Anti mouse il 4

Total single cells were isolated from spleen and lymph nodes and were stimulated with leukocyte activation cocktail (1:500 in RPMI-1640 containing 10% FBS, BD bioscience, #550583) at 37 °C in a humidified incubator with 5% CO2. After 4 h of stimulation, cells were washed and incubated with TruStain FcX™ (anti-mouse CD16/32) antibody for Fc receptors blocking (BioLegend, #101320) and then exposed at 4 °C to a mixture of the following antibodies: Fixable viability stain 780 (1:1000, BD bioscience, #565388), anti-mouse CD4 (1:40, BD bioscience, #566407), anti-mouse CD3 (1:100, Biolegend, Clone 145-2C11). For cytokine secretion staining, after cell surface marker and viability staining, the cells were fixed and permeabilized. And then, the cells were incubated with anti-mouse IL17A (1:40, BD bioscience, #564169), anti-mouse IL-4 (1:40, BD bioscience, #564006), anti-mouse IFNγ(1:40, BD bioscience, #554413). Cells were analyzed with BD FACS Celesta (BD Bioscience). Flow cytometry analysis was performed using FlowJo software (TreeStar, Ashland, OR). The gating strategy is shown in Supplementary Fig. S3.

CD4⁺ T cells were isolated from the MLN using a commercial cell isolation kit and following the manufacturer’s instructions (Miltenyi, Biotec Inc). Cells from each animal were cultured and polarized to Th17 cells in RPMI supplemented with: 2-β Mercaptoethanol (50 μM); immobilized anti-mouse CD3ε; anti-mouse CD28; anti-mouse IFN-γ; anti-mouse IL-4 (all from BD biosciences); hTGF-β and recombinant murine IL-6 (from Peprotech); and recombinant murine IL-23 (BioLegend). The cells were stimulated 3 days later for 5 hours with PMA (100 ng/ml) and ionomycin (500 ng/ml, both from Tocris Bioscience). The supernatants were collected and stored at −20°C for IL-17 measurement by solid phase sandwich ELISA using Quantikine kits (M1700; R&D systems Inc), according to the manufacturer’s instructions. The assay’s sensitivity was 5 pg/ml.

T and B cells were purified from spleens of 6- to 20-week old WT or Mst1^−/− mice by immunomagnetic negative selection (EasySep mouse T and B cell negative selection enrichment kits) to greater than 95% purity, according to the manufacturer's protocol (STEMCELL Technologies, San Diego, CA). The cells were plated in triplicates in 96-well round-bottom plates at a concentration of 1×10⁵ cells per well in RPMI 1640 medium supplemented with 10% FBS, 10 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 µM 2-ME (c-RPMI; all Gibco Life Technologies, Grand Island, NY).
Th1 and Th2 polarized in vitro cultures were generated from naive (CD62L^highCD44^low) CD4⁺ splenic T cells isolated as above by culturing at 37°C and 5% CO₂ in 6-well plates pre-coated with 2 µg/ml CD3ε mAb (145-2C11 clone) in the presence of soluble 2 µg/ml CD28 mAb (37.51 clone) and 25 U/ml mouse IL-2 for 6 days. For generation of Th1 cells, 10 ng/ml mouse IL-12 and 10 µg/ml anti-mouse IL-4 (clone 11B11; BD Pharmingen) were added to the cultures. For Th2 cells, 20 ng/ml mouse IL-4, 10 µg/ml anti-mouse IL-12, and 10 µg/ml anti-mouse IFN-γ (clone R46A2) were added to the cultures.

The following anti-mouse fluorescently labeled antibodies were used: CD19 (clone 1D3), CD11b (clone M1/70), CD5 (clone 53-7.3), CD80 (clone 16-1OA1), MHCII (clone 2G9), CD86 (clone GL1) (BD Biosciences, Germany), and CD23 (clone B3B4) (eBiosciences, Germany). Mitomycin-c was obtained from Sigma-Aldrich, Germany. CD19 MicroBeads isolation kit, CD5 Microbeads isolation kit, and regulatory T cells isolation kit were obtained from Miltenyi Biotec, Germany. Anti-mouse IL4 and anti-mouse IFNγ were from BD, Biosciences, Germany. TGFβ was purchased from R&D System, Germany. IL23 and IL6 were obtained from eBiosciences, Germany. Cytometric bead array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit was obtained from BD, Biosciences, Germany.