PROJECT SUMMARY PROJECT 1 The main objective of our U19 proposal is to define the contribution of airway epithelial cells to age-related dysfunction of the tissue-resident immune system, particularly in the context of lung responses to viral infections. Indeed, age is a risk factor for increased susceptibility to infectious diseases, and was most recently brought to focus by the COVID-19 pandemic, with poor outcomes disproportionately affecting individuals 65 years and older. Recently, we found that differentiated airway epithelial cells from older individuals have a baseline transcriptional program that is characterized by enhanced inflammation and is very distinct from young adult airway epithelium. Moreover, in response to influenza virus infection, young tissues induced a more potent interferon antiviral signature compared to old epithelium. Thus, we hypothesize that age-related epithelial changes contribute to chronic inflammation and altered tissue-resident lung immunity. We will leverage ex vivo air-liquid-interface (ALI) cultures, derived from airway epithelial progenitors, to study the effects of aging on airway epithelial viral responses and ensuing anti-viral immunity. We will determine how age impacts the response of broncho- epithelial cells (BECs) to virus, the fate of viral antigen, and how CD8+ T-cell immunity is modulated in aging tissues (Aim 1). Through mechanistic studies of antigen presentation and immune responses, coupled with deep analysis of epithelial-cell intrinsic mRNA isoform repertoire (Aim 2), our goal is to understand the molecular mechanisms that shape immunological status and early response to virus in epithelial cells and how their alterations in older individuals predispose to excessive inflammation and disease during viral infection. We will apply innovative technologies including 3D human lung tissues, functional studies based on precision-cut lung slices, spatial transcriptomics, as well as long-read RNA-sequencing (LR-seq) to address our hypothesis. Isoforms and functional endpoints will be analyzed in the context of the epigenetic status of ALI cultures influenced by the microbiome (Project 2) to identify genes and pathways dysregulated with aging. Integrative, multimodal data analyses will be carried out by the Data Science Core. Genes and isoforms selected during studies herein will be edited in Tech Dev Aim 3 in progenitor cells or induced pluripotent stem cells (iPSCs), thereby enabling functional validation in subsequent ALI cultures. Ultimately, our results will guide functional validation of a fully developed breathing lung model in Tech Dev Aim 1 for future studies of human lung immunology. The proposed studies will enable us to uncover the molecular mechanisms underlying immune dysfunction in the lung of older adults, potentially identifying new targets for preventive intervention.