Figure 7. Blockade of glycolysis improves the scurfy phenotype of Foxp3^ΔEGFPiCre mice.

(a) ECAR under glycolysis stress test conditions and OCR under mitochondrial stress test conditions of ΔT_reg cells isolated from Foxp3^ΔEGFPiCre or Foxp3^ΔEGFPiCrePfkfb3^Δ/Δ males (n=3 per group). Results are expressed as mean ± SEM and represent 1 of 2 independent experiments. (b) Frequencies of CD62L^loCD44^hi CD4⁺ T_eff cells, and IFN-γ and IL-4 expression by CD4⁺ T_reg and T_eff cells in spleens of Foxp3^EGFPCreR26^YFP, Foxp3^ΔEGFPiCreR26^YFP, Foxp3^ΔEGFPiCrePfkfb3^Δ/ΔR26^YFP, Foxp3^ΔEGFPiCreRictor^Δ/ΔR26^YFP, Foxp3^ΔEGFPiCreRictor^Δ/ΔPfkfb3^Δ/ΔR26^YFP mice. Results are expressed as scatter plot and mean and represent a pool of 3 independent experiments. (c) In vitro suppression of CD4⁺ T_eff cell (T_resp) proliferation by Foxp3^EGFPCre, Foxp3^ΔEGFPiCre or Foxp3^ΔEGFPiCrePfkfb3^Δ/Δ T_reg cells (T_supp) (n=3 per group). Results are expressed as mean ± SEM and represent 1 of 2 independent experiments. (d) Frequencies of IFN-γ⁺ and IL-4⁺ Foxp3^ΔEGFPiCreR26^YFP and Foxp3^ΔEGFPiCreRictor^Δ/ΔR26^YFP sorted T_reg cells that were either sham treated or treated ex-vivo with 2-deoxyglucose (2DG) (n=3 per group). Results are expressed as scatter plot and mean and represent 1 of 2 independent experiments. (e) In vitro suppression of CD4⁺ T_eff cell (T_resp) proliferation by Foxp3^ΔEGFPiCre ΔT_reg (T_supp) cells that were pre-treated for 12h with either PBS (Veh ΔT_reg cells) or 2DG (2DG ΔT_reg cells) (n=3 per group). Results are expressed as mean ± SEM and represent 1 of 2 independent experiments. (f, g) Representative hematoxylin and eosin-stained tissue histological sections (f) and tissue histological scores (g) of ears, livers and lungs of Foxp3^ΔEGFPiCreR26^YFP mice treated with PBS (n=) or 2 μg/g 2DG (n=) every other day from day 14 to day 28. Results are expressed as scatter plot and mean and represent a pool of 2 independent experiments. (h) Frequencies of CD62L^loCD44^hi CD4⁺ and CD8⁺ T_eff cells, IFN-γ⁺ and IL-4⁺ T_reg and CD4⁺ T_eff cells and T-Bet⁺ and Gata-3⁺ T_reg and CD4⁺ T_eff cells in Foxp3^ΔEGFPiCreR26^YFP mice treated with PBS (n=6) or 2DG (n=7). Results are expressed as scatter plot and mean and represent a pool of 2 independent experiments. Statistical significance was determined unpaired t-test (g, h), one-way ANOVA with Tukey’s multiple comparisons (b) or two-way ANOVA with Sidak’s multiple comparisons (a, d) or with Tukey’s multiple comparisons (c, e) (P values as indicated).