Novel regulatory therapies for prevention of Graft-versus-host disease

Graft-versus-host disease is one of the major transplant-related complications in allogeneic hematopoietic stem cell transplantation. Continued efforts have been made to prevent the occurrence of severe graft-versus-host disease by eliminating or suppressing donor-derived effector T cells. Conventional immunosuppression does not adequately prevent graft-versus-host disease, especially in mismatched transplants. Unfortunately, elimination of donor-derived T cells impairs stem cell engraftment, and delays immunologic reconstitution, rendering the recipient susceptible to post-transplant infections and disease relapse, with potentially lethal consequences. In this review, we discuss the role of dynamic immune regulation in controlling graft-versus-host disease, and how cell-based therapies are being developed using regulatory T cells and other tolerogenic cells for the prevention and treatment of graft-versus-host disease. In addition, advances in the design of cytoreductive conditioning regimens to selectively target graft-versus-host disease-inducing donor-derived T cells that have improved the safety of allogeneic stem cell transplantation are reviewed. Finally, we discuss advances in our understanding of the tolerogenic facilitating cell population, a phenotypically and functionally distinct population of bone marrow-derived cells which promote hematopoietic stem cell engraftment while reducing the risk of graft-versus-host disease

Regulatory T Cells Expanded from Hiv-1-Infected Individuals Maintain Phenotype, Tcr Repertoire and Suppressive Capacity

While modulation of regulatory T cell (Treg) function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4+ Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-β) repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region), characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection.Elizabeth Glaser Pediatric AIDS Foundation (Pediatric HIV Vaccine Program Award MV-00-9-900-1429-0-00)Massachusetts General Hospital. Executive Committee on Research (MGH/ECOR Physician Scientist Development Award)National Institutes of Health (U.S.) (NIH NIAID (KO8 AI074405))National Institutes of Health (U.S.) (NIH NIAID AI074405-03S1)Massachusetts General Hospital (William F. Milton Fund)Harvard University. Center for AIDS Research (CFAR Scholar Award)Massachusetts General Hospital. Center for the Study Inflammatory Bowel Disease (P30DK043351)Harvard University. Center for AIDS Research (NIH funded program (5P30AI060354-09

Moving to tolerance: clinical application of T regulatory cells.

Decreasing the incidence of chronic rejection and reducing the need for life-long immunosuppression remain important goals in clinical transplantation. In this article, we will review how regulatory T cells (Treg) came to be recognized as an attractive way to prevent or treat allograft rejection, the ways in which Treg can be manipulated or expanded in vivo, and the potential of in vitro expanded/generated Treg for cellular therapy. We will describe the first regulatory T cell therapies that have been or are in the process of being conducted in the clinic as well as the safety concerns of such therapies and how outcomes may be measured

Restimulation After Cryopreservation and Thawing Preserves the Phenotype and Function of Expanded Baboon Regulatory T Cells

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

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells

Regulatory T cell (Treg)-based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, weshow that large numbers of FOXP3(+) Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood