PROJECT SUMMARY Tuberculosis (TB), caused by the bacterium, Mycobacterium tuberculosis, currently 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. In turn, development of efficacious vaccines is complicated by incomplete understanding of the correlates and mechanisms of protective immunity to M. tuberculosis and by the limited knowledge that has been gained from studies in C57BL/6 (B6) mice. The development of Collaborative Cross mice, together with initial studies that reveal that CC001 and CC002 mice are more able to clear M. tuberculosis than are B6 mice, provides the opportunity to better understand the mechanisms of immunity to TB in a highly tractable and economical experimental model. Four elements provide the basis for our proposed approach to using CC001 and CC002 mice to better understand the mechanisms of protective immunity to TB. They are: 1) M. tuberculosis resides in macrophages and other antigen-presenting cells (collectively termed mononuclear phagocytes) in the lungs; 2) the superior control of M. tuberculosis in CC001 and CC002 mice is observed after development of adaptive (T cell) immunity; 3) CD4 T cells are essential for protective immunity to TB in mice and humans; 4) CD4 T cells must interact with macrophages and other mononuclear phagocytes (MNP) to provide protective immunity to TB. Therefore, we propose a two-part working hypothesis: i) CD4 T cell responses are more effective against M. tuberculosis in CC001 and CC002 than in B6 mice; and ii) CD4 T cell responses are more effective due to superior antigen-presenting and/or antimycobacterial activities of MNP in CC001 and CC002, compared with B6, mice. To test that two-part hypothesis, we will use established and innovative tools and methods to perform comparative studies of specific subsets of MNP in the lungs of M. tuberculosis-infected CC001, CC002, and B6 mice. Our studies will include determining whether specific subsets of MNP in the lungs of CC001 and CC002 mice are more capable of killing M. tuberculosis in vivo and whether they are more capable of activating M. tuberculosis-specific CD4 T cells than are their counterparts in B6 mice. Our studies are designed to generate quantitative data on the immunological phenotypes of CC001 and CC002 mice that account for their superior control of M. tuberculosis, and to determine whether the mechanism(s) of superior immunity in the two strains of mice are similar or are distinct. Furthermore, our studies are designed to provide quantitative data that will facilitate phenotyping of mice generated during future studies to map and identify the causal genetic variants that account for superior TB immunity in CC001 and CC002 mice, to ultimately define molecular mechanisms that contribute to TB immunity. We anticipate that our discoveries will provide a basis for translational studies in humans, and that they will contibute to development of host-directed therapies and efficacious vaccines for TB.