Benjamin Piegari

Introduction: Mixed chimerism (MC) induction via non-myeloablative conditioning and bone marrow transplantation (BMT) is a promising approach to achieve long-term kidney transplant tolerance. Prior data has demonstrated transient multilineage chimerism lasting 20-60 days in cynomolgous monkeys, with Tcell chimerism of upto 5%. The proposed induction protocol was able to yield higher and longer lasting mixed chimerism in the rhesus macaque as an operational tolerance model to solid organ transplantation. Methods: Rhesus macaques developed multilineage peripheral blood macrochimerism starting day 5, that was high in peak percentage (≥90%) and showed Tcell chimerism ≧10%. Bone marrow engraftment was evident after immunosuppression withdrawal and persisted significantly longer compared to cynomolgous controls. One animal developed persistent multilineage chimerism ≥30% for over 6 months. During the chimeric period, recipients showed donor-specific hyporesponsiveness in MLR and Elispot. No animal developed graft-versus-host disease. Animals accepted their donor kidney graft for over 180 days. Conclusion: While a similar induction regimen in other species has historically failed to achieve permanent chimerism, this tolerance protocol demonstrated significant findings in the rhesus macaque: higher chimerism overall, Tcell chimerism ≥10%, bone marrow engraftment after immunosuppression withdrawal and persistent macrochimerism for ≥5months. The rhesus macaques is thus a suitable model to study tolerance induction in solid organ transplantation with nonmyeloablative induction protocols.

Introduction: Cell Therapy with regulatory Tcells (Tregs) is a promising tool for tolerance induction in solid organ transplantation. Despite encouraging results in humans and monkeys, questions about Treg phenotype, function, survival and migration after in vivo infusion remain. To better understand Treg fate, we have used a CFSE-labeled tracking method for autologous, polyclonal expanded Tregs in cynomolgus macaques. Methods: Polyclonal Tregs were grown from sorted (CD4+CD25hi) PBMCs and ex vivo expanded on allogeneic CD40-expressing Bcells and cryopreserved. 10-30x106 Tregs/kg were thawed and labeled with CFSE dye, then Tregs were administered intravenously. Phenotype and function of Tregs were specified via flow cytometry and suppression assays prior to in vivo infusion into recipient cynomolgus macaques. Labeled Tregs were then tracked in vivo with flow cytometry on peripheral blood and iliac crest bone marrow aspirates over the first month. At the time of sacrifice (Day 30), lymphoid organs (thymus, mesenteric and inguinal lymph nodes, spleen) and bone marrow were harvested and analyzed. Results: The first cynomolgus macaque received 12x106 CFSE-labeled Tregs/kg that retained their phenotype (CD4+CD25hi, FoxP3+ expression ≥95%) and suppressive capacities (1:16 suppression titer) throughout the expansion and restimulation process. CFSE-labeled Tregs were detected in circulation at Day 0, 2, 3, 6, 9 and 14 and were undetectable by day 15 post infusion. Dividing CFSE-labeled Tregs were seen at day 2,6 and 9 in the bone marrow. At day 30, no CFSE-labeled cells were found in any tissues. Treg infusions are upcoming in 4 more cynomolgus macaques. Conclusion: CFSE labeling of autologous, ex vivo expanded polyclonal Tregs is a valid model that allows in vivo tracking of migration and survival of Treg infusions in cynomolgus macaques.

