Fun in the Dunn 1

Fun in the Dunn 1

IMMUNOGENETICSIMMUNOGENETICS

Variation in immune receptorsDiversity generation and MHC polymorphisms

Simon HuntDunn School of Pathology

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Objectives

Describe the chain structures of Ig, Tcr and MHC molecules Relate these structures broadly to function Understand the difference between somatic and germ-line

alterations in genetic information Know the order of magnitude of the numbers of different

kinds of antigen-recognising molecules on person can make Know the mechanism of diversity generation by gene

rearrangement Know the mechanism of diversity generation by somatic

mutation Know the gene changes caused by isotype switching

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Reading and e-reading

• Benjamini, Sunshine and Leskowitz, “Immunology, a short course”, chapters 6 & 10 (introductory)

• Roitt, Brostoff, Male “Immunology” (4th ed) chapters 5&6 (introductory)

• Klein , J. and Horejsi, V, “Immunology”, chapter 6 (advanced discussion of polymorphism of MHC)

• http://www.umass.edu/microbio/rasmol/ (molecular graphics of MHC molecules)

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• How are the genes for Antigen-recognising molecules (Ig, Tcr, MHC) organised, shuffled and mutated?

• To what extent is their variation due to what you inherited from Mum and Dad (germ-line)?

IntroductionIntroduction

• or to what you create during your lifetime (somatic)?

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• Is the variation due to changes over evolutionary timescales, like other ordinary God-fearing genes– are all variants available to everyone, i.e. are the

genes organised in tandem array along the genome?– or is there at least a contribution from genetic

polymorphism, with some allelic variants in each of us to broaden our repertoires?

• Is some of the variation due to unique changes occurring during lymphocyte development?

• Is the number of variants in the repertoire ever a limiting factor in specific immune responses?

Source and Nature of variation

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• The germ-line genetic information is not affected by the somatic genetic information– Somatic mutations don’t pass to next generation– (Somatic gene therapy is OK)

Weissman’s DoctrineWeissman’s Doctrine

R.I.P.soma

germ-line

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What is a “gene”?What is a “gene”?

• A heritable particle - Mendel. Defined by its alleles.• That germ-line combination of exons needed to be assembled to make one polypeptide chain. The exon combination is

altered by:– mRNA splicing - conventional– genomic DNA hanky-panky to create new “genes” - unique

• gene segment rearrangement for V regions (Ig and Tcr)

• then V gene somatic mutations (Ig only: VH and VL)

• switch recombination for CH genes (isotype switching)

• The finished product after this alteration is finished

Don’t confuse gene segments with genes!

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Antigen-recognising molecules

Binding molecule Ligand

• Antibodies = Immunoglobulins Epitope of non-denatured molecules

(protein, CHO, etc)• T cell receptors MHC + peptide• MHC molecules Linear

peptides

Mike Clark’s animated molecules are at:http://www.path.cam.ac.uk/~mrc7/movies/igg1y.htmlhttp://www.path.cam.ac.uk/~mrc7/movies/mhc_tcr.html

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IgG1from X-ray

crystallography H chain

L chain

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T cell receptor

MHC class I

peptide

©Mike ClarkPathology Cambridge

Alpha chain

ß2 micro-globulin

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• Rearrangement of DNA of genome• Example - Heavy chain gene segments.

V gene diversification: 1V gene diversification: 1

Combinatorial diversity by picking one V, one D, one J segment from many available

Hypervariable 3 (CDR3)

VH DH JHVariable Diversity Joining

1

2

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• Junctional diversity– Imprecise joins - frameshift or intra-codon recombination

V gene diversification: 2V gene diversification: 2

•Nucleotide insertion: N region diversity–Enzyme TdT (terminal deoxynucleotidyl transferase)

nibbles at joins; inserts bases - no template. DNA repair.

Errors!! Non-productive rearrangements!!

EITHER

C A T T A G G T C A T

OR

C A T T C A G T C A T

D J

N

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Cells in which primary diversification occurs

Cells in which primary diversification occurs

• Lymphocyte progenitors: DH to JH– Then seeding to Thymus; or stay in Bone Marrow

ThymusTry D to J then V to D

might make gamma geneIf not, try other allele

another go at gamma geneTry D to J then V to D

etc, etc

Tcr or Tcr

Try V to DJ

might make Heavy chain geneIf not, try other allele

another go at Heavy chain geneTry V to J; if no good

another go at kappa chain geneTry V to J; etc, etc

Ig or Ig

Bone Marrow

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One cell, one specificityOne cell, one specificity

• Postulate of Clonal Selection Theory– cell is committed to recognising just one antigen before it encounters it

• B cell has two Light chain isotypes ( and ), each with two alleles (maternal and paternal origin)– could make 4 Light chain V genes

• Therefore it stops when one productive rearrangement is made:either - feedback to prevent any more: “allelic and isotype exclusion”; or - possibly happens accidentally if high proportion of non-productive rearrangements

– observe in heterozygote with distinguishable alleles– likewise H chain genes and Tcr genes

