Activation and Isolation of Mouse and Human B Cells

For patient and healthy donor in vitro B cell activation, peripheral blood mononuclear cells were isolated from fresh blood using Lympholyte gradient (Cedarlane). For mouse in vitro B cell activation, single-cell suspensions were obtained by passing cells through 70-µm cell strainers. Red blood cells were lysed in lysis buffer (BD). Highly pure B cells were isolated via magnetic sorting using AutoMACS (Miltenyi Biotec). AffiniPure F(ab′)2 Fragment Goat Anti-Mouse IgM, μ Chain Specific (Jackson ImmunoResearch), AffiniPure F(ab′)2 Fragment Goat Anti-Human IgA + IgD + IgM (H + L) (Jackson ImmunoResearch), mouse anti-CD40 (1 µg/ml; Becton Dickinson), CXCL12 (25 nM; Peprotech), CCL21 (25 nM; Peprotech), AMD3100 (10 µg/ml; Sigma), and anti-CD95 (250 ng/ml, Clone Jo2; BD) were used to stimulate the B cells. For in vivo activation, WHIM knock-in or WT mice were immunized by intraperitoneal injection of alum-precipitated 4-hydroxy-3-nitrophenylacetyl (NP)-CGG (NP ratio >40 for high avidity immunization or 1–9 for low-avidity immunization, 100 µg per mouse; Santa Cruz Biotechnology). A boost immunization was performed 56 days after the primary immunization, using intraperitoneally injected NP_>40-CGG or NP_1–9-CGG (50 µg per mouse) resuspended in PBS, adapted from Ref. (26 (link)).