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Genética Molecular em Análises Clínicas MHC Prof.Doutor José Cabeda Genetic basis of transplant rejection Inbred mouse strains - all genes are identical Transplantation of skin between strains showed that rejection or acceptance was dependent upon the genetics of each strain ACCEPTED Skin from an inbred mouse grafted onto the same strain of mouse REJECTED Skin from an inbred mouse grafted onto a different strain of mouse Immunological basis of graft rejection Primary rejection of strain skin e.g. 10 days Transfer lymphocytes from primed mouse Naïve mouse Lyc 6 months Secondary rejection of strain skin e.g. 3 days Primary rejection of strain skin e.g. 10 days Transplant rejection is due to an antigen-specific immune response with immunological memory. Immunogenetics of graft rejection Parental strains X A F1 hybrid (one set of alleles from each parent) B ACCEPTED AxB AxB REJECTED A B Mice of strain (A x B) are immunologically tolerant to A or B skin Skin from (A x B) mice carry antigens that are recognised as foreign by parental strains Major Histocompatibility Complex – MHC In mice the MHC is called H-2 Rapid graft rejection segregated with a cell surface antigen, Antigen-2 Inbred mice identical at H-2 did not reject skin grafts from each other MHC genetics in mice is simplified by inbred strains In humans the MHC is called the Human Leukocyte Antigen system – HLA Only monozygous twins are identical at the HLA locus The human population is extensively outbred MHC genetics in humans is extremely complex MHC molecules MHC class I MHC class II Peptide Peptide binding groove Cell Membrane 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 Overall structure of MHC class I molecules MHC-encoded -chain of 43kDa 2 1 3 2m -chain anchored to the cell membrane Peptide antigen in a groove formed from a pair of -helicies on a floor of anti-parallel strands 2-microglobulin, 12kDa, non-MHC encoded, non-transmembrane, non covalently bound to chain 3 domain & 2m have structural & amino acid sequence homology with Ig C domains Ig GENE SUPERFAMILY MHC class I molecule structure Peptide -chain 2-microglobulin Chains Structures Structure of MHC class I molecules 1 and 2 domains form two segmented -helicies on eight anti-parallel -strands to form an antigen-binding cleft. Chains Structures Properties of the inner faces of the helicies and floor of the cleft determine which peptides bind to the MHC molecule Overall structure of MHC class II molecules MHC-encoded, -chain of 34kDa and a -chain of 29kDa 1 1 and chains anchored to the cell membrane No -2 microglobulin 2 2 Peptide antigen in a groove formed from a pair of -helicies on a floor of anti-parallel strands 2 & 2 domains have structural & amino acid sequence homology with Ig C domains Ig GENE SUPERFAMILY MHC class II molecule structure Peptide -chain -chain Cleft is made of both and chains MHC-binding peptides Each human usually expresses: 3 types of MHC class I (A, B, C) and 3 types of MHC class II (DR, DP,DQ) The number of different T cell antigen receptors is estimated to be 1,000,000,000,000,000 Each of which may potentially recognise a different peptide antigen How can 6 invariant molecules have the capacity to bind to 1,000,000,000,000,000 different peptides? Anchor residues and T cell antigen receptor contact residues Cell surface MHC anchor residue side-chains point down MHC class I Sliced between -helicies to reveal peptide T cell antigen receptor contact residue side-chains point up MHC molecules can bind peptides of different length Arched peptide P S S I A K S Y A I MHC molecule K S I P S Y I MHC molecule Complementary anchor residues & pockets provide the broad specificity of a particular type of MHC molecule for peptides Peptide sequence between anchors can vary Number of amino acids between anchors can vary Peptide antigen binding to MHC class II molecules Negatively charged Hydrophobic I S N Q L T L D S N T K Y F H K I P D N L F K S D G R I K Y T L N A T K Y G N M T E D H V N H L L Q N A G K F A I R P Y K K S N P I I R T V V F L L L L A Y K V P E T S L S T F D Y I A S G F R Q G G A S Q P P E V T V L T N S P V Y L R E P N V Y G Y T S Y Y F S W A E L T G H G A R T S T Y P T T D Y • Anchor residues are not localised at the N and C termini • Ends of the peptide are in extended conformation and may be trimmed • Motifs are less clear than in class I-binding peptides • Pockets are more permissive How can 6 invariant molecules have the capacity to bind to 1,000,000,000,000,000 different peptides with high affinity? MHC molecules • Adopt a flexible “floppy” conformation until a peptide binds • Fold around the peptide to increase stability of the complex • Use a small number of anchor residues to tether the peptide this allows different sequences between anchors and different lengths of peptide MHC molecules are targets for