Chapter 8

The Major Histocomptibility Complex(MHC) and Ag Presentation

Chapter 9

T-cell Receptor

Chapter 10

T-cell Maturation, Activation, and Differentiation

Chapter 8

The Major Histocomptibility Complex (MHC) and Ag Presentation

Nov 21, 2006

你需要瞭解的課題：

1. MHC 是如何發現的？

2. MHC 的生理功能是什麼？

3. MHC 包含哪些基因及分子？

4. MHC polymorphism 有什麼生物意義？

5. Self-MHC restriction of T cells 是什麼意思？

6. 抗原是如何被呈現 (presented) 到 MHC 分子上的？

7. 與 MHC 結合的胜肽有什麼性質？

TCR and MHC-peptide

TCR (on a T cell)

peptide (embedded in an MHC molecule)

MHC (on an Ag-presenting cell or on a virus- infected cell)

Gorer (1930s):

1. Rejection of foreign tissue is the result of an immune response to cell-surface molecules.

2. Identification of I, II, III and IV blood-cell Ags and genes in mice.

Gorer and Snell (1940s & 1950s):

1. Antigens encoded by the genes in the group II took part in the rejection of transplanted tumors and other tissues.

2. Snell called these genes “histocompatibility ( 組織相容 )

genes” (or H-2 genes)

3. Snell was awarded the Nobel Prize in 1980.

Human MHC: human leukocyte antigen (HLA) - the major reason for transplantation rejection Mouse MHC: H-2

The MHC Encodes 3 Major Classes of Molecules

Class I MHC:- Expressed on the surface of nearly all nucleated cells; the major

function of the class I gene products is presentation of peptide Ags to CD8+ T cells.

Class II MHC:- Expressed primarily on Ag-presenting cells (macrophages, dendritic

cells, and B cells), where they present processed antigenic peptides to CD4+ T cells

Class III MHC: - no function in Ag presentation

- Generally encode various secreted proteins that have immune functions, including components of the complement system and molecules involved in inflammation, e.g., C2, C4, factor B,

21-hydroxylase enzymes, TNFα, TNFβ, heat shock proteins (HSP)

D as class I

D as class II

- The MHC loci are highly polymorphic - many alternative forms of the gene, or alleles, exist at each locus among the population.

- The MHC genes lie close together - the recombination frequency within the H-2 complex is only 0.5%. Thus, most individuals inherit the closely linked MHC genes as two sets, one from each parent. Each set of genes is referred to as a haplotype.

- The MHC genes are codominantly expressed; that is, both maternal and paternal gene products are expressed in the same cells.

(H-2k)

Inheritance of MHC Haplotypes

inbredhomozygous

Acceptance or Rejection of Skin Grafts is Controlled by the MHC Type of the Inbred Mice

Inheritance of HLA Haplotype in a Hypothetical Human Family

outbredheterzygous

Congenic MHC Mouse Strain

- Inbred mouse strains are syngeneic or identical at all genetic loci.

- Two strains are congenic if they are genetically identical except at a single locus or region.

- Congenic strains can be produced by a series of crosses, backcrosses, and selections.

Production of Congenic Mouse Strain

Strain A.B

Genetically identical to strain A except for the MHC locus or loci contributed by strain B.

if a/a = C57BL/10 (B10) b/b = A A.B = B10.A

Examples of Recombinant Congenic Mouse Strains

MHC Molecules and Genes

Class I and Class II Molecules

45kDa

12 kDa

28 kDa33 kDa

Both class I and II molecules are glycoproteins.

Organization of Class I MHC Genes

2M gene

Organization of Class II MHC Genes

= IA

IA IA

= IA

3-D Structure of the External Domains of a Human Class I HLA Molecule

peptide-binding cleft of MHC class I molecule:

- 25Å x 10Å x 11Å

- can bind a peptide of 8-10 a.a.

Superimposition of the peptide-binding cleft of class I and class II MHC molecules

red: HLA-A2 (Class I) blue: HLA-DR1 (Class II)

Peptide Binding by MHC Molecules

- Several hundred different allelic variants of class I and II MHC molecules have been identified in humans.

