Contains S1-S11 Figs.

S1 Fig. Generation of mice harboring a conditional IFNγR2 allele. (A) Schematic representation of the IFNγR2 targeting strategy. (B) Immunoblot analysis of IFNγR2 in naive Tconv (TN) and Treg (TR) cells isolated from Foxp3creIFNγR2fl/fl and WT control mice. (C) FACS analysis of phosphorylation of Stat1 in Treg or Teff cells from in Foxp3creIFNγR2fl/fl and WT control mice.in response to IFNγ stimulation. FACS data are representative of three independent experiments. S2 Fig. No difference in total Treg cell numbers was observed in mice with DC- or myeloid cell-specific ablation of IFNγR2. Frequencies of Foxp3+ Treg cells from spleen in CD11ccreIFNγR2fl/fl, LyzcreIFNγR2fl/fl or WT control mice. Data are representative of two experiments and each dot represents an individual mouse. S3 Fig. Deletion of IFNγR in DCs does not lead to dysregulated IFNγ-mediated Th1 responses. FACS analysis and frequencies of T-bet+ or IFNγ+ Foxp3-CD4+ T cells isolated from (A) spleen or (B) LP of small intestine in CD11ccreIFNγR2fl/fl or WT control mice. FACS data are representative of three independent experiments and each dot represents an individual mouse. S4 Fig. IFNγ signaling in DCs is essential to drive the expression of IL-12. (A) FACS and (B) qRT-PCR analysis of IL-12 expression in CD11c+ DCs isolated from CD11ccreIFNγR2fl/fl mice or WT control mice in response to IFNγ stimulation. Data are representative of two independent experiments. (*pS5 Fig. Comparable effector Th1 cell responses in mice harboring IFNγ-insensitive DCs during early phase of T. gondii infection. (A) Frequencies of total Foxp3+ Treg cells and (B) FACS analysis and frequencies of T-bet+ cells in Foxp3+CD4+ Treg cells and IFNγ+ cells in Foxp3-CD4+ Teff cells from LP in CD11ccreIFNγR2fl/fl or WT control mice at days 4 after infection. FACS data are representative of two independent experiments and each dot represents an individual mouse. (**pS6 Fig. Acquisition of IFNγ-producing capacity by Treg cells from CD11ccreIFNγR2fl/fl mice with collapse in total Treg cell population during T. gondii infection. FACS analysis and frequencies of IFNγ+ cells in Foxp3+CD4+ Treg cells from LP in WT control mice and CD11ccreIFNγR2fl/fl mice with or without Treg cell collapse at days 8 after infection. FACS data are representative of three to four independent experiments and each dot represents an individual mouse. (**pS7 Fig. Deletion of IFNγR in Treg cells did not lead to reduced Th1-Treg cell frequencies and dysregulated IFNγ-mediated Th1 responses during T. gondii infection. (A) FACS analysis and frequencies of T-bet+Foxp3+CD4+ Treg cells and (B) FACS analysis and frequencies of IFNγ+Foxp3-CD4+ Teff cells isolated from spleen or LP of small intestine in Foxp3creIFNγR2fl/fl or WT control mice at days 8 after infection. FACS data are representative of three independent experiments and each dot represents an individual mouse. S8 Fig. Gene expression profiling analysis in IFNγ-unresponsive DCs isolated from T. gondii infected mice. (A) Schematic of mixed BM chimeras with T. gondii infection. (B) Gene expression volcano plot, with—log 10 of the p value on the y axis and log 2 fold change on the x axis. (C) Hierarchical clustering and heat map analysis with genes that were differentially regulated 2-fold or greater and p (D) Top 20 genes that were either upregulated or downregulated were shown. S9 Fig. Cell-type specific deletion of IFNγR2. qRT-PCR analysis of IFNγR2 expression in CD11c+ DCs or CD11b+ myeloid cells in CD11ccreIFNγR2fl/fl mice, LyzcreIFNγR2fl/fl mice or their corresponding WT littermates. Data are representative of two independent experiments. (***pS10 Fig. Impaired IL-27 production by IFNγ-insensitive DCs did not result in reduced IL-10 secretion by effector T cells during T. gondii infection. (A) FACS analysis and (B) frequencies IL-10+ cells in Foxp3-CD4+ Teff cells isolated from CD11ccreIFNγR2fl/fl and WT control mice day 8 post T. gondii infection. FACS data are representative of two independent experiments (n = 5). S11 Fig. Treg cell-intrinsic IL-27 signaling is essential to maintain normal T-bet+CXCR3+ Treg cell population at both physiological and T. gondii infection settings. FACS analysis and frequencies of T-bet+ cells within each donor-derived Foxp3+CD4+ T cell population from spleen and LP in IL-27Rα-/-/Ly5.1 B6 mixed BM chimeras and control chimeric mice (A) at steady state or (B) 8 days after T. gondii infection. FACS plots are representative of three independent experiments. (*p (PDF)