In this article we will discuss about:- 1. Introduction to Major Histocompatibility Complex (MHC) 2. Structure of Major Histocompatibility Complex (MHC) 3. Nomenclature and Inheritance 4. Expression of MHC Molecules 5. Classification of MHC Molecules.
Contents:
- Introduction to Major Histocompatibility Complex (MHC)
- Structure of Major Histocompatibility Complex (MHC)
- Nomenclature and Inheritance of Major Histocompatibility Complex (MHC)
- Expression of MHC Molecules
- Classification of MHC Molecules
1. Introduction to Major Histocompatibility Complex (MHC):
MHC complex is a large genomic region or group of genes found in most vertebrates on a single chromosome that codes the MHC molecules which plays a vital role in immune system. Major histocompatibility antigens (also called transplantation antigens) mediate rejection of grafts between two genetically different individuals. HLA (human leukocyte antigens) were first detected on leukocytes and so they are called MHC antigens of humans. H-2 antigens are their equivalent MHC antigens of mouse. A set of MHC alleles present on each chromosome is called an MHC haplo-type.
Monozygotic human twins have the same histocompatibility molecules on their cells, and they can accept transplants of tissue from each other. Histocompatibility molecules of one individual act as antigens when introduced into a different individual. George Snell, Jean Dausset and Baruj Benacerraf received the Nobel Prize in 1980 for their contributions to the discovery and understanding of the MHC in mice and humans MHC gene products were identified as responsible for graft rejection.
MHC gene products that control immune responses are called the immune response genes. Immune response genes influence responses to infections. The essential role of the HLA antigens lies in the induction and regulation of the immune response and defence against microorganisms. The physiologic function of MHC molecules is the presentation of peptide antigen to T lymphocytes.
There are two general classes of MHC molecules – Class I and Class II. Class I MHC molecules are found on all nucleated cells and present peptides to cytotoxic T cells. Class II MHC molecules are found on certain immune cells themselves, chiefly macrophages, B cells and dendritic cells, collectively known as professional antigen-presenting cells (APCs). These APCs specialize in the uptake of pathogens and subsequent processing into peptide fragments within phagosomes. The Class II MHC molecules on APCs present these fragments to helper T cells, which stimulate an immune reaction from other cells.
2. Structure of Major Histocompatibility Complex (MHC):
The MHC complex resides in the short arm of chromosome 6 and overall size of the MHC is approximately 3.5 million base pairs. The complete three-dimensional structure for both class I and class II MHC molecules has been determined by x-ray crystallography. The class I gene complex contains three loci A, B and C, each of which codes of α chain polypeptides.
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 β chain polypeptides. Class III region is not actually a part of the HLA complex, but is located within the HLA region, because its components are either related to the functions of HLA antigens or are under similar control mechanisms to the HLA genes. Class III antigens are associated with proteins in serum and other body fluids (e.g., C4, C2, factor B, TNF) and have no role in graft rejection.
3. Nomenclature and Inheritance of Major Histocompatibility Complex (MHC):
HLA specificities are identified by a letter for locus and a number (A1, B5, etc.), and the haplotypes are identified by individual specificities (e.g., A1, B7, Cw4, DP5, DQ10, DR8). Specificities which are defined by genomic analysis (PCR), are named with 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 inherited as a group (haplotype), one from each parent. Thus, MHC genes are co-dominantly expressed in each individual. 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. Each individual inherits a maximum of two alleles for each locus.
The maximum number of class I MHC gene products expressed in an individual is six; that for class II MHC products can exceed six but is also limited. Thus, as each chromosome is found twice (diploid) in each individual, a normal tissue type of an individual will involve 12 HLA antigens. Haplotypes, normally, are inherited intact and hence antigens encoded by different loci are inherited together.
However, on occasions, there is crossing over between two parental chromosomes, thereby resulting in new recombinant haplotypes. There is no somatic DNA recombination that occurs for antibodies and for the TCR, so the MHC genes lack re-combinational mechanisms for generating diversity. Many alleles of each locus permit thousands of possible assortments. There are at least 1000 officially recognized HLA alleles.
4. Expression of MHC Molecules:
MHC class I molecules are widely expressed, though the level varies between different cell types. MHC class II molecules are constitutively expressed only by certain cells involved in immune responses, though they can be induced on a wider variety of cells.
