Anti il 17 apc

For the analysis of intracellular cytokine expression T-cells were stimulated with phorbol myristate acetate (PMA), Ionomycin for 1 hour and Brefeldin A for 4.5 hours. All chemicals were obtained from Sigma-Aldrich (Munich, Germany). Cells were harvested and prepared for analysis using the Cytofix/Cytoperm kit (BD Bioscience, Heidelberg, Germany). For intracellular cell staining the following antibodies were used: anti-IL4-FITC, anti-IL17-APC, anti-IFNγ-PECy7, and anti-FOXP3-APC, obtained from eBioscience (San Diego, USA) and anti-Granzyme-PE (BD Bioscience, Heidelberg, Germany).

Anti-CD3 FITC, anti-CD3 APC, anti-CD4 PerCP-cy5.5, anti-CD8-PerCP-cy5.5, anti-CD8 FITC, anti-CD45RO FITC, anti-CD45RO PE, anti-IFN-γ FITC, anti-IFN-γ APC, anti-IL-4 PE and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-22 PE was purchased from R&D Systems (Abingdon, UK). Anti-IL-17 APC was purchased from eBioscience (San Diego, CA, USA). Phorbol myristate acetate (PMA), ionomycin, saponin and Brefeldin A (BFA) were purchased from Sigma-Aldrich (Fluka, Sigma, USA).

Anti-CD4 PerCP, Anti-CD4 PerCP-cy5.5, anti-CD3 APC, anti-CXCR5-Alexafluor 488, anti-CD45RO PE, anti-CCR7 PE, anti-CD28 PE, anti-CD69 PE, anti-HLA-DR PE, anti-IL-21 PE, anti-IL-4 PE, anti-IFN-γ APC and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-17 APC and ELISA for determining IL-21 was purchased from eBioscience (San Diego, CA, USA). Anti-IL-22 PE was purchased from R & D Systems (Abingdon, UK). PMA, ionomycin (INO), saponin and Brefeldin A were purchased from Sigma-Aldrich (Fluka, Sigma, USA).

Cell surface and intracellular phenotypes were determined by flow cytometry using the following reagents: anti‐CD4‐Alexa 488 (0.2 μg/mL, RM4‐5); anti‐ROR‐γT‐PE (2 μg/mL, Q31‐378); anti‐IL‐4‐PE ((2 μg/mL, BVD4‐1D11) BD Biosciences, UK); anti‐CD25‐eFluro450 (0.2 μg/mL, eBio3C7); anti‐IL‐17‐APC (0.2 μg/mL, eBio17B7), anti‐CD69‐PE (0.2 μg/mL, H1.2F3), and anti‐IFN‐γ‐PEcy7 ((0.2 μg/mL, XMG1.2) eBioscience, UK). For intracellular cytokine staining, cells were blocked with 200 μg/mL of rat serum (Sigma, UK) and fixed and permeabilized using Foxp3 staining kit (eBioscience) according to manufacturer's instructions. Data were acquired using FACS Canto‐II (BD) and analyzed by Flowjo software 8.7.1 (Treestar, USA). The following gating strategy was used (see also Supporting Information Fig. 1: (i) a ‘life’ gate was set on the FSC/SSC plot; (ii) a gate was set on CD4+ cells, based on an isotype‐matched control plot; (iii) marker of interest‐positive cells were gated on the basis of a fluorescence‐minus‐one plot – unstained and isotype‐matched control samples were used to exclude nonspecific binding of mAbs used.

5-Azacytidine was obtained from Sigma-Aldrich (Munich, Germany) and used at a final concentration of 5 μM or 20 μM. All flow cytometry experiments were performed using a FACS Canto II (BD Bioscience, Heidelberg, Germany). For flow cytometry the following antibodies were used: anti-CD3-PE, anti-CD4-FITC, anti-CD8-APC, anti-CD25-PE, anti-CD45-FITC, anti-CD45RO-PE, anti-CD45RA-FITC, anti-CD62L-PE, anti-CCR7-PE, anti-HLA-DR-APC, anti-Granzyme-PE antibodies (obtained from BD Bioscience) anti-CD127-PC5, anti-IFNγ-PE/Cy7, anti-IL17-APC, anti-IL4-FITC, and anti-FoxP3-APC antibodies obtained from eBioscience (San Diego, USA).

Popliteal lymph nodes were removed and macerated as described earlier. Cells were quantified by light microscope using trypan blue and then plated at 1 × 10⁶ cells/well in a 96-well plate. Staining of intracellular and extracellular markers was performed following manufacturer's instructions. Briefly, cells were re-stimulated ex vivo for 4 h with phorbol 12-myristate 13-acetate (PMA; 20 ng/mL) plus ionomycin (1 μg/mL) in the presence of a Golgi complex inhibitor (brefeldinA) for intracellular cytokine analysis. Extracellular markers were stained, the cells were fixed and permeabilized to enable intracellular staining. Cells from lymph nodes of infected and control mice were phenotyped according to the criteria described by Cossarizza et al. (10 (link)). The antibodies used in this work were: anti-CD3-Pacific Blue (eBioscience) (1:200), anti-CD4-PeCy7 (eBioscience) (1:200), anti-B220-PercP 5.5 (eBiosciences) (1:200), anti-TCRγδ-FITC (eBioscience) (1:200) and anti-CD8-PercP (eBioscience) (1:200) for the extracellular markers, and anti-IL-17-APC (eBiosciences) (1:100) and anti-IFN-γ-APC (eBiosciences) (1:100) for the intracellular markers. Analysis was performed in the FlowJo software. Gate strategy for IFN-γ-producing cells on Supplementary Figure 7 and for IL-17- producing cells on Supplementary Figure 8.

CD4⁺ phenotype was analyzed by flow cytometry as previously described [22 (link)]. Briefly, 300 μl peripheral blood were stained with anti-CD4 APC-Cy7 (Immunostep) and anti-CD25 FITC for 30 minutes at 4°C. Then, erythrocytes were lysed and cells were fixed and permeabilized (FOXP3 transcription factor staining kit, eBioscience) and intracellularly stained with anti-FOXP3 PE, anti-IFNγ PerCP-Cy 5.5 and anti-IL17 APC (all eBiosciences) or isotype controls (eBioscience). Samples were analyzed and 50,000 CD4⁺ lymphocytes were acquired in a FACS Canto II (BD) cytometer. CD4⁺ cells were gated and their intensity of IFNγ and IL-17 intracellular staining (measured as mean fluorescence intensity, MFI) was analyzed with FACS Diva v. 2.6 (BD) by subtracting the MFI in the isotype control tube to that of the specific antibody-stained one. Regulatory T cells (Treg) were identified as CD4⁺CD25^highFOXP3⁺.