Fig 1: Treg-derived IFN-γ reinforces Th1-Treg differentiation in vitro. (A) Flow cytometry analysis of YFP– CD4+ CD25+ cells that were derived from spleens of B16F10-bearing Foxp3-Cre/Tbx21-Flp/VeDTR(LF) mice and indirectly cocultured with RFP+ (Arg1+) TAMs from MC38-bearing Arg1-RFP mice by using transwell plates in the presence or absence of anti–IFN-γ monoclonal antibody. Gates show YFP+ cells. Representative FACS plots and the percentages of YFP+ cells are pooled from three independent experiments. (B) Flow cytometry analysis of YFP– CD4+ CD25+ cells that were derived from spleens of B16F10-bearing Foxp3-Cre/Tbx21-Flp/VeDTR(LF) mice and subsequently stimulated with rPF4 in the presence or absence of anti–IFN-γ monoclonal antibody. Gates show YFP+ cells. Representative FACS plots and the percentages of YFP+ cells pooled from four independent experiments. (C) Volcano plots from bulk RNAseq analysis showing differential expression of genes in PF4 treated YFP– CD4+ CD25+ from spleens of B16F10-bearing Foxp3-Cre/Tbx21-Flp/VeDTR(LF) mice. Data are mean ± SEM and pooled from two to three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.