Immunology

L A D E N SA L E H

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 Chapter 1

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 Introduction
Immunity: is the resistance to pathogens, noninfectious substances including
harmless environmental molecules, tumors, allergy, and consists of two things:-

1- Immune system: collections of cells, tissues, and molecules that mediate

immune response.
1- White blood cells.
2- Lymphocytes.
3- Bone marrow.
4- Thymus gland.
5- Spleen.
6- Vessels.
2- Immune response: reaction to microbes, as well as to other molecules that are
recognized as foreign.
Role of the immune system Implications
Defense against infections Deficient immunity results in
increased susceptibility (‫ )قابلية‬to
infections, exemplified by AIDS
(acquired immunodeficiency
syndrome)
Vaccinations boots immune
defenses and protects against
Infections.
Defense against tumors Potential for immunotherapy
Control of tissue regeneration and Repair of damaged tissue
scarring
The immune system can injure cells Immune responses are the cause
and induce pathologic inflammation of allergic, autoimmune, and other
inflammatory diseases.
The immune system recognizes Immune responses are barriers to
and responds to tissue grafts and transplantation and gene therapy
newly introduced proteins

 The reason why AIDS is more dangerous than influenza, is because AIDS
attacks the immune cells directly.
 Immunotherapy is a type of cancer treatment that helps your immune system
fight cancer, it is made up of white blood cells and organs and tissues of the
lymph system.

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  Since our immune system attacks cancer cells, why does it give no good results?
First, the growth of cancer cells is much faster than the growth of immune cells.
Second, in some cases the cancer can spread into the bone marrow and
weaken the immune system.
 The most important physiologic function of the immune system is to prevent or
eradicate infections.
 Stimulating immune responses against microbes through vaccination is the most
effective method for protecting individuals against infections; this approach has
led to the world wide eradication of smallpox, the only disease that has been
eliminated from civilization by human interventions.
Principal mechanisms of innate andadaptive immunity
Innate immunity Adaptive immunity
First line of defense for the body. Specific immunity or acquired
immunity.
Provides immediate protection Includes antigen recognition
against microbial invasion. molecules and specific sets of
lymphocytes.
Uses nonspecific cells such as Uses cells and molecules of the
phagocytes, complement innate immune system to eliminate
components microbes.
Required to initiate adaptive
immune responses against the
infectious agents.

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 Antigen & Antigen Recognition

 Antigen: Any molecule that is specifically recognized by lymphocytes or

antibodies, it can be Protein or non-protein.
Another definition: any foreign body enters the human body and stimulate an
Immune response.
It is derived from invading pathogens (virus, bacteria, and parasites), harmless
foreign substance (Dettol), derived from the self (self-antigen: produced by the
immune cells and attacked by the body).

 Epitopes: are the parts of the antigen

that binds to a specific antigen receptor
on the surface of a B-cell.
Slides definition: Small areas of the
molecular structures of the foreign
antigens.
The binding between the receptor and
epitope occurs only if their structures
are complementary.

 Antigen receptors or antigen recognition molecules.

 Antibodies (B-cell antigen receptors (immunoglobulin cells receptors)).
 T-cell antigen receptors. (TCR)
 Protein products of a genetic region (major histocompatibility complex
(MHC).

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 Cells of the immune system
 Lymphoid cells:
 Mostly are mediators of adaptive immune.
 Include B and T cells.
 Circulate through lymphoid organs and non-lymphoid tissues.
 Myeloid Cells:
 Tissue resident dendritic cells act as antigen presenting cells (APCs).
 Mast cells (considered as lymphocytes but its related to innate immunity).
 Phagocytes: neutrophils, macrophages and monocytes.
 Macrophages act as sentinels, destroys microbes, and in tissue repair.
Note: The primary source of the cells of the immune system is the
Hematopoietic stem cell (‫)الخاليا الجذعية‬.
Note: Dendritic cells are monocytes when it’s in the skin.

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 Types of Adaptive Immunity
 Humoral immunity (B-cells) )‫(المناعة السائلة‬
 Antibodies enter the circulation, extracellular tissue fluids, and lumens of
mucosal organs such as the gastrointestinal and respiratory tracts.
 Recognize proteins, carbohydrates, nucleic acids, and lipids (non-protein).
 Neutralize toxins.
 Enhance the uptake of extracellular microbes into phagocytes.
 Midterm question: A 16 years old girl got hepatitis B vaccine. Years later she got
exposed to hepatitis B, which immunity fights it?
The answer is: Humoral immunity
 Cell-mediated immunity (T-cells) )‫(المناعة الخلوية‬
 Most T cells recognize only peptide fragments of protein antigens presented on
cell surfaces, which means it doesn’t recognize non-protein.
 Defend against intracellular organisms.
 Kill any type of host cells (including non-phagocytic cells) that harbor
infectious microbes in the cytoplasm or nucleus.
 Defend against extracellular microbes by recruiting large
numbers of phagocytes to sites of infection.

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- In humoral immunity, B lymphocytes secrete antibodies that eliminate extracellular
microbes. In cell mediated immunity, some T lymphocytes secrete soluble proteins
called cytokines that recruit and activate phagocytes to destroy ingestes microbes, and
other T lymphocytes kill infected cells.

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 Types of Adaptive Immunity
 Active immunity
 There has to be immune response.
 Formed after exposure to the antigens.
 Natural: following infection.
 Artificial: by vaccination.
 Passive immunity
 No immune response needed
 Receiving antibodies or cells (e.g., lymphocytes) from another individual
already immune to an infection.
 Receiving protective antibodies synthesized using modern bioengineering
techniques.
 Natural: from mother across placenta or by breast feeding.
 Artificial: treating some immunodeficiency diseases, following snakebites,
antibodies and T cells designed to recognize tumors (Immunotherapy of
cancer).

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 Properties of Adaptive Immunity
 Specificity and Diversity
 Clonal selection hypothesis :
 Total population of B and T lymphocytes consists of many different
clones.
 Clones of lymphocytes specific for different antigens develop before
an encounter with these antigens.
 Each antigen elicits an immune response by selecting and activating
the lymphocytes of a specific clone.
 B cells: the daughter clones produce large amounts of soluble
receptor.
 T cells: produce large numbers of specific effector cells bearing the
appropriate receptor on their cell surface.
 TH1 T cells: usually produced in response to viral infections.
 TH2 T cells: produced in response to worms.
 TH17 T cells: respond to fungi and extracellular bacteria.
 Notes taken in the lecture:-
 Adaptive immunity is present in :
1. Lymph nodes (mostly).
2. Blood.
3. Spleen.
 Each clone has its own Antigen.
 Each lymphatic clone has almost at
least 1000 lymphocytes.
 When the Antigen links with the
clone, a proliferation happens,
increasing the number of cells, It’ll
keep increasing till the number of
cells is able to kill the Antigen, and
these cells split into group, and
each group has its’ own function.

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  Immunologic memory.
 The immune system remembers every encounter with antigen.
 Repeated exposure to the same antigen mounts faster, larger and more
effective responses.
 Optimizes the ability of the immune system to combat persistent and
recurrent infections.
 Depending on memory, vaccines confer long- lasting protection against
infections.
 Notes taken in the lecture:-
 Innate immunity doesn’t have memory cells.
 Memory cells are lymphocytes fought the Antigen but didn’t die, so it
became a memory cell.

- The primary immune response, is initiated by lymphocytes called naïve lymphocytes

that are seeing antigen for the first time.
- Subsequent encounters with the same antigen lead to responses called secondary
immune responses that usually are more rapid, larger, and better able to eliminate the
antigen that primary responses.
- Secondary responses are the result of the activation of memory lymphocytes.

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  Specificity.
 Ensures that immune responses are precisely targeted to microbial
pathogens.
 Clonal expansion.
 Rapidly increases the number of cells specific for the antigen encountered.
 Ensures that adaptive immunity keeps pace with rapidly proliferating
microbes.
 Self-limited.
 Declines as the infection is eliminated.
 Allows the system to return to a resting state (homeostasis).
 Prepare the system to respond to another infection.
 Stopping the immune system from attacking the body.
 Saving energy.
 The cells responsible for it is the regulatory T-cells.
 Immunological tolerance.
 Does not react against self-antigens.
 The cells responsible for it is the regulatory T-cells.

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 Cells of Adaptive Immune System
- Lymphocytes are the only cells that produce clonally distributed receptors
specific for diverse antigens and are the key mediators of adaptive immunity.

Note: Macrophages and granulocytes are present in both adaptive and innate immunity
Note: Cytotoxic T-cells are responsible for killing cancer and virus cells.

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 Classes of Lymphocytes

Note: Helper T lymphocyte is the most important immune cell in the human body.
Note: The Complement system is related to innate immunity, and it is a group of
proteins that help the antibodies.
Note: Helper T lymphocyte determine the type of antibodies produced by the B-cells.
Note: Natural killer is a lymphocyte related to innate immunity.

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 Classes of Lymphocytes

Note: MHC is a protein receptor present on the cell surface, transports the antigen to
lymphocytes in the lymph nodes.
Note: CD: cluster of differentiation.
Note: MHC class 1 is present in all of the cells except RBCs (red blood cells).
Note: αβ heterodimers is a protein complex composed of 2 peptides.
The meaning of the (-) is not present and (+) is present.
Class I – means CD8 is present.
Class II – means CD4 is present.

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 Maturation and Tissue Distribution of
Lymphocytes

- B lymphocytes mature in the bone marrow, and T lymphocytes mature in the thymus.
- Some B lymphocytes leave the bone marrow immature and complete their maturation
in the secondary lymphoid organs.
- Naïve lymphocytes express receptors for antigens but do not perform the functions
that are required to eliminate antigens. These cells reside in and circulate between
peripheral lymphoid organs and survive for several months up to a few years, waiting
to find and respond to antigen. If they are not activated by antigen, naïve lymphocytes
dies by the process of apoptosis and are replaced by new cells that have arisen in the
generative lymphoid organs.

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 Stages in the Life History of Lymphocytes

Naive cells are present in lymph organs, mainly lymph nodes and spleen.
Activated or effector lymphocyte, leave the lymph organs to meet the antigen at entry
point.
Memory lymphocytes could be anywhere, and they have no function till they get
exposed to the same antigen.

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 - Naïve lymphocytes recognize foreign antigens to initiate adaptive immune responses.
Naïve lymphocytes need signals in addition to antigens to proliferate and differentiate
into effector cells. Effector cells, which develop from naïve cells function to eliminate
antigens. The effector cells of the B lymphocyte lineage are antibody-secreting plasma
cells. The effector cells of the CD4 lymphocyte lineage produce cytokines. The effector
cells of CD8 lineage are CTLs (cytotoxic T-cells). Other progeny of the antigen-
stimulated lymphocytes into long-lived memory cells.

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 Antigen-Presenting Cells
Common portals of entry for microbes.
Specialized cells in the epithelium of skin, gastrointestinal, respiratory, and
genitourinary tracts.
 Capture protein antigens, transport them to peripheral lymphoid tissues.
 Present fragments of proteins for recognition by T- lymphocytes. Include:
 Dendritic cells: present antigens and stimulate naïve T-cell to
proliferate and differentiate into effector cells by production of
co-stimulators. The most specialized antigen presenting cells
in the immune system.
 Macrophages: present antigens to differentiated effector T-cells.
 B-cells: present antigens to differentiated effector T-cells.
 Follicular dendritic cell (FDC): resides in the germinal centers of
lymphoid follicles in the peripheral lymphoid organs and
displays antigens that stimulate the differentiation of B cells in
the follicles. FDCs do not present antigens to T cells and differ
from the dendritic cells.

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 Peripheral (Secondary) Lymphoid Organs and Tissues
Lymphoid organs are divided into two main groups:
1. Primary lymphoid organs (generative lymphoid organs).
 Bone marrow.
 Thymus gland.
2. Secondary lymphoid organs.
 Lymph nodes.
 Spleen.
 Mucosal lymphoid tissue.
 Cutaneous lymphoid tissue.

The most lymphocytes

The lease lymphocytes

The least lymphocytes

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 1. Lymph Node.
 Encapsulated nodular aggregates of lymphoid tissues located along lymphatic
channels throughout the body.
 Responses to lymph-borne antigens.
 Contain a fluid called lymph.

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Segregation of T and B lymphocytes in Different Regions of Peripheral Lymphoid
Organs

1. Follicle
 Located around the periphery, or cortex, of each node.
 Site of B-cell segregation inside the lymph node.
 Contain the FDCs (follicular dendritic cells) that secrete chemokine to
activate B cells and express(B-cell receptors) CXCR5.
 B cells don’t need to migrate out of the lymph node.
2. Germinal center
 A central lightly staining region in the follicle.
 Contains B cells that have recently responded to a protein antigen and
received signals from helper T cells.
 Contain plasma cells which create antibodies and leave the lymph node
into circulation to reach the sites of inflammation.
3. Paracortex (T-Cell zone)
 Contains dendritic cells that present antigens to T lymphocytes.
 Dendritic cells activate naïve T-cells to express CCR7.
 After the T cells are activated by dendritic cells, proliferation and
differentiation happens, and T cells differentiate into effector T cells.
 Effector T cells leave the lymph node via efferent lymphatic vessel to
react with microbes outside at site of entry.
Effector T cells are divided into:-
1. Helper T cells.
2. Cytotoxic T cells.
3. Regulatory T cells.
Notes:
1. High endothelial venule (HEV): the site of entry for lymphocytes.
2. The antigen together with antigen presenting cells enter the lymph node
through afferent lymphatic vessel.

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 2. Spleen
 consists of two main things:
1. Red pulp: called red pulp because it contains RBCs.
2. White pulp: contains WBCs and lymphocytes.
The space between Red pulp and White pulp is called
marginal zone.
Periarteriolar lymphoid sheaths (T-Cell zone):
 The site in the spleen where T -lymphocytes are concentrated.
 Dendritic cells activate naïve T-cells to express CCR7.

 Highly vascularized abdominal organ responses to blood-borne antigens.

 Sinusoidal macrophages destroy old RBCs.
 The spleen contains abundant phagocytes that line the sinusoids, which
ingest and destroy microbes in the blood. These macrophages also ingest
and destroy old red blood cells.

Two types of Antigen:

1. Blood borne antigen, carried by antigen presenting cells
through the blood (directly) to the spleen.
2. Lymph borne antigen, goes to lymph nodes through lymph cannels.

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 3. Cutaneous immune system (Skin) 4. Mucosal immune system

 These two types have many different cells, either innate immune cells
oradaptive immune cells.
 Most of the immune cells in these tissues are diffusely scattered beneath the
epithelial barriers.
 There are discrete collections of lymphocytes and APCs organized in lymph
nodes like tonsils in the pharynx and Peyer patches in the intestine.
 The only difference between CIS and MIS, is Langerhans cells, which are
immature dendritic cells present in CIS

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 Immune cells Interaction
 Lymphocyte are kept apart from each other until it is useful for them to interact,
after exposure to an antigen.
 Antigen activated B cells and T cells migrate toward each other and meet at the
edge of follicles.
 Helper T cells interact with and help B cells to differentiate into antibody
producing cells.
 Many of effector T cells exit the node through efferent lymphatic vessels and
leave the spleen through veins and end up in the circulation where they can go to
distant sites of infection.
 Some activated T cells remain in the lymphoid organ and migrate into lymphoid
follicles, where they help B cells to make high-affinity antibodies.
 Any antigen enters the body, simple or dangerous, not one cell interact, many
cells interact with each other to get the job done.

Lymphocyte Recirculation and Migration into Tissues

 High endothelial venules (HEVs) is
not present in spleen.
 Plasma cells do not need to migrate to
sites of infection.
 Antibodies enter the blood bind
pathogens or toxins in the blood, or
intissues into which the antibodies
enter.
 Effector T cells have to locate and
eliminate microbes at sites of
infection.

The darkest nights make the brightest stars.

- Witt Lowry

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 Chapter 2

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 Innate immunity
 Innate immunity: The first line of host defense, always present, functional, ready
to eliminate microbes and dead cells.
 Innate immune system: cells and molecules responsible for innate immunity.
Components of Innate Immunity
 Epithelial barriers: First line of defense.
1. Physical barrier: Physical barrier: keratin (in the skin) or secreted
mucus (in the gastrointestinal, Broncho pulmonary, and genitourinary
systems).
Keratin and mucus, are local antibiotics produced by epithelial cells that
kills the microbe immediately.
2. Chemical barrier: produce antimicrobial substances like defensins and
cathelicidins.
3. Intraepithelial T lymphocytes: express γ and δ receptors with limited
diversity.

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 General Features and Specificity of Innate
Immune Responses
 Innate immune response performs two types of reactions :
1. Inflammation. (Extracellular)
 Accumulation and activation of leukocytes and plasma proteins at
sites of infection or tissue injury.
 Action of cells and proteins together to kill mainly extracellular
microbes and to eliminate damaged tissues.
2. Antiviral defense. (Intracellular)
 Defense against intracellular viruses mediated by natural killer
(NK) cells.
 Kill virus-infected cells and release type I interferons (IFNs), which
block viral replication within host cells.
 The same immune response, no primary response nor secondary
response.
 Does not remember prior encounters with microbes and responds the
same:
 Macrophages and natural killer cells are altered by encounters with
microbes so they respond better upon repeat encounters.
 Recognizes structures that are shared by various classes of microbes not
in the host:
 Pattern recognition receptors recognize pathogen associated
molecular patterns (PAMPS).
 e.g.: LPS, peptidoglycan structures in bacteria, mannose residues in
bacteria and fungus, viral dsRNA , unmethylated CpG….etc.
Epitope (Adaptive immunity) •> PAMP (Innate immunity)
PAMP: a part of the microbe the innate immunity recognizes, the microbe itself
can’t give up its’ PAMP, it’s essential for the microbes’ life, various microbes
share the same PAMP.
Some antigens can give up its epitope without dying, so it can escape the
immune response.
 Receptors are specific for structures of microbes that are essential for their
survival and infectivity :
- A microbe cannot evade innate immunity by mutating or not
expressing the targets of innate immune recognition microbes.

