Single-cell sequencing reveals enrichment of cross-presenting dendritic cells in fibrotic hypersensitivity pneumonitis

<bold>Introduction:</bold> Dendritic cells (DC) are hypothesized to play an important role in antigen-presentation in hypersensitivity pneumonitis (HP). Single-cell RNA sequencing (scRNA-seq) may provide insight on DC involvement in HP. <bold>Objectives:</bold> To identify distinct DC subpopulations, present in the bronchoalveolar lavage fluid (BALF) of fibrotic HP (fHP) patients using scRNA-seq. <bold>Methods:</bold> We performed scRNA-seq on BAL samples from 4 patients with fHP, 3 with idiopathic pulmonary fibrosis (IPF), 2 with nonspecific interstitial pneumonia (NSIP), and 1 control. Cell ranger and Seurat pipelines were used to perform computational analysis. <bold>Results:</bold> Overall, 43976 cells were subjected to unsupervised clustering (Fig 1A). Five distinct DC clusters were identified (Fig 1B). FHP patients had higher proportions of CLEC9A+ (Fig 1C) and Langerhans (Fig 1D) DC, compared with IPF and/or NSIP. Macrophages of fHP patients exhibited increased expression of chemokines known to recruit these DC, including CCL20, CXCL9, CXCL10, CXCL11 (Fig 1E). Across all ILD patients, CLEC9A+ DC highly correlated with regulatory T cells levels (r=0.66, p<0.01), suggesting a possible connection to T cell response (Fig 1F). <bold>Conclusion:</bold> Langerhans and CLEC9A+ DC, were enriched in fHP BALF. Interestingly, these DC cells are capable of cross-presenting exogenous antigens to both CD4+ and CD8+ T cells, which may be important in HP pathobiology.