
Direct alloantigen recognition

a Alloreactive
CD8+ T cell
Donor Cytokines
dendritic
cell
Alloreactive Cytotoxic T cell Direct

CD4+ T cell recognizes donor MHC killing of
on graft tissue cell graft cells
T cells recognize donor MHC
and bound peptides on DC from graft
Indirect alloantigen recognition

b +
T cell recognizes
donor MHC peptide bound to
host MHC on host B cell
antibodies
Self MHC
Host
Allogeneic host’s antigen-
B cell
MHC presenting cell
Antibody-mediated
injury to graft cells
Alloreactive
CD4+ T cell
Host dendritic cells CD4+ T cell regonizes

takes up and processes donor MHC peptide
donor MHC molecules bound to host MHC
cytokines
on host
dendritic cells
+
T cell recognizes
donor MHC peptide bound to
host MHC on host injury to graft
marcophage in graft
Fig. 9.5 Direct and indirect alloantigen recognition. Direct alloantigen recognition involves
dendritic cells of the donor, which interact with MHC-I and MHC-II proteins of the host’s cytotoxic
T cells and TH cells (a). The cytotoxic T cells then recognize and directly kill MHC-I protein
carrying cells of the graft. Indirect alloantigen recognition happens when host’s dendritic cells take
up MHC proteins of the donor, process them and present the resulting peptides on their own MHC-
II proteins (b). This activates TH cells that stimulate B cells to produce MHC-specific antibodies
and macrophages to secrete pro-inflammatory cytokines. The cells of the graft are then harmed by
antibody-mediated phagocytosis and complement protein recruitment as well as by inflammatory
reactions
bind in each cell thousands of different self-peptides, i.e., they offer thousands of
different epitopes to be recognized by T cells. For comparison, during a virus infection
only 1 of 105 –106 T cells are specifically recognize peptides originating from the
microbe. Many of the host’s responding T cells are memory cells that react faster and
stronger than naïve T cells. These memory T cells derive from previous exposure
with foreign environmental or microbial proteins and cross-react with allogeneic
MHC proteins.
During indirect alloantigen recognition dendritic cells of the host migrate to
the graft and take up membrane fragments of donor’s cells including MHC proteins
(Fig. 9.5b). The host’s dendritic cells then move back to lymph nodes and present
via their MHC-II proteins peptides originating from the donor’s MHC proteins to
host’s CD4+ T cells. Since the donor’s MHC proteins have a different amino acid
sequence and structure than the host’s MHC proteins their processed peptides acti-
vate CD4+ T cell clones of the host. After proliferation and differentiation these
TH cells stimulate host’s B cells to produce antibodies specific for donor’s MHC
proteins as well as host’s macrophages to secrete pro-inflammatory cytokines. In
this way, the cells of the graft are injured through antibody-triggered activation of
neutrophils, macrophages and NK cells as well as of complement proteins that result
in a strong inflammatory response. Since endothelial cells express MHC proteins,
alloantibody-mediated damage concerns in particular the graft’s vasculature. The
indirect alloantigen recognition and the following effector reactions resemble those
of proteins originating from microbes. Thus, the immune response to man-made
transplantations is based on established mechanisms used for the fight against
microbes.
Based on histopathology, graft rejections are classified as hyperacute, acute and
chronic. Hyperacute rejections can happen within minutes to hours after surgery and
manifest by thrombotic occlusion of the graft’s vasculature. This reaction is based on
pre-existing IgM-type antibodies that circulate in the host. The alloreactive antibodies
bind to endothelial cells of the graft and activate via complement proteins platelets
that aggregate to form an intravascular thrombus, which harms the graft by ischemia.
Acute rejections start a few days to weeks after transplantation and are mediated by
alloreactive T cells and antibodies that cause injury to the parenchyma and vasculature
of the graft. The mechanisms behind acute rejections are primarily those of the direct
alloantigen recognition pathway (Fig. 9.5a), i.e., cytotoxic T cells kill parenchymal
and endothelial cells of the graft and TH 1 cells initiate via secretion of cytokines,
such as IFNγ and TNF, an inflammatory response. In addition, alloantibodies that are
created via the indirect alloantigen recognition pathway (Fig. 9.5b) cause endothelial
injury and intravascular thrombosis of the graft. Finally, chronic rejections develop
during months or years after transplantation and result in arterial occlusions due
to the stimulation proliferation of intimal smooth muscle cells by IFNγ and other
cytokines. This results in reduced blood flow to the graft parenchyma, which over
time is replaced by non-functional fibrous tissue. In part this interstitial fibrosis is a
repair response to parenchymal cell damage.
A major strategy to reduce the immunogenicity of a graft is to selected donors
that have minimal genetic differences, in particular concerning their MHC proteins.
Before a transplantation, by HLA typing the alleles of HLA genes (primarily that
of the highly polymorphic HLA-A, HLA-B and HLA-DR genes) are determined by
PCR (polymerase chain reaction), targeted sequencing or gene expression analysis
for both the donor and the host. The less mismatches in HLA alleles are found, the
lower is the risk of an acute graft rejection. Moreover, in order to avoid hyperacute
graft rejections, hosts are routinely screened using flow cytometry for the presence
of preformed antibodies that may have been produced due to previous pregnancies,
transfusions or transplantations.
Blood transfusion is one of the oldest and most common form of transplanta-
tion, which is primarily used to replace blood lost by hemorrhage. For a successful
transfusion the blood groups of donor and host need to be compatible. Blood group
antigens differ in the carbohydrates that are added by specific glycosyltransferases
to glycoproteins on the surface of erythrocytes. The 0 allele gene product has no
enzymatic activity, while in the A allele N-acetylgalactosamine and in the B allele
N-acetylglucosamine is added (Fig. 9.6). In AB heterozygotes both types of carbohy-
drates are added. Individuals, who do not express a particular blood group antigen,
produce natural IgM antibodies against that antigen, e.g., persons of blood group
Group A Group B Group AB Group 0

Type A Type B Type AB Type 0
Red blood
cell type
Anti-B Anti-A Anti-A and anti-B
Antibodies
None
present
A antigen B antigen A and B antigen

None
Antigens
present
N-Acetylgalactosamine N-Acetylglucosamine Fucose Galactose
Fig. 9.6 AB0 blood groups. The antigens of blood groups A, B and 0 are indicated carbohydrates on
the surface of erythrocytes added by specific glycosyltransferases. The blood group of an individual
indicates against which antigen they are tolerant and to which they produce natural antibodies that
react with other blood group antigens
9.3 Immunosuppression 167
A have antibodies against blood group B. Accordingly, AB individuals can tolerate

transfusions from all potential donors, while 0 individuals can tolerate transfusions
only from 0 donors but can provide blood to all possible hosts. In case of a contact
of blood from two different blood groups, antibody binding to erythrocytes activates
complement proteins and life-threatening immediate hemolytic reactions are initiated
causing kidney failure, a cytokine storm and excessive bleeding. In order to prevent
such reactions, during routine blood typing erythrocytes of a given individual are
mixed with standardized sera containing anti-A or anti-B antibodies. Agglutination
is observed, when the person expresses respective blood group antigens.
9.3 Immunosuppression
Allograft hosts that have a functional immune system are expected to show graft
rejection reactions. Therefore, in addition to select a donor that is identical to the host
in most HLA alleles, immunosuppressive drugs are routinely applied as a prophylaxis.
Thus, the host’s immune system is substantially weakened over a prolonged
period, in order to prevent graft rejection. This treatment carries substantial risks
of serious side effects, since in long-term the drug treatment rather results in immune
dysfunction than in desired tolerance against the graft.
Since T cells are the main mediators of graft rejection (Sect. 9.2) as well as the
key cell types to be affected for obtaining immunological tolerance (Sect. 9.1), a
modulation of their reaction and function is the first target of immunosuppressive
drugs. The molecular understanding of T cell signaling indicates that the transcrip-
tion factors NFκB, NFAT and AP1 may be prime targets for immunosuppressive
drugs (Sect. 6.1). However, NFκB and in particular AP1 have key functions in a
large variety of cell types suggesting that their functional modulation by drugs will
result in substantial side effects. Therefore, the lymphocyte-specific transcription
factor NFAT is the preferential target. Since cytosolic NFAT is activated by the
Ca2 - and calmodulin-dependent serine/threonine protein phosphatase calcineurin,
the enzyme is a promising target for T cell-specific immunosuppressive drugs. The
natural product cyclosporin (a peptide isolated from fungi) was introduced some
40 years ago in transplantation medicine and since then significantly reduced the
risk for graft rejections. Cyclosporin binds with high affinity to the cytosolic protein
PPIA (peptidylprolyl isomerase A, also called cyclophilin) that inhibits the enzy-
matic activity of calcineurin (Fig. 9.7). In this way, the translocation of NFAT to
the nucleus is prevented and the induction of its major target gene IL2 is majorly
reduced. Due to the key role of IL2 in the proliferation and differentiation of T
cells, the functionality of alloreactive TH cells and cytotoxic T cells is significantly
reduced. Tacrolimus (FK506) is also a natural product that binds to peptidylprolyl
isomerases of the FKBP (FK506 binding protein) family. It also inhibits calcineurin,
i.e., it acts very comparable to cyclosporin but has better efficacy and safety.
Another natural immunosuppressive drug is rapamycin, which like FK506 binds
to FKBPs but functions via the inhibition of the cytosolic serine/threonine protein
Antigen-presenting cell
Anti-TCR
(OCT3, Thymo-
globulin)
CD80
MHC-II
IL2
Anti-IL2R
IL2
CTLA4-Ig
TCR
IL2RA
CD28
Rapamycin Cyclosporin
Tacrolimus
MTOR Calcineurin
Azathioprine Co-stimulation
Mycophenolate
IL2 production
Proliferation
T cell
Fig. 9.7 Common immunosuppressive drugs. Different categories of immunosuppressive drugs

and their molecular targets are schematically indicated. More details are provided in the text
kinase MTOR (mechanistic target of rapamycin kinase). MTOR is critical for the
translation of proteins that promote cell survival and proliferation, i.e., rapamycin
inhibits T cell proliferation (Fig. 9.7). Since MTOR is widely expressed, rapamycin
affects also other tissues and cell types, but compared to activated T cells all other
cells are growing slowly or not at all. A few signal transduction pathways, such as
those of TCR/CD3, CD28 and IL2RA (CD25), promote the proliferation of T cells
and act via MTOR. Thus, inhibitors of these membrane-receptor based pathways,
such as specific monoclonal antibodies (Sect. 11.3), act as immunosuppressive drugs.
Furthermore, metabolic toxins, such as azathioprine and mycophenolates, inhibit the
proliferation of immature and mature lymphocytes. These anti-metabolites inhibit
key metabolic pathways, such as the de novo synthesis of guanine nucleotides by
mycophenolates. Anti-inflammatory drugs, in particular glucocorticoids, are often
used, in order to reduce the inflammatory reaction associated with reactions of the
Further Reading 169
immune system to allografts (Sect. 9.2). Glucocorticoids act as ligands of the nuclear
receptor GR (glucocorticoid receptor) and inhibit in macrophages the expression of
pro-inflammatory cytokines, such as TNF and IL1, and the production of inflamma-
tory mediators, such as prostaglandins, ROS and NO. In this way, the damage to the
graft is reduced due to lower numbers of recruited phagocytes causing inflammation.
Immunosuppressive and anti-inflammatory therapy largely prevents acute graft
rejection. However, immunosuppressive drugs are far less effective in the prevention
of chronic graft rejections. Since immunosuppression reduces the function not only
of alloreactive T cells but of all TH cells and cytotoxic T cells, the risk of the graft host
for infections and neoplasia substantially increases (Sect. 11.1). For example, latent
viruses, such as CMV, HSV, VZV and EBV (Sect. 8.2), are often reactivated due
to immunosuppression, so that anti-viral prophylaxis is recommended. Moreover,
the risk for a number of opportunistic infections, e.g., with fungi, is significantly
increased. In addition, immunosuppressive drugs can act toxically to non-immune
cells, which limits their application. Thus, new generations of more specific and less
toxic drugs need to be developed.
The final goal of a post-transplantation therapy is the development of toler-
ance of the host’s immune system against the graft. This would not only reduce
the risk of side effects of immunosuppressive drugs but also avoids chronic graft
rejections. This may be achieved by a blockade of co-stimulation via specific mono-
clonal antibodies or through adoptive therapy (Sect. 11.4) with donor-specific Treg
cells cultured ex vivo.
Further Reading
1. Bluestone JA, Anderson M (2020) Tolerance in the age of immunotherapy. N Engl J Med
383:1156–1166
2. ElTanbouly MA, Noelle RJ (2021) Rethinking peripheral T cell tolerance: checkpoints across a
T cell’s journey. Nat Rev Immunol 21:257–267
3. Loupy A, Lefaucheur C (2018) Antibody-mediated rejection of solid-organ allografts. N Engl J
Med 379:1150–1160
4. Yang JY, Sarwal MM (2017) Transplant genetics and genomics. Nat Rev Genet 18:309–326
5. Zeiser R, Blazar BR (2017) Pathophysiology of chronic graft-versus-host disease and therapeutic
targets. N Engl J Med 377:2565–2579
Chapter 10
Immunological Hypersensitivities:
Allergy and Autoimmunity
Abstract In this chapter, we will discuss about different types of hypersensitivi-

ties of the immune system, which are either mediated primarily by IgEs and TH 2
cells (type I), IgGs and IgMs (type II), immune complexes (type III) and T cells
(type IV). These diseases are caused by the failure of mechanisms responsible for
maintaining self-tolerance in B cells, T cells or both. We will outline the effector
mechanisms, responsible for tissue injury and disease in the context of these hyper-
sensitivities, which are based on genetic susceptibility and environmental triggers,
such as infections. While type I hypersensitivity causes different forms of allergy,
type II-IV hypersensitivities are the basis for distinct autoimmune diseases. Organ-
specific autoimmune diseases, such as type 1 diabetes and multiple sclerosis are
distinguished from those that are systemic, such as rheumatoid arthritis and SLE.
We will summarize these principles in the equilibrium model of immunity.
Keywords Immunological hypersensitivities · Allergy · IgEs · Autoimmune

