iNKT Cells, gating as GFPhi cells, were sorted from livers or spleens of Vα14 Tg.cxcr6gfp/+ mice by FACSAria (BD Biosciences). The purity of iNKT cells was more than 90% (fig. S1B). CD4 T cells gating out iNKT cells were sorted from the spleens. Purified iNKT cells were stimulated with plate-coated mCD1d-PBS57 tetramer (1 μg per well) or plate-coated antibodies against CD3 (1 μg per well) and CD28 (1 μg per well), with or without 2-DG (5 mM), G6P (25 mM), or HKVBD peptide (20 μM). Cytokines in supernatants were measured by CBA kit (BD Biosciences) after overnight activation. In proliferation assays, cells were stimulated with plate-coated mCD1d-PBS57 tetramer for 2 days, and cell proliferation was measured by Ki67 staining (BD Biosciences). To compare the expression of activation markers, iNKT cells were stimulated with plate-coated mCD1d-PBS57 tetramer overnight. In pyruvate supplementary assays, pyruvate (10 mM) was added to iNKT cells stimulated with plate-coated mCD1d-PBS57 tetramer overnight with or without 2-DG. In TCR blocking assays, enriched iNKT cells were stimulated with αGC-pulsed RBL.CD1d, and TCR signaling was blocked by antibodies against CD1d (10 μg/ml) at the indicated time points. To investigate the in vivo effect of 2-DG on iNKT cells, mice were administered intraperitoneally with 6 mg of 2-DG or PBS buffer, 1 hour before injecting 2 μg of αGC per mouse. Mice were sacrificed 3 hours later, and intracellular IL-4 and IFN-γ in hepatic iNKT cells were measured by flow cytometry. 2-DG, G6P, and pyruvate were purchased from Sigma-Aldrich. AKT inhibitor MK2206 and PKCθ inhibitor sotrastaurin were purchased from Selleck. HKVBD peptide was synthesized according to previous studies (52).

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