Zstk474

T cells were activated and cultured in RPMI-1640 medium containing 10% FCS, 50 U/ml Penicillin, 50 µg/ml Streptomycin, 50 µg/ml Gentamycin, 1 nM Sodium Pyruvate, 10 µM Hepes, 2 mM L-glutamine, and 50 µM 2-Mercaptoethanol (complete RPMI). Purified naive T cells were stimulated in 24-well plates (3–5 × 10⁶ cells/well) precoated with the indicated concentrations of anti-CD3 (2C11; Bio-XCell) and anti-CD28 (37.51; Bio-XCell) in PBS in complete medium containing 50–100 U/ml recombinant murine IL-2 (eBioscience) for the indicated times. In some experiments, the class I PI3K inhibitor ZSTK474 (Sigma) was added as indicated. For restimulation experiments, cells were stimulated for 2 d with 1 µg/ml plate-bound anti-CD3/anti-CD28 + IL-2, then removed from stimulus and placed in complete media (without IL-2) overnight before restimulation with plate-bound anti-CD3 as indicated. For polarization of CD4⁺ T cells into TH1 and TH2 cells, total CD4+ T cells were activated with plate-bound anti-CD3 and anti-CD28 in media containing 200 U/ml IL-2 supplemented with 10 ng/ml IL-12 (eBioscience) and 20 µg/ml anti–IL-4 (11B11; Bio-XCell) for TH1 polarization or 100 ng/ml IL-4 (eBioscience) and 40 µg/ml anti–IFN-γ (XMG1.2; Bio-XCell) for TH2 polarization.