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  Recognizes molecules called damage-associated molecular patterns
(DAMPs) released from damaged or necrotic host cells:
- Example: include high mobility group box protein 1 (HMGB1), a
histone protein that is released from cells with damaged nuclei,
extracellular ATP.
Innate immunity doesn’t have auto-immune disorder, the reason behind that is:
1. There are no Self-PAMP.
2. Present of regulatory molecules.
3. Present of endosomal PRR.
 Receptors are encoded by inherited genes that are identical in all cells :
- Identical receptors are expressed on all the cells of a particular type.
- Many cells may recognize and respond to the same microbe.
 Does not react against healthy cells:
- Receptors are specific for microbial structures (and products of
damaged cells) only.
- Some pattern recognition receptors that recognize nucleic acids
present in normal cells in cellular compartments (such as
endosomes) from where components of healthy cells are
excluded.
- Mammalian cells express regulatory molecules that prevent innate
immune reactions.

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 Specificity and receptors of innate immunity and adaptive immunity

In adaptive immunity one cell recognizes the antigen.

In innate immunity many cells recognize the same PAMP.
PRR present in:
1. Endosomal PRR.
2. Cytosol.
3. Skin surface.

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 Cellular Receptors for Microbes and Damaged Cells
 Expressed on phagocytes, dendritic cells, and many other cell types.
 Located on the surface, in vesicles (endosomes) and in the cytosol.
 Five major families of receptors: TLRs (Toll-Like Receptors), CLRs (C-type
lectin receptors), NLRs (NOD- like receptors), RLRs (RIG-like receptors),
and CDSs (cytosolic DNA sensors).

- Some receptors, such as certain Toll-like receptors (TLRs) and lectins, are located on
cell surfaces; other TLRs are in endosomes. Some receptors for viral nucleic acids,
bacterial peptides, and products of damaged cells are in the cytoplasm. NOD and RIG
refer to the founding members of families of structurally homologous cystollic receptors
for bacterial and viral products, respectively.

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 Specificities of Toll-like Receptors
 There are 10 different TLRs specific for different components of microbes.
 TLRs specific for microbial proteins, lipids, and polysaccharides are located
oncell surfaces.
 TLRs that recognize nucleic acids are in endosomes.
 You need to know each type and what it recognizes.

Examples of:-
1. dsRNA virus: herpes simplex virus:
2. ssRNA: corona virus.

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 Signaling Functions of Toll-Like Receptors
 TLRs generate signals that activate transcription factors
 Stimulation of transcription factors lead to expression of cytokines and other
proteins involved in the inflammatory response and in the antimicrobial functions
of activated phagocytes and other cells.
 Mutations in TLR-3 increase susceptibility to herpes simplex virus encephalitis.
 Mutations in MyD88 (the adaptor protein downstream of several TLRs) make
individuals susceptible to bacterial pneumonias.
2 videos to watch to understand everything.
1. https://www.youtube.com/watch?v=NNOnQdgWGR8
2. https://www.youtube.com/watch?v=cKSWHMRPOoI
To watch, click Ctrl + the link to open.

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 NOD-Like Receptors
 Innate receptors that sense DAMPs (mainly NOD-PYD (inflammasome)) and
PAMPs (mainly NOD-CARD) in the cytosol of cells.
 Initiate signaling events that promote inflammation.
 Contain a nucleotide oligomerization domain (NOD).
 Two NLRs:NOD1& NOD2:
- Have N-terminal caspase related domains (CARDs).
- Expressed in several cell types including mucosal barrier epithelial cells and
phagocytes.
- Recognize peptides derived from bacterial cell wall peptidoglycans.
- Generate signals that activate NF-κB transcription factor, which promotes
expression of genes encoding inflammatory proteins.
- NOD2 is highly expressed in intestinal Paneth cells in the small bowel stimulating
expression defensins (chemical barrier) in response to pathogens.
- NOD2 gene polymorphisms are associated with inflammatory bowel disease.

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 Inflammasomes
 Multiprotein complexes that assemble in the cytosol of cells in response to
microbes or changes associated with cell injury.
 Generate active forms of the inflammatory cytokines IL-1β and IL-18 under
the influence of caspase1.
 Composed of oligomers of a NLR-family proteins as sensor, caspase-1 and
an adaptor.
 Lead to generation of IL-1 that induces acute inflammation and causes
fever.
 Gain-of-function mutations in NLRP3, and less frequently, loss-of-
function mutations in regulators of inflammasome activations, are the
cause of auto-inflammatory syndromes. IL-1 antagonists are effective
treatments for these diseases.
 Cause pyroptosis: programmed cell death of macrophages and DCs.
- Gout disease: is caused by deposition of urate crystals and subsequent
inflammation mediated by inflammasome recognition of the crystals and
IL-1β production.
Video to watch
- https://youtu.be/xJPOCnbZrQ8?t=1834

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 Cytosolic RNA and DNA Sensors
 Innate immune system is able to recognize microbial RNA or DNA and respond
by generating signals that lead to the production of inflammatory and antiviral
cytokines:
- RIG-like receptors (RLRs) are cytosolic proteins sense features of viral.
- RLR interact with protein called mitochondrial antiviral- signaling (MAVS)that
needed to activate transcription factors that induce the production of type I
IFNs.
- Cytosolic DNA sensors (CDSs) mostly engage the stimulator of IFN
genes (STING) pathway to induce type 1 IFN production.
- Proteins recognize microbial double-stranded (ds) DNA in the cytosol.
- They activate signaling pathways that initiate antimicrobial responses,
including type 1 IFN production and autophagy.

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 Other Cellular Receptors of Innate Immunity
 Dectins (‫)بروتين يتعرف على السكريات‬: lectins in the plasma membrane specific for
fungal glucans.
 Mannose receptors: lectins for the terminal mannose residue.
- Involved in fungal and bacterial phagocytosis and inflammation.
 Formyl peptide receptor 1:
- Recognizes bacterial proteins with an N-terminal formylmethionine
polypeptides.
Components of Innate Immunity:
 Phagocytes:
- Neutrophils (polymorph nuclear leukocytes (PMNs):
 The most abundant leukocytes in the blood
 Count 4,000 to 10,000 per μL
 Produce in response to certain bacterial and fungal infections.
 The dominant cells of acute inflammation.
 Express receptors for complement products activation and for antibodies
that coat microbes.
- Monocytes/macrophages:
 Less abundant in the blood than neutrophils.
 Count 500 to 1000 per μL.
 During inflammation they enter extravascular tissues and differentiate into
macrophages.
 Survive in for long periods.
 Macrophages are resident in different tissues derived from progenitors in
the yolk sac or fetal liver
Notes:
1. Neutrophils is the most important cell in inflammation, because it’s the first
cell to arrive at the inflammation site, and the most abundant
leukocyte in the blood. However, it has a low effect, it lasts for 1-2 days.
2. Macrophages’ effect comes after the death of Neutrophil, and its’ effect is
longer.
3. Neutrophils does cytosis only on the microbe, macrophages swallow
everything in the site (site of inflammation).

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 Neutrophil Monocyte
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  Dendritic Cells:
 Initiate inflammation and stimulate adaptive immune responses.
 Capture protein antigens and display fragments of these antigens to T
cells
 Mast Cells:
 Present throughout the skin and mucosal barriers.
 Activated by microbial products, complement components or antibody.
 Release histamine, proteolytic enzymes, and heparin.
 Synthesize prostaglandins and leukotrienes, cytokines, TNF.
 Provide protection against helminthes (‫)الديدان‬, snake and insect
venomes.
 Responsible for symptoms of allergic diseases.

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 Maturation of mononuclear phagocytes

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 Activation and Functions of Macrophages
Macrophages are activated in four ways:
1. By TLR or PRR
2. Cytokines IL-2 produced by NK cells.
3. Complement system.
4. Activation of T-helper.
 Classical macrophage activation of M1 macrophages:
- Destroy microbes and trigger inflammation.
- Involve TLRs & NK cell–derived interferon-γ IFN-γ.
 Alternative macrophage activation of M2 macrophages:
- Occurs in tissue repair and terminating inflammation
- Involve IL4, IL13.
Notes:
1. When the macrophages are unable to kill the microbe it asks for help from
NK cells.
2. Neutrophil produces mainly reactive oxygen species (ROS).
3. Macrophages produce mainly Nitric oxide.

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Phagocytosis and Intracellular Killing of Microbes
 Phagocytosis:
- Ingestion of particles larger than 0.5 μm in diameter.
- Oxidative burst (or respiratory burst): formation of reactive oxygen species
(ROS) by phagocyte oxidase.
- Inducible nitric oxide synthase (iNOS): produced mainly in macrophages and
catalyzes the conversion of arginine to nitric oxide (NO), also a microbicidal
substance.
- Lysosomal proteases: break down microbial proteins.
- Neutrophil extracellular traps (NETs): network of chromatin formed following
death of neutrophils, contain antimicrobial substances.
- NETs: kill bacteria, fungus and other organisms.
- Tissue repair: involve macrophages, ILCs, and helper T cells.
- Chronic granulomatous disease (CGD): Inherited deficiency of the
phagocyte oxidase enzyme, inability of neutrophils to kill microbes and
accumulation of macrophages forming granulomas.
Notes:
1. Phagosome: a room inside the cell containing the microbe.
2. To kill the microbe, phagosome and lysosome fuse together to form
phagolysome, and by using lysosome enzymes, the microbe is killed
3. To make sure the microbe is killed, toxic materials are produced (nitric
oxide, reactive oxygen species (ROS)).

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Innate immune response react with microbes by one of two methods:-
1. Inflammation.
Mainly involving extracellular microbes.
Innate immune cells involved in inflammation are 4 types:-
1- Dendritic cells.
2- Macrophages.
3- Mast cells.
4- Neutrophils.
Inflammatory process:-
1. Entry of the microbe, stimulation of dendritic cells (indicating factor for
inflammatory process), it produces other cytokines that induce other immune
cells like mast cells.
2. Mast cells (sentinel cells/ ‫ )خاليا حارسة‬secrete vasoacting agents like histamine,
heparin, prostaglandins, the end product of these vasoacting agents is
vasodilation and increased capillary permeability, allowing plasma proteins
mainly complement system to enter the tissues.
3. Plasma proteins and complement system reach the inflammation site and
work on killing the microbe, but it doesn’t kill it completely, for the inflammation
to continue, we have other requirements for inflammatory process, which are
leukocytes.
Leukocytes mainly neutrophil and macrophages, macrophages are already
present in the tissue, but neutrophil is in the blood, neutrophils reach the tissue
by a process we call Migration of neutrophil.

- Cytokines and other mediators are produced by sentinel cells (macrophages, dendritic
cells, and mast cells), and other cells in tissues in response to microbial products and
damaged host cells.
- Histamine and prostaglandins increase the permeability of blood vessels, leading to the
entry of plasma proteins (mainly complement system) into the tissues.
- IL-1 and TNF increase expression of endothelial adhesion molecules and chemokines that
promotes the movement of leukocytes from the blood into the tissues.
- Leukocytes destroy microbes, clear damaged cells, and promote more
inflammation and repair.

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 Sequence of Events in Migration of Blood Leukocytes (Neutrophils) to Sites of
Infection
The goal of this process is to get the neutrophil from intravascular space to the
extravascular space.
1. The neutrophil is in the blood, and its speed is the same as the blood, the first step
is to slow neutrophil by selectin/selectin ligand.

Selectin: protein receptor present on the surface of endothelial cells.

For the selectin to appear on endothelial cells it needs to be induced by cytokines
(TNF, IL-1, and thrombin)
2. Selectin bind with selectin ligand, and now the speed of neutrophil is reduced, we
call this step “rolling”.
3. There is something called integrin, but it has a low-affinity state )‫(ال يوجد قابلية لالنجذاب‬
, also there is integrin ligand present on the endothelial surface.

Now the integrin can’t bind with integrin ligand because it has a low-affinity state, to
make it high-affinity we have something called chemokines.

Chemokines: small protein molecules present on the surface of

endothelial cells, because it’s small, it is carried by proteoglycans.
Proteoglycans function is just to show the chemokines.
4. Once chemokine binds with the chemokine receptor present on the neutrophil, the
integrin becomes high-affinity state, then interactions between integrin and integrin
ligand happens.
5. The cells stops at intercellular space, which is a gate, but it needs a powerful strain,
so the complement system pull it by C5a and C3a (chemoattractants) in a process
called chemoattraction.

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  Neutrophils and monocytes migrate to extravascular sites of infection or
tissue damage by binding to venular endothelial adhesion molecules in
response to chemoattractants produced by tissue cells reacting to infection or
injury.
 Integrins: Lymphocyte function- associated antigen (LFA-1), Very late antigen-4(
VLA-4), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion
molecule–1 (ICAM-1).
- Inherited deficiencies in integrins and selectin ligands lead to disorders
Called leukocyte adhesion deficiencies (LADs).
The baby reaches 40 days and the umbilical cord hasn’t fallen yet, because
for the umbilical cord to fall there has to be inflammation and then it stops
and tissue repair happens and necrosis, but nothing happens why?
Because of Inherited deficiencies in integrins and selectin ligands.

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 Regulation of Innate Immune Responses
 Anti-inflammatory cytokines:
These anti-inflammatory cytokines stop the inflammation process, to prevent
exhaustion of lymphocytes.
As soon as the microbe dies, anti-inflammatory cytokines are secreted and stops
the inflammatory process, also macrophages (M2), start tissue repairing.
1. Interleukin-10:
- Inhibits the microbicidal and proinflammatory functions of classical
pathway of macorphages.
2. IL-1 receptor antagonist:
- Blocks IL-1 actions.
3. Suppressors of cytokine signaling (SOCS):
- Stimulated by TLR signaling.
- Blocks the responses of cells to various cytokines, including IFNs.
4. Inflammasome intracellular regulators.

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2. Antiviral defense.
4 ways to kill viruses by innate immune response:

1. Apoptosis (‫موت الخلية المبرمج‬/‫)انتحار الخلية‬: when the virus enters the cell and
there is no other way to kill it but to kill the whole cell )‫ ( خيار نهائي‬.

2. Autophagy (‫)أكل الخلية لنفسها‬: the virus enters the cell and CDSs (cytosolic DNA
sensors) is induced, activation of lysosome, lysosome engulfs the endosome
containing the virus.

Difference between Apoptosis and Autophagy

Apoptosis, the whole cell dies.
Autophagy, just the part containing the virus dies.

3. Antiviral actions of type I interferons(IFN-α and one of IFN-β) by

Plasmacytoid dendritic cells.
Morphologically resemble plasma cells. Functionally resemble dendritic
cells.

Plasmacytoid dendritic cells are present in:-

1. Tissues.
2. Blood.
The main function of Plasmacytoid dendritic cells is to kill the virus by producing
cytokines called interferon type 1.
Viruses consists of two things:-
1. RNA or DNA.
2. Envelope (which is bunch of proteins).
How do Plasmacytoid dendritic cells kill the virus? By
three ways:-
1. Prevent the formation of the envelope.
2. Degradation of viral RNA or DNA.
3. If it can’t perform the two ways above, it prevents
the assembly of nucleic acids

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 4. NK cells killing of virus infected cells under the influence of type I
interferon (the most important innate immune cell to kill viruses)
You can differentiate (‫ )ت ُ َميّز‬NK cells from other immune cells by two receptors
presenton the surface on NK cells.
1. Activating receptor (NKG2D).
2. Inhibitory receptor (KIR).

Activating and Inhibitory Receptors of Natural Killer Cells

A- Inhibitory receptor engaged ITAM = Domain

Inhibition is mediated by activation of ITIMs, tyrosine phosphatase, de- phosphorylation

of tyrosine molecules
Every cell in our human body contains MHC I (except RBCs), NK cells are always
active and looking for viruses in our body, it finds a normal human cell, and finds on
its surface MHC I. MHC I binds with inhibitory receptor, which result in activating it,
ITIM is activated. Then activating receptor binds with activating ligand on the
normal cell, ITAM is activated.
ITIM (Immunoreceptor tyrosine-based inhibitory) prevent ITAM
(Immunoreceptor tyrosine-based activation) from activating NK
cell.
B- Inhibitory receptor not engaged
Activation is mediated by cytoplasmic ITAMs and tyrosine kinase and phosphorylation
of tyrosine molecules.
NK cell finds a virus cell, and it doesn’t find MHC I,
so inhibitory receptor is not activated, so ITIM is not
activated, only activation receptor is activated,
therefore ITAM is activated and NK cell is activated.
Activating receptors of NK:
 NKG2D: recognizes molecules that resemble
class I major histocompatibility complex
(MHC) proteins and are expressed in
response to many types of cellular stress.
 CD16: is specific for IgG bound to cells.

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Inhibitory receptors on NK:
 Killer cell immunoglobulin-like receptors (KIRs).
 Receptors consisting of CD94 and a lectin subunit called NKG2.

Lymphocytes

Lymphocytes

Innate lymphoid cells B cells T cells

Unconventional (innate Conventional

Cells involved in killing
Bacteria and Fungi. specificity and limited
Cells involved in killing
Parasites.
immunity), has no
diversity.
follicular because its present in
B-1 cells, present in
plural space and
peritoneal cavity
Unconventional (innate (adaptiveimmunity),
Conventional (adaptive immunity). has high specificity
immunity), another name is and broad diversity.
follicular B cells, called
follicles. Mucosal associated
invariant T cells. (MAIT)
CD4 (MHC II)
mainly in the human
liver

Marginal zone B cells

Cells involved in killing NK-T Cells.
(spleen), kills blood- Regulatory T cells
viruses, mainly NK cells.
borne antigen.

Intraepithelial T
lymphocytes CD8 (MHC I)

NK-T cells, the reason behind naming it NK cells:-

1. It has protein receptors on its surface the same as NK cells.
2. Help NK cells.
The reason behind naming it T cells.
Because it has TCR (unspecific).

