ZOOLOGY Immunology Structure and Function of Major Histocompatibility Complex

Paper No. : 10 : Immunology Module : 07 : Structure and function of Major Histocompatibility Complex

Development Team

Principal Investigator: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi

Co-Principal Investigator: Prof. D.K. Singh Department of Zoology, University of Delhi

Paper Coordinator: Prof. Anju Srivastava Department of Zoology, University of Delhi

Content Writer: Dr. Soma M. Ghorai Hindu College, University of Delhi

Content Reviewer: Prof. Sukhmahendra Singh Banaras Hindu University

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

Description of Module

Subject Name ZOOLOGY

Paper Name Zool 010: Immunology

Module Name/Title Structure and function of Major Histocompatibility Complex

Module ID M07 Structure and function of Major Histocompatibility Complex

Keywords MHC haplotypes, histocompatible genes, MHC Class-I, MHC-Class- II, MHC-Class-III, Antigen presentation, CD4+ T cells, CD8+ T cells, MHC polymorphism, Natural Selection, Immunoglobulin superfamily, MHCII transactivator (CIITA), HLA typing, Transplantation

Contents

1. Learning Objectives 2. Introduction 3. MHC Haplotypes 3.1 MHC Class-I 3.1.1 Structure of MHC Class-I 3.2 MHC Class-II 3.2.1 Structure of MHC Class-I 3.3 MHC Class-III 4. Structure of Peptide Binding Cleft 4.1 Peptide –MHC interaction 5. MHC Class-I and MHC Class-II Antigen Presentation 5.1 Class-I MHC Antigen Presentation 5.2 Class-II MHC Antigen Presentation 6. Role of MHC in Self/Nonself Recognition 7. The Genetic Architecture of MHC 8. Natural Selection and MHC Genes Polymorphisms 9. Epigenetics and MHC 10. MHC in Transplantation 10.1 Allogenic Immune Responses 10.2 HLA- Testing and Clinics 11. Summary

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

1. Learning Objectives

After studying this module, you shall be able to

 Recognize the various haplotypes of MHC and their structures.  Differentiate between the Class-I MHC and Class –II MHC antigen presentation.  Learn the role of MHC in self/non-self recognition.  Ascertain about the genetic architecture of MHC.  Learn about MHC polymorphism and their basis on Natural selection process.  How epigenetics has a role in MHC polymorphism.  Know the role of MHC in autoimmunity and other disease-conditions.  Why MHC (HLA) gene typing is essential for transplantation. 2. Introduction

For the body to rein an attack on any pathogen; it must be recognized as self or non-self and this is accomplished by the set of cell surface receptors which not only distinguishes self from non-self but also plays a pivotal role in tissue transplantation and rejection, autoimmunity and susceptibility to infections. These cell surface receptors are encoded by the most diverse gene loci in vertebrates known as the major histocompatibility complex (MHC Class I and II loci). In humans, MHC spans a considerable part of the DNA i.e. about 0.1% of total genome containing 200 coding loci. These are highly polymorphic genes that can be attributed to the natural selection over long periods of evolutionary time.

The history behind MHC genes dates back to 1916 when Little and Taylor showed that tissue could be transplanted only in the same strain mice; followed by George D. Snell’s observation in 1935 that several closely linked histocompatible genes are responsible for tissue rejection. He discovered that in tumor transplants on mice from their congenic cousins were immediately rejected but were successful in syngeneics trains. The mystery was solved in 1975 by Zinkernagel and Doherty, who found that T- cell responses were restricted to antigens and the MHC’s presented on the cell surface. The 1980’s and 90’s finally revealed the structure of MHC molecules and their encoded proteins. The Nobel Prize in Physiology or Medicine in 1980 was awarded jointly to Baruj Benacerraf, Jean Dausset and George D. Snell for their contribution in understanding that similar antigens are controlled by dominant autosomal genes termed immune response (Ir) genes located on the MHC loci of all the vertebrates.

