The Role of Induced Regulatory T cell Populations in Graft Versus Host Disease Biology

Allogeneic bone marrow transplantation (BMT) is curative for many hematological malignancies. However, its clinical application is limited by the high frequency of graft versus host disease (GVHD) which develops as the major complication of allogeneic BMT. This proinflammatory syndrome is driven by donor T cells that initiate a cytokine cascade, resulting in tissue damage of the GVHD target organs and a loss of tolerance. The generation of regulatory T cells (Tregs) is one tolerance mechanism that appears to be diminished during GVHD. However, to date, all studies examining the role and generation of Tregs in GVHD have focused on a subset of these suppressive cells, termed natural Tregs (nTregs): CD4+Foxp3+ cells that develop in the thymus. Adoptive transfer of nTregs into murine models of GVHD has been shown to ameliorate disease; however, this strategy would be difficult to implement in the clinic due to the low frequency of nTregs in the peripheral blood.;A second subset of Tregs is induced in the periphery (iTreg) in response to antigen, and can be easily generated and expanded in vitro. Thus, iTregs may provide a clinically relevant strategy to preventing GVHD. However, the ability of these cells to suppress GVHD has not been described. Therefore, the overall goal of this work was to define the role of iTregs in GVHD biology. The first aim of this project sought to determine whether in vitro-derived CD4+ iTregs were able to mitigate GVHD severity. To address this, we first generated CD4+ iTregs in vitro using cells from Foxp3EGFP reporter mice, and determined that they had a similar phenotype to nTregs and were equally potent at suppressing alloreactive T cell proliferation in vitro. However, when these cells were adoptively transferred into completely MHC-mismatched transplant recipients (B6[H-2b]→Balb/c[H-2 d]), they were unable to prevent lethal GVHD. Using a congenic marker to identify the adoptively transferred iTregs, we determined that the iTregs lost Foxp3 expression early post transplantation. Since Foxp3 expression has been shown to be both necessary and sufficient for the suppressive function of Tregs, we attributed the inability of CD4+ iTregs to ameliorate GVHD to Foxp3 instability. Furthermore, neither interleukin-6 receptor (IL-6R) signaling blockade nor retinoic acid were able to stabilize Foxp3 expression in in vitro-derived iTregs. Identifying an approach for stabilizing iTregs in vivo will be necessary in order to implement iTreg adoptive therapy clinically.;During the course of these studies, we observed a novel population of CD8+Foxp3+ cells that develop during GVHD. We determined that these cells were induced from CD8+Foxp3 - conventional T cells and were able to suppress alloreactive T cell proliferation in vitro, so we named them CD8+ iTregs. Therefore, the second aim of this work was to determine the role of CD8+ iTregs in GVHD biology. We found that both in vivo and in vitro-derived CD8 + iTregs had similar phenotypes to the corresponding CD4+ iTreg populations. To assess the extent of CD8+ iTreg induction during GVHD, studies were performed in three murine BMT models with varying degrees of major histocompatibility (MHC) disparity. CD8+ iTreg generation decreased as the degree of MHC disparity decreased. In order to determine the role of CD8+ iTregs in vivo, BMT studies were performed using T cells from mice with nonfunctional Foxp3. In these experiments, mice that lacked the ability to generate both CD4+ and CD8 + iTregs in vivo exhibited exacerbated GVHD, whereas mice that lacked only one functional iTreg population displayed similar survival kinetics as a control group that had both populations of iTregs. Furthermore, T cell activation and proinflammatory cytokine production were significantly increased in mice lacking both populations of iTregs. Together, these data reveal a novel functional role for CD8+ iTregs in reducing GVHD severity.;Overall, this project characterized the role of CD4+ and CD8+ iTreg populations in the biology of GVHD. Further studies should focus on stabilizing Foxp3 expression in these cells as well as defining the mechanisms by which these iTreg populations suppress alloreactivity in vivo. Understanding the relative contribution of CD4+ and CD8 + iTregs to ameliorating GVHD will allow for the development of therapeutics that augment Treg function and induce tolerance in a GVHD setting.