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Pharm-Immunology 7 & 8
7. Major Histocompatibility complex (MHC)
8. Cytokines &Cell Mediated ImmunityDr. Hussein
7. MHCObjectives
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 structure of class I MHC molecule
• The schematic diagrams and models of the crystal structures of MHC I and 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 II MHC molecules
• The schematic 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)
Fig.3-8Properties 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
• MHC II pathway
converts protein
Ags that are
endocytosed into
vesicles of APCs
into peptides that
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 3-11
MHC II pathway
MHC I pathway
Fig 3-12Features of the pathways of antigen
processing
Fig 3-12Features of the pathways of
antigen processing
MHC I pathway of processing of cytosolic antigens
• Proteins enter the cytoplasm of cells either from: – phagocytosed microbes or – from endogenous synthesis
by microbes, such as viruses, that reside in the cytoplasm of infected cells
• 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
Fig 3-14
Transporter associated with antigen processing (TAP) & class II invariant chain peptide (CLIP)
TAP MHC I• TAP1 & TAP2 are proteins encoded
by genes in the MHC “II” locus• TAP is necessary for the proper
assembly of MHC I• TAP transport peptides actively into
the ER where MHC I is assembled• MHC I without TAP molecule cannot
be loaded with the peptide to be displayed on cell surface
• MHC I without peptide is instable & would be destroyed by proteases
CLIP/Ii MHC II• Newly synthesized MHC II carries
CLIP or Ii peptide (class II invariant chain peptide)in the ER
• If MHC II is found without peptide it will be degraded
• DM (HLA-DM) is a peptide exchange molecule looks like MHC II
• DM in the endosome removes CLIP from the cleft of MHC II
• 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 cellsto 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
Ubiquitination of proteins
FYI
Ubiquitin • Ubiquitin is a small (8.5kD) 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
FYI
Pharm-Immuno 8
Cytokines &Cell Mediated Immunity Dr. Saber Hussein
Objectives1.Define: Cytokine, lymphokine, chemokine2.Biological characterization and Sources of
cytokines3.Role of cytokines in lymphocytes activation,
growth and differentiation4.Role of cytokines in immune-mediated
inflammation5.T-cell independent defense mechanisms:
Phagocytosis & chemotaxis6.Central role of T helper cells in T-cell-dependent
cell-mediated immunity
Objectives
7.Cytotoxic T cells function & relation to Th
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
Regulated on activation, normal T expressed and secreted
CXCL8 (IL-8, )
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+ TH Differentiation
1. IL-12, IFN, TGF favor:
TH0 TH1
2. IL4 favors: TH0 TH2
The cytokine pattern influences the effector functions that are activated
Ab B cell
Tc activation
IL-8 (CXCL8, RANTES)• A cytokine (chemokine) derived from:
– endothelial cells, – fibroblasts, – keratinocytes, – macrophages, and – monocytes
• IL-8 causes chemotaxis of – neutrophils and – T-cell lymphocytes.
• It is also called – monocyte-derived neutrophil chemotactic factor, – neutrophil-activating factor, – neutrophil chemotactant factor, – anionic neutrophil-activating peptide– Regulated on Activation, Normal T Expressed and Secreted
CCL5RANTES
Chemokine (C-C motif) ligand 5
Immune-mediated inflammation
1.Recruitment of inflammatory cells via cytokine network
2.Specific receptors on target cells3.Ag-activated CD4 & CD8 lymphocytes are
main producer of cytokines that regulate immune-mediated inflammation
4.CD4 & CD8 cytokines are regulators & effectors
These cytokines are involved in Immune-mediated Inflammation
(LT, TNF-β)
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
Th activates Tc in a receptor specific manner
Step 1 Step 2 Step 3
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
T cell activation
Fig 5-3
T cell activation
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 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 molecules
Tyrosinephosphorylation
Activation Inhibition
Tyrosinedephosphorylation
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
Fig 5-3: Ligand-receptor pairs involved
in T cell activation
• Major surface molecules of CD4+ T cells involved in their activation and the ligands on APCs
• CD8+ T cells use most of the 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
Lymphocyte functional Ag
Very late AgIntercellular Adh.Mol
Vascular Adh.Mol
Zeta
ADCC: Antibody-Dependent Cell-
mediated Cytotoxicity
NK
Neutrophil
Eosinophil
M
Ig
FcR
Granule-associated killing mechanisms• Cytotoxic cell vesicles release
– Perforin
– 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
(Tc, NK) Granzymes
Granule-associated killing mechanisms
(Tc, NK) Granzymes
Receptor-mediated killing mechanisms
• FasL (Fas ligand)– a molecule on the
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
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