NKG2A-HLA-E axis contributes to an immunosuppressive checkpoint network in hepatocellular carcinoma.

BACKGROUND: The immunosuppressive tumor microenvironment in hepatocellular carcinoma (HCC) limits the efficacy of current immunotherapies. The interplay between the emerging inhibitory receptor NKG2A and its activating counterpart NKG2C, both binding HLA-E, and its interaction with the PD-1-PD-L1 pathway in cancer remains unclear. This study aims to delineate the functional network formed by these pathways in shaping the immunosuppressive landscape of HCC. METHODS: We analyzed public databases and performed immunohistochemistry on HCC tissues to assess HLA-E expression and prognostic relevance. Tumor-infiltrating lymphocytes from patient samples were examined by flow cytometry to evaluate the functional impact of NKG2A, NKG2C, and PD-1 expression on T and NK cells. Single-cell RNA and T cell receptor (TCR) sequencing were employed to characterize immune subset heterogeneity. Functional assays and mouse models were used to evaluate the effects of NKG2A and PD-1 blockade. RESULTS: In the absence of CD8+ T cell infiltration, elevated HLA-E expression in HCC tissue correlated with poorer overall survival in non-immunotherapy-treated patients. Conversely, in the presence of CD8+ T cells, high HLA-E expression was linked to an inflammatory tumor microenvironment characterized by IFN-γ-producing T cells, and increased major histocompatibility complex I, which was associated with improved patient response to immunotherapy. Tumor-infiltrating CD8+ T and NK cells from HCC patients upregulated NKG2A, conferring tissue-resident memory and tumor-reactive characteristics while simultaneously impairing cytotoxic function. Single-cell RNA and TCR sequencing revealed differential expression patterns of NKG2A, NKG2C, and PD-1 in different T and NK cell subsets, with NKG2A+ PD-1+ T cells relevant to antigen-specific clonal expansion. Mechanistically, the inhibitory effect mediated by the NKG2A-HLA-E axis contributed more prominently than the stimulatory role of the NKG2C-HLA-E axis in suppressing antitumor immunity. Furthermore, the NKG2A-HLA-E axis acted cooperatively with the PD-1-PD-L1 axis to promote CD8+ T cell dysfunction. Combined blockade of NKG2A and PD-1 enhanced the cytotoxic activity of tumor-infiltrating CD8+ T and NK cells, resulting in potent tumor eradication in vitro and in vivo, outperforming single-agent treatments. CONCLUSIONS: Our findings identify the NKG2A-HLA-E axis as an important immunoregulatory pathway associated with immune suppression in HCC and provide a rationale for further evaluation of combined NKG2A and PD-1 blockade strategies.