Anti mouse cd3 clone 145 2c11

Anti-IDH2 was from Abcam (# ab131263), anti-IDH1 was from Cell Signaling Technology (#8137S), and anti-HIF1α is from Novus biologicals (#NB100–105). Anti-mouse CD3 (clone 145–2C11, cat#16–0031), anti-mouse CD28 (clone 37.51, cat#16–0281), anti-mouse IFNγ (Clone XMG1.2,Cat#16–7311), anti-mouse IL-4(Clone 11B11, cat#16–7041), anti-mouse FOXP3 (clone FJK-16s, cat#17–5773) are from eBioscience. Anti-mouse CD4 (clone RM4–5, Cat#550954), anti-mouse CD25 (clone 7D4, Cat#558642), anti-mouse CD44 (Clone IM7, Cat#559250), anti-mouse CD62L (clone MEL-14, Cat#560507), anti-mouse IL-17 (clone TC11–18H10, Cat#559502), anti-mouse IFNγ (Clone XMG1.2, Cat#561040, for staining) and anti-CD90.1 (Thy1.1) (clone OX-7, cat#561406) were from BD Bioscience.

CD4 naïve T cells (CD4+CD25-CD62^highCD44^low) were sorted from IL-17F-RFP/FOXP3-GFP mice, which were characterized previously or wide-type C57/BL6 mice (6–10 weeks old male or female mice were used unless specified). 0.4 million cells were plated into 48 wells coated with anti-mouse CD3 (clone 145–2C11, eBioscience) (2 μg/ml) and anti-mouse CD28 (clone 37.51, eBioscience) (1 μg/ml). The differentiation for T cells are as followed: 0.5 ng/ml (or indicated) TGFβ, 200 U/ml mouse IL-2, 2 μg/ml anti-IFNү and 2 μg/ml anti-mouse IL4 for iTreg, 2.5 ng/ml TGFβ, 10 ng/ml mouse IL-1β, 10 ng/ml mouse IL-6, 10 ng/ml mouse IL-23, 2 μg/ml anti-mouse IFNү, and anti-mouse IL-4 for T_H17. All the cytokines are from R&D systems. The cells were supplemented with new medium at day 4. In cases with which small-molecule compounds present, fresh medium containing the same concentration of compounds was used. When necessary, the individual metabolite was added into the T-cell culture 6 hours later after initial cell plating. On day 6, the cells were analyzed for IL17F-RFP and FOXP3-GFP or the cells were collected and restimulated for 4–6 hours with PMA, inomycin, and Golgi-stop for intracellular staining in absence of indicated compounds.