Clone 37

MSC-EVs were labeled with the fluorescent red dye PKH26 (Sigma) following the manufacturer’s instructions and subjected to ultracentrifugation for the washes required to remove excess of dye. Labeled EVs were added to the culture of naive CD4⁺ T lymphocytes purified by FACS sorting (FacsAria-BD) and activated with anti-CD3 (BD—clone145-2C11) and anti-CD28 (BD—clone 37.51) for evaluation of the internalization through the imaging by confocal microscopy (Zeiss LSM 780-NLO). Lymphocytes were monitored overnight for approximately 15 h.

Naïve CD4+ T cells (CD62L+ CD44low) were isolated from mouse spleens using the naive CD4⁺ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol and stimulated (2 × 10⁶ cells/ml) with plate-bound anti-CD3 (3 μg/ml; clone 145-2C11; BD Biosciences) and soluble anti-CD28 (1 μg/ml; clone 37.51; BD Biosciences) for 3 days.

Naïve CD4 T cells were isolated from spleens using the Naive CD4⁺ T Cell Isolation Kit (130–104–453, Miltenyi Biotec). Next, 96-well plates were activated overnight with plate-bound mouse anti-CD3 at 5 μg/ml (BD Pharmingen Clone 145-C11 cat. no. 553058) and mouse anti-CD28 at 2 μg/ml (BD Pharmingen Clone 37.51 cat. no. 553295) at 4 °C. Purified CD4⁺ T cells (2 × 10⁵) were cultured 200 μl cDMEM in the activated plate. To induce Th1 polarization in vitro, the media was supplemented with anti-IL-4 (10 μg/ml) (ebioscience clone 11B11 cat. no. 16–7041–85) and IL-12 (20 ng/ml) (R&D Systems cat. no. 419-ML-010). Th1 cells were used at 4–6 days polarization.
For Th17 polarization, T-cell media was supplemented with Th17-polarizing cytokines: IL-23 (10 ng/ml, Protein R&D Systems, 1886-ML), IL-6 (100 ng/ml, Protein R&D Systems, 406-ML/CF), rTGF-β1 (2 ng/ml, Protein R&D Systems, 7666-MB-005), anti-IL-4 (10 μg/ml, eBioscience, 16–7041–85), and anti-IFN-γ (10 μg/ml, eBioscience 16–7312–85). Th17 cells were ready for analysis on day 5 of polarization.

Animals were sacrificed on Day 30 ± 5 post immunization, and splenocytes were treated with MOG_35–55 at 50 μg/ml to measure antigen-induced T-cell proliferation and cytokine production. Cell proliferation was measured using the BrdU Cell Proliferation Assay Kit (K306–200, Biovision).
In certain experiments, mouse T cells were activated with anti-CD3 (5 μg/ml; clone 145-C11, BD) and soluble anti-CD28 (2 μg/ml; clone 37.51, BD); human CD4⁺ T cells and Jurkat cells were treated with ImmunoCult™ Human CD3/CD28 T Cell Activator (Cat. 10991, STEM CELL technologies) prior to the BrdU cell proliferation assay.

The binding of ArtinM to CD3 receptor was determined on CD4⁺ T cells isolated from spleen cell suspensions using CD4⁺ T cell isolation kits II and MS columns from Miltenyi-Biotec according to the manufacturer’s instructions. In the first assay, the cells were fixed and incubated with ArtinM (25 μg/mL) for 40 min at 4 °C. After two washes with phosphate-buffered saline (PBS), the cells were incubated with an anti-CD3 PE-Cy5 antibody (10 μg/mL; clone 17A2; BD Biosciences) for 40 min at 4 °C. The percentage of labeled cells was determined using flow cytometry (Guava EasyCyte). A functional inhibition assay was carried out by pre-incubating CD4⁺ T cells with either the anti-CD3 antibody (5 μg/mL) or an anti-CD28 antibody (5 μg/mL; clone 37.51; BD Biosciences) for 40 min at 4 °C. After washing with RPMI medium, the cells were stimulated for 48 h with jArtinM (78 nM). The culture supernatants were tested by means of ELISA for IL-2.