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52-OR: Targeted Enrichment for Complete Characterization of 1.4Mb of the MHC With Next Generation Sequencing
S144
Abstracts
51-OR
HLA CLASS I INDUCED SIGNAL TRANSDUCTION AND CELL PROLIFERATION IS DEPENDENT UPON MOLECULAR ASSOCIATION AND PHOSPHORYLATION OF INTEGRIN  4. Xiaohai Zhang, Elaine F. Reed. UCLA Immunogenetics Center, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA. Aim: Patients developing anti-donor HLA antibodies following transplantation are at increased risk of transplant vasculopathy and graft loss. Several studies suggest that the signaling events that occur in endothelial cells during interactions with MHC-I antibodies can contribute to the process of transplant vasculopathy. Previous studies have demonstrated that crosslinking of HLA-I molecules with antibodies activates intracellular signaling cascades and stimulates cell proliferation through the formation of a molecular complex with integrin 4. This finding led us to investigate the mechanisms underlying how integrin 4 transduces proliferation signals upon anti-HLA antibody ligation of HLA-I molecules. Methods: Primary cultures of human aortic endothelial cells were stimulated with F(ab’)2 fragments of the HLA-I monoclonal antibody W6/32 or control IgG. Cell lysates were immunoprecipitated with integrin 4 antibody and the immunecomplexes were assessed for tyrosine phosphorylation of integrin 4 and activation of SHC and ERK by Western blot. Results: Treatment of endothelial cells with HLA-I antibodies stimulated molecular association with integrin 4 and cell proliferation. siRNA knockdown of integrin 4 blocked anti-HLA-I mediated cell proliferation. Ligation of HLA-I molecules with anti-HLA antibodies triggered phosphorylation of integrin 4 on tyrosine residues and increased complex formation between integrin 4 and an adapter protein SHC. Conclusions: Crosslinking of HLA-I molecules stimulates signal transduction by associating with integrin 4. HLA-I ligation stimulates phosphorylation of integrin 4 which in turn recruits the adaptor protein SHC and activates ERK signaling. Elucidation of the HLA-I signaling pathway has the potential to identify novel therapeutic strategies to prevent transplant vasculopathy.
52-OR
TARGETED ENRICHMENT FOR COMPLETE CHARACTERIZATION OF 1.4Mb OF THE MHC WITH NEXT GENERATION SEQUENCING. Deborah Ferriola,1,2 Katarzyna Mackiewicz,1 Curt Lind,1 Xiaowu Gai,3 Monica D’Arcy,3 Johannes Dapprich,2 Dimitri Monos.1,4 1Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2Generation Biotech, Lawrenceville, NJ, USA; 3 Bioinformatics Core Facility, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 4Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA. Aim: Comprehensive, detailed characterization of the MHC region, which harbors a large number of immunologically relevant genes, is needed because of its importance in transplantation and many diseases. Our objective was to evaluate the use of an enrichment technology and Next-Generation Sequencing (NGS) to fully sequence the MHC. Methods: We enriched and sequenced a contiguous 1.4Mb region including HLA-C through DQB1. The homozygous PGF cell line was used, which is the established reference sequence for the MHC of the human genome. Region Specific Extraction, an enrichment technology was used by first hybridizing oligos to the region of interest and then enzymatically extending the oligos with biotinylated nucleotides. The DNA was then captured and sequenced on a single lane of an Illumina platform, producing 72 base paired-end reads. The reads were aligned with Burrows-Wheeler Aligner and differences between the experimentally determined sequence and the reference were identified with Sequence Alignment/Map tools. Results: A total of 170,730,300 bases were mapped to 738,945 unique bases in the target region. 99.8% of the targeted region of the MHC was covered with a mean depth of 68X. 92 % of the target region had read depth of at least 10. A comparison between the consensus sequence and the reference revealed 32 discrepancies. Sanger sequencing revealed that 3 were in agreement with NGS, 23 were in agreement with the reference and 6 were heterozygotes, differing from both reference and NGS. Overall, 99.996% of bases in the target region were correctly assigned with NGS. Conclusions: These new technologies allow detailed characterization of the MHC with significantly less cost and time, opening new opportunities for the comprehensive evaluation of this region. This in turn may identify the inter-relationship between genes that makes this genomic region so unique and important to immune response. Dapprich: Generation Biotech: Employee.
