Interleukin-35 expression promotes hepatocellular carcinogenesis by inducing γδ T-cell exhaustion

Interleukin (IL)-35, a new member of the IL-12 family, exerts immunosuppressive effects in the hepatic microenvironment. Innate immune cells, such as γδ T cells, have vital roles in hepatic diseases, including acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). In the current study, we focused on the effects and mechanisms of IL-35 on the local immune status of γδ T cells, especially in liver tumors. Based on CCK8 assay and immunofluorescence results, we showed that exogenous IL-35 stimulation of γδ T cells attenuated proliferative ability and killing functions against Hepa1-6 or H22 cells. Flow cytometry results showed that exogenous IL-35 stimulated the expression of programmed cell death 1 (PDCD1) and lymphocyte activation gene 3 (LAG3) in γδ T cells. The exogenous IL-35 stimulated group also showed impairment of cytotoxic cytokine secretion. In addition, stat5a revealed a significant increase after IL-35 stimulation of γδ T cells screened via transcription factor based on PCR array analysis. Furthermore, bioinformatics analysis revealed that stat5a-related tumor-specific genes were mainly involved in immune regulatory pathways. Correlation analysis indicated that stat5a expression was significantly and positively correlated with tumor immune cell infiltration, and pdcd1 and lag3 expression. Finally, bioinformatics analysis using the TCGA and GSE36376 HCC datasets corroborated the significant positive correlation between IL-35 and stat5a. Taken together, overexpressed IL-35 promoted exhaustion and impaired the anti-tumor function of γδ T cells in HCC. Targeting IL-35 might be a promising means to enhance the antitumor therapy of γδ T cells, which would significantly improve prognosis.