Pharm-Immunology 7 & 8

7. Major Histocompatibility complex

(MHC)
8. Cytokines &Cell Mediated Immunity
Dr. Hussein
 7. MHC
Objectives
1. Extracellular antigens (bacterial infections) enter the
MHC II pathway and presentation
2. Intracellular and cytoplasmic antigens such as virus,
intracellular bacteria and tumor antigens enter the
MHC I pathway and presentation
3. Role of class II invariant chain peptide (CLIP, Ii) in
the development of MHC II
4. Role of the specialized transport molecule, transporter
associated with antigen processing (TAP: TAP1 &
TAP2), in MHC I development and presentation
The Role of MHC in Ag Presentation to T Cells
1. Following Ag processing, the Ag is presented to
lymphocytes in a form they can recognize
2. Ag presentation to CD4+ helper T cells is associated
with MHC II
3. Ag presentation to CD8+ cytotoxic T cells is associated
with MHC I.
4. APCs are usually macrophages, but any nucleated cell
may serve as an APC
5. Ag processing is the series of events that occur between
exposure to an Ag and eventual immune response: Ab
production or T-cell activity. It includes fragmentation
of the protein Ag into small peptides in the
macrophage and the presentation to T cells as above.
 Fig. 3-6: Human HLA (MHC) Genes

• Schematic maps of the human MHC (HLA complex)

illustrating the major genes that code for molecules
involved in immune responses
• Sizes of genes and distances between them
are not drawn to scale.
 • The schematic diagrams
The structure of class I and models of the crystal
structures of MHC I and
MHC molecule MHC II (next slide)
molecules illustrate the
domains of the molecules
and the fundamental
similarities between them
• MHC I molecule contains:
– Peptide-binding clefts
– Invariant portions that
bind:
• CD8 (3 domain) or
– ß2m: ß2-microglobulin
The structure of class • The schematic
II MHC molecules diagrams and models of
the crystal structures of
MHC II molecules
illustrate the domains
of the molecule and the
fundamental
similarities to MHC I
• MHC II molecules
contain:
– Peptide-binding cleft
– Invariant portion that
binds:
• CD4 (ß2 domain)
 Figure 3-8
Properties of MHC molecules and genes
 Fig.3-8
Properties of MHC molecules and genes
Fig 3-10: Features of peptide binding to
MHC molecules
Fig 3-10
Pathways of intracellular processing of protein Ags
Fig 3-11
• MHC II pathway
MHC II pathway
converts protein
Ags that are
endocytosed into
vesicles of APCs
into peptides that
MHC I pathway
bind to MHC II
molecules for
recognition by
CD4+ T cells
• MHC I pathway converts proteins in the cytoplasm into peptides
that bind to MHC I molecules for recognition by CD8+ T cells.
• ER, endoplasmic reticulum
 Fig 3-12
Features of the pathways of antigen
processing
 Fig 3-12
Features of the pathways of
antigen processing
MHC I pathway of processing of• Proteins enter the cytoplasm of
cytosolic antigens cells either from:
– phagocytosed microbes or
– from endogenous synthesis
by microbes, such as
viruses, that reside in the
cytoplasm of infected cells
Fig 3-14 • Cytoplasmic proteins are un-
folded, ubiquitinated, and
degraded in proteasomes
• The peptides that are
produced are transported by
the TAP transporter into the
ER, where the peptides bind to
newly synthesized MHC I
• The peptide-MHC I complexes
are transported to the cell
surface and are recognized by
CD8+ T cells
 Transporter associated with antigen processing
(TAP) & class II invariant chain peptide (CLIP)
TAP  MHC I CLIP/Ii  MHC II
• TAP1 & TAP2 are proteins encoded • Newly synthesized MHC II carries
by genes in the MHC “II” locus CLIP or Ii peptide (class II invariant
• TAP is necessary for the proper chain peptide)in the ER
assembly of MHC I • If MHC II is found without peptide it
• TAP transport peptides actively into will be degraded
the ER where MHC I is assembled • DM (HLA-DM) is a peptide
• MHC I without TAP molecule cannot exchange molecule looks like MHC
be loaded with the peptide to be II
displayed on cell surface • DM in the endosome removes CLIP
• MHC I without peptide is instable & from the cleft of MHC II
would be destroyed by proteases • DM is not polymorphic, MHC II is
• Ii will be replaced by the presentable,
processed peptide and becomes
stable, otherwise it will be degraded
 The role of MHC-associated Ag presentation in the
recognition of microbes by CD4+ T cells
• Protein antigens of microbes that are endocytosed from the extracellular environment by macrophages and B lymphocytes enter the
MHC II pathway of antigen processing.
• As a result, these proteins are recognized by CD4+ helper T cells, whose functions are to activate macrophages to destroy phagocytosed
microbes and
activate B cells
to produce Abs
against
extracellular
microbes and
toxins
Fig 3-15A
The role of MHC-associated antigen presentation in
the recognition of microbes by CD8+ T cells
• Protein Ags of microbes that live in the cytoplasm of
infected cells enter the MHC I pathway of Ags processing
• As a result, these proteins are recognized by CD8+ CTLs,
whose function is to kill infected cells

