Generated LCs acquire mature DC features in defined serum-free 2° cultures.

Cells were generated in serum-free medium in the presence of TGF-β1, as shown in Figure 1. They were resuspended and plated in serum-free 2° cultures and further stimulated in the presence of cytokines (see “Materials and methods”). As shown in (A), 2° cultures were supplemented with either GM-CSF (GM) plus TNFα, GM-CSF plus CD40L, or GM-CSF alone. Control cultures represent primary cultures in which cell clusters were left undisturbed and which were maintained over the 48-hour secondary culture period. Diagrams show representative flow cytometric analyses of cells from these 2° cultures or from primary control cultures for the expression of CD86 or CD83 versus CD1a (see “Materials and methods”). (B) Representative CD86 or CD83 expression profiles of cells after resuspension and further stimulation in 2° cultures supplemented with fresh medium containing the cytokines TGF-β1 plus GM-CSF, TNFα, SCF, and FL (lines). Overlay diagrams (filled) show parallel cultures set up in identical fresh cytokine-supplemented growth medium without resuspension (ie, representing undisturbed cell clusters). Markers were set according to negative control cultures.