Comprehensive Single-Cell Chromatin and Transcriptomic Profiling of Peripheral Immune Cells in Non-Segmental Vitiligo

Abstract Introduction Non-segmental vitiligo (NSV) is an autoimmune condition characterized by melanocyte loss. While skin-specific mechanisms are well-studied, systemic immune dysregulation contributing to NSV pathogenesis remains unclear. Objective This study employs a multi-omic single-cell approach to investigate circulating immune cells in NSV, integrating transcriptional and chromatin accessibility data. Methods An integrative scRNA-seq/scATAC-seq analysis was conducted on PBMCs from NSV patients (n=11) and controls (n=5), identifying transcriptional markers, cell-cell interactions, chromatin accessibility, and transcription factor (TF) dynamics. Key findings were validated in an expanded cohort (NSV, n=16; controls, n=9) using spectral flow cytometry, with additional stratification by sex, age, disease activity, severity, and duration. Results Analysis of 59,192 PBMCs identified 8,204 gene expression markers and 13,925 ATAC peaks across 25 immune cell subtypes. A broadly activated immune response was observed, characterized by cytotoxicity, antigen presentation, cell exhaustion, and stress, predominantly in monocytes, NK cells, CD8+ T cells, and dendritic cells (DCs). Multi-omic integration revealed Th1/Th17 polarization and dysfunctional regulatory T cell (Treg/mTreg) responses. Chromatin accessibility highlighted enriched TF binding sites for FOXO3, SP1, AP1, STAT1/STAT3, IRF1, and IRF4, regulating pathways linked to cytotoxicity, antigen processing, NF-κB, Toll-like receptor, and JAK-STAT signaling. Flow cytometry validated these findings, showing that disease activity and shorter duration were associated with heightened immune dysregulation. Robust TCR activation drove Th1/Th17 polarization and elevated IFN-γ and TNF-α production in CD4+ and CD8+ T cells. CLA+ skin-homing Th1/Th17-polarized CD4+ T cells, CD8+ T cells, and mTregs exhibited persistent activation, marked by basal PD1+ expression. OX40/OX40L-mediated interactions between monocytes and effector T cells amplified inflammation. Regulatory dysfunction, including reduced IL-4 and IL-13 production in mTregs, was prominent in moderate-severe and active disease. Conclusion This is the first multi-omic single-cell study in PBMCs of NSV patients, revealing systemic immune dysregulation driven by cytotoxicity, antigen presentation, exhaustion, and regulatory failure. Disease severity, activity, and evolution influence these pathways, highlighting the OX40/OX40L axis as a potential therapeutic target to mitigate immune dysregulation and relapse risk.