diseases · SLE · Multiple sclerosis · Rheumatoid arthritis · Type 1 diabetes ·
Genetic susceptibility · Equilibrium model of immunity
10.1 Classification of Hypersensitivities
The effector mechanisms of immunity are very important for the efficient elimina-
tion of pathogenic microbes without creating major injuries to host tissues (Chaps. 7
and 8). Moreover, the immune system has an essential role in the development and
maintenance of tissues, such as wound healing after a trauma (Sect. 2.1). For the
latter reason, many common disorders, such as atherosclerosis, type 2 diabetes,
Alzheimer’s disease and cancer, are associated with inflammation that is initially
meant for supporting the regeneration of disturbed tissues but often gets chronic and
causes more harm than benefit, such as the support of tumor growth (Sect. 11.1). The
main role of immune cells is to keep an individuum in homeostasis by dynam-
ically responding to any kind of disturbance. For example, the ability of CD4+
T cells to differentiate into either TH 1, TH 2, TH 17 and Treg cells and to perform
specific effector responses depending on which type of microbe has invaded the
https://doi.org/10.1007/978-3-031-04025-2_10
172 10 Immunological Hypersensitivities: Allergy and Autoimmunity
body (Sect. 6.3), demonstrates why this part of immunity is referred to as adap-
tive. Moreover, also the variation of the relative numbers of cell types of the innate
immune system indicates adaptive responses. This is exemplified by macrophages
that respond, depending on the physiological situation, as M1-type and the production
of pro-inflammatory cytokines or as M2-type and the secretion of anti-inflammatory
cytokines. Thus, a functional immune system should provide an optimal response
in the continuum between tolerance and inactivity at one end and their over-
reactions that may create a cytokine storm, a septic shock or the destruction
functional cells of the host at the other end (Fig. 10.1).
The responses of innate and adaptive immune cells are in most cases interde-
pendent. This has the advantage that a possible disturbance of the system can be
internally corrected, but bears also the risk that a major dysregulation can cause a
cascade of multiple dysfunctions. Therefore, there are a number of situations, such
as insufficient control of immune cells, their targeting towards incorrect tissues and
cell type, the modulation of their response by the microbiome (Sect. 7.4) or the cell’s
inappropriate reaction to usually harmless antigens of the environment, that result
in significant tissue injuries and cause so-called hypersensitivity diseases. Based
on the type of immune response and the resulting effector mechanisms causing
the diseases, they are classified into four different types (Table 10.1). Immediate
hypersensitivities (type I) involve IgEs that are specific for environmental anti-
gens, such as grass pollen. In today’s society these diseases are very common and
grouped under the term allergies (Sect. 10.2). Allergies are mediated by IL4, IL5
and IL13 producing TH 2 cells (Sect. 6.3), the natural role of which is the defense
Emergine immune “accommodation” archetypes
Regulate com- Assist tissue Manage tissue Manage tissue Control chronic
mensal sym- Heal wounds
development metabolism repair infection
biosis
Treg / TH17 cells, T cells, Treg cells Neutrophils, Treg cells, Myeloid cells,
dendritic cells macrophages macrophages macrophages TH17 cells, ILCs exhausted T cells
Active nondestructive
immune responses
Tolerance Destruction
Immune Immune elimina-
inactivity tion of host cells
Classical spectrum of immune reactivity
Fig. 10.1 Continuum of immune responses. The indicated varieties of immune responses act in
a continuum between tolerance and host cell destruction. When one or several of these varieties are
dysregulated or dysfunctional, they contribute to immune-related diseases
10.1 Classification of Hypersensitivities 173
Table 10.1 Different types of hypersensitivities

Type of hypersensitivity Main mediators Mechanisms of tissue injury
I IgEs, TH cells Mast cells, eosinophils and their
mediators
II IgMs, IgGs Opsonization and phagocytosis of
cells
III Immune complexes involving Complement- and FcR-mediated
IgMs and IgGs recruitment and activation of
leukocytes
IV CD4+ (TH 1 and TH 17 cells), Cytokine-mediated inflammation
CD8+ (cytotoxic T cells) and macrophage activation; direct
target cell killing
against helminths. In contrast, type II hypersensitivities involve IgGs and IgMs

that specifically recognize self-antigens on the surface of cells or at the extracellular
matrix (Sect. 10.3). In the resulting humoral adaptive immune response (Sect. 4.3)
the antigens are either neutralized or opsonized. In the response to the latter process,
complement proteins are activated, phagocytes are recruited and inflammation is
induced. Also type III hypersensitivities engage IgGs and IgMs but these are parts
of larger antigen complexes that circulate in the blood (Sect. 10.3). This leads to a
systemic response that involves inflammation, thrombosis and blood vessel injury.
Finally, type IV hypersensitivities are mediated by both TH 1 and TH 17 cells as
well as cytotoxic T cells that secrete cytokines and induce inflammation or directly
kill target cells (Sect. 10.4). Types II–IV hypersensitivities cause different forms of
autoimmune diseases, major examples of which are listed in Box 10.1. Thus, hyper-
sensitivities are predominantly based either on self-reactive antibodies or on
self-reactive T cells. However, in many diseases these humoral and cell-mediated
responses act together.
Box 10.1: Major autoimmune diseases In contrast to microbial antigens

self-antigens are persistent, i.e., there is no chance to eliminate them. For this
reason, autoimmune diseases tend to be chronic. In addition, due to amplifica-
tion mechanisms, such perpetuating the immune response through additional
tissue injury, the diseases are often progressive. There are in total some 80
distinct autoimmune diseases that affect with a prevalence of 5–9% the popu-
lation of industrialized countries. The most common autoimmune disease is
psoriasis (2–3%), which causes skin redness, thickening and scales. It is based
on abnormal keratinocyte proliferation in the dermis and epidermis. The main
autoimmune disease of the CNS is multiple sclerosis (0.1%), is characterized
by visual loss, numbness, tingling, paresis and spasticity and caused by destruc-
tion of myelin. Autoimmune diseases of endocrine organs are type 1 diabetes
(0.4%) affecting β-cells of the pancreas as well as Graves’ disease (0.5%) and
Hashimoto’s disease (0.1%) of the thyroid gland. Type 1 diabetes is based

on destruction of INS producing β-cells. Graves’ disease is a hyperthyroidism
that leads to anxiety, irritability, hand tremors, weight loss, enlarged thyroid
gland, bulging eyes and palpitations, which is due to autoantibodies against the
thyrotropin receptor on thyroid cells leading to increased synthesis of thyroid
hormone. In contrast, Hashimoto’s disease comes with fatigue, sensitivity to
cold, puffy face, constipation, pale and dry skin and is based on fibrosis and
atrophy of thyrocytes causing decreased synthesis of thyroid hormone. Autoim-
mune diseases of the gastrointestinal tract are Crohn’s disease (0.2–0.3%),
ulcerative colitis (0.2–0.4%) and celiac disease (0.7%). Common to these
diseases is diarrhea, abdominal pain and discomfort, bloody and loose stools,
fever and fatigue, which are due to transmural inflammation, mucosal inflam-
mation, flattening of villi and elongation of crypts. The main autoimmune
disease of joints is rheumatoid arthritis (0.5–1%) that leads to joint swelling,
morning stiffness and tenderness based on synovial hyperplasia damaging
cartilage and bone. Myasthenia gravis has no high prevalence (0.02%), but
it is still the main autoimmune disease of muscles, which leads to muscle
weakness, dropping of upper eyelid and double vision. The disease is based
on autoantibodies that block acetylcholine receptors in post-synaptic muscle
membranes.
The mechanisms of tissue injury in hypersensitivity diseases are the same

as those that are used to eliminate infectious pathogens. These processes are
primarily mediated by phagocytes, mast cells, T cells and antibodies as well as by
mediators of inflammation. However, in contrast of pathogenic microbes the stimuli
for abnormal immune responses in the context of hypersensitivity diseases, such
as self-antigens, commensal microbes and environmental antigens can often not be
eliminated. For this reason, hypersensitivity diseases, such as allergies (Sect. 10.2)
and autoimmune diseases (Sect. 10.4), are mostly chronic and progressive.
10.2 Immunity of Allergies
Non-microbial environmental antigens, referred to as allergens (Box 10.2), are

detected during their first contact by the BCR of B cells (Fig. 10.2). These B cells
act as antigen-presenting cells that display via MHC-II proteins allergen-derived
peptides to CD4+ T cells, which differentiate then to TH 2 cells (Sect. 6.3). Like
ILC2s (Sect. 2.5) TH 2 cells produce primarily the cytokines IL4, IL5 and IL13. IL4
induces isotype switching of the allergen-specific B cells towards the production of
IgEs (Sect. 4.3). The constant domain of the allergen-specific IgEs are captured with
high affinity by FcεRIs (K d = 0.1 nM) on the surface of mast cells in tissues and
eosinophiles in the circulation, i.e., the cells are sensitized. In this way, both cell
10.2 Immunity of Allergies 175
Immediate hyper-
sensitivity reaction
(minutes after repeat Vasoactive amines,
exposure to allergen) lipid mediators
Activation of TH2 Binding of Activation of

Repeated
cells and stimulation Production IgE to FcεRI mast cell: re-
exposure to
of IgE class switching of IgE on mast cells lease of
allergen
in B cells mediators
IgE
FcεRI Mediators
B cell TH2 cell IgE secreting Mast cell

B cell
Late phase reaction
Cytokines
First exposure
(2 - 4 hours after
Allergen to allergen repeat exposure to
allergen)
Fig. 10.2 Immediate hypersensitivity reactions. Immediate hypersensitivity is initiated by an

allergen that stimulates TH 2 cell response and the production of allergen-specific IgEs. Mast cells
and eosinophiles (not shown) bind via FcεRIs these IgEs and obtain in the way high specificity for
the allergen. After re-exposure to the allergen the cells release within minutes molecules, such as
vasoactive amines and lipid mediators, that cause pathologic reactions. In addition, within 2–4 h
cytokines are released, genes of which are expressed as the result of signal transduction induced by
the activation of FcεRIs
types of the innate immune system gain high affinity and specificity to allergens.
After repeated exposure to the allergen, mast cells and eosinophiles rapidly release
molecules, such as vasoactive amines and lipid mediators, that promote increased
vascular permeability, vasodilation as well as bronchial and visceral smooth muscle
contraction. Thus, allergic reactions occur only in individuals that had been previ-
ously sensitized to the allergen. These reactions are called immediate hypersensi-
tivity and have their origin in the clearance of helminths (Box 10.3). Therefore,
they belong to the most powerful responses of the immune system. In parallel
to the very rapid hypersensitivity, in a so-called late-phase reaction (after 2–4 h)
the secretion of cytokines causes acute inflammation that leads to the accumulation
of eosinophils and neutrophils. This reaction lasts less than 24 h and is primarily
mediated by TH 2 cells.
Box 10.2: Allergens Most allergens are common environmental proteins

derived from animals or plants, such as animal dander, house dust mites or
pollen. A master example of a natural allergen explaining dust allergy is the
cysteine protease peptidase 1, which is found in the feces of mites, i.e., small
insects living in house dust. This enzyme destroys the integrity of the tight junc-
tions between epithelial cells and gains in this way access to subepithelial mast
cells and eosinophils. Thus, peptide fragments of peptidase 1 can be presented
to T cells, the enzyme itself gets easily access to mast cells, the globular protein
is rather stable and soluble and the rather low molecular weight allows diffusion
of the allergen. In addition, also chemicals that bind and modify self-proteins,
such as drugs, can act as self-proteins. The key example of a drug causing
allergy is that of the antibiotic drug penicillin. Penicillins can act as haptens
that bind covalently to lysines of serum proteins and cell surface proteins, i.e.,
penicillins structurally modify proteins so that they are recognized as foreign
by the immune system. Interestingly, penicillins can lead to allergies (type I
hypersensitivities) targeting erythrocytes, leukocytes and platelets with clinical
symptoms like hives, bronchospasm, hypotension and anaphylaxis (Box 10.5)
but also can cause type II and type III hypersensitivities (Sect. 10.3).
Box 10.3: Clearance of helminthes Helminthic parasites are too large to

be eliminated by phagocytosis and are rather resistant against microbicidal
products of neutrophils and macrophages. Therefore, the immune system uses
primarily antibodies, eosinophils and mast cells to kill and expulse the worms.
Major basic protein (MBP) is a cationic protein that is stored in granules within
eosinophiles. When the cells recognize via FcεRIs IgEs that cover the surface
of helminths, they secrete MBP and other aggressive molecules that are toxic
to the worms. Moreover, the IgEs also initiate the degranulation of mast cells,
which induces bronchoconstriction as well as increased intestinal motility, so
that the worms are expulsed from the lung and intestine, respectively.
The most common forms of allergy are atopic dermatitis, hay fever (also called
allergic rhinitis) and asthma (Box 10.4). Depending on the affected tissues, allergies
result in skin rashes, itchy eyes, sinus and nasal congestion, inflamed conjunctiva,
bronchial constriction abdominal pain and diarrhea (Table 10.2). In the extreme case
of an anaphylactic shock (Box 10.5), mast cell-derived molecules restrict airways
up to asphyxia and produce a collapse of the cardiovascular system, which can be
fatal. Allergies normally occur at sites where commonly mast cells are found, such
as connective tissues and under epithelial barriers. Allergic individuals have signif-
icantly higher levels of IgEs compared with non-allergic persons, i.e., the amount
Table 10.2 IgE-mediated forms of allergy

Syndrome Common allergens Route of entry Response Comment
Contact Animal hair, insect Through skin Local increase in Milder symptoms
dermatitis bites, allergy blood flow and
testing vascular
permeability
Allergic Pollen, dust mite Inhalation Edema of nasal Milder symptoms
rhinitis feces mucosa, irritation
of nasal mucosa
Asthma Dander (cat), Inhalation Bronchial Therapy should
pollen, dust mite constriction, start at least in this
feces increased mucus stage
production, airway
inflammation
Food allergy Tree nuts, peanuts, Oral Vomiting, diarrhea, Everyone should
shellfish, milk, itching, uticaria, be aware of
eggs, fish anaphylaxis his/her risk
Systemic Drugs, serum, Intravenous Edema, increased Dangerous!
anaphylaxis venoms, peanuts (directly or vascular
following oral permeability,
absorption into tracheal occlusion,
the blood) circulatory
collapse, death
of circulating IgEs is one biomarker of allergy. Individuals often have different

types of allergies starting with atopic dermatitis in childhood, developing into allergic
rhinitis and later bronchial asthma. Thus, these forms of allergy are closely related,
since they are based on similar mechanisms, such as a local accumulation of mast
cells and TH 2 cells.
Box 10.4: Asthma Asthma is the pulmonary form of immediate hypersensi-

tivity and associated with recurrent reversible airflow obstruction, increased
production of thick mucus and bronchial smooth muscle cell hyperresponsive-
ness. Respiratory viral and bacterial infections are often a predisposing factor
in the development of asthma. Moreover, asthma coexists with chronic obstruc-
tive pulmonary disease, e.g., caused by life-long smoking. However, some 70%
of asthma patients have increased IgE levels, i.e., their response is primarily
due to allergy. Asthma is associated with chronic inflammation of the lungs,
which is due to the late-phase reaction of the allergic response, which leads
to the recruitment of eosinophils and basophils. These cells secrete mediators
like LCT4 that constrict airway smooth muscle. Inhaled corticosteroids that
block the production of inflammatory cytokines are used as therapy (Sect. 9.3).
Furthermore, bronchial smooth muscle cell relaxation is achieved by inhaling

