ALCAM-CD6 interactions are essential for DC-induced T-cell proliferation. (A) Human PBLs were triple-stained and gated for T cells, NK cells, and B cells. CD6 and ALCAM staining is represented by shaded histograms; isotype-matched controls are represented by open histograms. One representative experiment of 3 is shown. Percentage of positive cells and MFI values are indicated in the top right corner of each subpanel. (B) PBLs (10⁵) were stimulated in vitro with 1.7 × 10³ mature monocyte-derived DCs, in the presence or absence of antibodies against LFA-1 (NKI-L15), ALCAM (A2N-L50), and CD6, or recombinant ALCAM-Fc (CD6-Fc). Total mouse IgG (mIgG) and a control Fc-chimeric protein (L-SIGN–Fc) were used as negative controls. Antibodies and Fc-chimeras were added at coculture onset. Proliferation was assessed at day 5 by [³H]-thymidine incorporation for 16 hours. Results are expressed as the percentage of medium control and are the mean ± SD of 3 independent experiments. (C) PBLs (10⁵) were stimulated with 1.7 × 10³, 4.5 × 10³, or 1.0 × 10⁴ allogeneic mDCs in the presence of anti-CD6. Antibodies were added at coculture onset. Results are expressed as the percentage of proliferation in the presence of control mouse IgG and are the mean ± SD of 3 independent experiments. (D) Mature DC-induced proliferation in the presence of anti–LFA-1 (□), anti-CD6 (▦), or control mIgG (▪) was measured at different time points (days 3, 5, or 7) by [³H]-thymidine incorporation for 16 hours. Antibodies were added at coculture onset. Results are mean ± SD of 3 independent experiments.