Project Summary: Project 2 Pulmonary tuberculosis (TB) is caused by infection with Mycobacterium tuberculosis (Mtb) and is a leading cause of death from a single infectious agent worldwide. Here we will characterize immune signatures (IMS) of Mtb antigen-specific CD4 T cells in individuals with different states of Mtb infection and BCG vaccination. Specifically, we will utilize our established Mtb peptide epitope pools and assay systems to identify antigen- specific CD4 T cells and their T cell receptors (TCRs). The IMS will be further characterized in cytometry and single-cell RNA-seq assays after antigen stimulation and ex vivo. For Aim 1, we will enroll cohorts of individuals with active tuberculosis (ATB), characterized by uncontrolled symptomatic Mtb infection, who will be receiving anti-TB therapy. We will longitudinally track the IMS over the course of treatment, and identify markers predictive of treatment outcomes. For Aim 2, we will enroll individuals with ‘latent’ TB infection (LTBI), which is characterized by immune reactivity to Mtb but no symptoms of disease (i.e. Mtb reactive). LTBI is heterogeneous, consisting both of individuals who have successfully cleared their infection and display a memory immune response, as well as others with subclinical infection at increased risk of developing active disease. There is currently no test available to distinguish subclinical from cleared infection. We will determine the IMS in prophylactically-treated LTBI longitudinally, and determine who displays a treatment response most similar to ATB, which we consider most likely to have had subclinical disease. For a separate cohort of LTBI, we will utilize a unique human challenge model to obtain samples with antigen-specific resident T cells from the lung and use them to evaluate overlap and differences between the IMS in the blood vs. lung from the same set of individuals. For Aim 3, we will obtain samples taken pre- and post-BCG (re)vaccination in adults to characterize the vaccine induced IMS. Revaccination of Mtb uninfected adults has been shown to be protective from disease, thus, comparing this IMS with that in LTBI (controlled infection) vs. ATB (uncontrolled infection) can reveal protective components of vaccine induced immunity.