Aberrant activation of tissue-resident lymphocytes and pro-fibrotic GZMK⁺ CD8⁺ T cells in IPF

Tissue-resident immune cells play a crucial role in chronic lung diseases, yet a comprehensive profile of these cells in human lungs is lacking. Here, we explore alterations of resident immune cells in idiopathic pulmonary fibrosis (IPF), a fatal lung disease characterized by tissue inflammation and progressive scarring. Utilizing ex-vivo human lung perfusion with single-cell RNA-sequencing, we successfully segregate the resident immune cells. By analyzing approximately 100,000 resident immune cells from seven patients with IPF and five healthy control lungs, we identify 13 distinct cell types. Previously unrecognized aberrant lymphocyte phenotypes are uncovered. Specifically, among T lymphocytes, we observe an enrichment of GZMK+ CD8+ T cells in IPF lungs, possessing a potential pro-fibrotic function. The fraction of pro-inflammatory HSPhi CD4+ conventional T cells increases in IPF lungs, while the quiescent subset decreases. Despite an increased presence of Tregs in IPF lungs, these cells show reduced expression of genes associated with immune suppression. Moreover, significant B cell expansion and activation occur, with continuous differentiation into IgG-producing plasma cells. Stromal niche interaction analysis shows that IPF fibroblasts, especially the CTHRC1hi subset, exert stronger effects on lymphocytes. These findings provide evidence of dysregulated immune cell populations in IPF, advancing our understanding of its immunopathology. EVLP coupled with scRNA-seq of IPF lungs reveals aberrant resident lymphocytes, including pro-fibrotic GZMK⁺CD8⁺ T cells, pro-inflammatory HSPhi CD4⁺ conventional T cells, dysfunctional Tregs, and activated B cells.