PROJECT SUMMARY Mycobacterium bovis is a member of the M. tuberculosis complex and the causative agent of tuberculosis (TB) in cattle and zoonotic infections in humans. Bovine TB is an excellent model for understanding TB in humans, as the diseases are parallel in many aspects of pathogenesis and innate and adaptive immune responses. Further, the study of bovine and human TB exemplifies the One Health approach as discoveries in both species have been closely intertwined throughout history. γδ T cells are a set of non-conventional CD3+ T cells that share important characteristics of both the innate and adaptive arms of the immune system. γδ T cells are particularly recognized for their ability to respond robustly to Mycobacterium infection. Several characteristics of γδ T cells, predominantly those functions commonly shared with αβ T cells, such as IFNγ production have been identified and characterized. However, it is increasingly apparent that γδ T cells have the capacity for a diverse array of immune functions including chemokine production, antigen presentation and regulatory cytokine production. These alternative immune functions are not well described in the context of TB infection and their role in vivo, particularly at the site of infection, remains poorly defined. Aim 1 of the proposed project employs a novel γδ T cell-alveolar macrophage co-culture system to model interactions that occur between immune populations at the site of M. bovis infection in the lungs. Next-generation RNA-Sequencing and transcriptome analysis will be performed on samples from the co-culture system to identify novel and alternative functions for γδ T cells responding to Mycobacterium infection. γδ T cells are hypothesized to play a critical role in granuloma formation and immune cell recruitment; however, given the difficulty of studying the immune response to TB in vivo, little is known about their capacity to promote improved disease outcome during TB infection. Therefore, Aim 2 proposes to utilize RNAScope, a commercial technology similar to in situ hybridization to identify local γδ T cell responses that correlate with increased resistance to infection, or improved outcome during TB. If successful, the RNA-Seq analysis in Aim 1 will further inform studies of γδ T cell functions in infected tissues. The proposed studies will be amongst the first to correlate γδ T cell responses in the peripheral blood, with those that occur in the local tissues during M. bovis infection. Effectively engaging γδ T cells in vaccine-induced protection from TB is expected to enhance the efficacy of candidate TB vaccines. Aim 3 proposes to test two vaccine platforms that specifically target unique γδ T cell-specific pattern recognition receptors, to determine if these platforms effectively engage γδ T cells in vaccine-induced immunity to TB. The knowledge gained from these studies will contribute to our understanding of the basic biology of γδ T cells an...