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 - Innate Lymphoid Cells (ILCs):
- Produce cytokines similar to those secreted by helper T lymphocytes.
- Do not express T cell antigen receptors (TCRs).
- Divided into three groups.
- Stimulated by cytokines produced by damaged epithelial and other cells at sites of
infection.
1. Natural Killer Cells (Cells involved in killing viruses):
 Developmentally related to group 1 ILCs.
 Make up approximately 10% of lymphocyte in the blood and
peripheral lymphoid organs.
 Contain cytoplasmic granules and express some unique surface proteins.
 Do not express immunoglobulins or T cell receptors, the antigen receptors
of B and T lymphocytes.
 Eliminate cellular reservoirs of infection and eradicate infections by
obligate intracellular microbes,
 Contribute to destruction of tumor.
 Need IL15, IL12, and IFNs for development and maturation.

2. Cells involved in killing Parasites.

3. Cells involved in killing Bacteria and Fungi.

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 - T cells (unconventional):
1. NK-T cells:
- Express TCRs with limited diversity.
- Their surface molecules typically similar to NK cells.
- Present in epithelia and lymphoid organs.
- Recognize microbial lipids bound to a class I MHC– related molecule CD1.
2. Mucosal associated invariant T (MAIT) cells:
- Express TCRs with limited diversity.
- Do not express CD4 or CD8.
- Present in mucosal tissues.
- Most abundant in the human liver.
- Account for 20% to 40% of all T cells in that organ.
- Specific for bacterial vitamin B metabolites.
- Contribute to innate defense against intestinal bacteria.
3. Intraepithelial T lymphocytes (epithelial barrier).
- γ δ T cells.

- B cells (Unconventional):
1. B-1 cells:
- Found mostly in the peritoneal cavity and mucosal tissues.
- Produce antibodies in response to microbes and microbial toxins that pass
through intestinal walls.
- Produce circulatory IgM antibodies specific for ABO blood group antigens.
2. Marginal-zone B cells:
- Present at the edges of lymphoid follicles in the spleen and other organs.
- Involved in rapid antibody responses to blood-borne polysaccharide-rich
microbes.

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 - Cytokines
Cytokines are communication messages between innate immune cells. (Immune
hormones).
Cytokines are secreted by dendritic cells, cytokines induce other immune cells like
mast cells, and some cytokines induce hepatocytes by endocrine to produce
plasma proteins, mainly complement system.

- Soluble proteins that mediate immune and inflammatory reactions.

- Responsible for communications between leukocytes and other
cells.
- Stimulated by recognition of cell wall components by TLR or cellular
components by TLR, RLR, CDSs mediated by different cells.
- Have paracrine, autocrine and endocrine actions.
- TNF promotes thrombus formation on the endothelium and reduces blood
pressure by a combination of reduced myocardial contractility and vascular
dilation and leakiness. Severe, disseminated bacterial infections sometimes
lead to a potentially clinical syndrome called Septic shock.

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 - Dendritic cells, macrophages, and other cells (such as mast cells and ILCs (not shown)) respond
to microbes by producing cytokines that stimulate inflammation (leukocyte recruitment) and
activate natural killer (NK) cells to produce the macrophage-activating cytokine interferon-γ
(IFN-γ).
- Some important characteristics of the major cytokines of innate immunity are listed in the table
above. Note that IFN-γ and transforming growth factor beta (TGF-β) are cytokines of both
innate and adaptive immunity.

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 Acute Phase Response Proteins
- Acute phase reactants: plasma proteins secreted by hepatocytes
in response to activation of cytokines.
1. Mannose binding lectin(MBL):
- Belongs to collectins family protein.
- Recognizes microbial carbohydrates and can coat microbes for
phagocytosis
- Activate lectin complement pathway.
2. Surfactant proteins:
- Protect the airways from infection.
3. C-reactive Protein (CRP) ‫معامل االلتهاب‬
- Acute phase reactant secreted during acute inflammation.
- Binds to phosphorylcholine on microbes and opsonizes the
microbes for phagocytosis.
- Activates proteins of the classical complement pathway.
- If CRP levels are high in blood, that means there is an inflammation.

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 Complement System (Part of the Acute Phase Reactants)

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Complement system is composed of 9 proteins (C1-C9). C1 = Complement factor 1.

We have 3 pathways for the complement system, classical pathway, .‫وعلى ذلك قِس‬
lectin pathway, and alternative pathway, which pathway will activate
the complement system? Depends on the microbe itself, specifically PAMP.

1. The alternative pathway is triggered when some complement proteins are activated on microbial
surfaces and cannot be controlled, because complement regulatory proteins are not present on
microbes (but present on host cells). The alternative pathway is a component of innate
immunity.
2. The classical pathway is most often triggered by antibodies that bind to microbes or other
antigens and is thus a component of the humoral arm of adaptive immunity.
3. The lectin pathway is activated when a carbohydrate-binding plasma protein, mannose binding
lectin (MBL) bind to its carbohydrates ligands on microbes. This lectin activates proteins of the
classical pathway, but because it is initiated by a microbial product in the absence of antibody, it
is a component of innate immunity.
- Strep pneumonia PAMP activates classical pathway.
- Polysaccharides PAMP activates alternative pathway.
- Mannose PAMP activates lectin pathway.
When either of the pathways gets activated, we have initial steps specific for each pathway, all
of them assemble at the same point (common point), which is C3 convertase, which is an
enzyme consists of C1, C2, and C4.
C3 convertase convert C3 into 2 parts, C3a (inflammation specifically at chemoattraction for
neutrophils), and C3b.
C3b has two functions:-
1. Opsonization, marking of the microbe.
2. C5 convertase, C5 will divide into two parts, C5a (inflammation specifically at
chemoattraction for neutrophils), and C5b.
C5b assemble C6-C9 and produces a complex called MAC (membrane attack complex), it
has a cylindrical shape, why? Because its function is to make a hole through the wall of
the cell, and lysis of microbe.

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 Microbial Evasion of Innate Immunity

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 Chapter 3

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 Antigen Capture and Presentation to Lymphocytes:
What Lymphocytes See
- Antigens enter the human body in two ways:-
1. Blood borne antigen (spleen).
2. Tissue born antigen (lymphnodes).
It enters through afferent lymphatic vessel.

Antigens Recognized by T-Lymphocytes

- The majority of T lymphocytes recognize peptide antigens bound to and displayed by
the MHC molecules of antigen-presenting cells (APCs) .

Capture and Display of Microbial Antigens

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 Antigen Uptake, Maturation and Migration of Dendritic Cells to Activate.
This is an immature dendritic cell, which responsible for capturing the antigen
(first step)

Here the dendritic cell reached the lymphnode and became mature
What is the differences between mature DC and immature DC:-
1. Loss of adhesiveness )‫(تفرعات أكثر‬.
2. Highly expressed MHC molecules.
3. Expression of costimulatory molecules such as CD40, CD80 and CD86.
For activation of any lymphocytes we need two signals:-
1- Signal one produced by interactions between MHC and TCR.
2- Signal two produced by costimulatory molecules.
4. Secretion of cytokine called CCR7 (‫)دليل سياحي للخلية‬.Induction of DC migration to lymphoid
organs to present the processed antigens for T cell activation.

- All DCs express PRRs on their surface including TLRs and C-type lectin receptors (CLRs).
- Dendritic cells mature upon pathogen recognition or in response to cytokines such as
TNF, IL1.

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 Structure of MHC Molecules
- MHC has two functions:-
1. Principle determinant of acceptance or rejection in tissue or organ
transplantation (‫)مطابقة األعضاء‬.
2. Antigen presentation for lymphocytes (CD4 or CD8).

- Class I MHC molecules:

- Composed on only one peptide chain called α chain, it is composed of 3 parts called
domain, α1 domain, α2 domain, α3 domain, each domain is connected to the other
domain by disulfide monomer.
- Ig domain is very similar to α3 domain, it has the same disulfide bond.
- α1 domain and α2 domain are very similar, they are polymorphic domains, because
they consist of two walls and peptide binding cleft.
- α1 domain and α2 domain form a group called peptide binding cleft.
- α3 domain is invariant (constant), main function is interactions with TCR (contains a
site that binds the CD8 T cell coreceptor).
- β2-microglobulin, a very small protein molecules present here for stabilization (to
stabilize MHC I), it’s not connected with the α chains, it stays stable by electromagnetic
interactions.
- Class II MHC molecules:
- Composed of two chains, α chain, β chain.
- α chain is composed of two domains, α1 domain (polymorphic), α2 domain
(invariant/constant).
- β chain is composed of two domains, β1 domain (polymorphic), β2 domain
(invariant/constant).
- β1 and α1 form a group called peptide-binding cleft, it
will be compatible with the peptide.
- β2 and α2 for TCR interactions (binding site for the CD4
T cell coreceptor).

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 Major Histocompatibility Complex
Genetic structure of MHC I and MHC II
- MHC I is a protein molecule, has 3 genes present on chromosome 6:-
1. B.
2. C.
3. A.
To get a MHC we need to get the 3 genes from both father and mother.
To form B gene we need to get B allele from the mother and B allele from the father.
The mothers B allele is different from the fathers B allele, they differ in the
arrangement of nucleic acids, and they are the same number and same kind, these
allele are co-dominant.
In the whole world we have only 620 B genes, they’re composed of the same nucleic
acids but differ in the arrangement.
The C gene the same as B gene, we need to take C allele from the mother and C allele
from the father, it is found that almost in the whole world the arrangement doesn’t
differ, it is constant.
The A gene the same as B and C gene, we need to take A allele from the mother and
A allele from the father, the arrangement differ.
How many alleles we need from the mother and father to form MHC I molecule? We
need 2 allele for B, and 2 for C and 2 for A, so we need 6 alleles.
- MHC II, very similar to MHC I.
We have two chains, α and β, 3 genes are responsible for the formation of α chain:
1. DP.
2. DQ.
3. DR.
It is found the genes that form α chain is the same as the genes that form β chain,
the only difference is in DR.
To form α chain, we need DP, DQ, and DR α.
To form β chain, we need DP, DQ, and DR β.
In some people, β chain is composed of 4 genes not 3, the one who has 4 genes
they are DP, DQ, DR β1, and DR β2.
α chain is composed of 6 alleles.
β chain is composed of 6-8 alleles.

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 MHC (HLA):
- Genetic locus that is the principal determinant of acceptance or rejection of
tissue grafts exchanged between individuals.
- Membrane proteins on APCs that display peptide antigens for recognition by T
lymphocytes.
- MHC contain genes for antigen processing (Transporter associated peptide
(TAP)).
- MHC also contain gene loci encode for complement and cytokines.

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 Properties of major histocompatibility complex (MHC) molecules and
genes.
- The normal value is that every person has his own MHC, we can find identical MHC in
identical twins, but very rarely in non-identical twins.
- Polymorphism: each individual has his own MHC.
- There are many different alleles in the population (B-620, C-90, A-200), and this gives wide
variation between MHC molecules among people.

 Each person inherits one of HLA class I genes (A, B, C) from each parent.
 Each person inherits two of HLA class II genes (DP, DQ) encoding for α chain and the β
chain from each parent.
- One or two for DRβ (HLA-DRB1 always and sometimes HLA-DRB3, HLA-DR4, or HLA-
DR5), and one for DRα.
- Each HLA allele is given a numeric designation: e.g: HLA-A2, B5, and DR3 --- etc.

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 Antigen Capture and Presentation to Lymphocytes
- The same MHC molecule can bind many different peptides, can carry only peptide antigen,
but carry one peptide at a time, it can’t carry more than one at the same time.
- If the person is sick, it carries foreign peptide, if not, it carries self-peptide.
- When the MHC is empty it is not stable and easily degraded, and immediately disappears.
- Very slow off-rate: peptide is carried in MHC for a long time so the lymphocyte have the time
identify it, it can be carried for maximum a week.

 MHC may be involved in the reactions of T cells to some nonpeptide antigens, such as
small molecules and metal ions.
 Class I MHC molecules acquire peptides from cytosolic proteins and class II molecules
from proteins that are taken up into intracellular vesicles.
 A single T cell need to see a peptide displayed by only as few as 0.1% to 1% of the
approximately 105 MHC molecules on the surface of an APC.(each cell contains
100thousand MHC molecule, for the T lymphocyte to see the peptide, 0.1%-1% of the
100thousand needs to be displayed).
 MHC molecules are unable to discriminate between self and foreign antigens, but T cells
specific for self-antigens are either killed or inactivated.

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 Antigens are two types:-
1. Protein antigen.
2. Non-protein antigen.
Will be expressed on antibodies and be killed by Humoral immunity.

Protein antigen

Intracellular Extracellular
microbes microbes

APCs (antigen
Any nucleated cell presenting cells) - Any nucleated cell APCs (antigen
Dendritic cells presenting cells)

MHC I Pathway - Killed by Humoral

MHC I pathway - MHC II pathway -
CD8 Cytotoxic (cross immunity mainly
CD8 Cytotoxic CD4 helper T cells
presentaion) antibodies

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 Class I MHC Pathway of Processing of Cytosolic Antigens
- Regarding cytosolic proteins or intracellular peptides.
- The first enzyme to work on the protein is Ubiquitin, its function is to unfold the protein.
- The unfolded protein enters the proteasome, and it gets degraded by proteasome into
peptide fragments, these peptide fragments enters via TAP (transported associated with
antigen processing) into RER.
- MHC is produced in the ER, but its unstable, to make it stable we need β2-microglobulin,
which is connected to the MHC by Chaperone.
- Tapasin connect the stable MHC with the peptide fragments, and pull them out of ER via TAB
to golgi apparatus to be expressed on cell surface.

Ubiquitin-proteasome pathway:
- Cytosolic Proteins are unfolded and covalently tagged with ubiquitin peptide.
- Unfolded proteins are threaded through proteasome which degrade them to
peptides.
 Transporter associated with antigen processing (TAP):
- Transporter molecule in the ER membrane binds proteasome-generated peptides on
the ER membrane, then actively pumps them into the interior of the ER.
 Chaperone:
- Stabilizes newly synthesized class I MHC molecule.
 Tapasin:
- Stabilizes MHC I by attachment to TAP

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 Class II Major Histocompatibility Complex Pathway of processing of
Internalized Vesicular Antigens
- We have extracellular protein, to enter the cell we have two methods:-
1. Endocytosis (fluid or small particles), pull it inside the cell as endosome or endocytic
vesicle.
2. Phagocytosis (big microbes), pull it inside the cell as phagosome.
- Fusing between lysosome and phgosome or lysosome and endosome, we get
phagolysosome, and it contains:-
1. Proteolytic enzymes of the lysosome.
2. Protein.
Proteolytic enzymes of the lysosome degrade the protein into peptide fragments.
- RER produces MHC II, this MHC II is empty.
- CLIP comes in, and make it stable so it doesn’t get degraded, and it holds the position
for the peptide (‫)حجز موقع‬, prevent any self-antigen from connecting with MHC II.
- They leave ER by exocytosis and meet with the phagolysosome in a specific organelle.
- When MHC II meet with the phagolysosome, HLA-DM (human leukocyte antigen)
removesCLIP so the peptide fragments can bind with MHC II.

- Extracellular microbes or proteins are ingested by phagocytosis or receptor mediated

endocytosis.
- B lymphocytes internalize proteins that specifically bind to the cells’ antigen
receptors.
- Proteins enter acidic intracellular vesicles (endosomes or phagosomes) which
fusewith lysosomes and broken by proteolytic enzymes.
 Invariant chain (Ii):
- Binds Class II MHC synthesized in ER.
- Contains class II invariant chain peptide (CLIP) sequence.
- Directs class II molecule to migrate to the endosomes and lysosomes.

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  CLIP:
- Binds to the peptide-binding cleft of the class II molecule.
 HLA-DM :
- Class II MHC–like protein.
- Exchange CLIP with other peptides available in the vesicles that can bind to
the MHCmolecule with higher affinity.

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 Class I MHC-Restricted Cross-Presentation of Microbial Antigens from Infected
Cells by Dendritic Cells
 Cross presentation:
- Subset of classical dendritic cells has the ability to ingest infected host
cells, deadtumor cells, microbes, and microbial and tumor antigens.
- Transport the antigens into the cytosol.
- Antigens are processed by the proteasome.
- The antigenic peptides enter the ER and bind to class I molecules.
- The antigens are displayed for recognition by CD8+ T lymphocytes.
- Differentiated CD8+ Cells into CTLs kill infected host cells or tumor cells
without theneed for dendritic cells or signals other than recognition of
antigen.

 Carried out when viruses infecting cells other than dendritic cells or when tumors
arise from different types of cells.

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 Role of MHC-Associated Antigen Presentation In
Recognition of Microbial Antigens by CD8+ and CD4+ Effector T Cells
 Immunodominant peptides:
- Only those peptides able to bind to the MHC molecules in that
individualcan be presented for recognition by T cells.
(Any peptide that bind on MHC molecule and recognized by T lymphocytes)

Antigen Recognition by B-Cells and Other Lymphocytes

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  Follicular dendritic cells (FDCs):
- Present in the B cell–rich lymphoid follicles of the lymph nodes and spleen.
- Display intact antigens to activated B cells.
1. They are not bone-marrow derived.
2. Not related to the dendritic cells that process and present antigens to T cells.
3. Express receptors that bind antigens coated with antibodies or
complement by-productssuch as C3b and C3d, with no role for MHC
molecules.

Functions of Antigen Presenting Cells

1. They display peptides for recognition by T cells in response to microbes.
2. Express additional signals for T cell activation.

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 Pre-quiz questions.

