Inflammatory monocytes (IMs) show IFN-γ-dependent activation and become a potent source of CXCL9 and IL-12 following vaccination with <i>S</i>. Typhimurium TAS2010.

Wild-type C57BL/6 mice were either naïve or i.v. injected with 200cfu TAS2010 or BRD509, and the splenic CD4+ T cells were analysed at the indicated time points post-vaccination, where Wk 0 denotes data from naïve mice. A-C) A) The frequency of MHC-II expression, B) geometric mean fluorescence intensity (gMFI) of CD64 expression, and C) the frequency of CXCL9 production was determined for IMs in the spleen. Data are shown as group mean ± SEM for each time point, with data pooled from 3–6 independent experiments per time point (n = 9–29). Two-way ANOVA with Bonferroni’s post-tests were used for statistical analysis between the two vaccination groups. D) Representative FACS plots showing staining of relevant markers in CXCL9-producing cells, using CXCL9-negative (CXCL9-) cells as the gating control. Plots show the vast majority of CXCL9-producing (CXCL9+) cells are MHC-II+ IMs. E) Representative FACS plots show IMs from mice that were either naïve or at day 7 post-vaccination with TAS2010. The expression of MHC-II and CXCL9 depends on IFN-γ. F) The percentage of IL-12p35-producing IMs was determined using intracellular staining for IL-12p35 after 4hr incubation with brefeldin A at 37°C ex vivo. Data are pooled from 2 independent experiments. One-way ANOVA with Bonferroni’s post-tests were used for statistical analyses. G) Representative spleen sections from C57BL/6 wild-type mice that were either naïve or vaccinated with TAS2010 for 2 or 3 weeks. Sections were stained with the indicated antibodies. White bars represent 50μm.