NKG2D costimulation induces 4-1BB expression, reversing TGF-β1–mediated inhibition. (A) Freshly purified CD8⁺ T cells from cord blood (CB) were stimulated for 3 days with plate-coated anti-CD3 (0.1 μg/mL) plus either (i) isotype control IgG, (ii) anti–4-1BB, or (iii) anti-NKG2D (10 μg/mL each) in the presence or absence of TGF-β1 (10 ng/mL). All cell cultures contained IL-15 (10 ng/mL). Average percentage of cells expressing 4-1BB was calculated from 4 different CB samples by flow and plotted in a bar graph as mean plus or minus standard deviation (SD). (B) Surface expression of 4-1BB and NKG2D on CD8⁺ T cells after stimulation as described in panel A was accessed by flow and is displayed in dot plots. Numbers in each quadrant represent percentage of cells expressing NKG2D and/or 4-1BB. Dot plots show one of the representative results from 4 different CB samples. (C) Cytotoxic activity of CD8⁺ T cells costimulated with isotype control IgG, anti–4-1BB, or anti-NKG2D in the presence and absence of TGF-β1 was measured using the MHC class I chain-related protein A (MICA)–expressing C1R cell line as target cells. All cell cultures contained anti-CD3 (0.1 μg/mL) and IL-15 (10 ng/mL). Percent specific lysis at different effector-to-target ratios was calculated as described in “Redirected cytotoxic assay.” Averaged percentages of specific lysis calculated from quadruplet assay data were further processed to obtain the mean plus or minus SD from 3 independent experiments using 3 CB samples. P values were calculated by Student t test. All the P values at each effector-to-target ratio for the percent specific lysis by the CD8⁺ T cells costimulated without TGF-β1 were less than .05 indicating the significance of anti-NKG2D effects.