Anti mouse cd40

Primary (CD11b⁺F4/80⁺) murine macrophages were derived from the bone marrow of BALB/c mice, by culturing with M-CSF (50ng/ml; eBioscience) for 6 days, and then seeded in RPMI supplemented with 10%v/v FCS and 50 μM beta mercaptoethanol at a density of 1 × 10⁶ cells/ml. Macrophages were untreated or incubated with FhHDM-1 (50 μg/mL) or LPS (100 ng/ml) overnight. For detection of MHCII expression only, macrophages were left untreated or incubated with FhHDM-1 (50 μg/ml) or IFNγ (10 ng/ml) for 2 h. Cells were blocked with Mouse Fc Block (BD Biosciences), and then stained with antibodies: anti-mouse CD80 (BD; clone 16-10A1), anti-mouse CD86 (BD; clone GL1), anti-mouse CD40 (BD; clone 3/23), anti-mouse I-A/I-E (BD; clone M5/114.15.2) or anti-mouse H-2K[d] (BD; clone SF1-1.1) to identify expression of CD80, CD86, CD40, MHCII and MHCI, respectively. Samples stained with corresponding isotype controls were included. Data was acquired using the BD LSR II (BD Biosciences) using FACS Diva software (BD Biosciences). Analysis was performed using FCS Express 4 Flow Research Edition (De Novo Software).

The Caspase-Glo 3/7 Assay (Promega) was performed according to manufacturer’s instructions. Briefly, purified B cells were plated in IMDM medium at the concentration of 100,000 cells/100 μl with stimuli [AffiniPure F(ab′)2 Fragment Goat Anti-Mouse IgM, μ Chain Specific (Jackson ImmunoResearch), mouse anti-CD40 (1 µg/ml, Becton Dickinson), and CXCL12 (25 nM, Peprotech)] for 24 h or left untreated. After stimulation, cells were counted and plated at 20,000/well in a white-walled multiwell plate. An equal volume of Caspase-Glo 3/7 Reagent was added to each well for 30 min. Luminescense signal was acquired on a Biotek Synergy 2 luminometer. Background readings were determined from wells containing culture medium without 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)).