Anti mouse ifn γ

Single-cell suspensions from BT-II or OT-II mouse lymph nodes were incubated with anti-CD4 MicroBeads isolated on a MACS LS column (Miltenyi Biotec, Singapore). Splenic dendritic cells (DCs) were digested with Liberase Cl (Roche, Switzerland) at 37°C for 30 min and isolated by centrifugation over OptiPrep Density Gradient Medium (Sigma-Aldrich, St Louis, MO), incubated with anti-mouse CD11c MicroBeads, and passed through a MACS LS column (Miltenyi Biotec). BT-II and OT-II CD4 T cells and splenic DCs were cocultured in 48-well plates with 10 μg/ml Blo t 5_55–70 peptide (IIRELDVVCAMIEGAQ) or OVA_323–339, respectively (AnaSpec, Fremont, CA); 20 ng/ml IL-4 (PeproTech, Rocky Hill, NJ)1; 20 μg/ml anti-mouse IFN-γ; 20 μg/ml anti-mouse IL-12/IL-23 p40 (eBioscience, ImmunoCell, Singapore); and 20 μg/ml mouse IFN-γ R1/CD119 Ab (R&D Systems, Minneapolis, MN). Cells were restimulated on days 3, 7, and 11 with Blo t 5_55–70, cytokines, and neutralizing Abs. Fresh DCs were added on day 7. Th2-polarized BT-II cells were harvested on day 14.

Flow cytometry was performed as previously described (33 ). Briefly, single-cell suspensions were isolated from spleens or lymph nodes by crushing with forceps, or from sciatic nerves by mincing and digestion in 2 mg/ml collagenase. Cells were stained with live/dead fixable yellow dye (Life Technologies), anti-mouse CD4 (Biolegend clone GK1.5), anti-mouse CD44 (eBioscience clone IM7), anti-mouse CD62L (eBioscience clone MEL-14), anti-mouse S1PR1 (R&D clone 713412), anti-mouse IFN-γ (eBioscience clone XMG1.2), and anti-mouse IL-10 (BD clone JES5-16E3) antibodies. For intracellular cytokine staining, cells were stimulated with PMA/ionomycin for 4 hours at 37°C 5% CO₂, and permeabilized using BD Cytofix/Cytoperm according to manufacturer’s instructions. Cells were analyzed on a CyAn ADP Analyzer (Beckman Coulter). Data were analyzed using FlowJo X.