β2-adrenergic agonists.
Box 10.5: Systemic anaphylaxis Anaphylaxis is an allergic reaction due to

the systemic presence of an allergen that reached the blood stream via an
injection like by an insect sting or resorption by intestinal mucosa. This imme-
diate hypersensitivity reaction is associated with edema in many tissues and a
decrease in blood pressure due to vasodilation and vascular leak. Anaphylaxis
is caused by drugs like penicillins (Box 10.2) but also by specific proteins in
peanuts, tree nuts, fish, shellfish, milk, eggs and bee venom. The significant
decrease in blood pressure is called anaphylactic shock and can lead to death.
Anaphylaxis occurs within seconds to an hour after exposure to an allergen.
It is treated by epinephrine injection, which reverses the bronchoconstrictive
and vasodilatory effects of mediators secreted by mast cells and improves the
cardiac output.
Most of its existence homo sapiens was (and in part still is) populated by helminths,
such as hookworms. These parasites are far larger than other types of microbes, such
as bacteria, and therefore require a different form of immune response. The central
event of type 2 immune response is the direct and indirect activation of TH 2 cells
(Fig. 10.3). This is often initiated by epithelial barrier injury, in the context of which
damaged epithelial cells secrete the cytokines IL25, IL33 and TSLP (Sect. 2.5). These
cytokines activate local dendritic cells, which have taken up allergens passing the
epithelium but also other antigens derived by damaged cells, parasites and bacteria,
to move to a neighboring lymph node and to present there the processed allergen
protein as peptides to naïve CD4+ T cells. In parallel, the same cytokines induce in
local mast cells, basophils and ILC2s the early production of the cytokines IL4, IL5
and IL13. This set of cytokines triggers primed CD4+ T cells to differentiate into
TH 2 cells with specificity to different types of presented antigens. Moreover, platelets
at the site of injury secrete the protein DKK1 (Dickkopf WNT signaling pathway
inhibitor 1) that also contributes to the development of TH 2 cells. The TH 2 cells
then move to the tissue site of allergen exposure, secrete themselves IL4, IL5 and
IL13 and activate a number of cell types in their environment, such as macrophages,
eosinophils, epithelial cell and myofibroblasts. This leads to inflammation, eosinophil
recruitment and activation, intestinal mucus production and peristalsis as well as to
fibrosis (Sect. 6.3).
Mast cells, basophils and eosinophils are cells of the innate immune system that all
derive from bone marrow. The cells have similar properties but also differ functionally
in significant ways (Table 10.3). Under the influence of KITLG (KIT ligand) mast
cells mature and found in tissues throughout the body, in particular in connective
Allergens
Parasites
Mucus
Tuft cell
Basophil Platelets Eosinophil

Mast cell Damaged
tissue
IL25, IL33, IL4, IL5,

Translocated blast
ILC2 TSLP IL13
bacteria DKK1
Dendritic naïve CD4+

cell T cell TH2
IL4, IL5
IL13
Fig. 10.3 Initiation of type 2 immune response. The central event in the initiation of type 2
immune response is the development of TH 2 cells. More details are provided in the text
and mucosal tissues. In contrast, eosinophils and basophils are found in circulation
but can be recruited under influences of IL3 and IL5, respectively, to tissue sites of
allergen exposure. Mast cells survive up to months, i.e., far longer than basophils
and eosinophils (days). Furthermore, mast cells and basophils express high levels
of FcεRIs and their granules contain similar molecules, such as histamine, heparin,
chondroitin sulfate and proteases. In contrast, eosinophils express lower amounts
of FcεRIs and their granules release MBP, eosinophil cationic protein, peroxidases,
hydrolases and lysophospholipase.
The binding of cross-linked IgEs to FcεRIs activates at its cytosolic carboxy-
terminus an ITAM (Fig. 10.4). This initiates a signal transduction cascade that
involves binding of the tyrosine kinases SYK and LYN to the ITAMs of FcεRIs,
the activation of PLCG2, which catalyzes phosphatidylinositol bisphosphate break-
down to yield IP3 (inositol trisphosphate) and DAG, which in turn increases Ca2+
levels and PRKC activity. PRKC phosphorylates the myosin light chain protein,
which promotes SNARE (soluble NSF attachment protein receptor) complex forma-
tion and fusion of granules with the membrane, so that their content is released
Table 10.3 Properties of mast cells, basophils and eosinophils

Characteristic Mast cells Basophils Eosinophils
Major site of Bone marrow Bone marrow Bone marrow
maturation precursors mature in
connective tissue and
mucosal tissues
Location of cells Connective tissue and Blood (0.5% of blood Blood (2% of blood
mucosal tissues leukocytes), recruited leukocytes), recruited
into tissues into tissues
Life span Weeks to months Days Days to weeks
Major growth and KITLG, IL3 IL3 IL5
differentiation factors
FcεRI expression High High Low
Major granule Histamine, heparin, Histamine, chondroitin Major basic protein,
contents chondroitin sulfate, sulfate, proteases eosinophil cationic
proteases protein, peroxidases,
hydrolases,
lysophospholipase
to the environment of the mast cell. Furthermore, Ca2+ and MAPKs together acti-
vate the enzyme PLA2 (phospholipase A2) that hydrolyzes membrane phospholipids
to release arachidonic acid, which is converted by the enzymes cyclooxygenase or
lipoxygenase into different lipid mediators. Finally, the expression of the cytokine
genes IL4, IL5, IL6, IL13 and TNF is regulated by the same signal transduction
pathway as in B and T cells (Sects. 4.2 and 6.1) involving the transcription factors
NFAT, NFκB and AP1.
The effector functions of mast cells, basophils and eosinophils on their
surrounding tissues and cell types are mediated by soluble molecules being spread
from the activated cells (Fig. 10.5). Some of these mediators, such as biogenic amines
like histamine and enzymes like tryptase, can be secreted very rapidly, i.e., within
minutes, since they are preformed and stored in granules that after activation of the
cell fuse with the membrane and release their content. In contrast, other mediators,
such as lipids and cytokines, need first to be synthesized. While the formation of lipids
is fast (i.e., takes only minutes), since it requires only a number of enzymatic reac-
tions, does the production of cytokine proteins involve gene activation, transcription
and translation that in total need 2–4 h.
The vasoactive amine histamine is released from granules of activated mast cells
and binds to specific receptors on blood vessel and smooth muscle cells. Histamine
causes the contraction of the endothelial cells, which leads to increased vascular
permeability and leakage of plasma into the tissues. Moreover, the molecule acts
as vascular smooth muscle cell relaxant causing vasodilation and produces the
wheal-and-flare response of immediate hypersensitivity (Box 10.6). Neverthe-
less, the actions of histamine are only short-term, since it is rapidly removed by
specific transporters from the extracellular milieu. The rapid de novo synthesis and
FcRI
FcRγ FcR1β
LYN
FYN P P
P P P P DAG
P P
P SYK
P PIP2
P PRKC
PLCG2 P
GAB2 Pi3K GRB2 P P
IP3
S P
SO P
PLA
Myosin light chain
Arachidonic acid RAS/MAP kinases
phosphorylation,
granule movement
Ca2+
FOS
Target genes
JUN
AP1 SNARE complex
formation,
membrane fusion Granule
Nucleus
PGD , LTC
Cytokines
Secretion
Mast cell plasma membrane

Exocytosis
TNF, others Histamine

PGD , LTE
Lipid mediators Cytokines Granule contents
Fig. 10.4 Signal transduction in mast cells. When cross-linked allergen-bound IgEs are recog-
nized by FcεRIs, signal transduction via the protein tyrosine kinases SYK and LYN is initiated
that via the pathways of MAPK and PLCG2 activate cytokine gene expression. In parallel, Ca2+
and MAPK activate PLA2 that initiates the synthesis of lipid mediators and SNAREs that mediate
degranulation. More details are provided in the text
Vasodilation
Vasoactive amines
(e.g., histamine) Vascular leak
Bronchoconstriction
Lipid mediators
(e.g., PAF, PGD , LTC )
Intestinal hyper-
mobility
Cytokines (e.g., TNF)

Lipid mediators
(e.g., PAF, PGD , LTC )
Enzymes (e.g., eosinophil Tissue damage

peroxidase)
Cationic granule proteins Killing of para-

(e.g., major basic protein, sites and host
eosinophil cationic protein) cells
Enzymes (e.g., eosinophil Tissue damage

peroxidase)
Fig. 10.5 Mediators of immediate hypersensitivity. Mast cells and basophils act via vasoactive
amines and lipids on blood vessels, airways and the gastrointestinal track, cause via cytokines and
lipids inflammation and via enzymes tissue damage. Eosinophils use cationic granule proteins for
killing parasites and cause via enzymes tissue damage. More details are provided in the text
release of lipid mediators affects blood vessels, bronchial smooth muscle and leuko-
cytes, i.e., they act as vasodilators, bronchoconstrictors or inducers of inflamma-
tion. Most of the lipids are derived from the polyunsaturated fatty acid arachidonic
acid, which is metabolized by cyclooxygenases to PGD2 (prostaglandin D2) or
by lipoxygenases to LTC4 (leukotriene C4). Moreover, on the basis of membrane
mast cells and basophils synthesize PAF (platelet-activating factor), which causes
bronchoconstriction, retraction of endothelial cells and relaxation of vascular smooth
muscle. Granules also contain preformed enzymes, such as neutral serine protease
tryptase that cleaves and activates collagenase causing tissue damage as well as
chymase that degrades epidermal basement membranes and stimulates mucus secre-
tion. Moreover, the granules of mast cells and basophils contain proteoglycans
like heparin and chondroitin sulfate that control via the binding of histamine the
kinetics of immediate hypersensitivity reactions. Mast cells produce cytokines, such
as TNF, IL1, IL3, IL4, IL-5, IL6, IL9, IL13, CCL3, CCL4 and CSF2 (colony-
stimulating factor 2), that contribute to the late-phase reaction of allergic inflamma-
tion. Eosinophils are recruited to late-phase reaction sites and release upon activa-
tion cationic granule proteins, such as MBP and eosinophil cationic protein, that
damage the envelop of helminths, bacterial cell walls as well as cells in normal
tissues. Furthermore, the granules of eosinophils contain an eosinophil peroxidase
that produces hypochlorous or hypobromous acid are toxic to helminths, protozoa as
well as host cells. Moreover, eosinophils also release lipid mediators and cytokines.
Box 10.6: Wheal-and-flare reaction A simple test of individual for possible

allergic reactions is the wheal-and-flare reaction. It involves the intradermal
injection of one or multiple allergens. Individuals who have previously encoun-
tered the one or other of these allergens will have mast cells residing in the
dermis that are loaded with IgEs specific for them. Then the injection site will
first become red as a result of locally dilated blood vessels and then rapidly
swells due to leakage of plasma from the venules. The swelling is called a
wheal and may get an extension of several centimeters. In the following blood
vessels at the margins of the wheal dilate and become engorged with red blood
cells, which produces a red rim referred to as flare. The full reaction normally
appears within 5–10 min and disappears within 1 h. It demonstrates immediate
hypersensitivity and depends on IgEs and mast cells discharging histamine.
Since mast cells in the skin produce only lower amounts of lipid mediators the
wheal-and-flare response subsides rapidly.
During recent centuries increased hygiene as well as cooking of meals substan-

tially decreased the exposure helminths, while the ability of IgE- and TH 2 cell-
mediated response persisted. This seems to be the main reason why today’s 30% of
the population of industrialized countries have allergies and this prevalence is further
increasing. Thus, the exposure to microbes in the training phase of the immune
system during childhood seems to be critical not to develop allergies. This is
the basis of the so-called hygiene hypothesis suggesting that early-life exposure to
environmental and commensal microbes allows a better maturation of the immune
system including sufficient amounts of Treg cells. This may reduce the frequency and
intensity of type 2 immune responses later in life.
Nevertheless, it is not fully understood, why proteins from common environmental
sources, such as dust mites and pollen, cause a type 2 immune response and not a
type 1 or type 3 response like against intra- and extracellular microbes, respectively,
which involves the activation of macrophages, dendritic cells, TH 1 or TH 17 cells
(Sect. 10.5). An important difference is the repeated exposure that are an essential
condition for initiating the type 2 immune response. Another important factor is the
genetic susceptibility to allergies, i.e., relatives of persons with allergy also have
more likely immediate hypersensitivities. The genetic risk for allergies is based on
complex gene-environment interactions, i.e., changes in the environmental exposure
with microbes and/or chemicals (including drugs and air pollution) are so rapid that
they are not counterbalanced by genetic adaptations. One cluster of genes, variations
of which show a significantly increased risk for allergies, is in chromosome 5 and
contains the cytokine genes IL4, IL5, IL9 and IL13. In addition, genetic variations of
the IL33 gene are strongly associated with asthma. All these cytokines importantly
contribute to the type 2 immune response.
10.3 Type 2 and 3 Hypersensitivities
Autoimmune diseases that are primarily mediated by antibodies are mediated either
by antibodies that bind to particular cell types (type 2 hypersensitivity) or by
antigen–antibody complexes in the circulation that attach to vessel walls (type 3
hypersensitivity). Diseases that are based on type 2 hypersensitivity are mostly
organ-specific, since the respective autoantibodies (mainly IgMs and IgGs) have
a specific target. The three major effector mechanisms that antibodies use against
pathogens (Sect. 7.2) do apply also to antibody-mediated diseases:
• Opsonization and phagocytosis (Fig. 10.6a): Autoantibodies that bind surface
molecules of circulating cells can opsonize them, which either recruits phago-
cytes, such as neutrophils, directly by FcRs or indirectly via the activation of
proteins of the complement system and receptors for C3b. In both cases the cells
are eliminated by phagocytosis. This mechanism of cell destruction is observed in
autoimmune hemolytic anemia and autoimmune thrombocytopenia, in which
autoantibodies against erythrocytes or platelets, respectively, circulate in respec-
tive patients. Complement proteins can also directly kill the cells via the membrane
attack complex, as observed in hemolysis after transfusion of inappropriate blood
groups (Sect. 9.2).
• Inflammation (Fig. 10.6b): Autoantibodies that recognize tissues, basement
membranes and extracellular matrix activate complement proteins, breakdown
products of which recruit macrophages and neutrophils. These leukocytes bind
via FcRs the autoantibodies or by complement receptors the tissue-bound
complement proteins and cause local inflammation and tissue injury. Glomeru-
lonephritis is an example of such an antibody-mediated autoimmune disease.
• Abnormal cellular functions (Fig. 10.6c): Autoantibodies that specifically
recognize either membrane-bound hormone receptors or neurotransmitter recep-
tors interfere with the normal function of these receptors. For example, in Graves’
disease (Fig. 10.6c, left) autoantibodies against the receptor of the peptide
hormone TSH (thyroid stimulating hormone) activate the receptors even in the
10.3 Type 2 and 3 Hypersensitivities 185
a Opsonization and phagocytosis

