Single-cell multi-omic analysis of post-transplant mesenchymal cells reveals molecular signatures and putative regulators of lung fibrosis

Survival after lung transplantation is limited by chronic progressive graft failure, termed chronic lung allograft dysfunction (CLAD). Graft-resident mesenchymal cells (MCs) drive CLAD pathogenesis and exhibit stable, dysregulated signaling; however, the transcriptomic and epigenomic drivers behind this fibrogenic transformation remain elusive. Here, we utilize single-cell multi-omic technologies to study gene expression and chromatin accessibility of MCs from the lavage fluid of lung transplant recipients with and without CLAD, obtained either early post-transplantation or after disease onset. MCs obtained after CLAD onset (CLAD-MCs) demonstrated a unique transcriptomic signature compared to non-CLAD controls; a logistic regression model trained on these profiles classified the disease status of individual cells with > 98% accuracy using a set of signature genes. Chromatin accessibility and motif scan analysis identified the CCAAT-enhancer-binding proteins family of transcription factors, specifically CEBPD, as a key marker of the CLAD-enriched subtype. Footprint analysis of early time-point MCs revealed minimal differences in accessibility, suggesting that CEBPD-associated regulatory changes emerge over time after transplantation. Integration and unsupervised clustering identified 8 distinct cell states, and a compositional shift was noted uniquely in CLAD-MCs. Knocking down CEBPD with siRNA in CLAD-MCs partially reverted the CLAD transcriptomic signature, confirming its importance in the dysregulated molecular state of CLAD-associated MCs. scRNA-seq analysis on human lung CLAD tissue provided in situ validation of key genes and CEBPD expression changes noted in CLAD-MCs. Our results provide deeper insights into the transcriptomic and epigenomic changes in post-transplant MCs, nominating biomarkers and disease-associated factors with implications for future therapeutic efforts.