7. PROJECT SUMMARY/ABSTRACT Rates of late renal allograft loss due to chronic rejection have not significantly changed over the past three decades despite marked improvements in controlling acute rejection. While the immune pathogenesis is incompletely understood, a significant proportion of allografts contain infiltrated host B cells and the presence of B cells correlates with poor prognosis that may be independent of the presence of a donor specific antibody. While T cell recruitment to allografts is well understood and may be initiated by either chemokines or antigen presentation within the vascular lumen, much less is known about B cell recruitment. We propose to study these processes with the goal of identifying pathways and molecular targets that may inhibit B cell infiltration of grafts. To study if and how this process can be induced by chemokines, we will employ both high dimensional cyclic tissue immunofluorescence microscopy of human allograft tissue and in vitro assays of transendothelial migration (TEM) by human peripheral blood B cells. Tissue analyses will provide information re the subtypes of B cells found within graft infiltrates, the chemokine and adhesion receptors they express, and their proximity to activated endothelial cells that are the likely point of entry. In vitro assays will use human microvascular endothelial cell monolayers in microfluidic chambers to characterize and manipulate B cells capable of undergoing TEM using approaches we developed in our T cell studies. To study if and how this process can be induced by antigen, we will use our high dimensional IF approach to determine if B cells within the graft are interacting with relevant T cell populations and we will use laser capture microscopy to isolate individual B cells from which we will clone and express the antigen combining regions to generate antibodies and then test specificity for different types of graft antigens. We will also modify our in vitro TEM assays to allow endothelial cells to cross-link and signal through the B cell receptor for antigen by display of anti-human kappa antibody, again employing a technique we used to mimic T cell receptor signaling with anti-human CD3. We anticipate that these studies will lead to identification of targets, possibly inhibitable by currently approved agents, that can limit B cell recruitment into allografts, an intervention that may reduce late graft loss without the risk to immunosuppressed patients mediated by total B cell ablation.