PROJECT 3 - SUMMARY/ABSTRACT Ischemia and reperfusion injury (IRI), the inevitable consequence of the transplant process, is a key limitation to human liver transplantation. The cellular damage incurred by IRI can lead to primary non-function necessitating re-transplantation. Liver IRI also predisposes the recipient to both acute and chronic rejection and graft loss, as well as, decreasing the pool of transplantable organs. Key observations in murine models indicate that liver IRI is mediated by both the innate and adaptive immune systems. Little is known whether similar mechanisms are at play in human liver transplantation. Our central hypothesis is that identifying the DAMPs and their associated PRRs driving myeloid cell plasticity during human OLT-IRI will permit us to develop therapeutic interventions to improve OLT outcomes. Aim 1, we will determine the TLR9 and TLR7/NOD2 ligands mediating differential polarization of inflammatory vs. regulatory macrophages and crosstalk with T cells. We will characterize the cell- free DNA/TLR9 ligands mitochondrial DNA, neutrophil extracellular traps, and HMGB1/RAGE in pre/post reperfusion blood to establish their contributions to human OLT-IRI, ability to polarize macrophages and develop alloreactive T cells. We will also determine the tolerogenic effect of TLR7 and NOD2 ligands for their regulatory potential to mitigate TLR4 and/or TLR9 polarization and suppression of alloimmunity. Characterization of longitudinal changes in myeloid and T cell phenotypes of recipient blood and donor allograft biopsies obtained pre- and post-reperfusion will identify new targetable interactions along the endotype-specific pathways TLR4/TLR9 and TLR7/NOD2 in situ. Aim 2 will determine the therapeutic potential of PRR preconditioning to mitigate myeloid activation and OLT-IRI. We will assess the therapeutic potential of TLR4/9 antagonists to mitigate IR mediated-injury, macrophage inflammatory polarization and T cell alloreactivity in vitro using blood from human OLT recipients, and in vivo in rodent models of IRI. In situ assessment of the spatial immune profile of donor vs. recipient myeloid cells in control vs. tolerized organs will explore the IRI-mediated immune cell and parenchymal changes contributing to a regulatory vs inflammatory state in the graft. Aim 3 will determine the impact of DAMP/PRR endotypes on generation of alloimmunity and graft outcome. We will assess the DAMP/PRR endotypes that direct the generation and breadth of alloreactive T cells and donor specific antibodies. Data will be integrated with clinical metadata, blood and graft immunophenotypes, IRI and transplant outcomes to determine DAMP/PRR endotypes responsible for susceptibility to IRI and worse outcomes.