TISSUE TRANSPLANTATION IS RESTRICTED BY MHC MOLECULES 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 T-LYMPHOCYTES RECOGNIZE ANTIGEN-DERIVED PROTEIN FRAGMENTS (PEPTIDES) EXPRESSED ON THE SURFACE OF SELF ANTIGEN PRESENTING CELLS

ANTIGEN PRESENTING CELLS Synthesize antigens – endogenous antigens (virus, tumor) Internalize antigens – exogenous antigens (any protein) Degrade protein antigens to peptides – processing 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

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

THE STRUCTURE OF MHC GENES AND PROTEINS

MOLECULES CONTAINING ONE OR MORE Ig DOMAIN(S) MEMBERS OF THE IMMUNOGLOBULIN SUPERGENE FAMILY MOLECULES CONTAINING ONE OR MORE Ig DOMAIN(S) V or C domain related 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

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

FROM THE HUMAN GENOME PROJECT MAP OF THE HUMAN MHC FROM THE HUMAN GENOME PROJECT 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

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

Differential distribution of MHC molecules Tissue MHC class I MHC 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

INHERITANCE OF MHC CLASS I AND CLASS II GENES HUMAN LEUKOCYTE ANTIGEN HLA EVERY CELL CLASS I CLASS II α1β1 α2β2 PROFESSIONAL APC

POLYMORPHISM OF MHC MOLECULES IN THE HUMAN POPULATION 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 ~6 x 1015 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

DISTRIBUTION OF MHC ALLELES IN THE HUMAN POPULATION Polymorphism (number of alleles) Class I 657 alleles 185 91 381 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 Class II 492 alleles a b 2 317 19 89 20 45 DR DP DQ Alleles are not distributed evenly in the population Alleles are segregated among races SELECTION

Inheritance of MHC haplotypes B C A DP DQ DR Children 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 Inheritance of MHC haplotypes B C A DP DQ DR X Parents B C A DP DQ DR 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 B C A DP DQ DR B C A DP DQ DR

FUNCTIONS OF MHC MEMBRANE PROTEINS

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

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

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

MHC – PEPTIDE COMPLEXES

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

Cleft geometry b2-M a-chain Peptide a-chain b-chain Peptide MHC class I accommodate peptides of 8-10 amino acids MHC class II accommodate peptides of >13 amino acids

Most polymorphisms are point mutations 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-DPb allele sequences between Nucleotides 204 and 290 (amino acids 35-68) Polymorphic nucleotides encode amino acids associated with the peptide binding site Y-F A-V Silent A-D A-E E-A I-L

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

Peptides can be eluted from MHC molecules Acid elute peptides

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 A common sequence in a peptide antigen that binds to an MHC molecule is called a MOTIF R T Y Q L V N C P E I Y S F H Amino acids common to many peptides tether the peptide to structural features of the MHC molecule ANCHOR RESIDUES A V T Y K Q L P S A Y I K Tethering amino acids need not be identical but must be related Y & F are aromatic V, L & I are hydrophobic R G Y V Q L S I F N E K L A P G Y N L Side chains of anchor residues bind into POCKETS in the MHC molecule Different types of MHC molecule bind peptides with different patterns of conserved amino acids

PEPTIDE MOTIFS IN SELF AND FOREIGN PROTEINS

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

BENEFITS OF MHC POLYMORPHISM IN THE POPULATION

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

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

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

Molecular basis of MHC types and variants POLYGENISM Several MHC class I and class II genes encoding different types of MHC molecule with a range of peptide-binding specificities. 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

Peptides recognized by an individual Foreign peptides Peptides recognized by an individual

CHANGES OF GENETIC POLYMORPHISM IN THE POPULATION

Multiple heterozygous allele Homozygous

FUNCTIONS OF MHC CLASSICAL MHC GENE PRODUCTS NON CLASSICAL MHC GENES 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

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