We, and others, have shown that the negative T cell costimulatory pathways play a critical role in regulating alloimmune responses and tolerance. Our overall hypothesis is that the integration of positive and negative signals delivered by allogeneic hematopoietic and/or parenchymal cells under inflammatory conditions dictates tolerance vs tissue destruction. Our main goal is to define the relationship between these signals at the tissue level and outcome of alloantigen exposure during graft-vs-host disease (GVHD) and organ grafting models. Lessons learned from these distinct but interrelated and complementary models will further our understanding of the mechanisms of GVHD and organ allograft rejection and tolerance. These studies will lead to development of novel therapeutic strategies by harnessing the physiologic mechanisms that regulate immune responses. We propose to address three questions: 1) What is the role of hematopoietic vs parenchyma cell expression of negative costimulatory molecules in alloimmunity and tolerance? 2) What are the important and unique interactions between positive and negative costimulatory pathways that determine the fate of alloimmune response in an inflammatory environment? 3) What are the effects and mechanisms of targeting of these pathways in preclinical models of alloimmune responses? Our specific aims are: AIM 1: To investigate the functions of the PD-1:PD-L1,-L2 negative costimulatory pathway in GVHD and solid organ transplants. 1A. Study the role of the PD-1 pathway in GVHD focusing on the parenchymal vs hematopoietic cell expression of PD-1 ligands in regulating alloimmunity. 1B. Study the role of PD-1 pathway interactions with the CD28/B7 positive T cell costimulatory pathway that augments and donor Tregs that suppress GVHD. 1C: To investigate the functions and mechanisms of PD-1:PD-L1 and B7-1/PD-L1 interactions in alloimmune responses in models of solid organ transplantation. AIM 2: To investigate the functions of B7-H3 expression on hematopoietic vs parenchymal cells during GVHD (2A). In order to define the exact functions of this novel pathway in alloimmunity we plan to characterize the B7-H3 receptor, TLT2, and clone a putative second and inhibitory B7-H3 receptor (2B). We have a number of unique tools that will enable us to dissect the functions, mechanisms, and interactions of negative and positive costimulatory pathways and the microenvironment that will permit the optimal targeting of costimulatory pathways to inhibit detrimental T cell alloresponses that limit hematopoietic and solid organ allograft acceptance.