Anti-4-1BB costimulation restores TGF-β1-suppressed expression of granzyme B and IFN-γ. IL-15-stimulated CD8⁺ T cells were activated with plate-coated anti-CD3 (1 μg/mL) plus anti-CD28 (10 μg/mL) or anti-4-1BB (10 μg/mL) in the presence (filled bars) or absence (open bars) of TGF-β1 (10 ng/mL) for 3 days. Cells were analyzed for intracellular expression of granzyme B (A) and IFN-γ (B) by 2-color immunostaining. Granzyme B and IFN-γ levels were plotted against CD8 and FSC, respectively. Means ± SD of percentage of granzyme B⁺ and IFN-γ⁺ cells from 3 separate experiments are shown in the histograms. P values are shown between cells treated with and without TGF-β1 in the presence of anti-CD28 or anti-4-1BB. (C) IL-15-stimulated CD8⁺ T cells were costimulated with anti-CD28 or anti-4-1BB for 3 days as in panels A and B and then treated with TGF-β1 (10 ng/mL) for 0, 10, and 60 minutes. Phosphorylation of Smad2 induced by TGF-β1 was detected by Western blot using phospho-Smad2-specific antibody. Equivalent total amounts of Smad2 levels in each sample were demonstrated by reprobing the stripped same blot with the Smad2-specific antibody. One representative result of 2 experiments with similar results is shown. Relative phospho-Smad2 intensities are shown as relative numbers normalized to 0 and 1 for anti-CD28 costimulated cells treated with TGF-β1 for 0 and 10 minutes, respectively, as described in “Materials and methods.”