EphB6 crosslinking enhances the T cell response to TCR stimulation. Representative results are shown. (a and b) MaxiSorp 96-well plates were coated overnight at 4°C with a suboptimal concentration of αCD3 mAb (clone OKT3, 0.2 μg/ml) along with various concentrations of αEphB6 mAb (clone 4F12) (a), or were coated with an optimal concentration of αEphB6 (4 μg/ml) along with various concentrations of αCD3 (b). MACS-purified T cells were cultured in these plates for 72 hours, and 3H-thymidine was added for the last 8 hours of culture. (c) MASC-purified T cells were cultured in these wells in the presence or absence of soluble EphB6-Fc or a control protein (control-Fc) (10 μg/ml) as inhibitor. (d and e) MaxiSorp 96-well plates were coated with α-CD3 plus α-EphB6, or with α-CD3 plus αCD28 (4 μg/ml) as described above. T cells were purified from PBMCs by MACS and subsequently fractionated into CD4+ cells, CD8+ cells, CD45RO+ cells, and CD45RO– cells (pure T cells depleted of CD45RO+ cells) using MACS beads. The cells were cultured for 1–3 days, and their thymidine uptake was measured for the last 8 hours of culture. (f) Costar 96-well plates were first coated with α-CD3 (0.2 μg/ml) overnight. After washing, the wells were incubated with mouse ephrinB2-Fc or a control recombinant protein with an Fc tag (control-Fc) (5 μg/ml or 10 μg/ml, as indicated) for 2 hours at 37°C followed by 2 hours on ice. MACS-purified T cells were cultured in the wells for 48 hours, and thymidine uptake was measured for the last 8 hours of culture. α, anti.