Reduced expression of NKG2D on SEB-activated CD8⁺ T cells cultured in the presence of CD4⁺ cells. (A) PBMCs were CFSE-labeled, depleted or not (TOT) of CD4⁺ cells, and then stimulated with SEB. After 3, 5, and 7 days, cells were harvested and stained with anti-CD8, anti-CD3, and anti-NKG2D mAbs, in a 4-color FACS analysis. Data shown are from CD3⁺CD8⁺ cells. Numbers represent the NKG2D median of fluorescence intensity (MFI) values derived from unstimulated CD3⁺CD8⁺ T cells, or from the same cells that had divided at least once, as measured by loss of CFSE intensity (gate R1). Dot plots are from a representative donor, of 18 tested. Percentage of proliferating cells (gate R1) was as follows: 50 plus or minus 15 (TOT) and 37 plus or minus 19 (CD4⁻) at day 3; 73 plus or minus 17 (TOT) and 69 plus or minus 14 (CD4⁻) at day 5; and 78 plus or minus 17 (TOT) and 78 plus or minus 13 (CD4⁻) at day 7. (B) Summary of all donors. The NKG2D MFI values obtained on dividing cells from total or CD4-depleted PBMCs were divided per NKG2D MFI of unstimulated cells. IgG isotype control MFI values were always subtracted. *P less than .0002; **P less than .00001; and ***P less than .000001, with paired Student t test. (C) CD3⁺ T cells were isolated from PBMCs by negative selection and further purified to obtain subpopulations of CD4⁺CD3⁺ and CD8⁺CD3⁺ T cells. CD8⁺ T cells were cultured alone (CD8) or with CD4⁺ T cells (CD8 + CD4) and stimulated with SEB in the presence of irradiated autologous PBMCs. After 3, 5, and 7 days, cells were harvested and stained as described in panel A. Numbers represent NKG2D MFI values derived from unstimulated CD3⁺CD8⁺ T cells (NT), or from the same cells that had divided at least once, as measured by loss of CFSE intensity (gate R1). IgG isotype control MFI values were always subtracted. Dot plots are from a representative donor of 5 tested. Percentage of proliferating cells (gate R1) was as follows: 30 plus or minus 21 (CD8 + CD4) and 25 plus or minus 16 (CD8) at day 3; 47 plus or minus 20 (CD8 + CD4) and 49 plus or minus 19 (CD8) at day 5; 53 plus or minus 20 (CD8 + CD4) and 54 plus or minus 20 (CD8) at day 7. (D) Summary of 5 different donors. Mean plus or minus SE of NKG2D MFI values obtained on dividing cells from CD4 plus CD8 or CD8 T lymphocytes. *P value less than .04 with paired Student t test.