Fig 3-15B
FYI

Ubiquitination of proteins
FYI
• Ubiquitin is a small (8.5kD)
Ubiquitin protein present in all
eukaryotic cells.
• Its 76 amino acid sequence is
so highly conserved that
nearly identical versions exist
in a variety of organisms
– yeast and human ubiquitin
differ at only 3 of the 76
residues
• It is involved in multiple
cellular functions:
– protein degradation
– chromatin structure
– heat shock
 Pharm-Immuno 8
Cytokines &Cell Mediated Immunity
Dr. Saber Hussein
 Objectives
1.Define: Cytokine, lymphokine, chemokine
2.Biological characterization and Sources of
cytokines
3.Role of cytokines in lymphocytes activation,
growth and differentiation
4.Role of cytokines in immune-mediated
inflammation
5.T-cell independent defense mechanisms:
Phagocytosis & chemotaxis
6.Central role of T helper cells in T-cell-dependent
cell-mediated immunity
 Objectives
7.Cytotoxic T cells function & relation to T h
8.Cell-mediated cytotoxicity:
a. Ab-independent
i. MHC-presentation dependent
ii. MHC unrestricted: NK, LAK
(Lymphokine Activated Killer)
b. Ab-dependent cell-mediated cytotoxicity
(ADCC)
9. Role of macrophages in immune response
Types of intracellular microbes combated by
T cell-mediated immunity
A. Microbes may be ingested by phagocytes and
survive within vesicles (phagolysosomes) or
escape into the cytoplasm where they are not
susceptible to the microbicidal mechanisms of
the phagocytes
B. Viruses may bind to receptors on many cell
types, including nonphagocytic cells, and
replicate in the cytoplasm of the infected
cells. Some viruses establish latent infections,
in which viral proteins are produced in infected
cells
 Definitions
• Cytokine:
– Small protein, secreted by cells to influence
behavior of other cells.
– The effect is receptor-mediated
• Lymphokine:
– Cytokine made by lymphocytes; interleukins
• Chemokines:
– Chemotactic cytokines; bind heparin; lymphocytes
& phagocytes migration; inflammatory responses
• Monokine:
– Cytokine produced by monocytes
 Biology of cytokines
• Antiviral interferons:
– IFN- as T-cell-derived antiviral protein or
– activator of macrophage (Macrophage-activating factor)
• Pyrogens:
– IL-1 in association with bacterial infection
• Cytokines as:
– Regulators
– Effectors
– need receptors
– work at low concentrations like hormones
• Exocrine
• Paracrine
• Autocrine
 Some common cytokines

CXCL8 (IL-8, )

Regulated on activation, normal T expressed and secreted

Actions of IL2
NK
 Lymphocyte activation
• Regulators of lymphocytes:
– IL-2
– IL-4
– TGF-
• TH produce cytokines involved in
regulation of acquired, specific immune
response
 IL-2 & IL-4 receptors
• IL-2 receptor is
high-affinity,
composed of 3
polypeptides:
– α & β bind to IL-2
– γ is involved in
signaling to the cell
in both receptors.
• IL-4 has only α
chain with a binding
site.
 Cytokine Action
• Cytokine binds to its
Receptor  Ligand-
induced aggregation
 Activation of
intracellular signaling
pathways (kinase
cascade)  activation
of transcription
factors  Into
nucleus  Binding to
promoter or
enhancer  Gene
transcription
 Cytokines & CD4+ ct i va tion
Tc a
TH Differentiation

1. IL-12, IFN, TGF Ab  B cell

favor:
TH0  TH1

2. IL4 favors:
TH0  TH2
The cytokine pattern
influences the effector
functions that are
activated
 IL-8 (CXCL8, RANTES)
• A cytokine (chemokine) derived from:
– endothelial cells,
CCL5
– fibroblasts,
RANTES
– keratinocytes,
– macrophages, and
– monocytes
• IL-8 causes chemotaxis of
– neutrophils and
– T-cell lymphocytes.
• It is also called Chemokine (C-C motif) ligand 5
– monocyte-derived neutrophil chemotactic factor,
– neutrophil-activating factor,
– neutrophil chemotactant factor,
– anionic neutrophil-activating peptide
– Regulated on Activation, Normal T Expressed and Secreted
Immune-mediated inflammation
1.Recruitment of inflammatory cells via
cytokine network
2.Specific receptors on target cells
3.Ag-activated CD4 & CD8 lymphocytes are
main producer of cytokines that regulate
immune-mediated inflammation
4.CD4 & CD8 cytokines are regulators &
effectors
 (LT, TNF-β)

