Vaccination with <i>S</i>. Typhimurium TAS2010 leads to robust Th1 activation in CD4⁺ T cells.

Wild-type C57BL/6 or IFN-γ.eYFP reporter mice were either naïve or i.v. injected with 200cfu BRD509 or TAS2010, and the splenic CD4+ T cells were analysed at the indicated time points post-vaccination, where Wk 0 denotes data from naïve mice. A, D, G) Representative FACS plots show total viable CD4+ T cells in mice that were either naïve or at A) week 1 or D) week 2 post-vaccination with BRD509 or TAS2010; or G) week 2 post-vaccination with TAS2010. The frequency of gated cells is shown. B, E, F, H) The frequency of CD4+ T cells that B) expressed IFN-γ.eYFP reporter, E) secreted IFN-γ protein as determined by ex vivo secretion assay using diabodies, or H) stained for indicated activation/memory markers is shown. F) The number of CD44hi CD4+ T cells was calculated per spleen. The mean or mean ± SEM is shown for each group at the indicated time points post-vaccination, and data are pooled from 2–4 independent experiments at each time point. Two-way ANOVA with Bonferroni’s post-tests were used for comparing the two vaccination groups at each time point. C) Spleen cells were identified as natural killer (NK) cells (CD3-NK1.1+), natural killer T (NKT) cells (CD3+NK1.1+), B cells (CD19+B220+), CD11b+ myeloid cells (CD3-CD19-CD11b+), CD4+ T cells (TCRβ+CD4+) or CD8+ T cells (TCRβ+CD8+) based on surface markers. The proportion of each cell subsets that expressed IFN-γ-eYFP was determined at day 7 post-vaccination. Shown are symbols for individual mice with group mean, data pooled from 2 independent experiments. Two-way ANOVA with Bonferroni’s post-tests were used twice: first for comparing the two vaccination groups for each immune cell subsets, then between cell subsets from TAS2010-vaccinated mice.