Figure 7. Rapamycin reverses the abnormal profile of the PP2A^flox T_reg cells.

(a, b) FACS-sorted PP2A^wt and PP2A^flox T_reg cells were stimulated with CD3 plus CD28 and then expanded for 4 days in the presence of 100 IU/mL of IL-2. During the final 24 hours, they were treated with 100nM of Rapamycin or DMSO. (a) The T_reg cells (gated on FoxP3-YFP⁺CD4⁺ T cells) were re-stimulated with PMA/Ionomycin for 6 hours and stained for IL-17 and IL-2. (b) The ECAR and OCR of PP2A^wt and PP2A^flox T_reg cells (sorted again as Foxp3-YFP⁺CD4⁺ T cells at the end of the culture) were measured (n=3 mice per group, unpaired, two-tailed t-test). Data are from one experiment representative of two independent experiments with similar results. (c) Left: H&E staining of the lungs and the salivary glands of PP2A^wt and PP2A^flox mice treated or not with rapamycin (scale bar represents 100 μm). Images are from one experiment representative of two independent experiments with similar results. Right: combined clinical score of inflammation in the liver, skin, stomach, salivary glands, lungs and pancreas of PP2A^wt and PP2A^flox mice treated or not with rapamycin (one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test). (d, e) Ex vivo amounts of p-S6 (d) and CD98 expression (e) in T_regs (FoxP3-YFP⁺) from mice in (c). (f) In vitro suppression assay using effector CD45.1⁺CD4⁺ (T_eff) T cells and T_reg cells from PP2A^wt or PP2A^flox rapamycin-treated and vehicle-treated mice. Representative histograms showing CFSE dilution and percentage of divided cells (ratio T_reg/T_eff: 1/1). Data are from one experiment representative of two independent experiments with similar results. Mean ± s.e.m., *P<0.05, **P<0.01, ***P<0.001.