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P056 Simultaneous HLA class I and II sequencing by long read-independent circular consensus SMRT technology
Abstracts / Human Immunology 77 (2016) 40–156
P055
DEVELOPMENT OF IMMUNE RESPONSES TO TISSUE-RESTRICTED SELF-ANTIGENS IN SIMULTANEOUS KIDNEY-PANCREAS TRANSPLANT RECIPIENTS WITH ACUTE REJECTION Muthukumar Gunasekaran 1, Neeta Vachharajani 2, Joseph Gaut 3, Donna Phelan 4, Thin Thin Maw 5, Rowena Delos Santos 5, Surendra Shenoy 6, William Chapman 6, Jason Wellen 6, Thalachallour Mohanakumar 1. 1 St. Joseph’s Hospital & Medical Center, Norton Thoracic Institute, Phoenix, AZ, United States; 2Washington University School of Medicine, Surgery, St Louis, MO, United States; 3Washington University School of Medicine, Pathology, St. Louis, MO, United States; 4Barnes-Jewish Hospital, HLA Laboratory, St. Louis, MO, United States; 5Washington University School of Medicine, Medicine, St. Louis, MO, United States; 6 Washington University School of Medicine, Surgery, St. Louis, MO, United States. Aim: Simultaneous kidney-pancreas transplantation (SKP Tx) is a treatment option for patients with Type I diabetes mellitus and end-stage kidney disease, but acute and chronic rejection remain obstacles to long-term allograft function. This study aimed to determine the role of immune responses to mismatched donor human leukocyte antigen (HLA) and tissue-restricted self-antigens (kidney and pancreas) in allograft rejection after SKP Tx. Methods: Sera were collected from 39 SKP Tx recipients. Preexisting and post-transplant de novo antibodies specific for kidney-associated self-antigens (KSAgs) (i.e., collagen-IV, fibronectin) and pancreas-associated self-antigens (PSAgs) (i.e., insulin, islet cell, glutamic acid decarboxylase, pancreas-associated protein-1) were measured by ELISA. The sample was considered positive if values exceeded mean + 2 standard deviations of healthy normal subjects samples. Donor-specific antibodies to HLA class I and II were determined using single antigen beads using Luminex assay. Results: Acute rejection was confirmed on biopsy in 23/39 patients (59%). Of these, 6/23 (26.1%) had kidney and pancreas rejection, 8/23 (34.8%) had kidney-alone rejection and 9/23 (39.1%) had pancreas-alone rejection. De novo development of antibodies to KSAgs and PSAgs was significantly higher in combined kidney-pancreas rejection patients compared with stable SKP Tx patients (p < 0.05) and normal healthy subjects (p < 0.05). SKP Tx recipients with kidney-alone rejection had significantly increased antibodies against KSAgs compared to stable SKP Tx recipients (<0.05) and normal healthy subjects (<0.05), but showed no increase in antibodies against PSAgs. Similarly, SKP Tx recipients with pancreas-alone rejection had significant increase in antibodies against PSAgs compared to stable SKP Tx recipients (<0.05) and normal healthy subjects (<0.05), but showed no increase in antibody development against KSAgs. Conclusions: Development of antibodies to KSAgs and PSAgs is associated with combined kidney-pancreas rejection after SKP Tx. Kidney-alone rejection showed increased antibodies to KSAgs, and pancreas-alone rejection showed increased antibodies to PSAgs. This suggests that immune responses to tissue-specific antigens play a critical role in allograft rejection.
P056
SIMULTANEOUS HLA CLASS I AND II SEQUENCING BY LONG READ-INDEPENDENT CIRCULAR CONSENSUS SMRT TECHNOLOGY Chee Loong Saw 1, Haig Djambazian 2, Ioannis Ragoussis 2. 1McGill University Health Centre, HLA Lab, Montreal, QC, Canada; 2McGill University Genome Quebec and Innovation Centre, Montreal, QC, Canada. Aim: PacBio SMRT sequencing technology is considered new approach to HLA typing. HLA class I genotyping has been reported using PacBio SMRT technology but little has been published to investigate the feasibility and the performance of the SMRT sequencing technology for both HLA class I and class II genes. Methods: We report here genotyping of 16 DNA samples, 5 loci (HLA-A, B, C, DRB1 and DQB1). The amplicons corresponding to the different genes were first pooled for each sample. The pooled genes from different samples were then pooled again after barcoded SMRTbell PacBio library construction. After sequencing an independent molecule consensus approach was applied. Whole gene amplicons were generated for both HLA class I and class II genes using Omixon Holotype protocol and were sequenced by PacBio SMRT sequencing as well as using Illumina. Bioinformatic algorithm and analysis were important to result in quality consensus sequences and allele assignment. Results: PacBio consensus sequences using at least 75 independent molecules were obtained for each locus and each sample. Balance between class I and class II genes of different length is important as imbalanced multiplexing may affect the typing results. We have shown for the first time that concurrent HLA typing of both class I and class II genes using the same sequencing SMRTcell is possible. Conclusion: Using in house allele assignment method, our PacBio analysis was in perfect agreement with the reference samples sequenced by Illumina (Omixon Twin protocol) to the 3rd field. The in house allele assignment method has the potential to resolve up to the 4th field of HLA genotyping.
