Ifn γ clone xmg1

Inflammatory cells from the CNS were isolated from each individual mouse at the peak of disease (day 16–20 post-transfer of Th17 cells). Anesthetized mice were perfused with 20 ml of cold PBS (4°C) and brain and spinal cord were dissected from each individual mouse. Tissues were digested with 0.4 mg/ml Collagenase VIII (Sigma-Aldrich) and 2 U/ml DNaseI (Sigma-Aldrich) at 37°C for 30 min. Infiltrated cells were isolated by passing the cell suspension through a 70-μm cell strainer and 33% Percoll density gradient centrifugation. After removal of myelin layer, the infiltrated cells were collected, washed and resuspended in culture medium for further analysis. Cells were resuspended in Fc-block (anti CD16/32 purified from hybridoma supernatants; clone 2.4G2) diluted in PBS for 15 min on ice. Surface staining of T cells was performed with antibodies to CD45 (clone; 30F11, BioLegend), CD4 (clone; RM4-5, BD Pharmingen) in the presence of Fixable Viability Dyes (FVD). After stimulation with PMA/Ionomycin/Golgi stop, the intracellular staining was done using the antibodies to IFN-γ (clone XMG1.2, eBioscience), IL-17 (clone: TC11-18H10, BD Bioscience) and GM-CSF (clone: MP1-22E9, BioLegend). Gating strategy used is shown is Supplementary Figure 6E.

Blood counts were analyzed using an automated hematology analyzer (KX-21N, Sysmex, Germany). For FACS analyses, tissues were passed through a 70 μm filter (BD Biosciences, Germany) to obtain single-cell suspensions. Whole blood was combined with a red blood cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) to allow the isolation of leukocytes. Whole aortae and aortic roots were excised, flushed with PBS, and enzymatically dissociated using Liberase Blendzyme TL (Roche, Germany) solution for 30 minutes at 37°C. The resulting single cell suspensions were resuspended in Hanks Buffered Saline Solution (HBSS), enumerated using a Neubauer counting chamber. Cells were stained for 30 minutes on ice using combinations of specific antibodies from BD biosciences (CD45, clone 30-F11; CD3, clone 500A2; CD8a, clone 53-6.7; IFNγ, clone XMG1.2) and eBioscience (TCRβ, clone H57-597; CD44, clone IM7; CD62L, clone MEL-14; CD4, clone RM4-5; Foxp3, clone FJK-16s; CD25, clone PC61.5; IL-17a, clone eBio17B7). Intracellular labelling of IL17A and IFNγ was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences). Intracellular labeling of Foxp3 was performed using the Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer's instructions. Probes were analyzed using a FACSCanto II (Becton Dickson, USA) and FlowJo 7.6 software (Treestar Inc., USA).

eWAT and liver was isolated from obese WT and IFNAR^−/− mice. eWAT-isolated SVF cells and liver immune cells were stimulated for 4 h with PMA (50 ng/ml; Sigma-Aldrich) and Ionomycin (1 μg/ml; Calbiochem). Briefly, cells were stained with Live/dead stain (Zombie UV Dye; 1:250; Biolegend), B220 (clone RA3-6B2; 1:100; Biolegend), CD45 (clone 104; 1:500), CD11b (clone M1/70; 1:100), F4/80 (clone BM8; 1:100), Gr1 (clone RB6-8C5; 1:100), CD4 (clone GK1.5; 1:50), CD8 (clone 53-6.7; 1:100), IFNγ (clone XMG1.2; 1:100) TNF (clone MP6-XT22; 1:100), and IL-6 clone (MP5-20F3; 1:100) (all ebioscience). Data were collected using a LSR Fortessa flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Spleens and mesenteric lymph nodes (mLNs) were removed from mice and disaggregated through a 100 μm sieve. Small intestines were excised and lamina propria lymphocytes (SILP) were prepared essentially as described [80 (link)] with slight modification in the tissue digestion step (digestion medium used was RPMI with 10% Foetal calf serum, 0.1% w/v collagenase type I and Dispase II (both Invitrogen), and tissue was digested for 30 min at 37°C). Cell suspensions were blocked with anti-FcγR antibody (clone 24G2; eBioscience) before labelling with antibodies specific for CD3 (eBio500A2), CD4 (clone GK1.5; eBioscience), Foxp3 (clone FJK-16s; eBioscience), IL-13 (clone eBiol13A; eBioscience), IFNγ (clone XMG1.2; eBioscience), IL-17(eBio17B7; eBioscience), IL-9 (RM9A4e; Biolegend) or p-Smad 2/3 (Santa Cruz). For intracellular cytokine analysis cells were incubated for 12 hours with 1x Cell stimulation cocktail (plus protein inhibitors) (ebioscience). Cells were then stained with antibodies using the eBioscience Foxp3 permibilization kit according to the manufacturer's instructions. For pSmad2/3 staining, an Alexa Fluor 594-labelled donkey anti-goat secondary antibody was used (Invitrogen). All samples were analysed on a FACS LSRII.