Characterization of NK cell cytotoxicity, cytokine production, and NKR expression by MNCs, CD56⁺CD3⁻, and CD56⁻CD3⁻ cells generated from CD34⁺ HPCs in various conditions. (A) Cytotoxicity assay. MNCs were harvested after 21-day culture with indicated cytokines, washed, and tested for cytotoxic activity against the NK-sensitive K562 target cell line at an 8:1 E:T ratio. (B) Cytokine (IFN-γ) and chemokine (MIP-1) production. MNCs were harvested after 21-day culture with indicated cytokines, sorted into CD56⁺CD3⁻ and CD56⁻CD3⁻ fractions, and costimulated with IL-15 plus IL-12 for 48 hours. Cell-free supernatants were harvested and tested for IFN-γ and MIP-1 by ELISA. For both (A) and (B), results represent the mean ± SEM of four separate experiments. The asterisk in (A) denotes a value of P ≤ .01 compared with culture in FL alone. (C) NKR expression. NK cells generated in vitro by culture in FL for 3 weeks, and then in IL-15 for 2 weeks, were stained with anti-NKR and anti-CD56 MoAbs and analyzed by flow cytometry as described in Materials and Methods. Flow cytometry data are representative of 3 separate donors, which are summarized in Table 1.