- Peptide binding by class I and class II molecules does not exhibit the fine specificity characteristic of Ag binding by Ab and TCR.

- A given MHC molecule can bind numerous different peptides, and some peptides can bind to several different MHC molecules.

Binding Affinity of MHC to Peptides

- The association of peptide-MHC molecule is very stable under physiological conditions.

- The Kd values range from ~10-6 to 10-10.

- Most of the MHC molecules expressed on the membrane of a cell are associated with a peptide of self or nonself origin.

MHC class I molecules bind to 8 ~ 10 a.a., most commonly 9.

Peptide-binding Cleft Is Blocked at Both Ends in Class I Molecules

HLA-A2 with

β2M

HIV RT 309-317 a.a.

Anchor ResiduesAnchor Residues in Nonameric (9) Peptides Eluted from Two Class I MHC Molecules

Anchor residuestend to be hydro-phobic a.a.

In general, any peptide of correct length that contains the same or similar anchor residues can bind to the same class I MHC molecules.

carboxyl-terminal anchor

amino-terminal anchor:2nd or 2nd & 3rd positions

Conformational Difference in Bound Peptides of Different Lengths

class I

An Influenza Virus Peptide and an Endogenous Peptide Bound to a Class I MHC Molecule

influenza virus endogenous

Peptide-binding Cleft is Open at Both Ends in Class II Molecules

MHC class II molecules can bind to 13 ~ 18 a. a. A central coreof 13 a.a. determines the ability of a peptide to bind class II.

Influenza virushemagglutinin306-318 a.a.

DRα

DRβ

HLA-DR1

Interacting T cells presented to CD8+ T cells presented to CD4+ T cells

Source of peptides endogenous exogenous

Ag processing pathway cytosolic pathway endocytic pathway

Polymorphism of class I and class II molecules

- The diversity of the MHC within a species stems from polymorphism, the presence of multiple alleles at a given genetic locus within the species.

- The MHC possesses an extraordinarily large number of different alleles at each locus and is one of the most polymorphic genetic complexes known in higher vertebrates.

HLA-A : 370 alleles, HLA-B : 660 alleles, HLA-C : 190 alleles

- The theoretical diversity possible for humans : class I : 370 (A) x 660 (B) x 190 (C) = 4.6 x 107

class II : ~ 8 x 1011

Total : 4.6 x 107 x 8 x 1011 ~ 4 x 1019

實際上你認為人類的 MHC polymorphism 比

4 x 1019 多？還是比 4 x 1019 少？

Antibody diversity

MHC polymorphism

Variability in the amino acid sequence of allelic class I MHC molecules

Location of Polymorphic Amino Acid Residues

Most of the residues with significant polymorphism are located in the peptide-binding cleft

Genomic Map of MHC Genes

Mouse H-2 is on the chromosome 17

Class IClass I Nonclassical Nonclassical Class IIClass II Class IIIClass III Class I Class I Nonclassical Nonclassical

Human HLA is on the chromosome 6

class IIclass II nonclassicalnonclassical class IIclass II class IIIclass III class Iclass I nonclassical nonclassical

Cellular Expression of MHC Molecules

Cellular Distribution of MHC Class I Molecules

- In general, the classical MHC class I molecules are expressed on most nucleated cells.

- The highest level of class I molecules are expressed on lymphocytes: 1 % of the total plasma membrane proteins or 5 x 105 molecules / cell.

- Fibroblasts, muscle cells, hepatocytes and neural cells express very low levels of class I molecules.

- A few cell types (e.g., neurons and sperm cells at certain stages of differentiation) appear to lack class I MHC molecules altogether.

Cellular distribution of MHC class II molecules

- Class II molecules are expressed constitutively only by Ag-presenting cells (APC), e.g., macrophages, mature dendritic cells, and B cells.

- Thymic epithelial cells and some other cell types can be induced to express class II molecules and to function as APC under certain conditions and under cytokine stimilation.

- In some cases, class II expression depends on the cell’s differentiation stage.

