Anti cd3 clone 2c11

CD4⁺ T cells were purified from spleens of naïve DBA/1 mice by positive selection using microbeads against CD4 (Miltenyi Biotec) following the protocol provided by the manufacturer. Then, the purified naïve CD4⁺ T cells were seeded at 2 × 10⁶ per well into 96-well U-bottom microplates with RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone, Carlsbad, CA), 100 units/ml penicillin, and 100 μg of streptomycin (all from Invitrogen-Gibco). Th1 differentiation was driven by the of naïve CD4⁺ T cells with 1 μg/ml plate-bound anti-CD3 (clone 2C11, eBioscience), 1 μg/ml soluble anti-CD28 (clone PV1.17, eBioscience), 10 ng/ml IL-12 (PeproTech), 5 ng/ml IL-2 (PeproTech), and 2 μg/ml anti-IL-4 antibody (clone 11B11, eBioscience). Th17 differentiation was driven by the stimulation of naïve CD4⁺ T cells with 1 μg/ml plate-bound anti-CD3, 1 μg/ml soluble anti-CD28, 50 ng/ml IL-6 (PeproTech), 10 ng/ml TGF-β1 (PeproTech), 2 μg/ml anti-IFN-γ antibody (clone R4-6A2, eBioscience) and 2 μg/ml anti-IL-4 antibody. DXM was used at the dose indicated at the beginning of the Induction. Cells were harvested on day 4 of DXM treatment and analyzed for intracellular cytokines while culture supernatants were examined for cytokine levels by ELISA assay.

CD4+CD25- and CD8+ T cells were isolated from spleen and lymph nodes to ≥90% purity by magnetic bead negative selection (Miltenyi Biotec). Cells were cultured in RPMI 1640 (MediaTech) supplemented with 10% FBS (Gemini BioProducts), penicillin-streptomycin (Gibco), L-glutamine (Gibco), and 50 µM β-ME (Sigma). Where indicated, cells were maintained in a quiescent, viable state using 10 ng/ml IL-7 (eBioscience). Alternatively, cells were stimulated on plates coated with 10 µg/ml anti-CD3 (clone 2C11) and 10 µg/ml anti-CD28 (both eBioscience) in the presence of 20 ng/ml IL-2 (Novartis), with a starting density of 0.5–1.5×10⁶ cells/ml. Cell concentration and size were measured using a Z2 particle counter (Coulter Corp.). Where indicated cells were activated in the presence of 2-deoxyglucose (Sigma) or rotenone (Seahorse Bioscience).