MHC Molecules – Major Histocompatibility Complex

Major Histocompatibility Complex (MHC Molecules)

• The term “histocompatibility” is self-explanatory: “histo” means “tissue” and “compatibility” means “getting along.”
• The initial investigations to identify the major histocompatibility complex (MHC) genes investigated why tissues and organs from one member of a species were destroyed when transplanted into another member of the same species.
• For instance, a patch of skin transplanted from a donor mouse to a recipient is sometimes accepted (histologically compatible) and sometimes rejected (histo-incompatible).
• If the recipient mouse and donor mouse share the same genetic background (same inbred strain), the transplanted skin graft is accepted and permanently fuses with the recipient’s skin.
• However, if the recipient and donor have distinct genetic histories (different inbred mice strains), there are antigens in the graft that the recipient’s immune system recognises as foreign, resulting in the destruction of the transplanted tissue.
• Antigens responsible for this form of response are the MHC proteins of the donor’s peptide display molecules.
• The genes influencing the histocompatibility of tissue transplantation have been mapped to a wide genomic area with several loci, hence the word “complex.”
• In order to distinguish them from other molecules (encoded elsewhere in the genome) that had very little impacts on histocompatibility, these molecules are referred to as “major” histocompatibility molecules.
• Consequently, the genes encoding these molecules were given the name “major histocompatibility complex.” Due to the presence of numerous loci in the MHC, each individual was found to express a variation of MHC molecules on his/her cells.
• MHC haplotype is the collective term for a collection of MHC alleles (see later). The MHC’s role in tissue acceptance and rejection was recognised early on, as were its transplantation-related clinical applications.
• However, since tissue transplantation is not a normal occurrence, experts questioned the physiological function of these molecules within the host’s body.
• This question proved to be substantially more difficult to answer than anticipated. The physiological function of the MHC in T cell antigen recognition was not clarified until decades of speculation and many key investigations.

Nomenclature and Inheritance of Major Histocompatibility Complex (MHC)

• The HLA specificities are defined by a letter and a number (A1, B5, etc.), whereas the haplotypes are identified by the specificities of each individual (e.g., A1, B7, Cw4, DP5, DQ10, DR8).
• Genomic analysis (PCR)-defined specificities are denoted by a letter for the locus and a four-digit number (e.g., A0101, B0701, C0401, etc.).

Inheritance of Major Histocompatibility Complex (MHC)

• Histocompatibility genes are passed down as a group (haplotype) from each father. Thus, MHC genes are expressed codominantly in each individual.
• A heterozygous individual receives one paternal and one maternal haplotype, with each haplotype comprising three Class-I (B, C, and A) and three Class II (DP, DQ, and DR) sites.
• Each individual can inherit up to two alleles per locus.

Characteristics of MHC Molecules

• MHC molecules are glycoproteins encoded on chromosome 6 by a vast cluster of genes.
• Their powerful effect on the immunological response to transplanted tissue led to their discovery (see later). This is why the gene complex is known as the major histocompatibility complex.
• In 1937, MHC genes (known as the H-2 complex in mice) were identified as a barrier to transplanting in mice. In humans, these genes are typically referred to as human leukocyte antigens (HLA), as they were initially identified based on antigenic variations between different people’ white blood cells.
• MHC refers to the region found on the short arm of human chromosome 6p21.31 and mouse chromosome 17 respectively. It contains more than 200 genes in humans.

• MHC’s primary role is to provide antigen to T lymphocytes so that they can distinguish between self (our cells and tissues) and nonself (the invaders or modified self).
• Two primary properties of the MHC make it challenging for viruses to elude immune responses: First, the MHC has several genes. It has many MHC-I and MHC-II genes, so that each individual possesses a unique set of MHC molecules with varying peptide-binding specificities. The MHC is also exceedingly polymorphic. MHC genes exhibit the highest level of variation in the human genome. There are numerous variants of each gene throughout the entire population. Alleles are the genetic variants that an individual inherits from his or her parents. Table 10-1 displays the number of alleles recognised at the classical loci.
• The majority of polymorphic sites are located in the domains of the MHC-I and MHC-II molecules.
• Although each HLA molecule has a slightly different amino acid sequence, resulting in a slightly altered three-dimensional structure in the peptide-binding cleft, the basic structures of MHC-I and MHC-II molecules are extremely similar; however, the manner in which the peptide is bound and presented in the binding cleft differs between Classes I and II.
• The groove’s charge properties determine which peptides can be delivered. Due to the fact that different antigenic peptides have different shapes and charge characteristics, it is crucial that the human population as a whole possesses a wide variety of HLA molecules, each with differently shaped peptide-binding areas (clefts), in order to deal with the abundance of self and nonself peptides presented.

• Three areas make up the MHC: MHC-I, MHC-II, and MHC-III.
• HLA-A, -B, and -C are the conventional HLA antigens encoded in the MHC-I region, while HLA-DR, -DQ, and -DP are encoded in the MHC-II area.
• The MHC-III region contains multiple genes involved in the complement cascade (C4A, C4B, C2, and FB) (see section 6, Complement), the TNF-a and TNF-b (LTa) genes, the CYP21 gene that encodes an enzyme in steroid metabolism, the HSP70 gene that encodes a chaperone, and numerous other genes with unknown immunological function.
• Typically, when we refer to MHC molecules, we mean either MHC-I or MHC-II.
• MHC-I molecules are composed of two polypeptide chains, the larger a chain encoded on chromosome 6 in the MHC region and the smaller b2 microglobulin encoded on chromosome 15.
• The class I a chains are a single polypeptide with three extracellular domains designated a1, a2, and a3, a transmembrane region that attaches it to the plasma membrane, and a short intracytoplasmic tail.
• The b2 microglobulin is encoded on chromosome 15 and consists of a single non-polymorphic molecule noncovalently attached to the alpha chain. The a1 and a2 domains fold into a single structure composed of two segmented a helices resting on an eight-stranded antiparallel b sheet.
• The lengthy cleft or groove created by the folding of the a1 and a2 domains is where peptide antigens attach to the MHC-I molecule and are delivered to the CD8 lymphocyte. MHC-II molecules consist of two polypeptide chains, a and b, which are noncovalently linked and encoded in the MHC-II region on chromosome 6. Similar to the MHC-I a chain, the a and b chains of the MHC-II molecule have a transmembrane region and cytoplasmic tail.
• The a2 and b2 domains close to the extracellular membrane are similar to immunoglobulinconstant domains.
• The crystallographic structure of the MHC-II molecule reveals that it is folded in a manner remarkably similar to that of the MHC-I molecule.
• MHC-II molecules possess more open peptide-binding clefts than MHC-I molecules. The cleft of the MHC-II molecule is composed of an association between the a1 and b1 domains that is noncovalent and that binds the peptide via numerous van der Waals forces and hydrogen bonds.
• The primary result of this distinction is that the ends of peptides attached to MHC-I molecules are buried within the molecule, while those bound to MHC-II molecules are not.
• This variation permits MHC-II molecules to bind a wider range of peptide lengths and kinds. Peptides that bind a certain class II molecule will share the same central anchor residues, but other residues may vary in length and sequence.

