Abstract

BACKGROUND Immune checkpoint inhibitors (ICIs) show promise in hepatocellular carcinoma (HCC) treatment, but clinical responses remain limited due to immune evasion. CD8+ T-cell dysfunction is a key mechanism of immune evasion in HCC. Interferon-stimulated gene 15 (ISG15), a ubiquitin-like modifier, is implicated in tumor progression, but its role in immune suppression in HCC is unclear. METHODS ISG15 expression was analyzed in the TCGA-LIHC cohort, and its correlation with prognosis, immune infiltration, and checkpoint-related gene expression was evaluated. Single-cell RNA sequencing identified ISG15 expression in CD8+ T-cell subsets. Immunohistochemistry and multiplex immunofluorescence were performed on clinical HCC samples, focusing on tumor recurrence. In vitro, ISG15 overexpression and knockdown models in HepG2 and MHCC97L cells assessed effects on CD8+ T-cell viability, cytokine production, and cytotoxic activity. PD-L1 blockade assays were performed in the ISG15-overexpression group to assess the functional involvement of PD-L1. RESULTS ISG15 was upregulated in HCC and was associated with poor prognosis, higher TIDE scores, and increased immune checkpoint gene expression. Single-cell analysis identified ISG15-enriched CD8+ T-cell subsets with IFN/ISG-related and exhaustion-associated features. In co-culture assays, tumor-cell ISG15 overexpression was associated with reduced CD8+ T-cell viability, cytokine secretion, and cytotoxicity, together with increased PD-L1 expression and anti-apoptotic proteins. PD-L1 blockade partially restored CD8+ T-cell effector function. CONCLUSIONS ISG15 is associated with an immunosuppressive phenotype in HCC and may represent a candidate biomarker and potential target for further investigation in HCC immunotherapy.