Other than any region of the genome, the MHC genes are the most associated with autoimmune and infectious diseases. These disease-associated variations in the MHC regions are due to very subtle and minute changes in the gene loci. There is still more to learn about MHC like (i) what mechanism drives MHC polymorphism, (ii) which are naturally and positively selected variations having functional importance, (iii) which signaling pathways does the MHC genes follow at the molecular level. 3. The MHC Haplotypes

The MHC genes encode three classes of molecules:

3.1 MHC Class I

The MHC class-I heavy chains are encoded by three genes (HLA-A, HLA-B, HLA-C) and presents small peptides to CTLs (cytotoxic T-lymphocytes) enabling each cell of the body to the immune 3

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

system with possible chances of infection, therefore every nucleated cells of the body present MHC-I molecules. Mostly these peptides are self-peptides, but in case of virus-infected cell or cancer cell or parasite infected cell, the foreign peptides are presented resulting in their elimination by CTLs. The CTLs are very unique cells with highly specific TCR (T-cell receptor) that scan for MHC-antigen complexes and CD8+ molecules to stabilize and help bind the cells to MHC – antigens. The cells with foreign antigens help activate the CTLs, resulting in the clonal selection of these specific CTLs, thus eliminating other infected cells. But it is still very difficult to envisage that how MHC molecules achieve the specificity in immune recognition with such small pool of TCR. In fact, MHC molecules can bind to a varied range of peptides rather than specific peptides.

3.1.1 Structure of MHC Class-I

It is composed of α and β polypeptide chains held together with non-covalent bonds with a short cytoplasmic tail (Figure 1). The α-chain is the MHC-encoded integral membrane glycoprotein of ~45000 Da; while β2-microglobulin is non-MHC encoded extracellular ~12kDa peptide. The α-chain is of 350 aminoacids with three globular domains (α1, α2, and α3). α2 and α3, both have intra-chain disulphide linkages. It also has a 26 hydrophobic amino acids transmembrane segment spanning the plasma membrane. The α1 and α2 forms the platform as the peptide-binding unit mainly comprising of β-sheets. The groove formed between them is blocked at both the ends and is enough to hold 8- 10amino acids, so it is imperative for the foreign residues to be processed and presented before they bind to MHC-I. The β2-m chain is non-polymorphic ~90a.a. peptide is encoded by chromosome 15 and interacts non-covalently with α3. This part of the MHC molecule binds to CTL. Class-I MHC molecule is a glycoprotein with one or two N-linked oligosaccharide.

Peptide-binding groove (β-sheets)

Peptide (8-10 a.a.)

Hydrophobic stretch (25 a.a.)

Cytoplasmic tail (10-15 a.a.) Figure 1. Structure of MHC-1 molecule

3.2 MHC Class-II

Class II peptides are expressed on specialized cells called antigen presenting cells (APCs); mainly the macrophages, dendritic cells and B-lymphocytes. MHC –II molecules are encoded by three polymorphic genes (HLA-DR, HLA-DQ and HLA-DP) that bind to different peptides. The immunodominant epitopes are largely confined to the pathways dependent on lysosomal degradation and peptide binding of newly synthesized MHC-II molecules in the acidic endosomal compartment.

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

The MHC-II molecules that are involved in the CD 4+ T-cell response against intracellular pathogens may trigger immunity towards infection, thymic selection, autoimmunity and tumor immunity

3.2.1 Structure of MHC Class -II

Like MHC-I, the class-II MHC molecules have an extracellular domain, a transmembrane domain and an intracellular carboxy terminal tail. The extracellular domain is composed of two non-covalently associated domains (α1, α2 or β1, β2) of ~90 a.a anchored to the cell membrane. The heavy chain (α 1 and α 2) has a molecular weight of ~30-34 KDa and the light chain (β 1 and β 2) has a molecular weight of ~26-29 KDa. α2, β1 and β2 domain contains disulphide linkages, while α1, α2 and β1 domains are glycosylated but β2 domain is not. The transmembrane domain is comprised of ~25 hydrophobic amino acids and the cytoplasmic tail of ~10-15 hydrophilic amino acids. Peptide antigen binds in a groove formed from a pair of α-helices (on a floor of anti-parallel β-strands) involving α1 or β1 segments, respectively. The antigenic peptide – binding domain can hold 13-18 a.a. amino acid residues as it is open at both the ends of the peptide binding groove (Figure 2).

Peptide-binding groove

Peptide binding cleft (13-17 a.a.)

Transmembrane stretch Cytoplasmic tail

Figure 2. Structure of MHC-II molecule.