HLA Complex

• The MHC in the human genome is known as the HLA complex (for human leukocyte antigen complex).
• The HLA complex encompasses approximately 3,500 kb on chromosome 6 and is composed of 12 main sections.
• Each region contains dozens of genes, of which only a few are functional and the majority do not seem to be involved in antigen presentation.
• The HLA-A, HLA-B, and HLA-C regions all belong to MHC class I. Each has a single gene expressing a human MHC class I α chain.
• The DP, DQ, and DR regions all belong to MHC class II. Each has numerous functional genes encoding the and chains of MHC class II  α and β chains.
• MHC class Ib proteins are encoded by a single gene in each of the HLA-E, -F, and -G regions, while MHC class IIb proteins are encoded by many genes in the DM and DO regions.
• MHC class Ib and IIb proteins are structurally similar to MHC class I and II proteins, respectively, but are not directly involved in the presentation of antigens to T cells.
• Therefore, MHC class Ib and IIb proteins are regarded as “non-classical” MHC molecules.
• The MHC class III area is not known to encode any peptide-binding presentation molecules, although it does contain numerous genes involved in immunological responses, including as those encoding complement components, HSPs, and the cytokines TNF and lymphotoxin (LT).

HLA Typing

• Since HLA antigens are identified on practically all of the body’s tissues (with few exceptions), their identification is also known as “Tissue Typing.”
• A donor-to-recipient HLA match is desirable for allogenic (distinct) transplantation.
• Class I typing procedures include microcytoxicity (for typing A, B, and C loci) and CML cellular approaches (for HLA-DPw typing).
• Class II typing utilises cellular techniques like MLR/MLC (for DR typing) and molecular procedures like PCR and direct sequencing (for DR, DQ typing).

Significance of HLA Typing

• Anthropology: The fact that HLA types vary greatly amongst ethnic populations has allowed anthropologists to establish or corroborate the relationship between populations and migration patterns. HLA-A34, which is present in 78% of Australian Aborigines, is less than 1% prevalent in both Australian Caucasoids and Chinese.
• Paternity Testing: If a man and a child share an HLA haplotype, there is a potential that the man is the father, but this cannot be verified. If they do not match or share a haplotype, however, it is determined that he is not the father.
• Transplantation: Because HLA plays such a prominent role in transplant immunity, histocompatibility testing prior to organ transplantation is crucial. Results with closely related living donors matched for one or both haplotypes with the recipient are superior to those obtained with unrelated cadaveric donors. i. Transfusion. ii. Forensic science. Iii. Disease Association. 

Disease Associations with HLA

Several diseases have been discovered to be more prevalent in persons with particular MHC haplotypes. Ankylosing spondylitis (B27), celiac disease (DR3), and Reiter’s syndrome are the most prevalent (B27).

• Disease Associations with Class I HLA: Ankylosing spondylitis (B27), Reiter’s disease (B27), acute anterior Uveitis (B27), and psoriasis vulgaris are diseases associated with Class I HLA (Cw6).
• Class II HLA is associated with the following diseases: Hashimoto’s disease (DR5), Primary myxedema (DR3), Graves thyrotoxicosis (DR3), Insulin-dependent diabetes (DQ2/8), Addison’s disease (adrenal) (DR3), Good pasture’s syndrome (DR2), Rheumatoid arthritis (DR4), Juvenile rheumatoid arthritis (DR8), Sjo (DR3). There is no recognised explanation for this relationship. Nonetheless, other ideas have been presented, including antigenic similarity between pathogens and MHC and antigenic hypo- and hyper-responsiveness mediated by class II genes.

Nomenclature of HLA

• Historically, the HLA nomenclature evolved from the original serological identifiers. Antibody response patterns were originally used to define protein polymorphisms. Modern definitions incorporate DNA sequences to define alleles. During the Tenth International Histocompatibility Workshop in 1987, the current nomenclature was recommended, with minor adjustments added in 1990.
• Each individual possesses two copies of each chromosome; hence, a normal tissue type will include twelve HLA antigens (three HLA class I loci [A, B, and C] from each parent and three class II loci [DR, DQ, and DP] from each parent).
• HLA-DM and HLA-DO are neither extremely polymorphic nor typed. These twelve antigens are co-dominantly inherited.
• A person’s MHC phenotype specifies the alleles they have without reference to heredity. A person may be typed as HLA-A1, -A3; B7, B8; Cw2, Cw4; DR15, DR4, DQ3, DQ6, DP4, DP4; or DR15, DR4, DQ3, DQ6, DP4, DP4.
• A haplotype is the collection of HLA antigens that are inherited from a single parent. For instance, the mother of the individual whose HLA type is shown above may have HLA-A3, -A69; B7, B45; Cw4, Cw9; DR15, DR17; DQ6, DQ2; DP2, DP4; or DR15, DR17. Therefore, the A3, B7, Cw4, DR15, DQ6, and DP4 genes were transmitted from the mother to the child in the preceding sentence. This set of antigens is known as a haplotype. Despite the vast number of alleles at each expressed locus, the number of haplotypes observed in the population is substantially lower than what would be predicted theoretically. This is because specific alleles tend to co-occur on the same haplotype, as opposed to segregating at random. The term for this is linkage disequilibrium.

