Persistent and progressive acute lung allograft dysfunction is linked to cell compositional and transcriptional changes in small airways.

BACKGROUND Acute lung allograft dysfunction (ALAD) is a clinical syndrome of forced expiratory volume in 1-second (FEV1) decline concerning for chronic lung allograft dysfunction (CLAD) onset. Novel diagnostic tools are needed to identify those with ALAD who will progress to CLAD and to target appropriate therapies. We hypothesized that progressive ALAD would be associated with changes in small airway cell composition and cell-specific transcription. METHODS We prospectively identified recipients with undifferentiated ALAD and controls with stable allograft function for small airway brushing and single-cell RNA sequencing analysis. ALAD outcome group was categorized as (1) control (n = 8), or ALAD with (2) recovered (n = 4), (3) persistent (n = 5), or (4) progressive (n = 3) FEV1 decline. Cell compositional changes, pseudobulk Reactome pathways, and the AI2 score, previously linked to CLAD in airway brush transcriptomes, were assessed as a function of ALAD outcome group. RESULTS Across 68,140 cells, the distribution of cell composition was linked to ALAD outcome group (PERMANOVA, p = 0.004). Worse ALAD outcomes correlated with loss of basal cells, changes in club and ciliated subsets, a loss of macrophages, and expansion of cytotoxic T cells. The AI2 gene score was positively associated with ALAD outcome group, particularly in epithelial cell subsets (p < 0.001). Pathway analysis showed increased interferon signaling and inhibition of cell proliferation in epithelial cells. CONCLUSIONS In this pilot study, persistent and progressive ALAD was associated with changes in bronchiolar cell composition and transcriptional programs. Molecular phenotyping may help identify and characterize individuals with ALAD at increased risk for progression.