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KIR POLYMORPHISM Christian Garcia, MD

Anthony Nolan Research Institute. KIR POLYMORPHISM Christian Garcia, MD. NK cell. Target. Image by Dr. G. Arancia and K. Malorni, Rome. Critical component of the innate immune system to a great variety of pathogens including virus, fungi, parasites and bacteria.

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KIR POLYMORPHISM Christian Garcia, MD

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  1. Anthony Nolan Research Institute KIR POLYMORPHISM Christian Garcia, MD

  2. NK cell Target Image by Dr. G. Arancia and K. Malorni, Rome Critical component of the innate immune system to a great variety of pathogens including virus, fungi, parasites and bacteria. Important mediators of anti-tumoral immunity. Comprise approximately 10% of all peripheral blood lymphocytes. Cytologically known as Large Granular Lymphocytes. Lack clonally expressed antigen receptors encoded by rearranging genes.

  3. Natural Killer cells have evolved several receptor systems to carry out their function of detecting and eliminating pathogen infected cells and tumour cells during the prolonged period required for the clonal expansion of antigen-specific B and T cells • NKG2s (C-type lectin-like) • NCR (Natural Cytotoxicity Receptors) • KIRs (Immunoglobulin-like)

  4. NKG2s (C-type lectin-like)

  5. NCR (Natural Cytotoxicity Receptors)

  6. KIRs (Immunoglobulin-like) Killer Immunoglobulin-like receptors (KIR) are polymorphic cell surface molecules present on Natural Killer (NK) cells and a small subset of T cells (NKT cells).

  7. KIRs (Immunoglobulin-like) They recognise classical HLA class I molecules with allotype specificity. They are the most polymorphic receptor system expressed by NK cells.

  8. First lymphocytes to engraft after a HSCT. Present 7 days post-transplant, their cytolytic and proliferative activity peaks 30-50 days post-transplant. One month after the HSCT, they make up 50-90% of the total circulating lymphocyte population.

  9. The existence of NK cell receptors capable of mediating specific allorecognition was established in 1990 by Moretta A. et al. Studies regarding NK cell roles in HSCT have produced controversial results in relation to the level of KIR matching necessary to allow for optimal engraftment and potent GvL while maintaining the level of GvH reactions to a minimum.

  10. KIR3D KIR2D KIR genes and proteins are classified and named on the basis of structure and sequence homology.

  11. KIR3D KIR2D KIR proteins can have two or three extracellular Immunoglobulin(Ig)-like domains. KIR gene & protein names start with “KIR” followed by a digit indicating the number of Ig-like domains followed by a “D” (domain)

  12. KIR2DL KIR3DS KIR3DL KIR2DS Each of these KIR proteins can also have dimorphic cytoplasmic tails. In nomenclature KIR2D or KIR3D is followed by a letter “L” or “S” indicating the presence of a “Long” or “Short” cytoplasmic tail. *Pseudogenes have a “P” instead of an “L” or “S” (KIR2DP1 & KIR3DP1)

  13. KIR3DL KIR2DS KIR2DL KIR3DS Long cytoplasmic tails have ITIMs which generate inhibitory signals.

  14. KIR3DL KIR2DS KIR2DL KIR3DS Short cytoplasmic tails generate activating signals.

  15. KIR2DL1 KIR2DL2 KIR2DL3 A final digit indicates the number of the gene encoding a protein with this structure in the order it was discovered. This constitutes the GENE NAME or LABEL

  16. KIR2DL1 KIR2DL2 KIR2DL3 Although the nucleotide and protein sequence between these three KIR genes may be very similar, genetic segregation in families has shown them to be different genes.

  17. KIR2DL1*001 KIR2DL1*002 KIR2DL1*003 KIR2DL1*004 KIR2DL1*005 KIR2DL1 Because these KIR genes are known to be polymorphic, the presence of allelic variants which lead to a protein change are further detailed by adding to the gene name a separator followed by a digit.

  18. KIR2DL1 KIR2DL1*00301 KIR2DL1*00302 KIR2DL1*001 KIR2DL1*002 KIR2DL1*003 KIR2DL1*004 KIR2DL1*005 Finally, if two different nucleotide sequences code for the same protein, such synonymous differences are indicated with two final digits.

  19. KIR2DL1 KIR2DL1*001 KIR2DL1*002 KIR2DL1*003 KIR2DL1*004 KIR2DL1*005 KIR2DL1*00301 KIR2DL1*00302 This KIR diversity is the result of several levels of organisational complexity.

  20. KIRs are encoded within a 150 Kb stretch of the leukocyte receptor complex (LRC) on chromosome 19 (19q13.4). The LRC constitutes a large (1 Mb), dense cluster of rapidly evolving immune genes of relatively recent evolutionary origin.

  21. First level of complexity: multiple KIR genes encoding different proteins. The KIR gene family consists of 17 genes. Different KIR genes are organised within this region as haplotypes.

  22. Second level of complexity: several haplotypes containing unique combinations of different KIR genes. KIR haplotypes show extensive diversity characterised by variability in the number of genes present.

  23. Third level of complexity: Each of the different KIR genes is polymorphic leading to multiple allelic variants.

  24. 15 KIR genes (plus 2 pseudogenes). Third level of complexity: Each of the different KIR genes is polymorphic leading to multiple allelic variants.

