Anti il 17a

For additional characterization of intracellular markers PBMCs were suspended in culture medium consisting of RPMI 1640 (Biochrom), 5% human AB serum (CC pro), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Biochrom). PBMCs were stimulated with 10 ng/ml phorbol myristate acetate (PMA, Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich) in the presence of 0.2 µM Monensin (Biomol) for 6 hours prior to analysis. First, cell surface markers were stained as described above. Before the characterization of intracellular markers, cells were fixed with freshly prepared fixation concentrate, and permeabilized with wash-permeabilization concentrate (Fixation/Permeabilisation Buffer Set, eBioscience). Subsequently, cells were stained using fluorescence-labeled anti-IFN-gamma, anti-IL-17A (BioLegend), anti-IL-4 (BD Bioscience), anti-FoxP3 antibody (Miltenyi Biotec) or isotype-matched irrelevant antibody (BD Biosciences) (Suppl. Fig. 6). After the staining procedure, cells were evaluated by flow cytometry. Cells were measured on a LSR-Fortessa (BD Bioscience) and evaluated by FACS-Diva Software (BD Bioscience).

Spleens and lymph nodes were isolated from mice in each group, and 1 × 10⁶ cells were incubated with PMA (50 ng/mL) plus ionomycin (500 ng/mL) for 5 h at 37 °C. The harvested cells were fixed and permeabilized (CytoFix/CytoPerm kit; BD Biosciences, Franklin Lakes, NJ, USA) and stained with fluorescence-labeled anti-CD4 (BD Biosciences, Franklin Lakes, NJ, USA), anti-IL-17A (BioLegend, San Diego, CA, USA), anti-TNF-α (BioLegend, San Diego, CA, USA), anti-IFN-γ (BD Biosciences, Franklin Lakes, NJ, USA), anti-Foxp3 (eBioscience, San Diego, CA, USA), anti-CD11b (BioLegend, San Diego, CA, USA), and anti-Gr-1 (BioLegend, San Diego, CA, USA) monoclonal antibodies. The expression of these markers was analyzed using a FACSVerse instrument and FlowJo 10.6.1 software (FlowJo LLC, Ashland, OR, USA).

We isolated T lymphocytes from spleens. After preparing a single cell suspension and counting the cells, we used magnetic beads (Miltenyi Biotec) to isolate CD4⁺ cells. Isolation was performed in the dark, and the total number of cells added to the LS column was restricted to 1 × 10⁸. Anti-CD4, anti-CD25, anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-Foxp3 antibodies were purchased from BioLegend. Intracellular straining for IFN-γ, IL-4, IL-17A and Foxp3 was performed using the Prem/Fix Buffer set, according to the manufacturer’s instructions [24 (link)]. After staining, cells were suspended in PBS + 1% FBS and analyzed using an Accuri C6 flow cytometer from BD Biosciences (San Jose, CA, USA). We also detected the serum IFN-γ, IL-4, and IL-17 levels using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA).

To characterize CNS-infiltrating cells at day 30 post-EAE induction, mice were euthanized by exsanguination by transcardial perfusion with PBS under isoflurane anesthetization. Lymphocytes were isolated from the spinal cord by Dounce homogenization to generate a single-cell suspension that was filtered with a 70-μm strainer followed by Percoll gradient (37%/70%) centrifugation and collection of the interphase. For intracellular cytokine analysis, cells were stimulated with 20 ng/ml PMA, 1 μg/ml of ionomycin and 1 mg/ml brefeldin A (Golgi Plug reagent, BD Bioscience) for 4 h. Cells were stained with the UV-Blue Live/Dead fixable stain (Thermo Fisher, USA) followed by surface staining with antibodies against CD45, CD11b, CD19, TCRβ, CD4, CD8, and TCRγδ (Biolegend, USA). For intracellular cytokine staining, cells were fixed, permeabilized with 0.05% saponin, and labeled with anti-IL-17A, anti-IFNγ, and anti-GM-CSF antibodies (Biolegend, USA). Cells were analyzed using a Cytek Aurora (Cytek Biosciences, USA). Spectral unmixing was performed with appropriate single-color controls using autofluorescence correction from an unstained group control. Data were analyzed using FlowJo software, version 10.8.1 (Tree Star Inc, Ashland, OR).

ELISA plates were coated with 2 µg/ml capture (purified) antibodies overnight at 4°C. Wells were washed three times with TBST and blocked for 1 h with 1% bovine serum albumin. Block was removed and recombinant protein standards and samples were incubated for 2 h. Wells were washed three times and 2 µg/ml of biotynilated antibody was incubated for 1 h. Wells were washed three times and a 1:1000 dilution of HRP‐Avidin antibody was incubated for 40 min. Wells were washed three times and 75 µl TMB was added. The reaction was stopped with 75 µl 10% sulfuric acid and plates read at 450OD. Cytokine concentrations were determined from recombinant protein standard curve. Interleukin‐17A (IL‐17A) (576002), interferon‐γ IFN‐γ (575304), IL‐12 (572109), tumor necrosis factor α (TNF‐α) (570109), and IL‐6 (570809), anti‐IL‐17A (506901), anti‐IFN‐γ (505701), anti‐IL‐12 (501801), anti‐TNF‐α (502801), anti‐IL‐6 (79026)biotinylated anti‐IL‐17A (507001), biotinylated anti‐IFN‐γ (505803), biotinylated anti‐IL‐12 (508801), biotinylated anti‐TNF‐α (502903), and biotinylated anti‐IL‐6 (79027); and HRP‐Aviden (405103) antibodies were purchased from Biolegend.

Lacrimal and salivary glands were removed from NOD or recipient NOD scid mice, fixed in 10% neutral-buffered formalin and embedded in paraffin blocks. Tissue sections (4 μm) were stained with hematoxylin and eosin, and images were analyzed with a light microscope. The histological score was determined as the number of infiltrated foci as follows:score 0, no foci; score 1, less than 1 focus; score 2, 2-5 foci; score 3, 6-9 foci; score 4, over 10 foci. A focus was defined as an infiltrated region (0.04 mm²) seen within the histological section. For immunohistochemical analysis, cryosections (5 μm) of lacrimal and salivary gland tissue were stained with PE anti-CD3 (BD Pharmingen, San Diego, CA, USA), anti- LPAR1 (Santa Cruz Biotechnology, Dallas, TX, USA) or anti-IL-17a (Biolegend, San Diego CA, USA) antibodies and 4′-6-diamidino-2-phenylindole (DAPI, Life Technologies, Carlsbad, CA, USA). Images were captured using a laser scanning confocal microscope (Carl Zeiss, Munich, Germany).