Human il 21

The ELISPOT assay was performed using a human IgG B cell ELISpot development module (R&D Systems GmbH), and all procedures were performed according to the manufacturer’s instructions. In brief, purified human B cells were stimulated in complete RPMI 1640 with 5 μg/ml anti-IgM, 1 μg/ml anti-CD40, and 100 ng/ml human IL-21 (R&D Systems GmbH). After 4 days, the cells were washed, and 2000 cells were seeded in each well in quintuplicate in a MultiScreen HTS IP filter plate (Millipore) precoated with goat anti-human IgG polyclonal antibody (R&D Systems GmbH). After 16 h, the cells were washed off, and the plate was incubated overnight with biotinylated goat anti-human IgG polyclonal antibody (R&D Systems GmbH) at 4 °C. ELISPOTs were then developed by an ELISpot blue color module (R&D Systems GmbH). The spot number and size were measured by AID EliSpot/FluoroSpot reader systems (Autoimmun Diagnostika GmbH).

The antibodies for flow cytometry and western blot assays were purchased from Biolegend (San Diego, CA, USA), eBioscience (San Diego, CA, USA), BD Bioscience (San Jose, CA, USA) and Cell Signaling Technology (Danvers, MA, USA). Antibody information can be found in Supplementary Table 1. All microbeads (CD3ɛ, CD11b, CD19, CD14, B220, EpCam and Biotin) for lymphocyte depletion or selection were purchased from Miltenyi Biotec (Bergish Galdbach, Germany). The antibodies for in vivo depletion (Asialo GM-1, NK1.1 (PK136), CD4 (GK1.5), CD8 (2.43), IL-21R (4A9) and IFN-γ (XMG 1.2)) were prepared in the laboratory or purchased from Biolegend or BioXcell. The recombinant mouse IL-2, mouse IL-21, human IL-2, human IL-21, ULBP2 and human NKp46 antibodies were purchased from R&D Systems. Chemical inhibitors, including S31-201, Fludarabine, LY294002 and PD98059, were purchased from Selleckchem (Houston, TX, USA).