 Peptide produced by processing of cytosolic proteins largely:

1. Are presented at the cell surface with MHC class II CD4 Helper T-cells.
2. Are presented at the cell surface with MHC class I CD8 cytotoxic T-cells. ✓
3. Enter the endoplasmic reticulum by diffusion.
4. Are generated in late endosomal vacuols.
 Expression of MHC gene is:
1. Dominant for maternal genes.
2. Totally dependent on the antigenic exposure of the individual.
3. Codominant. ✓
4. 4Dominant for paternal genes.
 All of the followings are true regarding MHC molecules except:
1. MHC class II display antigens to be recognized by CD4 T helper lymphocytes.
2. Each lymphocyte recognize only one peptide bound to MHC molecule.
3. MHC class I expressed on antigen presenting cells APCs & display antigens
to be recognized by CD8 cytotoxic T lymphocytes. ✓
4. Each MHC molecule can bind only one peptide at a time.
 In class I MHC pathway, proteins are converted to peptides in:
1. Mitochondria.
2. ER.
3. Endosome.
4. Cytosol. ✓
 Which statement is not correct regarding follicular dendritic cells:
1. Select B-cells that bind antigens with high affinity.
2. They are bone marrow derived cells display antigens to T-cells. ✓
3. Express receptors that bind antigens coated with c3b complement system.
4. Cells located in follicular region of spleen and display antigens to B-cells.
 Blood-borne antigens are captured by antigen presenting cells that present in:
1. All.
2. Bone marrow.
3. Mucus membrane.
4. Thymus.
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  The main function of HLA-DM molecule is to:
1. Bind class II MHC synthesized in ER.
2. Exchange peptides available in the vesicles for efficient binding. ✓
3. Direct class II molecule to migrate to the endosomes and lysosomes.
4. Bind peptide-binding cleft of the class II molecule.
 Peptides that bind to class I MHC molecules are derived from cytosolic proteins
following digestion by:
1. TAP.
2. Lysosome.
3. Cathepsin.
4. Proteasome. ✓
 Both MHC class I & II are heterodimers consisting of two Alfa peptide chains and two
beta peptide chains.
1. True.
2. False. ✓
 All nucleated body cells can express MHC class I molecules on their surfaces to display
antigen to CD8+ cytotoxic T lymphocytes.
1. True. ✓
2. False.
 In the class I MHC pathway of antigen processing, peptides generated in the cytosol
are translocated into the endoplasmic reticulum by which of the following?
1. Class II invariant chain peptide (CLIP).
2. Transporter associated with antigen processing (TAP). ✓
3. Proteasome.
4. Ubiquitin peptide.
 β2-microglobulin is non-covalently associated with:
1. α chain of MHC-II.
2. α3 domain of MHC-I.
3. α chain of MHC-I. ✓
4. β2 domain of MHC-II.
 The principle inducer of T dependent response is:
1. Dendritic cell. ✓
2. B-cell.
3. All.
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 Chapter 4

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 Antigen Recognition in the Adaptive Immune System: Structure of
Lymphocyte Antigen Receptors and Development of Immune Repertoires

- Antibody is formed of two parts:

1. Main peptide chain (monomer), mainly involved in antigen recognition.
2. Accessory chains, called immunoglobulin α and β, their main
function is signaltransduction.
- TCR is formed of two parts:
1. Main part, which is the receptor itself, and its main function is antigen
recognition.
2. Accessory molecules for signal transduction (CD3, ζ (zeta)).
Accessory chains/molecules have a similar function to the domain, which is signal
transduction.

Which is stronger? Antibodies or TCRs?

Antibodies can be in two forms:
1. Receptor.
2. Soluble proteins secreted in the plasma.
So the antibody can do effector function while being separated from the cell,
the TCRs on the other hand, if they get separated from the cell, they won’t
function.
So Antibodies are stronger.

- Antibodies recognize extracellular protein and non-protein antigen.

- TCRs, mainly involved in peptide, because it is displayed by MHC, and MHC only bind to
peptide.
- Both antibodies and TCRs are specific for only one antigen.
- TCRs have more diversity that antibodies.

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  Variable region (V): antigen recognizing domains.
 Constant region(C): conserved domain.
 B cell receptor (BCR) complex /T cell receptor (TCR) complex: The set of
plasma membrane antigen receptor and signaling molecules.
 Functions of lymphocyte receptor:
1. Antigen recognition.
2. Signal transduction causing lymphocyte to divide, differentiate and perform
effector functions.

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 Antibodies Structure
Reminder: When we say humoral immunity we mean Antibodies.
When we say cell-mediated immunity we mean TCRs.

- We have two chains, light chain and heavy chain:

- Two Light chains, composed of two domains:-
1. Variable domain (outermost).
2. Constant domain (innermost).
- Two Heavy chains, composed of two domain:-
1. Variable domain (outermost).
2. Constant domain (innermost).
- We also have something called Fab (Fragment, antigen-binding), which is the light chain
(variable and constant domain) and the upper part of the heavy chain (variable and
constant), it is mainly involved in antigen binding.
- Also we have Fc region (fragment, crystalline), which is the lower part of heavy chain,
composed of only constant domains, and it’s mainly involved in combination with
complement system.
- Hinge: The area between Fab and Fc, its function it to give free movements to the Fabs, to
allow antigen recognition.
- On each variable domain, we have very small hypervariable molecules we call CDRs.
- CDR: complementarity- determining regions.
 CDR3: located at the junction of the V and C regions contains the greatest variability
and contributes most to antigen binding.

- Even though the antibody is specific for only one antigen, the antibody can bind to another
antigen but it has to be very similar structurally to the antigen that the antibody is specific
for. We call this “Cross reaction”.
- Reminder: The epitope: are the parts of the antigen that binds to a specific antigen
receptor on the surface of a B-cell. The paratope: antigen-binding site, is the part of an
antibody which recognizes and binds to an antigen.
The antigen has more than one epitope, if 3 small antigens came across their antibody, all
of them will bind to 3 CDRs, and one big antigen can come across an antibody and bind to 3
CDRs.

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 Features of the major isotypes (classes) of antibodies
- There are 5 types of antibodies:
Captain MAGED
IgM, IgA, IgG, IgE, IgD
- They are named after their heavy chain.
There are five types of mammalian Ig heavy chain denoted by Greek letters: α (alpha),
δ (delta), ε (epsilon), γ (gama) and μ (Mu). These chains are found in IgA, IgD, IgE, IgG
and IgM antibodies, respectively.
Note: If the heavy chain changes, the whole antibody changes.

- IgG extra function: transports adaptive immunity to the neonates from the mother
through the placenta.
- There are two types of light chains:
 κ and λ differ in the C region.
 κ or λ remains fixed throughout the life of each B cell clone, regardless of whether or
not heavy-chain class switching has occurred.
 The light chains do not participate in effector functions, except binding and
neutralizing microbes and toxins

- IgG has the highest concentration in the plasma.

- IgG has the highest half-life.
- IgE has the lowest concentration in the plasma.
- IgE has the lowest half-life.

- Question:
How many CDRs does IgM has?
IgM is a pentamer, it has 5 antibodies, and each antibody has 12 CDRs, so 12 x 5 = 60
IgM has 60 CDRs.

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 Binding of Antigens to Antibodies
- Reminder: The epitope: are the parts of the antigen that binds to a specific antigen
receptor on the surface of a B-cell. The paratope: antigen-binding site, is the part of an
antibody which recognizes and binds to an antigen
- The binding strength of the antigen to the antibody is called affinity or avidity.
When do we call affinity and when do we call avidity?
Affinity of interaction: The strength with which one antigen-binding site of an antibody
binds to one epitope of an antigen.
Avidity of the interaction: Binding strength to all binding sites between antibody and
antigen.
- Dissociation constant (Kd): how quickly the antibody will be dissociated from the
corresponding antigen.
The lower the Kd, the higher the affinity.
- Affinity maturation: The increase in antigen-binding strength.
- Cross-reaction: Antibodies produced against one antigen may bind other, structurally
similar antigens
- Secreted IgM is a pentamer, with 10 antigen-binding sites, while IgG, IgE, IgD are
monomers have two binding sites, IgA is dimer has 4 binding sites.

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 Generation of Hybridomas and Monoclonal Antibodies
Video: https://www.youtube.com/watch?v=kcxQyIfca4I
You can also watch it on Moodle.
The videos text:
Monoclonal antibody preparations contain only one type of antibody, derived from a
single cloned B cell, consequently they are highly specific for a single epitope and have
applications, for example, in diagnostic microbiology and cancer therapy.

The first step in producing a monoclonal antibody is to inject an animal with an

antigen containing the specific epitope of interest, each B cell produces a single type
of antibody. B cells are isolated from the spleen and then mixed with myeloma cells, a
type of cancer cell that grow continuously.
Addition of polyethylene glycol causes the two types of cells to fuse together to form
cells called hypridomas, the mixture of B cells, myeloma cells and hybridomas are
cultured under conditions with permit growth of only the hybridoma cells, each
hybridoma cell will produce a single type of antibody against a single epitope.
The single hybridoma cells are then separated into individual wells of a microtiter
plate and tested for ability to produce the desired antibody, the one producing the
desired monoclonal antibody are then cultured.
Monoclonal antibodies are isolated and purified

- Monoclonal Antibodies )‫(الطريقة التقليدية‬

 Used as therapeutic agents and diagnostic reagents for many diseases in humans.
 Mouse monoclonal antibodies induce immune response when injected into human.
 Genetic engineering: the antigen-binding V regions of the mouse are retained and
the rest of the antibody with is replaced by human Ig.

- Recombinant DNA technology )‫(الطريقة الحديثة‬

The antibodies needed are encoded by a specific gene, this way, the gene is amplified,
and therefore more antibodies produced (Gene amplification).

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 T Cell Receptors for Antigens
Reminder:
- TCR complex is composed of two parts:
1. Main part, which is the receptor itself, mainly involved in antigen recognition.
2. Accessory chains, (CD3, ζ) mainly involved in signal transduction, we call them
signaling molecules.

- The receptor is composed of two chains:

1. β chain.
2. α chain.
Each chain is composed of two domains:
1. Variable domain (outermost).
2. Constant domain (innermost).
Constant transmembrane region: an extension of the constant domain, has no
function but to bind )‫ (تثبيت‬the receptor on the cell surface.
TCR ---------- Antibody
α chain ---------- Light chain
β chain ---------- Heavy chain
- On each variable domain we have 3 CDRs, therefore, a single TCR has 6 CDRs.
- CDR3 is the most variable among different CDRs.
- TCR interacts with both the MHC molecule and the
peptide presented.
- TCR activation requires:
1. CD3 and ζ chains: crucial for the initiation of
signaling when the TCR recognizes antigen.
2. CD4 or CD8: coreceptor which recognize
nonpolymorphic portions of MHC molecules

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Features of Antigen Recognition by Immunoglobulins and T cell Antigen
Receptors
Reminder:
- The higher the affinity, the lower the Kd.

- kD of TCR is higher than that of the antibody

- Affinity of TCR is lower than that of the antibody.
- On-Rate and off-rate: how fast the antigen bind to the antibody or TCR.

 Other Subsets of T-cells

- γδ T cells:
 Constitute 5% to 10% of the total T cells.
 γδ TCR recognize a variety of protein and nonprotein antigens.
 Usually antigens not displayed by classical MHC moleculs.
 Abundant in epithelia.
- Natural killer T cells (NK-T cells):
 Express αβ TCRs with limited diversity.
 Recognize lipid antigens displayed by non-polymorphic class I MHC–like molecules
called CD1.
- Mucosal associated invariant T (MAIT) cells:
 Express αβ TCRs with limited diversity.
 Some are specific for bacterially derived vitamin B metabolites bound to an MHC-
like protein called MR1.
 Account for only about 5% of blood T cells, and 20%– 40% of human liver T cells.

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 Y= 12 CDRs TCR = 6 CDRs

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 Production of Diverse Antigen Receptors: Germline Organization of Antigen
Receptor Gene Loci
- Receptor protein, each protein will be encoded by a gene, we have 107 receptors in
our body, if we want to encode all of these receptors, we would need 107 genes,
which is a very big number. The total human genes are from 25-30 thousand genes,
so there has to be a process in our body which created the receptors needed.
- In the picture, we have a hematopoietic stem cell, and this cell has 23 pairs of
chromosomes, 21 pairs are somatic chromosomes.

Each of these chromosome bellow is responsible for encoding a part of these

receptors:
• Chromosome 14, is responsible of encoding immunoglobulin heavy chain
(IgH).
• Chromosome 2, is responsible of encoding immunoglobulin light chain
mainly κ chain (Ig κ).
• Chromosome 22, is responsible of encoding immunoglobulin light chain
mainly λ chain (Ig λ).
• Chromosome 7, is responsible of encoding TCR β chain.
• Chromosome 14, is responsible of encoding TCR α chain.

- Somatic recombination: random assembly of gene segments.

We have 4 gene segments:
1. V – (V1-V65), V = Variable.
2. D – (D1-D27), D = Diversity.
3. J – (J1-J6/7), J = Joining.
4. C – (C1-C10), C = Constant.
- To create the heavy chain or β chain.
We need to create the constant domain and variable domain. To create the variable
domain we need to assemble V, D, and J. To create the constant domain, we need C
gene segment, in creating heavy chain, we need the constant domain to be created
by Cμ gene segment.

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 - To create the light chain (λ or κ) or α chain.
We need to create the constant domain and variable domain. To create the variable
domain we need to assemble V, and J. To create the constant domain, we need C
gene segment.
To create the variable domain we don’t need D segment.

 Hematopoietic stem cells in the bone marrow and early lymphoid progenitors contain Ig
and TCR genes.
 All antigen receptor gene loci contain V, J, and C gene segments.
 Only the Ig heavy chain and TCR β chain loci also contain D gene segments.

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Production of Diverse Antigen Receptors: Somatic Recombination and Expression
of Immunoglobulin (Ig)

Recombination and expression of immunoglobulin (Ig) genes. The expression of an Ig heavy

chain involves two gene recombination events (D-J joining, followed by joining of a V region to
the DJ complex, with deletion of intervening gene segments). The recombined gene is
transcribed, and the VDJ complex is spliced onto the C region exons of the first heavy-chain
RNA (which is μ), to give rise to the μ messenger RNA (mRNA). The mRNA is translated to
produce the μ heavy-chain protein. The recombination of other antigen receptor genes—that
is, the Ig light chain and the T cell receptor (TCR) α and β chains—follows essentially the same
sequence, except that in loci lacking D segments (Ig light chains and TCR α), a V gene
recombines directly with a J gene segment.
- This diversity is limited, we call it combinatorial diversity.
- Producing very different number of receptors by somatic recombination.

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 Mechanisms of Junctional Diversity
In the picture we have a double stranded DNA, and the squares are nucleotides
(adenine, thymine, guanine, and cytosine)

- The first step of connecting V to D, is cutting the area between them, by an enzyme
called exonuclease: Exclusion of the nucleotides between V and D segments.
- After the cut, the V and D segments are unstable, to stabilize them, we have to connect
them by hairpin loops.

- Another enzyme called recombinzae, creates the hairpin cleavage.

- Now we have a defect, the double stranded DNA should be symmetrical, to make it
symmetrical we have repair process.
- Repair process: Adding new nucleotides called “P nucleotides”, this is a templated
process. P = Palindromic
A palindrome is a word, number, phrase, or other sequence of characters which reads the same
backward as forward, such as madam or racecar.
- After the repair process, we still have a gap, we fill it by adding “N nucleotides”, and this
process is a non-templated process, and from this process we get the almost unlimited
diversity.
N nucleotides are added by an enzyme called TdT (Terminal deoxynucleotidyl
transferase).
N nucleotides are connected with P nucleotides by an enzyme called ligase.
 These junctional sequences and the D and J segments encode the amino acids of the
CDR3 loop that form the most variable of the CDRs and the most important for antigen
recognition.

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 Combinatorial vs Junctional Diversity

Creation of light chain or α chain

doesn’t need D segment.

 Combinatorial diversity is limited by the number of available V, D, and J gene segments,

but junctional diversity is almost unlimited.
 Somatic recombination of V and J, or of V, D, and J, gene segments is mediated by a
lymphoid specific enzyme, the VDJ recombinase:
- It is expressed only by immature B&T lymphocytes.
- Composed of the recombination activating gene 1 and 2 (RAG-1 and RAG-2) proteins

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 Development of T and B Lymphocytes
Reminder:
- Hematopoietic stem cells are the main source of T/B cells.
- The first cell arriving from lymphoid progenitor are called Pro B/T cells, these cells don’t
have receptors on their surfaces.
They will be proliferated under the effect of growth factors secreted by stromal cells
from the bone marrow.
These cells will try to express receptors on their surfaces, if they succeed, they become
Pre B/T cells, if they fail the cells die.
- Pre cells have receptors expressed partially on their surfaces.
Ig --- Heavy chain.
TCR --- β chain.
So, heavy chain or β chain are expressed first.
- When the receptors are fully expressed on the surface of the cell, they become
immature B/T cell.
This immature cell, will undergo selection process, which is the selection of positive
cells, which are the cells that recognize the MHC molecule/Antigen weakly and bind
with it with low affinity, this will form mature cells (Positive selection). If the cell
doesn’t recognize the MHC molecule/Antigen, or recognize it strongly with high
affinity (self-reactive lymphocytes), it’ll die
by Apoptosis (Negative selection).
This process (selection process) prevent the
development of auto-immune response.
 107–109 different clones of lymphocytes arise
before encounter with antigen. Each clone has
distinct specificity.
 IL-7 maintains and expands the number of T
lymphocyte progenitors before they express
antigen receptors.

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 Maturation and Selection of B Lymphocytes
(Same process as in the previous page)

The maturation of B lymphocytes proceeds through sequential steps, each of which is

characterized by particular changes in immunoglobulin (Ig) gene expression and in the
patterns of Ig protein expression.
Pro-B cells begin to rearrange Ig heavy-chain genes and large pre-B cells are selected to
survive and proliferate if they successfully rearrange an Ig heavy chain gene and assemble
a pre-BCR.
The pre-BCR consists of a membrane-associated Ig μ protein attached to two other
proteins called surrogate light chains because they take the place of the light chain in a
complete Ig molecule.
Small pre-B cells initiate Ig light-chain gene rearrangement, immature B cells assemble a
complete membrane IgM receptor, and mature B cells coexpress IgD, with the same V
regions and specificity as in the first Ig produced.

