1. Major Histocompatibility Complex 1. To give an overview of the role of the major histocompatibility complex in immune responses. 2. Genetics of the class I and class II MHC molecules. 3. To describe the structure and function of class I and class II MHC molecules. 4. To discuss the nature of polymorphisms in class I and class II MHC molecules. 5. The role of polymorphism in MHC in a population. 6. Methods for detecting MHC antigens (tissue typing).

2. MHC (major histocompatibility complex) A multigene family encoding molecules that plays a unique role in the regulation of immune response, tissue compatibility and disease association. In all vertebrates, there is a genetic region, product of which is involved in the regulation of immune response (e.g., Ag presentation, recognition) having major influence on graft survival Individuals identical for this region can exchange grafts more successfully than non-identical combinations in this region. Allelic differences can be associated with the most intense graft rejection within a species. in the association with the development of certain diseases. Polymorphisms in this region have been found to be associated as risk factors for certain diseases. This genetic region is referred to as Major Histocompatibility Complex.

3. These genes are expressed in most tissues as antigens, to which the immune system responses. Histocompatibility (transplantation) antigens: Antigens, on tissues and cells, which determine their rejection when grafted between two genetically different individuals Major histocompatibility (MHC) antigens: Histocompatibility antigens encoded by the MHC complex which cause a very strong immune response and are most important in rejection of organs and tissues MHC antigens of man: HLA (human leukocyte antigens) MHC antigens of mouse : H-2 antigens Xenograft: Grafts between members of different species Allograft: Grafts between two members of the same species Isograft/syngeneic: Grafts between members of the same species with identical genetic makeup (identical twins or inbred animals). Haplotype: a group of genes on a single chromosome.

4. The Human MHC gene complex The human MHC is located on chromosome 6. The MHC region is divided into three gene regions: class I, class II, and class III Products of class I gene, class I antigens (MHC class I molecules), are expressed on all nucleated cells. Products of class II gene, class II antigens (MHC class II molecules), are expressed only on antigen presenting cells (APC’s). Class III antigens are associated with proteins in serum and other body fluids (e.g.C4, C2, factor B, TNF). Antigens from class I and class II gene products play a critical role in in the regulation of immune response and transplantation; those from class III gene products have no direct role in immune responses that determine graft survival.

5. The human MHC gene complexClass I MHC: The class I gene complex contains three major loci of highest significance, B, C and A and some undefined loci of less significance. Each these loci codes for a polypeptide, α-chain that contains antigenic determinants that are polymorphic (has many alleles). Each α-chain associates with a β-2 microglobulin molecule (β-chain), encoded by a gene outside the MHC complex. The α-β-chain complex is expressed on the cell surface as the class-I MHC antigen. Without a functional β-2 microglobulin chain, the class I antigen will not be expressed on the cells surface. Individuals with defective a β-2 microglobulin gene do not express any class I antigen and hence they have a deficiency of cytotoxic T cells.

6. The human MHC gene complexClass II MHC: The class II gene complex also contains at least three loci, DP, DQ and DR. Each of these loci codes for one α- and a variable number of ß-chain polypeptides which associate together to form the class II antigens. The class II antigens are also polymorphic. The DR locus contains more than one, possibly 4, functional β-chain genes.

7. Structure of Class I MHC Molecules Class I MHC molecules are composed of two polypeptide chains, along α chain and a short β chain called β2 microglobulin. The α chain has four regions: a cytoplasmic region, containing sites for phosphoylation and binding to cytoskeletal elements. a transmembrane region containing hydrophobic amino acids by which the molecule is anchored in the cell membrane. a highly conserved α3 immunoglubilin-like domain to which CD8 binds. a highly polymorphic peptide binding region formed from the α1 and α2 domains. The β2- microglobulin associates with the α chain and helps maintain the proper conformation of the molecule.

8. Most variable part of the class I MHC molecules: α1 and α2 domains -- the peptide binding region. the peptide binding groove, revealed: The groove is composed of two α helices forming a wall on each side and eight β-pleated sheets forming a floor. The peptide is bound in the groove and the residues that line the groove make contact with the peptide. These are the residues that are the most polymorphic. The groove will accommodate peptides of approximately 8-10 amino acids long. Whether a particular peptide will bind to the groove will depend on the amino acids that line the groove. Because class I molecules are polymorphic, different class I molecules will bind different peptides. Thus, for every class I molecule, there are certain amino acids that must be a particular location in the peptide before it will bind to the MHC molecule.

9. Structure of Class II MHC Molecules Class II MHC molecules are composed of two polypeptide chains an α and a β chain of approximately equal length. Both chains have four regions: a cytoplasmic region containing site for phosphorylation and binding to cytoskeletal elements a transmembrane region containing hydrophobic amino acids by which the molecule is anchored in the cell membrane a highly conserved α2 domain and a highly conserved β2 domain to which CD4 binds and a highly polymorphic peptide binding region formed from the α1 and β1 domains.

10. Most variable part of the class II MHC molecules: the α1 and β1 domains-- the peptide binding region. the peptide binding groove, revealed: The groove is composed of two α helices forming a wall on each side and eight β-pleated sheets forming a floor. Both the α1 and β1 chain contribute to the peptide binding groove. The peptide is bound in the groove and the residues that line the groove make contact with the peptide. These are the residues that are the most polymorphic. Class II molecules is open at one end so that the groove can accommodate longer peptides of approximately 13-25 amino acid long with some of the amino acids located outside of the groove. For every class II molecule, there are certain amino acids that must be a particular location in the peptide before it will bind to the MHC molecule.

