Generation of NK1.1⁺ TCRαβ⁺cells from NK1.1⁺ CD3⁻ thymocytes of ZAP-70^−/− mice in neonatal thymic organ culture.

(A) A representative flow cytometric profile of inductive generation of the NK1.1⁺ TCRαβ⁺ cells in the dGuo-treated thymi in the presence of PMA plus ionomycin. The sorted NK1.1⁺ TCRαβ⁻ cells for the culture were demonstrated in the square of the left panel. Proportions of sorted NK1.1⁺ TCRαβ⁻ cells were 98.8% to 99.5%. Collected cells from cultures were stained with either PE-mouse IgG2a (isotype control of PK136)/FITC–anti-TCRαβ/propidium iodide (middle panel) or PE–anti-NK1.1/FITC–anti-TCRαβ/propidium iodide (right panel) and analyzed with FACScan. The proportions of NK1.1⁺ TCRαβ⁺ cells are indicated. (B) Mean proportion of NK1.1⁺ TCRαβ⁺ cells. The NK1.1⁺ CD3⁻ (about 5 × 10³/lobe to 8 × 10³/lobe) were seeded to the dGuo-treated neonatal thymic lobes and cultured in hanging-drop setup in the absence (left column) or presence (right column) of PMA and ionomycin. Five days later, total cells were stained as described for panel A. The net proportion of induced NK1.1⁺ TCRαβ⁺ cells were calculated as follows: [percentage of NK1.1⁺TCRαβ⁺ cells minus percentage of control IgG2a⁺ TCRαβ⁺ cells]. The data indicate mean net proportion and SD.