immune evasion by pathogens • T cells can only be activated by interaction between the antigen receptor and peptide antigen in an MHC molecule • Without T cells there can be no effective immune response • There is strong selective pressure on pathogens to evade the immune response • The MHC has evolved two strategies to prevent evasion by pathogens More than one type of MHC molecule in each individual Extensive differences in MHC molecules between individuals Example: If MHC X was the only type of MHC molecule MHC XX Pathogen that evades MHC X Survival of individual threatened Population threatened with extinction Example: If each individual could make two MHC molecules, MHC X and Y Pathogen that evades MHC X but has sequences that bind to MHC Y MHC XX MHC YY MHC XY Impact on the individual depends upon genotype Population survives Example: If each individual could make two MHC molecules, MHC X and Y……and the pathogen mutates MHC XX Pathogen that evades MHC X but has sequences that bind to MHC Y ….until it mutates to evade MHC Y MHC YY MHC XY Survival of individual threatened Population threatened with extinction The number of types of MHC molecule can not be increased ad infinitum 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 Variant MHC molecules protect the population Pathogen that evades MHC X and Y …but binds to the variant MHC XR and MHC YR MHC YYR MHC XX XX XXR XYRYXR YYR YY XRXR MHC YY YRYR XRYR XY MHC XY From 2 MHC types and 2 variants……. 10 different genotypes MHC XXR Variants of each type of MHC molecule increase the resistance of the population from rapidly mutating or newly encountered pathogens without increasing the number of types of MHC molecule 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 Simplified map of the HLA region DP DM LMP/TAP DQ 1 3DR 4 5 MHC Class II B C A Class III MHC Class I Polygeny CLASS I: 3 types HLA-A, HLA-B, HLA-C (sometimes called class Ia genes) CLASS II: 3 types HLA-DP HLA-DR. 3 extra DR genes in some individuals canHLA-DQ allow 3 extra HLA-DR molecules Maximum of 9 types of antigen presenting molecule allow interaction with a wide range of peptides. Detailed map of the HLA region http://www.anthonynolan.org.uk/HIG/data.html July 2000 update Map of the Human MHC from the Human Genome Project 3,838,986 bp 224 genes on chromosome 6 The MHC sequencing consortium Nature 401, 1999 http://webace.sanger.ac.uk/cgi-bin/ace/pic/6ace?name=MHC&class=Map&click=400-1 Other genes in the MHC MHC Class 1b genes Encoding MHC class I-like proteins that associate with -2 microglobulin: HLA-G interacts CD94 (NK-cell receptor). Inhibits NK cell attack of foetus/ tumours HLA-E binds conserved leader peptides from HLA-A, B, C. Interacts with CD94 HLA-F function unknown MHC Class II genes Encoding several antigen processing genes: HLA-DM and , 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 AND Immunologically irrelevant genes Genes encoding 21-hydroxylase, RNA Helicase, Caesin kinase Heat shock protein 70, Sialidase Polymorphism in the MHC Variation >1% at a single genetic locus in a population of individuals Each polymorphic variant is called an allele In the human population, over 1,200 MHC alleles have been identified No of polymorphisms 381 317 Class I Class II 657 alleles 492 alleles 185 89 91 2 A B C 19 DR DP 20 45 DQ Data from http://www.anthonynolan.org.uk/HIG/index.html July 2000 Allelic polymorphism is concentrated in the peptide antigen binding site Class I 2 3 1 1 1 2m 2 2 Class II (HLA-DR) Polymorphism in the MHC affects peptide antigen binding Allelic variants may differ by 20 amino acids Diversity of MHC molecules in the individual DP DQ DR 1 B C A Polygeny DP DQ DR 1 B C DP DQ DR 1 B C A Variant alleles polymorphism HAPLOTYPE 1 HAPLOTYPE 2 A Additional set of variant alleles on second chromosome MHC molecules are CODOMINANTLY expressed Two of each of the six types of MHC molecule are expressed Genes in the MHC are tightly LINKED and usually inherited in a group The combination of alleles on a chromosome is an MHC HAPLOTYPE Inheritance of MHC haplotypes DP-1,9 DQ-3,7 DR-5,5 B-7,3 C-9,1 A-11,9 Parents DP-1,2 DQ-3,4 DR-5,6 B-7,8 C-9,10 A-11,12 DP DP DQ DR DQ DR BC BC A A Children X 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 DQ DR BC A DP DQ DR BC A 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 DP DQ DR BC A DP DQ DR BC A DP DQ DR BC A DP DQ DR BC A DP DQ DR BC A DP DQ DR BC A DP DQ DR BC A DP DQ DR BC A Errors in the inheritance of haplotypes generate polymorphism in the MHC by gene conversion and recombination A B A C Multiple distinct but closely related MHC genes A B C B A C During meiosis chromosomes misalign A B C B C Chromosomes separate after meiosis DNA is exchanged between haplotypes GENE CONVERSION A B C RECOMBINATION between haplotypes In both mechanisms the type of MHC molecule remains the same, but a new allelic variant may be generated