Mil 4

Pooled cells from spleen and lymph nodes were first enriched for CD4⁺ T cells with Magnisort mouse CD4 T cell enrichment kit (eBioscience 8804-6821-74) and then stained and sorted for CD4⁺ CD25⁻ CD44^lowCD62L^high naive T cells. Sorted naive CD4⁺ T cells were used for in vitro cultures in IMDM (Life Science 12440-053) supplemented with β-mercaptoethanol, 10% fetal bovine serum and 1% penicillin-streptomycin. For survival experiment, naive T cells were cultured in the presence or absence of mIL-7 (20 ng/ml). For T cell activation, naive T cells were plated on cell culture plates pre-coated with goat anti-hamster IgG (MP Biomedicals, 0856984) and in the presence of hamster anti-mouse CD3ε (eBioscience 16-0031-86) and hamster anti-mouse CD28 (eBioscience 16-00281-86) antibodies and differentiated into different subsets using reagents and recipes listed in the table below: mIL-2 (Biolegend 575404), mIL-12 (Biolegend, 577004), mIL-4 (Biolegend 574304), mTGFβ1 (Biolegend 736102), mIL-6 (Biolegend 575702), αIFNγ (Biolegend 505812), αmIL-4 (Biolegend 504108), αmIL-6 (Biolegend 501110), mIL-1 (Peprotech 211-11B), mIL-23 (R&D systems 1887 CF), mIL-7 (Biolegend 577806). For the culture of pathogenic Th17 cells see EAE induction by adoptively transfer of Th17 cells.
Final Concentrations of Differentiation Antibodies and Cytokines.