Induction of CD4⁺ T-cell responses. (Top panel) Polarization of naive CD4⁺CD45RA⁺ T cells. CD4⁺CD45RA⁺ naive T cells were isolated via magnetic bead-based techniques and restimulated once after 5 days with allogeneic DC (1:10) that were matured as indicated. After 10 days, cells were harvested and stimulated with PMA and ionomycin. For detection of IFN-γ and IL-2, cells were stimulated for 6 hours. For detection of IL-4 cells were stimulated for 16 hours. Brefeldin A was added during the last 2 hours of incubation. For detection of intracellular cytokines, cells were permeabilized with 0.1% saponin, stained with the indicated antibodies and analyzed by FACS. (Bottom panel) Allogeneic mixed lymphocyte reactions. T cells (10⁵/well) that had been isolated using magnetic bead-mediated negative selection were cocultured at the indicated stimulator to responder ratios with allogeneic DCs that had been matured with CC or CD40L in the presence or absence of LTC₄ or PGE₂ for 24 hours. After 5 days, cell proliferation was determined using the WST-1 reagent according to the manual provided by the manufacturer. The data are shown as the mean ± SEM of triplicate cultures and represent 1 of 2 experiments that yielded similar results.