IL-12 amplifies anti-4-1BB blocking effects on TGF-β1 inhibition of 4-1BB expression. (A) IL-15-stimulated CD8⁺ T cells were activated with plate-coated anti-CD3 (1 μg/mL) for 3 days in the absence or presence of IL-12 (2 ng/mL) or TGF-β1 (10 ng/mL). Cells were analyzed for the expression of 4-1BB⁺CD8⁺ T cells as shown by percentages in the dot plots. Means ± SD of the percentage of 4-1BB⁺CD8⁺ T cells from 4 experiments are shown in the histograms. P values between effects of absence (open bars) or presence (filled bars) of TGF-β1 in the presence and absence of IL-12 are shown. (B) IL-15-stimulated CD8⁺ T cells were activated as in panel A with different concentrations of anti-4-1BB in the presence or absence of IL-12 (2 ng/mL). All activation was undertaken with TGF-β1 (10 ng/mL). Percentages of 4-1BB⁺ cells produced with (filled squares) or without IL-12 (open squares) from 4 independent experiments, each performed in triplicate, were plotted as mean ± SD against the concentrations of anti-4-1BB.