Inhibition of T reg's by anti-OX40 and anti-GITR mAbs restores IL-2 production by effector T cells. (A) CD4⁺CD25⁺ (CD25⁺; 5 × 10⁴ cells) were preincubated or not with anti-OX40 (a-OX40) or anti-GITR mAb (a-GITR), both at 30 μg/mL, and washed twice before adding CD4⁺CD25^- (CD25^-; 1 × 10⁵ cells) and 1 μg/mL of anti-CD3 plus ACs (1 × 10⁵ cells) in 30 wells of a 96-well plate for every experimental condition. After 72 hours, cells were pooled to isolate mRNA and to perform semiquantitative PCR. Relative IL-2 mRNA levels are reported in percentages of the amount of mRNA produced by CD4⁺CD25^- cells stimulated with anti-CD3. Data shown are the mean (±SD) of 2 independent experiments (^**P < .05; ns indicates nonstatistically different). (B) CD4⁺CD25^- (5 × 10⁴) cells were cultured at different ratio with CD4⁺CD25⁺ cells preincubated or not with anti-OX40 (a-OX40), anti-GITR mAb (a-GITR), or the relative isotype control (all at 30 μg/mL), in the presence of anti-CD3 (1 μg/mL) and ACs (5 × 10⁴). After 60 hours, supernatants were collected and IL-2 content was measured by ELISA. Results are from 1 representative out of 3 independent experiments. The significance of the data was evaluated by Student t test (^**P < .01).