Functional DC profiles are not predicted by the strength and persistence of NFκB signaling pathways. MoDCs were activated using intact E coli and inhibitors added as in Figure 3. (A) Strength of phosphorylation of p65 NFκB. Activation was assessed by quantitative immunoblotting. Data represent the means ± SD of n = 4 separate donors. (B) Relative number of viable MoDCs 36 to 48 hours following activation (no inhibitor = 1). Immature MoDCs were washed and resuspended in culture medium at a concentration of 1 to 3 × 105 cells/mL. Cells were either continued in GM-CSF + IL-4 (immature DCs) or stimulated with GM-CSF + TNF-α + IFN-α + PGE₂ or with CD40L trimers + IFN-γ as previously described.2 Supernatants and cells were harvested after 36 to 48 hours, and viability was assessed by trypan blue cell count (means ± SEM of 3 separate donors). (C) Migration toward CCL21 by MoDCs activated with GM-CSF + TNF-α + IFN-α + PGE₂ (means ± SEM of 3-6 separate donors). (D) Secretion of IL-12p70 by MoDCs activated with CD40L trimers + IFN-γ (means ± SEM of 3-6 separate donors).