The Axl/Gas6 pathway is important for generation of functionally competent NK cells from human CD34⁺ HPCs. (A) CD34⁺ HPCs from human peripheral blood were cultured for 21 days under different experimental conditions as indicated. On day 21, equal numbers of CD3⁻CD56⁺ NK cells were plated into each well, followed by stimulation with IL-12 and IL-15 for 48 hours. Supernatants were harvested, and the concentration of IFN-γ was measured by ELISA. Results shown are the compilation of data from 2 donors, and each experiment was performed in triplicate wells. Error bar represents SD. *P < .05; **P < .01; ***P < .001. (B) On day 21, NK cells that were derived from CD34⁺ HPCs under different experimental conditions as described in panel A were plated in equal numbers into a 96-well culture plate. After being treated with IL-12 and IL-15 for 6 hours, cells were lysed, total RNA was isolated, and IFN-γ or T-BET mRNA was quantified by Taqman real-time RT-PCR. 18S rRNA was used for normalization. Error bar represents SD. These results are representative of 3 experiments. *P < .05; **P < .01. (C) After a 21-day culture of CD34⁺ cells under the indicated experimental conditions, the same number of differentiated CD3⁻CD56⁺ NK cells were plated in each well and mixed with ⁵¹Cr-labeled NK-sensitive K-562 target cells at a 2:1 ratio of effector to target cells (triplicate wells). Results are representative of 3 experiments and show mean plus or minus SD. No significant differences in cytotoxicity were noted.