Adoptive protection from week 2 splenocytes is IFN-γ and CD4⁺ T cell-dependent.

B6 or IFN-γ-eYFP^in/in mice were either naïve or infected with 200CFU TAS2010 i.v. for 2 weeks. (A) Weight loss (expressed as a percentage of initial weight, group mean shown) and (B) percentage survival of Rag2^-/- Il2rg^-/- recipient mice after receiving 5×10⁷ splenocytes from naïve or week 2 p.i. B6 donor mice, i.p. injected with either isotype control, anti-Thy1.2, anti-CD4, anti-CD8 or anti-NK1.1 i.p. at the time of cell transfer, then challenged with 200CFU wild type SL1344 24 h after transfer (n = 9–10). Data are pooled from 2–4 independent experiments. Within week 2 p.i. donor splenocytes, (C) representative FACS plots and (D) percentage of the expression of IFN-γ-eYFP in different Thy1.2⁺ sub-populations and E) level of IFN-γ expression (expressed as MFI) identify CD4⁺ T cells are the main producer of IFN-γ. Each symbol represents a pool of donor cells from an independent experiment, data from 4–7 experiments are shown here with horizontal bar representing the mean for each sub-population, and one- or two-way ANOVA with Bonferroni post-tests was used for statistical analysis. F) Representative FACS histogram and graph show the majority of eYFP⁺ CD4⁺ T cells in week 2 p.i. mice secreted IFN-γ upon ex vivo restimulation with heat-killed TAS2010. Symbols in the graph represent data from individual animals (n = 8 per each group) with the group mean shown as horizontal bars. For survival data (B), The Mantel-Cox log-rank test was used for statistical analysis. For all other panels, one- two-way ANOVA with Bonferroni post-tests was used for statistical analysis, *, p<0.05; ***, p<0.001; ****, p<0.0001.