Targeting a tolerogenic HLA-G genotype to tackle immune evasion and adaptive resistance in HBV-driven HCC

BACKGROUND: HBV-associated hepatocellular carcinoma (HCC) is characterized by immune evasion and heterogeneous responses to immunotherapy. However, the mechanisms driving tumor immune tolerance and their impact on checkpoint inhibitor-based therapies remain poorly understood. To address this gap, we generated a patient-derived HBV-induced HCC cell line that preserves a clinically relevant immune evasion mechanism mediated by HLA-G expression. METHODS: Whole-exome sequencing (WES) was performed on two spatially separated tumor regions to define genomic signatures with emphasis on the HLA-G locus. Patient-derived tumor cells were modified using Upcyte® technology, generating the up-LC14A1 cell line. Molecular stratification included an in-house HCC cohort (n=13), healthy donors (n=4), serum samples from early-stage liver disease (n=4), diagnosed HCC (n=10), and global datasets (TCGA biopsies (n=24); HCC (n=120)). Functional characterization included NK92 co-culture assays (1:1, 1:5, 1:10) and orthotopic transplantation in mice (HUH7 (n=3), Hep3B (n=4), HepG2H1.3 (n=4), up-LC14A1 (n=14)). RESULTS: WES revealed a clonally coherent HCC carrying an HLA-G 3'UTR haplotype associated with immune tolerance. Cohort stratification confirmed elevated soluble HLA-G in patient #14. The derived up-LC14A1 line retained hepatobiliary and viral features and showed stable proliferation with low-level HBV DNA production. Transcriptomic profiling positioned up-LC14A1 within the HCC landscape. In-vivo, orthotopic transplantation generated structured liver tumors and prolonged survival compared to immortalized HCC lines. Up-LC14A1 resisted NK92 cytotoxicity through dynamic HLA-G induction, while HLA-G silencing restored immune killing and induced compensatory PD-L1 expression. CONCLUSIONS: The up-LC14A1 model represents a patient-derived HBV-HCC system capturing a clonally stable but dynamically regulated HLA-G-mediated immune-tolerant state. This platform enables mechanistic investigation of immune escape and provides a translational model for testing targeted immunotherapies in HBV-associated HCC. IMPACT AND IMPLICATIONS: Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) exhibits a profound immune evasion capacity and a insufficient response to immunotherapy. Nevertheless, pre-clinical models that mimic viral persistence and tumor resistance are underrepresented. The immune modulatory molecule HLA-G has emerged as a potent target for immunotherapy in HCCs, due to its direct suppression of T and NK cells. Here, we established a novel patient-derived HBV-HCC cell line (up-LC14A1) that mediates immune evasion via HLA-G upregulation.