CD4 T cells provide immune defense against Mycobacterium tuberculosis infection. Control of M. tuberculosis infection also requires TNF, IFN-?, iNOS and TLR-mediated innate immune signals. CD4 T cells orchestrate cellular and cytokine-mediated effector mechanisms that inhibit M. tuberculosis growth in the mammalian host. Vaccine induced priming of long-term memory CD4 T cells specific for M. tuberculosis, therefore, is an important but, to date, incompletely met goal. The proposed experiments focus on an important question: Why are CD4 T cells induced by natural infection unable to eliminate M. tuberculosis from the host. We hypothesize that natural infection primes mixed populations of effector and regulatory CD4 T cells that restrict M. tuberculosis growth but do not lead to sterile immunity. To test this hypothesis, we generated T cell receptor transgenic mice specific for ESAT-6, an immunodominant M. tuberculosis antigen, and tracked ESAT-6 specific CD4 T cells during infection. Adoptively transferred, Th1 differentiated ESAT-6 specific CD4 T cells provide protection against aerosol infection, enabling mechanistic studies of CD4 T cell mediated protective immunity. Our first aim is to investigate the kinetics of clonal CD4 T cell activation, expansion and contraction at different times during the course of TB infection. These studies will determine whether the inability of clear M. tuberculosis infection results from too few specific T lymphocytes, attrition of specific T cells or loss of T cell effector functions during chronic infection. Our second aim is to determine which CD4 T cell effector functions are required for protective immunity. These experiments will determine whether IFN-3 and/or TNF production by TB-specific CD4 T cells are essential for protection, and will determine the impact of innate immune responses on activation and differentiation of ESAT-6-specific T cells. Our final aim is to identify approaches that optimize in vivo, CD4 T cell-mediated protection. We will induce migration of CD4 T cells to lung parenchyma and airways, optimize in vivo differentiation of ESAT-6 specific CD4 T cells and determine the contribution of Th17 CD4 T cells to protective immunity. These studies will provide unprecedented views of CD4 T cell responses to M. tuberculosis infection and will likely suggest new and practical approaches to optimize immunity against this pathogen.