Protein kinase C theta (PKC?) is a PKC family member most abundant in T cells, where it plays critical roles in effector T (Teff) cell activation PKC? uniquely localizes to the center of the T cell immunological synapse (IS) via physical association with CD28, and this localization is required for downstream PKC?-mediated signaling. In vivo analysis of PKC?-deficient mice revealed a selective requirement of PKC? in some (Th2- and Th17- mediated inflammation, allograft rejection and graft vs. host disease) but not other (Th1/CTL antiviral and graft vs. leukemia) immune responses. Intriguingly, PKC? was recently found to be excluded from the IS of induced Treg cells (iTregs) and to inhibit their suppressive function, including in humans. Our preliminary studies show that strategies, which disrupt the PKC?-CD28 interaction and displace PKC? from the IS, including novel, previously uncharacterized PKC? allosteric inhibitors (AI), enhance the in vitro differentiation and suppressive function of mouse iTregs. Our new preliminary data suggest that the same strategies have similar effects on human T cells. These findings point to the high promise of PKC? as a selective drug target for immunosuppression of harmful T cell-mediated autoimmunity, inflammation, and GvHD, where inhibitory strategies are predicted to act synergistically to inhibit pathogenic T cells and promote Treg function, without interfering with beneficial antiviral protective immunity. Here, we will investigate in detail the potential therapeutic value of blocking the PKC?-CD28 interaction in a mouse model of Th2 cell-mediated allergic lung inflammation, and the selective and seemingly opposite roles of PKC? in the in vitro differentiation and function of human Teffs and Tregs, respectively. These studies will address the working hypothesis that blockade of the PKC?-CD28 interaction inhibits the differentiation and function of pathogenic T cells and promotes the function of Tregs in both mice and humans, thereby achieving a beneficial synergetic effect against harmful T cell- mediated inflammatory responses. In Aim 1, we will employ a dominant negative PKC? mutant and novel PKC? AI to study the resulting effects on the development and manifestation of Th2-mediated allergic lung inflammation in intact mice, bone marrow chimeric mice, or by using adoptive T cell transfers, including the use of TCR-transgenic and FoxP3eGFP reporter mice. In Aim 2, we will use, for the first time, the same blocking strategies of Aim 1 to disrupt the TCR/CD28-induced PKC?-CD28 interaction in human CD4+ T cells, and evaluate their effect on the in vitro differentiation and function of human blood-derived Teff and Treg cells. This project will extend our understanding of the importance of the PKC?-CD28 interaction, which was discovered in our laboratory, and its biological relevance for the differentiation and function of both Teff and Treg cells, importantly extending these findings to human T cells. It will also represent the first detailed analysis of novel PKC? AI on T cell responses, and may pave the way to the development of new, highly selective therapeutic strategies to treat T cell-mediated allergies, autoimmune, diseases, and GvHD.