Rules that dictate the nomenclature of HLA

• HLA is prepended to all antigens and alleles. A capital letter signifies a specific site (A, B, C, or D).
• All genes in region D are prefixed with the letter D and a second letter signifying the subregion of D. (DR, DQ, DP, DM, or DO). Next, loci encoding specific class II peptide chains are discovered (A1, A2, B1, and B2).
• Greek letters are used for protein designations while Latin capital letters are used for gene/allele designations, e.g. DRp1 as opposed to DRB1.
• Specific alleles are denoted by a “*” followed by a two-digit number indicating the most closely connected serologic specificity, and then a two-digit number defining the unique allele.
• For instance, the serologically determined HLA-A2 specificity consists of seventy-seven different alleles. These alleles are currently designated as HLA-A*02:01 to *02:99.
• Some alleles carry a third two-digit number (HLA-B*35:01:01 and B*35:01:02), which indicates that the two variants differ by a silent nucleotide change but do not differ in amino acid sequence 6.

H-2 Complex

• The MHC in the mouse genome is referred to as the H-2 complex. On chromosome 17, the H-2 complex spans 3000 kb and has 12 main areas.
• The K, D, and L sections each contain a single gene encoding the mouse MHC class I α chain.
• Each of the A and E sections encodes a single functional gene for an MHC class II α chain and one or more functional genes for an MHC class II β chain.
• The S section of the H-2 complex encodes MHC class III proteins, including again complement proteins, HSPs, TNF, and LT.
• The Q, T, and M sections of the H-2 complex contain genes for class Ib proteins, while the P, DO, and DM regions encode class IIb proteins.

Distribution of MHC

MHC I Molecules

• The classical class I MHC molecules are expressed on the majority of nucleated cells, however the level of expression varies between cell types.
• Class I molecules are expressed at the highest quantities in lymphocytes, where they account for approximately 1% of all plasma membrane proteins, or 5×105 molecules per cell.
• In contrast, fibroblasts, muscle cells, hepatocytes of the liver, and brain cells express negligible amounts of class I MHC molecules.
• The low amount on liver cells may contribute to the high success rate of liver transplants by decreasing the probability of graft identification by the recipient’s Tc. A few cell types (such as neurons and sperm cells at specific stages of development) appear to be devoid of class I MHC molecules entirely.
• As stated previously, a single MHC molecule can bind many peptides. Since the MHC alleles are codominantly expressed, a heterozygous individual expresses the gene products at each MHC locus that are encoded by both alleles.
• On each of its nucleated cells, an F1 mouse expresses the K, D, and L from each parent (six distinct class I MHC molecules). In humans, the A, B, and C alleles from each parent (six distinct class I MHC molecules) are expressed on the membrane of each nucleated cell by a heterozygous individual.
• Due to the production of so many class I MHC molecules, each cell is able to present a high number of peptides in the peptide-binding clefts of its MHC molecules.
• In normal, healthy cells, class I molecules will exhibit self-peptides as a result of the natural turnover of self proteins.
• In virus-infected cells, both viral peptides and selfpeptides will be expressed. On the membrane of a single virus-infected cell, there are many class I molecules displaying diverse sets of viral peptides.
• Due to individual allelic variances in the peptide-binding clefts of class I MHC molecules, different people within a species will be able to bind distinct sets of viral peptides.

MHC II Molecules

• In contrast to class I MHC molecules, class II molecules are expressed constitutively only by antigen-presenting cells, primarily macrophages, dendritic cells, and B cells; however, thymic epithelial cells and other cell types can be induced to express class II molecules and to function as antigen-presenting cells under certain conditions and in response to stimulation by certain cytokines.
• Among the numerous cell types that express class II MHC molecules, there have been reported to be significant expression discrepancies.
• In some instances, class II expression is dependent on the stage of cell development. Class II molecules, for instance, cannot be found on pre-B cells but are constitutively produced on the membrane of mature B cells.
• Until they are triggered by contact with an antigen, monocytes and macrophages express only modest quantities of class II molecules. α
• Due to the fact that each of the classical class II MHC molecules consists of two distinct polypeptide chains that are encoded by different loci, a heterozygous individual expresses not only the parental class II molecules, but also molecules including α and β chains from different chromosomes. For instance, an H-2k mouse expresses class II IAk and IEk molecules; an H-2d mice expresses class II IAd and IEd molecules.
• Four parental class II molecules and four molecules having one parent’s chain and the other parent’s chain are expressed in the F1 offspring of mice with these two haplotypes.
• Since the human MHC contains three classical class II genes (DP, DQ, and DR), an individual who is heterozygous expresses six parental class II molecules and six molecules containing α and β chain combinations from any parent.
• The existence of numerous β-chain genes in mice and humans, and multiple α-chain genes in humans, increases the number of various class II molecules expressed by one individual.
• This diversity likely increases the amount of antigenic peptides that can be presented, which is helpful for the organism.

What is Linkage Disequilibrium?

• Linkage disequilibrium is a genetic phenomena in which two alleles are more frequently observed together than would be predicted.
• It is the relationship between alleles at distinct loci that is not random. For instance, if 16% of the population has a particular HLA-A antigen (A1) and 10% of the population has a certain HLA-B antigen (B8), the probability of discovering A1 genetically connected to B8 on the same chromosome is given by the product of their gene frequencies (16% x 10% = 6%).
• In actuality, this is not always the case. Certain combinations of A and B specificities occur more frequently than would be predicted by chance alone. The frequency of the combination A1 and B8 in human populations is 8.8 percent, compared to the expected frequency of 1.6 percent. These coupled specificities are referred to as being in linkage disequilibrium.
• Haplotypes HLA-A1, B8, DR3 (DRB1*0301), and DQ2 (DQB1*0201) are highly preserved in the Caucasian population.
• This effect is so prevalent in HLA class II that the existence of specific HLA-DR alleles can be used to accurately predict the HLA-DQ allele prior to testing.
• The order of the HLA alleles on chromosome 6 is DP-DQ-DR-B-Cw-A. Typically, linkage disequilibrium is strongest between alleles that are physically nearest to one another. It is plausible that certain haplotypes are favourable from an immunological standpoint, giving them a selective advantage.

