ICOS Defines an Early Regulatory Exhaustion Immune Axis in HBV-Associated Hepatocellular Carcinoma

Abstract HBV-associated hepatocellular carcinoma (HCC) develops within a chronically inflamed tumor microenvironment. It is defined by persistent T-cell activation and checkpoint remodeling. The biological role of inducible T-cell co-stimulator (ICOS) in this context is still incompletely defined. The aim of this study was to evaluate whether ICOS defines a distinct T-cell transcriptional state, its position in T-cell differentiation, and its tumor-level immune relevance in HBV-related HCC. We performed a two-phase integrative transcriptomic analysis combining single-cell RNA sequencing of tumor-infiltrating T cells (GSE149614; 20,633 cells) with independent validation in TCGA-LIHC bulk RNA-seq data (n = 729 tumors). ICOS-based stratification was followed by module scoring, differential expression analysis, Hallmark pathway enrichment, diffusion pseudotime inference, multivariable regression, mediation modeling, and survival analysis. ICOS expression was confined to a minority subset (10%) and non-randomly enriched in discrete CD4-dominant clusters (up to 39.8%). ICOS high cells exhibited amplified regulatory transcriptional activity (Treg_score 0.375 vs − 0.041; ρ = 0.165; p = 1.06 × 10⁻¹²⁵) with more moderate exhaustion enrichment. On the other hand, CD8-restricted analysis showed no significant difference in exhaustion (p = 0.384). Differential expression revealed extensive transcriptional reprogramming (98.4% of tested genes at FDR < 0.05). Hallmark analysis exhibited coordinated enrichment of 30/50 pathways in ICOS low cells, showing relative transcriptional specialization rather than global activation in ICOS high cells. Diffusion pseudotime positioned ICOS high cells in early differentiation states (61.5% lower median pseudotime; p = 6.47 × 10⁻⁵²), with exhaustion intensity declining along trajectory (ρ = −0.3601; p < 10⁻³⁰⁰). At the tumor scale, ICOS strongly correlated with exhaustion (ρ = 0.727; p = 8.52 × 10⁻⁸⁷) and remained the dominant independent predictor (β = 0.710; R² = 0.542), with partial CD8 mediation (33.7%). ICOS did not independently predict overall survival, and it defines an early-positioned, transcriptionally coherent, regulatory-biased immune state that coordinates activation–regulation–exhaustion dynamics in HBV-associated HCC rather than marking terminal dysfunction.