Antibodies directed at non-gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into non-human primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgVH genes that encode antibody responses to non-gal pig xenoantigens. Immunoglobulin genes derived from the IGHV3-21 germline progenitor encode xenoantibodies directed at non-gal xenoantigens. Transduction of GalTKO cells with lentiviral vectors expressing the porcine α1,3 galactosyltransferase gene responsible for gal carbohydrate expression results in a higher level of binding of “anti-non-gal” xenoantibodies to transduced GalTKO cells expressing the gal carbohydrate, suggesting that anti-non-gal xenoantibodies crossreact with carbohydrate xenoantigens. The galactosyltransferase 2 gene encoding isoglobotriaosylceramide synthase (iGb3 synthase) is not expressed in GalTKO pig cells. Our results demonstrate that anti-non-gal xenoantibodies in primates are encoded by IgVH genes that are restricted to IGHV3-21 and bind to an epitope that is structurally related to but distinct from the Gal carbohydrate.

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.

Summary Synthetic anti‐idiotypic antibodies represent a potentially valuable tool for the isolation and characterization of B cells that produce xenoantibodies. An anti‐idiotypic antibody that binds to a subset of B cells producing antibodies encoded by the variable‐region heavy chain 3 (VH3) germline genes DP35 [immunoglobulin variable‐region heavy chain 3‐11 (IGHV3‐11)], DP‐53 and DP‐54 plus a small number of VH4 gene‐encoded antibodies in humans has recently been identified. These germline progenitors also encode xenoantibodies in humans. We tested whether the small, clearly defined group of B cells identified with this anti‐idiotypic antibody produce xenoantibodies in non‐human primates mounting active immune responses to porcine xenografts. Peripheral blood B cells were sorted by flow cytometry on the basis of phenotype, and cDNA libraries were prepared from each of these sorted groups of cells. Immunoglobulin VH gene libraries were prepared from the sorted cells, and the VH genes expressed in each of the sorted groups were identified by nucleic acid sequencing. Our results indicate that xenoantibody‐producing peripheral blood B cells, defined on the basis of binding to fluorescein isothiocyanate (FITC)‐conjugated galactose α(1,3) galactose–bovine serum albumin (Gal‐BSA) and the anti‐idiotypic antibody 2G10, used the IGHV3‐11 germline gene to encode xenoantibodies and were phenotypically CD11b+ (Mac‐1+) and CD5–. This novel reagent may be used in numerous applications including definition of xenoantibody‐producing B‐cell subsets in humans and non‐human primates and immunosuppression by depletion of B cells producing anti‐Gal xenoantibodies.

Technical advances in transplant surgery and the development of powerful and effective immunosuppressive drugs have contributed to the success of organ transplantation as a medical treatment for patients with end-stage diseases. Associated with this procedure, however, is a dependence on life-long immunosuppressive drugs, which are required to prevent graft rejection. These agents render the patient susceptible to infections, tumors and various side affects. The ability to achieve tolerance to organ grafts would free transplant patients from lifelong dependency on pharmacological agents with harmful side effects. Several laboratories have shown that tolerance can be achieved by the induction of mixed cell chimerism and/or by molecular chimerism achieved by gene transfer techniques prior to graft placement. Molecular chimerism, induced by transplantation of autologous bone marrow expressing either allo- or xenoantigens has the potential to induce tolerance without the development of graft vs. host disease. The application of gene transfer techniques to induce chimerism has been shown to reshape the immune repertoire by mechanisms that include clonal deletion, the induction of central tolerance or generation of regulatory T cells that would eliminate the need for immunosuppressive drugs. Optimization of this methodology for clinical use could therefore revolutionize the field of transplantation. This review summarizes the recent studies which have compared the efficacy of different vectors, conditioning regimens, and transduction conditions leading to new and improved techniques for the application of gene therapy to induce chimerism and transplant tolerance to both allografts and xenografts.