AbstractBackgroundWe investigated in vitro whether HLA-highly-sensitized patients with end-stage renal disease (ESRD) will be disadvantaged immunologically after a genetically-engineered pig kidney transplant.MethodsBlood was drawn from patients with a cPRA 99-100% (Gp1, n=10) or cPRA 0% (Gp2, n=12), and from healthy volunteers (Gp3, n=10). Serum IgM and IgG binding was measured (i) to Gal and Neu5Gc glycans by ELISA, and (ii) to pRBC, pAEC, and pPBMC from GTKO/CD46 and GTKO/CD46/CMAHKO pigs by flow cytometry. (iii) T and B cell phenotypes were determined by flow cytometry, and (iv) proliferation of T and B cells CFSE-MLR.Results(i) By ELISA, there was no difference in IgM or IgG binding to Gal or Neu5Gc between Gps1 and 2, but binding was significantly reduced in both groups compared to Gp3. (ii) IgM and IgG binding in Gps1 and 2 was also significantly lower to GTKO/CD46 pig cells than in healthy controls, but there were no differences between the 3 groups in binding to GTKO/CD46/CMAHKO cells. (iii and iv) Gp1 patients had more memory T cells than Gp2, but there was no difference in T or B cell proliferation when stimulated by any pig cells. The proliferative responses in all 3 groups were weakest when stimulated by GTKO/CD46/CMAHKO pPBMC.Conclusions(i) ESRD was associated with low anti-pig antibody levels. (ii) Xenoreactivity decreased with increased genetic engineering of pig cells. (iii) High cPRA status had no significant effect on antibody binding or T and B cell response. Background We investigated in vitro whether HLA-highly-sensitized patients with end-stage renal disease (ESRD) will be disadvantaged immunologically after a genetically-engineered pig kidney transplant. Methods Blood was drawn from patients with a cPRA 99-100% (Gp1, n=10) or cPRA 0% (Gp2, n=12), and from healthy volunteers (Gp3, n=10). Serum IgM and IgG binding was measured (i) to Gal and Neu5Gc glycans by ELISA, and (ii) to pRBC, pAEC, and pPBMC from GTKO/CD46 and GTKO/CD46/CMAHKO pigs by flow cytometry. (iii) T and B cell phenotypes were determined by flow cytometry, and (iv) proliferation of T and B cells CFSE-MLR. Results (i) By ELISA, there was no difference in IgM or IgG binding to Gal or Neu5Gc between Gps1 and 2, but binding was significantly reduced in both groups compared to Gp3. (ii) IgM and IgG binding in Gps1 and 2 was also significantly lower to GTKO/CD46 pig cells than in healthy controls, but there were no differences between the 3 groups in binding to GTKO/CD46/CMAHKO cells. (iii and iv) Gp1 patients had more memory T cells than Gp2, but there was no difference in T or B cell proliferation when stimulated by any pig cells. The proliferative responses in all 3 groups were weakest when stimulated by GTKO/CD46/CMAHKO pPBMC. Conclusions (i) ESRD was associated with low anti-pig antibody levels. (ii) Xenoreactivity decreased with increased genetic engineering of pig cells. (iii) High cPRA status had no significant effect on antibody binding or T and B cell response. Address Correspondence to: Martin Wijkstrom, MD, Thomas E Starzl Transplantation Institute, Montefiore University Hospital, MUH 756.2, University of Pittsburgh Medical Center, 200 Lothrop Street, Pittsburgh, PA 15261, USA. Email: wijkstrommn@upmc.edu Author Contributions: All authors participated in revising and approving the manuscript. ZZ, HQ, HH, MW, DKCC designed and initiated this study. ZZ, CL, IH, HH participated in laboratory assay. Data were collected and analyzed by ZZ, CL, HH, CM, MM, AZ, ME, MW, DKCC. The manuscript was prepared by ZZ, HH, ME, CM, AZ, MW, DKCC. Genetically-engineered pig cells were provided by DA. Conflicts of interest: David Ayares is an employee of Revivicor, Inc. No other author has a conflict of interest. Funding: Zhongqiang Zhang was supported by the China Scholarship Council (File No. 201506370112). Work on xenotransplantation in the Thomas E. Starzl Transplantation Institute of the University of Pittsburgh is, or has been, supported in part by NIH grants #U19 AI090959, #U01 AI068642, and # R21 A1074844, and by Sponsored Research Agreements between the University of Pittsburgh and Revivicor, Inc., Blacksburg, VA. Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.
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