B cells are an important pathogenic component of autoimmunity, but our current knowledge of the phenotype of pathogenic B cells in these diseases is limited. While most B cells express only one type of antibody and are specific for one antigen, rare B cells co-expressing two different antibodies (i.e., dual-reactive B cells) exist in mice and are present in humans. Our goal is to understand whether dual-reactive B cells represent a relevant B cell subset in autoimmunity and to characterize their biology. Our novel findings demonstrate that autoimmune mice generate dual-reactive B cells more frequently than nonautoimmune mice. The majority of these cells react with self-antigens and they are highly enriched in the antigen-activated B cell subsets of autoimmune mice. Furthermore, we have identified a molecular pathway that drive the differentiation of nonautoreactive immature B cells into transitional and mature B cells and that, we propose, is also used for the generation of B cells that co-express autoreactive and nonautoreactive antibodies. In the previous grant cycle we laid the foundation for the proposed research by generating the data on which we base our new hypotheses and by creating the mutant mice and methodologies with which to follow development and selection of dual-reactive B cells in autoimmune and nonautoimmune mice. We believe, therefore, that we are uniquely poised to carry out the proposed studies. Our hypothesis is that dual-reactive B cells evade mechanisms of central and peripheral B cell tolerance to become an important component of autoimmune diseases. The goal of the proposed research is to deepen our understanding of the relationship between dual-reactive B cells and autoimmunity in mice. We will investigate the mechanisms that cause increased generation, selection and enrichment of dual-reactive B cells in autoimmune-prone mice and whether these B cells contribute to the development of autoimmunity or are rather a diagnostic marker. We will also translate our mouse studies by examining the prevalence of dual-reactive B cells in humans with autoimmunity. To achieve our goals we will develop the following specific aims: 1) to determine whether activation of Ras and/or of tonic BCR signaling inhibits negative selection and promotes positive selection of autoreactive immature B cells; 2) to establish the B cell-extrinsic signals that drive the increased activation and positive selection of dual-κ B cells in autoimmune mice; 3) to determine whether dual-reactive B cells contribute to autoimmunity in mice and are enriched in autoimmune patients. The studies proposed will establish dual-reactive B cells as a diagnostic and/or pathogenic B cell subset in autoimmunity and will be of value for the development of methods that identify and target these B cells while preserving those that fight infections. Overall, these studies are important for understanding B cell-mediated mechanisms of autoimmune development and to uncover novel targets for therapeutic intervention.