Single-cell atlas of axial skeletal immune-matrix interaction in spondylitis.

Axial skeletal involvement is central to ankylosing spondylitis, yet the in situ interplay between immune effectors (especially cytotoxic T lymphocytes) and the bone matrix microenvironment remains poorly resolved. Prior single-cell studies in peripheral compartments revealed exhaustion-resistant CTLs and dysregulated transcriptional regulators (e.g. NFKB, FOS, JUN) in AS patients, but lacked spatial and stromal context. We obtained axial skeletal tissue (vertebral bone marrow, enthesial fibrocartilage, adjacent endplate stromal tissue) from 24 AS patients with active disease and 12 age-matched controls undergoing spine surgery (total 108,752 single cells). We performed joint single-cell RNA-seq and ATAC-seq on matched samples, followed by integrative clustering, differential gene expression, chromatin accessibility analysis, pseudotime trajectories, and ligand-receptor network inference. Correlations with clinical indices (BASDAI, MRI inflammation scores) and imaging structural progression were assessed. We identified a discrete CTL subset in axial tissues with high expression of checkpoint molecules (PD-1, TIM-3), cytotoxic genes (GZMB, PRF1), and chromatin accessibility favoring NFKB/FOS motif enrichment, consistent with resistance to exhaustion. This subset increased by about 2.8 times in AS compared to controls (p < 0.001). Simultaneously, enthesial stromal fibroblasts displayed the upregulation of matrix remodeling genes (MMP9, COL1A1, COL3A1) and osteogenic drivers (RUNX2, BMP2). Ligand-receptor modeling demonstrated robust interactions between CTLs and stromal fibroblasts via TNF, TGFβ, integrin, and Notch signaling pathways. It is important to note that CTL-stromal interaction scores were related to MRI inflammation grade (r = 0.62, p = 0.003) and structural progression (r = 0.54, p = 0.01). This integrated single-cell atlas identifies a pathogenic CTL-stromal axis in the axial skeleton of AS patients, linking immune cytotoxic mechanisms with matrix remodeling and osteogenesis. These findings delineate actionable molecular crosstalk nodes that could inform precision therapeutic strategies aimed at immune-matrix interactions in situ.Clinical trial numberNot applicable.