NKP30-B7-H6 interaction contributes to the NK cell-mediated hepatocyte toxicity.

<p>Cytotoxicity assays were performed to confirm the role of NKP30-B7H6 recognition in NK cell-mediated cytotoxicity against hepatoma cells and hepatocytes. NK-92 cells expressing high levels of NKp30 were used as effector cells. Hepatoma cell line Huh7 transfected with or no B7-H6 siRNA and LO2 cells transfected with or no pIRES2-EGFP-B7-H6 plasmid were used target cells. (A) representative FACS analysis of NK cells receptors on NK-92 cells. (B) A statistic analyses of the frequencies of NK-92 cells expressing NK cell receptors. Results are the mean ± SD. (C) B7-H6 mRNA expression in Huh7, HepG2, HepG2.215, LO2, K562, and HeLa cells. Results are the mean ± SD. *<i>P</i> < 0.05 as comparisons with HepG2, HepG2.215 and LO2 groups. (D) Soluble NKp30-Fc inhibited the killing of NK-92 cells against Huh7 cells. A 4-h cytoxicity assay was performed in the presence or absence of 2ug/ml NKp30-Fc. Transfection of Huh7 cells with B7-H6 siRNA resulted in silencing of B7-H6 gene as assessed by quantitative real time PCR (E) and western blotting (F). Results are the mean ± SD. <i>P</i> values are shown. (G) Effect of B7-H6 gene knockdown on the cytotoxicity of NK-92. Huh7 cells were transfected with non-targeting control siRNA or B7-H6 siRNA by lipofectamine and then incubated for 48 hr, thereafter co-cultured with NK-92 cells at different effector/target ratios for 4 h. The cytotoxicity of NK-92 cell was evaluated by the CCK8 assay. Results are the mean ± SD. *<i>P</i> < 0.05 as comparisons with non-transfection and control siRNA groups. The recombinant pIRES2-EGFP-B7-H6 plasmid was transfected into LO2 cells, B7-H6 expression was detected under fluorescence microscope (H) and confirmed by quantitative real time PCR (I) and western blotting (J). Results are the mean ± SD. <i>P</i> values are shown. (K) Effect of over-expression of the B7-H6 gene on the cytotoxicity of NK-92 cells. LO2 cells were transfected with control pIRES2-EGFP or pIRES2-EGFP-B7-H6 plasmid by lipofectamine and then incubated for 48 h, thereafter co-cultured with NK-92 cells at different effector/target ratios for 4 h. The cytotoxicity of NK-92 cell was evaluated by the CCK8 assay. Results are the mean ± SD. *<i>P</i> < 0.05 as comparisons with non-transfection and control pIRES2-EGFP plasmid groups.