Phagocytosis of whole cell
Complement
C1 activation
C3b receptor
C3b
Antigen FcR
Complement
activation
Leukocyte
activation
Complement by-products
Antigen (C5a, C3a)
c Abnormal physiologic responses without cell / tissue injury

Nerve ending
Antibody against
TSH receptor Acetylcholine (ACh)
TSH
receptor Antibody against
Thyroid ACh receptor
epithelial
ACh receptor
cell
Thyroid hormones
Antibody stimulates receptor Antibody inhibits binding

without hormone of neurotransmitter to receptor
Fig. 10.6 Type 2 hypersensitivities. Antibody-mediated diseases act either via opsonization and
phagocytosis (a), complement- and FcR-mediated inflammation (b) or abnormal physiologic
responses (c). More details are provided in the text
absence of TSH, which causes hyperthyroidis, i.e., too high thyroid hormone
release. In myasthenia gravis (Fig. 10.6c, right), autoantibodies against the
receptor of the neurotransmitter acetylcholine block the activation of muscle cells,
i.e., the muscles are paralyzed. Furthermore, autoantibodies against the protein
CBIF (cobalamin binding intrinsic factor), which is essential for vitamin B12
absorption, cause pernicious anemia.
Moreover, type 2 hypersensitivity can occur due to:
• a so-called innocent bystander damage in connection to an immune response
to a foreign antigen
• a foreign antigen that binds to host tissue like drug hypersensitivity reactions
• a molecular mimicry where microbial antigens have a similar molecular structure
as host tissue.
For example, rheumatic fever is caused by an antibody against Streptococcus
pyogenes, which also recognizes heart, joint and brain tissue.
Type 3 hypersensitivities, i.e., autoimmune diseases caused by immune
complexes, often affect multiple tissues and organs, since the target of the anti-
bodies are circulating protein complexes, so that disease symptoms like vasculitis
occur at multiple sites of immune complex deposition. However, capillaries in renal
glomeruli and synovia are the most common sites of immune complex deposition,
since plasma is ultrafiltered there, i.e., kidneys are often targets of immune complex-
mediated diseases. Most normal immune responses produce immune complexes,
but only when they are produced in excessive amounts and not efficiently cleared
by phagocytosis, they may cause disease. Small immune complexes more likely
escape phagocytosis. Moreover, when immune complexes contain cationic anti-
gens, they bind more efficiently anionic components of blood vessels and tissues,
which increases the risk to produce severe and long-lasting tissue injury. In addition,
the deposition of immune complexes activates leukocytes and mast cells to secrete
cytokines and vasoactive mediators, which causes more immune complex deposition
in vessel walls. The immune complexes contain either self-antigens or foreign anti-
gens that are specifically recognized by antibodies. Examples of respective diseases
are:
• Serum sickness: Animals, such as rabbits, sheep or horses, are often exposed to
large doses of a foreign protein antigen, in order to produce polyclonal antibodies.
The antibodies bind to the circulating antigen and form immune complexes, some
of which are deposited in the walls of blood vessels. At these sites neutrophils
recognize via FcRs the immune complexes and cause local inflammation. This
damages endothelial cells lining the vessels and promotes thrombus formation,
i.e., the blood flow to tissues and organs is impaired so that they may undergo
necrosis. This man-made serum disease of animals is a master example of an
immune complex-mediated disease. In human, the clinical symptoms of acute
serum sickness are arthritis and nephritis as well as skin rashes, which occur only
for shorter time until lesions heal (or the antigen is injected again to animals).
• SLE (Fig. 10.7): In this chronic systemic autoimmune disease immune complexes
of nuclear antigens, such as DNA, histones and ribonucleoproteins, and anti-
bodies against these protein deposit in small arteries and capillaries throughout
the body and are responsible for glomerulonephritis, arthritis and vasculitis. The
10.3 Type 2 and 3 Hypersensitivities 187
Susceptibility genes External triggers

(e.g., UV radiation)
Apoptosis
Defective clearance
of apoptotic bodies
Increased burden of nuclear antigens
Anti-nuclear antibody,
antigen-antibody complexes
Endocytosis of
antigen-antibody
complexes
TLR engagement
of nuclear antigen
in endosomes
Stimulation of
B cells and Type 1 IFNs
dendritic cells
Persistent high-level
anti-nuclear IgG
antibody production
Fig. 10.7 SLE: an example for type 3 hypersensitivity diseases. The onset of SLE is explained by
the interference of susceptibility genes with environmental triggers, such as UV radiation, leading to
apoptosis of cells and the persistence of nuclear antigens. The inadequate clearance of the nuclear
antigens results in an antibody response against them, which is amplified by the activation of
dendritic cells and B cells with TLRs that recognize DNA and RNA (TLR9 and TLR7, respectively)
disease affects 10-times more likely females than males. Like in other autoim-
mune diseases, genetic and environmental factors contribute to the breakdown of
tolerance of self-reactive B and T cells in SLE. Key risk genes are HLA-DR2 and
HLA-DR3 as well as genes encoding for complement proteins C1 C2 and C4.
10.4 T Cell-Mediated Autoimmunity
Type IV hypersensitivity describes autoimmune diseases that are caused primarily by

the reaction of T cells against host tissue. This tissue injury is either mediated by TH 1
cells and/or TH 17 cells, which via the secretion of cytokines stimulate macrophages
and neutrophils to initiate an inflammatory reaction like fighting against microbes
(Fig. 10.8a), or by cytotoxic T cells, which directly target tissues like eliminating
virus-infected or tumor cells (Fig. 10.8b). Although inflammation is defense mecha-
nism of innate immune cells (Sect. 2.4), it becomes more severe and chronic when T
cells are involved. For example, this is used in the fight of T cells against intracellular
bacteria, such as Mycobacterium tuberculosis (Sect. 7.3), where a strong T cell and
a
Cytokines Tissue injury
Neutrophil
enzymes, ROS
CD4+ T cell
Tissue antigen-
presenting cell
Normal tissue
b T cell-mediated cytotoxicity
Cell killing and

tissue injury
Cytotoxic T cells
Fig. 10.8 T cell-mediated autoimmune diseases. Type 4 hypersensitivity reactions, CD4+ T cells
(and sometimes CD8+ cells) respond to tissue antigens by secreting cytokines that stimulate inflam-
mation and activate phagocytes, which leads to tissue injury (a). In some diseases, cytotoxic T cells
directly kill tissue cells (b).
10.4 T Cell-Mediated Autoimmunity 189
macrophage response results in granulomatous inflammation and fibrosis. The inten-

sive tissue destruction in the lungs caused by T cells is more severe than the effects of
the bacteria themselves. TH 1 cells secrete IFNγ that activates macrophages and TH 17
cells produce IL17 for the recruitment of neutrophils. Aggressive molecules, such
as lysosomal enzymes and ROS, which are secreted by activated macrophages and
neutrophils, then cause tissue injury. The damage is extended and becomes chronic
through the recruitment of additional immune cells to the lesions via cytokine secre-
tion by the phagocytes. These inflammatory reactions, which are also referred to as
delayed-type hypersensitivity (because in contrast to immediate hypersensitivity
(Sect. 10.2) it occurs 24–48 h after antigen challenge) are the mechanistic basis
of autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, type 1
diabetes and psoriasis (Box 10.1). Cytotoxic T cells contribute to the tissue injury in
these diseases, e.g., in type 1 diabetes, where specifically INS-producing β cells in
pancreatic islets are destroyed.
In the context of SLE (Sect. 10.3) we already started to discuss the interfer-
ence of environmental and genetic factors for the onset of autoimmune diseases. An
important environmental trigger for autoimmunity are infectious diseases (Fig. 10.9).
The adaptive immune system has developed with central and peripheral tolerance
(Sect. 9.1) protection mechanisms to avoid harm to the host by self-reactive B and T
cells. This includes the elimination of these cells by the induction of apoptosis, their
down-regulation by Treg cells and the induction of functional anergy (Fig. 10.9a). The
latter is achieved primarily by preventing the presentation of co-stimulatory proteins
within the immune synapse, so that the self-reactive lymphocyte is not sufficiently
activated that it may cause harm. However, when antigen-presenting cells that display
self-peptides to self-reactive lymphocytes experience the infection with an unrelated
microbe, they express co-stimulatory proteins like CD80 or CD86. Although these
co-stimulators are meant in response to the microbe infection, they also active self-
reactive lymphocytes, which then cause autoimmunity (Fig. 10.9b). It may be that
even commensal microbes (Sect. 7.4) have effects on the development of autoimmune
diseases. Interestingly, there is the possibility that peptides deriving from a microbial
antigen resemble self-peptides. Due to this molecular mimicry self-reactive T cells
will be fully activated both by the presentation of the microbial peptide on MHC
proteins as well as by the presence of co-stimulatory proteins (Fig. 10.9c).
The observation that some 50% monozygotic twins but only 5% of dizygotic
twins develop both type 1 diabetes indicates that autoimmune diseases have a strong
genetic component. However, most autoimmune diseases are based on the variations
of multiple genes, each of which contributing only to a few percent of the disease
risk. Some genomic risk loci, such as the HLA gene cluster, are shared between
many autoimmune disorders, since they encode for proteins that mediate general
mechanisms of immune regulation and self-tolerance. For example, the HLA locus
contribute in some autoimmune disease to some 50% of the genetic risk. An extreme
example is that of the inflammatory and autoimmune disease ankylosing spondylitis
a
“Resting” tissue
Self antigen Self-tolerance
dendritic cell
T cell
Self-
tolerance
b
CD80 CD28
Autoimmunity
Expression of
Activation co-stimulators
of antigen- on dendritic cell
presenting
cell self-reactive
Self antigen T cell
Self tissue
c
Microbial peptide Autoimmunity
Self-reactive T cell that
recognizes microbial peptide
Activation of T cells
Molecular
mimicry Self antigen
Peptide
Microbial protein Self tissue

Self protein
Fig. 10.9 Infections and the onset of autoimmunity. Peripheral tolerance mechanisms, like the
lack of co-stimulatory molecules on antigen-presenting cells, inactivate mature self-reactive T cells
by inducing their anergy (a). However, when additional stimuli, such as microbe infections, induce
the expression of co-stimulatory molecules, the self-reactive T cells are activated and harm host
tissue (b). When by chance microbial proteins result in the same peptides as self-proteins, this
molecular mimicry can activate self-reactive T cells and cause autoimmunity (c)
affecting vertebral joints, where carriers of the HLA allele B27 (encoding for MHC-
I) have a 100-time higher risk than non-carriers. In contrast, other loci are disease-
specific, such as the INS gene, and may primarily affect organ-specific proteins that
yield self-antigens.
10.4 T Cell-Mediated Autoimmunity 191
The major T cell-mediated autoimmune diseases are:

• Psoriasis: is a chronic IL17-mediated inflammatory disease that primarily
involves the skin, but sometimes also affects joints. Possible self-antigens are
the anti-microbial protein CAMP and a keratin. The IL23R gene, which is critical
for TH 17 development, has the most significant association with the disease and
further promotes the inflammatory response.
• Rheumatoid arthritis: is an inflammatory disease affecting small and large joints
of arms and legs. The synovitis leads to the destruction of cartilage, ligaments
and tendons of the joints involving cytokines produced by TH 1 and TH 17 cells.
However, since activated B cells and plasma cells are often present in the synovia
of affected joints, also self-reactive antibodies contribute to the disease. Some of
these are specific for citrullinated proteins, i.e., proteins that in the inflammatory
environment have arginine residues converted to citrulline. This protein modifi-
cation leads new self-peptides, which had not been negative selected by central
tolerance. Genetic susceptibility to rheumatoid arthritis is linked to the HLA-DR4
allele, encoding for MHC-II protein presenting these neoantigens.
• Multiple sclerosis: is the most common neurologic disease of young adults, in
which TH 1 and TH 17 cells react against the protein myelin basic protein. This
results in inflammation that via macrophages destroys the myelin insulation of
nerves leading to abnormalities in nerve conduction (Fig. 10.10). The clinical
symptoms of the disease are weakness, paralysis and ocular symptoms. The HLA
allele DRB1 ∗ 1501 shows the strongest linkage to the disease, but more than
100 risk gene variants are known. For example, HLA-DR15 allows myelin basic
protein peptide-specific T cells to escape negative selection in the thymus.
• Type 1 diabetes: is a metabolic disease resulting from inflammation mediated
by TH 1 cells reactive to INS and cytotoxic T cells destroying the INS-producing
β cells. Continuous INS replacement therapy is needed, otherwise it leads to
microvascular obstruction causing damage to the retina, renal glomeruli and
peripheral nerves. The HLA alleles DR3 and DR4 are the strongest genetic risk
factors for type 1 diabetes.
• IBD: is a heterogeneous group of disorders, like Crohn’s disease and ulcerative
colitis, that are associated with chronic remitting inflammation in the small or
large intestine as a result of abnormal responses of TH 1 and TH 17 cells to the gut
microbiome due to insufficient suppression by Treg cells.
• Celiac disease: is an inflammatory disease of the intestinal mucosa that leads
to atrophy of villi, malabsorption and nutritional deficiencies. It is caused by the
responses of TH 1 and TH 17 cells against the protein gliadin of the gluten group that
is present in wheat and other grains and cytotoxic T cells that kill epithelial cells
of the intestine. The gut microbiome contributes to the disease, which is treated
by avoidance of dietary gluten. The risk for celiac disease is strongly associated
with the HLA alleles DQ2 and DQ8.
The therapy of these diseases is discussed in Box 10.7.
EBV and mononucleosis

