Tolerance of kidney allografts has been achieved in non-human primates (NHPs) and in humans using a combination of non-myeloablative conditioning and donor bone marrow transplantation (DMBT) that results in transient donor chimerism. However, until now, mixed-chimerism protocols that achieve long-term tolerance of kidney allografts in NHPs have consistently failed to induce tolerance in recipients of heart or lung allografts. It is well known that some organs, such as kidney and liver, are tolerance-prone while others, such as heart and lung, are tolerance-resistant. It has been hypothesized that only protocols that result in durable donor chimerism would be able to achieve tolerance in resistant heart and lung allografts. Remarkably, however, the results obtained during our current funding cycle demonstrate, for the first time, that tolerance of heart allografts can be achieved in NHPs via transient mixed-chimerism as long as host regulatory mechanisms are enhanced in the host. Indeed, our results suggest that it is the ability of a mixed-chimerism protocol to augment or expand regulatory T cells (Tregs), rather than its durability, that determines its ability to induc tolerance to resistant organs. Thus, the unifying goal of this program project is to combine mixed chimerism with novel strategies designed to amplify the contributions of Tregs in order develop a clinical tolerance protocol that can be rapidly translated to human recipients of heart and lung allografts. The Program will be organized in such a way that early advances in Project 3 (examining molecular pathways required to stabilize and promote Treg function in mice), will inform and refine the aims of Project 1 (optimizing mixed chimerism to induce NHP heart allograft tolerance) and Project 2 (optimizing mixed chimerism to induce NHP lung allograft tolerance). The availability of tissue and blood samples for longitudinal analyses will aid the study of immune mechanisms by assessing the consequences of T and B cell regulation, T and B cell memory and pro-inflammatory cytokines on graft survival (Cores A & B). The diverse yet complementary models and approaches that will be used to achieve heart and lung tolerance is a unique strength of this program project. Collaboration between all projects will be greatly enhanced by the fact that most all the investigators have worked together closely for many years. Together, our studies should result in a better understanding of tolerance and contribute to the successful application of tolerance to the full spectrum of deceased donor organ and tissue transplantation.