PROJECT SUMMARY This R21 application is based on exciting and novel preliminary data supporting the hypothesis that the orphan nuclear receptor NR4A1 restrains the protective function of CD8+ T cells in tuberculosis (TB). We found the NR4A1-/- mice control pulmonary infection with Mycobacterium tuberculosis (Mtb) significantly better than wildtype (WT) mice. A similar effect was observed in WT mice treated with the NR4A1 antagonist DIM-C. In an adoptive transfer experiment, NR4A1-/- CD8+ T cells protected Rag1-/- mice from aerosol Mtb infection better than transferred CD8+ T cells from NR4A1-/- or WT donors. This is remarkable since prior studies found little or no protection from transferred WT CD8+ T cells, which was confirmed in our experiment. Immunohistochemistry of lung TB lesions showed greater infiltration of CD8+ T cells in NR4A1-/- mice compared to WT. Analysis of bulk RNA sequencing data from purified lung CD8+ T cells from Mtb-infected mice identified upregulation of genes linked to cellular infiltrative capacity and cytotoxicity in NR4A1-/- compared to WT hosts. If true, these findings will advance the field of cellular immunity in TB and offer translation potential for NR4A1 inhibition as host-directed therapy. Expression of NR4A1 is induced by T cell receptor signaling, which is the predominant mechanism for regulating its activity. We found that NR4A1 mRNA is increased in the lungs of Mtb-infected mice, raising the fundamental question whether NR4A1-regulated T cell tolerance supports a pathogen-permissive environment in TB. The goal of this R21 application is to confirm and extend key findings of the TB phenotyping, adoptive transfer, and DIM-C treatment experiments, and to enhance these studies with more detailed outcome measures. We also plan to perform single cell RNA sequencing of lung leukocytes and spatial transcriptomic studies in Mtb-infected NR4A1-/- and WT mice as a first step towards identifying and prioritizing mechanistic hypotheses on which to base a future RO1 proposal. Accordingly, we describe a research plan that is feasible to complete within two years with the deliverables of an initial publication and a competitive RO1 application.