Reagents

Commercial betulin was purchased from Tauto Biotech Co, Ltd (purity ≥ 98%, Cat E-0164, CAS 473-98-3, Shanghai, China). Concanavalin A type IV (Con A, Cat C2272), carboxyfluorescein diacetate succinimidyl ester (CFSE, Cat 21888) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Cat M5655) were obtained from Sigma (St Louis, MO, USA). A cytometric bead array (CBA) kit for mouse T helper (Th)1/Th2/Th17 cytokines was obtained from BD Biosciences (Cat 560485, San Diego, CA 92121, USA). Alanine aminotransferase (ALT, Cat C009-2) and aspartate aminotransferase (AST, Cat C010-2) microplate assay kits were acquired from the Jiancheng Bioengineering Institute (Nanjing, China). GolgiStop brefeldin A solution (BFA, Cat 00-4506-51), IC fixation buffer (Cat 00-8222-49) and 10×permeabilization buffer (Cat 00-8333-56) were procured from eBioscience, Inc (San Diego, CA 92121, USA). The following monoclonal antibodies (mAbs) were used for cell surface marker staining: FITC-conjugated anti-CD3 (Cat 11-0031, eBioscience), APC/Cy7-conjugated anti-CD3 (Cat 100222, BioLegend) and CD8 (Cat. 100714, BioLegend), APC-conjugated anti-CD8 (Cat 17-0081, eBioscience), PE-Cy7-conjugated anti-CD4 (Cat 25-0041, eBioscience), and PE-conjugated anti-CD69 (Cat 12-0691, eBioscience). APC-conjugated mCD1d/PBS57 tetramers were provided by the National Institutes of Health (NIH) tetramer core facility (30082, USA). The following mAbs against intracellular cytokines were obtained from eBioscience: anti-interferon-γ (IFN-γ, Cat 11-7311), anti-tumor necrosis factor-α (TNF-α, Cat 12-7321), and anti-interleukin-4 (IL-4, Cat 25-7042).