Tuberculosis remains an important clinical problem throughout the world. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), is a facultative intracellular pathogen, residing primarily in macrophages. Protective immunity to tuberculosis depends upon the coordinated response of the cellular immune system. Because CD8+ T cells recognize and destroy target cells infected with intracellular pathogens, the CD8+ T cell response may be important for containment of Mtb and elimination of infected cells. Identification and characterization of human cytotoxic T cells may be essential for the understanding of immunity to tuberculosis, and hence may be required for the development of efficacious vaccines and improved therapeutic strategies. Work done by the PI and collaborators has defined mucosal associated invariant T cell (MAIT), T as an `innate' T cell population capable of recognizing the Mtb-infected cell. We find that while Mtb-reactive MAIT cells are enriched in the lung, they are nearly absent in those with TB. Recently, it has been proposed that MAITs recognize a vitamin B2 (riboflavin) metabolite presented in the context of MR1. However, by analyzing the repertoire of T Cell Receptor usage in response to a diverse array of pathogens, we find evidence of more diverse ligand recognition, finding confirmed via the direct analysis the known MR1 ligand with regard to T cell clones. Finally, we find that MR1 is found is discrete endosomal vesicles, and postulate that MR1 loading takes place via this endosomal environment. Indeed, given the presumed abundance of vitamin B and its metabolites, a critical question is the precise location and circumstances under which MR1 loading occurs. This proposal is predicated on developing an enhanced and comprehensive understanding of the role of MR-1 restricted MAIT cells in the human host response to infection with Mtb. Thus, this research proposal is focused on defining the full function and phenotype of human Mtb-reactive MAIT cells, and delineating the mechanisms by which MR1 becomes associated with its ligand. This project contains three Aims: AIM 1: Define the antigenic repertoire of MR1 ligands in the context of infection with Mtb. 1a. Determine the proportion of Mtb-reactive, MR1-restricted MAIT cells that respond to the known, salmonella ligand RL-6,7, diME. 1b. Define the role of MR1-ligands displayed by Mtb-infected cells. AIM 2: Define the phenotype of MAIT cells in the context of infection with Mtb and in the lung. AIM 3: Define the antigen processing pathway for MR1. 3a. Define the fate of newly synthesized and recycled MR1 and its relationship to antigen processing and presentation. 3b. Define the vesicular trafficking pathway used to present Mtb-derived MR1 ligands.