Figure 3.

Raptor coordinates T_reg proliferation and effector molecule expression by orchestrating cholesterol/lipid biosynthetic metabolism, especially the mevalonate pathway. a, CFSE-labeled T_regs from wild-type and CD4^creRaptor^fl/fl mice were stimulated with anti-CD3, anti-CD28 and IL-2 for 3 days, followed by analysis for CFSE dilution and CTLA-4 and ICOS expression (MFI presented above the plots). b, Celltrace violet-labeled T_regs from wild-type and Foxp3^creRaptor^fl/fl mice were transferred into unmanipulated CD45.1⁺, and Celltrace violet dilution and CTLA-4 expression were analyzed 7 days later. c, Gene-set enrichment analysis reveals the under-representation of cholesterol biosynthesis genes in Raptor-deficient T_regs. The lower part of the plot shows the distribution of the genes in the cholesterol biosynthesis signature gene set (‘Hits’) against the ranked list of genes, and the list on the right shows the top hit genes. d, Real-time PCR quantification of lipogenic gene expression in wild-type and Cd4^creRaptor^fl/fl freshly isolated T_regs or those stimulated with anti-CD3, anti-CD28 and IL-2 for 8 hours. e, De novo lipogenesis of T_regs from wild-type and Cd4^creRaptor^fl/fl mice after 24 hours of anti-CD3, anti-CD28 and IL-2 stimulation, with the final 4 hours labeled with ¹⁴C-glucose. f, Total cholesterol level was measured using lysates of activated T_regs from wild-type and Cd4^creRaptor^fl/fl mice. g, In vitro suppression assays mediated by T_regs previously activated by anti-CD3, anti-CD28 and IL-2 for 3 days in the presence of vehicle, simvastatin (2 μM), simvastatin and mevalonate (100 μM) or mevalonate alone. Error bars represent s.d. (n=3). Results represent 2 independent experiments except for c (n=4 from one experiment).