Active β₂ integrins on DCs modulate T-cell activation. (A) T cells (1 × 10⁵) from CD18^+/+ or CD18^−/− mice were cocultured with allogeneic BALB/c DCs at the indicated ratios. DCs were left untreated or were preincubated with 5 mM Mg²⁺ or with (B) 5 mM Mg²⁺ plus 10 μg/mL anti-CD18 antibody either of the GAME46 or M18.2, as indicated. After 3 days, 1 μCi (0.037 MBq) ³H-thymidine was added to the cultures for another 18 hours and thymidine incorporation was determined. Data are mean values (± SEM) of triplicates. One representative experiment of 3 is shown. (C) CFDA-labeled DCs and SNARF-labeled T cells were coincubated for 1 hour and sedimented on poly-l-lysine–coated slides. DC–T-cell contacts were analyzed by fluorescence microscopy. Bar graphs show the average number of DC–T-cell contacts in various concentrations of Mg²⁺, photomicrographs show representative pictures. (D) CFDA-labeled allogeneic BALB/c T cells (1 × 10⁵) were cocultured with either bmDCs from CD18^+/+ or CD18^−/− mice at a T/DC ratio of 10:1. Cells were left untreated or treated with 5 mM Mg²⁺. After 5 days of culture, cells were labeled with anti-CD3 and proliferation of T cells was measured by flow cytometry.