The Major Histocompatibility Complex

The Major Histocompatibility Complex Today we will discuss the MHC The study of MHC is necessary to understand how an immune response is generated. And these are the extra notes with respect to slides from 2 to 52. Slide # 2 - So it is very complex small body that governs the generation of immune response. Slide # 3 - The ability to present peptides to T cell and the recognition of these peptides by TCR is the function of gene product encoded by a locus called MHC system. The function is to present antigen to T cell in the generation of an immune response, express addresses to TCR. Slide # 4 - T helper o T cytotoxic recognize these peptide-mhc complexes. - The MHC resemble a cluster of closely linked genes present on the short arm of the chromosome number 6 in humans, number 17 in mice, and number 20 in rates.etc. -the human MHC us called HLA (Human Leukocyte Antigen ) because these antigens are expressed on WBCs. Slide # 5 - This represents the short arm of chromosome 6 where about 35000 kb forms the locus of MHC. By: Ibrahim Adnan 1

Slide # 6 - The system is composed of two types of molecules that are classified into class 1 and class 2, they differ in structure - Class 1 is composed o peptides ( α chain ) that composed of 3 domains (α1,α2,and α3), with a product produced by a another gene (not on chromosome 6 short arm ) the β microglobulin which is coded by a gene on chromosome number 15. - Class 2 is a hetrodimer composed of α and β chains. - The HLA, H2 in humans and mice respectively, they have similar structure and most of our knowledge about HLA came from studies carried on mice before the human system was discovered. - In class 1 (which is composed of α chain and β microglobulin) there are 3 major loci ( actually more than 3 loci but 3 MAJOR loci) which are HLA-A, HLA- B and HLA-C. - IN class 2 ( which is a hetrodimer) the major loci are the DR,DQ,DP and there are other loci of less importance. - The highly polymorphic loci are: 1. A,B and C in class 1. 2. DR,DQ, and DP in class 2. *Note that the structure of the loci is more complex than this as we will see later on. Slide # 7 - The system was discovered in the 50s and 60s where studies to explain the ability to mount an immune response were held, and it was discovered that this ability is encoded in the genetic makeup of horses and cats, and Ebn Abas agreed to call it MHC. - This system was studied by Antibodies first and the sources of these antibodies to MHC were: 1. The multiparous women, those who carry babies with different antigenic makeup as compared to themselves, that why this represent an source of transplantation which is associated with the production of antibodies against paternal antigens. 2. Actively immunized volunteers, volunteers which immunized against this antigens (the HLA antigens). 3. Recipients of blood transfusion, as the blood contain WBCs and these WBCs were the source of immunization against HLA antigens. 4. Transplanted patients, who have transplantation of various organs ( skin, kidney. etc). By: Ibrahim Adnan 2

Those were the sources of antibodies which facilitated the discovery of the system, at that time few antigens were known but now hundreds of antigens are recognized to the look to both class 1 and class 2. - These antigens were discovered in international workshops through which antisera where exchanged between scientists from different labs, and this led to the discovery of most antigens, and at the beginning a new antigen was given the name of the workshop, eg. The name W6 means it was discovered in workshop 6. But later when this was established it was given a letter and the name. - Initially the MHC antigens were identified by serology only, the remaining antigens were discovered by what called Multi lymphocyte culture, Serology result in the discovery of the most of antigens, however indivual who were matched with respect to the antigens that were defined by antibodies still rejected transplanted tissue which indicates that there are other antigens that were not identified by the antibodies so they investigated theses antigens and discover them by multi lymphocyte culture. - In the multi lymphocytes culture lymphocytes were taken from a donor and reacted with a recipient and vice versa,the donor and the recipient were matched in respect to the HLA antigens detected anti bodies,to recognize if the donor and the recipient carry antigenic determinant that are different from each other, but when we are mixing two lymphocyte populations together and measuring response we can't determine which lymphocyte is responding unless we inactivate one of these lymphocytes population, so they took the lymphocytes from the donor and inactivated them (by treating them with cytomycin or by irradiation to inhibit their ability to mount a response ) & made the recipient lymphocytes active only, so when these were mixed together if the lymphocytes from the recipient and the donor were matched no response will take place, but the result in many cases was that the donor lymphocytes were activated (responded) indicating that these lymphocytes recognizing antigenic determinant that were not present on the recipient and that is why these cells mounted an immune response, and the same process held to the donor lymphocytes (by inactivating the recipient lymphocytes and mix them with active donor lymphocytes) - This response could be measured by using tritiated thymidine where the proliferating lymphocytes will incorporate the tritiated thyamidine in their DNA and that can be measured using a β counter. - If the response were double than a negative control reaction this was considered as positive result. - So individuals, who were serologically matched still responding to cell from each other. Indicating that there were antigen which were not identified by By: Ibrahim Adnan 3

