MAJOR HISTOCOMPATIBILITY COMPLEX (MHC). THE MHC GENE COMPLEX The human MHC is located on chromosome 6.The MHC complex contains a number of genes that.

MAJOR HISTOCOMPATIBILITY

COMPLEX (MHC)

THE MHC GENE COMPLEX

The human MHC is located on chromosome 6.The MHC complex contains a number of genes that control several antigens, most of which influence allograft rejection. These antigens (and their genes) can be divided into three major classes : class I, class II and class III.

The class I and class II antigens are expressed on cells and tissues whereas as class III antigens are represented on proteins in serum and other body fluids (e.g.C4, C2, factor B, TNF). Antigens of class III gene products have no role in graft rejection

A. History 1.In transplantation studies, MHC gene products were identified

as responsible for graft rejection 2.In studies on responses to antigens, MHC gene products

were found to control immune responses, called the immune response (Ir) genes

3.It was determined that antigen-specific T cells recognize protein antigens that are bound non-covalently to MHC gene

products a. Helper T cells recognize peptide bound to class II MHC

gene products,Cytolytic T cells peptide bound to class I MHC

4.The complete three-dimensional structure for both class I and class II determined by x-ray crystallography .

Class I MHCThe class I gene complex contains three major loci,

B, C and A and other undefined minor loci. Each major locus codes for a polypeptide; the alpha-chain that contains antigenic determinants, is polymorphic (has many alleles).

داراي MHC مولكولهاي مختلف افراد اما مشابه فرد يك . هستند ژنتيكي لوكوس هر از مختلفي اللهاي

It associates with beta-2 microglobulin (beta-chain), encoded by a gene outside the MHC complex, and expressed on the cell surface. Without the beta-2 microglobulin, the class I antigen will not be expressed on the cell surface. Individuals with a defective beta-2 microglobulin gene do not express any class I antigen and hence have a deficiency of cytotoxic T cells.

Structure of Class I MHC Molecules

The MHC class 1 molecule has three globular domains alpha 1 (yellow), alpha 2 (green) and

alpha 3 (blue) .

The alpha 3 domain is closely associated with the non-MHC -encoded beta 2 microglobulin (pink). The latter is stabilized by a disulfide bridge (red) and is similar to an immunoglobulin domain in three-dimensional

structure .

The allo antigenic sites which carry determinants specific to each individual are found in the alpha

1 and 2 domains .

The latter also has a carbohydrate chain (blue, CHO) .

There is a phosphate in the cytoplasmic domain .

Papain cleaves near the outer surface of the plasma membrane.

Class I MHC molecules contain two separate polypeptide chains

1) MHC-encoded alpha (or heavy) chain; 43 kDa

2) non-MHC-encoded beta chain ( microglobulin) 12 kDa

b.There are four separate regions 1.peptide-binding region is a groove formed from

the alpha1 and alpha 2 regions which interact to form a "floor" of an 8-stranded, beta-pleated sheet with two opposite "walls" consisting of parallel strands of an alpha-helix. (alpha 1 and alpha 2 each contribute 4 strands of beta-pleated sheet and one alpha-helix).

A peptide 8-10 amino acids long sits in the groove. The greatest variability in amino acids occurs in the alpha1 and alpha 2 sequences that form the groove that interacts with amino acids of the peptide fragment.

Thus, the polymorphism among class I MHC gene products creates variation in the chemical surface of the peptide-binding groove.

For any given MHC molecule, binding of a peptide usually requires the peptide to have one or more specific amino acids at a fixed position, frequently the terminal or penultimate amino acid of the

peptide .

Binding of the specific amino acid in the groove of the MHC molecule occurs in what is termed

anchor site(s) .

The other amino acids can be variable so that each MHC molecule can bind many

different peptides .

Other polymorphic residues of the MHC molecule are those in contact with the T cell receptor (TCR), which interacts with

both peptide and the MHC molecule itself .

2.immunoglobulin-like region is composed of an alpha3 segment that is highly conserved and is homologous to Ig constant domains and non-covalently bound to beta2 microglobulin, an invariant molecule, also homologous to Ig constant domains.

These two interact with alpha1 and alpha2 to maintain their proper conformation.

The importance of the highly conserved region of alpha3 is that CD8, a molecule expressed on cytotoxic T cells that recognize class I MHC

molecules, binds to this region .

)3 (Transmembrane region is a stretch of ~25 hydrophobic amino acids .

) 4 (Cytoplasmic region is the carboxy terminal 30 amino acids. Contains phosphorylation sites and

provides binding sites for cytoskeletal elements .

