Clone 4s b3

The following conjugated antibodies were used for cell-surface staining: CD3-Pacific Blue (BD Pharmingen; Clone UCHT1; Cat. No. 558117; San Diego, CA, USA), CD8-APC H7 (BD Pharmingen; Clone SK1; Cat. No. 641400), CD45RA-PE (BD Pharmingen; Clone HI100; Cat. No. 555489), CCR7-PE-Cy7 (BD Pharmingen; Clone 3D12; Cat. No. 557648), CD28-PerCP-Cy5.5 (BD Biosciences; Clone L293; Cat. No. 337181; San Jose, CA, USA), CD27-Alexa Fluor 700 (BD Pharmingen; Clone M-T271; Cat. No. 560611), CD95-APC (BD Pharmingen; Clone DX2; Cat. No. 558814) and CD127-FITC (BD Pharmingen; Clone HIL-7R-M21; Cat. No. 560549). Conjugated antibodies for intracellular staining included the following: IFN-γ-FITC (BD Pharmingen; Clone 4S.B3; Cat. No. 554551), IL-2-PerCP-Cy5.5 (BD Pharmingen; Clone MQ1-17H12; Cat. No. 560708) and TNF-α-AlexaFluor 700 (BD Pharmingen; Clone MAb11; Cat. No. 557996). To exclude dead cells, the Fixable Aqua Dead Cell Stain viability marker was used (Invitrogen; Cat. No. L34957; Eugene, OR, USA).

PBMCs were washed with FACS buffer and stained for cell viability with the fixable live/dead Aqua staining kit (Molecular Probes) in the presence of Fc-R blocking reagent (Miltenyi biotech), and the antibodies against the surface markers anti-CD3-APC, CD45-V450 and anti-CD11b-APC/Cy7 indicated above. Surface staining was carried out for 25 min at 4°C. Cells were washed, and intracellular staining was carried out with BD cytofix-cytoperm kit (BD Biosciences). Cells were fixed with Fix-Perm (BD Biosciences) for 20 min at room temperature and permeabilized with Perm-Wash (BD Biosciences) for 30 min at room temperature. Cells were stained with two combinations of antibodies diluted in Perm-Wash: anti-IFN-γ (PE, 1:100, clone 4S.B3, BD Biosciences), anti-IL-4 (FITC, 1:100, clone MP4-25D2, BD Biosciences), and anti-IL-17A (PE/Cy7, 1:100, clone BL168, BioLegend) or anti-TNF-α (AlexaFluor-488, 1:100, clone MAb11, BD Biosciences) and anti-IL-10 (PE, 1:30, clone JES3-9D7, BD Biosciences). Data were acquired on a FACSCanto II flow cytometer (BD Biosciences) with Diva software (BD Biosciences) and analyzed with FlowJo (v10, BD Biosciences).

PBL subsets were determined as described previously [2 (link), 5 (link)]. For analysis of determinants on the cell surface, PBL were incubated with fluorochrome-labelled monoclonal antibodies against CD3, CD4, CD25, CD28, CD62L, CD95, CD119, CD127, CD152, CD154, CD178, CD252, HLA-DR, and CD183 (CXCR3) (all from BD Biosciences). Intracellular determinants were stained with fluorochrome-labelled monoclonal antibodies against Foxp3, IFNγ (clone B27), IL4, IL10, Granzyme B, Perforin, T-bet (all from BD Biosciences), Helios (ebioscience, Frankfurt, Germany) and TGFß₁ (R&D systems, Wiesbaden). Briefly, PBL were incubated with combinations of monoclonal antibodies for 30 min as described and eight-color fluorescence was analyzed using a FACSCanto II triple-laser flow cytometer (BD Biosciences) [2 (link), 5 (link)]. When, in addition, intracellular proteins were studied, cell membranes were permeabilized using BD Perm/Wash buffer (BD Biosciences). At least 100,000 events were analyzed in the initial FSC/SSC dot plot. IFNγ monoclonal antibody used for cell separation (BD clone 4S.B3) and IFNγ monoclonal antibody used for cell staining (BD clone B27) were not competitive (data not shown).