All-trans retinoic acid induces leukemia resistance to NK cell cytotoxicity by down-regulating B7-H6 expression via c-Myc signaling.

The interaction between activating receptor NKp30 and its major tumor ligand B7-H6 is important for NK cell-mediated tumor rejection. However, the regulation of B7-H6 by tumor therapeutics remains largely unknown. In this study, we investigated the regulation of B7-H6 by all-trans retinoic acid (atRA), a terminal differentiation inducer of tumor cells that is extensively used for clinical leukemia therapy. We investigated the role of NKp30:B7-H6 axis in NK cell-mediated tumor lysis against leukemia cells and the influence of atRA treatment on the cytotoxicity of NK cells using NK cell lines (NK92 and NKG) and leukemia cell lines (U-937 and THP-1). We evaluated the effect of atRA treatment on the expression of B7-H6 using real-time PCR, flow cytometry and western blotting. We used CRISPR/Cas9 to knockdown B7-H6 expression and siRNA to knockdown c-Myc in U-937 cells to evaluate the role of B7-H6 and c-Myc in atRA-induced tumor resistance against NK cells. NK cell-mediated U-937 cell lysis was mainly dependent on NKp30/B7-H6 interaction. Blockade of B7-H6 by monoclonal antibody significantly impaired NK cytotoxicity. atRA treatment induced U-937 resistance to NK cell cytotoxicity by reducing B7-H6 expression, and showed no effect on NK cytotoxicity against B7-H6 knockdown U-937 cells. Epigenetic modifications, such as DNA methylation and histone deacetylase (HDAC), were not responsible for atRA-mediated B7-H6 down-regulation as inhibitors of these pathways could not restore B7-H6 mRNA expression. On the other hand, atRA treatment reduced c-Myc expression, which in turn inhibited the transcription of B7-H6 on leukemia cells. atRA treatment promotes tumor cell resistance against NK cell-mediated lysis by down-regulating B7-H6 expression via the c-Myc signaling pathway, suggesting that more attention needs to be paid to the immunological adverse effects in the clinical use of atRA treatment.