Pre-BCR )‫ (مستقبل كاذب‬consists of:-

1. Real heavy chains.
2. Surrogate light chains )‫(كاذبة‬
Pre-BCR, ‫( بشكل مؤقت‬light chains)‫قائم بأعمال الـ‬
And it disappears after finishing his job.

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 Role of the Pre-BCR Complex in B cell Maturation
The pre-BCR complex signals to shut off recombination of Ig heavy-chain genes on the
second chromosome, so each B cell can express an Ig heavy chain from only one of the
two inherited parental alleles. This process is called allelic exclusion, and it helps ensure
that each cell can only express a receptor of a single
To maintain the specificity adaptive immunity.
‫ماذا تعني هذه الفقرة؟‬
Let’s take an example, chromosome 14.
Chromosome 14 is responsible of encoding TCR α chain, now humans, have 2
chromosome 14, one paternal and the other is maternal, only one of them will encode
the TCR α chain, the role of the Pre-BCR, is to stop the other chromosome from
encoding another α chain.

- Expression of the pre-BCR is the first checkpoint in B cell maturation

Clinical importance of Pre-BCR:
Pre-BCR will be connected to Bruton’s tyrosine kinase enzyme, which is encoded by Btk
gene on X chromosome.
Pro/Pre cells will be still be under the effect of growth factors, to make sure these cells
don’t turn into cancer cells in case the proliferation speed increases, we have an
inhibitory effect, which is Bruton’s tyrosine kinase enzyme. It is activated to
downstream the pre-BCR and required for delivery of signals from this receptor that
mediate survival, proliferation, and maturation

Now mutation in any gene can cause:

1. No activity.
2. Hyperactivity.
Mutation of Btk gene in boys cause hyperactivity, which means the B cells will stop
proliferating (strong anti-proliferation), therefore no antibodies are produced. This
causes X- linked agammaglobulinemia(XLA), (another name Brutons disease).
We can treat this disease by:
1. Gene therapy (not available in our country)
2. Artificial antibodies for life.

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  Functions of Pre-BCR.
1. Inhibition of H chain recombination (allelic exclusion).
2. Proliferation of Pre-B cells.
3. Stimulation of κ light chain recombination.
4. Shut off of surrogate light chain transcription.

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 Mature B-cells
- In the normal case, cells getting negatively selected die immediately. However,
negatively selected B cells get another chance, it’ll be introduced to another receptor,
and will undergo selection process once again, but if it gets negatively selected in the
second time, it’ll die by apoptosis.

- Subsets of Mature B-cells:

 Follicular B-cells: found within lymph node and spleen follicles, develop from
bone-marrow–derived hematopoietic stem cells.
 Marginal B-cells: found at the margins of splenic follicles, develop from bone-
marrow–derived hematopoietic stem cells.
 B 1 cells: found at mucosal sites and the peritoneal cavity, develop earlier from
fetal-liver derived hematopoietic stem cells.

Maturation and Selection of Major Histocompatibility Complex (MHC)–

Restricted T Lymphocytes
(The process is the same as mentioned in the previous pages)

 Pro T cell in fact has a CD3 receptor.

 Pro T cell is called a double negative cell, because it lacks CD4 and CD8 receptors.
 We have a Pre-TCR, which has the same function as Pre-BCR, but it doesn’t have clinical
importance.
 Immature T cell it called double positive cells, because it contains CD4 and CD8
receptors.
 The T cell receptor (TCR) β chain is first expressed at the double-negative pre-T cell
stage

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 - There are 4 possibilities of selection process.

1. Weak recognition of class II MHC + peptide.

This causes down regulation of CD8 (‫)تقليل عدد‬
And up regulation of CD4 (‫)زيادة عدد‬
Will create a mature CD4- T helper cell.

2. Weak recognition of class I MHC + peptide.

This causes down regulation of CD4 (‫)تقليل عدد‬
And up regulation of CD8 (‫)زيادة عدد‬
Will create a mature CD8- T cytotoxic cell.

3. NO recognition of MHC + peptide

Failure of positive selection (death by apoptosis).

4. Strong recognition of either class I or class II MHC + peptide.

Failure of positive selection (death by apoptosis).

- Some of the positively selected CD4 cells differentiate into regulatory T cells.

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 Chapter 5

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 - Adaptive immunity is separated into:
1. Humoral immunity, which is mainly involved in protein and non-protein
antigen, and involved in extracellular microbes.
2. Cell-Mediated immunity, which is mainly involved only in protein antigen,
and involved in intracellular microbes.

- T-cell Mediated Immunity: Activation of T-Lymphocytes

Any type of microbe can enter the cell either by phagocytosis by phagocytic cell, or by
another mechanism by non-phagocytic cell.
T lymphocytes perform multiple functions in defending against infections by various
kinds of microbes.

- CD8+ T cells also destroy cancerous cells.

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 Phases of T-cell Responses

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 - Antigen presenting cells enter the lymph node while holding the microbe to meet
with the lymphocytes that are already distributed as clones, these clones are
composed of CD4 T cells and CD8 T cells. These cells are Naïve cells.
- Naïve cells: mature lymphocytes that are unable to do effector function (unarmed).
- Antigen recognition: interaction between MHC molecule and two receptors:-
1. TCR.
2. CD4 or CD8.
Reminder: MHC I is composed of α1, α2, α3.
α1, α2 (polymorphic) interact with TCR. α3 (constant) interact with CD8
MHC II is composed of α1, α2, β1, β2.
α1, β1 (polymorphic) interact with TCR. α2, β2 (constant) interact with CD4.
- To activate a lymphocyte we need two signals.
1. MHC/TCR interactions.
As a result of this interaction, signal transduction occurs, which is biochemical
signals transduced via the cytosol of the cell.
- Different T cell molecules recognize antigen and deliver biochemical signals to the interior
of the cell as a result of antigen recognition.
- CD3 and ζ: are transmembrane singaling proteins, non-convalently attached to the T cell
receptor (TCR) α and β chains.
- Lymphocyte-specific protein tyrosine kinase (Lck): phosphorylates tyrosine residues of
certain proteins in the intracellular singaling pathways of lymphocytes.

There is 3 molecules responsible for these signals:

1) CD4/CD8.
2) CD3: signaling molecules for TCR.
3) ITAM.
Difference between Domain and Motif.
Domian: peptide chain that has its own biological action independently.

)‫(يعني عند فصله عن المستقبل يعمل بشكل طبيعي‬

Motif: doesn’t have its own biological action independently.

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 2. Co-stimulatory molecules.
1) CD28: co-stimulatory molecule present on T cell, it binds with CD80
(B7-1) and CD86 (B7-2) which are present on APC, ensure that naive T
lymphocytes are activated maximally by microbial antigens and not by
harmless foreign substances or by self-antigens.
2) ICOS (inducible co-stimulator): binds with receptors mainly on B cells.
- Homologous to CD28.
- Expressed on T cells.
- Important in the development and function of follicular helper T cells
during germinal center responses.
3) CD40 ligand.
- Expressed on activated T cells.
- Binds to CD40 on APCs activating them to express more B7
costimulators and to secrete cytokines (e.g., IL-2) that enhance T cell
differentiation.
- CD40L on effector CD4+ T cells enhances activation of B cells and
Macrophages.

 The full activation of T cells depends on the recognition of costimulators on APCs in addition
to antigen
 The binding of only one co-stimulator is enough to activate the second signal.
 The need of two signals to activate lymphocytes is to help prevent auto-immune
disorders.

We also have adhesion molecules, which has a main function of strengthening and
stabilizing the TCR/MHC interactions.
Adhesion molecules:
1. Leukocyte function–associated antigen 1 (LFA-1): The major T cell
integrin involved in binding to APCs, whose ligand on APCs is called
intercellular adhesion molecule 1 (ICAM-1).
Integrin involved inleukocyte binding to endothelium and other cells
- Increases the affinity and stabilize binding of TCR to APC MHC molecule.
- Directs the migration of effector T cells and other leukocytes from the
circulation to sites of infection.
2. ICAM-1: intercellular adhesion molecule.

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 We also have co-inhibitory molecules, and have functions of:
1. Limitation of immune response.
2. Prevent auto-immune disorders.
3. Prevent reactions with cancers and chronic viral infections, to
prevent the exhaustion of lymphocytes.
Co-inhibitory molecules:-
1) Immunoreceptor tyrosine-based inhibitory motifs(ITIM)
2) Cytotoxic T-lymphocyte protein 4(CTLA-4)
3) Programmed death protein-1(PD-1):

- All of the molecules mentioned above will be present in Synapse, which is the space
between APC and T cell. Interactions of the molecules will happen in this space.
1. MHC will bind with TCR.
2. MHC will bind with CD4/CD8.
3. Interactions of co-stimulatory molecules.
4. Interactions of adhesion molecules.
5. After the immune response is finished, interactions of co-inhibitory molecules
happen.
The synapse was first described as the side of delivery of activating signals from
membrane receptors to the cell’s interior, it may serve other functions. Some effector
molecules and cytokines may be secreted through this region, ensuring that they do
not diffuse away but are targeted to the cell in contact with the T cell. Enzymes that
degrade or inhibit signaling molecules are also recruited to the synapse, so it may be
involved in terminating lymphocyte activation as well.

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Proteins of the B7 and CD28 families.

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 The explanation here took CD4 as an example, the response of CD8 is the same.

- After the antigen recognition and the synapse formation, biochemical signals
transduce into the cell and activate the first domain, Lck (lymphocyte kinase), the
activation of this domain will add a phosphate group to a small peptide chains at the
end of ζ and CD3 chain (ITAM), activated ITAM will add a phosphate group to another
large protein molecule called ZAP-70 (zeta-associated protein of 70kD), activated
ZAP-70 will recruit other adapter proteins in the cytoplasm to the stock.
- Each adapter protein will activate a specific pathway, but every pathway produce
cytokines mainly involved in:
1. Proliferation.
2. Differentiation.
3. Migration.

1. ITK (interleukin T-cell kinase).

ZAP-70 will give a phosphate group to ITK, and this activates it. The activation of
ITK will activate another adapter protein called PLC γ (Phospholipase C Gamma).
Activated PLCγ can lead to two things:
1) Pull out )‫ (يٌخرج‬calcium from ER into the cytoplasm, which leads to high
concentration of calcium ions in the cytoplasm, which results in activation of
calcineurin (intermediate protein), which activates the transcription factor
NFAT.
Calcineurin inhibitors (cyclosporine and tacrolimus) are drugs that block
the phosphatase activity of calcineurin, and thus suppress the NFAT-
dependent production of cytokines by T cells. These drugs are widely uses
as immunosuppressant to prevent graft rejection and other T cell-mediated
inflammatory conditions.
2) Activates Diacylglycerol (DAG) (intermediate protein), DAG activates PKC
(Protein kinase C) (intermediate protein). Activated PKC activates two
transcription factors, NF-κB and AP-1.
2. Ras and Rac (regulator proteins).
They contain 2 phosphate groups GDP, when they take a phosphate group from
ZAP-70, they become GTP. Ras-GTP, and Rac-GTP (activated intermediate
proteins) both of them activate ERK, and JNK, which results in activation of
transcription factors, NF-κB and AP-1.

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 3. PI3 Kinase.
It originates from PI2, after taking a phosphate group it becomes PI3, Activation of
PI3 activates PI3P, PI3P (activated intermediate protein) activates Akt (intermediate
protein) which activates mTOR (intermediate protein), which results in two things:
1) Activation of transcription factors, NF-κB and AP-1.
2) Protein synthesis: substrate intermediate proteins.
Rapamycin: a drug that binds to and inactivated mTOR, it is used to treat graft
rejection.

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 - Lymphocyte activation is associated with a profound change in cellular metabolism. In
naïve (resting) T cells, low levels of glucose are taken up and used to generate energy
in the form of ATP by mitochondrial oxidative phosphorylation. Upon activation,
glucose uptake increases markedly, and the cells switch to aerobic glycolysis. This
process generates less ATP but facilitates the synthesis of more amino acids, lipids,
and other molecules that provide building blocks for organelles and for producing
new cells. As a result it is possible for activated T cells to more efficiently manufacture
the cellular constituents that are needed for their rapid increase in size and for
producing daughter cells.

- After the activation, IL-2 is secreted within 1 to 2 hours and binds with IL-2R (receptor)
which is present onthe same cell that secreted IL-2, Expression of IL2 and IL2-R initiate
the autocrine pathway of cell proliferation.
- Scientists name IL-2 “T-lymphocytes food”, it is used in experiments.
- The receptor for IL-2 is a three-chain molecule. Naïve T cells express two signaling chains, β
and γ, which constitute the low-affinity receptor for IL-2, but these cells do no express the α
chain (CD25) that enables the receptor to bind IL-2 with high affinity. Within hours after
activation by antigens and costimulators, the T cells produce the α chain of the receptor, and
now the complete IL-2 receptor is able to bind IL-2 strongly. Thus IL-2 produced by antigen
stimulated T cells preferentially bind to and acts on the same T cells, an example of autocrine
cytokine action.
- After lymphocyte activation, proliferation happens (‫)تكاثر‬, T lymphocytes activated by
antigen and costimulation begin to proliferate within 1 or 2 days. The 1000 clones are
not enough to fight the microbe, so “clonal expansion” (expansion of antigen-
specific clones) occurs in the 3rd day after infection, and it reaches its limits in the 7th
day, and it goes back to its normal state in the 14th day.
- CD8+ cells directly affect infected cell while CD4+ cells secrete cytokines that activate
many effector cells
- Number of CD8 cells is higher than the number of CD4 cells, because CD4 doesn’t work
as a fighter (effector), it just gives orders to other cells.
Why HIV infected patients are more susceptible to viral infections and cancer?
HIV virus infect CD4 cells directly, so when the patient get infected with a simple virus, CD8
cells won’t function because CD4 cells are dead and didn’t give them an order. So the patient
die because of a simple virus.

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 - Next we have differentiation, the differentiation of naive CD8+ T cells into fully active
cytotoxic T lymphocytes (CTLs) and memory cells require the concomitant activation of
CD4+ helper T cells.
- CD4+ T cells produce cytokines or membrane molecules that activate the CD8+ T cells.
- CD4 T cells differentiate into:
1) Th1: involved in inflammation.
2) Th2: involved in allergic.
3) Th17: involved in fighting Bacteria and Fungi.
4) Tfh (follicular helper T-cells): helps follicular B cells.
Th1, Th2, and Th17 leave the lymph node while Tfh stays in it.
- After fighting the microbe, some of these cells will die in the process, and some of them
will stay alive, the one that stay alive become “Memory cells”

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 - Memory cells respond much more vigorously and rapidly than do naive lymphocytes.
- They can be found anywhere, in lymphoid organs, in peripheral tissues, especially
mucosa and skin, and in the circulation.
- They can be activated in lymphoid and nonlymphoid tissues, and their activation, does
not require high levels of costimulation or antigen presentation by dendritic cells.
- Not like other cells, memory cells are required to have a long life, IL-7 and IL-15
(produced by stromal cells in tissues) work on keeping the memory cells alive and
cycling slowly.

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 Migration of Lymphocytes in Cell Mediated Immune Reactions
- Naïve T cells express adhesion molecules L-selectin (CD62L) and the chemokine receptor
CCR7, which mediate the selective migration of the naïve cells into lymph nodes though
specialized blood vessels called high endothelial venules (HEVs).

1. Tfh: this cell remain in lymphoid organs, because they express a chemokine receptor (CXCR5)
that draws them into lymphoid follicles, where they can interact with resident B
lymphocytes.
First, CCR7 binds with CCL19 or CCL21 (present on the surface of endothelial cell), which
results in activation of integrin LFA-1 (change from low-affinity state to high-affinity state).
LFA-1 binds with ICAM-1 (present on endothelial cell), this results in stopping the cell, to
strengther the adhesion between the cell and endothelial wall, L-selectin bind with L-selectin
ligand (present on endothelial cell).

2. Th1, Th2,Th17, CTL

The phospholipid sphingosine 1-phosphate (S1P) plays a key role in the egress of T cells from
lymph nodes. The levels of S1P are higher in the blood and lymph (peripheral tissue) than
inside lymph nodes. When a naïve T cell enters the node, it is exposed to lower concentrations
of S1P, the cell leaves the node through efferent lymphatic vessels following the gradient of
S1P into the lymph.

After the cell reaches the peripheral tissue, CXCR3 binds CXCL10 (present on endothelial cell),
which results in activation of integrins LFA-1 and VLA-4 (change from low-affinity state to
high-affinity state), LFA-1 and VLA-4 binds with ICAM-1 or VCAM-1 (present on endothelial
cell), This results in stopping the cell at endothelial wall, to strengther the adhesion between
the cell and endothelial wall, E-selectin ligand and P-selectin ligand binds with E-selectin and
P-selectin (present on endothelial cell).
Chemokines CCR7 binding enhancing integrin-dependent adhesion and migration thought the
HEV (High endothelial venules) of naïve T-cells.

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 Superanitgens
- An antigen that connects the variable domain of β chain with MHC.
Now this thing is not normal, the normal way is to connect it with peptide-binding cleft.
Certain types of bacteria secrete toxins which act as Superantigens.
Superantigens cause hyper activation of lymphocytes, which results in production of
large amounts of cytokines, which affects the body and cause something like septic
shock. Cause systemic inflammatory disease.

Role of Adjuvants in Stimulating T-cell Response

- For every vaccine to be licensed, it has to undergo four stages, one of these stages it the
testing of Adjuvants.
- Adjuvants: Substances added to the vaccine it either:
1. Accelerates.
2. Potentiate.
3. Prolong immune response either by innate immune mechanism or
adaptive immune mechanism.
- Examples of Adjuvants:
1. Type A: interact with pattern recognition receptors (such as Toll like receptors)
and act as immune-potentiators of the immune response (e.g., MPL).
2. Type B: function as delivery systems by improving the recruitment of innate
immune cells and favoring the Ag capture (e.g., Alum).
3. Type C: Enhance the immune response, act through co- stimulatory molecules
(CD28) present in T cells.
(Type A, B work in innate immunity. Type C work on Adaptive immunity)
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 Decline of the Immune Response
- Many proliferated cells become deprived of survival signals after
eradication of infectionand die by apoptosis.
- The response subsides within 1 or 2 weeks after the infection is eradicated.
- Only pool of memory lymphocytes will survive.

