In vitro phenotypic and functional correction in immune cells after GE of XMEN CD34⁺ HSPCs. (A) Dot plots showing the gating strategy after in vitro T-cell differentiation of CD34⁺ cells using the ATO system; bar graph on the right shows NKG2D expression in CD3⁺ T cells at 6 weeks of differentiation (data are representative of 2-4 independent experiments; 300-20 000 events acquired in CD3⁺ gate). (B) Targeted integration measured by ddPCR analysis in ATO-derived cells at week 6 of in vitro differentiation (data are representative of 2 independent experiments). (C) Dot plot showing the gating for NK cells (CD3^–CD56⁺) and NKG2D expression after 35 days of in vitro NK-cell differentiation from CD34⁺ cells; bar graph shows the percentage of CD34-derived NK cells in each condition. (D) NKG2D expression (% positive cells), (E) level of expression determined as the percentage of mean fluorescence intensity (MFI) of HD NK cells, and (F) targeted integration by ddPCR analysis in NK cells at day 35 of in vitro differentiation (4 independent experiments for –i53, 5 for +i53, and 8 for +i53+hGSE). (G) Cytotoxic activity of CD34⁺-differentiated NK cells against K562 cells at an effector:target ratio of 2:1 (4 independent experiments for –i53, 9 for +i53, and 8 for +i53+hGSE). (H) Correlation between the NKG2D expression (%) and the killing activity (%) in NK cells at day 35 was calculated using Spearman’s correlation coefficient and 2-tailed P value (n = 33 pairs). Data are shown as mean ± SD. *P < .05; **P < .01; ***P < .001; ****P < .0001. FSC, forward scatter.