Induced Expression Of NKp30 Ligand B7-H6 And NK Cell Cytolysis

NK cells play a role in combating cancer. Unlike T. B cells, which express antigen specific receptors, NK cells express various receptors encoded by the genome on their surface. NK cell activating receptors, include NKG2D, NKp30 and so on, recognize their ligands expressed on tumor cells. The ligands are mostly encoded by the host genome and are induced upon stress, which are called stress induced ligands.B7-H6, a type I transmembrane protein, is recently identified as an NKp30 ligand. B7-H6 is exclusively expressed on tumor cells rather than normal tissue cells, which makes the study on B7-H6 of great importance.To investigate the expression and regulation of B7-H6, anti-B7-H6 polyclonal antibody was generated in our lab. Gene coding the extracellular domain of B7-H6 was cloned from Ho8910 cell cDNA and inserted to the pET22b expression vector. B7-H6-6His fusion protein was expressed in E.coli and purified with Ni NTA affinity chromatography. Rabbits were immunized with purified B7-H6-6His protein to generate anti-B7-H6 antiserum. Finally. anti-B7-H6 antibody was obtained from the antiserum by protein G affinity chromatography and B7-H6 antigen specific affinity chromatography. And the antigen specificity of the antibody was verified.To investigate the influence of tumor therapeutics on B7-H6 expression, HEK293 cells and HL60 cells were treated with various tumor treatments and B7-H6 expression was detected with real-time PCR, Western blotting and flow cytometry. Chemotherapy agents, cisplatin and 5-FU, irradiation, non-lethal heat shock and TNF-a treatment all promoted B7-H6 mRNA transcription, protein synthesis as well as cell surface expression. Tumor sensitivity to NK cell cytolysis was also enhanced by tumor treatments in a B7-H6-dependent manner as for B7-H6 knock-down dampened NK cell-mediated cytolysis. Our study shows that B7-H6 works as a stress induced ligand and enhance NK cell mediated tumor immune surveillance.Our results showed that all-trans retino acid down-regulated B7-H6 expression on cell surface in tumor cells. To investigate the mechanisms underlying, we found that CIN85 interacted with B7-H6 in the co-immunoprecipitation assay. CIN85 knock-down enhanced cell surface B7-H6 expression. To investigate whether CIN85 interacted with B7-H6 directly or indirectly, we applied the GST pull down assay and found that CIN85 interact directly with the intracellular region of B7-H6, however, in a manner independent of the proline-rich motif. So, our results indicated that CIN85 may work as a negative regulator to block the transportation of B7-H6 on to cell surface. More study is needed to show the exact mechanism through which CIN85 down-regulating B7-H6 expression.Our study shows that tumor therapeutics promote B7-H6 expression on tumor cells and enhance the sensitivity of tumor cells to NK cell cytolysis, and also explore the mechanism under which tumor cells inhibit the transportation of B7-H6 from plasma to the cell surface in a CIN85 dependent manner. Overall, our results suggest that B7-H6 may be a potential target in tumor treatment in the future.