Complete freund adjuvant (cfa)

C57BL/6J mice were immunized on day 0 with 20 mg/kg Chicken Ovalbumin (OVA, Sigma) emulsified 1:1 with Complete Freunds Adjuvant, (CFA, Chondrex) and draining lymph nodes (DLNs) were collected on day 14. Single cell suspension was prepared in culture medium and seeded in 96-well plates at 2 × 10⁵ per well. Cells were stimulated with 50 μg/ml OVA in the absence or presence of 1 μM JNJ-54271074 and cultured at 37 °C and 5% CO₂ for 48 hours, then IL-17A- and IL-22-producing cells were analyzed by intracellular staining and flow cytometry analysis.

Alum (Imject^® Alum) from Pierce, Al-lactate from
Sigma-Aldrich, Aluminum hydroxide from InvivoGen, CFA (Chondrex, USA), CFDA-SE
(Molecular Probes, OR), IFA (Sigma-Aldrich) Ovaprotein (chicken, ovalbumin,
grade V, Sigma-Aldrich), Calhex 231 was from Santa Cruz Biotechnology, Fc
receptor block (aCD16/CD32) was from Miltenyi. Antibodies were obtained from
Miltenyi Biotech (aCD3, aCD5, aCD11c, aCD49b, aCD19, aCD69, aCD86
aCD273 = PDL2, aMHC-II,
aCD279 = PD1, aCXCR5, aSiglecF), ebioscience (aLy6C,
aF480,aCD11b), R&D (aCCR2) Beckton Dickinson (aLy6G and aCD4) in
different conjugates of FITC, Alexa488, PerCp, Pe and APC. Recombinant murine
IL-4 was from Immunotools. Functional grade aCD40 for culture was obtained from
ebioscience.

CIA was induced as previously described32 (link). Briefly, 9-week-old sex-matched WT (C57BL6/J) and Eaf2^−/− mice (generated on a pure C57BL6/J background) were injected intravenously with 100 μl of a 1/1 (v/v) emulsion of 0.05 M acetic acid containing 100 μg of chick type II collagen (20012, Chondrex) and CFA (7023, Chondrex). The day of the first immunization was defined as day 0. Twenty days after the primary immunization, the mice were boosted with 100 μg of collagen II in IFA via the same route. The severity of arthritis was evaluated every 3 days after the boost by visual inspection. Each paw was scored for clinical signs of arthritis as described52 . The clinical score for each mouse was the sum of the four paw scores, which results in a maximum score of 16. On the final day of the experiments, all mice were anaesthetized and their blood was collected by cardiac puncture.

C57BL/6 mice aged 8– 12 weeks were immunized with the emulsion of complete Freund's adjuvant (CFA, catalog number: 7023, Chondrex, Redmond, WA) and 100 μg of chicken type II collagen (catalog number: 20012, Chondrex) in a 1:1 mixture (total 100 μl) subcutaneously into the base of the tail on day 0^{18 (link)}. The booster immunization of CII with incomplete Freund's adjuvant (catalog number: 7002, Chondrex) was performed on day 21. Recombinant human G-CSF (Filgrastim, Neupogen, Amgen, Inc., Thousand Oaks, CA) was injected at 10 μg peritoneally once daily from day 20 to day 23. Vehicle (water) or 50 mg/kg of JBI-589 was orally administered twice daily from day 20 to day 56. The severity of arthritis was evaluated using the following clinical scoring system for each limb: 0, normal; 1, swelling in one finger joint; 2, swelling in more than one finger joint or wrist or ankle joint; 3, swelling in the entire paw; and 4, deformity and or ankylosis. The maximum score was 16 per mouse. Two evaluators (one knew the group allocation, the other did not) independently scored arthritis and arrived at agreements on final scorings. At the end of the study, all mice were euthanized after giving anesthesia using 2% isoflurane (Patterson Veterinary, catalog number: 07–893-2374).