Il 21r fc chimera

Cytokine production was assessed with BD Cytofix/Cytoperm containing BD Golgi-Plug (BD Biosciences). Cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, Sigma), Ionomycin (1 μg/ml, Sigma), and GolgiStop (1 μl/ml, BD Biosciences) at 37°C in 5% CO₂ for 4 h. After surface staining, cells were fixed, permeabilized, and stained for IFN-γ (PE-anti-mouse IFN-γ, Biolegend), IL-4 (PE-anti-mouse IL-4, Biolegend), and IL-17 (PE-anti-mouse IL-17A, Biolegend). For intracellular staining IL-21, permeabilized cells were incubated with IL-21R/Fc chimera (R&D systems) for 1 h at 4°C. Cells were then washed and stained with PE-conjugated affinity-purified F(ab')₂ fragment of goat anti-human Fc γ antibody (Jackson ImmunoResearch Laboratories) for 30 min at 4°C. Viability was assessed using LIVE/DEAD Cell Viability Assays (Life Technologies).

For surface staining, Fc receptors (FcRs) were blocked using an antimouse CD16/32 antibody (BD) for 15 min on ice, and standard multiparameter flow cytometric analyzes was performed by staining cells with the antibodies listed in Table 1. Data were collected using FACS Diva software on a BD LSR II analyzer (BD), and analyzed using FlowJo_V10 software (TreeStar, Ashland, OR, USA). Doublet discrimination was performed and viable cells were analyzed using either 7AAD (Invitrogen, Carlsbad, CA, USA) or the fixable viability dye (Biolegend, San Diego, CA, USA) exclusion method. For intracellular cytokine staining, cells were initially stimulated with a cell activation cocktail (Biolegend) (6 h, 37°C, 5% CO₂) and staining for IFN-γ, IL-21, IL-17, and IL-4 was performed using the BD CytoFix/Perm Kit following the manufacturer’s instructions. Staining for intracellular IL-21 was done using an IL-21R/Fc chimera (R&D Systems) and PE-conjugated F(ab′)2 fragment of goat anti–human Fcγ antibody (anti-Fc PE; Jackson ImmunoResearch, West Grove, PA, USA). For all intracellular staining, unstained cells (permeabilized as well as non-permeabilized) as well as fluorescence-minus-one were used to set gates and as negative controls. Staining for transcription factors including FoxP3, Tbet, and Bcl6 was done using the FoxP3 staining kit from eBiosciences (ThermoFisher, Waltham, MA, USA).

Single-cell suspensions of splenocytes were stimulated with PMA (1 μg/ml) and ionomycin (1 μg/ml) (both from Sigma-Aldrich,) in the presence of GolgiStop™ (BD Biosciences, 1:1,000) at 37°C for 4 h. Cells were incubated with antibody for CD4, and PD-1 at 4°C, then were fixed and permeabilized with Foxp3 Fix/Perm buffer set (eBioscience) and incubated with antibody for IL-2 (BD Biosciences, JES6-5H4), IL-4 (BD Biosciences, 11B11), IL-10 (eBioscience, JES5-16E3), and IFNγ (BD Biosciences, XMG1.2). For IL-21 staining, cells were incubated with IL-21 R/Fc chimera (R&D Systems) for 1 h, washed and stained with PE-labeled affinity-purified F(ab') α-human IgG Fc Region antibody (R&D Systems) for 30 min.