3.3 MHC Class-III

These molecules are mainly comprised of serum proteases and complement enzymes and do not have any role in antigen presentation. The complement component includes C2 (serine protease), C4A and C4B (pro-proteins of the multichain forms) and factor B (component of alternate complement pathway). They also include the TNF-α and TNF-β, as well two heat-shock proteins. 4. Structure of Peptide Binding Cleft

MHC I and class II molecules fold into a highly similar conformations featuring a peptide‐ binding groove to present T-cell epitopes. Peptide binding grooves of MHC I molecules are composed of two α‐ helices and eight β‐ strands formed by one heavy chain, while MHC II uses two domains from 5

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

different chains to construct the peptide‐ binding groove. Peptides bind to MHC molecules through primary and secondary anchor residues protruding into the pockets in the peptide‐ binding grooves. Peptide preferences are dependent on the amino acids polymorphisms comprising the anchor pockets, which are related to the various alleles of MHC. The conformations of peptides presented by MHC I molecules are length dependent. The modified groups of post-translationally modified peptides have important roles in the peptide–MHC interaction (Figure 3).

Figure 3. Peptide binding domain and cleft of MHC Class-I and Class-II. Source : Liu et al., (2011). Major Histocompatibility Complex: Interaction with Peptides. DOI: 10.1002/9780470015902.a0000922.pub2

4.1. Peptide –MHC Interaction

Peptide binding to MHC class-I and Class-II molecules requires classic anchor peptides at the N and C-terminus of the peptide, these residues being termed as ANCHOR RESIDUES. These anchor peptides bind to the pocket of the binding cleft of the MHC molecules. Class-I MHC anchor residues are mainly comprised of hydrophobic amino acids. At C-terminus, the anchor residues are at the 9tha.a. position mainly comprising leucine and isoleucine, while the anchor at the N-terminus is mostly present at the 2nd or 3rda.a. position e.g. N-terminus anchor residue in H-2Kd is tyrosine, H-2Dd is glycine. The mid-portion arches away from the floor of the MHC complex to make a contact with the TCR.

Peptides that bind to Class-II MHC have 7 to 8 a.a. that provides maximum contact. It has hydrophobic residues at N-terminus, C-terminus as well as 3 a.a. in the middle, thereby providing a constant elevation from the floor of the binding cleft (Figure 4).

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

Figure 4. Peptide-binding grooves of MHC class I and II proteins. (A) Peptides binding in an MHC class I groove are usually 8–10 amino acids long. The residues at both the N-terminus and C-terminus end of the peptide are anchored in the groove. (B) Peptides binding in an MHC class II groove are usually 13–18 amino acids long. The residues bind at both the ends as well as in the middle. Source: Major histocompatibility Complex in Primer to the Immune Response (Second Edition), 2014. 5. MHC Class-I and MHC Class-II Antigen Presentation

Both MHC-I and MHC-II present peptides to the CD8+ and CD4+ T-cells, the sources of these peptides may differ. The MHC-I are presented with intracellular peptides and the exogenous peptides are presented by MHC-II. Recently, it was noted that “cross-presentation” also occur where the exogenous peptides are presented by MHC-I and endogenous by MHC-II as evident by their overlapping characteristics (Figure 5). Both classes have similar three-dimensional structure; they originate from the common parent gene by gene duplication and a similar function in antigen presentation to the T-lymphocytes. However, these classes differ in their tissue distribution and processing of antigenic peptides.

Figure 5. Antigen processing and presentation pathways in dendritic cells. MHC-I pathway: Intrinsic pathogens (like viruses) or endogenous proteins are either degraded in cytosol or nucleus by immunoproteasomes and are presented as peptides along with MHC-I to CD8+ T-CTL cells. MHC-II pathway: The exogenous peptides are endocytosed and via endomembrane system binds to MHC-II to be presented to CD4+Th cells. Cross- presentation pathway: Indicates that the exogenous peptide which has been sequestered via endocytic mechanism may compete for the peptide either to MHC class II or MHC class I pathway. Some DCs have a unique ability to deliver exogenous antigens to the MHC class I pathway. Source: Villadangos J. A. & SchnorreP. (2007). Intrinsic and cooperative antigen-presenting functions of dendritic-cell subsets in vivo. Nature Reviews Immunology 7, 543-555 (July 2007) | 7

