NK-cell activation and infection of PBMCs are associated with HCMV-induced down-regulation of NKG2D expression. (A) PBMCs were cultured with or without TB40/E for 2 days. The expression of surface NKG2D and CD69 in CD56⁺CD3⁻ NK cells was monitored by flow cytometry. Histograms correspond to the isotype controls (thin lines), mock-treated (gray profiles), and TB40/E-infected samples (bold lines). Results from a representative donor of 4 tested are shown. (B) IFNγ was detected by ELISA in supernatants of mock- and TB40/E-treated PBMCs (72 hours). Data correspond to experiments with samples from 3 representative donors (mean ± SD). Empty and gray bars correspond to mock- and HCMV-treated cultures, respectively. (C) Cell proliferation, assessed by the carboxyfluorescein succinimidyl ester (CFSE) dilution assay, and surface NKG2D expression on CD56⁺CD3⁻ cells from PBMCs cultured for 72 hours with TB40/E. Results from one donor of 3 tested are shown. (D) PBMCs were cultured alone and in the presence of either TB40/E, UV-TB40/E, or filtered TB40/E. NKG2D surface expression was assessed at 72 hours by 3-color flow cytometric analysis with CD3, CD56, and NKG2D (BAT221) specific mAbs. Histograms showing NKG2D surface expression on CD56⁺CD3⁻ cells correspond to samples from a representative donor of 4 studied. Geometric mean values for relative fluorescence intensity are included. (E) IFNγ was measured by ELISA in supernatants harvested at 72 hours from samples studied in panel D. (F) PBMCs cocultured with TB40/E included in the R1 gate, containing mainly myelomonocytic cells (not shown), were sorted, and cytospin preparations were labeled by indirect immunofluorescence with an anti–IE1/IE-2 mAb; nuclei were counterstained with DAPI (4′-6′-diamidine-2-phenylindole). Image acquisition information: 40×/0.75 HCX FLUOTAR; Dako fluorescence mounting medium; see also “HCMV preparation and infection of PBMCs.”