Background Infusion of recipient regulatory T cells (Treg) promotes durable mixed hematopoietic chimerism and allograft tolerance in mice receiving allogeneic BMT with minimal conditioning. We applied this strategy in a Cynomolgus macaque model. Methods CD4+CD25high Treg that were polyclonally expanded in culture were highly suppressive in vitro and maintained high expression of FoxP3. Eight monkeys underwent nonmyeloablative conditioning and MHC-mismatched BMT with or without Treg infusion. Renal transplantation (from the same BMT donor) was performed 4 months post-BMT without immunosuppression to assess for robust donor-specific tolerance. Results Transient mixed chimerism, without significant T cell chimerism, was achieved in the animals that received BMT without Treg (N=3). In contrast, the 2 of 5 recipients of Treg + BMT that were evaluable displayed chimerism in all lineages, including T cells, for up to 335 days post-BMT. Importantly, in the animal that survived long-term, >90% of donor T cells were CD45RA+CD31+, suggesting they were new thymic emigrants. In this animal, the delayed (to 4 months) donor kidney graft was accepted >294 days without immunosuppression, whereas non-Treg BMT recipients rejected delayed donor kidneys within 3-4 weeks. Early cytomegalovirus reactivation and treatment was associated with early failure of chimerism, regardless of Treg administration. Conclusions Our studies provide proof-of-principle that, in the absence of early CMV reactivation (and BM-toxic antiviral therapy), co-transplantation of host Treg can promote prolonged and high levels of multilineage allogeneic chimerism and robust tolerance to the donor.

Core award; Irving Pilot Translational Science award for new investigators Reliable in vitro expansion protocols of regulatory T cells (Tregs) are needed for clinical use. We studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed four in vitro Treg expansion protocols for translational studies. Tregs expanded 3000-fold when artificial antigen presenting cells (aAPCs) expressing human CD80, CD58 and CD32 were used throughout the culture. When donor peripheral blood mononuclear cells (PBMCs) were used as the single source of APCs followed by aAPCs, Tregs expanded 2000-fold. Tregs from all protocols suppressed the proliferation of anti-CD2CD3CD28 bead-stimulated autologous PBMCs albeit with different potencies, varying from 1:2-1:4 Treg:PBMC ratios, up to >1:32. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed greater suppression of stimulation by anti-CD2CD3CD28 beads of PBMCs from the same donor used for stimulation during the Treg cultures and of autologous cells than of third-party PBMC responders. Similar to humans, the Treg-specific demethylated region (TSDR) within the Foxp3 locus correlated with suppressive activity and expression of Foxp3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression. In summary, we have characterized MCM Tregs and developed four Treg expansion protocols that can be used for preclinical applications. K E Y W O R D S animal models: nonhuman primate, basic (laboratory) research/science, cellular transplantation (nonislet), graft survival, immune regulation, immunobiology, immunosuppression/immune modulation, tolerance: chimerism, translational research/ science | 2187 ALONSO-GUALLART eT AL.

Background. Although short-term outcomes for liver transplantation have improved, patient and graft survival are limited by infection, cancer, and other complications of immunosuppression. Rapid induction of tolerance after liver transplantation would decrease these complications, improving survival and quality of life. Tolerance to kidneys, but not thoracic organs or islets, has been achieved in nonhuman primates and humans through the induction of transient donor chimerism. Since the liver is considered to be tolerogenic, we tested the hypothesis that the renal transplant transient chimerism protocol would induce liver tolerance. Methods. Seven cynomolgus macaques received immune conditioning followed by simultaneous donor bone marrow and liver transplantation. The more extensive liver surgery required minor adaptations of the kidney protocol to decrease complications. All immunosuppression was discontinued on postoperative day (POD) 28. Peripheral blood chimerism, recipient immune reconstitution, liver function tests, and graft survival were determined. Results. The level and duration of chimerism in liver recipients were comparable to those previously reported in renal transplant recipients. However, unlike in the kidney model, the liver was rejected soon after immunosuppression withdrawal. Rejection was associated with proliferation of recipient CD8 T effector cells in the periphery and liver, increased serum interleukin (IL)-6 and IL-2, but peripheral regulatory T cell (Treg) numbers did not increase. Antidonor antibody was also detected. Conclusions. These data show the transient chimerism protocol does not induce tolerance to livers, likely due to greater CD8 T cell responses than in the kidney model. Successful tolerance induction may depend on greater control or deletion of CD8 T cells in this model.

Background Regulatory T cells (Treg) are being explored for their tolerance-inducing capabilities. Freezing and banking Treg for future use makes this strategy more clinically applicable. We aimed to devise an improved method of expanding and cryopreserving Treg to maximize yield, purity, and function for use in xenotransplantation. Methods Baboon peripheral blood mononuclear cells (PBMC) were isolated from whole blood. CD4+/CD25hi cells were isolated by flow cytometric sorting and expanded for 26 days in culture with IL-2, anti-CD3 antibody, artificial APCs transfected with human CD58, CD32, and CD80, and rapamycin with weekly restimulations. Expanded Treg were frozen for 2 months then thawed and cultured for 48 hours in medium plus 1) no additives, 2) IL-2, 3) anti-CD3 antibody, 4) IL-2 + anti-CD3 antibody, and 5) IL-2 + anti-CD3 antibody + L cells. Phenotype and suppression were assessed after expansion, immediately after thawing, and after culturing. Results We expanded purified baboon Treg more than 10,000-fold. Expanded Treg exhibited excellent suppression in functional assays. Cryopreservation decreased suppressive function without changing phenotype, but increasing amounts of reactivation after thawing produced significantly better viability and suppressive function with a trend towards greater Treg purity. Conclusions We produced numbers of expanded Tregs consistent with clinical use. In contrast to some previous reports, both Treg phenotype and suppressive function were preserved or even enhanced by increasing amounts of restimulation after thawing. Thus, banking of expanded recipient Tregs for in vivo infusion should be possible.

Background. Cytomegalovirus (CMV) infection is a serious complication in immunosuppressed patients, specifically transplant recipients. Here, we describe the development and use of an assay to monitor the incidence and treatment of CMV viremia in a Cynomolgus macaque model of bone marrow transplantation (BMT) for tolerance induction. We address the correlation between the course of viremia and immune reconstitution. Methods. Twenty-one animals received a nonmyeloablative conditioning regimen. Seven received cyclosporine A for 28 days and 14 received rapamycin. A CMV polymerase chain reaction assay was developed and run twice per week to monitor viremia. Nineteen recipients were CMV seropositive before BMT. Immune reconstitution was monitored through flow cytometry and CMV viremia was tracked via quantitative polymerase chain reaction. Results. Recipients developed CMV viremia during the first month post-BMT. Two animals developed uncontrollable CMV disease. CMV reactivation occurred earlier in cyclosporine A-treated animals compared with those receiving rapamycin. Post-BMT, T-cell counts remained significantly lower compared with pretransplant levels until CMV reactivation, at which point they increased during the viremic phase and approached pretransplant levels 3 months post-BMT. Management of CMV required treatment before viremia reached 10 000 copies/mL; otherwise clinical symptoms were observed. High doses of ganciclovir resolved the viremia, which could subsequently be controlled with valganciclovir. Conclusions. We developed an assay to monitor CMV in Cynomolgus macaques. CMV reactivation occurred in 100% of seropositive animals in this model. Rapamycin delayed CMV reactivation and ganciclovir treatment was effective at high doses. As in humans, CD8+ T cells proliferated during CMV viremia.