HCMV-infected moDCs trigger a preferential response of CD94/NKG2A⁺ NK cells associated with a down-regulation of HLA-E expression. (A) NK cells purified by negative selection from PBMCs stimulated overnight with IL-2 were cocultured for 5 hours with target cells as described in “NK-cell functional assays.” Surface CD107a expression on CD94/NKG2A⁺ and CD94/NKG2C⁺ NK cells was analyzed by flow cytometry. Dot plots from 2 representative donors of 6 analyzed are shown. The proportions of CD107a⁺ cells referred to total NKG2A⁺ or NKG2C⁺ cells are specified in bold. (B) moDCs were surface labeled 48 hours after virus exposure by indirect immunofluorescence with an HLA-E-specific mAb (3D12) (open histograms, isotype control; filled histograms specific staining). Fluorescence intensity (geometric mean) of selected populations is included. Results of a representative experiment (60% IE-1/IE-2⁺ cells) of 6 performed are shown. (C) Alignment of part of gpUL40 amino acid sequences from AD169, TB40/E, and the HCMV clinical isolate UL1271; predicted leader sequences are shaded, and HLA-E binding peptides are boxed. (D) moDCs were mock treated (filled histograms) or infected with the HCMV clinical isolate UL1271 (MOI 25: gray line; MOI 100: black line, open histograms) and surface labeled at 48 hours and 72 hours after virus exposure by indirect immunofluorescence with mAbs specific for HLA-E (3D12) and HLA class I molecules (HP-1F7). Results of a representative experiment (MOI 25: 25% IE-1/IE-2⁺ cells; MOI 100: 85% IE-1/IE-2⁺ cells) of 3 performed are shown.