SUMMARY Mycobacterium tuberculosis (Mtb) is the pathogenic bacteria which causes tuberculosis (TB) disease and was responsible for 1.6 million deaths worldwide in 2021. It is well-established through human and animal studies that alpha-beta (αβ) T cells are important for the control of Mtb infection, but the role of gamma-delta (γδ) T cells is less well-defined. γδ T cells express T cell receptor (TCR) heterodimers comprised of a γ-chain and δ-chain that mediate recognition of a variety of lipids or small molecules presented by major histocompatibility-like ligands, such as cluster of differentiation 1 (CD1) or butyrophilin molecules. There is growing evidence that γδ T cells are important in the early protective immune response against Mtb in the lung. The central hypothesis of this application is that γδ T cells contribute to control of Mtb infection through recognition of mycobacterial lipids and promotion of an enhanced adaptive immune response. A subset of γδ T cells, termed Vδ1 cells, are enriched in tissue and have known reactivity to mycobacterial lipids presented by CD1 molecules. In AIM 1 we will evaluate the molecular mechanisms underlying Vδ1 T cell lipid antigen recognition through analysis of in vitro expanded clones and computational analysis of lipid-specific Vδ1 TCR motifs. By investigating specific TCR motifs governing recognition of mycobacterial lipids by Vδ1 T cells, we will determine shared TCR characteristics that will enable their identification in clinical cohorts. A major challenge to the field has been the lack of a suitable animal model to understand the role of γδ T cells in mediating protective immunity to Mtb. We will develop a novel in vitro granuloma model using the palatine tonsil, a readily accessible human mucosal tissue, and investigate the role of γδ T cells in mechanisms of bacterial control. Our preliminary data reveal that the tonsil contains γδ T cell subsets which respond to in vitro mycobacterial infection and specialized dendritic cells subsets that can prime T cell responses. Thus, in AIM 2 we will use tonsil cells to study γδ T cell interactions with dendritic cells in response to mycobacterial infection and develop an in vitro granuloma model and investigate the role of γδ T cells in granuloma organization. A tractable tonsil-based in vitro granuloma system will be an important contribution to the field and provides a unique system to interrogate the function of γδ T cells in response to Mtb. Together, our proposal will lead to increased understanding of γδ T cells and profile their involvement in the protective immune response to mycobacteria.