Figure 4.

Decrease of the suppressive function of Tregs through OX40 costimulation. CTR-stimulated (primed) CD4⁺ T cells and naïve CD8⁺ T cells (2.5 ×10⁴ cells/well) were mixed with Tregs or CD4⁺CD25⁻ T cells (2.5 ×10⁴ cells/well) which had been stimulated with plate-bound anti-CD3 (5 μg/ml) in the presence of anti-OX40 mAb (5 μg/ml) or IL-2 (10 ng/ml) or rat IgG (5 μg/ml) for 3 days, and then plated onto 96-well tissue culture plates. The cells were incubated with irradiated APCs (2.5 ×10⁵ cells/well) and anti-CD3 (0.25μg/ml) at 37°C for 60 hours. For the last 10 hours of culture, 1 μCi [methyl-³H]Thymidine/well was added, and incorporation of thymidine was determined. (A) naïve CD8⁺ T cells proliferation cocultured with CD4⁺CD25⁻ T cells with rIgG, Tregs with rIgG, or Tregs with anti-OX40 mAb. (B) primed CD4⁺ T cells proliferation cocultured with CD4⁺CD25⁻ T cells with rIgG, Tregs with rIgG, Tregs with IL-2, or Tregs with anti-OX40 mAb. (C) IFN-g production of tumor antigen-specific primed CD8⁺ T cells. CD8⁺ T cells (1×10⁵ cells) primed with NT2 tumor cells and 3T3 neu/GM vaccine cells were cocultured for 2 hours with Tregs or CD4⁺CD25⁻ T cells (1×10⁵ cells) which had been stimulated with plate-bound anti-CD3 in the presence of anti-OX40 mAb (5 μg/ml) or rat IgG (5 μg/ml). They were incubated for 12 hours with T2D^q cells (1×10⁵ cells) that had been pulsed with either RNEU_420–429 (named T2p50) or NP_118–126. (named T2NP) and then intracellular cytokine staining (ICS) was performed. In this figure, “NP” indicates primed CD8⁺ T cells that were stimulated with T2NP cells as a negative control. In other groups, primed CD8⁺ T cells were stimulated with T2p50. In these figures, “OX40” indicates the agonistic anti-OX40 mAb. Results are from 1 representative out of 3 independent experiments. The significance of the data was evaluated with the Student’s t -test (*, p<0.05).