Clone 145 c11

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.