CD6 and ALCAM accumulate at the contact site between DCs and T cells in the presence of SEB. (A) Mature DCs were preincubated with 1 μg/mL SEB and subsequently mixed with PBLs of an unrelated donor in a 1:10 ratio for 15 minutes at 37°C. Cell conjugates were mounted on poly(l)lysine–coated slides and stained for ALCAM (red), CD6 (green), and LFA-1 (blue), or DC-SIGN (blue), CD6 (green), and CD3 (red). Scale bar represents 10 μm. (B) The percentage of CD6 accumulation at the contact site was scored relative to the total number of DC–T-cell contacts counted. Mean ± SEM of 6 independent experiments is shown. (C) Prior to mixing, DCs (SEB-preincubated) and PBLs were incubated with 20 μg/mL antibody against LFA-1 (L15), ALCAM (L50 and/or J4-81), anti-CD6, or control mouse IgG for 30 minutes on ice. Antibodies remained present during conjugate formation. The percentage of CD6 accumulation was calculated as described for panel B. Mean ± SEM of 6 independent experiments is shown. Asterisks indicate statistically significant differences (Student t test, *P < .001; **P < .05).