Generation of MOG-specific Th1 and Th17 cells

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.