Abstract Background Chronic Lung Allograft Dysfunction (CLAD) remains the leading cause of mortality following lung transplantation. We previously established a ferret orthotopic left-lower lung transplantation model that recapitulates both obstructive and restrictive forms of human CLAD. To define the molecular and cellular mechanisms driving inflammation, injury, dysfunction, and fibrosis across lung compartments, we performed single-cell RNA sequencing (scRNA-seq) with tissues spanning healthy to end-stage CLAD. Methods Using the ferret lung transplantation model, we conducted single-cell transcriptomic profiling on cells isolated from naïve (pre-transplant) lungs and lungs collected after CLAD development. On the day of transplantation, cells were isolated from donor (non-transplanted) and recipient (explanted) lungs, as well as from the transplanted allograft and the native contralateral lung from each experiment. To capture compartment-specific populations, tissues underwent differential enzymatic digestion—Pronase for surface compartments and collagenase/hyaluronidase followed by TrypLE for submucosal compartments. Samples from four independent transplant experiments over a period of 8 months were fixed, permeabilized, cryopreserved, and processed together using a split-pool combinatorial barcoding method (Parse Biosciences) without employing cell-type selection. Results We obtained over 250,000 high-quality single-cell transcriptomes, identifying 88 distinct cell identities across four major lineages: 14 endothelial, 18 mesenchymal, 32 immune, and 23 epithelial subtypes. Allograft samples exhibited increased hematopoietic (myeloid and lymphoid) cell abundance, reflecting inflammation and immune activation, while epithelial populations declined, consistent with tissue injury and loss. Endothelial and mesenchymal cell proportions were relatively stable. However, differential enzymatic digestion was effective for enriching samples with endothelial and mesenchymal cell types as well as with epithelial submucosal gland cells, which were more abundant in the submucosal compartment. Immune cells were broadly distributed across both surface and submucosal compartments. Supporting our prior findings of submucosal gland destruction in CLAD, mucus, serous, and myoepithelial cell transcriptomes were greatly reduced in allograft samples. Distinct molecular signatures emerged, including upregulation of antigen processing and presentation pathways in both endothelial and epithelial cell populations, suggesting enhanced immunogenicity within these allograft microenvironments. Conclusions This single-cell transcriptomic atlas of ferret lung allografts provides a high-resolution view of cellular dynamics and gene expression changes underlying CLAD progression. Our findings implicate epithelial and endothelial antigen presentation as potential drivers of tertiary lymphoid organ formation near airways and vessels, offering mechanistic insight into immune-mediated injury. Ongoing analyses aim to validate these signatures and delineate differential responses between transplanted allografts and native lungs, advancing understanding of CLAD pathogenesis and identifying potential therapeutic targets. This abstract is funded by: 5R00HL155843
Lynch et al. (Fri,) studied this question.