Type I IFN and IL-12 down-regulate NKG2D expression. (A) NK cells isolated by negative selection were left untreated (NT) or stimulated with recombinant IL-12 (30 ng/mL), IFNγ (50 ng/mL), and/or IFNβ1 (1200 U/mL). Surface expression of NKG2D (1D11) and CD69 was monitored at 48 hours by flow cytometry. Data correspond to a representative donor of 3 studied. (B) Isolated NK cells were left untreated or incubated with IFNβ1 (1200 U/mL) and IL-12 (30 ng/mL). A neutralizing antibody for IFNγ (20 μg/mL) was added at the beginning of the stimulation. Results from a donor of 3 tested are shown. Numbers in histograms indicate geometric mean values of mean fluorescence intensity for each parameter. (C) Real-time polymerase chain reaction was performed on mRNA isolated from purified NK cells treated with IFNβ1(1200 U/mL) and/or IL-12 (30 ng/mL) for 36 hours. Bar graphs indicate the fold change in the expression levels of NKG2D and DAP10 transcripts in each condition compared with nontreated NK cells (whose levels of gene expression have been assumed as 1). The endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to internally standardize the levels of gene expression. Bar graphs include results from 3 different donors (mean ± SD).