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P055
DEVELOPMENT OF IMMUNE RESPONSES TO TISSUE-RESTRICTED SELF-ANTIGENS IN SIMULTANEOUS KIDNEY-PANCREAS TRANSPLANT RECIPIENTS WITH ACUTE REJECTION Muthukumar Gunasekaran 1, Neeta Vachharajani 2, Joseph Gaut 3, Donna Phelan 4, Thin Thin Maw 5, Rowena Delos Santos 5, Surendra Shenoy 6, William Chapman 6, Jason Wellen 6, Thalachallour Mohanakumar 1. 1 St. Joseph’s Hospital & Medical Center, Norton Thoracic Institute, Phoenix, AZ, United States; 2Washington University School of Medicine, Surgery, St Louis, MO, United States; 3Washington University School of Medicine, Pathology, St. Louis, MO, United States; 4Barnes-Jewish Hospital, HLA Laboratory, St. Louis, MO, United States; 5Washington University School of Medicine, Medicine, St. Louis, MO, United States; 6 Washington University School of Medicine, Surgery, St. Louis, MO, United States. Aim: Simultaneous kidney-pancreas transplantation (SKP Tx) is a treatment option for patients with Type I diabetes mellitus and end-stage kidney disease, but acute and chronic rejection remain obstacles to long-term allograft function. This study aimed to determine the role of immune responses to mismatched donor human leukocyte antigen (HLA) and tissue-restricted self-antigens (kidney and pancreas) in allograft rejection after SKP Tx. Methods: Sera were collected from 39 SKP Tx recipients. Preexisting and post-transplant de novo antibodies specific for kidney-associated self-antigens (KSAgs) (i.e., collagen-IV, fibronectin) and pancreas-associated self-antigens (PSAgs) (i.e., insulin, islet cell, glutamic acid decarboxylase, pancreas-associated protein-1) were measured by ELISA. The sample was considered positive if values exceeded mean + 2 standard deviations of healthy normal subjects samples. Donor-specific antibodies to HLA class I and II were determined using single antigen beads using Luminex assay. Results: Acute rejection was confirmed on biopsy in 23/39 patients (59%). Of these, 6/23 (26.1%) had kidney and pancreas rejection, 8/23 (34.8%) had kidney-alone rejection and 9/23 (39.1%) had pancreas-alone rejection. De novo development of antibodies to KSAgs and PSAgs was significantly higher in combined kidney-pancreas rejection patients compared with stable SKP Tx patients (p < 0.05) and normal healthy subjects (p < 0.05). SKP Tx recipients with kidney-alone rejection had significantly increased antibodies against KSAgs compared to stable SKP Tx recipients (<0.05) and normal healthy subjects (<0.05), but showed no increase in antibodies against PSAgs. Similarly, SKP Tx recipients with pancreas-alone rejection had significant increase in antibodies against PSAgs compared to stable SKP Tx recipients (<0.05) and normal healthy subjects (<0.05), but showed no increase in antibody development against KSAgs. Conclusions: Development of antibodies to KSAgs and PSAgs is associated with combined kidney-pancreas rejection after SKP Tx. Kidney-alone rejection showed increased antibodies to KSAgs, and pancreas-alone rejection showed increased antibodies to PSAgs. This suggests that immune responses to tissue-specific antigens play a critical role in allograft rejection.
P056
SIMULTANEOUS HLA CLASS I AND II SEQUENCING BY LONG READ-INDEPENDENT CIRCULAR CONSENSUS SMRT TECHNOLOGY Chee Loong Saw 1, Haig Djambazian 2, Ioannis Ragoussis 2. 1McGill University Health Centre, HLA Lab, Montreal, QC, Canada; 2McGill University Genome Quebec and Innovation Centre, Montreal, QC, Canada. Aim: PacBio SMRT sequencing technology is considered new approach to HLA typing. HLA class I genotyping has been reported using PacBio SMRT technology but little has been published to investigate the feasibility and the performance of the SMRT sequencing technology for both HLA class I and class II genes. Methods: We report here genotyping of 16 DNA samples, 5 loci (HLA-A, B, C, DRB1 and DQB1). The amplicons corresponding to the different genes were first pooled for each sample. The pooled genes from different samples were then pooled again after barcoded SMRTbell PacBio library construction. After sequencing an independent molecule consensus approach was applied. Whole gene amplicons were generated for both HLA class I and class II genes using Omixon Holotype protocol and were sequenced by PacBio SMRT sequencing as well as using Illumina. Bioinformatic algorithm and analysis were important to result in quality consensus sequences and allele assignment. Results: PacBio consensus sequences using at least 75 independent molecules were obtained for each locus and each sample. Balance between class I and class II genes of different length is important as imbalanced multiplexing may affect the typing results. We have shown for the first time that concurrent HLA typing of both class I and class II genes using the same sequencing SMRTcell is possible. Conclusion: Using in house allele assignment method, our PacBio analysis was in perfect agreement with the reference samples sequenced by Illumina (Omixon Twin protocol) to the 3rd field. The in house allele assignment method has the potential to resolve up to the 4th field of HLA genotyping.
79