Fig. 5: Antigen-specific T cell responses.

ICR mice were immunized on days 0 and 21 with OVA/alum, PrsA/alum, OVA/CFA/IFA or PrsA/CFA/IFA. Spleens were harvest 1 week post last immunization. Total splenocytes (10⁶) were stimulated with media alone (−), PMA (20 ng/ml) plus ionomycin (1 µg/ml) (P + I), OVA or PrsA proteins (5 µg/ml) for 6 h, and cytokine (IL-4, IFN-γ and TNF-α) expression in CD4 T cells was evaluated by intracellular cytokine stain. a Flow cytometry contour plots representing the gating strategy used to identify IL-4⁺CD4⁺, IFN-γ⁺CD4⁺, TNF-α⁺CD4⁺ T cells after P + I stimulation. The frequency of IL-4⁺CD4⁺ (b), IFN-γ⁺CD4⁺ (c), TNF-α⁺CD4⁺ (d) T cells in each treatment group. e, f Antigen-specific cytokine production. Total splenocytes (10⁶) from mice immunized with OVA/Alum, PrsA/alum, OVA/CFA/IFA or PrsA/CFA/IFA were stimulated with OVA or PrsA proteins (1 µg/ml) for 72 h. The accumulated IL-4 and IFN-γ released to the culture supernatants were analyzed by ELISA. Each dot in the figure represents the corresponding value of each mouse. Bars represent means ± SD. Significance was determined by two-way ANOVA with Tukey’s multiple comparison post hoc test (b–d) or Mann–Whitney U test (e, f). * <0.05; ** <0.01; *** <0.005.