Mouse il 5

The following conditions were used for B1 cell and CD4+ T cell stimulation. Enriched B1 cells were activated using 2.5 mg/mL LPS (ultrapure, Invivogen) in the presence of mouse IL-4, mouse IL-5 and mouse IL-6 (each 100 ng/mL; Biolegend) for 24 h at 37 C. Naı ¨ve CD4+ T cells were activated with ionomycin (1 mM; Life technologies) and phorbol 12-myristate 13-acetate (PMA), (10 ng/mL; Sigma Aldrich) or by incubation with surface-bound antibodies against CD3ε and CD28 (Biolegend). For surface coating of 96-well plates, 100 mL antibody mix containing 1 mg of each antibody in PBS was used per well and incubated for 2-3 h at 37 C followed by a washing step with 200 mL PBS.
Naı ¨ve CD4+ T cells were differentiated into FoxP3-expressing T cells by cultivation in anti-CD3ε/CD28-coated 96-well plates in the presence of human IL-2 (20 ng/mL; Biolegend), mouse IL-7 (100 ng/mL; Biolegend), mouse IL-15 (100 ng/mL; Biolegend), human TGF-beta1 (5 ng/mL; Biolegend) and retinoic acid (10 nM; Sigma-Aldrich) at 37 C for at least 48 h. FoxP3-expression was confirmed by flow cytometry after intracellular staining using the BD Cytofix/Cytoperm Kit and anti-FoxP3 antibody (clone MF-14; Biolegend).