antibodies because we started, with donor and recipient who are matched by the antibody testing for HLA antigens that are serologically defined. So although they were matched with respect antibodies carry the same antigen they still mount a response to the lymphocytes that were added either to recipient or donor. Indicating that these antigenic determinant where not identified by antibodies (IE serologically identified). - So HLA are either serologically defined or cellular defined (T lymphocyte defined ) and these t lymphocyte defined detected only by the mixed lymphocyte culture. - It is not sufficient to match individuals by testing antigens defined by antibodies only because there is antigenic determinant can be recognized only by lymphocytes and these lymphocytes have the potential to mount a response and reject the transplanted tissue. - In summary, this is to say that we have two types of HLA antigens, group of them are serologically defined and the cellular (T lymphocyte defined). Slide # 8 - The binding cleft,on the extracellular segment of MHC molecule, to wich peptides derived from the antigen can be loaded in groove. - The MHC molecule is a peptide that has a cytoplasmic tail, an extracellular segement contains a groove that results from the association of α,β in class 2 and α1,α2 in class 1 HLA molecules to which the antigen is loaded. - The polymorphic amino acids residues of MHC molecules are located in or adjacent to the peptide binding cleft. - In HLA also there is variability nut it is limited, it is not as the variation that seen in TCR or BCR, the limited variation is the result of association of the peptide binding cleft which is made of α1,α2 in class 1 and α1,β1 in class 2. - The variation is remarkable in β chain than what seen in α chain. Slide # 9 - This is the structure of MHC 1 and MHC 2. - As mentioned before, class 1 molecules are composed of one polypeptide α chain which is divided to 3 domains (α1,α2 and α3) and β microglobulin as second non MHC encoded protein (it is encoded by a gene on chromosome 15), where as class 2 MHC molecules are made up of 2 MHC encoded polypeptides the α chain (which is the light chain ) and the β chain ( which is the heavy chain ). - In class 1 there is no need for variation in α3 domain because it is not involved in accommodating peptides (antigens) so if the variation exists it should be in the binding groove (in α1 and α2 domains of the α chain). By: Ibrahim Adnan 4

Slide # 10 - In class 2 we have 2 chains α,which is divided to α1 and α2 domains, and β chain,which is also divided to β1 and β2, the variation exists in α1,β2 domains, but β chain is remarkably variable compared with α chain as mentioned before. - No HLA antigens can be expressed on the surface of the cell without an antigen(peptide from the antigen) associated with at the binding cleft, so antigen expression by MHC molecules is necessary for their expression on cell surface. - There is a high turnover of MHC molecule because they continuously replaced to present newly engulfed by class1 or class 2. - Recently several genes have been found with the MHC locus that are not class 1 or class 2 some of the complement components are present in the MHC locus and they are not class 1 or class 2, that is why it was agreed to call these class 3. - As class 3 recently added now we have class 1,class 2 and class 3 in the system. Slide # 11 - Crossover is an additional source of polymorphism of the MHC. - In inherited MHC gene,which is composed of large number of alleles, crossover can take place creating more HLA antigens. Slide # 12 - TAP is a product that present with the MHC locus, and is essential for the antigen presentation by class 1 MHC molecule, it transports peptides from cytosol to ER to be loaded with class 1 MHC. - Proteasome degrades intracellular antigens to be presented by class 1. - The first molecule that is involved in processing of those antigens that will be presented in conjunction with class1 MHC molecules is proteasome which acts to fragments the substance into peptides and then TAP is responsible for loading of the antigenic peptides into class 1 MHC molecules. Slide # 13 - Regarding class2 there are another molecules that help class 2 to be loaded with antigenic peptides and those are the HLA DM which encodes for HLA- DMA HLA-DMB this a hetrodimer that is essential in peptide binding to MHC class 2 molecules. By: Ibrahim Adnan 5