Various MHC Molecules Expressed on APC of a Heterozygous H-2k/d Mouse

Nonclassical MHC Class I and Class II Molecules

- Structurally similar to class I or class II molecules

- Less polymorphic

- Expressed at lower level

- Tissue distribution is more limited

- Functions are being studied

Function of HLA-G (nonclassical class I)

The expression of HLA-G molecules on cytotrophoblasts ( 滋養層細胞 ) at the fetal- maternal interface has been implicated in protection of the fetus from being recognized as foreign and from being rejected by maternal TC cells.

Hunt, J.S., M.G. Petroff, R.H. McIntire & C. Ober. HLA-G and immune tolerance in pregnancy. FASEB J 19: 681-693, 2005

CTB: cytotrophoblast cell

Regulation of MHC Expression

- Defect in class II MHC transcriptional activator CIITA or RFX cause one form of bare lymphoyte syndrome (BLS).

- The expression of MHC molecules is regulated by various cytokines, e.g., interferons (α, β,γ) and tumor necrosis factors (TNF), etc.

- MHC expression on cell surfaces is decreased by infection with certain viruses, including human cytomegalovirus (CMV), hepatitis B virus (HBV), adenovirus 12 (Ad12), etc.

- Decreased expression of class I MHC molecules is likely to help viruses evade the immune response by reducing the presentation of viral peptide on virus-infected cells.

MHC and Immune Responsiveness

Important Roles of MHC in the Immune Response

1. MHC molecules act as antigen-presenting structure.

2. MHC molecules expressed by an individual influence the repertoire of antigens to which that individual’s TH cells and TC cells can respond.

3. MHC partly determines the response of an individual to antigens of infectious organisms.

4. MHC has been implicated in the susceptibility to disease and in the development of autoimmunity.

Self-MHC Restriction of CD4+ T Cells

T cellsAPC

measure the 2nd

response againstimmunized Ag

Class II MHC

Restriction

Self-MHC Restriction of CD8+ T Cells

lymphocytic choriomeningitis virus ( 淋巴性脈絡叢腦膜炎病毒 )

Class I MHC

RestrictionZinkernagel & Doherty (1974), 1996 Nobel prize

Self-MHC Restriction:

T cells can recognize Ag only when it is presentedby a self-MHC molecule on the membrane of an APC.

* This observation is actually the only case under physiological situations.

Role of Ag-Presenting Cells

Since all cells expressing either class I or class II MHC molecules can present peptides to T cells, strictly speaking, they all could be designated as Ag-presenting cells (APC). However,…………

- Ag-presenting cells (APC): Cells that display peptides associated with class II MHC molecules to CD4+ (mostly TH) cells are called APC.

- Target cells: Cells that display peptides associated with class I MHC molecules to CD8+ (mostly TC ) cells

are referred to as target cells.

can be induced to express class II MHC & co-stimulatory signal. They function only for short periods of time during a sustained inflammatory response.

A Variety of Cells Can Function as APC

costimulatory signal

TCR MHC

TH-cell Activation Requires A Costimulatory Signal Provided by APC

Dendritic cells are the most effective APC. They constitutively express a high level of class II MHC molecules and have co-stimulatory activity, and can activate naïve TH cells.

Macrophages must be activated by phagocytosis of particular Ags before they express a high amount of class II MHC molecules or the co-stimulatory B7 membrane molecule.

B cells constitutively express class II MHC molecules but must be activated before they express the co-stimulatory B7 molecule.

Processing of Ag Is Required for Recognition by TH cells

(paraformaldehyde, 三聚甲醛 )

(glutaraldehyde, 戊乙醛 )

Ag processing is a metabolic process that digests proteins into peptides, which can then be displayed on the cell membrane together with a class II MHC molecules.

Processing of Ag Is Also Required for Recognition by TC cells

- In influenza virus-infected cells, not only viral envelop proteins (more exposed), but also polymerase and nucleoproteins (internal proteins of the virus), can be recognized by TC cells.

- TC cells recognize short linear peptide sequences of viruses.