To live is to suffer, to survive is to find some meaning in the suffering.

- Friedrich Nietzsche

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 Chapter 6

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 Development and functions of CD4+ effector T-lymphocytes

Types of cell mediated immune reactions

- Two main types of cell mediated immune reactions eliminate different types of
microbes; CD4+ helper T cells express molecules that recruit and active other
leukocytes to phagocytose (ingest) and destroy microbes, and CD8+ cytotoxic T
lymphocytes (CTLs) kill infected cells containing microbial proteins in the cytosol, thus
eliminating cellular reservoirs of infection.

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 Development of Th1 cells
- In response to bacteria and viruses. Two scenarios.
1. The first scenario is the virus (antiviral defense). Nk cells, which are the most
important cells in killing the viruses in innate immunity, will try to kill the virus, will
try to produce IFN-γ which is toxic to the virus, but the amount of IFN-γ produced is
too insufficient to kill the virus, so NK cells call for help from activated CD4 cells by
IFN-γ cytokine (eliciting cytokine/‫)مسبب أو محفز‬. IFN-γ will bind with IFN-γ receptor
on the CD4 cell, which results in activation of STAT1 (transcription factor).
2. The second scenario is the bacteria (like mycobacteria). Dendritic cells will meet with
the bacteria and try to kill it by initiating inflammation, but cannot, so it will call for
help from CD4 by producing IL-12 cytokine (eliciting cytokine). IL-12 will bind with its
receptor, which results in activation of STAT4 (transcription factor).
Note: Dendritic cells are not a professional phagocytic cells, it is unable to produce
toxins necessary for killing the microbe. Unlike Macrophages and Neutrophils.

After the activation of transcription factor STAT1 or STAT4, this activates a

transcription factor called T-bet (final transcription factor involved in production of
IFN-γ), T-bet results is producing more IFN- γ which is necessary for helping dendritic
cells and macrophages.
IFN-γ activates macrophages to kill ingested microbes, promotes more Th1
development, and inhibits the development of Th2 and Th17 cells.

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 Function of Th1 Cells
- Classical macrophage activation.
Will produce M1 macrophages, which is involved in inflammation.
The inflammation here will be stronger and more prolonged than the inflammation
induced by innate immune system. Because the macrophages are stronger and larger
in number.

Th1 cells will produce more IFN-γ, which will bind on IFN-γ receptor present on
macrophage, which will induce another binding between CD40 ligand and CD40
receptor. IFN-γ will activate the macrophage, and the response of activated macrophage
is:
1- Producing large amounts of toxic materials such as nitric oxide and reactive oxygen
species, which increases phagocytic capability.
2- Secretion of cytokines TNF, IL-1, and IL-12. These cytokines will recruit other
macrophages, because one macrophage is not enough to kill the microbe.
3- Increased expression of B7 costimulators, MHC molecules.

Note: Dendritic cell is the strongest APC for the presentation, because it has the
highest expression of MHC 2. Here after the increased expression, macrophage will
be as strong as dendritic cell.

- The critical role of Th1 cells in defense against intracellular microbes is demonstrated by
the fact that individuals with inherited defects in the development or function of this
subset are susceptible to infections with such microbes, especially prevalent
nontuberculous mycobacterial species that do not infect immunocompetent individuals.
Essentially the same reaction (classical macrophage activation), consisting of leukocyte
recruitment and activation, may be elicited by injecting a microbial (or other) protein
into the skin of an individual who has been immunized with the protein or previously
infected with the microbe. This reaction is called delayed type hypersensitivity-DTH
(Type 4 hypersensitivity), called delayed because the symptoms do not appear before
24 hours.

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 - Hypersensitivity reaction: exaggerated (more that needed, kills healthy cells) and
undesirable immune response, it can be:
1- Autoimmune disorder, if the antigen is self-antigen (endogenous).
2- Allergic disorder, if the antigen is exogenous (allergen).

- Response against Leishmania majorand Mycobacterium leprae is mediated by Th1

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 Development of Th2 cells
- Th2 cells are induced by parasitic worm infections and promote IgE-, mast cell- and
eosinophil mediated destruction of these parasites.

Helminths or Allergen (‫ )مسبب للحساسية‬enter the body, they will meet with dendritic cells,
mast cells, and eosinophils. These cells unfortunately can’t kill the helminth or the
allergen on their own, so they call for help from CD4-T cell by secreting IL-4 (eliciting
cytokine). IL-4 will bind with IL-4 receptor and results in activation of transcription
factor STAT6. STAT6 activates GATA3 (final transcription factor) which stimulate CD4 to
produce cytokines IL-4, IL-5, and IL-13 (defining cytokines) that help with killing the
microbe.
IL-4 enhances further Th2 differentiation amplifying the Th2 response.

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 Functions of Th2 cells
1. IL-4 works on B cells, it helps them to produce antibodies, these antibodies get class
switches and differentiate into IgE. For now, mast cells didn’t do anything, but they have
IgE present on their surfaces. This is the first exposure.
In the second exposure, hyperactivation of mast cells occur, and the antigen binds with
the antibodies, this results in mast cell degranulation, and secretion of vasoacting
agents (heparin….).
This type of reaction is called immediate hypersensitivity reaction, or type 1
hypersensitivity.
2. IL-5 activates the eosinophil to fight the helminth. When the eosinophil is activated, it
get degranulated and secrete toxins that are able to kill the helminth.
3. Another mechanism to kill the helminth, is by IL-4 and IL-13. IL-4 and IL-13 increase the
peristalsis movement in the intestine, which results in expulsion of parasites from the
body.
4. To repair the tissue damage that resulted from the previous processes, IL-4 and IL-13
activate M2 macrophages (Alternative activation of macrophages). Macrophages
secrete growth factors that act on fibroblasts to increase collagen synthesis and induce
fibrosis
- Classical macrophage activation is inhibited by TH2.
- IL-4 and IL-13 shut down the activation of inflammatory macrophages.

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 - The relative activation of Th1 and Th2 cells in response to an infectious microbe may
determine the outcome of the infection. For example, the protozoan leishmania major
lives inside the phagocytic vesicles of macrophages, and its elimination requires the
activation of the macrophages by leishmania major specific Th1 cells.
Most inbred strains of mice make an effective Th1 response to the parasite and are thus
able eradicate the infection. However, in some inbred mouse strain, the response to
leishmania major is dominated by Th2 cells, and these mice succumb to the infection.
Mycobacterium leprae, the bacterium that causes leprosy, is a pathogen for humans
that also lives inside macrophages and may be eliminated by cell-mediated immune
mechanisms. Some people infected with Mycobacterium leprea are unable to eradicate
the infection, which, if left untreated, will progress to destructive form of the disease
called leporomatous leprosy. By contrast, in other patients, the bacteria induce strong
cell-mediated immune response with activated T cells and macrophages around the
infection site and few surviving microbes; this form of less injurious infection is called
tuberculoid leprosy.
The tuberculoid form is associated with the activation of Mycobacterium leprae
specific Th1 cells, whereas the destructive lepromatous form is associated with a
defect in Th1 cell activation and sometimes a strong Th2 response. The same principle-
that the T cell cytokine response to an infectious pathogen is an important determinant
of the outcome of the infection- may be true for other infectious diseases.

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 Development of Th17 cells
- Th17 cells develop in response to extracellular bacterial and fungal infections
(extracellular microbes) and induce inflammatory reactions that destroy these
organisms.
- Dendritic cells and macrophages cannot kill the extracellular microbe, so they call for
help from CD4-T cell by secreting IL-1, IL-6, IL-23, and TGF-β (transforming growth
factor beta), all these cytokines bind with their receptors present on CD4-T cell and
activate STAT3 transcription factor, which activates RORγT. RORγT induce CD4 to
produce cytokines IL-7 and IL-22 (defining cytokines) to help dendritic cell and
macrophage.

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 Function of Th17
- Extracellular microbe means big microbes. Immune system can strengthen the epithelial
barrier by IL-22, it also can strengthen the chemical portion by producing defensins.
IL-22 also may promote repair of damaged epithelia.
- If IL-22 couldn’t stop the microbe, IL-17 induce classical macrophage activating (same
as Th1).
- TH17 recruit neutrophils and monocytes and induce a stronger and more prolonged
inflammation than that of innate immune response.
- Inherited defects in Th17 responses develop chronic mucocutaneous candidiasis and
bacterial abscesses in the skin.
- Antagonists of IL-17 and IL-23 used to treat psoriasis (skin inflammatory disease).
- Antagonists that neutralizes IL-12 and IL-23 inhibits development of both Th1 and Th17
cells, and used to treat inflammatory bowel disease and psoriasis.

- Antagonists: Type of receptor ligand or drug that blocks or dampens a biological

response by binding to and blocking a receptor rather than activating it like an
agonist.

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 Differentiation and functions of CD8+ cytotoxic T-lymphocytes
- CD8+ T lymphocytes activated by antigen and other signals differentiate into CTLs that
are able to kill infected cells expressing the antigen. The differentiation of naïve CD8+ T
cells into fully active CTLs is accompanied by the synthesis of molecules involved in cell
killing, giving these effector T cells the functional capacity that is the basis for their
designation as cytotoxic. CD8+ T lymphocytes recognize class I MHC–associated peptides
on infected cells and tumor cells. The sources of class I– associated peptides are protein
antigens synthesized in the cytosol and protein antigens of phagocytosed microbes that
escape from phagocytic vesicles into the cytosol. In addition, some dendritic cells may
capture the antigens of infected cells and tumors, transfer these antigens into the
cytosol, and thus present the ingested antigens on class I MHC molecules, by the
process known as cross-presentation. The differentiation of naive CD8+ T cells into
functional CTLs and memory cells requires not only antigen recognition but also
costimulation and, help from CD4+ T cells.

- CD8+ CTLs recognize class I MHC–peptide complexes on the surface of infected cells and
kill these cells, thus eliminating the reservoir of infection. The T cells recognize MHC-
associated peptides by their TCR and the CD8 coreceptor. These infected cells also are
called targets of CTLs, because they are destroyed by the CTLs. The TCR and CD8, as
well as other signaling proteins, cluster in the CTL membrane at the site of contact
with the target cell and are surrounded by the leukocyte function–associated antigen
1 (LFA-1) integrin. These molecules bind their ligands on the target cell, forming an
immune synapse. Antigen recognition by CTLs results in the activation of signal
transduction pathways that lead to the exocytosis of the contents of the CTL’s granules
into the synapse between the CTL and the target cell. Because all nucleated cells
express class I MHC, and differentiated CTLs do not require costimulation or T cell help
for activation, the CTLs can be activated by and are able to kill any infected cell in any
tissue. CTLs kill target cells mainly as a result of delivery of granule proteins into the
target cells. Two types of granule proteins critical for killing are granzymes (granule
enzymes) and perforin.
- Perforin disrupts the integrity of the target cell plasma membrane and endosomal
membranes, thereby facilitating the delivery of granzymes into the cytosol.
- Granzymes (granule enzymes) cleave and thereby activate enzymes called caspases
(cysteine proteases that cleave proteins after aspartic acid residues) that are present
in the cytosol of target cells and whose major function is to induce apoptosis.

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 - Activated CTLs also express a membrane protein called Fas ligand, which binds to a
death-inducing receptor, called Fas (CD95), on target cells. Engagement of Fas activates
caspases and induces target cell apoptosis; this pathway does not require granule
exocytosis and probably plays only a minor role in killing by CD8+ CTLs.
- The net result of these effector mechanisms of CTLs is that the infected cells are killed.
Cells that have undergone apoptosis are rapidly phagocytosed and eliminated.

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 - CTLs themselves are not injured during the process of killing other cells, so each CTL can
kill a target cell, detach, and go on to kill additional targets.
- In addition to their cytotoxic activity, CD8+ effector cells secrete IFN-γ. This cytokine is
responsible for activation of macrophages in infections and in disease states where
excessive activation of CD8+ T cells may be a feature. It may also play a role in defense
against some tumors, this also helps cleaning the area from dead cells and tissues.

- CD4 and CD8 may function cooperatively to destroy intracellular microbes. If microbes
are phagocytosed and remain sequestered in macrophage vesicles, CD4+ T cells may be
adequate to eradicate these infections by secreting IFN-γ and activating the
microbicidal mechanisms of the macrophages. However, if the microbes are able to
escape from vesicles into the cytoplasm, they become insusceptible to the killing
mechanisms of activated macrophages, and their elimination requires destruction of
the infected cells by CD8+ CTLs.
- This process is called co-operation, mainly involved in killing certain types of bacteria
such as Mycobacterium tuberculosis, which causes Tuberculosis (‫)السل الرئوي‬.

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 Evasion of Cell Mediated Immunity
- Mycobacteria: a type of bacteria that prevents the fusion of phagosome and lysosome.
So it cannot be phagocytized. So it stays alive without any apparent toxic materials.
It is killed by cytotoxic T cells by co-operation.

- Herpes simplex virus (HSV): blocks the TAP enzyme, so no processing of the peptide
occurs. This is Inhibitions of antigen presentation.

- Cytomegalovirus (CMV): this virus will remove the newly formed MHC class I, and
therefore no molecules to hold peptide. This is Inhibitions of antigen presentation.

- Epstein-Barr virus (EBR): this virus create a defect in the proteasome activity, and
therefore the unfolded protein will not be degraded into peptide fragments. This is
Inhibitions of antigen presentation.

The end result of HSV, CMV, and EBR no expression of the antigen peptide on MHC class
I, no recognition by CTLs.
NK cells recognize the absence of class I MHC molecules on infected stressed cells.

- Epstein-Barr virus (EBR): this virus has another mechanism. Production of IL-10 (anti-
inflammatory cytokine), this cytokine will bind with effector cells and prevent them
from killing the virus.

- Pox virus: produce soluble cytokines, that function as receptor (IL-1 receptor, IFN-γ
receptor), theses cytokines bind with released cytokines that are secreted from immune
cells. Therefore, stopping the binding between cytokines and their receptors and
prevent the work of immune cells.

- HIV virus: kills immune cells (CD4+ T cells).

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 Evasion of cell mediated immunity
- T-cell exhaustion: Premature termination of immune response against chronic viral
infection by viral stimulated expression of inhibitory receptors on CD8+ T-cell.

- The virus, detach CD28 from CD80 and CD86, then binds CD28 with CTLA-4 (cytotoxic T-
lymphocyte-associated antigen 4) and PD-1 (programmed death 1) stopping the
second signal (costimulatory) and stopping and activation of T cells. This process helps
prevent the exhaustion of T cells (termination of immune response).
- Regulatory T-cells use the same mechanism.

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 Regulatory T-cell mechanisms
1. The First mechanism is the same as above (page 138).
2. The second mechanism is producing anti-inflammatory cytokines like IL-10 and
TGF-β, they function to terminate the immune response.
3. The third mechanism. Regulatory T-cells have IL-2 receptors; it pulls IL-2
produced by activated T-lymphocytes and bind it with IL2-R. IL-2 initiates
proliferation of regulatory T cells. This mechanism stops the proliferation of
activated lymphocytes and stops the immune response.

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 Important

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 Pre-quiz questions
 Patients with chronic mucocutaneous candidiasis have inherited defects in
which of the following cells:
1. Th1.
2. CTLs.
3. Th17. ✓
4. Th2.
 The transcription factor essential for Th1 differentiation and development is:
1. NF-κB
2. GATA-3
3. T-bet. ✓
4. RORγT.
 Which one of the following are antimicrobial peptides releases following
stimulation of Th17?
1. IFN- γ.
2. GF-β.
3. IL17.
4. Defensins. ✓
 Naïve CD4+ T-cell differentiation into Th2 is stimulated by:
1. IL1.
2. IL12.
3. IL2.
4. IL4. ✓
 Th17 mainly involved in:
1. Recruitment of neutrophils and monocytes thus inducing stronger and more
prolonged inflammation than that of innate immune response.
2. All of the above. ✓
3. Maintaining integrity of epithelial barriers and repairing damaged epithelium.
4. Defense against fungal and bacterial infections, especially in epithelial barrier
tissues.
 Activation of M1 macrophages mainly mediated by:
1. Th1 cells. ✓
2. Th2 cells.
3. Th17 cells.
4. Follicular helper cells.

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  Cytotoxic T cells kill virally infected cells by releasing:
1. Fas ligands.
2. Perforins.
3. All of the above. ✓
4. Granzymes.
 Stimulation of M2 macrophages function is mainly mediated by:
1. Follicular helper cells.
2. Th17 cells.
3. Th1 cells.
4. Th2 cells. ✓
 T-cell exhaustion is:
1. Premature termination of immune response against chronic viral infection by
viral stimulated expression of inhibitory receptors on CD8+ T-cell. ✓
2. Response to viral infection by T-cells lead to early elimination.
3. Inhibiting the function of CTLA-4 molecules on the surface of T-cells.
4. Increase use of T-cells during viral infection.
 Apoptosis of viral infected cell is mediated by expression of membrane protein
on cytotoxic T-cell which interacts with its ligand on the infected cell, this
interaction occurs between:
1. Fas and CD95. ✓
2. IL4 and GATA3.
3. B7 and CD28.
4. CD40 and DC40L.
 Which of the following is not a mechanism by which microbes evade adaptive
immune response?
1. Inhibition of MHC-I expression.
2. Inhibition of phagolysosome formation.
3. Block cytokine activation of effecter cells. ✓
4. Neutralizing IFN- γ activity.
 Cell mediated immunity involved in all of the followings except:
1. Defending against extracellular microbes by recruiting large numbers of
phagocytes to sites of infection.
2. Enhancing the uptake of extracellular microbes into phagocytes. ✓
3. Killing any type of host cells that harbor infectious microbes in cytoplasm or
nucleus.
4. Defending against intracellular phagocytozed organisms.

