ALCAM-CD6 interactions are important in the formation of DC–T-cell contacts. (A) Mature monocyte-derived DCs, preincubated with (right panel) or without (left panel) 1 μg/mL SEB, were incubated with PBLs of an unrelated donor in a 1:10 ratio for 15 minutes at 37°C, allowing for DC–T-cell cluster formation. After mounting on poly(l)lysine–coated slides DCs were stained for ALCAM (red), and PBLs were stained for CD6 (green) and LFA-1 (blue). Arrows indicate DC–T-cell conjugates. Magnifications of 3 mature DC–T-cell contacts are shown (subpanels i-iii). Scale bar represents 10 μm. (B) The number of DC–T-cell contacts in the presence or absence of SEB was determined by counting 20 randomly selected microscopic fields. At least 100 DCs per condition were analyzed. The percentage of DC–T-cell conjugates per total number of DCs is calculated. 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 (AZN-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 conjugates was calculated as described for panel B. Mean ± SEM of 6 independent experiments is shown. *Statistically significant differences (Student t test, P < .05).