Figure 1.

Direct HCMV recognition induced CD69 expression in NK cells and enhanced IL-12-induced IFNγ secretion. Purity of NK cells isolated by negative selection from peripheral blood of healthy donors was assessed by multicolor flow cytometry including anti-CD56-APC and anti-CD3-PE, -CD19-PE, -CD14-PE, -CD1a-PE, -CD123-PE as lineage exclusion cocktail (A). Purified NK cells were left untreated (NT) or co-cultured with HCMV (MOI 5–10) for 24 and 48 h in the presence or absence of IL-12 (10 ng/ml) and IL-2 (200 μ/ml). At 24 h, CD69 expression on NK cells was tested by a double staining with anti-CD56-APC and CD69-PE specific mAbs by flow cytometry (B). IFNγ was measured by ELISA in cell-free supernatants harvested at 24 and 48 h (C). Intracellular IFNγ was monitored on NK cells co-cultured for 24 h with the different stimuli. Brefeldin A was added for the last 5 h and IFNγ staining was analyzed by flow cytometry. Inserts depict the proportion of IFNγ positive NK cells (D). In all cases, data correspond to results from a representative donor out of at least three tested.