B100-02 BAL-Derived Airway Organoids Reveal Epithelial Changes Associated With Acute Lung Transplant Rejection

Abstract Rationale Acute cellular rejection (ACR) is a common complication after lung transplant that injures the lung allograft and worsens patient outcomes. ACR is treated with nonspecific immunosuppression, which carries many adverse effects. We sought to uncover mechanisms of epithelial injury, downstream of immune attack, that could suggest new approaches to treatment. We focused on the airway epithelium because it is the major site of injury in chronic rejection, for which acute rejection may represent a precursor. To obtain enough airway epithelial cells to study, we established lung organoids from transplant patients’ bronchoalveolar lavage (BAL) fluid. We then performed single-cell RNA-sequencing (scRNA-seq) on organoids to determine transcriptomic changes associated with ACR. Methods BAL was obtained from five lung transplant patients with biopsy-proven ACR and three patients without rejection. Using established methods, we generated airway organoids and expanded them for 2-5 passages. BAL-derived organoids were analyzed by scRNA-seq using standard Seurat packages. Results Airway organoids from patients with ACR contain four major cell types: basal, cycling basal, suprabasal, and secretory cells. Gene expression and pathway analysis across these four major clusters showed upregulation of inflammatory pathways in ACR and downregulation of homeostatic processes. We selected genes that were common across two or more clusters and validated their expression using RT-qPCR on seven additional organoid samples (5 rejection, 2 normal). ATP12A, POU6F2, HLA-DRB1, FMO2, and SLC44A5 were consistently upregulated. By RT-qPCR, we also observed an increase in KRT5 and decrease in MUC5B, which were not significantly changed in the scRNA-seq analysis. Conclusions BAL-derived airway organoids provide a source of live, primary lung epithelial cells to investigate gene expression changes associated with epithelial injury during ACR. RT-qPCR supported the upregulation of several key genes identified through scRNA-seq analysis. We will further refine our focus using immunofluorescence on transbronchial biopsy samples collected concurrently with BAL. By identifying molecular pathways involved in epithelial injury during ACR, we aim to test cellular phenotypes and novel interventions using our organoid models. This abstract is funded by: American Thoracic Society Unrestricted Research Grant 24-25U4; NIH KL2TR002374; NIH T32HL007633; NIH R35HL150876; UW Department of Medicine Critical Experiment Pilot Award; Gifts from the Virginia S. Bare family