Il 10 clone jes5 16e3

The induction of collagen induced arthritis (CIA) in the C57BL/6 strain was performed as previously published [17 ]. Male WT and Ptpn22^−/− mice of 10–14 weeks of age were injected intradermally at the base of the tail with 100 μg chicken type II collagen (Sigma) emulsified in complete Freund's adjuvant. Clinical signs of arthritis were assessed visually in the wrist and ankle joints 3 times weekly, using a previously described severity scale: 0 = no arthritis; 1 = 1 inflamed digit; 2 = 2 inflamed digits; 3 = more than 2 digits and footpad inflamed; 4 = all digits and footpad inflamed [17 ]. Scoring was conducted under blinded conditions for up to 96 days. At day 96 single cell suspensions from lymph nodes (LN) and spleens were restimulated for 6 h with PMA (Sigma; 50 ng/ml) ionomycin (Sigma; 10 ng/ml) and monensin (Biolegend; 1 in 1000) and expression of IFNγ (clone XMG1.2; Biolegend), IL-17 (clone TC11-18H10.1; Biolegend), TNFα (clone MP6-XT22; Biolegend), IL-4 (clone 11B11; Biolegend) and IL-10 (clone JES5-16E3; Biolegend) determined by intracellular flow cytometry.

Spinal cord and brain tissues were harvested and digested with collagenase type 4 (2 mg/ml, Worthington Biochemical) for 1 hour at 37 °C in RPMI medium supplemented with 10% FBS. For T cell characterization by flow cytometry, cells were stained with antibodies to CD4 (clone RM4-5, BioLegend). Intracellular staining of the cells was performed after activation with phorbol myristate acetate, ionomycin and monensin and stained with antibodies to IL-10 (clone JES5-16E3, BioLegend), IL-17A (clone TC11-18H10.1, BioLegend), or IFN-γ (clone XMG1.2, BioLegend).

Mice were perfused with PBS before the brains were harvested. Brain tissues were pretreated with 2 μg/ml collagenase D and 1 μg/ml DNAse I (both Roche Diagnostics), and total cells were isolated by cell straining (100-μm mesh). Brain homogenates were separated into neuronal and leukocyte populations by discontinuous density gradient centrifugation using isotonic Percoll (GE HealthCare, Uppsala, Sweden) as described by Pino and Cardona [40 ]. After isolation, cells were stimulated in vitro with PMA and Ionomycin plus BD GolgiStop™ (BD Biosciences, San Jose, CA, USA) for at least 4 h at 37 °C. After stimulation, the cells were fixed with BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit and intracellular staining was carried out following the manufacturer’s instructions (BD Biosciences). Flow cytometry was performed using a FACS Verse (BD Biosciences) with the specific antibodies listed: anti-CD45 clone A20, anti-CD4 clone GK1.5, CD8α clone 5H10-1, IFN-γ clone XMG1.2, TNF-α clone MP6-XT22, CD3 clone 17A2, IL-10 clone JES5-16E3, F4/80 clone BM8 (all from BioLegend Inc., San Diego, CA, USA), IL-33 clone 396118 (R&D Systems, Minneapolis, MN, USA), and anti-iNOS (NOS2) clone N-20 (Santa Cruz Biotechnology, San Diego, CA, USA).