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 T cell responses in immune-mediated diseases. NR4A 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 and others have shown that NR4A TF expression scales with the intensity of Ag stimulation, and are highly upregulated in thymocytes undergoing negative selection, in regulatory T cells (Tregs), as well as in self-reactive, anergic, or exhausted T cells. Due to an overlapping expression pattern and considerable structural homology in their DNA-binding domain, the NR4A TFs exhibit profound functional redundancy; thymic deletion of multiple - but not individual - NR4A family members (Nr4a1 and Nr4a3 >> Nr4a2, which is minimally expressed) results in severe Treg deficiency and a “scurfy-like” disease that phenocopies Foxp3-/- mice. Consequently, it has not been possible previously to unmask additional redundant functions of this family during thymic selection and in conventional mature CD4 T cells (Tconv). This represents a major gap in our knowledge that limits full therapeutic exploitation of these factors. Recently, we took advantage of both conditional genetic and bone marrow chimera strategies in order to preserve Treg homeostasis. Unexpectedly, mixed chimeras harboring both WT and Nr4a1-/- Nr4a3-/- (DKO) bone marrow rapidly develop anti-nuclear autoantibodies (ANAs) and a systemic inflammatory disease despite a replete Treg compartment of largely WT origin. This disease differs qualitatively from that seen in germline DKO mice with Treg-deficiency, and is B cell-extrinsic. We show that negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO CD4 Tconv cells expressing phenotypic and transcriptional markers of anergy accumulate in these chimeras. However, these self-reactive DKO cells nevertheless exhibit exaggerated proliferation and IL-2 production, suggesting that functional anergy is defective. We therefore hypothesize that the NR4A family play cell-intrinsic, but redundant, roles in both central and peripheral CD4 T cell tolerance. To test this hypothesis: In our first aim, we propose to define the role of the NR4A family in thymic negative selection in the face of both ubiquitous and tissue-restricted antigens, and to define the transcriptional targets that contribute to this function. In our second aim, we propose to isolate NR4A contributions to peripheral T cell tolerance, and to canonical features of mature CD4 T cell anergy - including impaired sig...