B cells play an important role regulating immune responses. B cell deficiency or depletion in mice can worsen autoimmunity and prevent allograft tolerance. We showed that TIM-1 is a broad marker for IL-10+ regulatory B cells (Bregs) and that anti-TIM-1 induces tolerance through induction of IL-10+ Bregs. Based on this discovery, our collaborator Dr. Kuchroo, has just shown that TIM-1 regulates various inhibitory molecules on B cells in addition to IL-10, and that mice specifically lacking B cell-TIM-1 develop spontaneous systemic autoimmunity. This unequivocally demonstrates a critical role for Bregs in maintaining tolerance. In general agreement, tolerant human renal allograft recipients demonstrate a “Breg profile” and peri-transplant depletion of B cells increased acute rejection and vasculopathy after renal and cardiac transplantation, respectively. Despite these advances, our understanding of Bregs remains in its infancy. First, there is no agreement even about which B cells carry out Breg function, why Bregs in different studies belong to different subsets, or even whether plasma cells (PCs), and not B cells per se, actually carry out Breg function. Second, little is known about how Bregs are regulated in vivo, or what signals can be used to expand them. Based on our and our collaborator’s advances, this proposal, will directly address both of these major gaps. The field has been hampered because Bregs are rare and their prior best marker, IL-10, was only detected after stimulation of B cells ex vivo. Transfer of various B cell subsets can inhibit inflammation in transplant and autoimmune models. However, these subsets are suppressive because they contain a relatively high proportion of IL-10+ B cells in a given model, rather than representing a true Breg phenotype. Only 5-15% of B cells in such subsets express IL-10, and each subset comprises <20% of all IL-10+ B cells. As a result, the field has identified a myriad of different Breg "subsets” (often minor or immature), and there is no consensus as to their function. However, using IL- 10-GFP reporter mice, we recently demonstrated that IL-10+ B cells can be directly identified without in vitro culture. Moreover, on a protein level, canonical Follicular B cells (FOB), Marginal Zone B cells (MZB) and PCs each account for ~30% of all B-lineage IL-10. We hypothesize that Bregs belonging to these canonical subsets that have distinct localization in the SLO, have different functions. In AIM 1, using purified IL-10+ or TIM-1+ Bregs belonging to these 3 subsets, we will now directly determine whether they regulate the same or different aspects of the immune response (e.g. humoral vs. cellular). While PCs were reported to be essential for Breg function, we found that mice unable to generate PCs due to B cell-specific deletion of BLIMP-1 exhibit a regulated phenotype with improved allograft survival and less severe EAE, and a marked increase in both frequency and number of IL-10+ and TIM-1+ Bregs. ...