• Clinical application: myeloma diagnosis– myeloma is a monoclonal tumour (one V region only)– unbalanced kappa/lambda ratio

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Antigen-recognising molecules:chain compositions

Antigen-recognising molecules:chain compositions

– MHC Class I: 1 alpha (+ 2 microglobulin) A few A few

• Dimers: combinatorial possibilities between the two chains– MHC Class II: 1 alpha, 1 beta chain A few A few

dozen– Immunoglobulin: 2 Heavy, 2 Light chains Thousands Millions– Tcr: 1 alpha, 1 beta chain Thousands Millions– Tcr: 1 gamma, 1 delta chain Thousands Millions

Numbers of variantsChains Molecule

types•Dimer: but only one chain varies

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The numbers: how many genes?The numbers: how many genes?

Segment Variable Diversity Joining CombinationsHeavy 44 25 6 6600Kappa >32 0 5 160

Lambda 29 0 4 ~120

If random, total no. theoretical H-L combinations = 1.9 million.These segment nos. vary from person to person - i.e. are polymorphic

• Underestimate because:– No allowance for junctional or N region diversification– Omits occasional D-D joins

– VH and possibly DH show polymorphic alleles, so most people are heterozygous

– Possible contributions from stray “orphons” or pseudogenes

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Organisation of segments: the mapsOrganisation of segments: the maps

• Chromosome assignments: separate locus for each chain• Normal intron-exon structure for C regions at 3’ end

– one domain per exon

• J cluster upstream (5’) of C regions– brought in apposition to C genes by conventional mRNA splicing

• Widespread DH segments– some sprinkled among VH segments

• V segments show patterns of homology into families– over 70% nucleotide homology defines a family– family usage diagnosed by pcr primers, or monoclonal antibodies

• Extended over long regions of genome, e.g. 1.1Mbp– huge loops of DNA must be excised during rearrangement. Sequence signals.– tendency for breakpoints at rearrangement sites - lymphomas

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Secondary DiversificationSecondary Diversification

• For B cells only; somatic point mutations clustered around CDR regions– during memory generation in germinal centres– shown by direct isolation of one cell at a time from a germinal centre and pcr the cDNA from the mRNA for sequencing– in 1 example of high-rate mutation, >80 mutant bases were found out of about 300 in V region

• Affinity maturation– Majority of mutants must be harmful to the antibody specificity, but a few higher-affinity ones are produced and these overgrow the rest selectively.

• Mutations found in non-coding (“silent”) as well as coding (“replacement”) bases S/R indicates antigen-induced selection pressure

• Needs special enzyme machinery - must control it!

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Immunoglobulin Heavy chain switchImmunoglobulin Heavy chain switch

to is not true switching

– simply differential splicing of long transcript to or to or to is true switching

– mainly by excision of intervening DNA• employs switch-recombination sequence signals to identify which part of gene should be excised• once lost it can’t be recovered (unless available from opposite chromosome)

– regulated by T cells– mainly post-antigen,

• but some believe a cell can precommit its isotype before antigen, e.g. to IgA production

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MHC diversityMHC diversity

• No somatic genetic hanky-panky. No allelic exclusion. All variation is inherited through germ-line.• Some evolutionary gene duplication and divergence, but also a huge polymorphism• Human Leukocyte Antigen A locus

– First discovery as antigens preventing grafting. Now HLA typing tries to match as well as possible: • serological methods• DNA methods• In vitro mixed lymphocyte reaction

– Class I Class IIHLA-A; HLA-B HLA-DP; HLA-DQ; HLA-DR+ some more almost fully described

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MHC polymorphismMHC polymorphism

• Polymorphic changes found along MHC groove which binds peptide: – strong evidence that the selective benefit to heterozygotes is due to the wider range of peptides which can be accepted into one person’s set of grooves.

Heterozygous advantage maintains wide polymorphism.– 3 to 6 residues form the “anchor” points

• Detectable alleles: Class I Class IIHLA-A HLA-B HLA-C HLA-D (several sub-loci)

in population 59 118 36 >700in one individual 2 2 2 probably >36

• Much less variation than V genes; hence more likely to be limiting factor in recognition: Immune Response Genes; prevalence of HLA-B53 in some malarial areas; Autoimmunity.

• Map shows all MHC loci within about 3.5 Mbp; closely linked on Chr 6p

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SummarySummary

• The factor limiting specific antigen recognition is not the Ig or Tcr repertoires but the MHC Class I and Class II. This is the basis for – HLA-associated diseases, such as autoimmunity– Associations of HLA haplotypes with resistance to infection– The key to understanding this properly is to find out how cross-reactive are the MHC grooves for a range of peptides. How sloppy is the fit?

• Creating the repertoires by somatic changes, including isotype switches, leads to weak points on the genome liable to translocations. These can lead to lymphoproliferative disorders or frank tumours.

• In both V gene and MHC systems, large numbers of pseudogenes form a reservoir of potential variants which might someday be useful