DN1 and pre-DN2 cells in LN are thymus-dependent NK-cell progenitors. (A) LN cells from B6 mice were stained with Lin antibody cocktail, anti-CD25, and anti-CD44 and analyzed by flow cytometry. Lin⁻ cells were gated, and the percentages of DN1 (CD44⁺CD25⁻) and pre-DN2 (CD44⁺CD25^lo) cells were determined. The numbers in the dot plots show the percentage of cells in each region. (B) LN cells from nude (Foxn1^−/−) B6, and wild-type B6 mice were analyzed for the presence of IL-7Rα (CD127) and c-Kit (CD117) expression on their DN1 and pre-DN2 populations. Lin⁻CD44⁺ LN cells were gated, and CD25 and CD127/CD117 expression was analyzed by flow cytometry. Numbers indicate percentages of positively stained cells. (C) DN1 (top) and pre-DN2 (bottom) LN cells were purified from IL-15–deficient B6 mice and cultured for NK-cell differentiation in vitro. Cells recovered from the cultures were analyzed by flow cytometry for the presence of NK cells (left), and expression of Ly49G on NK cells (right). (D) Limiting numbers of DN1 and DN2 thymocytes, DN1 and pre-DN2 LN cells, and CD127⁺ and CD127⁻ LN DN cells were sorted by FACS and directly deposited into 96-well plates with preestablished OP9 stroma. The cells were cultured for 3 weeks, as in the bulk cultures above, for NK-cell differentiation, and wells with growing cells were scored. The frequency of NK progenitors in each population was determined by L-Calc software (StemCell Technologies). The results are the averages of 3 independent experiments for LN DN populations and 2 independent experiments for thymus DN1, DN2, and BM NKPs. (E) LN DN1 and pre-DN2 cells from B6 mice were purified and cultured for NK-cell differentiation as in panel C. NK cells at the end of culture were purified and subjected to semiquantitative genomic PCR to determine the frequency of cells with rearranged Tcrγ (Vγ2-Jγ1) as in Figure 2B. Images from different parts of the same membrane are divided by lines.