Il4 clone 11b11

Single-cell suspensions obtained from spleen, sdLNs, and skin were analyzed by flow cytometry using fluorochrome-conjugated mAbs listed in Table S1. Zombie Aqua or Zombie NIR (Biolegend) was used to distinguish live and dead cells. Intracellular staining was carried out on cells incubated with Brefeldin A (Biolegend) in all tissue digestion and FACS staining buffers, approximately for 4 hours. Cells were permeabilized and fixed with transcription factor staining buffer (Invitrogen) or Cytofix/Cytoperm (BD Biosciences) and subsequently incubated with fluorochrome-conjugated mAb to mouse IFN-γ (clone XMG1.2, eBioscience), IL4 (clone 11B11, Biolegend), Tbet (clone 4B10, Biolegend), or GATA3 (clone TWAJ, eBioscience). Flow cytometric analysis was carried out using a Cytek Aurora, and analysis was conducted with FlowJo software 9.7.6 (TreeStar).

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.