NKG2D costimulation of CD3-induced functions is impaired in CD3⁺CD8⁺ NKG2D^low T lymphocytes. (A) Analysis of the proliferative response. Total or CD4-depleted PBMCs were stimulated with SEB for 5 days and CD8⁺ T cells were then purified. NKG2D^low and NKG2D^high CD8⁺ T cells were restimulated with the indicated combinations of plate-bound Abs (at 5 μg/mL) and T-cell proliferation was assessed 48 hours later. Wells were pulsed with ³H-thymidine for the final 18 hours of culture and incorporated activity was measured in a scintillation counter. Data are represented as the mean plus or minus SD (triplicates) and are representative of 1 of 4 different donors tested. (B) IFN-γ production. CD8⁺ T cells were obtained as described in panel A and stimulated with plate-bound mAbs for 24 hours. Supernatants were collected and tested for the presence of IFN-γ. (C) CD8⁺ T cells were obtained as described in panel A and they were mixed with P815 cells preincubated with anti-NKG2D (at 1 μg/10⁶ cells) and different amounts of the anti-CD3 mAb. After 2 hours at 37°C, cells were stained with anti-CD8/PE and anti-CD107a/FITC mAbs. Cell-surface expression of CD107a was analyzed on CD8⁺ cells.