Inhibitory IL-4 effect on cytotoxic activity in the presence of TGF-β1 results from inhibition of 4-1BB expression. (A) IL-15-stimulated CD8⁺ T cells were activated with plate-coated anti-CD3 (1 μg/mL) plus anti-4-1BB (10 μg/mL) for 3 days in the absence or presence of IL-4 (10 ng/mL) or IL-12 (2 ng/mL). The activation was undertaken with (filled bars) or without (open bars) TGF-β1 (10 ng/mL). Cells were analyzed for cytotoxic activity to an EBV-transformed B-cell line at a 2:1 effector-to-target cell ratio using a standard ⁵¹Cr release assay. Cells activated with TGF-β1 in the absence of anti-4-1BB costimulation demonstrated low (below 5%) specific lysis. Mean ± SD of the percentage of specific lysis from 4 separate experiments each performed in triplicate are plotted. (B) Similarly, cells activated in the presence and absence of IL-4 (10 ng/mL) with or without IL-12 (10 ng/mL each) were assayed for cytotoxic activity as in panel A at different effector-to-target cell ratios. The activation was undertaken with TGF-β1 (10 ng/mL). Mean ± SD of the percentage of specific lysis from 4 separate experiments each performed in triplicate are plotted against E/T ratios. P values for the inhibitory IL-4 effects in the presence (^*P < .01) and absence of IL-12 (^**P < .01) are shown. (Ci) IL-15-stimulated T cells were activated with plate-coated anti-CD3 (1 μg/mL) plus anti-4-1BB (10 μg/mL) for 3 days in the absence or presence of IL-4. The activation was undertaken with TGF-β1 (10 ng/mL). Cells prior to and after anti-CD3 activation were analyzed for the expression of 4-1BB and CD62L on CD8⁺ T cells as shown by percentages in the dot plots. Mean ± SD of the percentage of CD62L⁺4-1BB^- (□), CD62L⁺4-1BB⁺ (▨), and CD62L^-4-1BB⁺ (▪) cell types from 4 experiments before (Cii) and after (Ciii) activation with or without IL-4 are plotted in the histograms. P values for IL-4 effects on production of CD62L⁺4-1BB^- and CD62L^-4-1BB⁺ cell types are shown.