other viruses
risk genes
low vitamin D level
temperate latitude
toxins
trauma
smoking
obesity
early adulthood
female gender
Demyelination Axonal loss
Low High
few spinal cord lesions, many spinal cord lesions,

good endogenous repair, poor endogenous repair,
preserved axons and synapses, Chance of progression mitochondrial dysfunction,
early treatment, extensive axon and synapse loss,
younger age delay in treatment,
older age
Fig. 10.10 Multiple sclerosis. The T cell-mediated autoimmune disease multiple sclerosis is a
complex disorder, the onset of which is triggered by a combination of genetic and environmental
factors (top). Multiple sclerosis is associated with inflammatory, demyelinating lesions with hetero-
geneous axonal loss (center). The different features of the lesions and their consequences can be
salutary or deleterious and modify the risk of disease progression (bottom)
10.5 The Equilibrium Model of Immunity 193
Box 10.7: Treatment of autoimmune diseases A general therapy for autoim-

mune diseases are anti-inflammatory drugs (Sect. 9.3), such as corticosteroids
and non-steroidal compounds, that reduce the expression of cytokine genes
and other mediators of inflammation. A more specific approach is the use of
specific antagonists of cytokines like TNF, which are beneficial against rheuma-
toid arthritis, Crohn’s disease and psoriasis. In addition, monoclonal antibodies
(Sect. 11.3) directed against IL6 receptor are effective against arthritis and
others are approved against TH 2 cytokines for the treatment of allergies. More-
over, monoclonal antibodies are used to treat inflammatory diseases by the
depletion of different types of lymphocytes. For example, the depletion of B
cells by an anti-CD20 antibody (rituximab) is efficient in the treatment of
rheumatoid arthritis and multiple sclerosis. In addition, tolerance-inducing
therapies are in development, where the specific antigens of organ-specific
autoimmune diseases like myelin basic protein and INS in case of multiple
sclerosis and type 1 diabetes, respectively, are used to inhibit possible self-
reactive lymphocytes. In general, a serious side effect of blocking different
functions of the immune system is that the patient has higher susceptibility to
infectious disease or reduced cancer surveillance.
10.5 The Equilibrium Model of Immunity
Immune responses often represent equilibria between antagonizing cells and

molecules, such as activating and inhibitory co-receptors of the immune synapse
(Sect. 6.1), pro- and anti-inflammatory cytokines (Sect. 9.1) or macrophages of types
1 and 2 (Sect. 2.4). Moreover, the interaction of immune cells and antibodies at the
intestinal mucosa with symbiotic members of the gut microbiome (Sect. 7.4) is a
master example of constant actions and reactions, which are in an equilibrium. In
addition, in a healthy immune system, effector T cells are in an equilibrium with Treg
cells (Sect. 9.1) involving numerous activating and inhibitory cytokines and surface
proteins produced by both cell types. Finally, different types of effector T cells, such
as TH 1, TH 2 and TH 17 inhibit each other in their action (Sect. 6.3). Based on these
and other observation, the equilibrium model of immunity is proposed as an approach
to summarize all major immune responses in health and disease. The model is based
on the assumption that the immune system of a healthy person is constantly active, in
order to reach a dynamic equilibrium, i.e., a balance between four types of immune
responses (Fig. 10.11a). These are:
• Type 1 responses against viruses (Sect. 8.1) and intracellular bacteria (Sect. 8.3)
as well as against tumors (Chap. 10). This response is triggered by dendritic cells
and macrophages producing IL12, activates first NK cells and ILC1s as well as
a
Type 2 response Type 3 response
IL25,
IL33, IL1β,
Large extracellular TSLP IL23 smaller extracellular
microbes microbes
IL4, IL5, IL17,
Type 1 response IL13 IL22
IL12 TGFβ
INFγ Type 4 response
exclusion of
Intracellular viruses
microbes
and bacteria as
well as cancer cells
ILC1 T H1 Macrophage
ILC2 ILC3
type I Dendritic
NK CTL T H2 TH17 Macrophage cell
type II
b
Health Autoimmunity Allergy
1 2 3 4 1 2 3 4
Type of response
Fig. 10.11 The equilibrium model of immunity. The model assumes that the immune system
relies on a dynamic equilibrium between four types of competing and mutually inhibitory immune
responses (a). Accordingly, in health the four types of responses are in an equilibrium (b, left),
while in autoimmunity type 3 response it prominent (b, center) and in allergy type 2 (b, right)
later TH 1 cells and cytotoxic T cells. The main effector molecules are IFNγ,
perforin and ROS.
• Type 2 responses against large extracellular microbes, such as helminths
(Sect. 10.2). These responses are pushed by IL25, IL33 and TSLP leading first
to the activation of ILC2s and then to the development of TH 2 cells. The key
effectors are IL4, IL5 and IL13 as well as M2-type macrophages for tissue repair.
• Type 3 responses against smaller extracellular microbes, such as bacteria and
fungi (Sect. 7.1). This response is initiated by IL1β and IL23 that are secreted
by dendritic cells and macrophages and activates ILC3s and TH 17 cells. Effector
functions are the production of IL17 and IL22, the secretion of anti-microbial
peptides and the recruitment of neutrophils.
Further Reading 195
• Type 4 response blocks microbes before they reach sensitive tissues. This
response is started by the secretion of TGFβ1 and primarily mediated by secretory
IgA in the intestinal lumen as well as in tears, saliva, sweat and other secretions.
Other effectors are mucus and anti-microbial peptides.
The equilibrium depends on the immunophenotype (Box 8.6) of the individual and
his/her exposure to microbes of the environment. An individual gets ill, when the
system gets in a disequilibrium, and returns to health, when again homeostasis,
i.e., an equilibrium, is reached (Fig. 10.11b, left).
The activation of one type of immune response inhibits the other types, i.e., the
responses are competing with each other. With help of the equilibrium model complex
immune phenomena, such as autoimmunity, allergy and tolerance, may be better
understood:
• Through the production of IFNs type 1 responses are involved in SLE and type 1
diabetes, type 2 responses drive allergy and fibrosis via IL4, IL5 and IL13, while
type 3 responses mediate through IL17 autoimmune inflammatory diseases, such
as rheumatoid arthritis and multiple sclerosis (Fig. 10.11b, center).
• The hygiene concept (Sect. 10.2) suggests that via better hygiene, vaccines and
antibiotics the exposure to pathogenic as well as commensal microbes is reduced,
i.e., there are less type 1 and 3 immune responses. The immune system reacts
to this change by allowing type 2 responses to get dominant, which may cause
allergies (Fig. 10.11b, right).
• The loss of type 3 immune response by dysbiosis of the microbiome after excessive
use of antibiotics (Sect. 7.4) increases the risk for autoimmune diseases like SLE
and type 1 diabetes that are mediated by type 1 responses.
• The immune system uses type 1 responses via cytotoxic T cells to fight against
cancers (Sect. 11.1), while tumor cells often try to counterbalance this by inducing
a type 3 immune responses, e.g., through the activation of neutrophils that inhibit
the activation of tumor-specific cytotoxic T cells.
• Instead of understanding immune tolerance (Sect. 9.1) as a mechanism to avoid
inflammatory pathology, it may rather reflect the inhibition of one type of immune
response by another type.
Further Reading
1. Bach JF (2018) The hygiene hypothesis in autoimmunity: the role of pathogens and commensals.
2. Baecher-Allan C, Kaskow BJ, Weiner HL (2018) Multiple sclerosis: mechanisms and
immunotherapy. Neuron 97:742–768
3. Chang JT (2020) Pathophysiology of inflammatory bowel diseases. N Engl J Med 383:2652–
2664
4. Dendrou CA, Petersen J, Rossjohn J, Fugger L (2018) HLA variation and disease. Nat Rev
Immunol 18:325–339
5. Eberl G (2016) Immunity by equilibrium. Nat Rev Immunol 16:524–532
6. Lambrecht BN, Hammad H (2017) The immunology of the allergy epidemic and the hygiene
hypothesis. Nat Immunol 18:1076–1083
7. Renz H, Skevaki C (2021) Early life microbial exposures and allergy risks: opportunities for
prevention. Nat Rev Immunol 21:177–191
8. Yu W, Freeland DMH, Nadeau KC (2016) Food allergy: immune mechanisms, diagnosis and
immunotherapy. Nat Rev Immunol 16:751–765
Chapter 11
Cancer Immunology
Abstract A potent immune system is not only protecting us from infectious diseases
but also performs daily surveillance of our body for transformed cancer cells. In
this chapter, we will discuss the core mechanism of cancer immunity, which is the
recognition of neoantigens on the surface of cancer cells and the elimination of
these cells by cytotoxic T cells. Since immune checkpoints are often modulated by
negative regulators, such as CTLA4 and PDCD1, which prevent an efficient action
of the cytotoxic T cells, monoclonal therapeutic antibodies that block CTLA4 and
PDCD1 enhance the anti-tumor response of T cells and show remarkable clinical
effects. Moreover, we will learn that most recent immunotherapeutics are T cells
of the patient that have been engineered with a chimeric antigen receptor (CAR)
and are applied preferentially in hematological malignancies. Further advances in
the understanding of immunology and genetic engineering technologies will lead to
even more sophisticated immune therapies against different types of cancer with less
systemic side effects than classical chemotherapies.
Keywords Immune system · Cytotoxic T cells · Neoantigens · Immune

checkpoint · Monoclonal antibodies · CTLA4 · PDCD1 · Cellular immune
therapies · CAR T cells
11.1 Outline of Cancer Immunity
Cancer is one of multiple age-related diseases, i.e., the likelihood of getting cancer
significantly increases with age. For example, in industrialized countries the median
age for first cancer diagnosis is close to 70 years. This is related to the fact that
the main mechanism for cancer onset, the accumulation of damage in cells and the
genome, takes time. However, another important mechanism is the decline of immune
surveillance due to the aging immune system (Box 7.9). This immunosenescence
also causes increased low-grade chronic inflammation with age, which acts as an
additional cancer promoter. The huge count of some 70,000 daily lesions to our
genome per cell and 3 × 1013 cells forming our body makes it very likely that
every day transformed cells are established, even if potent DNA repair systems are
https://doi.org/10.1007/978-3-031-04025-2_11
198 11 Cancer Immunology
Table 11.1 Benefits and disadvantages of a potent immune system
Potent
immune immune
system system
Infectious agent Recurrent

immunity infections
Higher risk for Low risk for

Allergen
allergy allergy
Grafted organ Graft rejection Graft acceptance
Higher risk for Low risk for

Own organ
autoimmunity autoimmunity
Malignancies High risk for

surveillance cancer
constantly active. Thus, without an effective immune system each of us would

suffer in early life from different types of cancer.
As discussed in Chaps. 7 and 8, the prime reason, why we have a potent immune
system, is to effectively fight against infections by microbes, such as bacteria, fungi,
viruses and parasites (Table 11.1). However, the overreaction of the immune system
can make us severely ill or even kill us, as discussed for bacterial sepsis (Sect. 7.2) and
in context of COVID-19 (Sect. 8.4). Other non-desired malfunctions of the immune
system are allergy (Sect. 10.2), autoimmune diseases (Sect. 10.4) and graft rejec-
tions (Sect. 9.2). In fact, the molecular mechanisms that allow the immune system to
recognize cells from a genetically different person and eliminate them, is a special
case of the constant surveillance of our body for the appearance of transformed
cancer cells and their destruction. Thus, the detection and elimination of abnormal
cells by the immune system is part of its normal function. This is supported by the
observation that patients who are prescribed with immunosuppressive drugs, e.g.,
after an organ transplantation (Sect. 9.3), have a significantly higher risk of getting a
number of different cancers (Box 11.1). In particular, when the development or func-
tion of cytotoxic T cells, TH 1 helper cells or NK cells is compromised, the cancer rate
clearly increases. For example, HIV-1 positive individuals that are severely immuno-
compromised by the outbreak of AIDS often develop Kaposi sarcoma. Importantly,
11.1 Outline of Cancer Immunity 199
immune cells are a regular component of the microenvironment of malignant tumors

and are a sign that the immune system is responding to the presence of the malignant
tumor. Thus, a potent immune system should protect us throughout our life from
cancer.
Box 11.1: Cancer incidence of immunosuppressed patients Patients

receiving an organ transplant need to suppress their immune system by long-
term therapy, e.g., with the calcineurin inhibitor cyclosporin or comparable
drugs, in order to avoid rejection of the organ, referred to as host-versus-graft
disease. Moreover, some infectious diseases destroy the immune system, such
as the drastic reduction of TH cells by HIV-1. In both cases the immune system
is less able to detect and fight pre-malignant and cancer cells. This increases the
risk for many types of cancer. Moreover, immunosuppressed patients are prone
to infectious diseases and existing infections can become chronic. Accordingly,
not only the direct effect of the suppression of the immune system increases the
cancer risk but also the indirect effect via chronic inflammation plays an impor-
tant role in the tumorigenesis in those patients. Most of the cancers arising in
these immunosuppressed patients are non-Hodgkin lymphomas and cancers
in the lung, kidney and liver, i.e., they represent rather non-common cancer
types. Patients with a hematopoietic stem cell transplant, who developed graft-
versus-host disease also have an elevated cancer risk due to the misbalance of
their immune system. Thus, the immune system has an important role in
the fight and prevention of cancer.
The interaction of the immune system with cells of a malignant tumor is under-
stood as a series of events summarized as the cancer-immune cycle (Fig. 11.1). During
aging and in particular during tumorigenesis, cells are accumulating thousands of
passenger mutations and a few driver mutations. In a typical solid malignant tumor,
some 30–70 of these mutations affect the coding region of proteins and can lead to
new substructures in proteins. These structures are referred to as neoantigens,
since they are foreign to the immune system and have the potential inducing
its strong response. Accordingly, T cells that are specific for neoantigens are not
negatively selected in the thymus, since they are not known from normal cells (self).
In the regular turnover of proteins, neoantigens are presented as peptides bound
to MHC-I proteins on the surface of cancer cells or are secreted as a whole protein
when the cell is dying. Secreted neoantigens are taken up via phagocytosis by
tissue-resident dendritic cells, which then move via the lymph system to the closest
lymph node (referred to as sentinel lymph node). There, dendritic cells present the
neoantigen peptides via MHC proteins to a large number of T cells until one is found,
the TCR of which recognizes with high affinity the MHC protein-neoantigen peptide
complex (Sect. 11.2). This immune checkpoint is supported positively and nega-
tively via the contact of pairs of auxiliary membrane proteins, such as a member of
the B7 family of co-stimulatory proteins like CD80 and CD86 on dendritic cells as
T cells to tumors
Co-stimulating ligand on
receptor CD80 or CD86/CD28
Cytotoxic T cells
Blood vessel
Priming and Endothelial cells

