1. T-LYMPHOCYTES RECOGNIZE ANTIGEN-DERIVED PROTEIN FRAGMENTS (PEPTIDES) EXPRESSED ON THE SURFACE OF SELF ANTIGEN PRESENTING CELLS VIRUS-INFECTED CELLS ARE RECOGNIZED BY T- LYMPHOCYTES IN MHC-DEPENDENT MANNER TISSUE TRANSPLANTATION IS RESTRICTED BY MHC MOLECULES THE IMMUNE RESPONSE TO PROTEIN ANTIGENS IS REGULATED BY INDIVIDUALLY POLYMORPHIC MHC GENES

2. ANTIGEN PRESENTING CELLS endogenous antigens Synthesize antigens – endogenous antigens (virus, tumor) exogenous antigens Internalize antigens – exogenous antigens (any protein) processing Degrade protein antigens to peptides – processing antigen presentation Protein – derived peptides are presented by MHC (HLA) membrane proteins – antigen presentation MHC molecules present both self and non-self protein – derived peptides MHC class I molecules are expressed in all nucleated cells MHC class II molecules are expressed by professional antigen presenting cells

3. ANTIGEN RECOGNITION BY T-CELLS REQUIRES PEPTIDE ANTIGENS AND ANTIGEN PRESENTING CELLS THAT EXPRESS MHC MOLECULES Y T No T-cell response soluble Ag Native membrane Ag Peptide antigen Cell surface MHC- peptide complex T-cell response Cell surface peptides APC

4. PROFESSIONAL ANTIGEN PRESENTING CELLS Express MHC class I and class II proteins in the cell membrane Express co-stimulatory molecules (CD40, B7) B cells – specialized for soluble proteins, toxins ADAPTIVE Macrophages – extracellular pathogens (bacteria, yeast) Dendritic cells – viruses, apoptotic cells INNATE APC) T-lymphocytes with αβ TCR recognize MHC – peptide complexes expressed on the surface of professional antigen presenting cells (APC) T-cell recognition requires the physical contact of APC and T cell

5. THE STRUCTURE OF MHC GENES AND PROTEINS

6. MEMBERS OF THE IMMUNOGLOBULIN SUPERGENE FAMILY FUNCTION RECOGNITION Ig, TCR, MHC-I, MHC-II ADHESION ICAM-1, ICAM-2, VCAM-1, NCAM BINDING CD4, CD8, CD28, B7, IL-1RI, PDGFR, Fc  RII, poly-IgR MOLECULES CONTAINING ONE OR MORE Ig DOMAIN(S) V or C domain related

7. THE STRUCTURE OF MHC GENE PRODUCTS 33  2m 22 11 11 22 22 11  1  2  3  1  2  1  2  3 and  2m Ig supergene family  2 and  2

8. 3,838,986 bp 224 genes on chromosome 6 http://webace.sanger.ac.uk/cgi-bin/ace/pic/6ace?name=MHC&class=Map&click=400-1 The MHC sequencing consortium Nature 401, 1999 MAP OF THE HUMAN MHC FROM THE HUMAN GENOME PROJECT

9. Classical MHC genes POLYMORPHIC HLA – Human Leukocyte Antigen HLA –A,B, C class I EXPRESSED IN ALL NUCLEATED CELLS HLA – DR, DP, DQclass II EXPRESSED IN PROFESSIONAL APC Non- classical MHC genes E, G, F Chromosome 6 short armMHC Chromosome 15  2m LOCALIZATION OF MHC GENES Class III genes

11. Differential distribution of MHC molecules Tissue MHC class IMHC class II T cells+++ +/- B cells+++ +++ Macrophages+++ ++ Other APC+++ +++ Epithelial cells of thymus + +++ Neutrophils+++ - Hepatocytes + - Kidney + - Brain + - Erythrocytes -- Cell activation affects the level of MHC expression The pattern of expression reflects the function of MHC molecules Class I is involved in anti-viral immune responses Class II involved in activation of other cells of the immune system

12. INHERITANCE OF MHC CLASS I AND CLASS II GENES HUMAN LEUKOCYTE ANTIGEN HLA α1β1α1β1α2β2α2β2 PROFESSIONAL APC EVERY CELL CLASS I CLASS II EVERY CELL CLASS I CLASS II

13. POSTULATION every individual has 6 MHC class I molecules alleles of all MHC types are distributed randomly in the population every one of the 1 200 different alleles can be expressed with any other allele POLYMORPHISM OF MHC MOLECULES IN THE HUMAN POPULATION ~6 x 10 15 individual combinations Only monozygous twins are identical at the HLA locus The human population is extensively outbred MHC genetics in humans is extremely complex POLYGENIC & POLYMORPHIC