Types of Major Histocompatibility Complex (MHC)

The major histocompatibility complex is a group of genes arranged along a lengthy stretch of DNA on chromosome 6 in humans and chromosome 17 in mice. Humans and mice, the species in which these regions have been researched the most, refer to the MHC as the human leukocyte antigen (HLA) complex and H-2 complex, respectively. Although the order of genes in the two species differs slightly, the MHC genes in both cases are grouped into sections encoding three types of molecules.

• Class I MHC genes: Class I MHC genes encode glycoproteins expressed on nearly all nucleated cell surfaces; the primary function of class I gene products is presentation of endogenous peptide antigens to CD8+ T lymphocytes. 
• Class II MHC genes: Class II MHC genes encode glycoproteins that are principally expressed on APCs (macrophages, dendritic cells, and B cells), where they present exogenous antigenic peptides to CD4+ T lymphocytes.  
• Class III MHC genes: Class III MHC genes encode several proteins, some of which have immunological roles, such as complement components and molecules implicated in inflammation.

Class	Encoding	Expression
I	(1) peptide-binding proteins, which select short sequences of amino acids for antigen presentation, as well as (2) molecules aiding antigen-processing (such as TAP and tapasin).	One chain, called α, whose ligands are the CD8 receptor—borne notably by cytotoxic T cells—and inhibitory receptors borne by NK cells
II	(1) peptide-binding proteins and (2) proteins assisting antigen loading onto MHC class II’s peptide-binding proteins (such as MHC II DM, MHC II DQ, MHC II DR, and MHC II DP).	Two chains, called α & β, whose ligands are the CD4 receptors borne by helper T cells.
III	Other immune proteins, outside antigen processing and presentation, such as components of the complement cascade (e.g., C2, C4, factor B), the cytokines of immune signaling (e.g., TNF-α), and heat shock proteins buffering cells from stresses	Various

There are two primary classes of MHC molecules: I and II. Both of these MHC molecules are membrane-bound glycoproteins with structural and functional similarities. X-ray crystallography has been utilised to determine the three-dimensional structures of the extracellular domains of both classes of MHC molecules. These membrane glycoproteins serve as highly specialised antigen-presenting molecules with grooves that form exceptionally stable complexes with peptide ligands, displaying them on the cell surface for T-cell recognition via T-cell receptor (TCR) engagement. In contrast, class III MHC molecules are a collection of unrelated proteins that do not have structural or functional similarities with class I and II molecules, although many of them are involved in other elements of the immune response.

Class I and class II molecules have ultimate quaternary structures that are quite similar, despite the fact that their primary through quaternary protein configurations are distinct. MHC molecules assemble within the cell, where they bind with short peptide fragments generated from either the cell’s own proteins or proteins that have been absorbed via phagocytosis or pinocytosis. In addition, they differ in terms of the cells that express them and the antigens they deliver to T cells. Class I molecules are found on all nucleated cells in the body and are specialised for delivering cytosolic antigens, such as viral proteins (MHC class I molecules bind to peptides derived from proteins being synthesised within the cell). These are delivered to CD8+ T lymphocytes, which detect and destroy intracellular antigen-expressing cells. In contrast, class II MHC molecules are produced almost exclusively on antigen-presenting cells (APCs), a subpopulation of leukocytes that specialise in presenting antigens from extracellular spaces that have been ingested by these cells, such as fungus and extracellular bacteria (MHC class II molecules bind to peptides derived from proteins made external to the cell). Once produced on the cell surface, the MHC class II molecule transmits the antigenic peptide to CD4+ T cells, which are then activated and stimulate largely anti-extracellular invading immunity.

1. MHC class I molecules

Structure of MHC class I molecules

• A single class I MHC molecule is composed of a 45-kilodalton (kDa) chain and a 12-kDa β2-microglobulin molecule that are noncovalently linked.
• The chain is composed of three external domains (α1, α2, and α3), each consisting of approximately approximately 90 amino acids; a transmembrane domain consisting of approximately 25 hydrophobic amino acids followed by a short stretch of charged (hydrophilic) amino acids; and a cytoplasmic anchor segment consisting of 30 amino acids.
• The α chain is encoded by polymorphic genes known as HLAA, HLAB, and HLAC within the A, B & C regions of the human HLA complex located located on chromosome no. 6 and H2 K, H2D, and H2 L genes within the K, D/L region of the mouse H2 complex located on chromosome no. 17, resulting in the expression of at least three different class I proteins in each cell.  α3
• The 2β2-microglobulin domain is comparable in size and structure to the α3 domain. β2-microglobulin lacks a transmembrane region and is noncovalently associated with the MHC class I α chain.
• β2-microglobulin is a protein encoded by a gene situated on a separate chromosome that is highly conserved.
• The α3 domain of MHC class I, β2- microglobulin, and the constant-region domains of immunoglobulin molecules share a high degree of sequence homology; hence, MHC class I has been classified as a member of the immunoglobulin superfamily of proteins.

α-chain of MHC-I

• The α-chain is a transmembrane glycoprotein that is encoded by a polymorphic gene in the A, B, and C regions of the human HLA complex.
• Hydrophobic transmembrane segment and hydrophilic cytoplasmic tail bind the α-chain to the plasma membrane.
• The α-chain consists of three domains (α1,α2 and α3). Each domain has around 90 amino acids, with a transmembrane domain consisting of roughly 25 hydrophobic amino acids followed by a brief stretch of charged (hydrophilic) amino acids and cytoplasmic tails of 30 amino acids.
• α1 and α2 domains interact to generate a peptide-binding groove on the surface of the protein. It can bind antigens consisting of 8 to 10 amino acids.
• Immunoglobulin fold is the organisation of α3 and β2 into -pleated sheets, each generated by antiparallel β-strands of amino acids.α-chain and β2 microglobulin are designated as members of the immunoglobulin superfamily receptor according to their structure.