11. MHC Polymorphism: MHC complex is the most polymorphic in the genome. This means that there is an astonishing allelic diversity found within MHC. Within the MHC there are 6 genes that encode class I molecules HLA-A, HLA –B, HLA-C, HLA-E, HLA-F and HLA-G. Among these HLA-A, HLA–B, and HLA-C are the most important and are most polymorphic. The degree of polymorphism at each of these loci HLA- B 439 HLA- A 218

12. Within the MHC there are 5 loci that encode class II molecules, each of which contain a gene for an α chain and at least one gene for a β chain. The loci are designated as HLA-DP, HLA –DQ, HLA-DR, HLA-DM, and HLA-DO. Among these, HLA-DP, HLA –DQ, and HLA-DR are the most important and are most polymorphic. Table 2 shows the degree of polymorphism at each of these loci. The degree of polymorphism at each of these loci HLA-DRB1 269

13. In humans, the most conspicuously-diverse loci, HLA-A, HLA-B, and HLA-DRB1, have roughly 218, 439, and 269 known alleles respectively. Each individual inherits a different sets of Class I and Class II molecules. Interindividual variation due to these polymorphisms, give rise to the difference in response to infection, vaccination and from whom transplant is received. MHC ANTIGENS: Nomenclature: HLA specificities are identified by a letter for locus and a number e.g., A1, B5, etc. The haplotypes are identified by individual specificities e.g., A1, B7, Cw4, DP5, DQ10 DR8 Specificities which are defined by genomic analysis (PCR), are named with a letter for the locus and a four digit number e.g. A0101, B0701, C0401, etc.

14. Inheritance: MHC 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 therefore inherit a maximum of 6 class I specificities (Figure top). 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 (α and β), with some antigenic determinants (specificities) on each chain, and DR α- and β-chains can associate in cis (both from the same parent) or trans (one from each parent) combination, an individual can have additional DR specificities. Hence, many DR specificities can be found in any one individual.

15. MHC antigen expression on cells: MHC antigens are expressed on the cell surface in a co-dominant manner: products of both parental genes are found on the same cells. not all cells express both class I and class II antigens. class I antigens are expressed on all nucleated cells and platelets the expression of class II antigens is more selective. It is expressed on B lymphocytes, a proportion of macrophages and monocytes, skin associated (Langerhans) cells, dendritic cells and occasionally on other cells.

16. Genetic principles of the major histocompatibility complex (MHC): The MHC demonstrates Each person has two chromosomes and thus two MHC haplotypes, each inherited from one parent. Because the human leukocyte antigen (HLA) genes are autosomal and codominant, the phenotype represents the combined expression of both haplotypes. Each child receives one chromosome and hence one haplotype from each parent. Because each heterozygous parent has two different haplotypes, four different combinations of haplotypes are possible in the offspring. This inheritance pattern is an important factor in finding compatible related donors for transplantation. Thus, an individual has a 25% chance of having an HLA-identical or a completely dissimilar sibling and a 50% chance of having a sibling matched for one haplotype.

17. Molecular Tissue Typing (HLA Typing) Histocompatibility testing has particular relevance to transplant program. Determination of human leukocyte antigen (HLA) phenotype or genotype of an individual is referred to as HLA typing or tissue typing. The clinical application HLA typing most commonly include: a) typing for renal transplantation b) typing for hematopoetic stem cell transplantation c) typing as an aid in the diagnosis of HLA-associated diseases example: the association of the HLA-B27 type with ankyolosing spondylitis. Only genes that encode the classical transplantation antigens are considered in HLA typing. Transplantation antigens influence the outcome of transplantation. HLA typing can be done by both phenotype and genotype method. The HLA phenotype consists of the array of HLA-specific proteins, encoded by MHC genes, displayed on the surface of most cells of the body.

18. HLA phenotype (HLA Typing by serology): The detection of HLA antigens is based on a complement-dependent microlymphocytotoxicity (MLCT) test: Viable lymphocytes are isolated by Density gradient. Viable lymphocytes are incubated with panels of antisera, each of which are specific for one or more HLA antigens at class I or II loci. HLA antisera of known specificity are placed in wells on a “Terasaki microdroplet tray.” After initial incubation, complement is added. If the lymphocytes express an antigen recognized by specific antibody. Antigen-antibody complex will be formed. Complement will bind to the complex through C1q, and initiates the complement cascade that leads to cell lysis. In a negative reaction, the lymphocytes are alive; In a positive reactive reaction, the lymphocytes are dead. The lysis can be made visible in both using either a color stain (e.g. eosin) or fluorescent dye using either an inverse phase contrast or UV microscope.

19. A2 typing sera A2 Incubate, Wash, Add complement Dead cells Anti-A2 A11 Anti-A11 A11 typing sera Dead cells Live cells HLA phenotype for A locus: A2, A11

20. HLA genotype: The application of molecular biology techniques has revolutionized the HLA laboratory. Methods based on sequence detection can be categorized as a) PCR-SSP: Polymerase chain reaction with sequence specific primers is the method in which DNA amplification is performed by PCR employing a panel of pair pairs and amplicons are visualized by electrophoresis on agarose gel. b) PCR-SSOP:Polymerase chain reaction amplified product with locus specific (generic) primer pairs are identified by hybridization with panels of allele-specific or allele-group-specific oligonucleotide probes. c) Sequencing based HLA typing: Sequencing involves isolation of DNA, locus-specific PCR, sequencing reactions, and analysis with sequencing instrument and software.

21. DNA-based methods for HLA typing offer significant advantages over standard serological analysis of HLA because of: PCR based detection is more accurate because of an increased sensitivity and specificity. Testing of DNA sequences permits detection of many more subtypes or "splits" of HLA antigens or alleles. DNA typing can define antigens at the allele level, assuring no ambiguity in interpretations. In serotyping, some antigens are difficult to identify and antigens may mask the presence of others. DNA typing does not require live blood cells, permitting more flexible sample requirements.