PROJECT SUMMARY Immune system dysfunction has been implicated in the pathogenesis of several age-related lung diseases. Interestingly, multiple recent studies have identified the tissue microenvironment as playing a critical role in the pathogenesis of some of these age-related immune system changes. The lung mesenchyme provides a supportive niche for the resident immune system. Therefore, to identify aging-related changes in the lung mesenchyme that might contribute to immune system dysfunction, we performed single-cell RNA-sequencing of lung mesenchyme from aged versus young mice, identifying evidence of nuclear-factor kappa-B (NF-kB) activation in aged relative to young adventitial fibroblasts. NF-kB is an important regulator of inflammation and cellular senescence, a multifaceted cellular stress response characterized by several features including permanent cell cycle arrest, expression of the cell cycle inhibitor 16INK4a, and a complex secretory profile known as the senescence-associated secretory phenotype. Using a novel p16INK4a reporter mouse to identify cells with senescent features in vivo, we found that p16INK4a+ lung fibroblasts were enriched for NF-kB activation and accumulated in the parenchyma with age. Therefore, to study the effects of mesenchymal NF-kB activation in regulating the immune system, we used a genetic model to conditionally delete Tnfaip3, which encodes an important negative regulator of NF-kB signaling, from the mesenchyme. Preliminary data in this proposal demonstrates that mesenchymal deletion of Tnfaip3 leads to the accumulation of CD8+ T cells within the lung adventitial mesenchyme of young mice, and that these T cells transcriptionally resemble an aging-associated CD8+ T cell population (Taa). Therefore, the central hypothesis of this proposal is that NF-kB activation within senescent fibroblasts plays a direct role in driving lung Taa accumulation. Aim 1 is to identify the contribution of lung fibroblast senescence to Taa formation using our novel p16INK4a+ reporter mouse. Aim 2 focuses on identifying the mechanism by which mesenchymal NF-kB drives Taa formation, and Aim 3 will determine the functional consequences of Taa formation in the setting of viral pneumonia. The training plan for this proposal has been developed to achieve Dr. Allen’s goal of becoming an independent physician-scientist studying the interactions between the lung mesenchyme and immune system in health and disease. The plan involves formal didactics, conferences, mentorship, and hands-on experience though which Dr. Allen will develop expertise in cellular senescence, adaptive immunity, mouse models of pulmonary infection, the lung mesenchyme and associated epithelial stem cell nice, as well as responsible and effective research practices. She will be supported by a strong mentorship team with expertise spanning lung biology, cellular senescence, tissue resident lymphocytes, and NF-kB signaling. With completion of this Career Development ...