Evidence for Different Ag-processing and Presentation Pathways

Processing and Presentation of Exogenous and Endogenous Antigens

- requires synthesis of viral protein- inhibited by protein synthesis inhibitor, emetine

- new protein synthesis is not required- inhibited by chloroquine & protease inhibitors

interacting with CD4+

T cell

interacting with CD8+T cell

endocytic or exogenous pathway

cytosolic or endogenous pathway

Endogenous Ags: the cytosolic proteolytic system for degradation of intracellular proteins

misfolded or defective proteins:

intact proteins:

Peptides are Transported from the Cytosol to the Rough Endoplasmic Reticulum (RER)

- by Transporters Associated with Ag Presentation (TAP)

Both TAP1 and TAP2 belong to the family of ATP-binding cassette (ABC) proteins found in the membranes of many cells, including bacteria. These proteins mediate ATP-dependent transport of amino acids, sugars, ions, and peptides.

a heterodimer

TAP Genes in Mouse H-2

Class IClass I Nonclassical Nonclassical Class IIClass II Class IIIClass III Class I Class I Nonclassical Nonclassical

TAP: 1. has affinity for peptides containing 8 to 16 amino acids. 2. favors peptides with hydrophobic or basic C-terminal a.a., the preferred anchor residues for class I MHC molecules. 3. Thus, TAP is optimized to transport peptides that will interact with class I MHC molecules. 4. different allelic forms of TAP genes exit within the population.

Peptides Assemble with Class I MHC Aided by Chaperone Molecules

molecular chaperones: facilitate the folding of polypeptides- calnexin: a resident membrane protein of the ER, promoting the folding of class I α chain.- calreticulin: a chaperone - tapasin (TAP-associated protein) : brings the TAP transporter into proximity with class I MHC and allows it to acquire a peptide.- ERp57: allows for the release of class I α/β2m after acquisition of peptide.

ERAP1 and ERAP2:ER aminopeptidase

Exogenous Ags: the endocytic system for degradation of internalized molecules

- APC can internalize Ag by phagocytosis or endocytosis (pinocytosis or receptor-mediated endocytosis).

- The endocytic pathway involves 3 increasingly acidic compartments: early endosome (pH 6.0 - 6.5), late endosomes (endolysosomes) (pH 5.0 - 6.0),

lysosomes (pH 4.5-5.0).

- Lysosomes contain > 40 acid-dependent hydrolases, including proteases, nucleases, glycosidases, lipases, phospholipases and phosphatases.

- Ag is degraded into oligopeptides of about 13 – 18 a.a., which bind to class II MHC molecules and are thus, protected from further proteolysis.

B Cells Can Act as APC through Internalization of Ag by Receptor-mediated Endocytosis

- using mAb as receptors

The Invariant Chain Guides Transport of Class II MHC Molecules to Endocytic Vesicles

catalyzing the exchange of CLIP with antigenic peptides into class II

a negative regulator, binding to HLA-DM and lessening the efficiency of the exchange reaction facilitated by HLA-DM

CLIP : class II-associated invariant chain peptide

Functions of Invariant (Ii, CD74) chain

1. Prevent the binding of endogenous peptides to the class II molecules.

2. Proper folding of the class II and chains

3. Exit of class II from the RER

4. Direct the transport of the class II MHC complex from the trans-Golgi network to the endocytic compartment.

Cross-Presentation of Exogenous Ags

APC may present exogenous Ag to TC cells in the context of class I MHC molecules.

Presentation of Nonpeptide Ags

Non-MHC, Nonclassical Class I molecules – CD1 family of genes

(group 2) (group 1)

- CD1 genes are not located within the MHC.

- The CD1 family of molecules associates with 2m and has general structural similarity to class I MHC molecules.

- The Ag-binding groove of the CD1 molecules is deeper and larger than that of classical class I molecules.

Ags Presented by CD1 Molecules

Mycolic acid of the Mycobacterium tuberculosis cell wall ( 結核桿菌 )

Lipoarabinomannan (a glycolipid) of Mycobacterium leprae ( 痲瘋桿菌 )

CD1 molecules present glycolipids and phospholipids.

- CD1 Ag presentation is another pathway for the processing of Ags.

- TAP is not required.

- CD1 molecules locate and traffic differently from class I or class II MHC molecules.

- The distribution of CD1 molecules is different from the classical class I molecules.

- The mouse CD1d1 is found on T cells, B cells, dendritic cells, hepatocytes, and some epithelial cells.

- CD1 genes can be induced by GM-CSF or IL-3.

You should be able to explain how these happen now!