Induction of reactivity to the CD28 superagonist TGN1412 in human PBMCs by preculture. (A) Fresh PBMCs were cultured in 0.2 mL AB medium for 24 hours at 1 × 106 cells/mL in 96-well flat-bottom tissue culture plates. Cytokines in the supernatants were analyzed after 24 hours. mAb were used at 1 μg/mL. Isotype controls were negative (not shown). (B) PBMCs from the same donor as in Figure 1A were cultured in 1.5 mL AB medium for 2 days at 1 × 107 cells/mL in 24-well flat-bottom suspension culture plates before being washed and readjusted to 1 × 106 cells/mL. With these cells, the same experiment was performed as shown in Figure 1A. (C) Compiled data from 22 individual healthy donors. Conditions for antibody stimulation and for preculture were as in Figure 1A. Wilcoxon signed rank test: *P ≤ .05; **P ≤ .005; ***P ≤ .0001. − represents unstimulated cells. Data are mean ± SD of triplicate samples.
