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)

Figure 3-8 Properties of MHC molecules and genes

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