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P059 Using NGS in solid organ transplantation to resolve anti-HLA antibodies to self antigens
Abstracts / Human Immunology 78 (2017) 51–254
P058
RENAL REPLACEMENT THERAPY: VARIABILITY IN HLA-G EXPRESSION AS AN OUTCOME INDICATOR Uma Kanga a, Shweta Tyagi a, Manish Mourya a, Sanjay K. Agarwal b, Virinder K. Bansal c. aDept of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, India; bDept of Nephrology, All India Institute of Medical Sciences, New Delhi, India; cDept of Surgical Disciplines, All India Institute of Medical Sciences, New Delhi, India. Aim: The non-classical HLA class I molecule, HLA-G has unique features like restricted expression, limited polymorphism, and alternative splicing led isoforms. HLA-G plays crucial immunomodulatory role in allogenic situations like pregnancy and allo-transplantation. This molecule creates a tolerogenic microenvironment through interaction with receptors ILT-2/ILT-4/KIR2DL4 on immune cells. The posttranscriptional regulation and mRNA stability is altered due to 3’UTR region polymorphism impacting miRNA interactions. To investigate the clinical relevance of HLA-G in patients undergoing renal transplantation. For this study, 50 healthy controls and 39 recipient-donor pairs were enrolled of which, 11 recipients experienced graft rejection episodes. Methods: HLA-G exon8-14bp INDEL polymorphism was evaluated by PCR-SSP and sequencing. The 14bp genotype (ins/del, del/del and ins/ins) distribution was evaluated. Serum soluble HLA-G levels (pre and serial post transplant days) were measured by ELISA. Results: For analysis, recipients were segregated into rejection (R) and non-rejection (NR) or well functioning graft (WFG) groups. Higher percentage of NR recipients carried ins/ins genotype as compared to the R group (32.1% vs 20% respectively). In healthy controls, the median sHLA-G level was 31.7 U/ml and group with ins/ins genotype had highest levels. In transplant recipients, sHLA-G levels were significantly higher than controls. Median sHLA-G levels were lower (105.4 U/ml) among rejection group, in comparison to WFG (260.4 U/ ml). Throughout the post-transplant period, sHLA-G levels were better maintained in the WFG group. In the rejection group, the sHLA-G levels improved only after anti-rejection therapy, however it remained lower than WFG. Conclusions: The HLA-G 3’UTR genotyping and serial sHLA-G level monitoring are likely to predict adverse event like rejection. HLA-G expression on alloactivated recipient cells is underway to better understand functional relevance.
P059
USING NGS IN SOLID ORGAN TRANSPLANTATION TO RESOLVE ANTI-HLA ANTIBODIES TO SELF ANTIGENS Peter Jindra, Thuydung Tu, Holly Dane, Lauren E. Clark, Anna-Marie Young, Stacie Gray, Ronald H. Kerman. Baylor College of Medicine, Houston, TX, United States. Next generation sequencing (NGS) is an innovative technology that is redefining the landscape of human molecular genetic testing. NGS technology has entered the clinical lab space with validated machines and reagents to streamline the process of HLA typing. This case highlights the utility of HLA typing by NGS for a solid organ laboratory. A 45 year old African American male presented to our center for new patient evaluation. The patient had received a previous kidney transplant in June 2013 at another institution. We performed HLA typing by SSO method and screened the patient for anti-HLA antibodies by Flow PRA (FPRA) and reflexed to single antigen bead identification using One Lambda reagents. The patient was HLA typed as DRB1⁄ 10:XX, DRB1⁄ 14:04; DQA1⁄ 01:MV (01/04/05), DQA1⁄ 01:SXYS (01/04/05/12) with DQB1⁄ 05:XX, DQB1⁄ 05:XX by SSO. We confirmed the patient’s HLA typing with the previous transplant center. Due to previous sensitizing events, the patient’s FPRA was 99% class I and 100% class II. Examination of the class II single antigen bead profile revealed strong DR14 and DQ5 antibodies. (DRB1⁄ 14:54, MFI:10,640; DRB1⁄ 14:01, MFI:10299; DRB1⁄ 14:02, MFI: 9379) (DQB1⁄ 01:02-DQB1⁄ 05:02, MFI: 15707) (DQB1⁄ 01:01-DQB1⁄ 05:01, MFI: 7141). For further resolution of the patient’s DR and DQ typings we performed NGS high resolution typing using Illumina TruSight v2 sequencing panel. The patient was typed (DRB1⁄ 10:01:01, DRB1⁄ 14:04:01) (DQA1⁄ 01:04:02, DQA1⁄ 01:05:01) with (DQB1⁄ 05:01:01, DQB1⁄ 05:03:01). DQA1⁄ 01:04:02 was not common and well defined, differing from DQA1⁄ 01:04:01 at position 5433 A?C in exon 3 leading to a synonymous base substitution. The DR and DQA-DQB pairs of the patient found through sequencing differ from the DR14 and DQ5 single antigen beads used for calling unacceptables. The patient was not generating anti-HLA antibodies to self, but rather common DR DQA-DQB allele variants. We confirmed there was no self-reactivity by auto crossmatch which was negative by Flow Cytometry and Cytotoxicity. Given that deceased donor typing is performed at lower resolution and single antigen beads are unable to cover all HLA alleles, a combination of virtual XM and real time crossmatches will need to be performed to safely get this patient re-transplanted.