activation T cell
of T cells
CTLA4 T cells into tumors

Lymph node
Chemokine
gradient
Cancer antigen
presentation Recognition of
cancer cells by T cells
Peptide
Dendritic cells
and other antigen- Cancer cells
presenting cells Antigens Cytotoxic T cells
and epitopes Tumor and immune cells
MHC I TCR
Killing of cancer cells
Release of cancer cell

antigens (cancer cell death)
Co-inhibition ligand or receptor

(checkpoints) CD274/PDCD1
Fig. 11.1 The cancer-immune cycle. The interaction of the immune system with cancer cells
involves a number of consecutive steps: (i) cancer cells (bottom) present or release neoantigens
that are detected and ingested by dendritic cells, (ii) in lymph nodes dendritic cells present on
their MHC proteins neoantigen peptides to T cells (left), (iii) T cells carrying a TCR fitting to the
MHC protein-peptide complex undergo clonal expansion and circulate then in the bloodstream until
they are attracted by a chemokine gradient targeting them to the cancer cells (top) and (iv) T cells
specifically recognize cancer cells and kill them (right). Key molecules and cells are indicated,
more details are provided in the text
well as CD28 (activating) and CTLA4 (inhibiting) on T cells (Sect. 6.1). The purpose
of negative regulators of this immune checkpoint is to avoid possible overreactions of
the immune system, such as observed in allergy and autoimmune disease. Activated
T cells undergo clonal expansion and thousands of identical cells go into circulation
via the bloodstream until a chemokine gradient is directing them to cancer cells. The
specific recognition of cancer cells is again facilitated by the immune checkpoint
formed by TCR and MHC-II proteins presenting the neoantigen peptide, by which
the T cell clone had been selected. Activating and inhibitory auxiliary receptors, such
as CD274 on the cancer cells and PDCD1 on the T cells, modulate the efficiency of
the T cell-cancer cell contact. In case of an activating contact, the cytotoxic T cells
secrete granzymes and perforin 1, which induce apoptosis within the cancer cells
(Sect. 6.4).
In addition to the specific recruitment of anti-tumor cytotoxic T cells, the microen-
vironment contains a number of cells of the innate and adaptive immune system.
Importantly, the immune microenvironment of early pre-malignant tumors clearly
differs from that of aggressive carcinomas (Fig. 11.2). Main differences are a shift
from the dominance of anti-tumor immune cells, such as cytotoxic T cells, NK cells,
dendritic cells, neutrophils and pro-inflammatory tumor-associated macrophages
Early pre-malignant lesion Advanced pre-malignant lesion Cancer
IFNγ +++ IFNγ ++ IFNγ +++

TNF + TNF ++ TNF +++
IL6 + IL6 ++ IL6 +++
Arginase + Arginase +++
Cytotoxic T cell B cell MDSC
Treg cell Macrophage NK cell
TH cell TAM Dendritic cell
Fig. 11.2 The microenvironment changes during the tumorigenesis process. The immune
microenvironment of early pre-malignant tumors is composed of cells of the innate immune system,
such as NK cells, dendritic cells and macrophages, and of the adaptive immune system, such as B
and T cells, while immunosuppressive Treg cells and MDSCs are not often found (left). Advanced
pre-malignant tumors contain less T and B cells but more Treg cells and MDSCs (center). The
immune microenvironment of cancer cells is dominated by Treg cells and MDSCs as well as by
TAMs (right). The change in the production of major cytokines, such as INFγ, TNF and IL6 is
indicated below. The production of enzyme arginase is a biomarker for active MDSCs
(TAMs) of type 1 to a governance by tumor-supportive TAMs of type 2, immunosup-

pressive Treg cells and MDSCs. The change in the immune microenvironment is also
visible on the level of signaling molecules that are secreted by aggressive tumors,
such as the cytokines INFγ, TNF and IL6 as well as the chemokines CX3CL1,
CXCL5, CXCL9, CXCL10, CXCL11 and CXCL13 (Fig. 11.3). Cytokines create a
pro-inflammatory milieu of the malignant tumor that also affects neighboring organs,
while chemokines guide further immune cells to the malignant tumor. IL21 and
CXCL13 levels are used most often as biomarkers (Box 11.2) of a good immune
contexture predicting the patients’ survival. Accordingly, the immune contexture
provides an advance to the classical cancer classification system. Importantly, malig-
nant tumors with a high mutational rate, such as melanoma and non-small cell lung
cancer, have a large load with neoantigens and recruit far more immune cells than
cancers with lower numbers of mutations. Thus, the immune infiltrate is an impor-
tant characteristic for the respective cancer type as well as for its prognosis and
possible therapy.
IL21
CXCL5
CXCL13
CX3CL1,
CXCL9,
CXCL10
CXCL11
Cytotoxic factors
Pleiotropic cytokines Granulysin, perforin 1,
IL15, TNF & CSF2 IFNγ granzymes A, B, H, M & K
Cytotoxic T cell Mast cell TAM Epithelial cell
T reg cell Neutrophil

Fibroblast Cancer cell
T H cell NK cell
Invasive cancer
MDSC
cell
B cell Dendritic cell
Fig. 11.3 Main immune cells shaping cancer progression. Immune cell types are shown
belonging to the microenvironment of malignant tumors. Cytokines and chemokines with a positive
prognosis on cancer are in blue boxes, while those with negative impact are in red boxes. More
Box 11.2: Immunologic biomarkers of cancer In general, a biomarker is

a substance, structure or process that can be measured in the body or its
products and influences or predicts the incidence of outcome or disease. In
cancer, biomarkers are primarily used for prognosis, i.e., they are factors that
influence positively or negatively the clinical outcome of the patients. For
example, non-proliferating versus proliferating tumor-associated cytotoxic T
cells have a negative or positive prognostic value, respectively. Moreover, TH 1
cells and their main cytokine INFγ predict a good clinical outcome in all
cancer types. In addition, the presence of tumor-associated NK cells predicts
an increased survival in patients with colorectal and prostate cancer, while
increased numbers of tumor-associated neutrophils provide a negative prog-
nosis. Thus, the immune contexture contains a number of useful prognostic
biomarkers. However, in most cases, these biomarkers are not predictive for
therapeutic effectiveness, where other biomarkers need to be applied.
Taken together, immunity is a rapidly responding system that is capable to remove

not only a load of microbes but also kills huge numbers of cancer cells arising in
our body. This process of cancer immunosurveillance works perfectly for every
second of us and provides life-long absence of any detectable malignant tumor.
In the same way as pathogenic microbes find mechanisms to escape from immuno-
surveillance and elimination (Box 7.1), also cancer cells often adapt the hallmark
“avoiding immune destruction”. Examples for the latter are:
• genetic changes that make cancer cells less visible to the immune system, such
as the down-regulation of MHC proteins carrying neoantigens
• up-regulating the expression of membrane proteins that inhibit the immune
checkpoint, such as CD274
• induce a change in the pattern of tumor-infiltrating lymphocytes from anti-tumor
cytotoxic T cells to pro-tumor Treg cells.
However, even in cases when immunosurveillance has failed in removing a
malignant tumor in an early stage, immunotherapies, such as:
• boosting the recognition of cancer neoantigens and creating tumor vaccines
(Sect. 11.2)
• blocking immune checkpoint inhibitors by monoclonal antibodies (Sect. 11.3)
• the use of engineered CAR T cells (Sect. 11.4).
belong to the most promising anti-cancer treatments. Therefore, immunotherapy
may be the only effective strategy for the elimination of metastatic cells in
completeness.
11.2 Recognition of Cancer Antigens
Healthy adults have at every moment of life at least some 10 million different clones
of naïve T cells in their lymphoid organs and in circulation. As a whole these T cells
are able to recognize about this number of variant protein antigen structures, i.e.,
our immune system is well prepared to react on a huge number of putative antigens.
These antigens may be of environmental origin, such as proteins on the surface of
microbes that we got infected with, but they may also be created by our own cells.
The regular turnover of all intracellular proteins via the ubiquitin-proteosome system
in the cytosol results in a large number of smaller peptides, some of which are loaded
in the ER to MHC-I proteins and finally are exposed on the cell surface. In this way,
fragments of intracellular proteins are visible to the immune system and define self-
antigens. Central tolerance mechanisms taking place in the thymus prevent that any
naïve T cell gets into circulation, the TCR of which recognize by chance these self-
peptides. Accordingly, normal cells should not cause any response of cytotoxic T
cells (Fig. 11.4a).
a Normal cell
MHC-I Normal self peptides

displayed on MHC,
Self-peptide no responding T cells
due to tolerance
Cancer cell
b Cytotoxic T cell
TCR Mutation-generated
new epitope => new
TCR contact residue,
T cell response
Mutation
Cancer cell Oncogenic virus Neoantigen

c
Peptide from a protein

encoded by an
oncogenic virus,
T cell response
Viral gene
Fig. 11.4 Occurrence of neoantigens in cancer cells. T cells have learned via central tolerance
mechanisms during their schooling phase in the thymus not to react to peptides being presented on
MHC proteins, which originate from regular cellular proteins (self) in normal cells (a). In cancer
cells, neoantigens can be produced by non-synonymous driver or passenger mutations in the coding
region of regular proteins, which cause an amino acid exchange (b), or by new proteins, the genes
of which are introduced by oncoviruses (c). In the two latter cases MHC protein-presented peptides
cause a response of the TCR of cytotoxic T cells
11.2 Recognition of Cancer Antigens 205
In contrast, when somatic cells accumulate driver and passenger mutations, some
of these affect in a non-synonymous way the coding region of proteins and cause
amino acid exchanges. In addition, some of these mutations may create frameshifts,
splice variants, gene fusions and other processes that result in changes of endogenous
proteins. When the respective proteins are degraded, new types of peptides are formed
and presented by MHC proteins on the cell surface (Fig. 11.4b). These neoantigens
are exclusively recognized by those clones of cytotoxic T cells that fit with their
TCR to the MHC protein-neoantigen complex. The specific contact of cytotoxic T
cells starts an intracellular signal transduction cascade that results in the release of
apoptosis-inducing granzymes, i.e., the neoantigen carrying cancer cell is elim-
inated. Similarly, when cells are transformed by an oncovirus they produce large
amounts of viral proteins, some of which also get degraded, so that neoantigens can
be loaded on MHC proteins (Fig. 11.4c). The elimination of the oncovirus-infected
cells follows the same principles as described for neoantigens derived from mutated
proteins.
The elimination of oncovirus-transformed cells by cytotoxic T cells can be consid-
ered as a special case of immunosurveillance for cells infected by regular viruses,
i.e., for the immune system there is no mechanistic difference in its fight against
transformed cancer cells or virus infections. Since vaccination is an effective
therapy against virus infections, there may be also the chance for a vaccination
against cancer. Such an anti-cancer vaccination comprises not only a cancer preven-
tive therapy against tumor viruses, such as EBV, HBV and HPV, but also the applica-
tion of vaccines based on tumor neoantigens. Malignant tumors with a larger number
of mutations, such as melanoma, carry many neoantigens. These cancers are more
efficiently recognized by the immune system and are the best targets of cancer treating
and protecting vaccines. Interestingly, the characterization of neoantigens of malig-
nant tumors allows an optimized use of immune checkpoint inhibitors (Sect. 11.3)
and improves other forms of immunotherapy, such as CAR T cells (Sect. 11.4).
On the basis of results obtained with next-generation sequencing methods, such as
RNA-seq, exome sequencing and whole genome sequencing, a cancer patients’ muta-
tional burden can be determined and neoantigens that are specific for the respective
malignant tumor can be envisioned. Moreover, these data also allow the determina-
tion of variants of HLA genes encoding for MHC proteins (Sect. 5.2) and can predict
their neoantigen peptide affinity. This molecular profiling information allows the
design of precise cancer treating vaccines on the basis of DNA or RNA, peptides
or dendritic cells. Accordingly, the information about tumor-specific neoantigens
can be used in a number of different approaches, such as:
• dendritic cells presenting neoantigen peptides
• viruses encoding neoantigen peptides on their surfaces
• vaccines containing neoantigen peptides and antibodies
• T cells with reactivity directed against neoantigen peptides.
However, targeted therapies are very costly and in case of metastatic cancer they
may not be sufficient, i.e., they add to the substantial costs of other therapies. There-
fore, also from the economic point of view it would be more efficient to boost the
whole immune system.
11.3 Monoclonal Antibodies in Cancer Immunotherapy
Since most antigens have multiple epitopes, a number of different B cell clones
respond to the presence of the antigen and produce a polyclonal set of antibodies.
Polyclonal antibodies are often a challenge for a therapeutic application, which
requires high specificity, in order to minimize side effects. Thus, in oncology
only monoclonal antibodies are used for immunotherapy. Approved antibodies
(Table 11.2) use different mechanisms:
• direct action of the antibody via receptor antagonism, e.g., blockade of agonist
activity of a oncoprotein, such as the receptor tyrosine kinase HER2 (Trastuzumab,
trade name “Herceptin”) (Fig. 11.5a)
• immune-mediated cell killing, such as complement-dependent cytotoxicity, e.g.,
via binding to the B cell-specific surface protein CD20 (Rituximab) (Fig. 11.5b)
• immune-mediated cell killing, such as antibody-dependent cellular cytotoxicity,
e.g., via binding to the chemokine receptor CCR4 (Mogamulizumab) (Fig. 11.5c).
The blockage of the immune checkpoint inhibiting proteins CTLA4, PDCD1
or CD274 (Fig. 11.6) via the antibodies Ipilimumab, Nivolumab/Pembrolizumab
and Atezolizumab/Avelumab/Durvalumab, respectively, follows the first mecha-
nism. Thus, blocking the security mechanism for possible overactions of the
immune system is boosting the cancer surveillance potential of T cells. For
some cancer patients this antibody-based immunotherapies show impressive durable
responses, but unfortunately the majority of patients do not get long-term benefits,
when using these immune checkpoint inhibitors as monotherapy. Moreover, a side
effect of the therapeutic application of anti-CTLA4 antibodies is the development of
autoimmunity and inflammation in various organs.
Importantly, the efficiency of neoantigen presentation via MHC-I proteins, which
in part is based on the polymorphisms of the encoding genes HLA-A, HLA-B and
HLA-C (Sect. 5.2), correlates with the success of immune checkpoint blockage. Inter-
estingly, unfavorable microbiome signatures of the gut are linked to poor responses
to immune checkpoint blockade suggesting that a number of extrinsic factors modu-
late the response to immunotherapy. Moreover, the efficiency of immune checkpoint
blockade but also of other immunotherapies depend on the amount of immune cell
infiltration of cancer, which is low for brain tumors and uveal melanoma (an eye
cancer). In particular, the amount of professionally antigen-presenting cells, such
as dendritic cells and macrophages, in non-lymphoid tissues is critical. It is low in
pancreas and brain, mid-level in filtering organs, such as the kidney and liver, and
highest in skin, lung and gut that are heavily exposed to the environment.
11.3 Monoclonal Antibodies in Cancer Immunotherapy 207
Table 11.2 Therapeutic monoclonal antibodies approved for use in oncology

