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Characterisation of the KIR genes in HLA homozygous cell lines Christian Garcia

Anthony Nolan Research Institute Royal Free Hospital School of Medicine University College London. Characterisation of the KIR genes in HLA homozygous cell lines Christian Garcia. Introduction.

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Characterisation of the KIR genes in HLA homozygous cell lines Christian Garcia

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  1. Anthony Nolan Research Institute Royal Free Hospital School of Medicine University College London Characterisation of the KIR genes in HLA homozygous cell lines Christian Garcia

  2. Introduction • The availability of Homozygous Typing Cells for the HLA region has been of huge benefit in the study of the human MHC. • No such panel of well characterised material exists for the KIR genes. • In an attempt to provide such material we have analysed the KIR genes in the original 10th IHW cell line panel comprising 107 cell lines.

  3. Aims of this study • Characterise the KIR gene/allele profile of HLA homozygous cell lines. • Determine the KIR haplotypes present in HLA homozygous cell lines. • Define a set of cell lines which are homozygous for the KIR gene region, from those cells which are both homozygous for the HLA region and consanguineous.

  4. 19q13.4 Introduction Extended LRC LRC KIR FcR DAP CD66 SIGLEC FcGRT ILT LAIR ILT NKp46

  5. Introduction • Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex.

  6. Introduction • Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex. A

  7. Introduction • Killer cell Immunoglobulin-like receptors are encoded by a polygenic, polymorphic, and polyhaplotypic gene complex. B

  8. Introduction • KIR gene polymorphism is not restricted to a single exon, but distributed all along the gene. () 3 1 2 4 5 6 7 8 9

  9. Typing strategy • PCR-SSP approach for the subtyping of inhibitory KIRs: • 2DL1, 2DL3, 3DL1, 3DL2 • As well as for the typing of the following activating KIRs: • 2DS1, 2DS2, 2DS3, 2DS4 & 2DS5. HLA-C C2 specificity HLA-C C1 specificity HLA-B Bw4 specificity HLA-A

  10. Typing strategy () 3 1 2 4 5 6 7 8 9 ca 200 bp 34 bp 36 bp 327 bp 300 bp 294 bp 51 bp 53 bp 102 bp A 1.75 kb KIR 2DL1 B 0.26 kb A B C D E F G C 0.28 kb *001 A B C D E F G *002 A B C D E F G D 2.2 kb *005 A B C D E F G E 2.2 kb *00301 A B C D E F G *00302 A B C D E F G F 0.23 kb *004 A B C D E F G G 0.23 kb

  11. Typing strategy () 3 1 2 4 5 6 7 8 9 ca 200 bp 34 bp 36 bp 327 bp 300 bp 294 bp 51 bp 53 bp 102 bp A 1.75 kb AT Cell Line B 0.26 kb C 0.28 kb D 2.2 kb E 2.2 kb F 0.23 kb G 0.23 kb KIR2DL1: *002, *003

  12. Typing strategy () 3 1 2 4 5 6 7 8 9 ca 200 bp 34 bp 36 bp 327 bp 300 bp 294 bp 51 bp 53 bp 102 bp A 1.75 kb AT Cell Line B 0.26 kb C 0.28 kb D 2.2 kb E 2.2 kb F 0.23 kb G 0.23 kb KIR2DL1: *002, *003

  13. Strategy Our current SSP strategy allows us to detect 36 different KIR alleles and permits us to call unequivocally 26 of them. KIR2DL1 KIR2DL3 KIR3DL1 KIR3DL2 *005 *006 *007 *008 *001 *002 *003 *004 *005 *006 *007 *008 *009 *010 *011 *012 *001 *002 *00301 *00302 *004 *005 *001 *002 *003 *004 *005 *006 *00101 *00102 *002 *003 *00401 *00402

  14. KIR3DSs KIR2DL1 KIR2DL3 KIR3DL1 KIR3DL2 1 2 3 4 5 Results • Profiled the KIR repertoire of 107 cell lines. • Same cell lines are completely characterised for HLA.

  15. Results Gene/allele combinations consistent with previously published data. 2DS 1 2 3 4 5 2DL1 2DL3 3DL1 3DL2 Haplotype PF97387 003,002 002/6,001 002/3/6/7/8, 005 002,010 05,20 SCHU 003,- 001,- 002/3/6/7/8, 005 002,001 12,19 HOR 002,- 002/6,- NEGATIVE 006,007 24,35 RML 003,- 001,- 002/3/6/7/8,- 002,007 12,33 Gene/allele combinations novel or previously not known. 2DS 1 2 3 4 5 2DL1 2DL3 3DL1 3DL2 Haplotype CB6B 004,- NEGATIVE NEGATIVE 007,010 NOVEL WT47 004,- NEGATIVE NEGATIVE 007,010 NOVEL

  16. Results Cell lines homozygous for KIRs. 2DS Hp 1 2 3 4 5 2DL1 2DL3 3DL1 3DL2 SPO010 003,- 001,- 005,- 001,- 19,- SA 003,- 001,- 002/3/6/7/8,- 002,- 12,- SPL 003,- 001,- 005,- 010,- 20,- EMJ 003,- 001,- 001,- 001,- 10,- DUCAF 003,- 001,- 005,- 001,- 9,- KT17 003,- 001,- 005,- 006,- - TUBO 002,- 002/6,- 005,- 001,- 9,- HID 003,- 001,- 002/3/6/7/8,- 002,- 12,-

  17. Conclusions • The PCR-SSP technique is suitable for high resolution routine typing. • This study constitutes the most comprehensive characterisation of the KIR gene repertoire in cell lines that have previously been thoroughly characterised for their HLA profile.

  18. Conclusions • Most cell lines in this panel have a Caucasoid ethnicity with representatives of Oriental, Amerindian, Hispanic and Black origin also present. However, • Studies on non- Caucasoid populations should allow us to further characterise the complexity of the KIR gene polymorphism and haplotypes.

  19. Acknowledgements Anthony Nolan Research Institute HLA Informatics Group Steven G.E. Marsh James Robinson J. Alejandro Madrigal Stanford University, California US Department of Structural Biology Peter Parham Heather Schilling Libby Guethlein

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