PROJECT SUMMARY/ABSTRACT Tissue-resident memory CD8+ T cells have now been shown to form and persist in nearly every type of non- lymphoid tissue. Because these cells are permanently positioned at environmental barrier surfaces such as the skin, they are believed to be critically important for host defense against pathogens. In fact, tissue-resident memory T cells are more protective than memory T cells in the circulation against a variety of viral and bacterial infections. Infection of the skin with Vaccinia virus (VacV) has emerged as an attractive model system to interrogate the mechanisms that control the formation of highly functional tissue-resident T cell populations. We have recently demonstrated that local recognition of antigen in the tissue microenvironment is critical for the formation of tissue-resident memory CD8+ T cells in non-lymphoid tissues. Specifically, we showed that after effector CD8+ T cells enter the VacV-infected skin, a second antigen encounter causes rapid expression of CD69 and retention of these T cells in the non-lymphoid tissue. In this proposal, we will now define the molecular and transcriptional mechanisms that control the formation of tissue-resident memory CD8+ T cells in the skin microenvironment during a viral infection, as the fundamental immunology that regulates T cell function and memory differentiation in non-lymphoid tissue remains largely undefined. To address this question, we will 1) determine how T cell receptor affinity/avidity for antigen shapes the composition of the tissue-resident memory T cell compartment, 2) determine if a Blimp1/IL-10 signaling axis controls the formation of tissue-resident memory T cells in the skin, and 3) identify the relevant antigen- presenting cells (both hematopoietic and non-hematopoietic) that regulate tissue-residency by presenting peptide and/or other survival factors to CD8+ T cells in the skin during viral infection. The overall goal of this project will be to define the mechanisms that regulate both the formation and function of tissue-resident memory T cell populations, which will contribute to our long-term goal of improving vaccine design and identifying strategies for the treatment of inflammatory disorders of the skin.