Project Summary Autoreactive B cells play critical roles in many autoimmune diseases. Multiple immune tolerance checkpoints exist to remove autoreactive B cells or keep them under control. Defects in these checkpoints constitute the basis for the development of autoimmune diseases. Despite intensive study, our understanding of these checkpoints remains incomplete and fragmentary. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate a large diversity of biological processes. Hundreds of miRNAs are expressed in the immune system. While some miRNAs have been shown to play important roles in lymphocyte development and function, the roles of miRNAs in controlling immune tolerance remain poorly understood. We performed in vivo functional analysis of hundreds of miRNAs in the recently established IgMb-macroself mouse model and identified miR-148a as an important regulator of B cell central tolerance and autoimmunity (Nature Immunology 17:433-40, 2016). Further molecular analysis identified 119 target genes regulated by miR-148a in immature B cells. We examined 4 of these target genes and demonstrated that 3 of them, Gadd45a, Bim and Pten, regulate B cell central tolerance. In this proposal, we will 1) further investigate the cellular and molecular mechanisms underlying miR-148a regulation of immune tolerance and autoimmunity, focusing on its role in controlling various B cell tolerance checkpoints and plasma cell differentiation; 2) evaluate the possibility of treating systemic autoimmunity through miR-148a ablation and inhibition by genetic and chemical approaches, respectively, and 3) perform an in vitro functional screen of the other 115 miR-148a target genes to identify novel regulators of B cell tolerance. Our pilot screen has identified B4galt5 as a positive hit. As B4galt5 is a major enzyme in the glycolipid biosynthesis pathway, we speculate that this pathway plays important roles in immune tolerance. Therefore, we will elucidate the function and mechanism of B4galt5 and the glycolipid biosynthesis pathway in controlling B cell tolerance and autoimmunity.