Introduction:  In gene-edited (GE) pig-NHP xenotransplantation, the induction of de novo antibodies after surgery is still an important factor affecting the long-term survival of the xenograft. However, it is not clear whether the porcine SLA antigen can induce significant anti-SLA antibody production in primate recipients after kidney xenotransplantation.
Methods: We performed 14 pig-rhesus monkey kidney xenotransplantation using kidneys from GTKO/hCD55 (n=7) and GTKO/βGalNT2KO(DKO)/hCD55/TM (n=7) pigs. Five recipients (GTKO: 2 cases, DKO: 3 cases) developed high levels of circulating antibodies against non-Gal antigens and experienced acute humoral xenograft rejection on days 14 to 29. To investigate the role of anti-SLA antibodies in xenograft rejection, we used serum samples collected before transplantation and at the time of rejection to measure IgM/IgG antibody binding against the following cells by flow cytometry: 1) PBMCs from donor and GTKO/SLA-I/II KO pigs (to exclude interference from anti-Sda antibodies, serum from DKO group was adsorbed with WT pig RBC); 2) DKO/SLA-I⁺/ SLA-II^- and DKO/SLA-I⁺⁺/ SLA-II⁺⁺ pAECs (stimulated with pIFN-γ). Finally, a xenogeneic MLR system was established using PBMCs isolated from SLA⁺ (WT, GTKO, GTKO/ βGalNT2KO, TKO) and GTKO/SLA-I/II KO pigs as stimulator cells and human PBMCs as responder cells.
Results: In the GTKO group, the binding of IgM and IgG antibodies against GTKO pig PBMCs was significantly (5 to 10 times) higher in the serum of two monkeys at the time of rejection than in serum collected before transplantation. The levels of IgM and IgG binding decreased significantly after the additional deletion of SLA-I/II (IgM decreased by 45% and 83%, IgG decreased by 54.4% and 57.7%, respectively). In the DKO group, the serum IgM/IgG antibody levels against DKO pig PBMCs were also elevate significantly at the time of rejection. After SLA-I/II deletion, the IgM binding levels in 2 cases decreased by more than 50%, and the IgG level in 3 cases decreased by more than 60%. The above results were further confirmed by using DKO/SLA-I⁺/ SLA-II^- and DKO/SLA-I⁺⁺/ SLA-II⁺⁺ pAECs, as the binding of IgM and IgG antibodies against pAECs was significantly elevated with the increase in SLA-I and SLA-II expression. Moreover, when anti-SLA-I antibodies was adsorbed by DKO/SLA-I⁺/ SLA-II^- pAECs, the IgM/IgG binding to DKO/SLA-I⁺⁺/ SLA-II⁺⁺ pAECs was reduced by more than 60%, indicating that the de novo antibodies were mainly anti-SLA-I antibodies. Finally, when the SLA-I and SLA-II were deleted, the average T cell proliferation rates in the xeno-MLR decreased from more than 28% (SLA+) to 5% (SLA-), indicating the strong immunogenicity of the SLA antigens.
Conclusion: We conclude that induced anti-SLA-I and SLA-II antibodies play important roles in GE pig-to-rhesus monkey xenotransplantation, thus providing further evidence for SLAKO pigs as the preferred organ source for rhesus monkeys as a preclinical model of xenotransplantation.