Background Recent survivals of our pig-to-baboon kidney xenotransplants have been markedly shorter than the graft survivals we previously reported. The discovery of high levels of porcine CMV (pCMV) in one of the rejected xenografts led us to evaluate whether this reduction in graft survival might be due to the inadvertent introduction of pCMV into our GalT-KO swine herd. Methods Archived frozen sections of xeno-kidney grafts over the past 10 years were analyzed for the presence of pCMV, using real-time PCR. Three prospective pig-to-baboon renal transplants using kidneys from swine delivered by caesarian section (C-section) and raised in isolation were likewise analyzed. Results Kidney grafts from which 8 of the 18 archived samples were derived were found to be pCMV-negative, had a mean graft survival of 48.3 days and were from transplants performed before 2008. None had shown signs of DIC and were lost due to either proteinuria or infectious complications. In contrast, 10 of the archived samples were pCMV positive, were from kidney transplants with a mean graft survival of 14.1 days, had been performed after 2008 and had demonstrated early vascular changes and decreased platelet counts. Three prospective xenografts from swine delivered by C-section were pCMV negative and survived an average of 53.0 days. Conclusions Decreased survivals of GalT-KO renal xenografts in this laboratory correlate temporally with latent pCMV in the donor animals and pCMV in the rejected xeno-kidneys. Transmission of pCMV to swine offspring may be avoided by C-section delivery and scrupulous isolation of donor animals.

Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3–13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.

Our previous in vitro data have demonstrated that regulatory mechanisms are involved in tolerance of class I-mismatched renal allografts in miniature swine treated with 12 days of high dose Cyclsporin A. In the present study, we attempted to induce tolerance of class I-mismatched kidneys by adoptive transfer of cells and/or kidneys from long-term tolerant animals. Fifteen SLAdd miniature swine received 1.5 Gy whole body irradiation and class I-mismatched (SLAgg) kidneys from naïve pigs with or without co-transplanted kidneys and/or adoptively transferred cells from long-term tolerant (LTT) SLAdd recipients of SLAgg grafts. Additionally, three SLAdd miniature swine received class I mismatched kidney with adoptively transferred cells from LTT SLAdd recipients. Naïve kidneys transplanted without a LTT kidney were rejected within 9 days. All recipients of naive kidneys along with cells and kidney grafts from LTT animals showed markedly prolonged survival of the naive renal grafts (day 28, >150 and >150 days). These studies suggest that (1) tolerated kidneys have potent regulatory effects, and (2) cells from LTT animals infused in conjunction with kidney grafts augment these regulatory effects. To our knowledge, these studies represent the first demonstration of successful adoptive transfer of tolerance in large animals.