Project Summary/Abstract This proposal's long-term goal is to take advantage of the biology of the Nr4a family of orphan nuclear hormone receptors to selectively manipulate antigen-specific B cell responses. If successful, this could transform our approach to vaccination and treatment of immune-mediated diseases. Nr4a family members (Nur77, Nurr1, and Nor1) are encoded by three genes (Nr4a1-3, respectively) that are rapidly induced by antigen (Ag) stimulation in lymphocytes. Although they are thought to function as ligand- independent, constitutively active transcription factors, small molecule agonist and antagonist ligands have been developed, rendering them druggable. We have shown that a fluorescent reporter of Nr4a1 expression (Nur77- eGFP BAC Tg) scales with the intensity of Ag stimulation, and also correlates with self-reactivity in naturally occurring B cells in healthy mice and in three different BCR Tg models. We have recently shown that Nur77 curbs the survival and expansion of B cells in response to chronic and acute Ag stimulation, imposing a novel layer of B cell tolerance. Importantly, the Nr4a family harbor considerable structural homology in their DNA- binding domain and, as a result, exhibit profound functional redundancy in T cells, where they also play an important regulatory role. Such redundancy is likely highly relevant to their function in B cells (which predominantly express Nr4a1 and Nr4a3) yet has never been experimentally addressed. This represents a major gap in our knowledge that limits full therapeutic exploitation of these factors. Here we report that newly generated mice with B cell-specific deletion of Nr4a1 and Nr4a3 show a major break in B cell tolerance, including the development of spontaneous germinal centers and autoantibodies. These defects correlate with profound super-induction of novel target genes in response to Ag stimulation – including several genes required for recruitment of T cell help – that is largely masked when even one functional Nr4a allele remains. We therefore hypothesize that the Nr4a family mediates negative feedback downstream of Ag stimulation via transcriptional repression of key target genes. We propose that this serves two functions: (a) to restrain self-reactive clones that receive signal 1 (antigen) in the absence of signal 2 (co-stimulation), and (b) restrain immunodominant clones from monopolizing normal humoral immune responses, in order to preserve clonal diversity. In this grant, we propose: (1) To define the role of the Nr4a family in regulating central and peripheral B cell tolerance, and identify transcriptional mechanisms that mediate these functions in BCR Tg and naturally occurring self-reactive B cells. (2) To define the role of the Nr4a family in regulating B cell inter-clonal competition and clonal diversity during the primary T-dependent humoral immune response and its contribution to limiting immunodominance. (3) To define the role of the newly identified Nr4a tar...