PROJECT SUMMARY/ABSTRACT The success of heart transplantation is limited by ischemia reperfusion injury-mediated primary graft dysfunction and allograft rejection, two processes that may be immunologically linked. Current strategies to reduce graft rejection and improve survival are mostly based on ablation of recipient immune cell populations. These approaches are only modestly effective and carry high risks of life-threatening infections. An alternative and potentially safer approach is to target immune pathways and cell populations within the donor graft that initiate inflammatory responses and resultant alloreactivity. The ability to precisely control the initial immune response following heart transplantation represents a promising approach to increase allograft tolerance and improve clinical outcomes. Our recent work has identified that ferroptosis, a non-apoptotic form of inflammatory cell death mediates the early inflammatory response after reperfusion of heart grafts. We have discovered that graft endothelial cells and tissue-resident CCR2+ macrophages play important and complementary roles in promoting the recruitment of inflammatory immune cells to the transplanted heart. In this proposal, we will use state-of-the- art techniques, including intravital microscopy, single cell RNA sequencing and novel mouse strains to perform studies that will 1) define mechanisms of cell death (Aim1), 2) evaluate the role of cell-specific inflammatory cytokine signaling (Aim 2) and 3) examine the role of cardiac macrophage heterogeneity (Aim 3) in driving innate inflammatory and alloimmune responses after heart transplantation. Our studies will lay the foundation for novel therapy that will improve outcomes after cardiac transplantation.