PROJECT SUMMARY Accumulating evidence supports that B cell can regulate immune responses and are essential for limiting inflammation and autoimmunity by IL-10-dependent and -independent mechanisms. The processes and mechanisms by which B cells with regulatory properties are generated have also not been identified. We have shown that Tim-1 is also expressed on B cells that produce IL-10 and Tim-1 is required for apoptotic cell (AC) binding to B cells and induction of IL-10. We generated a mouse with a conditional deletion of Tim-1 selectively on B cells (Tim-1BKO). The Tim-1BKO mice show progressive loss of IL-10 production in B cells and, with age, developed severe multi-organ tissue inflammation including colitis, dermatitis, hepatitis and a paralytic disease with meningeal inflammation in the CNS. On the other hand, Tim-1BKO mice show strong anti-tumor immunity with inhibition of tumor growth. Therefore, in addition to serving as a marker for regulatory function of B cells, Tim-1 as a phosphatidylserine receptor is also functionally required for optimal regulatory B cell function. However, it is not clear what induces Tim-1 expression and what are the transcription factors that drive Tim-1 expression and regulatory B cell function. Our data clearly shows that Tim-1 does not define a subset of B cells but can be induced on B cell upon activation. Based on the preliminary data we hypothesize that Tim-1 is a functional marker on B cells that can induce IL-10 upon binding to AC and regulate tissue inflammation, autoimmunity, and anti-tumor immunity. To address this hypothesis we propose to: 1) Identify Transcriptional Regulators of Tim-1+ B cells, focusing on EHF as the positive regulator of Tim-1, which was identified by a focused Crispr-Cas9 screen; 2) Study the effect of loss of Tim-1 on B cells in generating pro-inflammatory effector B cells, as Tim-1BKO B cells are highly activated and produce proinflammatory cytokines including IL-23 and IL-6 which promote differentiation of pathogenic Th17 cells; 3) Study the role of Tim-1+ B cells in maintaining stem-like Th17 cells and limit differentiation of pathogenic CXCR6+ Th17 cells. We have found that Tim-1+ B cells produce Wnt10a, which promotes stemness, and other suppressive cytokines that keep Th17 cells in a stem-like state and limit their differentiation into pathogenic Th17 cells. The proposed studies will greatly increase our understanding of the role of Tim-1 in B cells and the generation of regulatory activity on B cells to promote immune tolerance and limit tissue inflammation and autoimmunity. These studies will provide a novel therapeutic strategy by targeting B cells for the treatment of autoimmune diseases and promoting anti-tumor immunity.