Anti il 17

CD4⁺ T cells-depleted splenic cells were cultured with IL-23 (20 ng/ml, Biolegend) with or without butyrate (0.5 mM) for 16 h, and then stimulated with ionomycin (750 ng/ml) and phorbol-12-myristate 13-acetate (50 ng/ml) for 2 h, followed by addition of brefeldin (BD Biosciences) for another 3 h. After Fc blocking, cells were stained with surface markers (PE/Cy7-anti-Thy1, 1:200, Clone#30-H12, Cat#105326; FITC-lineage (CD3, Clone#145-2C11, Cat#100306; CD11b, Clone#M1/70, Cat#101206; CD11c, Clone#N418, Cat#117306; B220, Clone#RA3-6B2, Cat#103206; F4/80, Clone#BM8, Cat#123108; NK1.1, Clone#PK136, Cat#108706; and Gr1, Clone#RB6-8C5, Cat#108419), 1:100), which were purchased from Biolegend. Cells were permeabilized using Foxp3/Transcription Factor Fixation/Permeabilization set (Cat#00-5523-00, ThermoFisher), followed by intracellular staining (anti-IL-22, 1:200, Clone#1H8PWSR, Cat#12-7221-82, Invitrogen; anti-IL-17, 1:200, Clone#TC11-18H10.1, Cat#506916, Biolegend). For ILC3, cells were also stained with RORγt (1:200, Clone#B2D, Cat#17-6981-82, Invitrogen).
All the events were collected by BD FACS Diva software. IL-22 and IL-17 production in ILCs (Thy1⁺ Lineage⁻ cells), or in ILC3 (Thy1⁺ Lineage⁻ RORγt⁺ cells) was analyzed using FlowJo. Gating strategies are included in Supplementary Fig. 15.

In the peak phase of EAE in mice, splenic MNCs and brain cells were harvested as described previously (29 (link)). For intracellular cytokine staining, splenic MNCs were restimulated in complete 1640 medium with a cell activation cocktail containing Brefeldin A (BioLegend) for 6 h. The cells were surface-stained with an anti-CD4 antibody for 30 min. After washing and fixation, the cells were stained with anti-IFN-γ (BioLegend) for Th1 cells, anti-IL-4 (BioLegend) antibody for Th2 cells, and anti-IL-17 (BioLegend) for Th17 cells. To quantify the Treg cells, the cells were surface-stained with anti-CD4 (BioLegend) and anti-CD25 (BioLegend) without the stimulation protocol. After fixation, permeabilization buffer was used as a washing solution (BioLegend), and the cells were then stained with anti-Foxp3 antibodies. The antibodies were tagged with phycoerythrin, allophycocyanin, or fluorescein isothiocyanate. Data were acquired on a FACSAria flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (Ashland, OR, USA).

We used the following antibodies: anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5, or -APC, anti-CD8 (clone: 53-6.7)-FITC, -PerCP-Cy5, or -APC, anti-CD44 (clone: IM7)-APC, anti-BrdU (clone: Bu20a)-PE, 7AAD, anti-IFN-γ (clone: XMG1.2)-APC, anti-IL-17 (clone: TC11-18H10.1)-PE, anti-IL-4 (clone: 11B11)-PE, anti-IL-6 (clone: MPS-20F3)-PE, anti-IL-12 (clone: C15.6)-PE, anti-IL-22 (clone: Poly5164)-PE, anti-IL-10 (clone: JES5-16E3)-PE, anti-IL-9 (clone: MH9A4)-PE, anti-TNF-α (clone: MP6-XT22)-PE (all from Biolegend, USA), anti-Active Caspase-3 (clone: C92-605)-FITC or -PE (from BD Pharmingen™, USA), and anti-CD69 (clone: H1.2F3)-PE (from eBiosciences, USA).