PROJECT SUMMARY/ABSTRACT The project goal is to understand how age-associated changes to the lymph nodes contribute to decreased survival of naïve T cells, a hallmark of immune aging. Naïve T cells are essential for resolving infections caused by newly encountered pathogens, but their number and frequency within the T cell population decrease dramatically with age. This contributes to reduced magnitude and delayed timing of the aged immune response against infectious disease. Naïve T cell survival requires constant signaling from the tissue environment, and it has been well-supported that the aged environment is defective in its ability to maintain naïve T cell homeostasis. Naïve T cells primarily interact with the tissues of the lymph nodes, organized tissue compartments strategically located throughout the body that serve as coordination centers for the immune response. Broad changes to the size and structure of the lymph nodes have been described with age, for both mice and humans; however, the mechanisms driving these changes, and how they impact naïve T cells, are unknown. Our data have revealed changes with age to the lymph node stromal cell type known as the fibroblastic reticular cell (FRC). Naïve T cells adhere to FRCs and crawl along the FRC network to navigate the lymph nodes. FRCs differentiate in situ in a process that requires continuous lymphotoxin-beta (LTβ) signaling. Mature FRCs express homeostatic cytokines that are essential for naive T cell survival and chemokines that promote interactions and motility within the lymph nodes. Homeostatic chemokines such as CCL21 have well-appreciated roles in promoting naïve T cell recruitment to and migration within the lymph nodes, but how their expression and distribution are impacted by age is not clear. In our preliminary work, we determined that lymph node stromal expression of Ltbr (lymphotoxin- beta receptor) and Ccl21 were significantly decreased with age. We also found evidence that FRC differentiation is impaired with age, in that early FRC precursors were decreased in frequency, and that there was an accumulation intermediate FRC precursors. The perturbation to FRC precursors was correlated to decreased expression of LTBR, suggesting that aged FRC precursors were unable to receive the LTβ signals necessary to complete maturation. We hypothesize that with impaired FRC differentiation, FRCs become fibrotic and spatially redistributed within the lymph node, which inhibits the interactions necessary for naïve T cell survival. In this project we will determine the role of age-associated inflammation in impairing signals through LTβR and promoting lymph node fibrosis (Aim 1), and whether increased fibrosis of the lymph node microenvironment impairs naïve T cell survival (Aim 2). Our experimental approach uses live-cell 2-photon microscopy, which enables us to visualize the interaction of naïve T cells with FRCs in real time within intact tissues. The completion of the project will suppo...