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

5.1 Class-I MHC Antigen Presentation

The endogenous antigens are first degraded by cytosolic and nuclear proteasomes. These are immunoproteasomes which are mainly involved in extending the peptide pool rather than be selective for MHC-I peptides, this thus explain the wide diversity of peptides presented. The peptides thus formed are transported via Transporter associated with antigen presentation (TAP) and assemble in ER with the MHC-I chaperone molecule (Tapasin). The binding of the peptide to MHC-I molecule is aided by two more chaperones proteins; calreticulin and ERp57. The complex thus formed after loading of the peptide along with TAP, tapasin, MHC-I, ERp57 and calreticulin is called peptide binding complex (PLC). The MHC class-I complex is finally released of the chaperones and is transported to plasma membrane (Figure 6).

Figure 6. In the MHC class I molecule pathway, endogenous proteins are broken down by proteasomes into smaller peptides. In the endoplasmic reticulum (ER), an antigenic peptide binds to the peptide binding site in an MHC class I molecule. The peptide-MHC complex then migrates through the Golgi apparatus to the cell surface. Source: Neefjes et al., (2011). Towards a systems understanding of MHC class I and MHC class II antigen presentation .Nature Reviews Immunology 11, 823-836.doi:10.1038/nri3084

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

5.2 Class-II MHC Antigen Presentation

MHC-II molecules are synthesized de novo and assembled in the ER. They are transported to golgi where they associate with the chaperone the invariant chain (Ii). MHC-II-Ii complex move to the endomembrane system, there together with other chaperones, DM and DO regulate the loading of peptide within the MHC-II peptide binding cleft. MHC class-II associated Ii peptide (CLIP) remains associated with the peptide binding groove of MHC heterodimer until DM catalyzes the dissociation of CLIP and binds the processed antigen at a low pH. HLA-DM then transfers the CLIP with the specific peptide from the exogenous pathogen brought in through the endosomal pathway to be + presented to CD4 TH cells. Thus, cytosol-to-endosome transport may lead to the processing of proteins translocated into the host cytosol by intracellular pathogens (Figure 7).

Figure 7. In the MHC class II molecule pathway, α and βsubunits of the MHC class II molecule bind to the invariant chain (Ii) in the ER. Ii is partially degraded in an endosomal compartment. The portion of Ii that occupies the antigenic peptide binding site on the MHC class II molecule (called CLIP) is removed with the help of HLA-DM, freeing the molecule for binding processed antigen. Once antigenic peptide has bound to an MHC class II molecule, the complex migrates to the cell surface. Source: cellular and molecular Immunology, 8th Edition (2015) by Abbas et al. 6. Role of MHC in Self/Nonself Recognition

T-cells cannot distinguish between self and non-self peptides. During the early development, most of the immature, undifferentiated T- cells from bone marrow that encounter self-antigens are eliminated. The maturation and proliferation of T cells happen in thymus via random gene rearrangements of TCR genes. This random generation of huge repertoire of T-cells also generates 95% of those cells that bind to self-antigens and are eliminated. The only T-cells that mature and enter the circulation are those that recognize peptide-MHC complex. Thus, through thymic selection, MHC genes influence the development of T-cell repertoire. 7. The Genetic Architecture of MHC

The MHC genes are located in autosomes, Y-chromosomes and even in mitochondrial genome. It is a large chromosomal region of chromosome # 6; that has over 200 coding regions and apart from specific immune functions; these genes also impact growth, development, reproduction, odor and

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

olfaction. The MHC in mice is termed as H-2 (Histocompatible-2) complex and in humans as HLA (human leukocyte antigens) complex. The immune response specific regions of MHC gene cluster (also called classical) are closely linked loci called class I and class II loci and are the most divergent among vertebrates. Among the most studied, humans have six class I loci (A, B and C) and eight class II loci (DP, DQ and DR), whereas mice have three class I loci (K, D and L) and 2 class II loci (A and E) (Figure 8). The polymorphism can be observed with over 170-100 alleles per locus with each locus having multiple coding genes. Close linkage among these loci means each individual inherits a particular combination of MHC alleles as a single haplotype; thereby there are little or no chances of recombination separating these loci resulting in highly polymorphic status. This condition suggests that the MHC are inherited from a common ancestor and is been maintained by natural selection (Figure 9). Till date, it is extremely difficult to unravel the functional aspect of each loci owing to high gene density, extreme polymorphism, strong linkage disequilibrium and clustering of genes with related functions. Hence, our understanding of MHC-related diseases are very poor because (i) natural selection should have eliminated the neutral and deleterious alleles and must have kept only the disease-protective allele (ii) which specific variation among the array of MHC gene variation is functionally relevant (iii) the MHC-gene related diseases may not be in the genes but the discrete changes in the amino-acids or the changes in the groove residues.

