Cpg odn 2006

Peripheral blood mononuclear cells (PBMCs) were isolated and cultured as described before (15 (link)). In short, for 79 GPA patients PBMCs were available and were cultured for 12 days with or without 3.2 μg/mL CpG-ODN 2006 (Hycult Biotech, Uden, the Netherlands), 100 ng/mL IL-21 (Immunotools, Friesoythe, Germany) and 100 ng/mL BAFF (PeproTech Inc., Rocky Hill, CT, USA). Stimulated and spontaneous PR3-ANCA (RU/mL) production was determined in the supernatant by Phadia ImmunoCAP® 250 analyzer using EliA PR3^S (Thermo Fisher Scientific) and total spontaneous and stimulated IgG production was assessed by ELISA. Five samples (2 F-R and 3 N-R) were excluded because of a culture infection.

Tryptic soy broth (TSB), N-acetylmuramic acid, TRI Reagent, l-benzoyl-Arg-pNA (l-BApNA), and Tos-Gly-Pro-Lys-pNA (Tos-GPK-pNA) substrates, gentamicin, LPS from Escherichia coli O111:B4, lipoteichoic acid (LTA), and carboxymethylcelullose were from Sigma-Aldrich. Yeast extract, l-cysteine, hemin, and bovine serum albumin (BSA) were from BioShop Canada, Inc. Dulbecco’s phosphate-buffered saline without Ca²⁺ and Mg²⁺ (PBS), fetal bovine serum (FBS), RPMI 1640, the BCA protein assay kit, reducing sample buffer, Hoechst 33342 stain, eosin, and decalcifier were from Thermo Fisher Scientific. Menadione was from ICN Biomedicals, KYT-1 and KYT-36 from PeptaNova, and saponin from Serva Electrophoresis GmbH. Toll-like receptor agonists were obtained as follows: Pam₃CSK_4, flagellin, and R848 from Enzo Life Sciences, macrophage-activating lipopeptide (MALP-2) from Imgenex, CpG ODN 2006 from Hycult Biotech, and human recombinant IL-1β from BioLegend. Their purity was estimated for CpG 85% (high-performance liquid chromatograph [HPLC]), flagellin 95% (SDS-PAGE), LPS 80%, MALP-2 95% (HPLC), and LTA 97%, according to the manufacturer’s statement. All agonists, except LPS, were endotoxin free.

RPMI-1640 media (Sigma Aldrich) supplemented with L-glutamine, Penicillin/Streptomycin and 10% fetal bovine serum (FBS) (Sigma Aldrich) was used throughout the study. Isolated memory B cells were set at different cell densities in 96-well U-bottom plates (2×10
³ cells/well in 250 μl) or in 24-well flat bottom plates (1×10
⁶ cells/well in 1 ml). Small-scale expansions were used for sequencing and Ig quantification, large-scale expansions were used for phenotyping of expanding cells by flow cytometry. Before adding memory B cells, each well was seeded with irradiated (2,000 cGray) HV13280 feeder cells (CD154
⁺ HEK-293T cells, kindly provided by L. Liao’s lab, Duke University, Durham, NC, USA) at ratios varying from 1:1 to 1:50 (HV13280:memory B cell). After the addition of memory B cells, cultures were stimulated with iterative combinations of the following stimuli: recombinant human interleukin (IL)-2 (100–1000 U/ml), IL-6 (10–100 ng/ml), IL-15 (10–100 ng/ml), IL-21 (10–100 ng/ml), APRIL (10–100 ng/ml), BAFF (10–100 ng/ml), CpG ODN
₂₀₀₆ (0.25–10 μg/ml) (HyCult Biotech, Uden, Netherlands); R848 (0.25–5 μg/ml; Invivogen, Toulouse, France); PWM (5–100 ng/ml; Sigma Aldrich). Cells were then cultured for 5 or 10 days at 37°C 5% CO
₂. All recombinant proteins were ordered from Peprotech (London, UK) unless otherwise stated.