Name (trade name) Antigen Antibody type Approved indications
(selection)
Rituximab (Rituxan and CD20 Chimeric IgG1, κ-chain Non-Hodgkin
MabThera) lymphoma, CLL
Ofatumumab (Arzerra) CD20 Human, mouse-derived CLL
IgG1, κ-chain
Obinutuzumab (Gazyva) CD20 Humanized, CLL, follicular
glyco-engineered IgG1, lymphoma
κ-chain
Ibritumomab tiuxetan CD20 Mouse IgG1, κ-chain; Non-Hodgkin lymphoma
(Zevalin) 90Y-containing
radioimmunoconjugate
Tositumomab (Bexxar) CD20 Mouse IgG2a, λ-chain; Non-Hodgkin lymphoma
131I-containing
radioimmunoconjugate
Inotuzumab ozogamicin CD22 Humanized IgG4, ALL
(Besponsa) κ-chain
Atezolizumab (Tecentriq) CD274 Human, phage-derived, a Urothelial cancer and
glycosylated IgG1, NSCLC
κ-chain
Avelumab (Bavencio) CD274 Human, phage-derived Merkel cell and
IgG1, λ-chain urothelial cancer
Durvalumab (Imfinzi) CD274 Human, transgenic Urothelial cancer
mouse-derived IgG1,
κ-chain
Brentuximab vedotin CD30 Chimeric IgG1, κ-chain Hodgkin lymphoma,
(Adcetris) systemic anaplastic large
cell lymphoma
Gemtuzumab ozogamicin CD33 Humanized IgG4, AML
(Mylotarg) κ-chain
Daratumumab (Darzalex) CD38 Human, transgenic Multiple myeloma
mouse-derived IgG1,
κ-chain
Ipilimumab (Yervoy) CTLA4 Human, transgenic Melanoma
mouse-derived IgG1,
κ-chain
Cetuximab (Erbitux) EGFR Chimeric IgG1, κ-chain Colorectal cancer, head
and neck cancer
Panitumumab (Vectibix) EGFR Human, transgenic Colorectal cancer
mouse-derived IgG2,
κ-chain
Necitumumab (Portrazza) EGFR Human, phage-derived Squamous NSCLC
IgG1, κ-chain
Nimotuzumab (TheraCIM, EGFR Humanized IgG1, Glioma, head and neck,
BIOMAb-EGFR) κ-chain nasopharyngeal and
pancreatic cancer
(continued)
Table 11.2 (continued)

Name (trade name) Antigen Antibody type Approved indications
(selection)
Trastuzumab (Herceptin) HER2 Humanized IgG1, Breast, gastric and
κ-chain gastroesophageal
junction cancer
Pertuzumab (Perjeta) HER2 Humanized IgG1, Breast cancer
κ-chain
Ado-trastuzumab emtansine HER2 Humanized IgG1, Breast cancer
(Kadcyla) κ-chain
Nivolumab (Opdivo) PD1 Human, mouse-derived Melanoma, Hodgkin
IgG4, κ-chain lymphoma, squamous
cell carcinoma
Pembrolizumab (Keytruda) PD1 Humanized IgG4, Melanoma, Hodgkin
κ-chain lymphoma, NSCLC
Olaratumab (Lartruvo) PDGFRA Human, transgenic Soft tissue sarcoma
mouse-derived IgG1,
κ-chain
Bevacizumab (Avastin) VEGFA Humanized IgG1, Non-squamous NSCLC
κ-chain and colorectal cancer
Ramucirumab (Cyramza) VEGFR2 Human, phage-derived NSCLC, gastric and
IgG1, κ-chain colorectal cancer
The safety and efficacy of therapeutic monoclonal antibodies largely depends on

the targeted antigen. Ideally, the latter should be expressed exclusively and abun-
dantly on the surface of cancer cells, while secreted antigens risk to prevent anti-
body binding to malignant tumor cells. Moreover, when complement- and antibody-
dependent cytotoxicity is chosen as mechanism of action, the antigen–antibody
complex should not be internalized, while the latter is desired, if the antibody delivers
a radioisotope or other toxins or down-regulates a protein on the surface of the cancer
cell.
11.4 Immune Cell Therapies
Red blood cell transfusions are used since some 70 years as cell therapies in the
context of trauma, surgery and bone marrow failure. Later, platelet transfusions as
well as hematopoietic stem cell transplantation were introduced, in order to improve
the survival of patients with hematological diseases. More recently, adoptive cell
transfer, also called immune cell therapy, has emerged for the treatment of solid
and hematological cancers. Adoptive cell transfer is mostly applied for T cells in
an autologous fashion, i.e., patients are getting back their own cells. In contrast, in
11.4 Immune Cell Therapies 209
a Antagonism
b Complement-
c Antibody-
IgG4 dependent dependent
IgG3 cytotoxicity cell-mediated
IgG1, IgG3, IgM cytotoxicity
IgG1, IgG3
Complement T cell
CD FcγR
molecule
Ligand
x Cell lysis Cell lysis
Omalizumab
Adalimumab Adalimumab
Natalizumab
Rituximab Rituximab
Daclizumab
Fig. 11.5 Application of monoclonal antibodies in oncology. Monoclonal antibodies are

produced in tissue culture using hybridoma technology, where a murine B cell producing an antibody
of desired specificity obtains unlimited growth potential by fusion with a myeloma cell. The type of
monoclonal antibody can be deduced from elements of its name: there are original murine antibodies,
chimeric antibodies with murine variable regions grafted onto human constant regions, human-
ized antibodies having human scaffold and murine CDRs and antibodies with complete human
sequences. The functions of monoclonal antibodies include antagonism (a), complement-dependent
cytotoxicity (b) and antibody-dependent cell-mediated cytotoxicity (c)
allogenic approaches the patient receives cells from a donor, who needs to match as
close as possible in the HLA profile, in order to avoid graft rejections.
In a process referred to as apheresis whole blood is removed from a patient or
donor, selected immune cells are extracted and the remaining blood components
are given back to the same person. The selected immune cells are often cancer-
specific T cells, which are cultured in vitro in the presence of IL2 and other growth
stimulating cytokines, in order to increase their number (Fig. 11.7a). The phase of
in vitro culture allows further purification of the cells and their genetic engineering
(Fig. 11.7b). When the cells are given back to the patient, they are expected to boost
the natural ability of the immune system fighting against the malignant tumor.
a Introduction of anti-tumor immune response in lymph node
Tumor peptide
MHC-II TCR
TCR
CD80 CD28
CD80 CTLA4
Primed cytotoxic T
CTLA4 cell capable of kill-
CD28 anti-CTLA4
ing tumor cells
No co-stimulation Co-stimulation
b Cytotoxic T cell-mediated killing of tumor cells

Tumor peptide
MHC-I
TCR
MHC-I
TCR
PDCD1
CD274
CD274 PDCD1
anti-CD274 anti-PDCD1 Dead tumor cell
Fig. 11.6 Immune checkpoint blockade. T cell responses to malignant tumors are often ineffi-
cient due to the up-regulation of inhibitory receptors of the immune checkpoint, such as CTLA4
and PDCD1 on the tumor-specific T cells and CD274 on the malignant tumor cells. Blocking these
immune checkpoint inhibitors via anti-CTLA4 antibodies (a) or anti-PDCD1 or anti-CD274 anti-
bodies (b) results in the activation of cytotoxic T cells and the induction of apoptosis to cancer
cells
In some solid cancers, such as melanoma, adoptive transfer of tumor-infiltrating

T cells or of T cells expressing recombinant TCRs recognizing tumor-specific
neoantigens showed impressive responses. This approach is most promising for
cancers with a large mutational burden attracting a high number of tumor-infiltrating
lymphocytes. In contrast, in case of other cancer types, where the number of
tumor-specific T cells is rather low, this adoptive cell transfer approach is not very
promising.
The therapy options of relapsed hematological malignancies, such as acute
lymphocytic leukemia, B cell lymphoma and multiple myeloma, had a significant
breakthrough by adoptive transfer of T cells, which had been engineered with CARs
(Fig. 11.8a) (Box 11.3). For example, CAR T cells targeting the B cell-specific
membrane proteins CD19 and CD22 have been approved first in 2017 by FDA and
EMA for the treatment of B cell malignancies, such as of relapsed or refractory B cell
acute lymphocyte leukemia, relapsed or refractory diffuse large B cell lymphoma
and primary mediastinal large B cell lymphoma. Other specific target proteins on
the surface of cancer cells are TNFRSF (TNF receptor superfamily member) 8
in Hodgkin lymphoma and anaplastic large cell lymphoma, TNFRSF17, SLAMF7
11.4 Immune Cell Therapies 211
b T cells with engineered TCR
T cells with CAR
Patient with leukemia

NK cells with CAR
Fig. 11.7 Immune cell therapies in cancer treatment. Immune cell therapies are performed either
without engineered receptors using tumor-infiltrating lymphocytes (a) or with T cells transduced
with engineered TCR or T cells and NK cells transduced with CAR (b). The engineered cell types
are grown in vitro before re-infusion into the patient. In all cases the cells specifically recognize
malignant tumor cells and eliminate them
(SLAM family member 7) and CD38 in multiple myeloma as well as CD33, IL3RA
(interleukin 3 receptor subunit alpha) and CLEC12A (C-type lectin domain family 12
member A) in acute myeloid leukemia.
Box 11.3: CAR T cells Through the expression of CARs, T cells can be
specifically directed against a given malignant tumor. CARs are recombinant
proteins that are composed of two extracellular antigen-targeting Ig domains
of an antibody, a so-called single chain variable fragment (scFv), which has
high specificity for a given tumor antigen, and cytosolic signaling domains
that activate T cells, such as the ITAM domain of the TCR co-receptors CD3
and ζ and co-stimulatory domains of the proteins CD28 and/or TNFRSF9
(Fig. 11.8b). Recombinant technologies allow to generate scFvs to any cell
surface molecule, such as modified proteins, lipids, sugars and neoantigens,
displaying a broad range of biochemical properties. Thus, the combination of
Cancer cell
a Isolate lymphocytes b Nucleus
from blood
MHC-I Cytoplasm
Expand in culture with

anti-CD3, anti-CD28 and
Patient with leukemia IL2, transduce with
CAR gene Tumor peptide
VH VL
Killing
CAR mechanisms
Transfer back
into patient
Cytoplasm
ITAMs
CD28 motif
TNFSF9
motif
Tumor Activation Nucleus

regression
Patient’s CAR T cell
Fig. 11.8 CAR T cell therapy. T cells are isolated from the patient’s blood expanded in vitro by
culture in the presence of the cytokine IL2. A specific lymphodepletion is achieved by removing
CD3 and CD28 positive cells via respective specific antibodies. The remaining cells are transduced
with CARs and transferred back to the patient (a). CARs are recombinant proteins composed of
two variable Ig domains specific for a tumor antigen and cytosolic signaling domains that activate
T cells (b). More details are provided in the text
immunotherapy and genetic engineering has created with CAR T cells a kind
of “living” drugs.
CAR T cells are mostly used in an autologous fashion, i.e., they derive from
patient’s own T cells. An individual manufacturing process is required for each
patient and causes a treatment delay of at least 3 weeks. In contrast, allogeneic CAR
T cells from donors are immediately available for treatment and can be produced as
standardized batches at far lowers costs. Nevertheless, the latter cells bear the risk of
causing life-threatening GvHD as well as host-versus-graft rejection, i.e., rapid elim-
ination of the therapeutic cells. Therefore, HLA matching between donor and patient
is of utmost importance. Furthermore, a well-known immunologic phenomenon, T
cell exhaustion due to chronic antigen stimulation, can limit the efficacy of CAR
Further Reading 213
T cells. Unfortunately, the present generation of CAR T cells did not show any
convincing efficacy in patients with solid malignant tumors. This is related to the
lack of high-level, homogeneous expression of membrane proteins on solid malig-
nant tumors compared to normal tissue. In addition, CAR T cells are inhibited by
the suppressive immune cells being part of the microenvironment of solid malignant
tumors. However, there is the chance that next-generation CAR T cells, which are
engineered to overexpress the cytokine IL12 for promoting survival or enzymes for
handling the tumor microenvironment, have a higher success rate.
Although NK cells belong to the innate immune system, i.e., they do not express
any high-specificity antigen receptor, they are able to kill malignant tumor cells and
are an integral part of our body’s tumor immunosurveillance system. Different types
of cancer have evolved mechanism inhibiting the function of NK cells and in this
way escape from being killed. Therefore, an attractive concept is to transduce NK
cells with CARs (Fig. 11.7b).
Taken together, most malignant tumors develop due to a failure of the cancer
surveillance mechanisms of our immune system. Thus, taking care on the potency
of our immune system is the best way of cancer prevention. Moreover, different
approaches of immunotherapy promise to be the most effective way of treating a
cancer.
Further Reading
1. Bruni D, Angell HK, Galon J (2020) The immune contexture and immunoscore in cancer
prognosis and therapeutic efficacy. Nat Rev Cancer 20:662–680
2. Carlberg C, Velleuer E (2021) Cancer biology: how science works. Springer, Berlin, ISBN
978-3-030-75699-4
3. Ferreira LMR, Muller YD, Bluestone JA, Tang Q (2019) Next-generation regulatory T cell
therapy. Nat Rev Drug Discov 18:749–769
4. Garner H, de Visser KE (2020) Immune crosstalk in cancer progression and metastatic spread:
a complex conversation. Nat Rev Immunol 20:483–497
5. Hackl H, Charoentong P, Finotello F, Trajanoski Z (2016) Computational genomics tools for
dissecting tumour-immune cell interactions. Nat Rev Genet 17:441–458
6. Havel JJ, Chowell D, Chan TA (2019) The evolving landscape of biomarkers for checkpoint
inhibitor immunotherapy. Nat Rev Cancer 19:133–150
7. Jhunjhunwala S, Hammer C, Delamarre L (2021) Antigen presentation in cancer: insights into
tumour immunogenicity and immune evasion. Nat Rev Cancer 21:298–312
8. Smith CC, Selitsky SR, Chai S, Armistead PM, Vincent BG, Serody JS (2019) Alternative
tumour-specific antigens. Nat Rev Cancer 19:465–478
9. Weber EW, Maus MV, Mackall CL (2020) The emerging landscape of immune cell therapies.
Cell 181:46–62
Glossary
Acute inflammation is a short-term immunological process occurring in response

