Activation marker expression and proliferation of EphB6⁺ versus EphB6^– T cells. All experiments were performed more than three times and were reproducible. Representative results are shown. T cells were first purified from PBMCs with negative selection using MACS beads. The T cells were cultured in medium for 48 hours to achieve high EphB6 expression, and then stained with biotin-conjugated anti-EphB6 mAb followed by PE-streptavidin. The cells with the highest 30% and lowest 30% EphB6 fluorescence intensity were sorted by high-speed flow cytometry. (a) The top row shows EphB6 expression of sorted EphB6⁺ and EphB6^– T cells. The lower two rows show CD25 and CD69 expression after stimulation with solid-phase anti-CD3 mAb alone (0.2 μg/ml during coating) or with anti-CD3 plus anti-CD28 mAb (4 μg/ml during coating) for 24 hours after sorting. Solid lines represent EphB6⁺ T cells and dotted lines represent EphB6^– T cells. The percentages represent cells in CD25⁺ or CD69⁺ regions after deducting background staining (isotypic mAb). For the last panel, region A represents all CD69-positive cells, and region B represents cells with high CD69 intensity. (b) Sorted EphB6⁺ (solid line) and EphB6^– (dotted line) cells were cultured in wells coated with anti-CD3 and anti-CD28, as described above, for an additional 1–3 days. Their thymidine uptake was measure for the last 8 hours of culture.