Persistence of different classes of stimuli modulates MoDCmigration. MoDCs were activated with the indicated stimuli. Cells were washed twice 3 hours or 12 hours after stimulation, and cell cultures were continued in RPMI plus 10% FCS without cytokines for a further 36 to 48 hours. MoDCs containing the various stimuli throughout the 36-hour culture period were cultured in parallel as controls. (A) Migration toward CCL21 of transiently or continuously stimulated MoDCs (mean ± SEM of 9 experiments, *P < .05; **P < .01 comparing CD40L₃ plus IFN-γ stimulation over 3 hours and 12 hours with 36-hour stimulation). (B) CD40L₁ plus IFN-γ was added either once or repeatedly at 0, 6, and 18 hours (+18h) or at 0 hours and for a second time 2 hours prior to the migration assay (–2h) (mean ± SEM of 4 experiments, **P < .05 as compared with single stimulation). (C) GM-CSF plus TNF-α plus IFN-α plus PGE₂ was added either once or for a second time 2 hours prior to the migration assay (–2h). Alternatively, CD40L₁ plus IFN-γ was added 2 hours prior to the migration assay into cultures initially stimulated with GM plus TNF-α plus IFN-α plus PGE₂ (mean ± SEM of 4 experiments, **P < .01 as compared with single stimulation). (D) Intact E coli was prepared as indicated in “Materials and methods,” and 20 μL of the E coli suspension was added to 1 mL of culture. Intact E coli was added either once or for a second time 2 hours prior to the migration assay (–2h). Alternatively, CD40L₁ plus IFN-γ was added 2 hours prior to the migration assay into cultures initially stimulated with intact E coli (mean ± SEM of 4 experiments, **P < .01 as compared with single stimulation).