Figure 8. The genetic architecture of MHC in humans and mice. Source: Bradford M. Elmer, A. Kimberley McAllister, (2012). Trends in Neuroscience; 35(11), p660–670.

Figure 9. The immunoglobulin multigene superfamily gave rise to various immunoglobulins, T-cell receptors, antibodies as well as MHC genes; suggesting that these molecules evolved from a common ancestor. Source: Dustin J. Penn, 2001. DOI: 10.1038/npg.els.0000919.

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

8. Natural Selection and MHC Genes Polymorphisms

There are many evidences that confirm that MHC polymorphism is maintained due to natural selection (Figure 10). First, high number of alleles in these gene segments or gene densities may arise when neutral alleles are not selected and are randomly fixed through genetic drift. Second, there is lot more gene density and uniformity in the proportions of allelic frequencies. Third, the selection pressure resulted in fewer MHC homozygotes which are not expected of a randomly mating population. Fourth, high linkage among allelic loci in individuals and their strong linkage disequilibrium are more often than chance expectations. Fifth, the high dS/dN (synonymous substitution to non-synonymous substitution) ratio in the antigen- binding site of MHC-genes to both neutrality and purifying selection, maintains diversity. Sixth, the divergence of MHC alleles is pretty ancient in evolutionary time scale, thus the persistence of Class-I and II MHC diversity is clarified by Darwinian Natural selection process.

Figure 10. Chromosomal location of the MHC locus in man depicting allelic polymorphism. Source: International Immunogenetics information systems (IMGT) (http:/www.imgt.org/)

MHC polymorphism though is important for disease resistance; it is also involved in promoting outbreeding. MHC polymorphism is aimed to provide “Herd Immunity” i.e. if large numbers of variables are present in a population then it is more likely that the infection may not get an escape route and the spread is restricted. Thus, it can be related to the potential of certain populations to their relationship with the pathogen-associated selection.

MHC polymorphism among humans can also be explained on the basis of admixture with archaic humans. Whenever a new species is territorially isolated, it acquired a new set of MHC alleles thus, evolving novel alleles. The best example may be given in human evolution where HLA-B*73 allele of HLA-B found in West Asian population has been traced to Denisovnas, who are related to Neanderthals. Hence, certain haplotypes of MHC were introgressed into Eurasian and Oceanian populations much before they were introduced into the African population. The polymorphism among MHC has been useful in tracing ancient population migration; for example, this can help deduce the divergence of early humans from their African origins. 9. Epigenetics and MHC

At times it is difficult to explain certain phenomena through conventional genetics and there are such cases where expression of MHC genes was shown to have an epigenetic effect. The famous example being that 11

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

the offspring’s of mothers carrying the affected allele of MHC-II, HLA-DRB1*15 for multiple sclerosis (an autoimmune disorder) are more likely to get infected rather than fathers carrying the affected gene. The master regulator of MHCII genes has been the MHCII transactivator (CIITA), which play the pivotal role in normal immune function. Significant epigenetic modifications such as methylation, acetylation and histone modification have been demonstrated to alter CIITA expression. Therefore, many diseases associated with aging and autoimmunity is linked to polymorphism of CIITA. 10. MHC in Transplantation

10.1 Allogenic Immune Responses

When a tissue transplant is performed, the HLA of the donor is recognized by the recipient’s immune system, triggering an allogenic immune response. The divergent alloantigens caused due to large numbers of genetic differences between donors and recipients are the easy targets for T-cell recognition, thereby increasing the risk of both graft rejection and graft-versus-host disease (GVHD). Allogenic immune responses may happen by three recognizing mechanisms: (1) Indirect recognition- the donor’s HLA is processed by APC and presented as peptides with MHC molecules to the recipient’s T-cells. This mechanism is mainly dominant in chronic rejection. (2) Direct recognition- the donor’s HLA molecules is directly recognized on the donor’s presenting cells; i.e. the receptor recognizes the foreign HLA as own molecule with foreign peptide. This causes acute rejection. (3) Semi-direct recognition- the Immunoglobulin-like receptors (KIRs) on NK cells recognize the polymorphic sequences of HLA-C, B or A in the target cells, causing acute rejection. It has been shown recently that the naïve and memory CD4+ and CD8+ T-cells are cross-reactive against allogenic HLA molecules presenting self-peptides (Figure 11).

