Epithelial and Immune Phenotyping of Distal Airway Cells in Lung Allograft Dysfunction

Abstract

<h3>Purpose</h3> Airway epithelial injury is thought to be the final common pathway leading to airway fibrosis in chronic lung allograft dysfunction (CLAD). While cumulative post-transplant inflammatory insults represent CLAD risk factors, we lack adequate methods to measure epithelial changes during such events. We hypothesize that early detection of allograft epithelial injury will reveal relevant pathways in CLAD pathogenesis and predict subsequent CLAD. We aimed to use transbronchial brushings (TBBr) to sample distal airway epithelial cells and assess their characteristics in the context of allograft dysfunction. <h3>Methods</h3> In this study, TBBr were collected from patients undergoing routine surveillance bronchoscopies at 3, 6, 9, and 12 months post-transplant, as well as patients with acute lung allograft dysfunction (ALAD), defined as a >=10% drop in FEV₁ compared to previous baseline. Cytospins were assessed with H&E and immunofluorescence stains (Figure 1A-G). Multi-colour flow cytometry panels were developed to identify populations of epithelial and immune cells (Figure 1H). <h3>Results</h3> In this cross-sectional analysis of TBBr from 22 patients (10 stable, 12 ALAD), we were able to identify important epithelial and mucosal immune cell subsets. Some patients with ALAD exhibited an increase in club cells, which act as epithelial cell progenitors and regenerate in response to epithelial damage (Figure 1I, p=NS). Additionally, a higher percentage of epithelial cells expressed the activation marker CD54 (ICAM-1) (Figure 1J, p=NS). In contrast, T and B lymphocytes were similar between patient groups. <h3>Conclusion</h3> This study shows that TBBr can successfully isolate airway epithelial and mucosal immune cells for relevant analyses of specific cell subsets and activation states. Trends were observed in club cells and epithelial cell populations at the time of ALAD as well as over time. Our multi-platform approach will generate new hypotheses on the role of epithelial cells in CLAD pathogenesis.