For costaining Foxp3 with GFP, cells were fixed by cytofix buffer (BD Bioscience), permeablized by ice-cold methanol and stained with indicated antibodies in the 1× Perm/Wash buffer (BD Bioscience). Splenocytes and lymph node cells were first stained with anti-CD4 biotin and CD4+ cells were magnetically purified using a Biotin-selection kit (Stem cell). Purified CD4+ T cells were stimulated with plate bound anti-CD3 (3 μg/mL; 2C11) and soluble anti-CD28 (2 μg/mL; 37N) in T-cell medium (RPMI 1640, 10% FBS, 1× antibiotics, 1× nonessential amino acid, and 50 μM β-mercaptoethanol).

When indicated, Selleckchem DAPT recombinant (r) cytokines were added into the culture: TH1: anti-IL-4 (5 μg/mL; 11B11) and IL-12 (10 ng/mL; PeproTech); iTreg-cell: anti-IL-4 (5 μg/mL, 11B11), anti-IFN-γ (5 μg/mL; R46A2), rhIL-2 (100 U/mL, PeproTech), and indicated concentration of rhTGF-β (Peprotech); TH17: anti-IL4 (5 μg/mL), anti-IFN-γ (5 μg/mL), IL-6 (20 ng/mL, Peprotech), and indicated concentration of rhTGF-β (Peprotech). When indicated, the following inhibitors were used in this study: Rapamycin (LC laboratories); pp242 [[19]]. Naive CD4+ T cells were activated with anti-CD3/anti-CD28

antibodies in the presence EPZ-6438 in vivo of IL-2 (50 U/mL) for 4 days. Activated cells were then split into fresh culture medium with IL-2 (100 U/mL) and expanded for an additional 4 days. Cultured T cells were rested in T-cell medium without PD184352 (CI-1040) IL-2 overnight and stimulated with either plate bound anti-CD3 (5 μg/mL) + anti-CD28 (2 μg/mL) for various time points. Stimulated T cells were washed with ice-cold PBS and lysed with RIPA buffer plus freshly added protease inhibitors and phosphatase inhibitors. Total cell

lysates were used for immunoblot analysis. To detect S6 and Akt S473 phosphorylation following TCR stimulation, CD4+ T cells were first stained with anti-CD3 (5 μg/mL) for 30 min on ice. After wash, T cells were cross-linked with anti-Hamster IgG for 3 min, fixed with Phosflow fix buffer I (BD Bioscience), and stained with anti-pS6 S235/236 or anti-pAkt S473 (Cell Signaling) in Phosflow perm/wash buffer (BD Bioscience) for 30 min at room temperature followed by Alex-fluor 647-conjugated anti-Rabbit IgG (Cell Signaling) in Phosflow perm/wash buffer for 15 min at room temperature. Purified CD4+ T cells were labeled with CFSE (3 nM) at 37°C for 10 min. CFSE-labeled cells were stimulated with plate bound anti-CD3 and anti-CD28 as described [[28, 29]]. We thank Drs. A. Di Lorenzo and W. Sessa (Yale University) for the Akt1 and Akt2 knockout mice, K.M. Shokat (UCSF) for providing the pp242. This work is supported in part by grants AI063348 (NIH) and PR093728 (DOD) (to B. Su). A.S. Lazorchak is a Leukemia and Lymphoma Society fellow, and X. Chang was a recipient of Gershon and Trudeau Fellowship from Yale University. The authors declare no financial or commercial conflict of interest.