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33-OR: Update on conserved extended haplotypes and possible identification of the ancestral MHC haplotype
Abstracts
S115
Thursday, October 30, 2008 4:00 PM - 5:30 PM Immunogenetic Polymorphisms and Disease ASHI Scholar 33-OR
UPDATE ON CONSERVED EXTENDED HAPLOTYPES AND POSSIBLE IDENTIFICATION OF THE ANCESTRAL MHC HAPLOTYPE. Esma Ucisik-Akkaya, Brittany A. Morrison, Charronne Davis, Thuy Do, M. Tevfik Dorak. Genomic Immunoepidemiology Lab, HUMIGEN LLC, The Institute for Genetic Immunology, Hamilton, NJ, USA. Aim: The HLA complex is the most gene-dense region of the genome. With the completion of the MHC Haplotyping Project, interest has focused on non-HLA genes in HLA-disease associations. Following the complete characterization of all conserved extended haplotypes (CEH) at conventional HLA complex loci, we aimed to examine 82 IHWG cell lines representing 36 of the CEHs, at genes encoding proto-oncogenes and proteins involved in transcriptional machinery, DNA repair, apoptosis and NK cell activity (ZNRD1, IER3, DDR1, TCF19, POU5F1, MICA, NCR3, BAT3, CLIC1, MSH5, HSPA1L/A/B, SKIV2L, CYP21A2, PBX2, NOTCH4, C6orf10, BTNL2, BRD2, RXRB, DAXX) as well as at HLA-C, DRA and DQA1 loci. Methods: 54 SNPs were genotyped. Mutations causing complement deficiencies were also typed. SNP selection was guided by disease associations and HapMap data. The IER3 gene, the C4d fragment and HLADQA1 3’UTR were sequenced in 31 cell lines. Most polymorphisms were also genotyped in a sample from a European population (n⫽389). Results: We were able to identify or confirm haplotype-specific markers in MSH5 and CYP21A2 (CEH65.1), CLIC1 and MSH5 (CEH8.1), NOTCH4 (CEH8.1 ⫹ CEH47.1) and DRA (DRB1*1501). Only CEH18.2 had the 28bp deletion in exon 6 of C2 causing C2Q0, and a number of cell lines contained C4AQ0 and C4BQ0 mutations. The cell line SA representing CEH7.2 (B*0702-DRB1*0101) contained the ancestral alleles (common alleles in the HapMap African population) in 33 of 37 SNPs centromeric to HLA-C suggesting it may be the ancestral MHC haplotype. Conclusions: This study provided an extensive catalog of HLA complex polymorphisms, their relationships with each other and with HLA alleles and haplotypes.
34-OR
CYTOTOXIC T LYMPHOCYTES OF BREAST CANCER SURVIVORS RECOGNIZE NOVEL EPITOPES PRESENTED BY THE HLA-A*0201. Oriana Hawkins,1 Rodney VanGundy,2 Annette Eckerd,1 Wilfried Bardet,1 Rico Buchli,2 Jon Weidanz,3 William Hildebrand.1 1University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Pure Protein, LLC, Oklahoma City, OK; 3Texas Tech University Health Sciences Center, Abilene. Aim: Detection and destruction of cancerous cells are mediated by the human leukocyte antigen (HLA) class I molecules which present peptides of intracellular origin on the cell surface. The aim of this study was identification of novel, tumor-specific epitopes to facilitate the development of immunotherapeutics for breast cancer. Methods: Secreted class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201.sHLA-A*0201 was expressed in breast cancer cell lines MCF-7, MDAMB-231, and BT-20 and the non-tumorigenic line MCF10A. Peptides eluted from purified sHLA were analyzed by mass spectroscopy. Cellular recognition of identified peptides was tested by tetramer stain, ELISPOT, intracellular cytokine stain, and CD107 cytotoxicity assays. Results: Comparative analysis of HLA-A*0201 peptides revealed one epitope each from Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC), Kinetochore Associated 2 (KNTC2), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6) specifically presented by the tumorigenic cell lines. Cellular recognition of these 5 peptides by the circulating CD8⫹ cells of 3 cancer survivors was demonstrated. Conclusions: We have demonstrated the ability to detect class I HLA presented peptide ligands specific to cancerous cells and verified their immunogenicity in breast cancer patients. The identification and characterization of immunogenic peptides presented specifically by the class I HLA of breast cancer cells may provide novel targets for the development of immune diagnostic tools and therapeutics.
