PROJECT 3- Abstract Naïve T cells (Tn) are produced in the thymus, but require peripheral mechanisms to maintain their homeostasis and function. The premise of this project is that, while thymic involution is a proximal cause of reduced Tn numbers with aging, defects in peripheral maintenance mechanisms in secondary lymphoid organs (SLO) also significantly contribute to immunosenescence. Indeed, in the past project period we have demonstrated that uncorrected defects in peripheral maintenance of Tn cells can powerfully undermine the benefits of reawakening T cell production by the rejuvenated thymus. We further pinpointed a precise chronological sequence of degenerative structural and functional changes in different SLO. This renewal application will continue to dissect mechanistic molecular and cellular basis of SLO aging and regeneration. Based on the order of changes and the evidence supporting cross-talk between Tn cells and SLO stroma, we hypothesize that reduced thymic production of Tn cells reduces trophic signals to SLO stroma, leading to initial stromal niche and network disorganization, that in turn deprives Tn cells of vital trophic signals and initiates a negative feed-forward loop of deterioration in both Tn cell and SLO stromal responses. Our specific aims are: SA1. To elucidate molecular changes in lymph node stroma with aging and IL-7 complex rejuvenation, using a hierarchy of in vitro and in vivo approaches to test a pipeline of candidates identified by miniarray and scRNASeq approaches in the past support period; and SA2. To dissect the intrinsic and extrinsic age-related defects in SLO stromal cells and the role of Tn:stromal crosstalk in LN homeostasis with aging, by examining the roles of oxidative stress, senescent cell accumulation, persistent Tn cell influx and natural polymicrobial exposure (to pet store mice) in LN/SLO aging. Once the defects are dissected, we will formulate interventions that improve peripheral T cell maintenance in aged organisms. These interventions will be tested by Core D, individually or combined with thymic rejuvenation treatments coming from P1&2, for the ability to improve protective immunity against infection. Together with powerful analytical pipelines from Cores A&B, as well as human molecular target verification by Core C, that will correlate age-related changes in mouse T cell and lymphoid organ aging to those in humans, this will provide direct preclinical data that are expected to pave the way for human T cell rejuvenation in older adults.