Persistent loss of intrahepatic IFN-γ in HBV is linked to selective impairment of liver-resident CXCR6+NK cells despite long-term NUC therapy

BACKGROUND & AIMS: Chronic HBV infection remains incurable in most patients despite long-term nucleos(t)ide analog (NUC) therapy. Interferon-γ (IFN-γ) is a cornerstone of antiviral immunity, but its in situ source and status in the human liver remain unknown. We aimed to define the primary source of IFN-γ in healthy liver and determine how this axis is altered in stably suppressed NUC-HBV. METHODS: Multiplex immunofluorescence for CD3, CD56, CXCR6, and IFN-γ was performed on liver biopsies from patients with NUC-HBV (n = 9) and healthy donors (n = 7), with whole-slide scanning and AI-based segmentation for unbiased in situ cell quantification. Single-cell RNA sequencing (scRNAseq) was conducted on paired blood and liver fine-needle aspirates (FNAs) from NUC-HBV (liver n = 9, blood n = 18) and integrated with control datasets (liver n = 5, blood n = 9). Differential gene expression was used for transcriptional readout, and cell-cell interaction analysis mapped altered signaling networks. RESULTS: In healthy liver, CXCR6-positive natural killer (CXCR6+NK) cells were the dominant IFN-γ producers (24.2%; IQR 14.1-67.2%). In NUC-HBV, these cells were reduced (675 vs. 1,918; p = 0.012) and rarely expressed IFN-γ (0.2%; IQR 0.0-1.4%), despite normal alanine aminotransferase and absence of fibrosis. To validate transcriptionally, we performed scRNAseq on FNA and paired blood. A single, liver-restricted CXCR6+NK cluster was identified, and showed selective downregulation of IFNG and chemokines (XCL1, CCL3, CCL4), while cytotoxic genes (GZMB, PRF1) were preserved. Interaction analysis revealed reduced pro-inflammatory signaling and enrichment of transforming growth factor-beta-associated pathways. CXCR6+NK frequency correlated with serum HBsAg (p = 0.037) but was unchanged after 24 weeks of NUC in a longitudinal dataset. CONCLUSIONS: Long-term NUC therapy does not restore intrahepatic IFN-γ. Loss and transcriptional reprogramming of CXCR6+NK may contribute to a stable, altered immune state, representing a target for immune-based HBV cure strategies. IMPACT AND IMPLICATIONS: Effective antiviral therapy for chronic HBV suppresses viral replication but does not provide cure, indicating persistent defects in intrahepatic antiviral immunity. By combining protein-level analysis of liver tissue from healthy living donors and NUC-treated HBV liver biopsies with single-cell RNA-seq of fine-needle aspiration-derived immune cells from clinically stable, long-term NUC-treated patients, we directly examined IFN-γ regulation in the human liver-an immune compartment that is largely inaccessible in this patient group. We demonstrate that IFN-γ is produced in situ predominantly by CXCR6+NK cells in healthy liver and is selectively reduced in the liver of NUC-treated patients, despite normal alanine aminotransferase levels, absence of fibrosis, and lack of inflammatory or exhaustion signatures, while no corresponding defect is observed in blood. These findings identify a liver-restricted, non-exhaustion-driven impairment of NK-cell function and suggest that future therapeutic strategies should focus on restoring intrahepatic immunity-potentially via modulation of TGF-β-associated pathways or CXCR6+NK cell IFN-γ production.