Activation marker expression and lymphokine secretion of EphB6-costimulated T cells. All experiments were carried out more than three times and were reproducible. Representative results are shown. (a) MACS-purified T cells were cultured in wells coated with anti-EphB6 mAb (4 μg/ml) plus anti-CD3 mAb (0.2 μg/ml) as described in Figure 2. Their CD25, CD54, and CD69 expression at 24 hours was assessed by one-color flow cytometry using FITC-labeled anti-CD25, PE-labeled anti-CD54, and FITC-labeled anti-CD69. Percentages represent positive populations in the gated regions after deducting background staining (isotypic Ab, shaded area). (b) MACS-purified T cells were cultured in wells coated with anti-EphB6 mAb (4 μg/ml), anti-CD3 mAb (0.2 μg/ml), or both, for 48 hours. IL-2, IL-4, IL-6, IL-10, IFN-γ, TGF-β, TNF-α and GM-CSF in supernatants were measured by ELISA, with samples in duplicate. Data are expressed as mean ± SD.