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

Transfer lymphocytes from primed mouse Lyc Primary rejection of strain skin e.g. 10 days

6 months

Naïve mouse 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.

Parental strains

Immunogenetics of graft rejection

X A B F1 hybrid (one set of alleles from each parent) A x B A x B ACCEPTED 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

Peptide binding groove

MHC molecules

MHC class I MHC class II Peptide 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

 2  1  3  2m MHC-encoded  -chain of 43kDa  -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

Chains

 2-micro globulin

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

 1  2  1  2 MHC-encoded,  -chain of 34kDa and a  -chain of 29kDa  and  chains anchored to the cell membrane No  -2 microglobulin 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



-chain

MHC class II molecule structure

Peptide



-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 class I Sliced between  -helicies to reveal peptide MHC anchor residue side-chains point down T cell antigen receptor contact residue side-chains point up

MHC molecules can bind peptides of different length

Arched peptide

S Y I P S S A K I MHC molecule A Y K S I P S MHC molecule I

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

P P E A V T I I S P D T K Y N N G Q L T L F K N M T G K V F T V L Y G H G F L T G A A I R L L L T F N S P Y T S R T S P A D V Y T L S E D D S G D H Y Y Y Y Y Y L F P K K I N T K R I K V N H K S N V P E A S G R E P S W A T T D Y Y L P T F N V E L Y I S R F T L H K L N Q N A I R T L S Q G G V A S Q

• 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 X R and MHC Y R MHC YY R MHC XX MHC YY MHC XY XX XX R XY R YX R YY R YY X R X From 2 MHC types and 2 variants…….

10 different genotypes Y R Y R X R Y R XY R MHC XX R 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  3 DR  5  B C A MHC Class II Class III MHC Class I Polygeny CLASS I: 3 types HLA-A, HLA-B, HLA-C (sometimes called class Ia genes) 3 extra DR  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

HLA-DM  Encoding several antigen processing genes: 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 TUMOUR NECROSIS FACTORS  and AND  FACTOR B

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 381 317 Class I 657 alleles Class II 492 alleles 185 A B 91 C 89 19 20 45 2       DR DP 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  2 m  1  2  1  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

 DR 1  B C A Polygeny  1  B C A HAPLOTYPE 1 Variant alleles polymorphism  1  B C A HAPLOTYPE 2 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

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 Inheritance of MHC haplotypes DP DQ DR B C DP DQ DR B C DP

Parents X

DQ DR B C DP DQ DR B C A A A A

Children

DP-1,9 DQ-3,7 DR-5,5 B-7,3 C-9,1 A-11,9 DP-1,8 DQ-3,6 DR-5,4 B-7,2 C-9,8 A-11,10 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 B C DP DQ DR B C DP DQ DR B C DP DQ DR B C DP DQ DR B C DP DQ DR B C DP DQ DR B C DP DQ DR B C A A A A A A A A

A

Errors in the inheritance of haplotypes generate polymorphism in the MHC by gene conversion and recombination

A B C B C A B C Multiple distinct but closely related MHC genes A B C During meiosis chromosomes misalign A 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