Extended Data Fig. 3: Transcriptomic analysis of Ifngr1-deficient T_reg cells verses control, during chronic infection.

a, Expression of the deleted region of Ifngr1, normalized to the intact region of Ifngr1, from uninfected Ifngr1^L/LFoxp3^Cre-YFP (n = 5) compared to Foxp3^Cre-YFP mice (n = 6). Thy1.2^– cells, CD8⁺ T cells, CD4⁺ T_conv cells and T_reg cells were sorted, gDNA was isolated and qPCR was performed. b, Expression of IFNGR1 in T_reg cells from uninfected Foxp3^Cre-YFP (n = 7) and Ifngr1^L/LFoxp3^Cre-YFP (n = 5) mice was measured by flow cytometry. c, d, Foxp3^Cre-YFP mice remained uninfected (n = 15, 5 independent experiments, control; n = 10, 4 independent experiments, Ifngr1-deficient) or were infected with Arm. (n = 9, 2 independent experiments, control; n = 8, 2 independent experiments, Ifngr1-deficient) or Cl. 13 (n = 15, control; n = 14 (c), n = 15 (d), Ifngr1-deficient) and percentage of Foxp3⁺ of CD4⁺ T cells (c), number of T_reg cells (d, upper) and CD4⁺ T_conv cells (d, lower) per spleen, were determined by flow cytometry. e-g, scRNA-seq of splenic T_reg cells from uninfected (n = 3) or Cl. 13 infected (n = 5) (d16) Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice. e, Distribution of cells amongst clusters. f, Top 50 ranked significant differentially expressed genes from T_reg cells in Foxp3^Cre-YFP and Ifngr1^L/LFoxp3^Cre-YFP mice infected with Cl. 13. g, GSEA overview illustrating pathways upregulated in the control T_reg cell gene set compared to Ifngr1-deficient T_reg cells during Cl. 13 infection. h, Foxp3^Cre-YFP or Ifngr1^L/LFoxp3^Cre-YFP mice remained uninfected and the expression of T-bet (n = 7), GATA-3 (n = 6, control; n = 5, Ifngr1-deficient) and RORγt (n = 3, control; n = 4, Ifngr1-deficient) by splenic T_reg cells were measured by flow cytometry. a-h, Data are presented as mean values and represents biologically independent mice and 2 (a, b, e-h and as indicated) or 3 (c, d) or 5 (as indicated) independent experiments. Statistical significance was determined by an unpaired two-tailed Student’s t-test (b), or by multiple unpaired t-tests (a, c, d, h), relative to Foxp3^Cre-YFP mice. Adjusted P values were determined by one-way ANOVA relative to Foxp3^Cre-YFP mice (e) or by Wilcoxon rank-sum test (f) or Kolmogorov-Smirnov test (g) relative to infected Foxp3^Cre-YFP mice (P values indicated when significant); NS, not significant.

Source data