Neutralizing antibodies

Naïve CD4⁺CD25^-CD62L⁺ T cells were isolated from spleen and lymph nodes using FACS Aria (BD bioscience, San Jose, CA, USA). PerCPCy5.5-conjugated anti-CD4 (GK1.5), fluorescein isothiocyanate (FITC)-conjugated anti-CD25 (7D4), phycoerythrin (PE)-conjugated anti-CD62L (MEL-14) were purchased from Biolegend. The naïve T cells were cultured in DMEM (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (Biowest, Nuaillè, France) and penicillin G and streptomycin. Naïve T cells were activated with anti-CD3/CD28 beads (Life Technologies, Oslo, Norway), anti-IL-4 (10 μg/mL) (Biolegend), and anti-IFNγ (10 μg/mL) neutralizing antibodies (Biolegend) and were polarized with cytokines to generate Treg (TGF-β: 0.125 or 0.5 ng/mL, Peprotech, Inc, Rocky Hill, NJ, USA) or Th17 (TGF-β: 2 ng/mL and IL-6: 100 ng/mL, Peprotech, Inc) in the presence or absence of LRG. Cells were harvested on day 3 for analysis.
For determination of the differentiation of CD4+ T cells, inguinal lymphoid cells were isolated on day 27, and cultured in the presence of type-2 collagen (Chondrex,Inc. Redmond, WA, USA) for 3 days. Then, T cell subsets were analyzed by flow cytometry.

Briefly, 1 × 10⁴ cells were plated into 96-well plate for 24 h and serum-starved for additional 24 h, before treated with complete media, control media (media containing 1% FBS) or fibroblast conditioned media (1.0 μg/μl) with or without chemicals, recombinant proteins or neutralizing antibodies (BioLegend, CA, USA) for subsequent 24-72 h. At the end of treatment, MTT solution (5 mg/ml) was added into each well, followed by 4 h incubation at 37°C. The formazan crystals were dissolved with sodium dodecyl sulfate before reading the absorbance using CHAMELEON™V plate reader (Hidex, Finland) at 570 nm with reference wavelength of 630 nm.
To measure cell proliferation, we determined BrdU incorporation into the cellular DNA using an ELISA-based approach (Cell Signaling Technology, MA, USA). Cells were seeded and treated as described above, and at the end of treatment, BrdU solution was added for 24 h. Following fixation and denaturation, the cells were labeled with antibodies and washed before added with TMB substrate. Color development was terminated with a STOP solution, and measured at 450 nm using CHAMELEON™V plate reader. In addition, the cells were also stained with 0.04% trypan blue to count for number of viable cells, using neubauer improved hemocytometer (Sigma Aldrich).