Project Summary Effector CD8+ T cells restricted by the non-classical Class I-like molecule MR1 (MAIT cells) are a conserved T cell subset prevalent in humans. Because CD8+ T cells recognize and destroy target cells infected with pathogens, lung-resident subsets like MAIT cells may be important for early containment of bacterial infections of the airway. In fact, we find that MAIT cells are highly enriched in the airways, positioning them to rapidly respond to bacterially infected airway epithelial cells (AEC). MAIT cells recognize small molecule ligands generated by bacterial metabolic pathways that are presented on MR1 by many cell types, including AEC. The prevalence of MAIT cells in mucosal tissues, the ubiquitous expression of MR1, and the presumed abundance of small molecule ligands suggest that MR1 is tightly regulated to prevent inappropriate MAIT cell activation. Interestingly, we found that AEC can present antigen from both intracellular and extracellular pathogens to MAIT cells. Furthermore, we demonstrate that ligands from intracellular infection are presented on MR1 by different pathways than those generated from extracellular pathogens. For this application, we present evidence that presentation of MR1 ligands from intracellular pathogens involves endosomal recycling and requires proteins that regulate endosomal trafficking like Rab6. In contrast, presentation of exogenously added ligands occurs through Rab6-independent mechanisms. This proposal is thus focused on defining distinct mechanisms for MR1 antigen presentation upon infection with intracellular versus extracellular lung pathogens. Defining distinct mechanisms for presentation of these ligands may be key to understanding how MAIT cells sample the airway environment and become activated. This project contains two Aims: Specific Aim 1. Define how Rab6 regulates presentation of MR1 ligands derived from the intracellular lung pathogen M. tuberculosis. We will determine how Rab6 regulates expression or localization of MR1 in human AEC. Additionally, we will determine the role of Rab6 in regulating the maturation of the Mtb endosomal compartment in human AEC. Specific Aim 2. Identify and validate Rab6-independent mechanisms for presentation of MR1 ligands from the extracellular lung pathogens Streptococcus pneumoniae and Staphylococcus aureus. We will identify trafficking molecules in AEC that are critical to presentation of ligands from extracellular pathogens and determine how these molecules impact bacterial infection and cellular localization of MR1.