Splenic NKp46highNK cells produced the highest levels of IFN-γ. (A + B) Intracellular cytokine staining for IFN-γ production in NKp46-defined NK cells within PBMC (upper graphs) and splenocytes (lower graphs) by four-colour FCM after 24 h in vitro stimulation with rhIL-2, rpIL-12 and rpIL-18 or medium. CD3- lymphocytes were gated (not shown) and NKp46/CD8α defined total NK cells were further subgated for IFN-γ production. IFN-γ producing NK cells were gated according to NKp46 expression levels (CD8α+NKp46-: blue, CD8α+NKp46+: green, CD8αdim/-NKp46high: red). (A) Numbers indicate the percentage of IFN-γ+ NK cells within respective gates. Results are representative for experiments with four different animals. (B) Percentage of IFN-γ+ cells (left graph) and mean fluorescence intensity of IFN-γ+ cells (right graph) within the different NK-cell subsets in blood and spleen of four animals are shown. (C) Supernatants of cytokine stimulated FACS-sorted CD3-CD8α+NKp46- and CD3-CD8α+NKp46+ NK cells from blood and CD3-CD8α+NKp46-, CD3-CD8α+NKp46+ and CD3-CD8αdim/-NKp46high NK cells from spleen were tested for IFN-γ production in ELISA following 24 h in vitro stimulation with rhIL-2, rpIL-12 and rpIL-18. Data on the left are from one representative animal and displayed as the mean of duplicates ± SD. IFN-γ production in experiments with four animals analysed is shown on the right. Mean values are represented by a black bar. (B + C) Significant differences between the subsets in blood or spleen are indicated (* = p < 0.05, ** = p < 0.01).