β2 microglobulin of MHC-I

• β2 microglobulin is a protein encoded by a chromosome-specific, highly conserved gene.
• β2 microglobulin is comparable in size and structure to α3 domain.
• β2 microglobulin is non-covalently connected to α-chain and lacks a transmembrane region.

Functions of different domains

• The α1 and α2 domains interact to generate a platform of eight antiparallel β strands spanned by two long α-helical regions to create a deep groove, or cleft, with the long α helices serving as the sides and the β strands of the β sheet serving as the bottom. This peptide-binding groove (-25 10 11) is positioned on the top surface of the class I MHC molecule and is large enough to bind an 8- to 10-amino acid peptide in a flexible, extended shape.
• The α3 domain and β2-microglobulin are structured as two -pleated sheets composed of antiparallel β-strands amino acid chains.
• The α3 domain of class I MHC molecules appears to be extremely conserved and contains a region that interacts significantly with the CD8 cell surface protein present on TC cells.
• All three components (class I α chain, β2-microglobulin, and a peptide) are required for the correct folding and surface expression of the MHC-peptide complex. In the absence of β2-microglobulin, the membrane does not express the class I MHC α chain.

Functions of MHC class I

• MHC-primary I’s role is to bind peptide antigens and present them to CD8+ T lymphocytes (T helper cells)
• MHC-I antigen is specific for CD8 T lymphocytes.
• MHC-I binds endogenous antigen and present to T helper cells.
• There are MHC-I molecules on the surface of all nucleated cells.

2. MHC class II molecules

Structure of MHC class II molecules

• Class II MHC molecules are composed of two distinct polypeptide chains, a 33-kDa α chain and a 28-kDa β chain, which connect via noncovalent bonds.
• Class II MHC molecules are membrane-bound glycoproteins with exterior domains, a transmembrane segment, and a cytoplasmic anchor segment, similar to class I α chains.
• Each chain of a class II molecule possesses two exterior domains: α1 and α2 domains on one chain and β1 and β2 domains on the other.
• Sequence similarity exists between the membrane-proximal α2 and β2 domains and the membrane-proximal α3/β2- microglobulin domains of class I MHC molecules with respect to the immunoglobulin-fold structure. Class II MHC molecules are therefore categorised within the immunoglobulin superfamily.
• Composed of α1 and β1 domains, the membrane distal regions of class II molecules provide the peptide-binding groove for processed antigen.
• Humans express three different forms of MHC class II α and β-chain genes, HLA DQ, HLA-DP, and HLADR, while mice express two pairs, H2A (IA) and H2E (IE). Therefore, humans can express at least three distinct class II molecules.

α-chain and β-chain of MHC-II

• α-chain and β-chain of MHC-II are membrane-bound glycoproteins with extracellular domains, a transmembrane segment, and a cytoplasmic tail.
• The α-chain and -chain each include two domains (α1 and α2) and (β1 and β2), respectively.
• The peptide bidding cleft is an open-ended groove developed at the proximal end between the α-chain and β-chain. The cleft can bind antigenic peptides consisting of 13 to 18 amino acids.

Functions of different domains

• The peptide-binding groove of class II molecules, like that of class I molecules, consists of a floor of eight antiparallel β strands and sides of antiparallel α helices, where peptides typically containing 13 to 18 amino acids can bind.
• CD4 is attracted to the β2 domain of class II MHC.
• The polymorphic residues are situated in and around the peptide-binding cleft in α1 and β1.
• The newly generated class II molecule is coupled with a nonpolymorphic polypeptide known as the invariant chain (Ii). 

Functions of MHC class II

• MHC-primary II’s role is to bind peptide antigens and present them to CD4 T lymphocytes.
• Antigen-presenting cells contain MHC-II at their surface (APCs).
• CD4+T-cells are MHC-II-specific
• Activates B cells for generation of antibodies
• Because the immunological response gene in humans is identical to MHC-II, MHC-II plays a crucial role in graft-versus-host response and mixed lymphocyte reaction (MLR).

Feature	Class I MHC	Class II MHC
Constituting polypeptide chains	α chain (45KDa in humans)β2 chain (12 KDa in humans)	α chain (30-34 KDa in humans)β chain (26-29 KDa in humans)
Antigen binding domain	α1and α2 domains	α1 and β1 domains
Binds protein antigens of	8-10 amino acids residues	13-18 amino acids residues
Peptide bending cleft	Floor formed by β sheets and sides by αhelices, blocked at both the ends	Floor formed by β sheets and sides by αhelices, opened at both the ends
Antigenic peptide motifsinvolved in binding	Anchor residues located at amino andcarbon terminal ends	Anchor residues located almost uniformlyalong the peptide
Presents antigenic peptide to	CD8+ T cells	CD4+ T cells

3. MHC class-III Molecules

• MHc-III are a broad set of molecules that provide a vast array of immune system functions.
• MHC-III are not cell surface markers.

Functions of MHC class-III

• Participant in complement activation Participant in inflammation induced by cytokines, tumour necrosis factors, etc.

Antigen Presentation and Processing

Two classical routes are used to digest and present peptides:

Antigen Presentation and Processing In MHC class II

• In MHC class II, phagocytes such as macrophages and immature dendritic cells take up entities via phagocytosis into phagosomes, whereas B cells exhibit the more general endocytosis into endosomes. The phagosomes then fuse with lysosomes, whose acidic enzymes cleave the protein into numerous peptides.
• By means of physicochemical dynamics in molecular contact with the specific MHC class II variations carried by the host, which are encoded in the host’s genome, a specific peptide displays immunodominance and loads onto MHC class II molecules. These are transported to the cell surface and externalised.