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  Th2 cells are mainly developed by IL-4 produced by:
1. All of the above. ✓
2. CD4 T-cells.
3. Eosinophils.
4. Mast cells.
 Th17 cells mainly involved in:
1. All of the above. ✓
2. Defense against fungal and bacterial infections, especially in epithelial barrier
tissues.
3. Recruitment of neutrophils and monocytes thus inducing stronger and more
prolonged inflammation than the innate immune response.
4. Maintaining integrity of epithelial barriers and damaged epithelium.

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 Chapter 7

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 Humoral Immunity
- Mainly involved in extracellular microbes (big microbes), proteinous and non-
proteinous.
- Therefore, humoral immunity can be divided according to the antigen to:-
1. T-dependent response.
The antigen will be proteinous, and the B-cell will need help from cell-mediated
immunity (T-cells) to kill the microbe
The cell involved in this response is conventional B cells (follicular B cells), which has
a high specificity and diversity (adaptive immunity).
Follicular B cells will differentiate into Plasma cells (long lived) and memory cells.
The antibodies produced will undergo class switching and affinity maturation.
Class switch (short explanation): IgM will change depending on the antigen and
cytokines produced to either one of IgE, IgA, or IgG.
Affinity maturation (short explanation): Antibodies will change from low affinity
state to high affinity state.
2. T-independent response.
The antigen will be non-proteinous, and B-cell will not need help from cell-mediated
immunity, it can kill the microbe on its own.
The cell involved in this response is unconventional B cells (marginal zone B cells
(only)), which has limited diversity and no specificity (innate immunity).
Marginal zone B cells will differentiate into Plasma cells (short lived) only (No
memory cells).
The antibodies produced do not undergo class switching and affinity maturation.
The antibodies will be low affinity and only produce IgM.
The IgM produced after the differentiation will change from insoluble (receptor) to
soluble (secreting).
- B-1 cells are also a part of unconventional B cells, but are not involved in
humoral immunity response (Comes from Fetal liver)
1. Respond to multivalent antigens in the mucosal tissues and peritoneum.
2. Express antigen receptors of limited diversity and make predominantly T-
independent IgM responses.
3. Produce natural antibodies of IgM type that clear cells die by apoptosis
during normal cell turnover.
4. Provide protection against some bacterial pathogens

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  Activation of Marginal zone B cells (T-independent response).
In Any activation, five processes occur:
1. Antigen recognition.
2. Lymphocyte activation.
3. Proliferation.
4. Differentiation.
5. Migration.
CR2 = complement receptor 2

- Antigen recognition and co-stimulatory signal.

- We know that we need two signals to initiate the activation. The binding of
BCR with the antigen induces the first signal. The second signal, C3d opsonizes
(‫ )يعلم‬the microbe; C3d binds with its receptor CR2 (this process is when the
antigen enters via tissues).
- If the non-proteinous enter via blood, no inflammation occurs, so it reaches
the marginal zone B cells with being opsonized by C3d. So signal two comes
from TLR-PAMP interactions.

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 - Lymphocyte activation.
- This is a multivalent antigen: meaning it
has multiple epitopes, and binds with
multiple antibodies (cross-linking).
Cross-linking:
1. More stimulation.
2. More proliferation.
3. Production of large amount of cytokines.
Lymphocyte activation has four processes.
1- Domain activation.
2- Adaptor protein recruitments.
3- Activation of intermediate proteins.
4- Activation of transcription factors.

- Three domains are activated Fyn, Lyn, and BIK. Their activation results in
phosphorylation of the motif at the terminal part of the accessory chains Igα and
Igβ (ITAM). Activated ITAM will phosphorylate protein Syk (Zap-70 ‫)يقابله‬. Now Syk
has multiple phosphate groups that will be given to the adaptor proteins
(recruitments of adaptor proteins).
1. Ras-Rac (adaptor proteins), 2 phosphate grouped proteins (GDP). Ras-Rac
will undergo phosphorylation and become 3 phosphate grouped proteins
(Ras-GTP/Rac-GTP). Ras-GTP/Rac-GTP (activated intermediate proteins)
will activate ERK and JNK (intermediate proteins), ERK and JNK will activate
transcription factors AP-1, Myc, NFAT, and NF-κB
2. BTK (adaptor protein). Activated BTK will activate PLCγ2 (intermediate
protein). Activated PLCγ2 can lead to two things.
- Activate Diacylglycereol-DAG (intermediate protein). DAG will
activate PKC (intermediate protein). Activated PKC will activate
transcription factors AP-1, Myc, NFAT, and NF-κB.
- Pull out (‫ )يخرج‬calcium from ER into the cytoplasm, which leads to
high concentration of calcium ions in the cytoplasm, which results in
activation of calcineurin (intermediate protein), which activates the
transcription factors AP-1, Myc, NFAT, and NF-κB.
- Transcription Factors will produce cytokines that perform autocrine, paracrine
mechanisms and initiate proliferation.

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  Activation of Follicular B cells (T-dependent response).

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 Interactions between T-cell and B-cell.
When the protein antigen enter. T cell will work alone and at the same time B cell
will work alone, after that, they will interact.
Note: 1. B cell zone: contain naïve B cells.
2. T cell zone: contain naïve T cells.

1. When the proteinous antigen enters the body, it will meet with the dendritic
cell (APC), dendritic cells will perform endocytosis, which will form endosomal
protein, then class II antigen processing occurs, thus expression on MHC class
II.
Dendritic cell will migrate to the lymph node and meet with CD4 T-cell in T-cell
zone. Interactions between dendritic cell and CD4 occurs and results in
activating CD4. As a result, cytokines will be produces, not to initiate
proliferation, but to:-
1- Induce the expression of CD40 ligand on the surface of T cells.
2- Induce the expression of CXCR5 receptor on the surface of T cells.
T cell zone will have a high concentration of CCR7 chemokines.
B cell zone will have a high concentration of CXCR5 Chemokines, and with
the expression of CXCR5 receptor on CD4, CD4 cell will be pulled closer to
the B cell zone, at marginal zone (at the rim of the follicle).

2. At the same time, the same proteinous antigen will meet with the B-cell.
The antigen will bind with Ig with a single epitope; B cell will take a small part
of the proteinous antigen. With a certain mechanism, the receptor will enter
the cell while holding some particles of the antigen (endocytosis). This forms
an endosome that contains the receptor with some particles of the antigen.
Then by exocytosis, only the receptor leave the endosome and re-expresses on
the cell surface. This process is called Receptor mediated endocytosis.

Animated video: https://www.youtube.com/watch?v=PjrH1dpCyyE

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 Now the small particles of protein antigen entered as endosomal protein, so
class II antigen processing occus, and this peptide will be expressed on MHC II.
Therefore, B cell under the effect of CCR7 receptor present on its surface will
be dragged outside the follicle to meet with the T cell.

Interactions between B cell and T cell occurs. This activates B cell by two
signals:
1- MHC II (B cell) with TCR (T cell) (signal 1).
2- CD40R (B cell) with CD40L (T cell) (signal 2).
This interaction is called Initial T-B interaction, Initial antibody response,
or Extrafollicular interaction.

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 The activation of B cell will induce the expression of another costimulatory molecule
called ICOS (inducible costimulator), which is a costimulatory molecule on the
surface of T cell. Then B cell proliferate and differentiate, but this differentiation and
proliferation is initial and limited. Therefore, this response is weak (weak antibody
response).
So initial B cell T cell interaction will result in:
1. Short-lived plasma cells.
2. Producing low affinity IgM.
However, we need a stronger response.
- Active B and T cells will migrate back into follicles to form germinal centers (light area
in the follicle in the lymph node and spleen) where the more specialized antibody
responses are induced.
- When the activated CD4 T cell enter the follicle, its name change into follicular helper
T cell (has co-stimulatory ICOS on its surface).
- Germinal center contain:
1. Follicular Dendritic cells (present antigen to B cells).
2. Activated B cells.
3. Follicular Helper T cells.
Follicular dendritic cells capture any proteinous antigen that enter the
germinal center; B cells will be next to them and follicular dendritic cells
present them the antigens repeatedly and prolongly. This presentation
process occurs in the dark area of the follicle.

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In the germinal center two processes occur:
1. Class Switch.
This process in initiated by interactions between follicular helper T cells and B cells.
Follicular helper T cells order B cell to do class switch, for this to occur follicular helper
T cells need to bind with B cells, by binding the antigen present on B cell with TCR
present on follicular helper T cell by interactions between MHC II on the surface of B
cells and the TCR on the surface of follicular helper T cells, this will be the first signal.
Interactions between ICOS present on follicular helper T cell with ICOS receptor
present on B cell will be the second signal.
This activation of B cells and follicular helper T cells help to maintain the life the cells.
Here there will be a third signal, which are interactions between CD40 ligand present
on follicular helper T cells and CD40 receptor present on B cells. This interaction is
very important in the class switch process.
This interaction will produce cytokines that determine which antibody will be
produced by B cells (IgG, IgA, and IgE).
1. IFN-γ cytokine will produce IgG subclasses (IgG1, IgG3).
2. IL-4 cytokine will produce IgE.
3. TNF-β (transforming growth factor beta) or BAFF (B cell activating factor) will
produce IgA.
4. IgM already present on the surface of B cells, it will change from insoluble
(receptor) form to soluble (secreted) form.
These cytokines are produced by follicular helper T cells.

Antibodies eliminate antigens by:

1- Complement activation.
2- Opsonization.
3- Antibody dependent cell mediated cytotoxicity.
4- Binding to neonatal Fc receptor (FcRn) providing protection to the newborn.
FcRn expresses on endothelial cells and phagocytes plays a special role in
protecting IgG from intracellular catabolism, prolonging its half-life in the blood.

Note: class switching occurs by changing the heavy chain of the antibody.

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 Mechanism of Class switching
- As we know from before, to produce an antibody we need to make light chain and
heavy chain. Here we will explain the mechanism on heavy chain only, because class
switch depends only on the constant domain of the heavy chain. Antibodies change
by changing the constant domain of the heavy chain.
IgA – Cα
IgG – Cγ
IgE – Cε
IgD – Cδ
IgM – Cμ

Note: the explanation below is about changing IgM to IgG, but keep in mind that the
same mechanism and explanation is applied to IgA and IgE.

- Notice that near (to the left) every gene segment of constant domain there is switch
region.
Sα – Cα
Sγ – Cγ
Sε – Cε
Interactions between T cell and B cell occur (CD40L with CD40L Receptor). This
interaction results in producing IFN-γ. IFN-γ bind with IFN-γ receptor present on B cell
surface, this induce B cell to create IgG.
- B cells will secrete activation induced-deaminase (AID). AID will make switch regions
close to each other by creating this loop.
Intervening C genes

So AID will induce the recombination of Sμ with Sγ and the deletion of intervening C
genes. Now we have Sμ and Sγ close to each other. The next step AID transforms
cytoseal nucleotides to uracil. This will create an unstable DNA.
- Other enzymes delete uracil nucleotides in switch regions, this will result in unstable
switch regions, and therefore, it will be deleted.
After this the gene is transcripted into RNA and translated to produce IgG.

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- In the absence of CD40 or CD40L,
B cells secrete only IgM and fail to
switch to other isotypes,
indicating the essential role of this
ligand-receptor pain in isotype
switching. A disease called
X-linked hyper-IgM syndrome.
This disease is caused by
mutations in the CD40L gene,
whitch is located on the X
chromosome, leading to
production of nonfunctional
forms of CD40L in males who
inherit the mutation.
- Patients with this disease also
have defective cell-mediated
immunity against intracellular
microbes, because CD40L is
important for T cell-mediated
activation of macrophages and for
the amplifications of T cell
responses by dendritic cells.

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 2. Affinity Maturation.
This process in initiated by interactions between follicular dendritic cells and B cells.
Affinity maturation is the process by which the affinity of antibodies produced in
response to a protein antigen increases with prolonged or repeated exposure to that
antigen.
This increase in affinity is caused by point mutations in the V regions, and particularly
in the antigen- binding hypervariable regions, of the genes encoding the antibodies
produced. This mutation occurs in the dark zone of the follicle.
Mutation is induced by AID (activation-induced deaminase) by transforming cytoseal
to uracil, this results in creating unstable DNA. Uracil nucleotides are either removed
or changed which both result in mutation.
Affinity maturation occurs in the germinal centers of lymphoid follicles and is the
result of somatic hypermutation of Ig genes in dividing B cells, followed by the
selection of high-affinity B cells by antigen
Named hypermutation because almost 1 thousand pairs of nucleotides are changed
from cytoseal to uracil.

- Inherited mutation in the ICOS gene cause some antibody deficiencies.

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 Selection Process
Somatic hypermutation will result in changing the form of variable region (new variable
gene segments). Producing a large number of antibodies with different forms of
variable domain. Some will bind with high affinity antigens, others will bind with low
affinity antigens, and the rest will not bind with antigens.
B cells producing high affinity antibodies will be positively selected (will be activated by
follicular helper B cells), other B cells that produce low affinity antibodies will die by
apoptosis. This process occurs in the light zone of the follicle.

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 Antibody feedback
- Antibodies are produced to perform specific effector functions. When the
function is performed, antibodies producing stops, how? When the antibody
finish its functions, it sends a signal to B cells to stop producing more antibodies.
B cells has FcγRIIB (named Fc because it binds with Fc region) receptor on its
surface. FcγRIIB binds with antibody antigen complex, now the cytoplasmic
domain of FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif
(ITIM) that binds enzymes ITAM (immunoreceptor tyrosine-based activation
motif) and results in inhibiting antigen-receptor mediated B cells activation. This
will result in stopping the production of antibodies.

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 Conjugate vaccines

The hapten-carrier concept

- A conjugate vaccine is a type of vaccine, which combines a weak antigen with a
strong antigen as a carrier so that the immune system has a stronger response
to the weak antigen.
- Conjugate vaccines have been developed to induce a robust immune response
against bacterial capsular polysaccharides (CPSs). This bacteria is considered a
basic pathogen and it is very dangerous. Like :
1. Meningococcal causes meningitis.
2. Pneumococcal causes pneumonia.
These diseases are very dangerous and deadly. So scientists developed
conjugate vaccines which is the treatment of these diseases.

- To create vaccines we need to add the capsule of the bacteria inside the body to
produce memory cells, but theses bacteria’s capsule is polysaccharides, and
adding polysaccharides will induce T-independent response, which will produce
no memory cells.
To protect the body from these diseases we need memory cells to produce a
more powerful, prolonged, and rapid response.
Therefore, scientists developed a way to create a vaccine by combining a weak
antigen with a strong antigen as a carrier.
- The carrier antigen here is Tetanus toxoid protein. Injection of tetanus toxoid
combined with bacterial capsular polysaccharides occur, this injection will induce
T-dependent response and T-independent response, why? Because there is
both proteinous antigen and non-proteinous antigen.
T-dependent response will produce long-lived plasma cells and memory cells.
- Conjugate vaccine idea came from a small molecule called Hapten. Hapten is
small chemical that is recognized by B cells, but stimulates a strong antibody
response only if it is attached to a carrier protein.

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 Generation of plasma cells and memory B cells
- Plasmablasts: the antibody secreting cells enter the circulation.
1. Survive for years as plasma cells.
2. Continue to produce high-affinity antibodies, even after the antigen is eliminated.
Antibodies produced provide immediate protection during re-exposure to antigens.

- Memory cells: fraction of the activated B cells, which often are the progeny of isotype
switched high-affinity B cells.
1. Do not secrete antibodies.
2. Circulate in the blood and reside in mucosal and other tissues.
3. Survive for months or years in the absence of additional antigen exposure.
4. Memory from a T-dependent antibody response can last for a lifetime.
5. Undergo cycling, and are ready to respond rapidly if the antigen is re-introduced.

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 Types and Features of Antibody Responses

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 Chapter 11

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 Hypersensitivity reactions
- Hypersensitivity reactions: exaggerated and undesirable immune response, it can be:-
1. Autoimmune disorder, if the antigen is endogenous (self-antigen).
2. Allergic disorder, if the antigen is exogenous (allergen).
- Require a pre-sensitized state of the host.
- Occur in response to three types of antigen:
1. Infectious agents.
2. Environmental substances.
3. Self-antigens.

- Hypersensitivity reactions affect some people and others not, why? Duo to atopy (affect
10-20% of people).
- Atopic people (individuals): people that have mutations in some genes, these
mutations make these people susceptible to these hypersensitivity reactions.

- Allergic diseases include:

1. Hay fever.
2. Food allergy.
3. Asthma.
4. Anaphylaxis.
- Factors contribute to allergy:
1. Genetic.
For example, mutations is flagrin gene lead to atopic dermatitis and asthma.
2. Environmental.
Duo to repeated and prolonged exposure of the same antigen.

- There are four types of hypersensitivity reactions.

1. Immediate hypersensitivity (type 1).
Against exogenous antigens result in allergic disorders.
2. Antibody-mediated diseases (type 2).
Against endogenous antigens (self-antigen) result in autoimmune disorders.
3. Immune complex-mediated diseases (type 3).
Against endogenous antigens (self-antigen) result in autoimmune disorders.
4. T cell-mediated diseases (type 4).

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 Immediate hypersensitivity (Type 1)
- As we know from before (chapter 6), that Th2 cell is involved in type 2 hypersensitivity
reaction (immediate hypersensitivity).
- There are four things important for this reaction:
1. Development of Th2.
2. IgE.
IgE will bind with Fcε receptor on the surface of mast cells.
3. Mast cells.
Mast cells will ask for help from eosinophils.
4. Eosinophils.

Mechanism of immediate hypersensitivity reactions.

