TFs and effector molecules expression coupled with the progression of sequential NK cell stages along the in vitro trajectory. (A) Representative FACS gating scheme to define S1/2 (Lin⁻CD34⁺CD161⁻CD56⁻CD94⁻), S3a (Lin⁻CD34⁻CD161⁺CD45RA⁺CD56⁻CD94⁻), S3b-NKp44⁻ (Lin⁻CD34⁻CD56⁺CD94⁻NKp44⁻), S3b-NKp44⁺ (Lin⁻CD34⁻CD56⁺CD94⁻ NKp44⁺DNAM-1^hiCD161^lo), ILC3 (Lin⁻CD34⁻CD56⁺CD94⁻NKp44⁺DNAM-1^loCD161^hi), and S4 (Lin⁻CD34⁻CD56⁺CD94⁺) populations generated in vitro from Lin⁻CD34⁺ progenitors collected from cultures at 2 weeks (S1/2 and S3a) and 4 weeks (S3b-NKp44⁺, S3bNKp44⁻, ILC3, S4). (B) Violin plots display intracellular expression of GZMB and PRF1 within the subsets S3a, S3b-NKp44⁻, S3b-NKp44⁺, S4, and ILC3, from a combined pool of 3 donors. Statistical significance was performed with Kruskal-Wallis test between pair-wise comparisons S4 NK vs all other subsets. (C) Representative FACS histograms of TBET, EOMES, and RORγt expression within the subsets S3a, S3b-NKp44⁻, S3b-NKp44⁺, S4, and ILC3. Values described mean fluorescence intensity (MFI) of each indicated population. (D) Secretion of IFN-γ and IL-22 by in vitro–generated NK-S3b, NK-S4, and ILC3 cells after 4-week culture in response to stimulations with either PMA/ionomycin, IL-12/15/18, or IL-2/1β/23 compared with untreated cells (UT). Statistical significance was performed with 1-way analysis of variance (ANOVA) between all subsets (n = 9). (E) Upregulation of CD107a by in vitro generated NK-S3b (left panels), NK-S4 (middle panels) and ILC3 (right panels) cells after 4-week culture in response to stimulations with either PMA/ionomycin or K562 target cell (with effector-to-target [E:T] ratio of 1:5), compared with UT cells as controls (n = 3 individual donors, with 3 replicates for each donors). (F) Upregulation of CD107a by in vitro–generated NK cells after 2 weeks of further expansion on mbIL21-41BBL-K562 feeder cells, stimulated with K562 target cells. Resting peripheral blood (PB) NK cells were used as reference control and CB-derived in vitro expanded NK cells were separated into NK-S3b and NK-S4 stages. FACS plots show representative CD107a expression and bar plots represent the proportion of CD107a positive NK cells from PB NK cells and in vitro generated expanded NK cells (n = 3 donors). (G) IFN-γ secretion in response to K562 target cell stimulation from the same experiment settings as described in panel F. (F-G) Statistical significance was performed with t test;∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; and n.s., not significant.