Most variability in amino acids at different positions along the alpha chain of class I MHC molecules occurs in the alpha 1 and alpha 2 regions. The greatest polymorphism is found for amino acids that line the wall and floor of the groove that binds the peptides .

The regions of variability of MHC I molecules and the interaction of the alpha chain with other subunits of the MHC I complex and the bound peptide

2.Class II MHC

MHC class II molecules comprise two non-identical proteins (alpha and beta) which are non-covalently associated and traverse the plasma membrane with the N terminus to the outside of

the cell .

The domains closest to the membrane in each chain are structurally related to immunoglobulins. With the exception of the alpha 1 domain, all

domains are stabilized by disulfide bridges (red) .

Both the alpha and beta chains are glycosylated .

The beta chain is shorter than the alpha chain (beta mol. wt = 28,000) and contains

the alloantigenic sites .

There is some polymorphism in the alpha chain of some MHC II molecules.

Class II MHCThe class II gene complex also contains at least

three loci, DP, DQ and DR; each of these loci codes for  one alpha- and one beta-chain polypeptide which associate together to form the class II antigens.

Like the class I antigens, the class II antigens are also polymorphic. The DR locus may contain more than one, possibly four, functional beta-chain genes.

MHC class II

a. contains two non-covalently associated polypeptide chains, both MHC-encoded

and polymorphic, especially the beta chain in human class II MHC

1.alpha chain; 34 kDa

beta chain; 28 kDa

b. There are four separate regions

1.peptide-binding region is formed by interaction of the alpha1 and beta1 segments.

There is a groove having a "floor" of 8 beta-pleated strands and two "walls" with alpha-helices; alpha1 and beta1 make equal contributions to this structure.

As in the case for class I MHC, the greatest polymorphic variability is in the amino acids facing the groove.

Thus, as for class I MHC, the genetic polymorphism determines the chemical structure of the groove and influences

the specificity and affinity of peptide binding and T cell recognition.

Peptides associated with class II MHC are 13-25 amino acids long;

the longer peptides =project from the ends of the groove.

As with class I MHC, anchor sites for one or more amino acids also exist in the groove of the class II MHC molecule, but these occur at more variable locations.

تر محدود يك س كال به نسبت دو كالس مولكولهاي توزيعبدون يعني مداوم و طبيعي طور به كه طوري به است

انفوسيتهاي در خارجي و Bتحريك دندريتيك، سلولهاي ،بعضي چه اگر گردند مي ظاهر تيموس تليال اپي سلولهاي

نيز ماكروفاژها و اندوتليال سلولهاي مثل ديگر سلولهاياينتر مثل اي كننده فعال عوامل تحريك اثر در است ممكن

ظاهر را دو كالس مولكولهاي شوند وادار گاما فرونسازند.

تفاوت دايمر MHCمهمترين فرم داشتن در دو و يك كالس ( - ) آن توانايي به شايد كه بتا آلفا جفت يك است دو كالس

لنفوسيت سطح گيرنده مولكول دو به اتصال در Tمولكوللنفوسيت شدن فعال به منجر كه گردد مي . Tبر گردد مي

The greatest polymorphism for the beta chain of class II MHC molecules is found for those amino acids in the beta I region that line the wall and floor of the groove that binds the peptide.

2. Immunoglobulin-like region formed by alpha 2 and beta 2 is folded into Ig-like domains. These are largely non-polymorphic.

The correlation of CD4 expression on helper T cells with a specific TCR for class II MHC molecules is due to binding of the CD4 molecules to the Ig-like non-polymorphic beta 2 domain of the class II MHC molecules.

3,4.Transmembrane and cytoplasmic regions have

proposed functions similar to their counterparts in class I MHC.

Similarities between class I and class II MHC molecules Despite differences in the two-chain composition of class I

and class II MHC molecules, they are quite similar structurally.

Important aspects of MHC 1. Because MHC molecules are membrane-associated and

not soluble, T cells must make cell to cell contact with cells expressing MHC molecules.

2 . In general, peptide fragments of proteins in the cytosol associate with class I MHC; those of vesicular proteins associate with class II MHC. Each is recognized by functionally distinct T cell populations. Tc recognize class I MHC-peptide; Th recognize class II MHC-peptide.

3.There are many different MHC gene products for class I

and class II in the human population (polymorphism), only some of which are found in an individual. The maximum number of class I MHC gene products expressed in an individual is six; that for class II

MHC products can exceed six but is also limited.

Whether or not a peptide fragment can associate with a given MHC product determines whether there will be an immune response. This is one level of control of immune responses.

4. Mature T cells respond to foreign antigens, but not self protein. The repertoire of antigen recognition is based on selection processes involving MHC molecules that occur

mainly in the thymus. This is another level of control of immune responses. 5. Only a single binding site exists on a class I or class II

MHC molecule; all peptides it is capable of binding must bind to the same site.