97
P058
RENAL REPLACEMENT THERAPY: VARIABILITY IN HLA-G EXPRESSION AS AN OUTCOME INDICATOR Uma Kanga a, Shweta Tyagi a, Manish Mourya a, Sanjay K. Agarwal b, Virinder K. Bansal c. aDept of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, India; bDept of Nephrology, All India Institute of Medical Sciences, New Delhi, India; cDept of Surgical Disciplines, All India Institute of Medical Sciences, New Delhi, India. Aim: The non-classical HLA class I molecule, HLA-G has unique features like restricted expression, limited polymorphism, and alternative splicing led isoforms. HLA-G plays crucial immunomodulatory role in allogenic situations like pregnancy and allo-transplantation. This molecule creates a tolerogenic microenvironment through interaction with receptors ILT-2/ILT-4/KIR2DL4 on immune cells. The posttranscriptional regulation and mRNA stability is altered due to 3’UTR region polymorphism impacting miRNA interactions. To investigate the clinical relevance of HLA-G in patients undergoing renal transplantation. For this study, 50 healthy controls and 39 recipient-donor pairs were enrolled of which, 11 recipients experienced graft rejection episodes. Methods: HLA-G exon8-14bp INDEL polymorphism was evaluated by PCR-SSP and sequencing. The 14bp genotype (ins/del, del/del and ins/ins) distribution was evaluated. Serum soluble HLA-G levels (pre and serial post transplant days) were measured by ELISA. Results: For analysis, recipients were segregated into rejection (R) and non-rejection (NR) or well functioning graft (WFG) groups. Higher percentage of NR recipients carried ins/ins genotype as compared to the R group (32.1% vs 20% respectively). In healthy controls, the median sHLA-G level was 31.7 U/ml and group with ins/ins genotype had highest levels. In transplant recipients, sHLA-G levels were significantly higher than controls. Median sHLA-G levels were lower (105.4 U/ml) among rejection group, in comparison to WFG (260.4 U/ ml). Throughout the post-transplant period, sHLA-G levels were better maintained in the WFG group. In the rejection group, the sHLA-G levels improved only after anti-rejection therapy, however it remained lower than WFG. Conclusions: The HLA-G 3’UTR genotyping and serial sHLA-G level monitoring are likely to predict adverse event like rejection. HLA-G expression on alloactivated recipient cells is underway to better understand functional relevance.
P059
USING NGS IN SOLID ORGAN TRANSPLANTATION TO RESOLVE ANTI-HLA ANTIBODIES TO SELF ANTIGENS Peter Jindra, Thuydung Tu, Holly Dane, Lauren E. Clark, Anna-Marie Young, Stacie Gray, Ronald H. Kerman. Baylor College of Medicine, Houston, TX, United States. Next generation sequencing (NGS) is an innovative technology that is redefining the landscape of human molecular genetic testing. NGS technology has entered the clinical lab space with validated machines and reagents to streamline the process of HLA typing. This case highlights the utility of HLA typing by NGS for a solid organ laboratory. A 45 year old African American male presented to our center for new patient evaluation. The patient had received a previous kidney transplant in June 2013 at another institution. We performed HLA typing by SSO method and screened the patient for anti-HLA antibodies by Flow PRA (FPRA) and reflexed to single antigen bead identification using One Lambda reagents. The patient was HLA typed as DRB1⁄ 10:XX, DRB1⁄ 14:04; DQA1⁄ 01:MV (01/04/05), DQA1⁄ 01:SXYS (01/04/05/12) with DQB1⁄ 05:XX, DQB1⁄ 05:XX by SSO. We confirmed the patient’s HLA typing with the previous transplant center. Due to previous sensitizing events, the patient’s FPRA was 99% class I and 100% class II. Examination of the class II single antigen bead profile revealed strong DR14 and DQ5 antibodies. (DRB1⁄ 14:54, MFI:10,640; DRB1⁄ 14:01, MFI:10299; DRB1⁄ 14:02, MFI: 9379) (DQB1⁄ 01:02-DQB1⁄ 05:02, MFI: 15707) (DQB1⁄ 01:01-DQB1⁄ 05:01, MFI: 7141). For further resolution of the patient’s DR and DQ typings we performed NGS high resolution typing using Illumina TruSight v2 sequencing panel. The patient was typed (DRB1⁄ 10:01:01, DRB1⁄ 14:04:01) (DQA1⁄ 01:04:02, DQA1⁄ 01:05:01) with (DQB1⁄ 05:01:01, DQB1⁄ 05:03:01). DQA1⁄ 01:04:02 was not common and well defined, differing from DQA1⁄ 01:04:01 at position 5433 A?C in exon 3 leading to a synonymous base substitution. The DR and DQA-DQB pairs of the patient found through sequencing differ from the DR14 and DQ5 single antigen beads used for calling unacceptables. The patient was not generating anti-HLA antibodies to self, but rather common DR DQA-DQB allele variants. We confirmed there was no self-reactivity by auto crossmatch which was negative by Flow Cytometry and Cytotoxicity. Given that deceased donor typing is performed at lower resolution and single antigen beads are unable to cover all HLA alleles, a combination of virtual XM and real time crossmatches will need to be performed to safely get this patient re-transplanted.
97