Down-modulation of NKG2D is dependent mainly on soluble factor(s) released by CD4⁺ cells. (A,B) PBMCs were CFSE labeled and depleted of CD4⁺ cells (CD4-depleted PBMCs) or not depleted (total PBMCs) before SEB stimulation. Cells were grown in 24-well or in Transwell plates. In Transwell plates, CD4-depleted PBMCs were cultured in the top chamber, whereas total or CD4-depleted PBMCs were plated in the bottom chamber, as indicated. After 5 days, NKG2D or CD48 expression was evaluated on CD3⁺CD8⁺ T cells derived from the top chamber. In panel A, progressive loss of CFSE fluorescence intensity in CD8⁺ T cells is indicated by the R1 region. The MFI relative to NKG2D and calculated on cells gated in R1 is shown. Dot plots presented derive from a representative donor of 5 tested. In panel B, results obtained from 5 different donors are shown and are presented as the mean plus or minus SD of NKG2D or CD48 (used as control) MFI calculated as described for panel A. In parallel, NKG2D and CD48 expression was evaluated on CD3⁺CD8⁺ T cells grown in 24-well plates.