Fig. 1.

Polarized DCs produce increased amounts of IL-12 as compared to equally mature, non-polarized DCs. CD11c⁺ DCs were positively isolated from day eight bulk cultures differentiated in presence of GM-CSF. a After 1 day of maturation with LPS or polarization with LPS, IL-4 and/or IFNγ, cells were harvested and co-incubated with a CD40L-transfected cell line (J558-CD40L) without co-stimulating cytokines for 24 h. IL-12 in the supernatant was measured by ELISA. b Spontaneous and induced IL-12 production measured for cytokine-matured (upper panels, LPS or LPS, IL-4, IFNγ) and TLR ligand-matured (lower panels, poly-(I:C)/CpG or poly-(I:C)/CpG/IFNγ) DCs. Supernatants were harvested after 24 h maturation (spontaneous) and after matured DCs were stimulated with J558-CD40L cells for an additional 24 h. c After 1 day of maturation with LPS or LPS, IL4 and IFNγ, cells were harvested and replated in medium containing only GM-CSF. J558-CD40L cells were added at the indicated times and supernatants were harvested 24 h later to measure IL-12 production after delayed CD40L stimulation. d Expression of maturation markers on immature DCs, non-polarized (LPS-matured) and polarized (LPS, IL-4 and IFNγ-matured) DCs. Representative histograms are shown. Data represent one of three independent experiments