MDSC functional assay

To investigate the suppressive function of MDSCs, T cells were stained with 10 μM 5-(and-6)-carboxy-fluorescein diacetate, succinimidyl ester (CFSE) according to the manufacturer`s instructions (Molecular Probes, Carlsbad, CA, USA). CFSE-labeled T cells were cultured with Dynabeads Mouse T-Activator CD3/CD28 (Life Technologies) in the absence or presence of sorted SSC^highCD11b⁺Gr1^dim cells or SSC^lowCD11b⁺Gr1^dim cells from the livers of NAFLD mice. After 60 h, T cell proliferation was analyzed by flow cytometry. Division indices were calculated using FlowJo software. To determine the roles of iNOS, ROS, and arginase 1 in T cell proliferation, 0.5 μM _L-N⁶-(1-iminoethyl) lysine dihydrochloride (L-NIL; Sigma-Aldrich, Gillingham, UK), 1000 U/mL catalase (Sigma-Aldrich), or 1 mM N-hydroxy-nor-arginine (nor-NOHA; Cayman Chemical, Ann Arbor, MI, USA), was added at the start of the cultures, respectively. In some experiments, T cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/mL; Sigma) and ionomycin (1 μg/mL; Sigma). Allogenic mixed lymphocyte reactions were used to confirm the suppressive ability of MDSCs. T cells from C57BL/6J mice were mixed with dendritic cells from C3H/HeN mice and co-cultured in the absence or presence of sorted SSC^highCD11b⁺Gr1^dim cells or SSC^lowCD11b⁺Gr1^dim cells from the livers of NAFLD mice at different ratios. [³H]-thymidine (1.0 μCi/mL; Amersham Biosciences, Buckinghamshire, UK) was diluted in sterile RPMI-1640 and added to the cultures for the last 16 h. The stimulation index was calculated using a formula described previously [20]. All culturing was performed in 96-well U-bottomed plates (Corning Inc., New York, NY, USA).