IL-12 and IL-4 affect NKG2D and 4-1BB expression. (A) CD8⁺ T cells were sequentially costimulated with isotype control IgG (i) or anti-NKG2D (ii) for 3 days, or with anti–4-1BB for an additional 3 days after 3-day anti-NKG2D costimulation (iii). Costimulation was undertaken in the presence and absence of IL-12 (10 ng/mL) or IL-4 (10 ng/mL). All the cell cultures contained anti-CD3, IL-15 and TGF-β1. Surface expression of NKG2D and 4-1BB is displayed in dot plots. Numbers in for each quadrant represent percentage of cells expressing NKG2D and/or 4-1BB. (B) Percent NKG2D⁺, NKG2D⁻4-1BB⁺ and NKG2D⁺4-1BB⁺ subsets in the respective CD8⁺ T-cell groups costimulated with (i) control IgG, (ii) anti-NKG2D, and (iii) anti–4-1BB after anti-NKG2D in the presence or absence of IL-12 or IL-4 are compared as mean plus or minus SD from 4 different CB samples. (C) CD8⁺ T cells consecutively costimulated with anti–4-1BB and anti-NKG2D with no cytokine (i) or with either IL-12 (ii) or IL-4 (iii) were measured for percentage of cell lysis using the MIC-expressing C1R cell line as target cells. Effector to target cell ratio was 2. These results are average plus or minus SD from 4 different CB samples.