Thymic-derived CD4+25+FoxP3+ regulatory T cells (Tregs) are required to suppress autoimmunity and limit foreign antigen responses. Our first-in-human phase I dose escalation trial of ex vivo expanded human Tregs proved efficacious in significantly reducing the incidence of acute graft-vs-host disease (GVHD). Two major limitations to uniform efficacy were identified: Problem 1 (achieving optimal suppression): The %suppression was variable. Problem 2 (maintaining adequate Treg:Teffector ratios): Ratios of 1:5.5 achieved were well below the desired ratios of e1:1 need for GVHD prevention in mice; no Tregs were detected by 2 weeks post-transfer. Aim 1 will focus on Problem 1. PKC-q coordinates immune function, which results in its movement to the immunological synapse (direct contact point between T cell and antigen-presenting cell) in Teffectors but not Tregs. PKC-q deficient murine Teffectors are unable to cause GVHD but can retain anti-tumor and anti- pathogen responses. Tregs treated with a small molecule PKC-q inhibitor had augmented suppressor function and were more effective in suppressing murine colitis. In aim 1, focused on Problem 1, we will use small molecule PKC-q inhibitors in vitro or in vivo to test the hypotheses that murine and human Treg potency for GVHD suppression is limited by movement of PKC-q into the immunological synapse. In aim 2, focused on Problem 2, we will test the hypothesis that sustained IL-2R signaling pathways will provide a PKC- q independent route to Treg expansion and GVHD suppression. We will use 3 strategies to improve Treg mediated GVHD suppression: (a) administer IL-2/anti-IL2 antibody complexes or low dose IL-2 to preferentially expand murine and human Tregs vs Teffectors; (b) Constitutively express STAT5b in murine and human Tregs based upon our findings that constitutive STAT5b promotes murine Treg generation, proliferation and function including GVHD suppression, bypassing the need for IL-2, present in limiting amounts in vivo; and (c) increase b-catenin signaling using a GSK-3b inhibitor in vitro or expressing a stabilized b-catenin in human Tregs since stabilized b-catenin reduces Treg dependency upon IL-2 and provides a marked enhancement of murine Treg survival and efficacy in preventing colitis. In aim 3, we will select the best 1-2 approaches, derived from studies in aims 1 and 2, for testing in preclinical GVHD models for the preservation of beneficial immune responses later post-transplant. Long-term chimeras that do not succumb to GVHD lethality, as a result of preferred approaches developed aims 1 and 2, will be challenged with leukemia cells or bacterial or viral pathogens to quantify the extent to which the immune system can respond to life-threatening complications post-transplant.