B6 LN Lin⁻CD49b⁺CD127⁺ cells differentiate into mature NK cells in vitro and in vivo. (A) LN Lin⁻CD49b⁺CD127⁺ and BM Lin⁻CD122⁺ cells from B6 mice were purified by FACS and cultured with OP9 stroma cells and cytokines for differentiation into NK cells. Cells were harvested after 12 days in culture and analyzed by flow cytometry for the expression of indicated cell surface markers. Data are representative of 5 independent experiments. The numbers show the average (± SD) percentages of positive cells. (B) The frequency of NK-cell precursors among LN Lin⁻CD49b⁺CD127⁺ cells (LN NKP) and control conventional BM Lin⁻CD122⁺ NKPs (BM NKP) was determined by limiting dilutions analysis. Both cell types were sorted in pools of 3, 10, 30, and 90 cells (n = 40) onto OP9 stroma layers and cultured for 12 days. The frequency ± SEM of NK progenitors in each population was calculated by L-Calc software (StemCell Technologies). (C) The cytotoxicity of NK cells generated in vitro from LN NKPs and BM NKPs was tested with the use of YAC-1 as target. (D) The in vitro–generated NK cells were stimulated overnight with 1 ng/mL IL-12 alone or IL-12 and IL-18 (0.5 ng/mL). The level of IFN-γ secretion was measured by ELISA. Shown is 1 representative experiment of 3. (E) Nonirradiated NOD/Scid/IL-2Rγ KO (NSG) mice were intravenously injected with FACS-purified LN NKPs (10³ cells per mouse). After 4 weeks, spleen cells were analyzed for the presence of CD45.2⁺ cells. CD45.2⁺ cells were gated for NKp46⁺ cells, and the expression of CD127 and CD49b was analyzed (top). CD45.2⁺ cells were analyzed for the expression of NK1.1 (NK cells), CD3 (T cells), and CD19 (B cells) (bottom).