Project Summary Although clinical approaches directed at T cells, B cells, and immune effector molecules prevent transplant rejection and slow the progression of autoimmune disease, these therapies do not induce durable immune tolerance as immune-mediated destruction recurs when therapy is stopped. This failure is due to an imbalance in regulatory and effector immunity that no current approach has been able to address and is particularly difficult in approaches like islet transplantation for T1D where the induction of durable immune tolerance would be curative alongside islet replacement. Islet loss following transplant and autoimmune attack in T1D are both heralded by the presence of anti-islet antibodies, which are so important in signifying incipient islet injury that they define new onset stage 1 T1D. Traditionally, it has been thought that the production of these antibodies is a sign of T-B collaboration that leads to effector cell activation. This proposal will investigate an alternative hypothesis that B lymphocytes also directly erode immune regulatory cell function. In the absence of B lymphocytes, we find that the T1D-prone NOD immune system becomes susceptible to inducible transplantation tolerance, which is not otherwise achievable in this background, and that tolerance following islet transplantation is also enhanced in healthy, non-autoimmune recipients. This successful transplantation was not due to loss of effector cell specificities in the absence of B cells but instead was associated with an increase in islet-protective Tregs and a recovery in their response to immune therapy. Thus, we have determined that B lymphocytes are the critical barrier to successful immune reprogramming and that they act by disrupting Treg function. We hypothesize that B cells interact with T cells during their development through both antigen-specific (signal 1) and co-stimulatory (signal 2) interactions to shape the balance of Tregs and Teffs in a way that depends on B cell tolerance. In this proposal, we will investigate this new paradigm in two aims. In Aim 1, we will determine the role of antigen presentation, acquisition, and tissue origin on the interaction of B lymphocytes and Tregs using transgenic systems to control and trace their relationships. In Aim 2, we will determine the mechanism of deleterious B cell-Treg contact including the roles of B cell development, B cell tolerance including anergy, and the intercellular interactions between B and T cells that modulate immunity. Together, these aims will reveal how B cells impede tolerance induction and how this barrier may be repaired to permit inducible immune tolerance in islet transplantation.