Human il 21

Peripheral blood was obtained from healthy donors and the affected patient after informed consent. Mononuclear cells were obtained following lymphoprep density centrifugation. B cell selection was carried out with a Miltenyi memory B-cell isolation kit, selecting for untouched total B cells using the first step of the manufacturer’s protocol which depletes non-B cells. Alternatively, naïve B cells were enriched by depletion of memory cells using anti-CD27 MACs beads (Mitenyi). B-cells were cultured as previously described [30 (link), 31 (link)] in 24-well plates at 1×10⁵ cells/ml in IMDM + 10% FBS with MEM amino acid solution (1:50) and Lipid Mixture 1, chemically defined (1:200) (all Thermo Fisher Scientific) and the addition of 20 U/ml human IL-2 (Roche), 50 ng/ml human IL-21 (Peprotech) and 10 μg/ml F(ab′)₂ goat anti-human IgM/IgG (Jackson Immunoresearch) on previously irradiated CD40L-expressing fibroblasts. After 3 days, B cells were removed from the CD40L-cells by gentle pipette mixing. B cells were reseeded at in media containing 20 U/ml human IL-2 and 50 ng/ml human IL-21. On day 6, cells were seeded in media containing human IL-6 (10 ng/ml), human IL-21 (50 ng/ml), 100 U/ml multimeric APRIL (Adipogen), and 100nM gamma secretase inhibitor (GSI) inhibitor (L-685458; Sigma) and re-fed at day 10.