- Between class 1 & class 2,we have class 3 molecules which include a complements (C4b, complement B factor and others ) in addition to cytokines like TNF, lymphoytoxin A and lymphotoxin B, as well as heat shock proteins & these called class 3 antigens. Slide # 14 - The class 1 like molecules are more stable product of the MHC molecules. Slide # 15 - This slide shows gene mapping of MHC complex. - In the MHC class 2 map which include DQ,DR,DP molecules in addition to the substances that are involved in antigen presentation like DMA,DMB this is followed by class 3 MHC molecules that map between class 1 and class 2 MHC and class 3 contains TNF,lymphotoxin A,lymphotoxin B in addition to MICA,MICB ( remember those are involved in the mannan binding lectin pathway ) in addition to complement components (C4,C42and factor B) - Class 1 MHC molecules the organizations B,C then A & in between there are other less variable more stable MHC molecules. - The locus in very complex containing substances that belong to class 1,class 2 &class3 molecules. Slide # 16 - As consequence of co-dominance expression the individual can inherit 3 HLA class 1 antigens from maternal source and 3 from paternal source but the locus is highly polymorphic locus hundreds of alleles exists for each of these loci for each A,B,C antigen and that is the source of polymorphism of the HLA system. - Other than being polygenetic because of the presence of multiple genes it also polymorphic because of the presence of large number of alleles at each locus of the system. Slide # 17 - The structure of the class 1 with the cytoplasmic tail, the transmembrane segment and the extra cellular segment with the binding groove. Slide # 18 - There is a significant difference between class 2& class 1 MHC molecules which is in the size of the peptide that can be accommodated by the antigen binding cleft. By: Ibrahim Adnan 6

- The size is 8-11 amino acids for MHC 1 of an average of 9 whereas the number is double for class 2 MHC which is can accommodate up to 25-30 amino acids (Peptide formed of 25-30). - If the individual can in inherit only 6 HLA class 1antigens but the number of antigens that can be presented with class 1 are billions because the TCR of D8 + cells, Which recognized antigens presented in conjunction with class 1 can recognized billions of antigens so the specificity of binding of MHC is not similar to the specificity of binding of TCR so the same antigen binding site of MHC 1 molecules can accommodate different antigens. - An individual can inherit a maximum of 6 different HLA antigen with respect to A,B & C because each alleles is inherited from a parent so one A one B one C from each parent. - Those class1 antigens that fold to make the antigen binding site can present billions of antigens to the TCR where recognition is very specific with respect to TCR, here the recognition is broad (the specificity is very broad with respect to MHC class 1 )they can accommodate many antigen.however within these concepts the antigens that can accommodate different in their binding to HLA molecules ( high affinity or low ). - γδ T cell recognize antigen in conjunction with such more stable non variable MHC class 1 molecules. Slide # 19 - That is the antigen presenting site and many antigens could be accommodated in this site. Slide # 20 - class 2 MHC molecules are more specialized antigens that present on APC, and on the surface of ACTIVATED T lymphocyte (never on a resting T cell). - After synthesis MHC class 2 α and β chains associate only with other within the same region and this is very important because the maximum number of class 2 MHC antigens could be more than 6 (6 in the case of class 1), and this is due to the fact that these antigens associate after synthesis so DPα from maternal source could be associated with DPβ from paternal source and vice versa creating more possibilities. - The number of antigen could reach up to 20 because of the ability of chain from different source within the cell to assemble together (different chromosomes within the same cell) - For example an DP molecules on the same cell could have all these combinations on it surface :- By: Ibrahim Adnan 7

