Cell growth inhibition of CD25⁺CD4⁺ T cells and CD8⁺ T cells by soluble OX40L and 4-1BBL. (A) CD4⁺ T cells were stimulated with anti-CD3 Ab (1 μg/mL) and anti-CD28 Ab (1 μg/mL) for 4 days. CD25⁺ cells were positively purified by anti-CD25 Ab and cell separation kit (StemCell Technologies). Enriched (∼90%) CD25⁺CD4⁺ cells were cultured with anti-CD3 Ab (1 μg/mL), anti-CD28 Ab (1 μg/mL), and IL-2 (50 U/mL). Cells were then stimulated with OX40L-GST (5 μg/mL) or 4-1BBL–GST (5 μg/mL) or both, as indicated. When cells were stimulated by one ligand only, additional GST (5 μg/mL) was added. After 3 days of stimulation [³H]thymidine incorporation assay (16 hours of pulsing) was performed. Data indicate the average and the SD of triplicate samples. Two results obtained from independent experiments are shown. (B) CD8⁺ T cells were stimulated with anti-CD3 Ab (1 μg/mL) and anti-CD28 Ab (1 μg/mL) for 4 days. These preactivated cells were cultured with anti-CD3 Ab (1 μg/mL) and IL-2 (50 U/mL). Cells were simultaneously then stimulated with OX40L-GST (5 μg/mL) or 4-1BBL-GST (5 μg/mL) or both, as indicated. When cells were stimulated by one ligand only, additional GST (5 μg/mL) was added. After 3 days of stimulation, [³H]thymidine incorporation assay (16 hours of pulsing) was performed. Data indicate the average and the SD of triplicate samples. The result represents 3 experiments with similar results.