Anti cd2 cd3 cd28 beads

Peripheral blood and synovial fluid mononuclear cells were isolated by density‐gradient centrifugation. Total or naive (CD45RO−) CD4+ T cells were separated by negative selection on magnetic beads (Miltenyi Biotec). CD4+ T cells were activated either with anti‐CD2/CD3/CD28 beads (Miltenyi Biotec) or with 125 ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin (Sigma).

Peptides were synthesized by Peptides and Elephants GmbH (Henningsdorf, Germany) and dissolved in DMSO at a stock concentration of 5 mM. The peptides and their sequences which we used are listed in Supplementary Table 2. The response of PHA-expanded CSF-infiltrating CD4⁺ T cells to citrullinated or non-citrullinated myelin and the CEF (CMV, EBV, influenza virus, tetanus toxoid) (Peptides and Elephants GmbH), peptide pool was tested by seeding 6 × 10⁴ of expanded CSF-infiltrating CD4⁺ T cells and 2 × 10⁵ irradiated autologous PBMCs in quadruplicates per each condition of peptide stimulation or in the absence of peptides. Stimulation with anti-CD2/CD3/CD28 beads (Miltenyi) was used as additional positive control.

To analyze the induction of regulatory T lymphocytes, we cocultured macrophages treated with MSCs without cellular contact in a 0.4-μm Transwell system (Corning) for 6 days in the presence of inducer medium M0, M1 or M2, with CD4⁺ T lymphocytes selected using CD4 MicroBeads (Miltenyi Biotec) in a 1:1 ratio (macrophages:CD4 T lymphocytes) in RPMI medium (HyClone) containing 10% FBS (Biowest) activated with anti-CD2/CD3/CD28 beads (1 bead for each T lymphocyte) (Miltenyi Biotec). After 5 days of coculture, T lymphocytes were collected, washed and blocked with FBS (Biowest) at 4 °C for 15 min. After blocking, FITC mouse anti-human CD4 (BD Biosciences) and PE mouse anti-human CD25 (BD Biosciences) antibodies were added. Then, the cells were stained following the instructions of the FoxP3 staining buffer set (Invitrogen), and PE-Cyanine7 mouse anti-human FoxP3 (eBioscience) was added. Acquisitions were made on a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo V10 software. CD4⁺ T lymphocytes cultured in the absence of macrophages were used as a control.

PBMCs were isolated from whole blood by means of centrifugation over a Ficoll gradient at 400g for 30 minutes at room temperature and stimulated in complete medium containing IMDM (HyClone), supplemented with 10% FBS, 2 mM L-glutamine, 1× nonessential amino acids (all from Thermo Fisher Scientific), and 57 μM β-mercaptoethanol in the presence of anti-CD2/CD3/CD28 beads (Miltenyi Biotec). Cells were split every 2–4 days with medium containing human recombinant IL-2 (40 U/mL) to maintain a cell concentration of 5 × 10⁵/mL. After 7 days of expansion to generate sufficient cells for experimentation, T cells were incubated with phorbol 12-myristate 13-acetate (PMA), ionomycin, and GolgiStop (BD Biosciences), according to the manufacturer’s instructions in complete medium for 4 hours. Cells were then fixed and permeabilized using BD Cytofix Fixation Buffer BD Perm/Wash Buffer, respectively (both from BD Biosciences), and stained with a fixable viability stain and monoclonal antibodies against the following human proteins: CD3 (clone 641406), CD4 (clone SK3), IFN-γ (clone Β27), IL-17 (clone N49-653), IL-4 (clone MP4-25D2), and IL-9 (clone MH9A3) (all from BD Biosciences). Data were collected with an LSR II cytometer (BD Biosciences) and analyzed using FlowJo software.