Single-Cell Analysis Reveals Novel Immune Perturbations in Fibrotic Hypersensitivity Pneumonitis

Rationale: Fibrotic hypersensitivity pneumonitis (FHP) is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in FHP in single-cell resolution. Methods: Single-cell 5' RNA sequencing was conducted on peripheral blood mononuclear cells and BAL cells obtained from 45 patients with FHP, 63 patients with idiopathic pulmonary fibrosis (IPF), 4 patients with nonfibrotic hypersensitivity pneumonitis, and 36 healthy control subjects in the United States and Mexico. Analyses included differential gene expression (Seurat), TF (transcription factor) activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3 and Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and control subjects and 88,336 BAL cells from 19 patients were profiled. Compared with control samples, FHP has elevated classical monocytes (adjusted-P = 2.5 × 10^-3) and is enriched in CCL3^hi/CCL4^hi and S100A^hi classical monocytes (adjusted-P < 2.2 × 10^-16). Trajectory analyses demonstrate that S100A^hi classical monocytes differentiate into SPP1^hi lung macrophages associated with fibrosis. Compared with both control subjects and IPF, cells from patients with FHP are significantly enriched in GZM^hi cytotoxic T cells. These cells exhibit TF activities indicative of TGFβ and TNFα and NFκB pathways. These results are publicly available at http://ildimmunecellatlas.com. Conclusions: Single-cell transcriptomics of patients with FHP uncovered novel immune perturbations, including previously undescribed increases in GZM^hi cytotoxic CD4⁺ and CD8⁺ T cells-reflecting this disease's unique inflammatory T cell-driven nature-as well as increased S100A^hi and CCL3^hi/CCL4^hi classical monocytes also observed in IPF. Both cell populations may guide the development of new biomarkers and therapeutic interventions.