TOX2 directly regulates TBX21(T-BET) transcription during NK cell development in vitro. (A) UCB CD34⁺ cells transduced with control or TOX2-KD constructs were subjected to in vitro NK cell development. Different developmental stages were gated for GFP expression and were sorted as described in Figure 1D. The expression of TOX2 and other NK cell developmental genes was analyzed by RT-qPCR (n = 3). (B) Schematic representation of PGL3-based luciferase reporter constructs containing the putative promoter of ETS1, IL-15RB, or T-BET. (C) IL-15RB, ETS1, or T-BET reporter construct was cotransfected with vector-control or TOX2-WT expression vector into 293T cells. Luciferase activities were determined 3 days after the cotransfection. Data shown are expressed as fold change relative to the luciferase activity observed in vector-control transfected cells ± SD (n = 3). (D) Total cell lysate collected from TOX2-WT–transiently transfected 293T cells was incubated with streptavidin microbeads with or without the biotinylated T-BET-, IL-15RB-, or ULBP2-promoter probe. Protein fractions pulled down by the microbeads were subjected to western blot analysis to detect the presence of TOX2. Data shown are representative of 3 independent experiments. *P < .05, **P < .005, ***P < .0005.