14. Alleles are not distributed evenly in the population Alleles are segregated among races SELECTION 15.18 28.65 13.38 4.46 0.02 5.72 18.88 8.44 9.92 1.88 4.48 24.63 2.64 1.76 0.01 CAU AFR ASI Frequency (%) HLA-A1 HLA- A2 HLA- A3 HLA- A28 HLA- A36 Allelic groups ABC Polymorphism (number of alleles) Class I 657 alleles 185 91 381 Class II 492 alleles   2 317 19 89 20 45 DRDPDQ DISTRIBUTION OF MHC ALLELES IN THE HUMAN POPULATION

16. Inheritance of MHC haplotypes BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR X Parents DP-1,2 DQ-3,4 DR-5,6 B-7,8 C-9,10 A-11,12 DP-9,8 DQ-7,6 DR-5,4 B-3,2 C-1,8 A-9,10 DP-1,8 DQ-3,6 DR-5,4 B-7,2 C-9,8 A-11,10 DP-1,9 DQ-3,7 DR-5,5 B-7,3 C-9,1 A-11,9 DP-2,8 DQ-4,6 DR-6,4 B-8,2 C-10,8 A-12,10 DP-2,9 DQ-4,7 DR-6,5 B-8,3 C-10,10 A-12,9 BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR Children

17. FUNCTIONS OF MHC MEMBRANE PROTEINS

18. PEPTIDE 11 33 22 2m2m STRUCTURE OF CLASS I MHC MOLECULES EXPRESSED BY ALL NUCLEATED CELLS

19. 22 11 22 11 PEPTIDE EXPRESSED BY PROFESSIONAL ANTIGEN PRESENTING CELLS CAN BE INDUCED IN OTHER CELLS (endothel, microglia, astocyte) STRUCTURE OF CLASS II MHC MOLECULES

20. MEMBRANE RECEPTORS Intracellular peptide binding capacity One binding site can accomodate multiple peptides

21. MHC – PEPTIDE COMPLEXES

22. 11 33 22 2m2m 22 11 22 11 Allelic polymorphism is concentrated in the peptide antigen binding site Polymorphism in the MHC affects peptide antigen binding Allelic variants may differ by 20 amino acids Class II (HLA-DR) Class I

23. MHC class I accommodate peptides of 8-10 amino acids Cleft geometry MHC class II accommodate peptides of >13 amino acids  -M  -chain Peptide  -chain  -chain Peptide

24. DPB1*01011 TAC GCG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GCT GCG GAG TAC TGG AAC AGC CAG AAG GAC ATC CTG GAG GAG DPB1*01012 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*02012 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*02013 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AC -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0202 CT- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0301 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*0401 -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0402 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0501 CT- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0601 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*0801 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0901 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- --- --- --- --- DPB1*1001 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*11011 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- DPB1*11012 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- DPB1*1301 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*1401 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*1501 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- DPB1*1601 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*1701 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- --- --- --- --- DPB1*1801 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*1901 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*20011 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*20012 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*2101 CT- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*2201 CT- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*2301 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*2401 -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*2501 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- C-- --- --- --- DPB1*26011 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*26012 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 30 HLA-DP  allele sequences between Nucleotides 204 and 290 (amino acids 35-68) Most polymorphisms are point mutations Y-F A-V Silent A-D A-E E-A I-L Polymorphic nucleotides encode amino acids associated with the peptide binding site

25. STRUCTURE OF THE PEPTIDE BINDING SITE P2 & P9 interact with hydrophobic pockets Amino acid side chains of the ‘core’ region occupy evenly distributed pockets

26. Peptides can be eluted from MHC molecules Acid elute peptides

27. Eluted peptides from MHC molecules have different sequences but contain motifs Peptides bound to a particular type of MHC class I molecule have conserved patterns of amino acids PEIYSFH I AVTYKQT L PSAYSIK I RTRYTQLV NC Tethering amino acids need not be identical but must be related Y & F are aromatic V, L & I are hydrophobic Side chains of anchor residues bind into POCKETS in the MHC molecule SIIFNEKL APGYNPAL RGYYVQQL Different types of MHC molecule bind peptides with different patterns of conserved amino acids A common sequence in a peptide antigen that binds to an MHC molecule is called a MOTIF Amino acids common to many peptides tether the peptide to structural features of the MHC molecule ANCHOR RESIDUES

