Project Summary Thymic T cell development produces peripheral T cell receptor repertoires with significant residual reactivity against our self-antigens. Several mechanisms have emerged to manage this self-reactivity and prevent overt auto-immunity, including T cell anergy and deletion, as well as the activities of regulatory T cells. Failure of such tolerance mechanisms is thought to underlie many autoimmune disease. In studying the naive CD8+ T cell pool in mice, we have recently observed that a fraction of naive T cells are – under homeostatic conditions – exposed to active TGF-b during MHC-I-dependent interactions with migratory dendritic cells in lymph nodes. This TGF-b exposure pre-conditions naive CD8+ T cells to efficiently form epithelial-resident memory T cells (eTRM) upon immune challenges, such as skin vaccination against viral pathogens. When this pre-conditioning process is disrupted, e.g. in the absence of lymph nodes, by preventing dendritic cell migration to lymph nodes, or by deleting TGF-b-activating aV-integrins in dendritic cells, formation of eTRM in skin is strongly reduced, causing diminished antiviral protection. In preliminary studies for this project we have discovered that homeostatic TGF-b conditioning occurs preferentially in weakly self-reactive CD8+ T cells, while more strongly self-reactive cells are not conditioned. Mechanistically, TCR-dependent ERK activity appears to restrict TGF-b signaling through an inhibitory phosphorylation of the linker region in Smad2 and Smad3 transcription factors that prevents their nuclear translocation and effects on gene transcription. Based on these observations we hypothesize that homeostatic TGF-b signals that condition naive CD8+ T cells for the formation of long-lived epithelial resident memory cells are restricted to cells that pose the lowest risk of causing autoimmunity. In this project we will test this hypothesis by comparing TCR self-reactivity in naive CD8+ T cells as well as the eTRM pools in skin and other barrier tissues, and by examining epigenetic changes that characterize conditioned naive T cells and their relation to gene programs activated during eTRM differentiation. If our hypothesis is confirmed, our studies could reveal a new mechanism by which auto- reactivity in the peripheral TCR repertoire is managed. Breakdown of this mechanism could underlie autoimmune diseases that manifest in barrier tissues such as the skin.