S115
Thursday, October 30, 2008 4:00 PM - 5:30 PM Immunogenetic Polymorphisms and Disease ASHI Scholar 33-OR
UPDATE ON CONSERVED EXTENDED HAPLOTYPES AND POSSIBLE IDENTIFICATION OF THE ANCESTRAL MHC HAPLOTYPE. Esma Ucisik-Akkaya, Brittany A. Morrison, Charronne Davis, Thuy Do, M. Tevfik Dorak. Genomic Immunoepidemiology Lab, HUMIGEN LLC, The Institute for Genetic Immunology, Hamilton, NJ, USA. Aim: The HLA complex is the most gene-dense region of the genome. With the completion of the MHC Haplotyping Project, interest has focused on non-HLA genes in HLA-disease associations. Following the complete characterization of all conserved extended haplotypes (CEH) at conventional HLA complex loci, we aimed to examine 82 IHWG cell lines representing 36 of the CEHs, at genes encoding proto-oncogenes and proteins involved in transcriptional machinery, DNA repair, apoptosis and NK cell activity (ZNRD1, IER3, DDR1, TCF19, POU5F1, MICA, NCR3, BAT3, CLIC1, MSH5, HSPA1L/A/B, SKIV2L, CYP21A2, PBX2, NOTCH4, C6orf10, BTNL2, BRD2, RXRB, DAXX) as well as at HLA-C, DRA and DQA1 loci. Methods: 54 SNPs were genotyped. Mutations causing complement deficiencies were also typed. SNP selection was guided by disease associations and HapMap data. The IER3 gene, the C4d fragment and HLADQA1 3’UTR were sequenced in 31 cell lines. Most polymorphisms were also genotyped in a sample from a European population (n⫽389). Results: We were able to identify or confirm haplotype-specific markers in MSH5 and CYP21A2 (CEH65.1), CLIC1 and MSH5 (CEH8.1), NOTCH4 (CEH8.1 ⫹ CEH47.1) and DRA (DRB1*1501). Only CEH18.2 had the 28bp deletion in exon 6 of C2 causing C2Q0, and a number of cell lines contained C4AQ0 and C4BQ0 mutations. The cell line SA representing CEH7.2 (B*0702-DRB1*0101) contained the ancestral alleles (common alleles in the HapMap African population) in 33 of 37 SNPs centromeric to HLA-C suggesting it may be the ancestral MHC haplotype. Conclusions: This study provided an extensive catalog of HLA complex polymorphisms, their relationships with each other and with HLA alleles and haplotypes.
34-OR
CYTOTOXIC T LYMPHOCYTES OF BREAST CANCER SURVIVORS RECOGNIZE NOVEL EPITOPES PRESENTED BY THE HLA-A*0201. Oriana Hawkins,1 Rodney VanGundy,2 Annette Eckerd,1 Wilfried Bardet,1 Rico Buchli,2 Jon Weidanz,3 William Hildebrand.1 1University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Pure Protein, LLC, Oklahoma City, OK; 3Texas Tech University Health Sciences Center, Abilene. Aim: Detection and destruction of cancerous cells are mediated by the human leukocyte antigen (HLA) class I molecules which present peptides of intracellular origin on the cell surface. The aim of this study was identification of novel, tumor-specific epitopes to facilitate the development of immunotherapeutics for breast cancer. Methods: Secreted class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201.sHLA-A*0201 was expressed in breast cancer cell lines MCF-7, MDAMB-231, and BT-20 and the non-tumorigenic line MCF10A. Peptides eluted from purified sHLA were analyzed by mass spectroscopy. Cellular recognition of identified peptides was tested by tetramer stain, ELISPOT, intracellular cytokine stain, and CD107 cytotoxicity assays. Results: Comparative analysis of HLA-A*0201 peptides revealed one epitope each from Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC), Kinetochore Associated 2 (KNTC2), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6) specifically presented by the tumorigenic cell lines. Cellular recognition of these 5 peptides by the circulating CD8⫹ cells of 3 cancer survivors was demonstrated. Conclusions: We have demonstrated the ability to detect class I HLA presented peptide ligands specific to cancerous cells and verified their immunogenicity in breast cancer patients. The identification and characterization of immunogenic peptides presented specifically by the class I HLA of breast cancer cells may provide novel targets for the development of immune diagnostic tools and therapeutics.