Figure 4

Suppressive mechanisms of MDSCs are dampened by VSSP. Mice were treated as described in Figure 2. FACS analyses of the down-regulation of CD3ζ chain on CD8⁺ (A) and CD4⁺ (B) T cells, as well as CD62L on T cells (C), were performed in splenocytes from five individual mice per group. Data were normalized by the percentage of MDSCs in each mouse. The reduction in Tregs frequency caused by VSSP was further corroborated and resulted more obvious after the normalization procedure (D). Statistical comparisons between groups were done with Student’s t test for CD62L expression and Tregs percentages, whereas Mann-Whitney’s U test was used to analyze down-regulation of CD3ζ chain. (E-F) MDSCs immunomagnetically enriched from the spleens of VSSP-injected or untreated MCA203 TB mice were cultured at 20% with splenocytes from OTI transgenic mice, in the presence of relevant peptide. Histograms show the expression of CD3ζ chain on CD8⁺ T cells specifically stimulated with SIINFEKL peptide in the presence or absence of MDSCs. Further characterization of MDSCs isolated from each experimental group was done by RT-PCR and the reduction of Arg1 (G) and Nos 2 (H) gene expression is represented in bar graphs. Three replicates of CD11b⁺Gr1⁺ cells isolated from pools of three mice per group were included in the RT-PCR analysis. (I) The capacity of MDSCs to behave as APCs during Con A-stimulated IFN-γ production by CD8⁺ T cells was measured through ELISPOT assay. CD8⁺ T cells and CD11b⁺Gr1⁺ cells were isolated from TB mice, either treated or not with the adjuvant, and cocultured for 72 h in the presence of Con A mitogen. Graph indicates the mean ± SD of the number of IFN-γ spots per 10⁵ CD8⁺ T cells from one experiment representative of two (T: tumor and V: VSSP). (G-I) The multiple comparisons of mean values were executed with ANOVA and Tukey’s tests.