Abstracts
51-OR
HLA CLASS I INDUCED SIGNAL TRANSDUCTION AND CELL PROLIFERATION IS DEPENDENT UPON MOLECULAR ASSOCIATION AND PHOSPHORYLATION OF INTEGRIN  4. Xiaohai Zhang, Elaine F. Reed. UCLA Immunogenetics Center, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA. Aim: Patients developing anti-donor HLA antibodies following transplantation are at increased risk of transplant vasculopathy and graft loss. Several studies suggest that the signaling events that occur in endothelial cells during interactions with MHC-I antibodies can contribute to the process of transplant vasculopathy. Previous studies have demonstrated that crosslinking of HLA-I molecules with antibodies activates intracellular signaling cascades and stimulates cell proliferation through the formation of a molecular complex with integrin 4. This finding led us to investigate the mechanisms underlying how integrin 4 transduces proliferation signals upon anti-HLA antibody ligation of HLA-I molecules. Methods: Primary cultures of human aortic endothelial cells were stimulated with F(ab’)2 fragments of the HLA-I monoclonal antibody W6/32 or control IgG. Cell lysates were immunoprecipitated with integrin 4 antibody and the immunecomplexes were assessed for tyrosine phosphorylation of integrin 4 and activation of SHC and ERK by Western blot. Results: Treatment of endothelial cells with HLA-I antibodies stimulated molecular association with integrin 4 and cell proliferation. siRNA knockdown of integrin 4 blocked anti-HLA-I mediated cell proliferation. Ligation of HLA-I molecules with anti-HLA antibodies triggered phosphorylation of integrin 4 on tyrosine residues and increased complex formation between integrin 4 and an adapter protein SHC. Conclusions: Crosslinking of HLA-I molecules stimulates signal transduction by associating with integrin 4. HLA-I ligation stimulates phosphorylation of integrin 4 which in turn recruits the adaptor protein SHC and activates ERK signaling. Elucidation of the HLA-I signaling pathway has the potential to identify novel therapeutic strategies to prevent transplant vasculopathy.
52-OR
TARGETED ENRICHMENT FOR COMPLETE CHARACTERIZATION OF 1.4Mb OF THE MHC WITH NEXT GENERATION SEQUENCING. Deborah Ferriola,1,2 Katarzyna Mackiewicz,1 Curt Lind,1 Xiaowu Gai,3 Monica D’Arcy,3 Johannes Dapprich,2 Dimitri Monos.1,4 1Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2Generation Biotech, Lawrenceville, NJ, USA; 3 Bioinformatics Core Facility, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 4Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA. Aim: Comprehensive, detailed characterization of the MHC region, which harbors a large number of immunologically relevant genes, is needed because of its importance in transplantation and many diseases. Our objective was to evaluate the use of an enrichment technology and Next-Generation Sequencing (NGS) to fully sequence the MHC. Methods: We enriched and sequenced a contiguous 1.4Mb region including HLA-C through DQB1. The homozygous PGF cell line was used, which is the established reference sequence for the MHC of the human genome. Region Specific Extraction, an enrichment technology was used by first hybridizing oligos to the region of interest and then enzymatically extending the oligos with biotinylated nucleotides. The DNA was then captured and sequenced on a single lane of an Illumina platform, producing 72 base paired-end reads. The reads were aligned with Burrows-Wheeler Aligner and differences between the experimentally determined sequence and the reference were identified with Sequence Alignment/Map tools. Results: A total of 170,730,300 bases were mapped to 738,945 unique bases in the target region. 99.8% of the targeted region of the MHC was covered with a mean depth of 68X. 92 % of the target region had read depth of at least 10. A comparison between the consensus sequence and the reference revealed 32 discrepancies. Sanger sequencing revealed that 3 were in agreement with NGS, 23 were in agreement with the reference and 6 were heterozygotes, differing from both reference and NGS. Overall, 99.996% of bases in the target region were correctly assigned with NGS. Conclusions: These new technologies allow detailed characterization of the MHC with significantly less cost and time, opening new opportunities for the comprehensive evaluation of this region. This in turn may identify the inter-relationship between genes that makes this genomic region so unique and important to immune response. Dapprich: Generation Biotech: Employee.