Il 23

Naive CD4⁺ T cells were cultured in 96-well round-bottomed plates (Corning, New York City, NY, USA) at a density of 5 × 10⁴ per well in X-VIVO 15 serum-free medium (Lonza, Walkersville, MD, USA) in the presence of Dynabeads CD3-CD28 T cell expander (one bead per cell; Life Technologies, Carlsbad, CA, USA) and indicated cytokines: IL-1β (10 ng/mL), IL-6 (20 ng/mL), TGF-β (1 ng/mL) and IL-23 (100 ng/mL) (Miltenyi) for Th17 differentiation, as previously described [11 (link),21 (link)]. After 5–6 days, cells were harvested and stained for flow cytometry analysis, or extensively washed, counted, and re-stimulated 1 × 10⁶ cells/mL with Dynabeads CD3-CD28 T cell expander (one bead per cell) for 24 h for cytokine quantification.

To provide conditions for TH9 and Th17 cell polarization, CD4⁺ T cells enriched by magnetic sorting were cultured at 1 × 10⁵ per well in flat-bottomed 96-well plates (Costar) pre-coated with 2 μg/ml αCD3 (eBioscience) and 2 μg/ml or 1 μg/ml αCD28 (eBioscience), for Th9 and Th17 conditions respectively. Cytokines for Th9 conditions were added in complete medium as follows 40 ng/ml IL-4, 20 ng/ml IL-2 and for Th17 conditions 100 ng/ml IL-6, 5 ng/ml IL-23 (all from Miltenyi Biotech) and both polarization cultures contained 10 μg/ml αIFN-γ (BioXcell) in addition to varying concentrations of hp-TGM or hTGF-β3. Cultures were left at 37 °C with 5% CO₂ and restimulated with 500 ng/ml PMA, 500 ng/ml ionomycin and 1 µg/ml BFA (Sigma Aldrich) for 4.5 h on day 4 (Th9) or day 5 (Th17) followed by staining for flow cytometry with the FoxP3 transcription factor buffer kit (eBioscience) as detailed below, as well as for intracellular IL-9 with anti-IL-9-PE (BioLegend 514104) and for IL-17 with anti-IL-17-PE (bioLegend 506904).

Murine IL-1β, IL-2, IL-6, IL-7, IL-12, IL-23, IL-27 were obtained from Miltenyi (Bergisch-Gladbach, Germany). Murine IL-4 and Human TGF-β1 were obtained from Biolegend (San Diego, CA, USA).

Purified naive CD4⁺ T cells were resuspended in RPMI medium supplemented with 10% FCS and cultured in the following conditions: TCR: stimulation with Dynabeads Human T-activator CD3/CD28 (4 beads per 10⁶ cells; Thermo Fisher scientific); TCR + TGFβ: TCR stimulation in the presence of TGFβ (10 ng/ml; Miltenyi); Th17 polarizing condition: TCR stimulation in the presence of TGFβ (10 ng/ml), IL-1β (10 ng/ml; Miltenyi), IL-23 (10 ng/ml; Miltenyi) and IL-21 (25 ng/ml; Miltenyi), plus anti-IFNγ and anti-IL-4 neutralizing antibodies (1 μg/ml; BD Biosciences); Th1 polarizing condition: TCR stimulation in the presence of IL-12 (2.5 ng/ml; Roche) and anti-IL-4 antibodies. The calcineurin inhibitor Cyclosporine A (CsA; 1 μg/ml; Sigma-Aldrich) was added to the cultures where indicated.

CD4⁺ T cells were isolated from healthy donors' PBMCs by human CD4 microbeads (Miltenyi Biotec) and resuspended in complete 1640 medium in the presence of anti-CD3/CD28 antibodies (5 μg/mL), anti-IL4 antibody (10 μg/mL) (eBioscience), and anti-IFNγ antibody (10 μg/mL) (eBioscience). MSCs were cocultured with CD4⁺ T cells (1 : 10) in different culturing systems. For Treg induction, recombinant human TGF-β1 (5 ng/mL) (R&D Systems) and IL-2 (5 ng/mL) (Miltenyi Biotec) were added. For Th17 induction, recombinant human TGF-β1 (5 ng/mL), IL-6 (50 ng/mL) (Miltenyi Biotec), and IL-23 (10 ng/mL) (Miltenyi Biotec) were added. In some experiments anti-CCL2 antibody (R&D Systems) was added for neutralization. After coculture for 5 days, floating cells were used to examine Treg and Th17 cell percentages by flow cytometry, and culture supernatant was collected for measuring IL-17A levels by enzyme-linked immunosorbent assay (ELISA) kits (Biolegend). The adherent MSCs were also collected for measuring the gene expression of transforming growth factor- (TGF-) β1, indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), interleukin- (IL-) 6, and chemokine (C-C motif) ligand 2 (CCL2) by real-time PCR.

Spleens were collected from 2D2 TCR transgenic mice, and a single-cell suspension was prepared. Spleen cells were cultured as previously described (24 (link)). Briefly, 20 × 10⁶ cells per well were cultured in a six-well plate for 7 days in the presence of 20 μg/ml MOG₃₅₋₅₅ peptide (GenScript Corporation). For Th1 polarization, we added 1 ng/ml IL-12 (R&D Systems) and 10 μg/ml anti-IL-4 antibody (clone 11B11, hybridoma provided by E. C. Butcher, Stanford University). For Th17 cell polarization, we added 5 ng/ml TGFβ, 20 ng/ml IL-6 and 20 ng/ml IL-23 (all from Miltenyi Biotech or R&D Systems), as well as 10 μg/ml anti-IL-4 antibody (as above) and 10 μg/ml anti-IFNγ antibody (clone HB170, R&D Systems). Th1 cells were supplemented with IL-2 (20 U/ml) and Th17 cells with IL-7 (10 ng/ml) after 4 days in culture. After a further 72 h, MOG₃₅₋₅₅-specific Th1 and Th17 cells were isolated using a Ficoll-Paque density gradient (GE Healthcare Life Sciences) and frozen in fetal calf serum (FCS, Lonza) containing 10% dimethylsulfoxide (DMSO, Sigma-Aldrich). Before use, the cells were thawed and re-stimulated for 3 days in the presence of irradiated splenocytes as APCs (APC:T cell ratio = 5:1) with the same peptide/cytokine cocktail as described above. Th1 and Th17 cells were then isolated using a Ficoll-Paque gradient and supplemented for one further day with IL-2 or IL-7, as appropriate.