Cd40 ligand

HEK-293T and HT1080 cells were cultivated in DMEM (Biowest, Nuaille, France) (10% FCS (Biochrom, Berlin, Germany)). Raji and SupT1 cells were cultured in RPMI (Biowest, Nuaille, France) supplemented with 10% FCS and 2 mM L-glutamine (Biowest, Nuaille, France). PBMCs were isolated from buffy coat derived from healthy donors via density centrifugation using Ficoll (Pan Biotech, Aidenbach, Germany) and cultured in TexMACS™ (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 12.5 ng/mL IL-7 and 12.5 ng/mL IL-15 (both Miltenyi Biotec, Bergisch Gladbach, Germany). For the selectivity assay, PBMCs were cultivated in TexMACS™ supplemented with 50 IU/mL IL-4, 12.5 ng/mL IL-7, and 12.5 ng/mL IL-15 (all Miltenyi Biotec, Bergisch Gladbach, Germany) and 1 µg/mL CD40-ligand (RD Systems, Minneapolis, MN, USA).

For BCR crosslinking 1 × 10⁷ cells were treated with goat anti‐human IgM F(ab′)2 (Jackson ImmunoResearch) at 10 μg/mL final concentration for 8‐24 hours. CD40 ligand (R&D Systems) was used at 50 ng/mL final concentration for 1 hour.