These cytokines are involved in Immune-mediated Inflammation

 Cytotoxic T cells kill infected cells
• Cytotoxic T cells kill infected cells, preventing
these cells from producing more pathogen.
• Receptors on the surface of cytotoxic T cells
detect fragments of the virus on the surfaces of
infected cells.
• A successful immune response against a virus
means that we will make large numbers of virus
specific cytotoxic T cells.
• In an EBV infection, cytotoxic T cells can make
up the vast majority of our white blood cells.
- T cells contain a T cell receptor that is like the antibody of B cells.
- Each T cell has only one kind of receptor with a unique specificity.
- Analogous to the genetic events of antibody production, T cells
rearrange a set of genes coding for the T cell receptor.
- Each T cell ends up with a unique receptor, but the population of T
cells contains billions of different receptors
Step 1 Step 2 Step 3

Th activates Tc in a
receptor specific manner
 Fig 5-2:Steps in the activation of T lymphocytes

• Naive T cells recognize MHC-associated peptide

antigens displayed on APCs and other signals
• The T cells respond by:
– Producing cytokines, such as IL-2, and
– Expressing receptors for these cytokines, leading to
an autocrine pathway of cell proliferation
• The result is clonal expansion of the T cells
• Some of the progeny differentiate into:
– Effector cells, which serve various functions in cell-
mediated immunity, and
– Memory cells, which survive for long periods
 TT cell activation
cell activation

Fig 5-3
 T-cell independent defense mechanisms

• Phagocytosis
• Chemotaxis
Functions of KIRs
• KIR receptors recognize
MHC I on the target cell
• They signal inhibition
of cytotoxicity
• Other NK receptors
identify the target cell
positively for killing
• Antigens recognizable
include:
– CD2
– CD69
– Antibody bound to the Fc
receptor (CD16)
 NK receptors crosslinking
• Crosslinking of the
activation receptors leads to
– phosphorylation of the ITAM
sequences on the associated Activation Inhibition
DAP12 molecule by a src-
family kinase
– The phosphorylated ITAMs
recruit and activate tyrosine
kinases of the ZAP70/syk
family
• Crosslinking of the inhibitor
receptor leads to
– ITIM phosphorylation and
recruitment of the tyrosine
phosphatase SHP-1, which then
dephosphorylates the
ZAP70/syk activation Tyrosine Tyrosine
molecules phosphorylation dephosphorylation
 NK & the missing-self hypothesis
• Kärre’s “missing-self hypothesis”
– The expression of MHC I protects against NK cell-
mediated lysis
• NK cells are constantly surveying tissues for normal
expression of MHC I. Because class I molecules are
expressed on all tissues, NK cell cytotoxic activity is typically
inhibited.
• NK cell is released from its inhibition when it finds a cell with
down-regulated or mutated MHC I
– the target cell is lysed
• This NK cell function is important because certain viruses are
able to down-regulate MHC I expression in the cell they’ve
infected, protecting themselves from detection by cytotoxic T
cells.
• Some tumors also have diminished MHC I expression, and
their recognition and lysis is the basis of “natural” killing
 • Major surface molecules of CD4+
Fig 5-3: Ligand- T cells involved in their activation
and the ligands on APCs
receptor pairs involved • CD8+ T cells use most of the
in T cell activation same molecules, except that the
– TCR recognizes peptide-
MHC I complexes, and
– the coreceptor is CD8, which
recognizes class I
• Immunoreceptor tyrosine-based
activation motifs (ITAMs) are
the regions of signaling proteins
that are phosphorylated on
tyrosine residues and become
docking sites for other signaling
molecules
• CD3 is composed of three
polypeptide chains
Fig 5-3

Zeta

Lymphocyte functional Ag

Intercellular Adh.Mol
Very late Ag
Vascular Adh.Mol
 ADCC:
Antibody-Dependent Cell-
mediated Cytotoxicity

Neutrophil Ig
FcR

NK

Eosinophil

M
 Granule-associated killing mechanisms
• Cytotoxic cell vesicles release
– Perforin (Tc, NK) Granzymes
– Enzymes
• Leading to
polymerization
of perforins 
Polyperforin
channels in the
in the
membrane
of the target
cell
• Granules with enzymes (Granzymes) release enzymes that enter the
target cell through polyperforin channels  death of target
Granule-associated killing mechanisms

(Tc, NK) Granzymes

 • FasL (Fas ligand)
Receptor-mediated – a molecule on the
killing mechanisms surface of cytotoxic
T cells that binds to
its receptor, Fas, on
the surface of other
cells initiating
apoptosis in the
target cell.
• TNF-mediated
killing
– TNF released by Tc
binds TNF-
Receptor on the
target cell surface
 Cell death