The antitumor effect of natural killer cells against hepatocellular carcinoma through CXCL9

Backgrounds: CXCL9, known as monokine-induced by interferon gamma (MIG), is one of the ligands of chemokine receptor CXCR3 that mediates the infiltration of lymphocytes to focal sites and suppresses tumor growth. Natural killer (NK) cells are lymphocytes belonging to the innate immunity, which can directly kill cancer cells. In addition, they have immunoregulatory activities by secreting multiple cytokines and chemokines. Objective: In the present study, we explored the role of CXCL9 in hepatocellular carcinoma (HCC) cell lines cocultured with NK-92. Two HCC cell lines, SK-Hep1 and Hep3B, were cocultured with NK-92. The cytotoxic effect of NK-92 against HCC was analyzed by CytoTox96 assay, and CXCL9 expression was measured using Human Cytokine Array, ELISA and RT q-PCR. Also, the expressions of CXCR3A and CXCR3B were analyzed by western blot. Blocking CXCL9 in Hep3B was performed using CXCL9 shRNA and recombinant human CXCL9 was used to examine the direct role of CXCL9 in NK-92. Results: The cytotoxicity of NK-92 was greater against Hep3B than against SK-Hep1, and the mRNA and protein expressions of CXCL9 were significantly higher in Hep3B when cocultured with NK-92. CXCR3B is significantly upregulated compared with CXCR3A in Hep3B and is positively affected by NK-92 coculture. CXCL9 blockade reduced the cytotoxicity of NK-92 against Hep3B. In addition, Recombinant human CXCL9 induced NK-92 chemotaxis along with increased CXCR3, PI3K (p110γ), and PLC-β3 expression and significantly increased the expressions of T-bet and STAT1, which are associated with NK cell activation. Conclusion: These findings suggest that CXCL9 can directly induce NK cells to be recruited to HCC and activate NK cytotoxicity against HCC.