Antigen Presentation and Processing In MHC class I

• Any nucleated cell typically contains cytosolic peptides in MHC class I, the majority of which are self-peptides produced from protein turnover and faulty ribosomal products.
• During viral infection, intracellular microbe infection, or transformation into cancer, proteosome-degraded proteins are loaded onto MHC class I molecules and displayed on the cell surface.
• T cells are able to identify peptides expressed on 0.1% to 1% of MHC molecules.

Characteristics of the antigen processing pathways

Characteristic	MHC-I pathway	MHC-II pathway
Composition of the stable peptide-MHC complex	Polymorphic chain α and β2 microglobulin, peptide bound to α chain	Polymorphic chains α and β, peptide binds to both
Types of antigen-presenting cells (APC)	All nucleated cells	Dendritic cells, mononuclear phagocytes, B lymphocytes, some endothelial cells, epithelium of thymus
T lymphocytes able to respond	Cytotoxic T lymphocytes (CD8+)	Helper T lymphocytes (CD4+)
Origin of antigenic proteins	cytosolic proteins (mostly synthetized by the cell; may also enter from the extracellular medium via phagosomes)	Proteins present in endosomes or lysosomes (mostly internalized from extracellular medium)
Enzymes responsible for peptide generation	Cytosolic proteasome	Proteases from endosomes and lysosomes (for instance, cathepsin)
Location of loading the peptide on the MHC molecule	Endoplasmic reticulum	Specialized vesicular compartment
Molecules implicated in transporting the peptides and loading them on the MHC molecules	TAP (transporter associated with antigen processing)	DM, invariant chain

Mechanism of Antigen processing & presentation by MHC class I molecule

Endogenous antigen & cytosolic pathway

• The proteasome, a cytosolic proteolytic mechanism present in all cells, converts intracellular proteins into small peptides. Attaching a tiny protein called ubiquitin to a large number of proteins targets them for proteolysis. These ubiquitin-protein conjugates are degraded by the proteasome complex.
• Each proteasome consists of fourteen subunits arranged in a barrel-like shape of symmetrical rings. Through a small channel at the 19S end, ubiquitin-protein conjugates enter the proteasome complex, which consists of the 20S core and an associated 19S regulatory component. In an ATP-dependent mechanism, the proteasome complex breaks peptide bonds. It is believed that ubiquitin-protein complexes are degraded within the core cavity of the proteasome.
• In addition to the regular 20S proteasomes found in all cells, pAPCs and infected tissue cells contain a unique proteasome of the same size. This specialised proteasome, known as the immunoproteasome, has unique components that can be stimulated by interferon-γ or TNF- α. LMP2 and LMP7 encode replacement catalytic protein subunits that turn ordinary proteasomes into immunoproteasomes, thereby enhancing the synthesis of peptides that bind efficiently to MHC class I proteins.
• TAP (transporter associated with antigen processing), a transporter protein, translocates peptides produced by the proteasome in the cytosol into the RER in a process that needs ATP hydrolysis. TAP is a heterodimer composed of two membrane-spanning proteins: TAP1 and TAP2. TAP is attracted to peptides having eight to sixteen amino acids. The ideal peptide length for class I MHC binding is approximately 9 amino acids, and enzymes found in the ER, such as ERAP, trim longer peptides (endoplasmic reticulum aminopeptidase).
• On ribosomes on the RER, the chain α chain and β2-microglobulin components of the class I MHC molecule are produced.
• The assembly of these components into a stable class I MHC molecular complex requires multiple processes and the participation of molecular chaperones that aid in the folding of polypeptides that can exit the RER. I also requires the presence of a peptide in the class I molecule’s binding groove.
• Calnexin, an ER-resident membrane protein, is the first molecular chaperone engaged in class I MHC assembly. ERp57, an enzyme-active protein, and calnexin bind to the free class I chain and facilitate its folding. When β2-microglobulin binds to the α chain, calnexin is released and the class I molecule binds to the chaperone calreticulin and the protease tapasin.
• Tapasin (TAP-associated protein) facilitates the acquisition of an antigenic peptide by bringing the TAP transporter into close proximity with the class I molecule. Before peptides are exposed to the luminal environment of the RER, the TAP protein facilitates their uptake by the class I molecule.
• As a result of efficient peptide binding, the class I molecule is more stable and is able to dissociate from the complex with calreticulin, tapasin, and ERp57. The class I molecule can then depart the RER and go via the Golgi complex to the cell surface.

Mechanism of Antigen processing & presentation by MHC class II molecule

Exogenous antigen & endocytic pathway

• Antigen-presenting cells (APCs) can internalise particulate material by simple phagocytosis (also known as “cell eating”), in which the material is ingested by pseudopods of the cell membrane, or by receptor-mediated endocytosis, in which the material attaches to specific surface receptors. Both methods are utilised by macrophages and dendritic cells to ingest antigen.
• Once an antigen has been internalised, it is digested into peptides within endocytic processing pathway compartments.
• Early endosomes (pH 6.0–6.5); late endosomes, or endolysosomes (pH 4.5–5.0); and lysosomes appear to be involved in the endocytic antigen processing pathway (pH 4.5). Antigen that has been internalised moves through these compartments, where it encounters hydrolytic enzymes and a decreasing pH in each compartment.
• In the MHC class II-containing compartment (MIIC) of antigen-presenting cells, final protein breakdown and peptide loading into MHC class II proteins take place. Within the endocytic pathway compartments, antigen is degraded into oligopeptides of around 13 to 18 residues that bind to class II MHC molecules in late endosomes.
• When class II MHC molecules are generated in the RER, class II αβ chains bind to a protein known as the invariant chain (Ii, CD74). This conserved, non-MHC-encoded protein interacts with the class II peptide-binding groove, inhibiting the binding of any endogenously produced peptides when the class II molecule is within the RER.
• The invariant chain appears to be involved in the folding of class II α and β chains, their escape from the RER, and the subsequent routing of class II molecules from the trans-Golgi network to the endocytic processing route. In its cytoplasmic tail, the invariant chain carries sorting signals that direct the transport of the class II MHC complex from the trans-Golgi network to endocytic compartments.
• Recent research indicates that the majority of class II MHC invariant chain complexes are transported from the RER, where they are formed, through the Golgi complex and trans-Golgi network, and then through the endocytic pathway, moving from early endosomes to the MIIC late endosomal compartments, and then to the lysosome.
• As the proteolytic activity in each successive compartment rises, the invariant chain degrades gradually. CLIP (class II-associated invariant chain peptide) is a small segment of the invariant chain that remains bound to the class II molecule after the majority of the invariant chain has been broken within the endosomal compartments.
• CLIP physically occupies the peptide-binding groove of the class II MHC molecule, thereby preventing antigen-derived peptide from binding prematurely.
• HLA-DM, a nonclassical class II MHC protein, is necessary to catalyse the exchange of antigenic peptides with CLIP.
• In the presence of HLA-DO, the interaction between HLA-DM and the class II CLIP complex, which facilitates exchange of CLIP with another peptide, is inhibited, as HLA-DO binds to HLA-DM and reduces the efficacy of the exchange reaction.

