TOX2 regulates NK cell maturation. (A) TOX2-WT or TOX2-INS overexpression construct was cotransfected with either control or TOX2-KD construct into 293T cells. TOX2 protein level was analyzed by western blotting. Results are representative of 3 independent experiments. (B) Total RNA was collected from GFP⁺ sorted developing cells on day 15 for TOX2 RT-qPCR analysis (n = 4). (C) UCB CD34⁺ cells transduced with control or TOX2-KD constructs were subjected to in vitro NK cell development. Different developmental stages appeared on day 17 and were analyzed by flow cytometry based on CD117 and CD94 staining in both GFP⁻ve and GFP⁺ve populations. Results are representative of 3 independent experiments. (D) Stage III (CD117⁺CD94⁻) cells from the GFP+ve populations were gated for the analysis of CD56 expression to distinguish early stage III (CD117⁺CD94⁻CD56⁻) and late stage III (CD117⁺CD94⁻CD56⁺) cells. (E) The quantity of CD56⁺ cells was compared between control and TOX2-KD GFP⁺ve populations on day 17 by flow cytometry. Bar charts display the mean percentage ± standard deviation (SD) of 8 independent experiments. (F) Different NK developmental stages among control, TOX2-KD^–, TOX2-WT^–, or TOX2-KD⁺TOX2-INS–transduced cells (GFP⁺ve sorted populations) were compared based on the surface expression of CD117 and CD94 by flow cytometry on day 17. FACS plots are representative of 4 independent experiments with (G) the relative production of CD56⁺ cells shown as the mean ± SD. (H) Control or TOX2-KD CD34⁺ cells were intravenously injected into irradiated (250 rad) NSG mice (0.5 × 10⁶ cells/mouse; 10 mice per groups). Six weeks after CD34⁺ cell transplantation, 2.5 μg of RLI by intraperitoneal injection was administered to mice every 5 days for 3 doses. BM and spleen cells were collected 3 days after the last dose of RLI to analyze GFP⁺CD3⁻CD56⁺ NK cell content by flow cytometry. *P < .05, **P < .005, ***P < .001, ****P < .0001.