Roles And Mechanisms Of Regulatory T Cells In Regulating The Pathogenesis Of Acute Graft-versus Host Disease

CD4+CD25+Foxp3+ regulatory T cells (Tregs) play an essential role in maintaining immunologic homeostasis. Forkhead box P3 transcription factor(Foxp3) is responsible for the differentiation and function in Tregs.. Fundamental research on regulatory T cells (Tregs) from our group have indicated that Tregs play an important role in treating varius autoimmune disease including autoimmune diabetes, experimental arthritis and so on. However, data showed that Tregs are not stable under inflammatory conditions, it lose Foxp3 quickly in autoimmune disease models especially in acute graft versus-host disease. Most importantly, our pre-experiments also indicated that pre-Tregs express high level of IL-17, thus may improve the serveity of the disease. During this paper, we will investigate the role of Tregs in GVHD; determine the best protocol for stable Treg generation and most importantly figure out the best marker for Treg stabiliy identification. The success of the project will be an important significance in transplantation disease, and provide a new insight to the cell therapy for organ of bone marrow transplantat patients. The subject includes the following three parts:The First Part:TGF-0-induced CD4+Foxp3+ T cells attenuate acute graft-verse-host disease via suppressing expansion and killing of effector CD8+ cellsTGF-β-induced CD4+Foxp3+T cells (iTregs) have been identified as important preventing and treating strategies for cell therapy to autoimmune diseases and other disorders. However, the prevention effect of these cells on acute GVHD has been unsatisfied because these cells are unstable and less suppressive in this disease. Here we restudied the ability of iTregs to prevent and treat acute GVHD in two different mouse models. Our results showed that iTregs, as long as an appropriate protocol is used, displayed a strong and consistent ability to control acute GVHD development and increase the survival in two different types of acute GVHD animal models. iTregs infusion markedly suppressed the engraftment of donor CD8+ cells, the expression of Granzyme A and B and cytotoxic effect of donor CD8+ cells, production of cytokines in T cells in acute GVHD. To valuate the stability of Tregs generated from different protocols, iTregs were induced from beads stimulated and anti-CD3 coating plate. Data showed that anti-CD3/CD28 stimulated Tregs showed better stability and in vivo protection function compared with Tregs induced by anti-CD3 coating plate. We therefore conclude that iTregs are able to prevent and even treat acute GVHD as long as the right methods for generating iTregs have been employed.The Second Part:All trans Retinoic Acid Favors Treg Development and Function in Liver Transplant PatientsGraft-versus-host disease (GVHD) is known as intractable complication in transplant patients. Tregs have been shown to have the ability to prevent GVHD. Treg consists of two subsets:natural Treg (nTregs) cells and induced Treg (iTregs) cells. iTregs also know as a condition Tregs originate in the peripheral and shows a better prolification and suppresive ability in an inflammatory milieu compared to nTregs. All-trans retinoic acid (atRA) is able to favor Treg expansion and Foxp3 expression in human Tregs. However, whether atRA would affect iTreg from transplant patients remains inconclusive. Therefore, we sorted naive T cells from liver transplantation patients and cultured them in vitro. Further analysis was performed to assess the suppressive function in vitro and vivo. We found that atRA favored Treg expansion and Foxp3 expression as well as suppressive ability in vitro and vivo in transplant patients. To conclude, our study shows that atRA has the potential to improve iTreg development and function in transplant-related patients and provide a novel insight into Treg cell therapy in GVHD clinical trials.The Third Part:CD39 is a key marker in stable nature regulatory T cells under inflammation conditionAutoimmune diseases are characterized by an imbalance between regulatory T (Treg) cells and effector T cell subsets such as Thl and Th17 cells. Recent studies have demonstrated that thymus-derived naturally-occurring CD4+Foxp3+ regulatory T cells (nTregs) in both human and mouse are unstable and dysfunctional in the presence of pro-inflammatory cytokines. In this study, human CD39hi nTregs and CD39low nTregs were sorted from nTregs 7 days after expansion. Foxp3 expression and suppressive activities were analyzed in vitro and in vivo using a standard assay in a humanized animal model. We find that in the presence of IL-1β and IL-6, CD4+CD39hi nTregs were stable, while CD4+CD39low nTregs lose Foxp3 expression and transdifferentiate into Thl or Th17 cells. Interestingly, human CD4+CD39hi nTregs, but not CD4+CD39low nTregs significantly protect xeno-graft versus host diseases and only these cells maintained functional activity against xGVHD. Finally, STAT1 and STAT3 activation and CPG methylation were evaluated in CD39low nTregs under pathological conditions. These results strongly implicated the physiological importance of CD39 expression in maintaining Foxp3 stability, and suggest that CD39hi nTregs may represent a novel treatment strategy to control established immune-mediated autoimmune and inflammatory diseases.