A93-02 The Bronchoalveolar and Systemic Immune Landscape in Post-Covid-19 Residual Lung Abnormalities (RLA)

Abstract Introduction Residual lung abnormalities (RLA) following severe COVID-19 are increasingly recognized, yet the underlying pathomechanisms remain poorly understood. Here, we present an integrated single-cell multi-omic atlas of the bronchoalveolar and peripheral immune landscape in patients with post-COVID-19 RLA, revealing compartment-specific immune dysregulation persisting months after acute infection. Methods Single-cell RNA sequencing, CITE-seq, and TCR sequencing were performed on paired bronchoalveolar lavage (BAL) and peripheral blood mononuclear cell (PBMC) samples from patients with post-COVID-19 RLA (n = 16) and healthy controls (HC; n = 6) in the U.K. Analyses included differential gene expression (Seurat), trajectory inference (Monocle3, Velocyto-scVelo), and cell-cell communication (CellChat, NicheNet). Functional bioassays evaluated the fibrogenic potential of BAL fluid (BALF) from HC (n = 6), post-COVID-19 RLA (n = 16), and idiopathic pulmonary fibrosis (IPF; n = 5). BALF was applied in serial dilutions to primary human lung fibroblasts pre-incubated with or without nintedanib. Fibroblast proliferation (MTS/DAPI) and differentiation/activation (αSMA/COL1 staining) were evaluated. Results We profiled 125,334 BAL and 108,650 PBMC cells. Post-COVID-19 RLA BAL, compared with HC, demonstrated expansion of profibrotic SPP1+ monocyte-derived alveolar macrophages (MoAMs). A transcriptionally distinct cycling SPP1+MMP9+ subset was found exclusively in the Post-COVID-19 RLA cohort. RNA velocity and trajectory analyses indicated that SPP1+ MoAMs likely arise from an expanded HLA-DRloPDE4Dhi classical monocyte population in blood, transcriptionally primed for recruitment and profibrotic differentiation. Cross-compartment ligand-receptor analyses revealed bidirectional macrophage-monocyte crosstalk and inferred macrophage-fibroblast/myofibroblast interactions mediated by PDGFC-PDGFRA and FN1-integrin signalling. Lymphocyte remodelling was also observed, including depletion of pulmonary NK and MAIT cells across compartments, alongside expansion of circulating cytotoxic CD8+ T cells and Tregs. scTCR-seq demonstrated clonal expansion of CD4+ and CD8+ T cells in post-COVID-19 RLA across BAL and blood, with disease-specific dominant clones enriched in the lung, consistent with localised, antigen-driven immune responses. BALF from post-COVID-19 RLA patients significantly induced fibroblast proliferation (p 0.0001), with a median effect size of 35.87%, which was significantly attenuated by nintedanib, similar to IPF. Post-COVID-19 BALF also induced fibroblast differentiation and collagen deposition. Conclusions Circulating HLA-DRloPDE4Dhi classical monocytes appear to be progenitors of the profibrotic SPP1+ MoAM population in the lung. Given their expansion in post-COVID-19 RLA, both may serve as biomarkers of impaired lung repair and potential therapeutic targets. The enhanced mitogenic potential of post-COVID-19 BALF, attenuated by nintedanib, suggests fibroproliferative processes contribute to post-COVID-19 RLA pathogenesis. Collectively, these findings reveal persistent immune activation and profibrotic myeloid remodelling in post-COVID-19 RLA, highlighting therapeutic opportunities targeting both immune and stromal compartments. This abstract is funded by: UKRI