CD4 T cells are essential for protection against tuberculosis (TB). But current approaches to TB therapy have not harnessed their potential benefit, and the mechanisms they use to control Mycobacterium tuberculosis (Mtb) have not been completely defined. At the peak of adaptive immunity to TB, millions of Mtb-specific CD4 T cells traffic to the lungs, but very few of them re-encounter their cognate antigen to become activated. The ability to clearly discriminate and isolate these few activated cells from the greater population of inactive CD4 T cells could enable the discovery of important new markers, effector functions, and clonally expanded T cell receptor (TCR) sequences closely associated with protective immunity. To study activated CD4 T cells from Mtb-infected lungs, we used a new approach that combines: (i) a reporter mouse to identify cells actively receiving TCR stimulation in vivo (Nur77-GFP); (ii) BSL3-contained fluorescence cell sorting to isolate them live from tissues; and (iii) single cell RNA sequencing (scRNA-Seq) with CITE-Seq and TCR immune-profiling to interrogate their function and antigen-specificity. In our preliminary work, we discovered that Nur77-GFPHI CD4 T cells express an array of activation markers and costimulatory receptors including OX40. This population was enriched for T regulatory cells and effector T cells with TCR clonotypes localizing to lung parenchyma. Using adoptive transfer, we found that Nur77-GFPHI cells are more protective than their Nur77-GFPLO counterparts. To therapeutically harness activated CD4 T cells in vivo, we treated Mtb-infected mice with a monoclonal antibody that agonizes OX40 and found that this treatment reduced the lung bacterial burden, prolonged survival of infected mice by >100 days and did not cause toxicity. Immunotherapy specifically targeting activated CD4 T cells is a novel approach to TB treatment that has not been thoroughly investigated. In Aim 1 of this proposal, we will define the function of activated CD4 T cells in the lungs of Mtb-infected mice using scRNA-Seq and CITE-Seq. With adoptive transfer and scTCR-Seq, we will uncover how antigen specificity shapes the function and fate of different CD4 T cell TCR clonotypes throughout chronic infection. We will use TCR sequences we generate to develop TCR retrogenic mice that can identify protective Mtb antigens and test the hypothesis that antigen specificity shapes T cell phenotype in TB In Aim 2, we will determine the mechanisms underlying activation marker immunotherapy mediated CD4 T cell control of TB. We will test the hypothesis that OX40 agonist treatment provides protection by selectively modulating the function of both activated T conventional and T regulatory cells. Finally, we will determine how OX40 agonism impacts activated CD4 T cell survival through chronic infection. The studies proposed here represent the first high-definition characterization of the small population of activated CD4 T cells at the site of M...