Cd8 amcyan

Mononuclear cells were stimulated with PMA and ionomycin for six hours in the presence of ‘Golgi stop’ (eBioscience, San Diego, CA) as described previously [33 (link)]. Briefly, cells were stained with surface antibodies CD8 Amcyan (BD Biosciences. Oxford, UK), CD3 APC-Cy7 (Cambridge Biosciences, Cambridge, UK), CCR4 PE-Cy7 (BD Biosciences, Oxford, UK) and CXCR3 PE (BD Biosciences. Oxford, UK) for 20 mins at 4°C. Samples were washed, re-suspended in RPMI with 10% human serum (TCS Biosciences, Buckingham, UK) and then stimulated with PMA and ionomycin for six hours in the presence of ‘Golgi stop’. Cells were washed, stained with a dead cell exclusion dye (Invitrogen, Oregon, USA) at 4°C for 20 mins and then washed again before fixation with 2% paraformaldehyde at room temperature for 10–15 mins. After further washing, cells were permeabilised with 0.5% saponin for 5 minutes followed by incubation with the intracellular antibodies IL-17A (BioLegend, London, UK) (2.5 ml), IFN-γ (BioLegend, London, UK) (0.5 ml) and isotype controls for 30 mins at room temperature. Samples were washed and analysed by flow cytometry using on LSR II flow cytometry. To define Th17 cells we measured the secretion of IL-17 using intracellular staining and FACS analysis.

Heparinized whole blood was kept at room temperature. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-gradient (Ficoll Paque PLUS, GE Healthcare, Uppsala, Sweden) and washed twice with cell wash (PBS, 0.5%BSA and 0.02%NaN₃). From each sample, 0.5×10⁶ PBMCs were fixed by Fixation/Permeabilization Concentrate and Diluent (eBioscience), permeabilized by Permeabilization Buffer (eBioscience) and stained for surface proteins using the following antibodies; CD3-Pacific Blue (BD Pharmingen), anti CD4-APC-H7 (BD Pharmingen) and CD8-AmCyan (BD Pharmingen). For intracellular staining of cytoplasmic and nuclear proteins the following antibodies were used: polyclonal rabbit IgG anti-AUF1-APC (LifeSpan BioSciences), monoclonal mouse IgG1 kappa anti-HuR-APC (LifeSpan BioSciences), APC mouse IgG1 kappa isotype control (BD Pharmingen) and APC rabbit IgG isotype control (Santa Cruz biotechnology). Results are expressed as mean fluorescence intensity (MFI) minus background provided by the isotype-matched negative control antibodies. Samples were run on an eight-colour FACSCanto II flow cytometer (Becton Dickinson). Data were analysed with Flowjo 10, Treestar.

BAL and blood cells were stained using the following antibodies; CD3-Pacific Blue (BD Pharmingen), anti CD4-APC-H7, CD8-AmCyan (11 of 15 patient samples and all control samles) (BD Bioscience). Normal mouse serum (Corning Life Science, Tewksbury, MA, USA) was used as Fc-Block. TCR Vβ was stained using an eight-tube panel, containing 24 monoclonal antibodies known to react with specific TCR-Vβ families (IO Test Beta Mark TCR Vβ Repertoire Kit)(Beckman Coulter, Brea, CA, USA). Each test, containing 1 million BAL cells or 0.5 million PBMC, permitted analysis of three Vβ families simultaneously as each of the eight tubes included a mixture of three antibodies conjugated to fluorescein isothiocyanate, phycoerythrin or fluorescein isothiocyanate and phycoerythrin. Due to limitations in cell numbers, all Vβ families were not analysed in all subjects. Nomenclature for the Vβ families used throughout this paper is from [17 (link)]. Flow cytometry was run on a BD FACS Canto II. Results were expressed as the percentage of CD4⁺ and CD8⁺ T cells, respectively, that expressed the various TCR Vβ families.