Xenoantibody response to porcine islet cell transplantation using GTKO, CD55, CD59, and fucosyltransferase multiple transgenic donors

Abstract

Background Promising developments in porcine islet xenotransplantation could resolve the donor pancreas shortage for type 1 diabetic patients. Using GTKO donor pigs with multiple transgenes should extend xenoislet survival via reducing complement activation, thrombus formation, and the requirement for exogenous immune suppression. Studying the xenoantibody response to GTKO/hCD55/hCD59/hHT islets in the pig-to-baboon model, and comparing it with previously analyzed responses, would allow the development of inhibitory reagents capable of targeting conserved idiotypic regions. Methods We generated IgM heavy and light chain gene libraries from ten untreated baboons and three baboons at 28 days following transplantation of GTKO/hCD55/hCD59/hHT pig neonatal islet cell clusters with immunosuppression. Flow cytometry was used to confirm the induction of a xenoantibody response. IgM germline gene usage was compared pre and post transplant. Homology modeling was used to compare the structure of xenoantibodies elicited after transplantation of GTKO/hCD55/hCD59/hHT pig islets with those induced by GTKO and wild type pig endothelial cells without further genetic modification. Results IgM xenoantibodies that bind to GTKO pig cells and wild type pig cells were induced after transplantation. These anti-nonGal antibodies were encoded by the IGHV3-66*02 (Δ28%) and IGKV1-12*02 (Δ25%) alleles, for the immunoglobulin heavy and light chains, respectively. IGHV3-66 is 86.7% similar to IGHV3-21 which was elicited by rhesus monkeys in response to GTKO endothelial cells. Heavy chain genes most similar to IGHV3-66 were found to utilize the IGHJ4 gene in 85% of V-D regions analyzed. However, unlike the wild type response, a consensus complementary determining region 3 was not identified. Conclusions Additional genetic modifications in transgenic GTKO pigs do not substantially modify the structure of the restricted group of anti-nonGal xenoantibodies that mediate induced xenoantibody responses with or without immunosuppression. The use of this information to develop new therapeutic agents to target this restricted response will likely be beneficial for long term islet cell survival and for developing targeted immunosuppressive regimens with less toxicity.

Early barriers to neonatal porcine islet engraftment in a dual transplant model

“…(42) The dual transplantation of islet xenografts and allografts with costimulation blockade generated a xenospecific IgM response, which was not prevented by our experimental regimen. Indeed, non-Gal natural antibodies exist,(43–45) and our findings may point to the need for immunosuppressive interventions addressing the initial immune activation and additional genetic modifications of porcine islets to minimize natural IgM binding to non-Gal xenoantigens. Screening for these antibodies and selecting recipients that are naturally low in these antibodies might also be warranted.…”

Pig BMSCs Transfected with Human TFPI Combat Species Incompatibility and Regulate the Human TF Pathway in Vitro and in a Rodent Model

“…Another possible option is breeding donor pigs transfected with human TFPI. However, transgenic pigs with other genes, such as CD46, CD55, CD59, and thrombomodulin, have also shown a potential to prolong survival following xenotransplantation [27][28][29][30]. In such cases, we developed donor human TFPI-transfected BMSCs as an alternative way to overexpress human TFPI in xenotransplantation when pigs transfected with other genes (without TFPI) were used as donors.…”

Rhesus monkeys and baboons develop clotting factor VIII inhibitors in response to porcine endothelial cells or islets

“…Those residues in the xenoantibody are of particular interest. The heavy chain amino acid sequence induced in baboons in response to transplantation of GTKO/hCD55/hCD59/hHT transgenic porcine NICC and in rhesus monkeys immunized with GTKO porcine endothelial cells are 92% similar [16]. Furthermore, the light chain germline variable genes utilized in response to wild-type porcine organs and GTKO/hCD55/hCD59/hHT are the same [16].…”