Combined Single-Cell and Spatial Transcriptomics Reveals the Cellular Landscape of Bronchiolitis Obliterans Syndrome in Chronic Graft-Versus-Host Disease Mouse Model

BACKGROUND: Chronic graft-versus-host disease (cGVHD) is an autoimmune-like syndrome that occurs after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The pathogenesis of the cGVHD involve complex cellular and molecular events network. Bronchiolitis obliterans syndrome (BOS) is one of the most severe complications of cGVHD, with a very poor prognosis.The lack of a detailed characterization of the network severely limits the development of targeted therapies for BOS cGVHD.

OBJECTIVE: This study aims to investigate the global transcriptional landscape of lung cell populations in the BOS cGVHD mouse model.

METHODS: By single cell RNA sequencing and spatial transcriptome, we profile lung cells from the BOS cGVHD mouse model to characterize cellular composition. Cells were processed using the 10× Genomics platform, and sequencing data were analyzed by Seurat. We identify distinct populations of epithelial, endothelial, mesenchymal, and immune cells, each containing distinct subpopulations. Furthermore, we compare changes in RNA expression patterns between cGVHD and control mouse to identify signaling pathways selectively activated in specific pulmonary cell types.

RESULTS: We present an interactive murine single-cell atlas of the mouse model(4 cGVHD mouse and 4 control mouse). Unsupervised analysis revealed distinct clusters, each characterized by specific signature genes. Notably, we observed a higher proportion of interstitial macrophages (IM) and CD8⁺ tissue-resident memory T(TRM) cells in cGVHD mice. To identify intrinsic alterations in potentially pathological cells, we performed differential gene expression analysis on all clusters, comparing cGVHD mice with control mice. Interestingly, several differential gene expression were shared across multiple immune clusters, indicating common molecular programs that might drive immune cell plasticity. Cell-cell communication and spatial transcriptomics analysis revealed crosstalk and co-localization between IM and CD8⁺CD69⁺Itgae^- TRM cells. Furthermore, we found that IM and CD8⁺CD69⁺Itgae^- TRM cells interact via the CXCL16 and CXCR6 signaling axis. Notably, neutralization of CXCL16 with anti-CXCL16 monoclonal antibody led to an alleviation in cGVHD symptoms.

CONCLUSION: Our study presents a comprehensive single-cell atlas of the in murine BOS cGVHD model. Differential gene expression analysis identified shared molecular signatures across multiple immune clusters, suggesting common pathways that may contribute to immune cell plasticity and disease progression. Notably, the interaction between CD8⁺CD69⁺Itgae^- TRM cells and IM through the CXCL16-CXCR6 axis plays a pivotal role in amplifying inflammatory signaling. Therapeutic targeting of this pathway led to a marked alleviation in cGVHD symptoms, highlighting its potential as a therapeutic strategy for cGVHD.

KEY WORDS: Single Cell RNA sequencing; Spatial Transcriptomics; Bronchiolitis Obliterans Syndrome, Chronic graft-versus-host disease.