PROJECT SUMMARY Poor long-term graft survival rates despite continuous immunosuppression still plagues transplantation in patients. This is primarily due to two undesired effects of chronic immunosuppression: 1) inadequate control of memory T cells, which can attack and reject the graft at any time, and 2) inhibition of regulatory CD4+ T cells (Tregs) that exhibit suppressor functions and can protect the graft from immune attacks by memory T cells. Thus, improved therapeutics that promote Treg suppressor function are critically needed to enhance long-term graft survival. However, the mechanisms of Treg suppressor function within allografts is unknown, and improved therapeutics will originate from a better understanding of these mechanisms. In this regard, our data identified dendritic cells (DC) as the likely cellular promoter and/or target of Treg suppression within allografts. Thus, we hypothesize that cognate interactions between Treg and DCs are key to Treg function within allografts, and that these interactions can be manipulated therapeutically to enhance Treg suppressor function in the graft. We will test this through the following AIMs: In AIM 1, we will investigate whether Treg-DC interactions are key to Treg function within allografts. Specifically, we will determine whether Treg-DC cognate interactions promote Treg function, and whether Treg then suppress effector T cells through the inhibition of DC function resulting in the inhibition of effector T-DC cognate interactions. In AIM 2, we will investigate whether Treg suppressor function within allografts can be enhanced by therapeutically promoting Treg-DC interactions. We recently showed that a tolerogenic agent (anti- CD45RB antibody) specifically promotes Treg proliferation in lymphoid organs by enhancing cognate Treg-DC interactions. However, it remains unknown whether anti-CD45RB would promote Treg-DC interactions within allografts to promote Treg function. Thus, this will be specifically addressed here. The visualization of Treg suppressor function using multi-photon intravital microscopy is crucial to testing these hypotheses. In addition, these AIMs will be accomplished through the collaborative efforts of an interdisciplinary group of experts, which includes a world-renowned microscopy expert. Therefore, this grant application is responsive to this RFA-AI-16-042: Emerging Science and Technology in Transplantation (U01) as it will investigate intragraft Treg function using intravital imaging. Overall, new knowledge generated from this grant will: 1) identify ? at the cellular level, in the living host, and in real time ? the mechanisms of action of Tregs in the setting of transplantation; and 2) identify cellular and molecular targets to promote Treg suppressor function in transplant recipients.