Figure 11. Direct recognition: recognition by both CD8+ and CD 4+ T-cells of an intact MHC molecule displayed by donor APC in the transplanted graft. Indirect recognition: donor’s MHC with the foreign peptide is processed and presented by the recipient’s APC to be presented to only the C4+ Tcells.

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

10.2 HLA-Testing and Clinics

Traditionally, HLA typing was performed by serologic testing by using antiserum in complement- dependent cytotoxic assays. Lately, more precise DNA-based HLA typing methods using molecular techniques have been developed. Techniques like sequence-specific oligonucleotide probe hybridization; sequence-specific primer amplification, sequencing-based typing, and reference strand- based conformation analysis are frequently used. The high polymorphism among HLA with more than 800 alleles have restricted to standard HLA typing at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 genetic loci. An HSCT donor is referred to as a "10/10 allele match" or "perfect match" when both HLA alleles are identical at each of the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci. While searching for an unrelated donor, high-resolution (4-digit) genetic typing of both the patient and the donor is necessary. 11. Summary

a) Major histocompatibility complex (MHC Class I and II loci) are the cell surface receptors encoded by the most diverse gene loci in vertebrates and spans about 0.1% of total genome containing 200 coding loci. b) The MHC genes are located in autosomes, Y-chromosomes and even in mitochondrial genome. It is a large chromosomal region of chromosome # 6; that has over 200 coding regions. The MHC in mice is termed as H-2 (Histocompatible-2) complex and in humans as HLA (human leukocyte antigens) complex. c) MHC not only distinguishes self from non-self but also plays a pivotal role in tissue transplantation and rejection, autoimmunity and susceptibility to infections. Apart from specific immune functions; these genes also impact growth, development, reproduction, odor and olfaction. d) The MHC class-I heavy chains are encoded by three genes (HLA-A, HLA-B, HLA-C) and presents small peptides to CTLs (cytotoxic T-lymphocytes) enabling each cell of the body to the immune system with possible chances of infection, therefore every nucleated cells of the body present MHC-I molecules. e) MHC Class II peptides are expressed on specialized cells called antigen presenting cells (APCs); mainly the macrophages, dendritic cells and B-lymphocytes. These molecules are encoded by three polymorphic genes (HLA-DR, HLA-DQ and HLA-DP) that bind to different peptides. f) Both MHC-I and MHC-II present peptides to the CD8+ and CD4+ T-cells, the sources of these peptides may differ. The MHC-I are presented with intracellular peptides and the exogenous peptides are presented by MHC-II. g) Both MHC classes have similar three-dimensional structure; they originate from the common parent gene by gene duplication and a similar function in antigen presentation to the T- lymphocytes. h) MHC genes influence the development of T-cell repertoire through thymic selection as only those T-cells that recognize peptide-MHC complex are able to mature and enter the circulation. i) There are many evidences that confirm that MHC polymorphism is maintained due to natural selection like high number of alleles, uniform allelic frequencies, deficiencies of homozygotes, linkage disequilibrium among loci, high dN/dS substitution, ancient allelic linkages and disassortative mating preferences. j) Expression of MHC genes was shown to have an epigenetic effect as many diseases associated with aging and autoimmunity is linked to polymorphism of MHCII transactivator (CIITA). 13

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex

k) Allogenic immune responses happen due to divergent MHC alloantigens between donors and recipients which are the easy targets for T-cell recognition, thereby increasing the risk of both graft rejection and graft-versus-host disease (GVHD). l) Precise DNA-based HLA typing methods using molecular techniques like sequence-specific oligonucleotide probe hybridization; sequence-specific primer amplification, sequencing-based typing, and reference strand-based conformation analysis are frequently used for HLA typing in clinics.

ZOOLOGY Immunology Structure and function of Major Histocompatibility Complex