Cytokine-producing CD4+ T-cells in the spleens of vaccinated mice after intravenous challenge.
(A) Representative plots show IFN-γ+, TNF-α+ and IFN-γ+TNF-α double-positive cytokine production by splenocytes of mice vaccinated subcutaneously with BCG or 3dBCG at 28 days after intravenous challenge with 2×107 CFU of BCG. Splenocytes were re-stimulated on IFN-γ-treated uninfected bone-marrow derived macrophages and BCG-infected macrophages. Percent values represent frequency of cytokine+ CD4+ T cells for each stimulation condition. (B) Number of IFN-γ+, TNF-α+, IL-2+ and dual cytokine+ CD4+ T cells in spleens 26 to 30 days after intravenous challenge with BCG. Each data point represents the number of BCG-specific cytokine+ T cells in one mouse spleen, calculated from percent values of cytokine+ CD4+ T cells, as in (a), and total number of splenocytes isolated from each mouse. The bars represent median values from 7 to 9 mice in each vaccinated group. Media/- mice are age-matched controls that were vaccinated with media but not challenged, C = challenged. The variances of the median values of the three challenged groups were compared by the Kruskal-Wallis non-parametric test: IFN-γ, P = 0.0031; TNF-α, P = 0.0006; IL-2, P = 0.0165; IFN-γ+TNF-α, P = 0.0007; and IFN-γ+IL-2, P = .0131. The Mann-Whitney P values for two group comparisons are displayed in the figure.