The hygiene hypothesis states that there is an inverse correlation between exposure to microbes and the development of autoimmunity, i.e., the “cleaner” your environment is, the higher probability autoimmunity will develop. The reason for this is unclear. Thus, there are outstanding gaps in our knowledge about the hygiene hypothesis, including what changes occur in the host after microbial exposure and how this affects self-specific immunity leading to decreased autoimmunity. We have replicated aspects of the hygiene hypothesis using tractable, antigen-specific mouse models of infection and autoimmunity. Our data show that previous induction of T cell immunity under different conditioning contexts (infection or interaction with self-antigen) drives decreased self-specific CD8 T cell activation months later. The presence of anergic OTI T cells decreases future autoimmune pathology, as both the proliferation and function of self-specific CD8 T cells is blunted. We observe the same outcome in LCMV immune mice and in `dirty' mice. Previous immune experience is linked to earlier induction of a tolerant signature in responding self-specific CD8 T cells. This is specific for self-antigen, as foreign-antigen-specific T cell responses are unaffected. These aims will test the hypothesis that previous T cell responses alter self-antigen production and/or presentation in lymphoid tissue to speed tolerance induction. Aim 1 will determine whether changes in stromal cells in lymph nodes contribute to decreased self-specific CD8 T cell activation in immune-experienced mice. OTI cell division in iFABP-ova mice is reduced 60h post priming in inguinal LN of immune-experienced hosts, which is driven by non-migratory cells. As stromal cells in LN can produce tissue-restricted antigens, changes in these cells may negatively affect self-specific CD8 T cell activation. We will evaluate the number, epigenetic and phenotypic signature of LN stromal cells in naïve and immune-experienced mice and test if type I IFN, IFN, IL-6 or TNF, are important for the observed changes. Aim 2 will establish whether increased self-antigen presentation on LN stromal cells is responsible for decreased autoimmunity. We will evaluate expression of tissue-restricted antigens in immune-experienced animals, as well as factors important for their presentation, such as AIRE and DEAF1. Expression of these will be quantified in conjunction with staining for Kb-SIINFEKL complexes, as well as in vivo imaging of T cells in live mice. Overall, this application will determine the novel mechanisms by which self-antigen production or presentation is altered due to previous environmental programming from the adaptive immune response. It also addresses the important, but mechanistically unresolved, hygiene hypothesis, a fundamental topic intersecting TCR responsiveness with environmental impact, leading to vastly different outcomes of tolerance or autoimmunity.