to tissue injury or microbe infection usually appearing within minutes or hours.
It is characterized by pain, redness, swelling and heat.
Acute rejection is an episode of sudden deterioration in allograft function as a result
of either antibody-mediated rejection or T cell-mediated rejection, which result
from different molecular processes.
Adaptive therapy is the application of cancer treatment in a manner that quickly
responds to changes in the disease rather than following a fixed protocol, with the
goal of managing the cancer and maintaining a limited tumor burden, rather than
attempting to totally eliminate the disease.
Affinity maturation is a process by which B cells in germinal centers increase their
affinity for antigen during an immune response.
Allogeneic is a term that describes tissues that are of distinct genetic origins and
thus often immunologically incompatible.
Allorecognition is the ability of a graft hosts to recognize allogeneic tissue as distinct
from its own.
Anaphylaxis is a severe and potentially life-threatening reaction to an allergen.
Anergy is a tolerance mechanism of T cells, in which after an antigen encounter
cells are inactivated via epigenomic reprograming.
Antigens are parts of the pathogen, such as proteins or polysaccharides, that are
recognized by the immune system and can be used to induce an immune response
by vaccination.
Antigenic drift is a process by which circulating virus genomes are constantly
changing. This allows the virus to cause annual epidemics.
Autophagy is a mechanism for degrading cellular proteins and recycling their
products as sources of nutrients during times of stress.
Carcinoma is a type of malignant tumor composed of epithelial cells.
Central tolerance is the mechanisms by which T cells and B cells are rendered
non-reactive to an antigen (typically a self-antigen) in primary lymphoid organs.
© The Editor(s) (if applicable) and The Author(s), under exclusive license 215
to Springer Nature Switzerland AG 2022
https://doi.org/10.1007/978-3-031-04025-2
216 Glossary
Chemokines is a group of small cytokine-like proteins that induce directed

chemotaxis to responsive neighboring cells, i.e., they act as chemoattractant
cytokines.
Chromatin is the molecular substance of chromosomes being a complex of genomic
DNA and histone proteins.
Chronic inflammation is long-term inflammation lasting for prolonged periods
of several months to years. Chronic inflammation plays a central role in most
common non-communicable diseases, such as cancer, type 2 diabetes, asthma
and Alzheimer’s.
Complement system is a component of the innate immune system that can be
activated by antigen-bound antibodies.
Cytokines is a group of small proteins (~5 to 20 kDa) that are important in
cell signaling involved in autocrine, paracrine and endocrine signaling as
immunomodulating agents.
Damage-associated molecular patterns (DAMPs) also known as alarmins, are
molecules often released by stressed cells undergoing necrosis that act as
endogenous danger signals to promote and exacerbate inflammatory responses.
DNA methylation is the covalent addition of a methyl group to the C5 position of
cytosine.
Dysbiosis is compositional and functional alteration of the gut microbiome.
Embryogenesis is also called embryonic development, i.e., the process by which the
embryo forms and develops. In mammals, the term is use exclusively to the early
stages of pre-natal development, whereas the terms fetus and fetal development
describe later stages.
Epigenetic programing is the process leading to stable and long-lasting alter-
ations of the epigenome based on specific covalent modifications of the DNA
and histones.
Epitope is a specific portion of the antigen specifically recognized by a TCR or
BCR.
Exome is the collection of exons in the human genome. Exome sequencing generally
refers to the collection of exons that encode proteins.
Gene expression is the process by which information from a gene is used in the
synthesis of a functional gene product. These products are often proteins, but can
also be non-coding RNAs.
Genome is the complete haploid DNA sequence of an organism comprising all
coding genes and far larger non-coding regions. The genome of all 400 tissues and
cell types of an individual is identical and constant over time (with the exception
of cancer cells).
Genotype represents the complete heritable genetic identity.
Graft-versus-host disease (GvHD) is a medical complication that is largely specific
to HSC transplantation, in which immune cells of the donor reject and attack cells
of the host.
Healthspan is the duration of disease-free physiological health within the lifespan
of an individual. In humans, this corresponds to the period of high cognitive
abilities, immune competence and peak physical condition.
Glossary 217
Hematopoietic stem cells (HSCs) are stem cells located in the bone marrow that
can develop into all types of blood cells.
HLA typing is the process for identifying the HLA receptor alleles of an individual.
Homeostasis is the state of steady internal physical and chemical conditions
maintained by living systems.
Human leukocyte antigen (HLA) is a protein encoded by genes that determine
an individual’s capacity to respond to specific antigens or reject transplants from
other individuals. HLA genes encode for MHC proteins.
Immune checkpoint blockage represents the inhibition of regulators of the immune
system being crucial for self-tolerance. Immune checkpoint blocking proteins,
such as CTLA4 and PDCD1, are targets for cancer immunotherapy.
Immunity refers historically to the protection of politicians from legal prosecu-
tion in the old Rome (immunitas in Latin). Applied to science, immunity means
protection from disease, in particular from infectious disease.
Immunosenescence is the age-associated gradual deterioration of the immune
system, in particular of the adaptive immune system. This involves capacity of
the host to respond to infections, to develop of long-term immune memory and
to perform efficient immune surveillance.
Inflammasome is a supramolecular complex responsible for the CASP1-dependent
maturation of IL1B and IL18 in response to microbial products or other danger
signals.
Macrophage is a leukocyte that engulfs and digests cellular debris, foreign
substances, microbes and cancer cells in a process called phagocytosis.
Missense mutation is a single-nucleotide substitution (e.g., C to T) that results in
an amino acid substitution (e.g., histidine to arginine).
Monocytes are leukocytes of the innate immune system that can differentiate into
macrophages and myeloid lineage DCs.
Naïve lymphocytes are B and T cells that have not been exposed to an antigen.
Neutrophils are the most abundant type of white blood cells and belong to the innate
immune system.
Non-communicable disease is a disease that is not transmissible directly from one
person to another, such as autoimmune diseases, CDVs, most cancers, diabetes
and Alzheimer’s disease.
Nonsense mutation is a single-nucleotide substitution (e.g., C to T) that results in
the production of a stop codon.
Non-synonymous mutation is a mutation that alters the encoded amino acid
sequence of a protein. These include missense, nonsense, splice site, translation
start, translation stop and indel mutations.
Neoantigens are antigens specific to cancer cells.
Oncogene is a gene that, when activated by mutation, increases the selective growth
advantage of the cell in which it resides.
Pandemic is an epidemic, in which a disease spreads worldwide.
Pathobiont is an organism that can exist as an innocuous member of the microbiota
but that in some circumstances can cause pathogenesis.
218 Glossary
Pathogen-associated molecular patterns (PAMPs) are small molecules derived

from microbes, such as LPS. They are recognized by TLRs and other pattern-
recognition receptors on the surface of cells of the innate immune system.
Pattern-recognition receptors are evolutionarily conserved receptors that elicit
inflammation and innate immunity upon recognition of conserved microbial
products (PAMPs) or endogenous danger signals (DAMPs).
Peripheral tolerance is the mechanisms by which T cells and B cells are rendered
non-reactive to an antigen outside the primary lymphoid organs.
Phenotype is the set of observable characteristics of an individual resulting from
the interaction of its genotype with the environment.
Plasma cells are large, terminally differentiated B cells that continually secrete
antibodies
Pyroptosis is a specialized form of programmed cell death that requires CASP1,
CASP4 or CASP5 in humans. It is characterized by cytosolic swelling, early
plasma membrane rupture, nuclear condensation and internucleosomal DNA
fragmentation.
Rearrangement is a mutation that juxtaposes nucleotides that are normally sepa-
rated, such as those on two different chromosomes.
Regulatory T cells (Treg cells) are a subpopulation of T cells that are generally
immunosuppressive rather than pro-inflammatory.
RNA sequencing (RNA-seq) is a method using massive parallel sequencing to
reveal the presence and quantity of RNA in a biological sample at a given moment.
Senescence is also called biological aging, the gradual deterioration of functional
characteristics. It can refer either to cellular senescence or to senescence of the
whole organism.
Signal transduction cascade is the process by which a chemical or physical signal
is transmitted through a cell membrane as a series of molecular events, such as
protein phosphorylation catalyzed by protein kinases. Mostly, signal transduction
cascades end in the activation of a transcription factor or a chromatin modifier.
Somatic hypermutation is a cellular process, in which proliferating B cells accumu-
late mutations in antibody CDRs, potentially impacting their ability to recognize
antigen.
Stem cells can differentiate into other cell types and also divide in self-renewal to
produce more of the same type of stem cells. There are embryonic stem cells,
which are isolated from the inner cell mass of blastocysts, and adult stem cells,
which are found in various tissues.
Tolerance is a state of immune unresponsiveness and quiescence towards specific
antigens. In the case of transplantation, tolerance is directed towards donor-
specific antigens.
Toll-like receptor (TLR) is a class of patter-recognition receptors that play a key
role in the innate immune system.
Trait is a distinguishing quality or characteristic belonging to a person.
Transcription factors are proteins that sequence-specifically bind to genomic
DNA. Our genome encodes approximately 1600 transcription factors, referred to
as trans-acting factors, since they are not encoded by the same genomic regions,
Glossary 219
which they are controlling. Accordingly, the process of transcriptional regulation

by transcription factors is often called trans-activation.
Transcriptome is the complete set of all transcribed RNA molecules of a tissue
or cell type. It significantly differs between tissues and depends on extra and
intracellular signals.
Tumor antigens are antigens produced by the malignant tumor cell, typically in the
setting of enriched or specific expression relative to normal tissue(s).
Tumor suppressor gene is a gene that, when inactivated by mutation, increases the
selective growth advantage of the cell in which it resides.
Virome is the viral component of the microbiome.
Virulence is he capacity to cause severe illness once a pathogenic microbe infects
a host.
Zoonotic pathogens are pathogens naturally transmitted between animals and
humans.

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Carlberg C. Molecular Immunology. How Science Works 2022

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Carsten Carlberg

ISBN 978-3-031-04024-5 ISBN 978-3-031-04025-2 (eBook)

Kuopio, Finland Carsten Carlberg

1 Cells and Tissues of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . 1

5 Antigen-Presenting Cells and the Major Histocompatibility

11 Cancer Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

HBV Hepatitis B virus

NASH Non-alcoholic steatohepatitis

TBX21 T-box transcription factor 21

Keywords Pathogenic microbes · Infectious diseases · Pandemics · Innate

1.1 Global Burden of Infectious Diseases

The immune system is meant to protect us against pathogenic microbes, such as

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 1

Table 1.1 The immune

Table 1.2 Major pandemics in human history

percentage of deaths related to diseases associated with problems of the immune

1.2 Innate and Adaptive Immunity

Cardiovascular diseases 17.79 million

Cardiovascular diseases 21,359

Table 1.3 Comparing innate and adaptive immunity

antigen receptors, antibodies and specialized lymphoid tissues, evolved coor-

The innate immune system is composed of (Fig. 1.2, left):

Innate immunity Adaptive immunity

Complement Naïve T cell

and to promote their elimination by cellular (phagocytes) and humoral (complement

pro-T cell pre-T cell

CFU-M Monoblast Monocyte Macrophage

CFU-G Neutro myelocyte

their adaptive response to several environmental challenges, such as microbe infec-

Box 1.2: The impact of epigenetics in hematopoiesis During hematopoiesis

Box 1.3: The impact of master transcription factors in hematopoiesis In

Table 1.4 Normal blood cell counts

1.4 Primary and Secondary Structures of the Immune

Generative Blood, Peripheral

activate signal transduction cascades that lead to their proliferation, referred to as

Lymph node Naïve

Lymph node lymphatic

Naïve as well as memory B and T cells cannot be distinguished morphologically

Abnormal (ectopic) lymph node-like structures that form in peripheral tissues at

Keywords Innate immunity · Pattern-recognition receptors · PAMPs · DAMPs ·

2.1 Pathogen- and Danger-Associated Molecular Patterns

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 19

2.2 Myeloid Cells of Innate Immunity

The myeloid compartment of innate immunity comprises circulating monocytes,

Monocyte Macrophage NK cell

Band neutrophil Mature neutrohil Aged neutrophil

Lymphocyte Active lymphocyte Plasma cell

microbe, they express the chemokine receptor CCR7, which is stimulated by

· Antigen presen- · Phagocytosis · NETs

Cytokine and Adhesion Co-stimulatory

NK cells, ILCs as well as lymphocytes, are mononuclear cells. Neutrophils, basophils

2.3 Mechanisms of Phagocytosis

Box 2.3: Tissue-resident macrophages In addition to macrophages that

Classically activated Alternatively activated

Anti-microbial Anti- Wound

macrophages. Tissue-resident macrophages have a wide variety of homeo-

Box 2.4: Cluster of differentiation Immune cells are regularly phenotyped

Phagocytosis is an active, energy-dependent multistep process that aims on a

Microbes bind to Lectin receptor

Phagocyte membrane Lysosome with

Killing of microbes by ROS, NO

On the cellular level, phagocytes like neutrophils and macrophages secrete a

reduction in K+ ion concentration. Realizing that crystalline substances potently acti-

Local Systemic Systemic