α chain β chain Combination # 1 maternal maternal Combination # 2 maternal paternal Combination # 3 paternal maternal Combination # 4 paternal Paternal Which create more variation and more polymorphism at MHC class 2. Slide # 22 - The number of amino acids presented sometimes is up to 30. Slide # 23 - Those up to 20 different HLA class 2 antigens have to deal with millions of antigens so also binding is not very specific, it is of broad specificity. Slide # 24 - An additional form of variation is available to class 2 molecules because of their hetrodimeric structure and the ability of peptides derived from maternal & paternal source to associate together forming the structure. Slide # 26 - Also lymphotoxins A & B are encoded in MHC class 3. Slide # 27 - Class 1MHC molecules expressed on all nucleated cells whereas class 2 are expressed on a limited number of cells those who could present antigens. Slide # 28 - Increased frequency of the inheritance of haplotypes more than what can be predicted by random assortment and this is a feature of HLA inheritance so within population certain haplotypes are common because the phenomenon of linkage disequilibrium and that could be explained that haplotypes inherited together so if an individuals are matched for DR this is indicates that they are matched for DP and DQ also, because DP,DQ and DR are inherited together as haplotypes rather than genes alone. Slide # 29 - This is indicates the polymorphism and the number increased over years as new antigens were detected the numbers in the slide back to 2007. By: Ibrahim Adnan 8

- The largest number of alleles discovered was 728 different have been detected at the B locus, 210 at c locus and 414 at A locus of MHC class 1 molecules. - DR is also variable and we can see that the DRα(with only 3 alleles) is more stable than DRβ ( with 503 alleles), also if we look to DPβ, and DQβ with 120 and 68 alleles respectively and compared them with the DPα and DQα with 23 and 32 alleles respectively we can conclude that the β chain shows remarkable variation compared with α chain in MHC class 2 molecules. - The number of detected antigens is huge and individuals could have any of these antigens and that is what we call it polymorphism. - Due to the extreme polymorphism in HLA system it is very rare to have two identical individuals carry the same HLA CLASS 1 and class 2 antigens,so these individual could be used as source of organs to be transplanted. Slide # 32 - The major function of the HLA molecule is to accommodate and present antigens to lymphocytes because these lymphocytes can't recognize antigen unless it was presented with MHC & MHC molecule are expressed on these cells(apc) where peptide is loaded &TCR recognize these antigens on the surface of an APC. Slide # 35 - Remember the first step in the development of T cells is to recognize MHC, so MHC expression is essential for self and non cell discrimination. Slide # 38 - these are very important in the expression of the class 1 & class 2 HLA antigens which are polymorphic. Slide # 40 - Why are these called "regions"? because they code a substance of genes that are expressed as groups. Slide # 41 - The inheritance of HLA antigen follow the mendelian fashion. - There is an additional source of variation that is the recombination. - Recombination can take place in class 1 as well as class 2 so in addition to AC,AD,BC,BD we have AR1 that is the result from the recombination of paternal antigen. By: Ibrahim Adnan 9

- R2C which is the result of the recombination of the maternal and R1R2 which are the result of recombination on each. - So in addition to the mendelian fashion of inheritance crossover between these antigens which take place at rate of 4% is another source for variation polymorphism in respect to MHC. Slide # 42 - During evolution human developed MHC system and it is first discovery was that MHC controls graft rejection that can't be the primary function of MHC because trans plantation is not commonly performed & later it was discovered that MHC primary function is to present antigens to T lymphocytes which control immune response & that is why the HLA system contains the genes that where referred to as immune response genes. - The MHC is actually the product of the immune response genes that control response to protein antigens by T lymphocytes. Slide # 47 - Slow on rate & very slow of rate means the binding slow and it is continued until is saturated however the dissociation between peptide & MHC is very slow. Slide # 49 - This mean that these are very common between population. Slide # 50 - Peptides like +A1,A24 and B44 have frequency up to 99% among population, so you can expect to find these HLA type among population they differ with DR DP, and DQ but these are common antigen that can be deleted among individuals. The End الطب طبي والمأساة مأساتي لتسقط شيتاتي فلتحيا الكتب و Done by: Ibrahim Adnan By: Ibrahim Adnan 10