Figure 1.
Figure 1. Anti-4-1BB but not anti-CD28 or anti-CD30 costimulation reverses TGF-β1-mediated inhibition of cytotoxic activity. (A) T cells isolated from CB were stimulated with IL-15 for 5 days and further purified for CD8+ T cells by FACS. These cells were activated with plate-coated anti-CD3 (1 μg/mL) plus increasing concentrations of anti-4-1BB at 0.1, 1.0, and 10 μg/mL in the presence (filled circles) or absence (open circles) of TGF-β1 (10 ng/mL) for 3 days. Cells were analyzed for cytotoxic activity by a standard 4-hour 51Cr release assay using an EBV-transformed B-cell line as target cells. The effector-to-target cell ratio was 2:1. Percentage of specific lysis was calculated as described in “Materials and methods.” Means ± SD of percentage of specific lysis obtained from data of 4 separate experiments each performed in triplicate are plotted. P values between cells treated with and without TGF-β1 in the presence of 0.1 and 10 μg/mL anti-4-1BB are shown. NS indicates not significant. (B) Similarly, IL-15-stimulated CD8+ T cells were activated with plate-coated anti-CD3 (1 μg/mL) plus control IgG, anti-CD28, anti-CD30, or anti-4-1BB at 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 cytotoxic activity as reflected by percentage of specific lysis. Mean ± SD obtained from data of 4 separate experiments each performed in triplicate are plotted, with P values calculated between mean percentage of specific lysis obtained with either anti-4-1BB and anti-CD28 or anti-CD30. (C) Cells were activated as in panel B with anti-CD3 (1 μg/mL) and anti-4-1BB (10 μg/mL; squares), or anti-CD28 (10 μg/mL; circles) in the presence (filled) or absence (open) of TGF-β1 (10 ng/mL) for 3 days at different effector-to-target cell ratios. Cells were analyzed for cytotoxic activity. Mean ± SD percentage of specific lysis from 4 independent experiments each performed in triplicate are plotted. P values between effects of anti-4-1BB and anti-CD28 in the presence (**P < .001) and absence of TGF-β1 (*P = .2, NS) are shown.

Anti-4-1BB but not anti-CD28 or anti-CD30 costimulation reverses TGF-β1-mediated inhibition of cytotoxic activity. (A) T cells isolated from CB were stimulated with IL-15 for 5 days and further purified for CD8+ T cells by FACS. These cells were activated with plate-coated anti-CD3 (1 μg/mL) plus increasing concentrations of anti-4-1BB at 0.1, 1.0, and 10 μg/mL in the presence (filled circles) or absence (open circles) of TGF-β1 (10 ng/mL) for 3 days. Cells were analyzed for cytotoxic activity by a standard 4-hour 51Cr release assay using an EBV-transformed B-cell line as target cells. The effector-to-target cell ratio was 2:1. Percentage of specific lysis was calculated as described in “Materials and methods.” Means ± SD of percentage of specific lysis obtained from data of 4 separate experiments each performed in triplicate are plotted. P values between cells treated with and without TGF-β1 in the presence of 0.1 and 10 μg/mL anti-4-1BB are shown. NS indicates not significant. (B) Similarly, IL-15-stimulated CD8+ T cells were activated with plate-coated anti-CD3 (1 μg/mL) plus control IgG, anti-CD28, anti-CD30, or anti-4-1BB at 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 cytotoxic activity as reflected by percentage of specific lysis. Mean ± SD obtained from data of 4 separate experiments each performed in triplicate are plotted, with P values calculated between mean percentage of specific lysis obtained with either anti-4-1BB and anti-CD28 or anti-CD30. (C) Cells were activated as in panel B with anti-CD3 (1 μg/mL) and anti-4-1BB (10 μg/mL; squares), or anti-CD28 (10 μg/mL; circles) in the presence (filled) or absence (open) of TGF-β1 (10 ng/mL) for 3 days at different effector-to-target cell ratios. Cells were analyzed for cytotoxic activity. Mean ± SD percentage of specific lysis from 4 independent experiments each performed in triplicate are plotted. P values between effects of anti-4-1BB and anti-CD28 in the presence (**P < .001) and absence of TGF-β1 (*P = .2, NS) are shown.

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