Introduction: Xenotransplantation poses a heightened immunologic barrier; therefore, tolerance induction strategies are needed. Mixed chimerism (MC) has successfully induced tolerance in xenogeneic humanized mouse models. In these models, porcine hematopoietic cytokines are needed to support porcine MC. Our study uses a transgenic pig with a humanized IL-3 receptor (Tg hIL-3R) in addition to hCD47 to support engraftment of mobilized porcine peripheral blood stem cells (PBSC) to promote MC in a pig-to-baboon model. Methods: A novel GalTKO/hCD46/hCD47/hCD55/hCD59 Tg, hCD123/hCD131/hCD116 knock-in pig underwent mobilization with porcine stem cell factor (p-SCF) and porcine IL-3 prior to leukapheresis. PBSC product, dosed at 1x10^9 mononucleated cells/kg, was infused into two P. hamadryas baboons on days 0, 49 and 63 after non-myeloablative conditioning with total body and thymic irradiation, rituximab, ATGAM, LoCD2b and anti-CD154. Tacrolimus and pSCF was given from day 0 to 28. Two control baboons received the same protocol, without PBSC. Donor skin from the donor pig was grafted at week 12 to test tolerance. Controls will be grafted this month. Grafts were serially monitored using punch biopsy and photography. Results: Mobilized porcine PBSC expressed the hGM-CSF and hIL-3R alpha chains, but only the hIL-3R was functional in vitro. Peripheral blood chimerism (PC) was elevated in the first 48-hours compared to current and historical controls that received PBSC from pigs not Tg for hIL-3R. The experimental animals demonstrated a relative increase in PC at day 15-23 compared to all controls. Xeno-antibody levels were reduced from pre-transplant levels until skin grafts rejected, while control animals maintained stable levels during the initial 30 days. Donor skin grafts survived 37 and 41 days, similar to historical MC controls receiving PBSC and skin from GalTKO hCD47hi pigs. After grafts were rejected, the two animals were euthanized and the bone marrow (BM), thymus, liver and lymph nodes were examined for potential engraftment. Porcine lymphocytes were found only in the liver and represented >20% of total live leukocytes for both baboons. These cells were further characterized to be CD3+CD4lo gamma delta T cells. Conclusion: Due to the remarkable percentage of porcine gamma delta T cells found in the liver at 173 post-infusion, we hypothesize that the liver is an immunoprivileged site for xenogeneic T cells, allowing for cell survival. We continue to explore the infusion of xenogeneic stem cells into protected compartments, such as intra-bone BM transplant, to support porcine stem cell survival. The increase in PC at days 15-23 demonstrates evidence for later stage hematopoiesis in these animals, which has not previously been seen in our studies. This can be attributed to the functional Tg hIL-3R, supporting the hypothesis that genetic engineering of cytokine receptors provides a strategy to enable prolonged MC to promote xenogeneic tolerance. NIH Award Project Number: 2P01AI045897-21A1- “A Tolerance Approach to Xenotransplantation”. ChoironeX.

Introduction: Achieving long-term xenograft survival is a major goal. We hypothesize that long-term survival will be achieved through induction of immune tolerance via co-transplantation of the donor thymus. While the use of pig donors with multiple transgenes has been described in both nonhuman primate (NHP) and brain-dead human models, we hypothesize that long-term survival can be achieved with minimal genetic modifications with the thymokidney approach to induce immune tolerance. Methods: Six baboons with low non-Gal anti-pig natural antibody levels received thymokidney transplants from alpha1,3-galactosyltransferase gene-knockout donors (GalTKO) pigs. Briefly, we implanted a piece of the donor’s thymus under its own renal capsule, allowed it to mature for 6 weeks in situ, and then harvested and transplanted the thymokidney into the baboon recipient. Induction and maintenance immunosuppression is illustrated in Figure 1A.Results: Survival exceeded one year (370 days) in Baboon 2, which was euthanized for reaching endpoint, with no clinical or histological signs of rejection. Two baboons are still under observation and are currently six months (Baboon 4) and five months (Baboon 5) post- transplant, showing no clinical or histological signs of rejection. Three baboons were euthanized shortly after the transplant due to non-immunological complications. Electron microscopy was performed on Baboon 2’s xenograft at the time of euthanasia (H&E and PAS pending), and showed endothelial injury manifested as swollen endothelium with loss of fenestration, and extensive but incomplete foot process effacement (Figure 2A). Baboon 4’s latest biopsy (Day 145) showed mild to moderate tubulointerstitial scarring, along with mild transplant glomerulopathy and minimal C4d staining in peritubular capillaries. Baboon 5’s latest biopsy (Day 98) showed mild to moderate tubulointerstitial scarring with focal acute tubular injury (Figure 2B) and minimal C4d staining in peritubular capillaries. Serum creatinine values for Baboons 2, 4, and 5 stabilized at 2-3 mg/dL, despite occasional spikes in serum creatinine, which were related to urine outflow problems and urinary tract infections, corrected by ureter stent exchanges and/or removal and treatment with antibiotics (Figure 1B).Discussion: Our data indicate that long-term survival greater than one year in NHPs can be achieved utilizing GalTKO pig donors via co-transplantation of the thymus and kidney. This model demonstrates that extensive genetic modifications of donor swine are not necessary for long term survival in recipients with low non-Gal natural antibody levels if additional strategies are incorporated, such as co-transplanting the thymus tissue.