28. PEPTIDE MOTIFS IN SELF AND FOREIGN PROTEINS

29. MHC MOLECULES ARE EXPRESSED IN THE CELL SURFACE MEMBRANE WITH BOUND PEPTIDES DERIVED FROM SELF OR NON- SELF PROTEINS B-cell, macrophage, dendritic cell Kidney epithelial cell Liver cell Present intracellular environment Present intra- and extracellular environment Class I MHC – code of 6 Immunological ID Peptides of restricted size, which derive from cytosolic or nuclear proteins Class II MHC Overlapping peptides of various sizes, which derive from membrane/exogeneous proteins 70% derives from MHC molecules

30. BENEFITS OF MHC POLYMORPHISM IN THE POPULATION

31. COMBINATION OF MHC ALLOTYPES IN INDIVIDUALS AND IN THE POPULATION * * *

32. Populations need to express variants of each type of MHC molecule The rate of replication by pathogenic microorganisms is faster than human reproduction In a given time a pathogen can mutate genes more frequently than humans and can easily evade changes in MHC molecules The number of types of MHC molecules are limited To counteract the flexibility of pathogens The MHC has developed many variants of each type of MHC molecule These variants may not necessarily protect all individuals from every pathogen, but will protect the population from extinction

33. THE OUTCOME OF INFECTION IN A POPULATION WITH POLYMORPHIC MHC GENESMHC-Gen v v v v v v v v v v v v v vvv v v v v v v v v v Example: If MHC X was the only type of MHC molecule Population threatened with extinction Pathogen that evades MHC X MHC XX Population is protected

34. Molecular basis of MHC types and variants POLYMORPHISM Variation >1% at a single genetic locus in a population of individuals MHC genes are the most polymorphic known The type and variant MHC molecules do not vary in the lifetime of the individual The diversity in MHC molecules exists at the population level This sharply contrast diversity in T and B cell antigen receptors which exists within the individual POLYGENISM Several MHC class I and class II genes encoding different types of MHC molecule with a range of peptide-binding specificities.

35. Foreign peptides Peptides recognized by an individual

36. CHANGES OF GENETIC POLYMORPHISM IN THE POPULATION Csökkenés KialakulásNövekedés Nagy csökkenés

37. Multiple heterozygous allele Homozygous

38. Molecular basis of MHC types and variants POLYMORPHISM Variation >1% at a single genetic locus in a population of individuals MHC genes are the most polymorphic known The type and variant MHC molecules do not vary in the lifetime of the individual The diversity in MHC molecules exists at the population level This sharply contrast diversity in T and B cell antigen receptors which exists within the individual POLYGENISM Several MHC class I and class II genes encoding different types of MHC molecule with a range of peptide-binding specificities.

39. FUNCTIONS OF MHC CLASSICAL MHC GENE PRODUCTS –Presentation of peptides derived from self proteins – continuos presentation of self for the immune system –Determination of immunological self Self MHC + self peptides –Presentation of peptides derived from foreign (antigenic) proteins –Determination of non-self Self MHC + non-self peptides –Allogeneic immune response against non-self MHC (transplantation) Recognition of non-self MHC + peptide combinations – consequence of MHC- restricted T-cell recognition –NK cell recognition –T-lymphocyte differentiation and selection in the thymus –T-lymphocyte survival in the periphery NON CLASSICAL MHC GENES –Specialized functions PROTEINS STRUCTURALLY RELATED TO CLASSICAL MHC MOLECULES

40. OTHER GENES IN THE MHC – not polymorphic MHC Class 1b genes Encoding MHC class I-like proteins that associate with  -2 microglobulin Restricted tissue expression HLA-G trophoblast, interacts CD94 (NK-cell receptor). Inhibits NK cell attack of foetus/ tumours HLA-E in the membrane of certain cells, binds conserved leader peptides from HLA-A, B, C. Interacts with CD94 HLA-F fetal liver, eosinophil surface, function unknown MHC Class II genes Encoding several antigen processing genes HLA-DM   and  in professional APC, proteasome components ( LMP-2 & 7 ), peptide transporters ( TAP-1 & 2 ), HLA-DO  and DO  Many pseudogenes MHC Class III genes Encoding complement proteins C4A and C4B, C2 and FACTOR B TUMOUR NECROSIS FACTORS-  Immunologically irrelevant genes Genes encoding 21-hydroxylase, RNA Helicase, Caesin kinase Heat shock protein 70, Sialidase