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 - Th2 is activated and will produce IL-4, IL-5, and IL-13. IL-5 will activate eosinophils and
IL-4 and IL-13 will increase peristalsis movement in the intestine and cause GI upset
which results in symptoms like abdominal pain, diarrhea, nausea, vomiting….
- IL-4 will act on B cells to produce IgE cells (class switch), IgE will leave the
lymphnode/spleen to meet with mast cells that are present at site of allergen entry.
IgE will bind with Fcε receptor on the surface of mast cells.
So far, no symptoms appear on the patient, we call this process sensitization (first
exposure).
- If second exposure of the same allergen occur, allergen will bind with IgE via Fab region,
and signal transduction occur (domain activation – adaptor protein recruitments –
activating of intermediate proteins – activating of transcription factor), which will lead
to one of three pathways.
1. The mast cell has granules that contain vasoacting agents/vasoactive amines. These
granules will leave the cell by exocytosis, once it leaves it will deregulated and its
contents will be released mainly histamine and proteases.
1- Proteases will lead to tissue damage.
2- Histamine will lead to two things:-
1. Vascular dilation.
2. Smooth muscle contraction (bronchoconstriction)
Most symptoms of type 1 hypersensitivity are related to histamine.
This reaction takes only minutes, maximum one hour, and is called early phase
(primary reaction).
2. Synthesis of lipid mediators. Arachidonic acid is transformed by specific enzymes to
prostaglandins, which results in vascular dilation, and leukotrienes, which results in
smooth muscle contraction.
Prostaglandins and leukotrienes are lipid mediators that are created from the
beginning, how? Phospholipids present on the cell membrane are transformed by
specific enzymes mainly Arachidonic acid to lipid mediators.
This process takes 4-6 hours maximum 24 hours. This reaction is named late phase
reaction (secondary reaction).
3. Mast cells produce cytokines that induce leukocytes mainly neutrophils and
esoniphils to help, such as IL-4 and TNF. When the leukocytes arrive at allergen
entry they will induce inflammation which results in tissue damage at allergen entry.
This reaction takes hours like the synthesis of lipid mediators and named late phase
reaction (secondary reaction).

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A patient who get affected by hypersensitivity reaction needs to be monitored for 24 hours to
make sure that late phase (secondary reaction) does not occur.

- The high-affinity receptor for IgE is called FcεRI, consists of three polypeptide chains,
one of which binds with the Fc portion of the ε heavy chain very strongly, with a Kd of
approximately 10-11M. (The concentration of IgE in the plasma is approximately 10-9 M,
which explains why even in normal individuals, mast cells are always coated with IgE
bound to FcεRI).
The other two chains of the receptor are signaling proteins.
The same receptor is also present on basophils.

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 - Basophils are very similar to mast cells. IgE bind with basophils because Fcε receptor is
present on its surface.
Mast cells are present in tissues, at site of allergen entry, but basophils are present in
the blood. So mast cells are faster and mainly involved in early phase reaction (primary
reaction), but basophils are mainly involved in late phase reaction (secondary reaction)
because they take time migrating from the blood.
- Basophils are low in number, about 1% of leukocytes.
- The amount of histamine secreted from basophils are very low in contrast of the
amount of histamine secreted from mast cells.
- Eosinophils are granulocytes; they contain granules that contain toxic materials. When
IL-5 activate eosinophils these toxic materials are secreted and result in damaging the
epithelial barrier. Which will induce inflammation.
- IL-13 work on mucous secretion.

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Treatment of type 1 hypersensitivity reaction.
1. Monoclonal antibodies that act against IL-4 (anti-IL-4).

2. Monoclonal antibodies that act against IL-5 (anti-IL-5).

3. Monoclonal antibodies that act against IL-13 (anti-IL-13).

4. Monoclonal antibodies that prevent IgE from binding with Fc epsilon receptor.

5. The main treatment, is anti-histamine drug (has 4 types).

6. Cromolyn (mast cell stabilizer): prevent histamine release from granules.

7. Montelukast act as leukotriene antagonists (inhibitor).

8. Corticosteroids are strong anti-inflammatory drugs, so it prevent late phase reaction.

9. Many patients benefit from repeated administration of small doses of allergens, called
desensitization or allergen-specific immunotherapy. This treatment may work by
changing the T cell response away from Th2 dominance or the antibody response away
from IgE, by inducing tolerance in allergen-specific T cells or by stimulating regulatory
T cells.

Note: any disease we mention bellow can be treated by these treatments except
anaphylaxis. Anaphylaxis is specifically treated by epinephrine.

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Clinical manifestations of immediate hypersensitivity reaction type 1
1. Hay fever (‫)ح ّمى القش‬
Production of mucus by the effect of histamine and IL-13 by Th2
Treated by antihistamines.
2. Food allergy.
Patients will food allergy will have symptoms different that vasodilation and smooth
muscle contraction, they will have GI upset, which result is diarrhea, nausea, and
vomiting, because of the increase in the peristalsis movement of the intestine by IL-13
and IL-4.
3. Asthma.
Leukotrienes, which cause repeated bouts of bronchial constriction and airway
obstruction.
Eosinophils accumulate in the bronchial mucosa lead to excessive secretion of mucus
occurs in the airways, and the bronchial smooth muscle becomes hypertrophied and
hyperactive.
Treated by beta2-agonist (ventolin mediolateral) and corticosteroids
4. Anaphylaxis.
The most severe form of immediate hypersensitivity.
Characterized by edema in many tissues including the larynx.
The patient will have hypotension and bronchoconstriction, because of the massive
involvement of mast cells all over the body, why? Because the allergen will migrate to
the circulation, so it can bind with mast cells anywhere in the body. It will bind to mast
cells all over the body (massive involvement of mast cells) which result in secretion of
large amounts of histamine, which will induce vasodilation all over the body, which will
cause drop of blood pressure (hypotension), and bronchoconstriction.
Some of the most frequent inducers of anaphylaxis include bee stings, injected or
ingested penicillin family antibiotics, and ingested nuts or shellfish.
To treat anaphylaxis we need epinephrine.

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 Type 2 hypersensitivity reaction
- Also called antibody mediated hypersensitivity reaction.
- The components involved in this reaction are:
1. Autoantibodies (IgM and IgG).
2. Antigens.
3. Complement system activation.
4. Effector cells, mainly neutrophils.
- The antigen attacked in hypersensitivity reaction is insoluble (tissue antigen present on
cell surface).

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 Mechanisms of hypersensitivity reactions type 2
- Three effector mechanisms for this reaction.
1. Inflammation
The body produces autoantibodies against self-antigens present on cell surface,
these antibodies react/bind with neutrophils via Fc receptors. Antibodies involved in
this mechanism either IgG or IgM.
IgG (IgG1 + IgG3) bind to neutrophil and macrophage via Fc receptors and activate
these leukocytes, resulting in inflammation, tissue injury by releasing toxic
materials.
IgM, activate the complement system by the classical pathway, resulting in the
production of complement by-products that recruit leukocytes and induce
inflammation and tissue injury by releasing toxic materials.

Examples of disease that happen with this mechanism (inflammation)

1. Goodpasture syndrome: rare disorder in which your body mistakenly makes
antibodies that attach the lungs (alveoli) and kidneys (glomeruli) which result in
glomerulonephritis and alveolar pulmonary hemorrhage.
2. Acute rheumatic fever: streptococcus bacteria causes’ pharyngitis, but it’s not
fully treated so the leftovers of this bacteria will enter the body and stays there.
Our body has a protein antigen (M protein) that is similar to the protein produced
by streptococcus, and this protein antigen is present in the myocardium and
joints. By this mechanism, the body produced antibodies against M protein, which
will cause inflammation in the myocardium (myocarditis) and joints (arthritis).

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 2. Opsonization and phagocytosis.
Healthy body cell but has a self-antigen on its surface, the body produced
autoantibodies (IgG or IgM) that attack this antigen. Antibody binding with antigen
will attract the complement system, and complement system bind with antigen,
which results in complement activation mainly classical pathway. One of the by-
products of this activation is C3b that has an opsonization function.
Now the healthy body cell has been opsonized, and this cell will circulate to the
spleen and meet with the macrophages, which will phagocytose it.

Examples of disease that happen with this mechanism

1. Autoimmune hemolytic anemia. ‫فقر دم بسبب تكسر خاليا الدم الحمراء‬
The healthy body cells (target cells) here are RBCs. RBCs has a self-antigen on its
surface (RH group). The body produces autoantibodies against RH group, so when
the RBCs circulate through the spleen, macrophages will phagocytose it. If the
number of RBCs killed is large, hemolysis (‫ )تكسر‬occur.
2. Autoimmune (idiopathic) thrombocytopenic purpura. ‫تكسير الصفائح الدموية‬
Has the same idea of autoimmune hemolytic anemia.
Platelets has self-antigen present on their surface (glycoprotein IIb/IIIa), the body
produces autoantibodies against this antigen, and will result in phagocytosis of
the platelets by macrophages in the spleen. Which will cause bleeding.

These two diseases can be treated by splenectomy (‫)خيار اخير‬.

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 3. Abnormal cellular responses.
Two diseases involved:
1. Graves’ disease (hyperthyroidism).
Hypersecretion of thyroid hormones mainly T3 and T4. Normally pituitary
gland secrete TSH hormones, TSH hormones act with TSH receptor present
on thyroid epithelial cells, which activates thyroid gland, which secrete T3
and T4.
In this disease, abnormal cell response occur, the body recognizes TSH
receptor as self-antigen, and produces autoantibodies against TSH receptor.
Autoantibodies will bind with TSH receptor and act like TSH hormones, which
results in hypersecretion of thyroid hormones mainly T3 and T4.
2. Myasthenia gravis (‫)وهن العضالت‬
The normal way of contracting muscles, neurotransmitters transmit
acetylcholine to the synapse between the nerve ending and muscle.
Acetylcholine will bind with its receptor present on the muscle and induce
the contraction of the muscle.
In the abnormal way, the body recognize acetylcholine receptor as self-
antigen, and will produce autoantibodies against it. These antibodies will
block acetylcholine receptor (prevent acetylcholine from binding).
Therefore, no muscle contraction occur. The first muscle affected is the
eyelid and gradually develops to paralysis.

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 - Diseases of type 2 hypersensitivity reactions
Explained during the explanation of the mechanisms.
- In pernicious anemia, autoantibodies specific for a protein required for absorption of of
vitamin B12 causes a multisystem disease duo to B12 deficiency.

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 Treatment of type 2 hypersensitivity reaction.
- Corticosteroids: strong anti-inflammatory drugs. The main treatment for type 2
hypersensitivity reaction.
- Plasmapheresis (‫غسيل بالزما‬/‫)غسيل دم‬: the main problem is that autoantibodies are
present in the blood/platelets, so getting rid of these antibodies is an effective way of
limiting inflammation. This is done by therapeutic plasma exchange machine.
- Splenectomy: in hemolytic anemia and thrombocytopenia inhibit opsonization and
phagocytosis.
- Intravenous IgG (IVIG): function the same as feedback mechanism (chapter 7). Activate
ITIM that will cancel the activation and production of antibodies.
Bind to inhibitory Fc receptor on myeloid or B-cell preventing their activation.
- CD20 antibodies.
CD20: cytokine that work on the development of B cells.
CD20 antibodies will prevent the work of CD20 and lower the level of B cells developed.
- Block of CD40 or its ligand, which prevent class switch mechanism, therefore, no
autoantibodies produced.
- Blocking cytokines that promote the survival of B cells and plasma cells.

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 Type 3 hypersensitivity reaction
Antigen antibody complex (immune complex)
- Very similar to type 2 hypersensitivity reaction. The only difference is that the antigen is
soluble (‫()في الدم‬secreted).
- Components of this reaction:
1. Autoantibodies (IgM and IgG).
2. Antigens.
3. Complement system.
4. Effector cells mainly neutrophils.
- The antibodies are also soluble (‫)في الدم‬, so the antigen and antibody will meet and react.
This complex is killed by macrophages in the spleen.
- In the case of autoimmune disorders a large amount of immune complexes are formed
and in different sizes. Macrophages can’t kill all of the immune complexes, so some
complexes are left alive and stay in the circulation and undergo deposition in the blood
vessels.
- Complement system will meet with the complexes in the blood vessels, react with it,
and induce classical pathway activation. One of by-products of this activation is C5a and
C3a, which function as chemoattractants and induce inflammation.
- Neutrophils will migrate to the blood vessels and bind with immune complex via Fc
region, which will activate neutrophils and secrete toxic materials. This will result in
inflammation of blood vessels (systemic vasculitis) and tissue damage.
- Immune complexes usually deposit in blood vessels, especially vessels through which
plasma is filtered at high pressure (renal glomeruli and joint synovium). Therefore, in
contrast to diseases caused by tissue antigen-specific antibodies, immune complex
diseases tend to systemic and often manifest as widespread vasculitits involving sites
that are particularly susceptible to immune complex deposition, such as kidneys and
joints.

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 Type 3 hypersensitivity reaction diseases
1. Systemic lupus erythematous (SLE). (‫)الذئبة الحمراء‬
Affects the females more than males.
Some people are allergic to ultraviolet radiation. When the allergic individual get
exposed to sun light for example for too long, lysis of the cell membrane occur,
which results in exclusion of nucleus contents into the circulation. The body
recognized these contents as antigens and produce autoantibodies (IgG or IgM)
against them, and these antibodies create immune complex with the antigens.
This disease affects almost every organ.
2. Serum sickness.
Very rare.
When a person is bitten by a snake, he gets injected with a serum. The Serum
contains antitoxins against the toxin. The toxin is eliminated. After Two weeks or so
that patient develops symptoms like rash and joints pain and fever, what happened
is that the body recognized the antitoxins as antigens and produced autoantibodies
against it which results in immune complex.
3. Arthus reaction.
When a person is vaccinated and the body recognizes the vaccine antibodies as
antigens and result in immune complex creation. This induces local vasculitis.

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 Type 4 hypersensitivity reaction
- Reactions of T lymphocytes specific for self-antigens or microbes in tissues.
- Purely cell-mediated immunity.
- Types on antigens involved:
1. Cellular antigens.
Intracellular protein antigen processed by class I pathway and expression on MHC
class I and presentation on CD8 T cells and activation of CTLs.
2. Environmental antigens.
Like poison ivy: toxic plant that contains urushiol (proteinous antigen). We also call
this contact dermatitis.
Killed by CD4 T cells by producing cytokines.
3. Certain microbes.
Like mycobacterium tuberculosis.
Killed by CD4 T cells by producing cytokines.

Effector mechanisms of type 4 hypersensitivity reaction

- According to the cells involved, it can be classified into two-effector mechanism.
1. CD4 T cells (delayed hypersensitivity reaction).
Named delayed, because for the CD4 to activate, proliferate, and differentiate it
takes 24-72 hours.
- Antigen presenting cells activate CD4. CD4 secrete cytokines that work on effector
cells such as macrophages and neutrophils, and these cells work on inflammation
by secreting toxic materials.
- Th1 and Th17 are mainly involved in type 4 hypersensitivity reaction.
- When mycobacterium tuberculosis enters the body, Th1 produce IL-1, IL-12, and
TNF that work on recruiting other leukocytes (macrophages) to the site of
inflammation. Mycobacterium can prevent the fusion of phagsome and lysosome
and stay in the phagosome without it dying. So to stop the replication of this
bacteria and the spread of it all over the body, macrophages assemble a wall
around it (granuloma).
- Granuloma: aggregation of macrophages around mycobacterium in order to
isolate this bacterium from the surrounding.
So mycobacterium tuberculosis has another name, chronic granulomatous
disease.

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 - Th17 work on killing poison ivy antigen, by producing IL-22 (maintenance of
epithelial barrier integrity) and IL-17 (recruitments of leukocytes) cytokines. This
result in inflammation at site of allergen entry (contact dermatitis).

2. Cytotoxic T-cell mediated hypersensitivity type 4, involves CTLs, which kills directly
and does not take time.
- The main cells involved are cytotoxic T cells.
- Abnormal cellular proteins are processed by Class I pathway and expressed on
MHC I and presented on CD8. Activated CTLs will migrate to the site of target cells
and direct kill of the cells that contain cellular antigen.
For example:
1. Type 1 diabetes.
Abnormal antigens (proteins) in beta cells of the pancreas that function to
secrete insulin. Beta cells are killed by CTLs. Killing of these cells will cause
Type 1 diabetes.
2. Rheumatoid arthritis.
Abnormal intracellular proteins present in join cells, this antigen will be
expressed on MHC I and activate CTLs. CTLs will come in contact with tissue
cells and kills them. This will cause joint destruction and joint deformation.

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 3. Multiple sclerosis.
The body recognizes myelin protein present in myelin sheath as abnormal
protein; CTLs will attack the cells that form the myelin sheath, which will cause
demyelination in the central nervous system, sensory and motor
dysfunction.

- Other diseases related to type 4-hypersensitivity reaction.

1. Crohn disease.
2. Psoriases.
However, these two diseases for now, the antigen causing them is still unknown.

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 These diseases are explained during the explanation of effector mechanisms.

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 Treatment of type 4 hypersensitivity reaction.
1. Drugs that inhibit T cell responses (T cell activation).
1- Inject regulatory T cells and injection of IL-2.
This injection will initiate the proliferation of regulatory T cells.
2- Preventing the second signal (costimulatory), by injecting drugs (monoclonal
antibodies) that block B7 (CD80 and CD86).
This treatment is dangerous, because we are preventing T cell mediated
immunity, this treatment is considered when the patient’s condition is very
serious (life threatening).
2. Anti-inflammatory cytokines.
1- Anti-TNF or anti-IL-6 receptor.
2- Inhibitor of inflammatory cytokines signaling molecules Janus kinase 3 (JAK3),
used to treat rheumatoid arthritis.
3- IL-17 blocking antibodies used to treat psoriasis.

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 ‫‪THE END‬‬
‫أسأل هللا أن يوفقنا جميعا لما فيه خير لنا وألهلنا ولبلدنا وأن يمأل‬
‫يومنا صالحا وفالحا وأن يسخرنا لعون من نستطيع مـن الفـقراء‬
‫والمكروبـين‬

‫‪L A D E N‬‬ ‫‪S A L E H‬‬

‫‪The tree that would grow to heaven must send its roots to hell.‬‬
‫‪-‬‬ ‫‪Friedrich Nietasche‬‬

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