  25. 91 sequences analysed and aligned. Third level of complexity: Each of the different KIR genes is polymorphic leading to multiple allelic variants.

  26. 1 2 3 4 5 6 7 8 9 Most KIR genes have 9 exons. KIR genes span approximately 1.2 Kb and are arranged in head to tail fashion and separated by 2 Kb from each other.

  27. KIR3D KIR2D (TYPE 1) KIR2D (TYPE 2) 1 2 3 4 5 6 7 8 9 KIR exons correspond approximately to the regions and domains of their respective KIR proteins.

  28. KIR3D KIR2D (TYPE 1) KIR2D (TYPE 2) 1 2 3 4 5 6 7 8 9 KIR exons 1 and 2 encode the leader peptide which is cleaved before expression.

  29. KIR3D KIR2D (TYPE 1) KIR2D (TYPE 2) 1 2 3 4 5 6 7 8 9 Exon 3 of KIR3Ds and type 2 KIR2Ds encodes for the membrane distal domain (in type 1 KIR2Ds it is represented by a pseudoexon).

  30. KIR3D KIR2D (TYPE 1) KIR2D (TYPE 2) 1 2 3 4 5 6 7 8 9 Exon 4 of KIR3Ds and type 1 KIR2Ds encodes for the middle domain (this exon is absent in type 2 KIR2Ds).

  31. KIR3D KIR2D (TYPE 1) KIR2D (TYPE 2) 1 2 3 4 5 6 7 8 9 Exon 5 encodes the membrane proximal domain of all KIRs.

  32. KIR3D KIR2D (TYPE 1) KIR2D (TYPE 2) 1 2 3 4 5 6 7 8 9 Exon 6 and part of exon 7 encode the stem region of all KIRs.

  33. KIR3D KIR2D (TYPE 1) KIR2D (TYPE 2) 1 2 3 4 5 6 7 8 9 Part of exon 7 encodes the transmembrane region of all KIRs.

  34. KIR3D KIR2D (TYPE 1) KIR2D (TYPE 2) 1 2 3 4 5 6 7 8 9 Part of exon 7, all of exon 8 and 9 encode the cytoplasmic region of all KIRs.

  35. 2 4 5 6 7 8 3 1 Class I HLA In classical Class I MHC most of the polymorphism is found in exons 2 and 3, which encode the peptide binding groove.

  36. 2 4 5 6 7 8 3 1 Class I HLA () 3 1 2 4 5 6 7 8 9 2DL1 () 3 1 2 4 5 6 7 8 9 2DL3 3 1 2 4 5 6 7 8 9 3DL1 3 1 2 4 5 6 7 8 9 3DL2 KIR gene polymorphism is evenly distributed along the entire length of the gene.

  37. LIGAND BINDING LOOPS In an attempt to establish the functional impact of the amino acid substitutions observed in KIR proteins we analysed the nature of the replacements.

  38. TRANSMEMBRANE REGION In an attempt to establish the functional impact of the amino acid substitutions observed in KIR proteins we analysed the nature of the replacements.

  39. CYTOPLASMIC TAIL In an attempt to establish the functional impact of the amino acid substitutions observed in KIR proteins we analysed the nature of the replacements.

  40. LIGAND BINDING LOOPS Crystallographic analysis have described the regions of KIR2Ds which directly interact with their cognate HLA ligands.

  41. LIGAND BINDING LOOPS KIR2DL1 with HLA-Cw4 Q.R.Fan, E.O.Long & D.C.Wiley 10-May-01

  42. LIGAND BINDING LOOPS KIR2DL2 with HLA-Cw3 J.C.Boyington, S.A.Motyka, P.Schuck, A.G.Brooks & P.D.Sun 10-Feb-00

  43. Twelve different KIR maps were generated, each comparing the polymorphic residues representing allelic differences within a gene.

  44. AMINO ACID REPLACEMENTS IN THE EXTRACELLULAR DOMAINS OF KIR PROTEINS Some KIR proteins have most of their extracellular polymorphic residues restricted to a single domain.

  45. In some cases two of these polymorphisms are found in a single allele and could potentially relate to differential HLA-binding properties, although this has yet to be shown through binding assays. KIR3DL1*004

  46. AMINO ACID REPLACEMENTS IN THE EXTRACELLULAR DOMAINS OF KIR PROTEINS • Some KIRs do have polymorphic residues in their HLA-binding region and in close proximity (<5Å) to the putative HLA-ligand. • This is the case of: • KIR2DS1 • KIR2DS4 and • KIR3DL1.

  47. 70% of the replacements observed & classified according to their charge were conservative. 53% of the replacements observed & classified according to their polarity & volume were conservative. 65% of the replacements observed & classified according to their hydrophobicity were conservative. This suggest a requirement to maintain a conserved overall charge distribution in KIR proteins.

  48. ANTHONY NOLAN RESEARCH INSTITUTE HLA INFORMATICS GROUP Steven G.E. Marsh, BSc PhD ARCS James Robinson, BSc MSc Christian Garcia, MD Prof. J. Alejandro Madrigal, MD PhD FRCP MRCPath DSc

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