Recombinant human il 21

The following fluorochrome conjugated antihuman monoclonal antibodies (MoAbs) were used for flow cytometry studies: ICOSBV421, ICOSAlexa488, CD40LBV605, CD69BV650, HLA-DRFITC, CD38APCCy7, and TNF-αAPCCy7 from BioLegend (San Diego, CA); CD3BUV395, CD4PerCPCy5.5, CD8Alexa-Fluor700, CCR7PECF594, IL-2BV711, CXCR5Alexa647, IFNγPE-Cy7, PD1BV650, CD45ROAPCH7, CD21PECy5, CD27PerCPCy5.5, IgDFITC, CD10PECy7, and CD20Alexa700 from BD Bioscience (San Jose, CA); IL-21PE, CD27PECy5, and IL-17Alexa488 from e-Biosciences (San Diego, CA); and CD45ROPE-TexasRed from Beckman Coulter (Fullerton, CA). Live/Dead Fixable Aqua Dead Cell Stain Kit and CellTrace Violet Cell Proliferation Kit were from ThermoFisher (Boston, MA). Recombinant human IL-21 (Cat #8879-IL-010) from R&D systems, purified antihuman IL-2 (Cat #3440-ON-500) and antihuman-IL-21 (Cat #MT216G/21.3m) from Mabtech, and antihumanTNF-α (Cat #502922) from BioLegend were used. Samples were acquired on a BD LSRFortessa (BD Biosciences, CA) flow cytometer and analyzed by FlowJo V10 (TreeStar, Inc).

Human naive B cells (>95% pure) were purified by negative selection, using the EasySep Human Naive B Cell Enrichment Kit (19254; StemCell Technologies), from healthy donor peripheral blood mononuclear cells, following the manufacturer's instructions. Naive B cells were then cultured in FBS–RPMI and stimulated with mCD154 (1, 2 or 4 U ml⁻¹), recombinant human IL-4 (20 ng ml⁻¹; R&D Systems) and recombinant human IL-21 (50 ng ml⁻¹; R&D Systems) for 24, 48, 72 and 96 h. RNA was extracted from stimulated and unstimulated B cells and used for qRT–PCR analysis.

CD4⁺ T cells were activated using a tetrameric complex of anti-CD3/CD28 beads (ImmunoCult human T cell activator, STEMCELL) with or without recombinant human IL-21 (25 ng/ml, R&D Systems). Recombinant human PD-L1 (rhPD-L1, R&D Systems) was incubated at a concentration of 100 ng/ml in 96-well plates overnight in the presence of a goat anti-human IgG Fc antibody for dimerization and plate immobilization. Tregs were generated by stimulating CD4⁺ T cells with anti-CD3/CD28 beads for 6 days with or without the presence of immobilized PD-L1 or IL-21. To study Treg generation, which may be influenced by the tumor microenvironment, tumor slices from clinical specimens (n= 10) were laid on the bottom of the wells of a 96-well plate and cocultured with CD4⁺ T cells isolated from PBMCs in the presence of anti-CD3/CD28 beads. For neutralization studies, recombinant human IL-21 R Fc Chimera (10 µg/ml, R&D Systems) and an anti-human PD-1 antibody (10 µg/ml, R&D Systems) were added at the start of the coculture. Cells were harvested at the indicated time and then analyzed with a Cytoflex flow cytometer.