Expansion of NKG2C⁺ NK cells is dependent on the HCMV infection of fibroblasts and on the time of their interaction. (A) The distribution of NKG2C⁺ cells in response to HCMV-infected MRC-5 cells at different MOI was analyzed. The percentage of NKG2C at MOI of 0.01 was comparable to mock-infected samples (not shown). (B) PBLs from HCMV⁺ donors were cocultured with mock- or Towne-infected MRC-5 fibroblasts; in parallel, PBLs were incubated with the virus alone (HCMV) or fibroblasts infected with UV-inactivated Towne (iHCMV). Two-color flow cytometry analysis was performed at day 10 with anti-CD3, -NKG2C, and -ILT2 mAbs. Data are representative of 5 different experiments. (C) PBLs were incubated with Towne-infected MRC-5 cells at different time points after infection (days 0, 1, and 3). At day 10, cells were harvested, counted, and analyzed by flow cytometry; the numbers of NKG2C⁺ and NKG2A⁺ cells recovered are shown. A similar pattern of response was observed in 3 different experiments.