Recombinant il 21

CD19 mAb-coated microbeads (Miltenyi Biotec, San Diego, CA) were used to purify splenic B cells by positive selection (> 95% purity) using the manufacturer’s instructions. Purified B cells, whole splenocytes or whole MLNs (1 × 10⁶ cells/ml) were cultured with recombinant IL-4 (50 ng/ml; Biolegend, San Diego, CA) plus LPS (40 μg/ml; Sigma) for 5d. In addition to IL-4 and LPS some cultures contained anti-CD40 (2 μg/ml; eBioscience, San Diego, CA), recombinant IL-10 (50 ng/ml; Biolegend), recombinant IL-21 (100 ng/ml, Biolegend), or anti-IgM (2 μg, Southern Biotech, Birmingham, AL) and 40 μg/ml of anti-IL-10 or IgG1 control (both from Biolegend) for the 5d culture.

Naïve CD4⁺ T cells were sorted from the spleens and peripheral lymph nodes of mice on day 0. Cells were then cultured with RPMI medium containing 10% FBS and 1% antibiotics in 24-well plates coated with 10 μg/ml anti-CD3 (145–2C11, BioXCell) and 10 μg/ml anti-CD28 (37.51, BioXCell). Cytokines for cells culture with the following concentration were used in the indicated conditions: 50 ng/ml recombinant IL-21 (Biolegend), 1 ng/ml TGFβ1 (R&D systems), 20 μg/ml anti-IFN-γ, 20 μg/ml anti-IL-4, and 100 ng/ml recombinant human activin (Biolegend). 10 μM TGF-β receptor I inhibitor SB525334 (Selleckchem) was used in indicated conditions. Cells were transduced with retrovirus on day 1 and analyzed by flow cytometry on day 4.