PROJECT SUMMARY/ABSTRACT Chronic rejection (CR) after heart transplant (HTx) is the principal barrier to long-term graft survival that results in nearly 50% graft rejection after 10 years post-transplant (Tx). The factors driving CR are complex, as current immunosuppressive regimes targeting lymphocytes fail to prevent progressive fibrotic scarring and occlusion of graft vasculature. However, a known protagonist to the development of CR is prolonged graft ischemia reperfusion injury (IRI) that triggers robust early inflammatory responses of graft infiltrating monocytes that react to local inflammatory signals. Recently, it was established that monocytes and macrophages (MΦs) distinguish non-self from self MHCI via paired-immunoglobulin-like receptors (PIRs), and direct innate recognition of allogeneic H2-Dd by PIR-A promoted HTx rejection. MΦs are essential for resolving inflammation to promote reparative responses that re-establishes graft homeostasis to preserve organ function, yet how MΦ alloantigen recognition shapes or dysregulates the repair of grafts is completely unknown. I tested how MΦ recognition of self or non-self coordinates their differentiation and demonstrated that mono-derived MΦ recognition of allogeneic materials locally transforms them into pro-inflammatory Ly6chiCD86hi MΦs in wildtype, Rag2-/-γc-/-, and Pira-/--Rag2-/-γc-/ B6 recipients-. My preliminary data further settled that recombinant H2-Dd promotes pro- inflammatory differentiation in bone marrow-derived MΦs (BMDMΦs) and there were again no differences between wildtype and Pira-/- BMDMΦs. Therefore, I tested whether MΦs express other H2-Dd receptors and my preliminary data shows that blocking the receptor Ly49d extinguished H2-Dd-associated pathways. Lastly, I determined the functional consequences of this MΦ allorecognition, and show that MΦ exposure to allogeneic, but not syngeneic, amplifies their repair of fibroblasts. Therefore, my hypothesis is that recipient MΦs differentiate into a pro-fibrotic subset due to Ly49d-induced signaling triggered by donor H2-Dd MHCI leading to dysregulated repair and accelerated CR. I will directly test this hypothesis by addressing the following Specific Aims: 1. Define how MΦ recognition of non-self H2-Dd shapes the transcriptome and metabolome to induce pro- inflammatory MΦ differentiation. 2. Determine if preventing MΦ non-self MHCI recognition limits CR after IRI in heart grafts. Completion of these aims will establish if Ly49d is a fundamental MΦ allorecognition receptor and define pathways mediating pro-inflammatory MΦ differentiation to H2-Dd. I will also precisely determine whether MΦ recognition of H2-Dd amplifies their pro-fibrotic roles, and if abolishing MΦ responses to non-self MHCI limits CR to deliver effective graft repair. Contribution to Training: This proposal entails an extensive training plan to elucidate MΦ alloimmune responses combined with superb mentorship in bioinformatics and biochemistry. Together, the ...