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Allogeneic hematopoietic stem cell transplantation (allo-HCT) has been a successful cellular therapy for patients suffering from hematological malignancies for many decades; however, the beneficial effects of graft-versus-leukemia (GVL) are classically offset by graft-versus-host disease (GVHD). GVHD occurs when major and/or minor human leukocyte antigen (HLA) mismatches between donor and recipient cause rapid expansion and activation of donor effector T cells (Teffs) resulting in end organ damage to the recipient’s epithelial tissues. Given the lymphoproliferative nature of this disease, the standard treatment option is broad immunosuppression, which can result in primary disease relapse, steroid refractory GVHD, and/or opportunistic infection. A more targeted therapy that can selectively suppress GVH responses with maintained GVL responses would achieve the optimal goal of allo-HCT. Regulatory T cells (Tregs) both natural (nTregs) or induced (iTregs) could be potential cellular therapies for the treatment of GVHD, given their innate suppressive function. Initial clinical trials using nTregs have yielded positive results; however, nTreg cellular therapy has been cumbersome due to the necessity for large scale ex vivo expansion given their low yield within an apheresis product and non-specific suppression. Conversely, iTregs can be generated from naïve T cells thus decreasing ex vivo culture times and can be educated with specific antigen thus providing targeted suppression, but a consensus on their efficacy for GVHD therapy has not been reached. Therefore, we investigated the efficacy of antigen specific iTreg therapy for the prevention of GVHD while maintaining GVL responses. In Chapter 2, we evaluated the effectiveness of monoclonal HY-specific iTregs in GVHD attenuation. We chose HY as a target antigen because it is a naturally processed, ubiquitously expressed minor mismatch antigen carried by only male donors/recipients cited to increase GVHD prevalence when donor and recipient are sex-mismatched. Utilizing HY-transgenic mice in which all T cells recognize HY antigen exclusively, we generated HY specific iTregs which effectively attenuating GVHD in male, but not female recipients in three murine bone marrow transplantation (BMT) models (major mismatch, parent to F1, and miHAg mismatch). We found HY specific iTregs lost stability in female recipients but remained stable and suppressive in male recipients suggesting expression of HY antigen was required for their suppressive function and stability. GVL responses were not compromised with the addition of HY specific iTregs in recipient mice using a pre-established tumor model. Thus, HY-specific iTregs can be generated and suppress GVHD in an antigen-dependent manner while sparing the GVL effect. In Chapter 3, we extend our findings in Chapter 2, which provided proof of principle that antigen specific iTregs effectively control GVHD; however, this therapy has a limited translational potential. Therefore, we generated alloreactive CD4 and CD8 iTregs and evaluated GVHD attenuation and GVL preservation in either full or haplo-MHC mismatched BMT models. We found alloreactive CD4 iTregs significantly suppress lethal GVHD, but completely abrogated the GVL effect against aggressive tumors. Conversely, alloreactive CD8 iTregs moderately attenuated GVHD and possessed direct cytotoxicity against tumor cells. Therefore, to rescue the impaired GVL effect mediated by CD4 iTregs, we established a combinational therapy with CD8 iTregs. Indeed we found combination CD4 and CD8 iTreg therapy significantly suppressed GVHD while sparing GVL responses compared to either CD4 or CD8 singular therapy. Mechanistically, this was achieved by potent suppression of both CD4 and CD8 Teffs coupled with preserved cytolytic molecule expression by both CD8 iTregs and Teffs. Taken together, we propose antigen specific iTreg therapy can effectively attenuate GVHD while preserving GVL responses. We further uncovered unique characteristics of CD4 and CD8 iTregs that can be exploited to achieve the optimal cellular therapy following allo-HCT.