Regulation of MHC Expression

• The study of the regulatory mechanisms that control the differential expression of MHC genes in various cell types is still in its infancy, although a great deal has been learnt.
• It is anticipated that the release of the whole genetic map of the MHC complex will considerably accelerate the identification and examination of coding and regulatory sequences, leading to new research avenues into how the system is controlled.
• Both class I and class II MHC genes have five promoter regions that bind sequence-specific transcription factors.
• For a number of MHC genes, the promoter motifs and transcription factors that bind to these motifs have been identified.
• Positive and negative elements both regulate the MHC’s transcriptional regulation. CIITA, an MHC II transactivator, and RFX, another transcription factor, have both been demonstrated to bind to the promoter region of class II MHC genes.
• These transcription factor deficiencies induce one variant of bare lymphocyte syndrome. Due to the crucial role of class II MHC molecules in T-cell maturation and activation, patients with this illness suffer from severe immunodeficiency due to the absence of class II MHC molecules on their cells.
• Various cytokines control the expression of MHC molecules as well. It has been demonstrated that the interferons (alpha, beta, and gamma) and tumour necrosis factor promote the expression of class I MHC molecules on cells.
• Interferon gamma (IFN-γ), for instance, seems to cause the production of a transcription factor that binds to the promoter sequence bordering the class I MHC genes.
• This transcription factor appears to coordinate the up-regulation of transcription of the genes encoding the class I chain, α chain, β2-microglobulin, the proteasome subunits (LMP), and the transporter subunits by binding to the promoter region (TAP).
• It has been demonstrated that IFN-γ induces expression of the class II transactivator (CIITA), thereby indirectly enhancing expression of class II MHC molecules on a range of cells, including non-antigen-presenting cells (e.g., skin keratinocytes, intestinal epithelial cells, vascular endothelium, placental cells, and pancreatic beta cells).
• Other cytokines only alter MHC expression in specific cell types; for instance, IL-4 promotes class II molecule expression in resting B cells.
• IFN-γ inhibits B cell expression of class II molecules; corticosteroids and prostaglandins also inhibit B cell expression of class II molecules.
• Infection with several viruses, including human cytomegalovirus (CMV), hepatitis B virus (HBV), and adenovirus 12, reduces MHC expression (Ad12).
• In some instances, decreased production of class I MHC molecules on cell surfaces is owing to decreasing quantities of a component required for peptide transport or MHC class I assembly, as opposed to transcription.
• In CMV infection, for instance, a viral protein attaches to β2-microglobulin, inhibiting the assembly and transport of class I MHC molecules to the plasma membrane.
• Infection with Adenovirus 12 significantly reduces the transcription of transporter genes (TAP1 and TAP2). As shown in the following chapter, the TAP gene products play a crucial function in the transport of peptides from the cytoplasm to the rough endoplasmic reticulum.
• Blocking the expression of the TAP gene reduces peptide transport, preventing class I MHC molecules from assembling with β2-microglobulin or being delivered to the cell membrane.
• By reducing the likelihood that virus-infected cells will show MHC–viral peptide complexes and become targets for CTL-mediated destruction, decreased production of class I MHC molecules, regardless of the underlying mechanism, may aid viruses in evading the immune response.

MHC and Immune Responsiveness

• Immunologists have recognised for a long time that some foreign proteins that elicit robust immune responses in some individuals do not do so in others. Those who did not mount a reaction were previously referred to as “non-responders,” while those who did were referred to as “responders.”
• There were subtle disparities in the level of reaction across respondents, leading to the classification of individuals as low or high responders.
• Immunologists quickly pinpointed the genes responsible for immunological responsiveness to the MHC and demonstrated that mice with distinct MHC haplotypes respond differentially to a given peptide.
• These variations in response levels to a given antigen might be read as changes in the ability of various MHC alleles to present antigen-derived peptides that T cells can detect.
• In an inbred population, there is a larger likelihood that an individual will be a non-responder; that is, a person will lack an MHC allele that can result in specific T cell activation in response to a specific antigen challenge.
• Two ideas, neither of which is mutually incompatible, have been offered to explain non-response: the determinant selection model and the hole in the T cell repertoire model.

a. Determinant Selection Model

• To mount a T cell response against a foreign protein, the host must have at least one MHC allele with a groove that can accommodate a peptide generated from that protein.
• The responsiveness is thus determined by the strength of binding between a certain MHC allele and a given determinant (peptide), which is in turn determined by structural compatibility. In other words, the MHC proteins in an individual “choose” the immunogenic factors and the magnitude of the immune response.
• Since a foreign protein is often digested into three to four immunogenic peptides, it is quite likely that an outbred individual will possess an MHC allele capable of binding to at least one of these peptides and eliciting an immune response.
• The individual is therefore a responder to this particular antigen, and his or her position as a high or low responder coincides with the peptide’s strong or weak binding to MHC, respectively.
• The individual is a non-responder to this antigen if none of his or her MHC molecules can bind to any of the peptides derived from the protein.
• Experiments demonstrating that a peptide is only immunogenic when coupled to a specific MHC allele have provided support for the determinant selection paradigm. For instance, a specific peptide of an influenza virus protein rapidly induces an immunological response in H-2k haplotype inbred mice.
• Specifically, T lymphocytes identify the determinant when it is given to them on the MHC class I H-2Kk molecule.
• However, this peptide fails to excite T cells in H-2b haplotype mice. When displayed on the MHC class I H-2Db molecule, a peptide from a different portion of the same influenza virus protein induces an antiviral response in H-2b mice.

b. Hole in the T Cell Repertoire Model

• Immune non-reactivity may potentially be caused by tolerance mechanisms.
• In non-responders, it is possible that a particular foreign peptide–MHC combination resembles the structure of a self peptide–MHC combination so closely that T cell clones capable of recognising the foreign peptide–MHC combination were eliminated as autoreactive during the establishment of central tolerance.
• This would result in a lack of T cell specificity or a “hole” in a non-T responder’s cell repertoire relative to a responder’s repertoire.

