Resiquimod r848

Polyclonal B–cell activation was carried out by stimulating 2 × 10⁶ PBMC/well in a total volume of 2 mL/well in 24–well plates with an activation cocktail consisting of 2.5 μg/mL Toll–like receptor 7/8 agonist (resiquimod [R848]; Sigma–Aldrich, St. Louis, MO) and 1000 IU/mL IL–2 (Proleukin, Novartis, the Netherlands).^{15 (link)} Peripheral blood mononuclear cell cultures were carried out in Iscove’s modified Dulbecca’s medium (Gibco Invitrogen, Paisley, UK) containing 10% fetal bovine serum (Gibco Invitrogen) and 100 U/mL penicillin with 100 μg/ml streptomycin (Gibco Invitrogen). Supernatants harvested at day 6 (d6) or day 10 (d10) were kept at −20°C until further use. Flow cytometry was performed before (d0) and after (d6, d10, and d14) polyclonal activation according to standard protocols using the following antibodies (clone): CD19 (A07770), CD27 (1A4CD27), CD38 (LS198–4–3) and CD45 (J33) (all from Beckman Coulter, Woerden, the Netherlands).

Antagonists for TLR7 (IRS 661), TLR9 (IRS 869), the TLR9 agonist (1018 ISS) and a control ODN were purchased from Eurofins Genomics (Eurofins Genomics, Ebersberg, Germany). The sequences were previously published.20, 21 Resiquimod (R‐848) served as TLR7 agonist (Sigma‐Aldrich, St. Louis, USA). For in vitro analysis by flow cytometry, IRS 869 and the control ODN had a fluorescein modification at the 5′‐end (Eurofins Genomics).

To obtain medium enriched for newly synthesized antibodies (MENSA) for the analysis of ex vivo antibody production, the isolated PBMCs were cultured in IMDM cell culture medium (IMDM supplemented with 10% FCS, 100 U/mL penicillin G sodium, 100 µg/mL streptomycin sulfate, 292 µg/mL L-glutamine (Penicillin-Streptomycin-Glutamine Gibco; Life Technologies, Carlsbad, California, USA) and 50 µmol/L β-mercaptoethanol (Sigma-Aldrich)) under non-stimulating and stimulating conditions. In detail, PBMCs were seeded at 2 × 10⁶ cells/mL in 200 µL IMDM cell culture medium into 96-well round-bottom cell culture plates. PBMCs were either left untreated to assess spontaneous antibody secretion (MENSA) or stimulated with 20 ng/mL IL-2 (Gibco, Life Technologies) and 2 µg/mL of the synthetic dual TLR7/8 agonist resiquimod (R848; Sigma-Aldrich) to assess antibody secretion by Bmems (MENSA+) (31 (link)). After 7 days of incubation (37°C, 5% CO₂), PBMCs were harvested (300 × g, 5 min) and the culture supernatants were collected, aliquoted and stored at -80°C until assayed.

CAL-1 cells, obtained under a material transfer agreement, were cultured in complete growth medium RPMI 1640 containing 2mM L-alanyl-L-glutamine (Pan Biotech Ref. P04-18500, Chemie Brunschwig, Basel, Switzerland), supplemented with 10% FCS (Gibco Ref. 10270-106; LuBioScience, Luzern, Switzerland) and 1% penicillin/streptomycin (BioWhittaker Lonza; VWR Scientific, Nyon, Switzerland) in suspension tissue culture flasks or plates (Greiner Bio-One; Huberlab, Aesch, Switzerland). Floating cells were passaged to new culture flasks for cell maintenance. Where indicated, CAL-1 cells (2x 10⁵ cells/ml) were exposed to 10ng/ml human GM-CSF (Peprotech, LuBioScience; catalog #300-03) for 3 days, referred to as GM/CAL-1 cells, washed, resuspended at a density of 1x 10⁶ cells/ml in complete growth medium without GM-CSF and then left untreated or matured for another 18-19h with either the TLR7/8 agonist Resiquimod R848 (10μg/ml; Sigma–Aldrich, Buchs, Switzerland; #SML0196) or the TLR9 ligand CpG-B ODN 2006 (1μM; InvivoGen; LabForce, Muttenz, Switzerland; #tlrl-2006-1).