5. Classification of MHC Molecules:
1. MHC Class I Molecule:
MHC Class I is a membrane spanning molecule composed of two proteins. The membrane spanning protein is approximately 350 amino acids in length, with about 75 amino acids at the carboxylic end comprising the trans-membrane and cytoplasmic portions. The remaining 270 amino acids, as shown in the diagram, are divided into three globular domains Alpha-1, Alpha-2 and Alpha-3 prime, with alpha-1 being closest to the amino terminus and alpha-3 closest to the membrane.
The second portion of the molecule is a small globular protein called Beta-2 Micro-globulin. It associates primarily with the alpha-3 prime domain and is necessary for MHC stability. The bound peptide sits within the groove. The MHC molecules ability to present a wide range of antigenic peptides for T cell recognition requires a compromise between broad specificity and high affinity.
The peptide main chain is tightly bound whilst peptide side chains show less restrictive interactions. It is primarily the peptide side-chain contacts and conformational variability that ensures that the peptide-MHC complex presents an antigenically unique surface to T cell receptors.
2. MHC Class II Molecule:
Although similar to Class I, the MHC Class II molecule is composed of two membrane spanning proteins. Each chain is approximately 30 kilodaltons in size, and made of two globular domains as shown in the diagram. The domains are named Alpha-1, Alpha-2, Beta-1 and Beta-2. The two regions farthest from the membrane are alpha-1 and beta-1. The two chains associate without covalent bonds.
The bound peptide is within the groove. The MHC molecules ability to present a wide range of antigenic peptides for T cell recognition requires a compromise between broad specificity and high affinity. The peptide main chain is tightly bound whilst peptide side chains show less restrictive interactions. It is primarily the peptide side-chain contacts and conformational variability that ensures that the peptide-MHC complex presents an antigenically unique surface to T cell receptors. Class II molecules are dimers consisting of an alpha and beta polypeptide chain.
Each chain contains an immunoglobulin like region, next to the cell membrane. The antigen binding cleft, composed of two alpha-helices above a beta-pleated sheet, specifically binds short peptides, about 15 to 24 residues long. The amino acid sequence around the binding site, which specifies the antigen binding properties, is the most variable site in the MHC molecule.
Differences between Class I and Class II structures can explain the different length requirements for the bound peptide. The ends of the antigen binding cleft of Class I molecules taper and are blocked by bulky tyrosine that bind the N terminus of the peptide. These conserved residues are not found in Class II molecules where smaller residues (glycine or valine) replace the larger tyrosine.
3. MHC Class III Molecule:
This class includes genes coding several secreted proteins with immune functions-components of the complement system (such as C2, C4 and B factor) and molecules related with inflammation (cytokines such as TNF-α, LTA, LTB) or heat shock proteins (hsp). Class-III molecules do not share the same function as class-I and II molecules, but they are located between them in the short arm of human chromosome 6. For this reason they are frequently described together.
HLA Typing:
HLA antigens are recognized on almost all of the tissues of the body (with few exceptions), the identification of HLA antigens is also described as “Tissue Typing”. HLA matching between donor and recipient is desirable for allogenic (distinct) transplantation. Class I typing methods include test such as microcytoxicity (for typing A, B, C loci) and cellular techniques such as CML (for HLA-DPw typing). Class II typing involves cellular techniques such as MLR/MLC (for DR typing) and molecular techniques such as PCR and direct sequencing (for DR, DQ typing).
Significance of HLA Typing:
1. Anthropology:
The fact that HLA types vary very widely among different ethnic populations has allowed anthropologists to establish or confirm relationship among populations and migration pattern. HLA-A34, which is present in 78% of Australian Aborigines, has a frequency of less than 1% in both Australian Caucasoids and Chinese.
2. Paternity Testing:
If a man and child share a HLA haplotype, then there is possibility that the man may be the father but not proven. However, if they don’t match or share a haplotype then it is agreed that he is not the father.
3. Transplantation:
Because HLA plays such a dominant role in transplant immunity, pre-transplant histocompatibility testing is very important for organ transplantation. Results with closely related living donors matched with the recipient for one more both haplotypes are superior than obtained with unrelated cadaveric donors.
i. Transfusion.
ii. Forensic science.
iii. Disease Correlation.
A number of diseases have been found to occur at a higher frequency in individuals with certain MHC haplotypes. Most prominent among these are ankylosing spondylitis (B27), celiac disease (DR3), Reiter’s syndrome (B27).
