Distinct epigenetic and transcriptional landscape of pathogenic CD44s-hiCD49dlo virtual memory CD8+ T cells in alopecia areata 2256633

Abstract Introduction Virtual memory (TVM) CD8+ T cells exhibit memory-like features without prior antigen exposure and provide protection. We previously identified a CD44s-hiCD49dlo subset that acquires pathogenic properties upon IL-12, IL-15, and IL-18 stimulation and mediates alopecia areata (AA) via NKG2D-dependent cytotoxicity. These findings reveal functional heterogeneity within the TVM compartment, suggesting that subsets can transition to a disease-promoting phenotype under inflammation. Based on these findings, we aimed to define the molecular and epigenetic heterogeneity of TVM cells and identify transcriptional regulators driving pathogenic CD44s-hiCD49dlo CD8+ T cell differentiation. Methods We performed joint single-cell RNA sequencing and ATAC sequencing on conventional TVM and CD44s-hiCD49dlo CD8+ T cells isolated from the lymph nodes and skin of AA mice. UMAP projection and clustering defined transcriptional heterogeneity, and pseudotime trajectory analysis inferred differentiation pathways. Integration of transcriptomic and chromatin accessibility data enabled the identification of transcription factors associated with pathogenic transition. Results Single-cell analyses revealed that conventional TVM and CD44s-hiCD49dlo CD8+ T cells form transcriptionally distinct populations. Clustering identified multiple subpopulations within the TVM pool, including a transitional subset located along a pseudotime trajectory leading toward the pathogenic state. Integrated gene expression and epigenomic profiling highlighted RUNX2, RUNX3, and RORα as candidate regulators orchestrating this differentiation program. Conclusion Taken together, these findings delineate a previously unrecognized trajectory from conventional TVM cells to CD44s-hiCD49dlo CD8+ T cells that drive inflammation in AA. The identified regulatory factors define the transcriptional and epigenetic framework underlying this pathogenic transition and represent potential molecular targets for therapeutic modulation of cytotoxic T-cell responses. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)