Project Summary Tuberculosis (TB), caused by the bacterium, Mycobacterium tuberculosis, kills more humans every year than does any other infectious disease, including HIV. Among the obstacles to eliminating TB is the lack of a sufficiently-efficacious vaccine. Despite strong evidence for essential roles of CD4 T cells in TB immunity, naturally-occurring T cell responses do not reliably eliminate the pathogen in TB, and we do not fully understand the mechanisms that limit the effectiveness of CD4 T cell responses to M. tuberculosis. In earlier work, we discovered that for optimal immune control, CD4 T cells must directly recognize and make intermolecular contacts with M. tuberculosis-infected cells at the site of infection in the lungs. In light of this requirement for intimate contact, we and others have found (in mice, nonhuman primates, and humans) that CD4 T cells are located at the periphery of granulomas, rather than in the core where infected cells reside. We therefore hypothesize that a major mechanism limiting immunity to TB is the failure of CD4 T cells to contact and engage with infected cells, and that overcoming this failure will increase the efficacy of T cell immunity to TB. Several potential mechanisms can explain the spatial separation of M. tuberculosis-infected cells and CD4 T cells in vivo, yet these are not amenable to analysis using fixed, static images or cells removed from the granuloma environment. Therefore, in this project, we propose to combine a unique multiphoton microscopy system for intravital imaging, contained in a Biosafety Level 3 facility at the IPBS in Toulouse, with unique reporter mice and M. tuberculosis strains developed and characterized at UCSF, to characterize interactions of antigen-specific CD4 T cells with M. tuberculosis-infected and bystander cells in the lungs of live mice. The combination of these innovative experimental systems allows us to test specific hypotheses that can account for the spatial and functional separation of CD4 T cells and M. tuberculosis-infected cells in the lungs. The knowledge gained from these studies will be used: 1) to inform design of experiments to define the molecular mechanisms that restrict CD4 T cell interactions with M. tuberculosis-infected cells in vivo; 2) to provide the basis for studies comparing distinct candidate TB vaccines, to determine which of them promote development of T cells that optimally access M. tuberculosis-infected cells in granuloma cores. In additional experiments, we will extend recent findings indicating important roles for innate lymphoid cells (ILC) in protective immunity to TB. Specifically, we will follow up on findings that type 2 ILC (ILC2) undergo dramatic phenotypic transformations to ILC1-like cells in vivo in response to M. tuberculosis infection, by live imaging of the interactions of ILC2 and ILC1-like cells with M. tuberculosis-infected cells and with CD4 T cells, during the initial and the chronic stages of infection. Our st...