ABSTRACT-Project 2 During the process of age-associated thymus involution, T cell output progressively declines, contributing to a reduction in naive T cells in older humans and mice. The resultant decline in new T-cell responses to pathogens, vaccines, and cancers, leads to compromised immunity and increased morbidity with age. Thus, it is important to elucidate mechanisms underlying reduced thymic function in order to improve human health throughout the lifespan. In the past Project period, we found that early T-cell progenitors (ETPs), which give rise to all downstream thymocyte subsets, decline in numbers surprisingly early, by 3 months (mo) of age. ETP cellularity remains proportional to downstream thymocyte subsets at all ages, indicating the early decline in ETPs sets the stage for diminished thymopoiesis during thymic involution. Because ETPs are the direct progeny of circulating thymic seeding progenitors (TSPs), we anticipated a decline in the number of functional TSP niches by 3mo of age. However, our data reveal that the available TSP niches do not decline through at least 12mo of age, raising the question of what mechanisms underlie the early reduction in ETPs. Our preliminary data suggest that changes in the BM and thymus microenvironments contribute to the early loss in ETPs. Although the underlying mechanisms are not yet resolved, preliminary data suggest diminished NOTCH signaling in BM lymphoid progenitors and thymic ETPs contribute. Thus, we will use genetic mouse models and advanced imaging approaches to test if reduced expression of Notch ligands, and/or changes in other signals identified through additional single-cell transcriptional profiling (Core B), are responsible for the early decline in lymphoid progenitors in the BM (Aim 1) and ETPs in the thymus (Aim 2), and whether comparable mechanisms impact the aging human thymus (Core C). We will also test if restoring NOTCH signaling rescues cellularity of BM lymphoid progenitors, ETPs and thymocytes, and downstream T cell function in aging mice (P3 and Core D). Changes in the cellular composition and organization of the thymic microenvironment during involution may impact not only the quantity, but also the quality of developing T cells with age. During the previous Project period, we found that by middle age, the thymus becomes impaired in its ability to support central tolerance through both negative selection and regulatory T cell generation, particularly in response to low avidity self- antigens. New onset autoimmunity also peaks at middle age in humans, suggesting a role for impaired central tolerance. Thus, we will analyze current single-cell transcriptional profiling datasets from P2 and P1 (Core B) and perform live 2-photon imaging and thymic slice assays to identify age-associated changes in the mouse and human (Core C) thymic microenvironments that impair central tolerance and increase autoimmune susceptibility by middle-age (Aim 3). P2 is tightly integrated with...