Integrated EVLP and single-cell profiling uncovers aberrant activation of tissue-resident lymphocytes and pro-fibrotic GZMK⁺ CD8 T cells in IPF

Tissue-resident immune cells are crucial in chronic lung diseases, yet a comprehensive profile in human lungs is lacking. Here, we defined alterations of resident immune cells in idiopathic pulmonary fibrosis (IPF), a fatal interstitial lung disease characterized by tissue inflammation and progressive scarring. Utilizing ex-vivo human lung perfusion coupled with single-cell RNA-sequencing, we successfully segregated the resident immune cells. Analyzing approximately 100,000 resident immune cells from 7 IPF and 5 control lungs, we identified 13 distinct cell types. Previously unrecognized aberrant lymphocyte phenotypes were uncovered. Specifically, among T lymphocytes, we observed an enrichment of GZMK ⁺ CD8 ⁺ T cells in IPF lungs, possessing a potential pro-fibrotic function. The fraction of pro-inflammatory HSP ^hi CD4 + T cells was increased in IPF lungs, while the quiescent subset decreased. Despite an increased presence of Tregs in IPF lungs, these cells showed reduced expression of genes associated with immune suppression. Moreover, significant B cell expansion and activation occurred, with continuous differentiation into IgG-producing plasma cells. Stromal niche interaction analysis showed that IPF fibroblasts, especially the CTHRC1 ^hi subset, exerted stronger effects on lymphocytes. These findings offer novel insights into dysregulated immune populations in IPF, advancing understanding of its immunopathology.