PROJECT SUMMARY/ABSTRACT Approximately one-third of the world’s population is infected with Mycobacterium tuberculosis. These numbers are compounded by the extraordinary prevalence of human immunodeficiency virus. Given the emergence of multidrug-resistant TB, there is an urgent need for new TB vaccines that can either prevent or therapeutically treat Mtb. Further, provided the overlap in Mtb and HIV prevalence, we must understand how HIV dismantles immunity to TB. CD8+ T cells are an important component of TB control, but vaccines targeting conventional CD8+ T cells face hurdles that may require tailored vaccines. Here, we propose to study two novel populations of unconventional Mtb-specific CD8+ T cells, mucosal-associated invariant T cells (MAITs) and MHC-E restricted CD8+ T cells (MERTs) in NHPs. We will use a multifaceted approach to establish these populations as new universal TB vaccine targets in a physiologically relevant animal model of HIV/TB co-infection. MAITs are enriched in the lung, have immediate adaptive and innate-like immune function upon thymic egress, and contain early Mtb replication. MAITs recognize vitamin B metabolites in the context of MHC-related protein 1 (MR1). MR1 is a highly conserved, MHC-Ib molecule that acts as a sensor of bacterial infection. Similarly, MHC-E is a highly conserved, non-classical, MHC class Ib molecule. In healthy cells, MHC-E binds the VL9 leader peptide of MHC class I molecules and presents them on the cell surface, signaling to natural killer cells that antigen processing and presentation is proceeding normally. However, in the context of certain viral and bacterial infections, MHC-E can present a surprising array of pathogen-derived peptides to MHC-E restricted CD8+ T cells (MERTs). The goal of this study is to characterize these two mycobacteria-specific, unconventional T cell populations and to understand how immunodeficiency virus impacts their function. We have generated exciting preliminary data demonstrating the existence of MAITs and MERTs in rhesus macaques (RM). RM are an excellent model for studying novel TB vaccines and the impact of HIV infection. We hypothesize that IV BCG will drive antigen-specific MAIT and MERT expansion and that SIV will lead to BCG-specific MAIT and MERT dysfunction. We will define BCG-specific MAIT and MERT TCR usage and function after IV BCG vaccination and determine the impact of SIV on these cells using flow cytometry and single cell RNAseq (scRNAseq). This multipronged approach will assess MAITs and MERTs as universal TB vaccine targets and interrogate their sensitivity to immunodeficiency virus-induced immune destruction.