6. The MHC polymorphism is determined only in the germline.

There is no somatic DNA recombination that occurs for antibodies and for the TCR, so the MHC genes lack recombinational mechanisms for generating diversity. As a result, the affinity and selectivity of MHC molecules for foreign proteins are considerably lower than those of

antibodies and T cell antigens. 7.Because each MHC molecule can bind many different

peptides, the binding is said to be degenerate. Compare with the restricted binding of a hormone to a receptor, for

example, where a difference in even one amino acid may impair binding.

8.cytokines, especially interferon gamma (IFN-gamma), increase the level of expression of class I and class II MHC molecules.

9. Alleles for MHC genes are co-dominant, i.e. each gene

product is expressed on the cell surface. (Contrast this with allelic exclusion.)

10. Why the high polymorphism of MHC molecules?

Although an individual may not possess MHC molecules capable of binding certain antigenic peptides, say for example against a virulent organism, the likelihood is great that other MHC in the species can do so.

Overall this affords an evolutionary protection for the continuation of that species.

MHC ANTIGENSHLA specificities are identified by a letter for locus and a

number (A1, B5, etc.) and the haplotypes are identified by individual specificities (e.g., A1, B7, Cw4, DP5, DQ10, DR8).

Specificities which are defined by genomic analysis (PCR), are names with a letter for the locus and a four digit number (e.g. A0101, B0701, C0401 etc). 

Specificities of mouse MHC (H-2) are identified by a number. Since laboratory mice are inbred, each strain is homozygous and has a unique haplotype. The MHC haplotype in these strains is designated by a 'small' letter (a, b, d, k, q, s, etc.); for example, the MHC haplotype of Balb/c mice is H2d.

InheritanceMHC genes are inherited as a group haplotype), one from

each parent (. Thus, a heterozygous human inherits one paternal and one

maternal haplotype, each containing three class-I (B, C and A) and three class II (DP, DQ and DR) loci.

A heterozygous individual will inherit a maximum of 6 class I specificities.

Similarly, the individual will also inherit DP and DQ genes and express both parental antigens.

Since the class II MHC molecule consists of two chains (alpha and beta), with some antigenic determinants (specificities) on each chain, and DR alpha- and beta-chains can associate in  either cis (both from the same parent) or trans (one from each parent) combinations, an individual can have additional DR specificities.

Also, there are more than one functional DR beta-

chain genes. Hence, many DR specificities can be found in any one individual.

MHC antigen expression on cellsMHC antigens are expressed on the cell surface in

a co-dominant manner: products of both parental genes are found on the same cells.

However, not all cells express both class I and class II antigens.

While class I antigens are expressed on all nucleated cells and platelets (and red blood cells in the mouse), the expression of class II antigens is more selective. They are expressed on B Cells, a proportion of MQs and monocytes, skin associated (Langerhans) cells, dendritic cells and occasionally on other cells.

MHC detection by serological testThe MHC class I antigens are detected by

serological assays (Ab and C) .

Tissue typing sera for the HLA were obtained, in the past, from multiparous women who were exposed to the child's paternal antigens during  parturition and subsequently developed antibodies

to these antigens .

More recently, they are produced by monoclonal antibody technology.

MHC detection by mixed leukocyte reaction (MLR)It has been observed that lymphocytes from one donor,

when cultured with lymphocytes from an unrelated donor are stimulated to proliferate.

It has been established that this proliferation is due to a disparity (differences, inequality) in the class II MHC (DR) antigens and T cells of one individual interact with allogeneic class II-MHC antigen-bearing cells. (B cells, dendritic cells, Langerhans cells etc.) 

This reactivity was termed mixed leukocyte reaction

(MLR) and has been used for typing some class II MHC antigens.  

The test lymphocytes were mixed with irradiated or mitomycin-C treated homozygous leukocytes, containing B-lymphocytes and monocytes (stimulator cells).

In culture (over 4 - 6 days), T-cells (responder cells) recognize the foreign class II antigen and undergo transformation (DNA synthesis and enlargement: blastogenesis) and proliferation (mitogenesis).

These changes can be recorded by the addition of radioactive (tritiated, 3H) thymidine into the culture and monitoring its incorporation into DNA. 

Most modern laboratories, however, are switching to PCR technology for tissue typing using specific probes for MHC specificities

مي صورت مرحله دو در پپتيدها به پروتئين تجزيهگيرد:

) به) اسيدي وزيكولهاي درون كالس MHCالف .( شوند مي دومتصل

) به) آندوپالسميك رتيكولوم و سيتوپالسم درون بMHC.( شوند مي متصل يك كالس