MHC and Disease Predisposition

• The MHC haplotype of an individual determines his or her susceptibility to immunogens. If a person is unable to establish an adequate immune response to an immunogen associated with an infection or malignancy, he or she will likely develop a disease.
• A disease in the form of autoimmunity or hypersensitivity (including allergy) may result from an improper immune response.
• Due to the direct association between immunological responsiveness and distinct MHC alleles, specific MHC haplotypes may predispose individuals to particular susceptibilities or diseases. Numerous illnesses associated with specific MHC alleles manifest as autoimmune disease in humans.
• When self-reactive T cell clones escape the tolerance mechanisms that would usually prevent them from entering or activating in the periphery, autoimmune illness occurs.
• The individual may then contain T cells that can recognise self-components and assault tissues that express them. For instance, type 1 (insulin-dependent) diabetes mellitus is believed to be caused by an autoimmune attack on antigens expressed by insulin-producing β cells in the pancreatic islets. The elimination of β islet cells by the immune system results in insulin insufficiency and diabetes.
• The HLA-DQ8 allele is eight times more frequent in human communities with type 1 diabetes than in healthy ones for unknown causes.
• Similarly, 90% of Caucasian patients with ankylosing spondylitis contain the HLA-B27 allele, compared to 9% of healthy Caucasian individuals.
• Note, however, that the mere presence of a predisposing HLA allele is typically insufficient to produce disease; it is believed that additional genetic and environmental variables are required.
• This finishes our examination of the MHC’s anatomy and physiology.
• In the following chapter, headed “Antigen Processing and Presentation,” we detail how antigenic peptides are produced and how MHC molecules bind to and present these peptides to T cells.

2003 marked the conclusion of the Human Genome Project (HGP), which found ∼25,000 genes in human DNA and sequenced 3 billion base pairs in the human genome. The HGP has enabled unparalleled precision in determining the chromosomal locations and DNA sequences of HLA alleles. By comparing the sequences of HLA alleles derived from distinct populations around the world, researchers have explored the relationships between certain HLA alleles and illness risk among individuals of various nationalities. Moreover, other non-HLA genes have been linked to illness occurrence. The term “Genome-Wide Association Studies” refers to research that utilises the extensive data of the HGP to identify the locations of genes associated with certain diseases (GWAS).

Polymorphism and MHC Restriction

• How did MHC genes get so polymorphic? In an earlier, antigenically simple environment, a primordial MHC molecule that showed endogenous and foreign protein fragments to T cells probably existed, but its diversity was extremely limited (or nonexistent).
• Individuals with many duplications of this primordial MHC gene likely survived as the world became more antigenically complex because they possessed more than one gene devoted to displaying protein fragments.
• Possibly concurrently, distinct alleles of each gene, each with a distinct sequence in the peptide-binding groove, also emerged.
• A wider variety of peptide-binding and -presentation molecules would have been produced. Today, the ensuing polymorphism at numerous MHC loci assures that each member of an outbred species is heterozygous at the majority, if not all, MHC loci, and hence has a high probability of harbouring at least one MHC allele capable of binding to any given antigenic peptide.
• MHC polymorphism indicates that a significant number of MHC alleles are distributed throughout the entire population. In the event of a devastating disease attack, a considerable portion of the population will be able to respond to the infection and survive in order to reproduce the species.
• However, the plurality of MHC molecules does not permit anarchy in terms of T-cell presentation. Exists a phenomena known as MHC limitation, which demands that the epitope detected by a given TCR is a combination of a certain peptide and a particular MHC molecule.

Important Aspects of MHC Molecules

• Although a species exhibits a significant degree of polymorphism, an individual possesses no more than six distinct class I MHC products and only slightly more class II MHC products (considering only the major loci).
• Each MHC molecule contains a single binding site. The various peptides that a particular MHC molecule can bind bind to the same location, but only one at a time.
• Because one MHC molecule can bind numerous distinct peptides, this type of binding is known as degenerate.
• MHC polymorphism is solely determined at the germline level. There are no processes for recombination that generate variety.
• MHC molecules are membrane-bound, and T cell recognition needs cell-cell interaction.
• Alleles for MHC genes are co-dominant. Each MHC gene product is expressed on the surface of a nucleated cell.
• A peptide must bind to a specific MHC of that individual in order for an immune response to occur. That is one control level.
• Mature T cells must have a T cell receptor capable of recognising the MHC-associated peptide. This is the second control level.
• Cytokines (particularly interferon-γ) boost the level of MHC expression.
• Tc cells identify peptides from the cytosol when they bind to class I MHC. Th cells identify peptides from inside vesicles that connect with class II MHC.
• MHC polymorphism is essential for the survival of the species.

How Do Peptides Get Into The MHC Groove?

• CTL cells identify cytosolic peptides through their association with class I MHC. The peptides penetrate the endoplasmic reticulum, where they bind to the MHC class I groove.
• Through the Golgi, this complex is then transferred to the cell surface. In the ER and Golgi, MHC class II molecules are synthesised using an invariant (Ii) chain as a placeholder.
• Once the complex enters a vesicle, the Ii chain is cleaved and eliminated. Within the vesicle, peptides bind to class II MHC before being ejected to the cell surface, where they are identified by Th cells.

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