The major barrier to long term survival following lung transplantation is a progressive loss of lung function, termed chronic lung allograft dysfunction (CLAD), for which constrictive fibrosis in small airways is a pathologic hallmark. CLAD affects over half of lung transplant recipients by 4 years post-transplant and negates much of the quality of life and functional improvements associated with transplantation. Pitt, Toronto, and UCSF Lung Transplant programs have refined transcriptional analysis of small airway brushings from lung transplant recipients as a novel technique to understand the gene expression changes at the anatomical site where CLAD pathology develops. We have published gene expression changes associated with CLAD validated across our centers. This proposal leverages this innovative approach to understand mechanisms of CLAD pathogenesis. Our preliminary data show an early upregulation of hypoxia pathways in airway brushings including genes that recruit and activate cytotoxic T lymphocytes using both airway epithelial cell culture in hypoxic conditions and pathway analysis of airway brush transcriptomes. This hypoxia signaling may reflect disordered microvasculature, absent bronchial circulation, and vascular inflammation associated with lung transplant. In recruited T lymphocytes, we also observed upregulation of tumor necrosis factor superfamily (TNFSF) genes, which are major drivers of apoptosis in lymphocyte targets. Our data show preferential apoptosis in airway club cells, the protectors and progenitors of small airways, in association with upregulated TNF-related apoptosis- inducing ligand (TRAIL) expression. Our single cell investigations in airway brushings and bronchoalveolar lavage (BAL) fluid show the segregation of these pathways across epithelial and lymphoid cell types. Based on these data, we hypothesize that airway hypoxia precedes TNFSF gene expression and T cell-mediated airway club cell apoptosis that drive CLAD pathogenesis. To test this hypothesis, we will generate parallel cohorts investigating bulk and single cell transcriptomes of CLAD versus controls across three centers, allowing for rigorous cross-validation of gene expression signatures. We will complement these studies with cell culture- based methods to determine mechanisms driving these gene expression changes. In Aim 1, we will quantify hypoxia-related transcripts in airway brush cells with respect to CLAD and determine how hypoxia can promote lymphocytic inflammation. In Aim 2, we will determine the cellular sources and kinetics of TNFSF co-stimulatory molecule expression in CLAD using airway brushes and BAL fluid. In Aim 3, we will investigate whether TRAIL preferentially induces club cell apoptosis. The synergy of three large lung-transplant translational research programs with world-class cross-institutional biostatistical infrastructures provides a unique opportunity to address this hypothesis rigorously. The cell-specific gene expression sig...