Despite recent advances in xenotransplantation, immunological hurdles remain a challenge. Investigations of strategies to improve tolerance across the xenogeneic barrier are ongoing. Our protocol combines intra-bone bone marrow transplantation (IBBMT) with autologous regulatory T cells (Tregs) expanded on donor pig cells to promote tolerance in a pig-to-baboon kidney model.

Tregs were obtained by cell sorting recipient baboon PBMCs for CD4+CD25hiCD127- cells. Tregs were cultured for 28 days with stimulation provided weekly by anti-CD3 antibody and pig PBMCs obtained from the bone marrow donor. Two baboons received IBBMT from hCD47/hCD55 transgenic GalT-KO pigs after conditioning with irradiation and B and T cell depletion using Rituximab and rATG, respectively. On day 0, 2, 5, and 7 of IBBMT and day 0 of delayed kidney transplantation, performed as a test of tolerance between day 90-110, the ex-vivo expanded Tregs were infused at 40 million/kg/dose. To track the Tregs in vivo, cells were CFSE labeled prior to the final infusion and tracking was performed using flow cytometry and fluorescent microscopy. Immunosuppression had been tapered between IBBMT and delayed kidney transplant. At the time of the delayed kidney transplants, baboons were on minimal immunosuppression with a plan to wean completely at day 30.

Expanded Tregs had strong in vitro suppression and expressed FoxP3. Peripheral blood pig chimerism waned after the initial 24 hours post-IBBMT. In vitro assays showed generalized hyporesponsiveness at 30-day intervals from day 0 to day 90 and there was no elicitation of anti-pig antibodies during this time. After kidney transplantation and adoptive transfer of CFSE labeled Tregs, the ex-vivo expanded Tregs were detectable in the periphery until POD5-8. Animal 1 was euthanized at POD13 for complications due to bowel adhesions. Serum creatinine was 1.0 the day prior to sacrifice. Animal 2 was euthanized on POD28 after creatinine rose from 2 to 10 beginning on POD20 (POD117 from IBBMT). Anti-donor antibodies (ADA) also rose in parallel to creatinine (Figure 1). He was only on oral MMF, anti-CD40 and CTLA-4 Ig at the time of xenograft loss. At sacrifice, the xenograft and draining lymph node were found to have a distinct population of CFSE+CD4+FoxP3+ cells on flow cytometry (Figure 2A). On fluorescent microscopy of unstained tissue samples, CFSE positivity was also demonstrated (2B, 2C).

Our protocol was able to suppress the ADA of both animals until at least POD13 after xenograft implantation. For Animal 2, the graft was lost between POD20 and POD28, which coincided with an increase in ADA and heightened response to donor in MLR.  Despite evidence for an effect of Treg administration on the post-transplant antibody response, tolerance was not achieved with this protocol.  Further refinements of the immune induction and Treg expansion protocols to promote more durable mixed chimerism and tolerance are ongoing.