The efficacy of allogeneic hematopoietic stem cell transplant (HSCT) is limited by graft-versus-host disease (GVHD), a severe immune-mediated multi-organ inflammatory disease that is the leading cause of morbidity and mortality after HSCT. Current globally immunosuppressive therapies to prevent GVHD are incompletely effective, and have unwanted consequences, such as increasing cancer recurrence and infection risk. Thus, new treatments for GVHD are needed. Regulatory T-cells (Tregs) play an indispensible role in controlling autoimmunity and responses to foreign antigens, including the allo-antigens that drive GVHD. As a result, Tregs hold immense promise for the treatment of GVHD. O ur group conducted the first-in-human clinical trial using ex vivo expanded Tregs for GVHD prevention. While this trial demonstrated a significant reduction in GVHD, it was not eliminated. We determined that infused Tregs had variable suppressor potency, which limited consistent efficacy. Therefore, the overarching goal of this project is to improve Treg suppressive function so that we can better prevent GVHD. To accomplish this, the specific aims of this application build on our lab's finding that protein kinase C-theta (PKC-?), a key molecule necessary for Effector T-cell (Teff) activation and proliferation, negatively regulates Treg function. We are taking advantage of this finding by using a clinically available PKC-? inhibitor (AEB071) as a means to enhance Treg suppressive potency. We now find that ex vivo treatment of murine Tregs with AEB071 results in a 300% increase in Treg function in vitro, and increases Treg-mediated suppression of murine GVHD. However, in vitro, PKC-? inhibition enhances Treg suppressive potency in a dose-dependent manner, whereas in vivo, only low dose treatment augments GVHD suppression. This suggests that while PKC-? inhibition enhances Treg suppression in a dose-dependent manner, treatment with higher doses restrains Treg function in vivo. In order to overcome this limitation and further advance the efficacy of Treg therapies, the aims proposed in this application are designed to identify the downstream mechanisms by which PKC-? inhibition mediates these modifications to Treg functionality in vitro and in vivo. Aim 1 will focus on Treg suppression and test the hypothesis that PKC-? inhibition augments Treg function by up-regulating known contact-dependent and possibly secreted suppressive molecules in a dose-dependent manner. Aim 2 will focus on diminished in vivo efficacy with higher dose AEB071 treatment, and test the hypothesis that PKC-? inhibition reduces Treg proliferation and GI tract homing in vivo. These studies will provide novel insights into the mechanisms by which inhibiting a negative regulator of Treg function (PKC-?) alters Treg biology. These results will help us overcome the limitation to Treg therapeutic efficacy identified in our group's clinical trial and inform a new wave of clinical studies. Therefore, this proposal has broad implications not only for the treatment of GVHD, but also for transplant rejection and autoimmunity.