The application proposes a career development plan for Dr. Kamal M. Khanna, a Damon Runyon post-doctoral Fellow in the laboratory of Dr. Leo Lefrancois. This proposal is centered upon understanding the mechanisms that mediate the migration and trafficking patterns of endogenous CD8 T cells after infection. Of the three specific aims, aim 1 will be completed in its entirety during the mentored phase. In addition, some preliminary experiments supporting aim 2 and 3 will also be conducted during the K99 phase, however both aim 2 and 3 will be largely completed during the independent phase of the award. Project summary: Although previous studies have examined splenic lymphoid architecture, the anatomical events driving primary and secondary CD8 T cell responses to infection have not been clearly delineated. Our work has begun to methodically examine large areas of splenic tissue without disturbing the individual structures and localization of cellular compartments within the organ, adding a new dimension to the analysis of immune responses. This type of imaging analysis allowed us to derive relevant and significant physiologic data concerning the movements, location and retention of an antibacterial endogenous CD8 T cell population. Our data reveal a step-wise progression of local cellular migration leading to CD8 T cell expansion, exit from the spleen and localization of resulting memory CD8 T cells in discrete splenic compartments. Imaging revealed previously unappreciated secondary encounters of daughter CD8 T cells with antigen-bearing dendritic cells in large clusters. Memory CD8 T cells did not undergo secondary activation events and large cluster formation but upon reactivation rapidly moved from the B cell follicles to the red pulp via bridging channels. Thus, these studies have set the stage for identification of the factors that control the processes driving each anatomical phase of the response. Therefore the overall goal of this proposal is to identify these factors and attempt to determine the roles they play in controlling the anatomy of a primary and secondary immune response. In doing so, we hope to explicate the underlying mechanisms that guide the complex movement of T cells during infection and recall responses in lymphoid and non-lymphoid tissues. The proposed studies will help us understand how a productive immune response takes place in vivo, and this information will provide clues to improving vaccine design.