C1q increases the production of IL-12p70 from DCs stimulated by CD40 ligation. (A) WT and C1qa^−/− GM-CSF DCs were cultured alone or stimulated with antibodies against CD40 (4 μg/mL) for 16 hours. The production of the cytokine IL-12 was evaluated by intracellular staining and flow cytometry. Numbers in the square gates represent the percentage of IL-12–positive DCs. The bar graph on the right represents the mean (± SD) of 3 independent experiments (the response of the WT DCs to CD40 Ab is normalized to 100). (B) GM-CSF DCs were stimulated using CD40L-transfected or mock-transfected fibroblasts. IL-12p70 from DCs was induced only by the CD40L-transfected fibroblasts (left panel). WT and C1qa^−/− GM-CSF DCs were cultured with CD40L-transfected fibroblasts and IL-12p70 was evaluated in the supernatants (middle panel). C1q (20-40 μg/mL) was added as indicated (middle panel). WT and C1qa^−/− GM-CSF/IL-4 DCs were cultured with CD40L-transfected fibroblasts and IL-12p70 was evaluated in the supernatants (right panel). Results depicted are from 1 experiment of 3 similar ones performed with DCs from different mice (mean ± SD of duplicate samples). (C) WT or C1qa^−/− GM-CSF DCs were cultured alone or in the presence of CD40Ab (4 μg/mL) for 48 hours and the expression of CD86 was evaluated by flow cytometry. The numbers adjacent to the rectangular gates represent the percentage of CD86⁺ cells. (D) DCs were cultured as described for panel C. The graphs display the percentage increase of CD86, CD80 (% positive cells), and class II (mean fluorescence intensity, MFI). Results represent mean (±SD) of at least 3 independent experiments performed with DCs from different mice. *P < .05, significantly different from control.