Immune dysregulation and exhaustion in EBV- associated hemophagocytic lymphohistiocytosis: insights from single-cell transcriptomics and TCR repertoire analysis.

INTRODUCTION: Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening hyperinflammatory syndrome paradoxically characterized by excessive cytokine production and immune activation despite failure to clear Epstein-Barr virus (EBV). The cellular mechanisms underlying immune dysregulation in EBV-HLH remain incompletely understood. METHODS: We performed single-cell RNA sequencing (scRNA-seq) and single-cell T-cell receptor sequencing (scTCR-seq) to characterize the peripheral immune landscape of patients with EBV-HLH. Key findings were validated by flow cytometry and enzyme-linked immunosorbent assay (ELISA) in an independent cohort of 37 patients with secondary hemophagocytic lymphohistiocytosis (sHLH). RESULTS: We identified EBV transcript-positive NK/NKT-like cells undergoing clonal expansion and proliferation as key contributors to immune dysfunction. These cells exhibited high CD160 expression, increased interferon-γ expression, and enrichment of IL-10-mediated signaling. In addition, monocytes acquired an immunosuppressive phenotype characterized by enhanced TGFB1 expression and increased migratory and phagocytic functions. This dysregulated immune environment may promote CD8+ T-cell exhaustion and abnormal clonal expansion of effector CD4+ T cells, ultimately contributing to EBV tolerance. DISCUSSION: This study delineates the peripheral immune landscape of EBV-HLH and identifies potential therapeutic targets for EBV-HLH.