Thymic NK cells have immature phenotype but are functionally mature. (A) Lymphocytes from various tissues of B6 mice were stained for CD3, NK1.1, and indicated NK-cell receptors and analyzed by multicolor flow cytometry. NK cells were gated by CD3⁻NK1.1⁺, and the percentages of NK cells expressing indicated NK receptors over control staining with isotype matched antibodies were determined. Dead cells were gated out by staining with propidium iodide. The results are average (± SD) of more than 3 independent experiments. (B) Thymocytes and spleen cells from Tcrβ^−/−δ^−/− mice were stimulated in vitro with IL-12 and IL-18 for 24 hours, stained for CD3⁻NK1.1, fixed and stained for intracellular IFNγ. The percentages of NK cells positively stained for IFNγ over isotype-matched control antibody staining were determined by flow cytometry. The results are averages of 3 independent experiments. (C) Thymocytes and spleen cells were stimulated with IL-12 and IL-18 as above, and the level of intracellular IFNγ in NK cells was determined by flow cytometry. Histograms of control staining with isotype matched control Ab (filled in gray), intracellular IFNγ staining of thymic NK cell (black), and that of spleen NK cells (gray) is shown. The mean fluorescence intensities of positively stained cells are also shown. The results are representative of 3 independent experiments. (D) Thymus and spleen NK cells were stimulated in vitro with IL-2, and killing of RMA-S cells was analyzed. The results are the average of quadruplicate experiments.