FIG. 3.

Production of (A) IL-5, (B) IL-6, (C) IL-12, (D) IFN-γ, and (E) GM-CSF from spleen cells and of (F) nitric oxide from peritoneal macrophages from mice stimulated with different B. pertussis antigens for 48 h (spleen cells) or 24 h (macrophages). A description of the immunization procedure is given in the legend to Fig. 1A. Groups of mice were immunized with ACV alone, CyaA* alone, or ACV plus a CyaA form (25 μg per mouse) or were given PBS only. Spleen cells and macrophages were obtained on day 42, and the cells from five mice were pooled. PAgs (white bars), PAgs plus CyaA* (gray bars), and CyaA* alone (black bars) were used as stimuli. Other cells were not stimulated and served as controls (striped bars). PAgs consisted of a mixture of formalin-treated PT, FHA, and PRN, used at final concentrations of 2, 2, and 5 μg/ml, respectively. CyaA* was used at a final concentration of 1 μg/ml. Results represent the means for duplicate assays (for spleens) or triplicate assays (for macrophages) with SEM (error bars). †, P < 0.05 (PAg-plus-CyaA*-stimulated group versus PAg-plus-CyaA*-stimulated, ACV-immunized group [ANOVA]); ††, P < 0.05 (CyaA*-stimulated group versus CyaA*-stimulated, CyaA*-immunized group [ANOVA]); ##, stimulation with PAgs or PAgs plus CyaA* was not tested.