DN2 thymocytes differentiate in vitro into TCRγ⁺ NK cells. (A) BM NKPs (top row), thymic DN1 (middle row), and DN2 progenitors (bottom row) were purified and cultured with OP9 stroma and cytokines for differentiation into NK cells. Cells were harvested after 2 weeks and analyzed by flow cytometry for the presence of NK cells (NK1.1⁺CD3⁻) as well as expression of indicated receptors on NK cells. Data are representative of 5 independent experiments. (B) NK cells generated in cultures from DN1 and DN2 thymocytes above were purified, and the percentages of Tcrγ⁺ NK cells were estimated by semiquantitative genomic PCR for rearranged Tcrγ (Vγ2-Jγ1) gene using γδT cell DNA and fibroblast DNA mixed at set ratios as controls. The total amount of DNA was kept consistent for the PCR. The PCR products were analyzed by Southern blot using Jγ1 probe. Short and long exposures of the same membrane are shown. Images from different parts of the same membrane are divided by lines. The results are representative of 3 independent experiments.