I. Disease Associations with Class I HLA:
Ankylosing spondylitis (B27), Reiter’s disease (B27), Acute anterior Uvietis (B27), Psoriasis vulgaris (Cw6).
II. Disease Associations with Class II HLA:
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), Sjogren’s syndrome (DR3), Chronic active hepatitis (DR3), Multiple sclerosis (DR2, DR6), Celiac disease (DR3), Dermatitis herpetiformis (DR3). No definite reason is known for this association.
However, several hypotheses have been proposed-antigenic similarity between pathogens and MHC, antigenic hypo- and hyper-responsiveness controlled by the class II genes are included among them.
Possible explanation for these associations is that the HLA antigen itself plays a role in disease, by a method similar to one of the following models:
(a) By being a poor presenter of a certain viral or bacterial antigen.
(b) By providing a binding site on the surface of the cell for a disease provoking virus or bacterium.
(c) By providing a transport piece for the virus to allow it to enter the cell by having a such a close molecular similarity to the pathogen that the immune system fails to recognize the pathogen as foreign and so fails to mount an immune response against it.
Serologic Methods:
Serologic techniques provide one of the simplest and fastest methods for histocompatibility testing. These methods use sera that contain specific antibodies to HLA antigens. Tissue typing sera for the HLA were obtained in the past, from multiparous women who were exposed to the child’s paternal antigens during the parturition and subsequently developed antibodies to these antigens. More recently they are being produced by the monoclonal antibody technology.
Microcytotoxicity Assay:
This is done by exposing the unknown lymphocyte to a battery of antisera of known HLA specificities. Lymphocytes are isolated from the peripheral blood (or from lymph node or spleen in cadavers) and separated from other cells by buoyant density gradient centrifugation. For HLA I antigens, T lymphocytes are chosen while for HLA II antigens, B lymphocytes are chosen.
An array of anti-HLA sera covering full range of HLA types is chosen. Individual serum is dispensed into microtitre wells. Approximately 2000 lymphocytes are dispensed per well and incubated. Complement is then added to each well and incubated. The duration of incubation is different for T and B lymphocytes.
If the antibodies bind to lymphocytes, complement gets activated and results in lysis of that lymphocyte. The damaged cells are not completely lysed but suffer sufficient membrane damage to allow uptake of vital stains such as eosin Y, Trypan Blue or fluorescent stains such as ethidium bromide. Live cells don’t stain but the dead cells take up the stain.
Antibody Screening:
This is used to detect the presence of HLA antibodies in the potential transplant recipients. A highly sensitive solid phase ELISA is used to detect antibodies in recipient’s serum. Purified preparations of HLA antigens are adsorbed on the solid phase of plastic plates. Recipient’s serum is then added to different HLA antigen coated wells.
After the removal of unbound antibodies by washing the wells are treated with enzyme-labeled anti-gamma globulin. The wells are washed and treated with color generating substrate. If the recipient is positive for the HLA type of the donor (that means recipient has antibodies directed against donor’s antigens), then transplantation is not possible.
Cellular Assays:
Lymphocytes from one donor, when cultured with lymphocytes from an unrelated donor, are stimulated to proliferate or become cytotoxic. This proliferation is due to a disparity in the class IIMHC (DR) antigens.
Mixed Leukocyte Reaction (MLR) or Mixed Leukocyte Culture (MLC):
T cells of one individual interact with allogeneic class-II MHC antigen bearing cells (e.g., B cells) of unrelated individual. When lymphocytes from individuals of different class II haplotypes are cultured together, blast cell transformation and mitosis occurs. The irradiated or mitomycin-C treated stimulator cells of recipient (usually containing B cells, macrophages, and dendritic cells) are mixed with CD4 cells of responder (donor).
The donor cells respond to different class II antigens on stimulator cells and undergo transformation (DNA synthesis and enlargement) and proliferation (mitogenesis). These changes were recorded by the addition of radioactive (tritiated, 3H) thymidine into the culture and monitoring its incorporation into DNA.
Cell Mediated Lympholysis (CML):
The responder cells not only undergo blast transformation and proliferation on contact with different MHC II molecules, they also give rise to cytotoxic cells. These cytotoxic cells in turn identify the HLA I antigen on the stimulator cells and kill them.
Molecular Techniques:
These methods involve detection of the genes coding for the antigens rather than detecting the antigen itself. These are Sequence-specific PCR, Restriction fragment length polymorphism and sequence specific oligonucleotide probe etc.