Overexpression of T-BET but not ETS-1 rescues the TOX2 knockdown phenotype in NK cell development. (A) CD34⁺ cells transduced with control, ETS-1, TOX2-KD, or TOX2-KD+ETS-1 were subjected to in vitro NK cell development. Representative FACS plots of different NK developmental stages based on GFP gating and the surface expression of CD117 and CD94 on day 17 were shown. (B) Stage III (CD117⁺CD94⁻) cells were gated for the analysis of CD56 expression to distinguish early stage III (CD117⁺CD94⁻CD56⁻) and late stage III (CD117⁺CD94⁻CD56⁺) cells. (C) The relative amount of GFP⁺CD56⁺ cells are calculated: Relative amount of CD56⁺ cells = (% of GFP⁺ CD56⁺ cells of the sample)/(% of GFP⁺ CD56⁺ cells of Control) and displayed as the mean ± SD of 5 independent experiments. (D) CD34⁺ cells transduced with control, T-BET, TOX2-KD, or TOX2-KD⁺T-BET were subjected to in vitro NK cell development. Representative FACS plots of different NK developmental stages based on GFP gating and the surface expression of CD117 and CD94 on day 17 were shown. (E) The relative amount of GFP⁺CD56⁺ cells are displayed as the mean ± SD of 4 independent experiments. *P < .01, **P < .0001.