Project Summary/Abstract My laboratory has contributed chiefly to the better understanding of the mechanisms by which lymph node (LN) stroma controls transplant tolerance. We have devoted much effort to defining specific compartments of the LN where Foxp3+ regulatory T cells (Treg) are induced and activated. We showed that during tolerance induction by costimulatory blockade, naïve T cells migrate to the LN, but not the spleen. During tolerance induction, naïve T cells specifically home to the cortical ridge (CR) of LN, entering via the nearby high endothelial venules (HEV). In the CR they are stimulated by alloantigen-presenting plasmacytoid dendritic cells to differentiate into induced Treg (iTreg). Our data indicate that in tolerogenic conditions iTreg are mostly formed within the CR region, whereas T cells which enter the medulla of LN experience anergy. More specifically, the ratio of stromal laminin α4:α5 (referred to as LAMA4/LAMA5) of the CR region critically determine the response to alloantigen and iTreg formation. A high LAMA4/LAMA5 ratio promotes tolerance, whereas a low LAMA4/LAMA5 ratio promotes transplant immunity. Mechanistically, LAMA4 promotes CD4 migration to the CR, promotes Foxp3 expression and iTreg maturation, and inhibits effector T cell differentiation. In contrast, LAMA5 inhibits migration of CD4 cells into HEV, yet costimulates T cell proliferation and maturation to inflammatory Th17. Abrogating this interaction with neutralizing antibodies enhances iTreg migration to the CR region and significantly prolongs heart allograft survival. Our overall hypothesis is that the LAMA4/LAMA5 ratio of the CR critically determines the fate of iTreg formation and transplant tolerance. Our overall goal is to define the key cellular (FRC) and molecular (LTβR) mechanisms which control the laminin composition of LN and thereby leverage this microstructure to manipulate immunity toward transplant tolerance. To investigate this hypothesis, we propose the following AIMS: Aim 1. Define the role of LN stromal cells in controlling the balance of LAMA4 and LAMA5 during alloimmune responses. Aim 2. Define the role of LTβR activation of FRC as a key pathway in inducing formation of LAMA5. Aim 3. Targeted delivery of costimulatory molecule anti-CD40L mAbs and anti-laminin α5 Abs to the LN to promote tolerance. Overall, key signaling molecules that regulate FRC function to remodel LN laminins and the LAMA4/LAMA5 ratio dictate the immune response toward inflammation and immunity (low ratio) or toward suppression and tolerance (high ratio). These Aims will achieve our overall goal to define the key cellular (FRC) and molecular (LTβR) mechanisms which control the laminin composition of LN. These data lay the groundwork for developing highly innovative targeted therapies to reprogram the microstructure of LN to promote transplant tolerance.