Gardiquimod

PBMC were stimulated with ligands for TLR3 [polyinosinic:polycytidylic acid, poly(I:C)], RNA-helicases [poly(I:C) liposome, lipopoly(I:C)] and TLR7-8 (gardiquimod) (InvivoGen, San Diego, CA). PBMC supernatants were collected at baseline and 24 h after stimulation. Levels of IL-4, IL-5 (Th2 cytokines), CXCL8, IL-17, IL-22 (R&D Systems, Abingdon, UK), IFN-γ, IL-1β, IL-6, IL-29 (IFN-λ) (eBiosciences, San Diego, CA) and IFN-β (Elabsciences Biot., Wuhan, China) in plasma, sputum fluids and supernatants from PBMC were measured by ELISA. On the whole population, P(I:C) increased the secretion of IL-4, IL-5, IL-6, IL-29, IFN-β, IFN-γ and CXCL8 as compared to cells in medium alone, whereas gardiquimod and lipoP(I:C) upregulated the levels of IL-1β, IL-6, IFN-β, IFN-γ and CXCL8 (data not shown).

We also evaluated effects of TLR‐7 on the production of interferon‐α from pDCs under the Th2 immune milieu in asthmatic subjects and non‐asthmatic normal controls ex vivo. This was achieved by isolating PBMCs, pretreating them with IL‐4 (4 ng/ml), IL‐13 (10 ng/ml), or PBS for 1 h, followed by TLR7 stimulation via the TLR7 agonist Gardiquimod (InvivoGen) (0.5ug/ml) for 6 h as well as Brefeldin‐A (Thermo Fisher Scientific) for the last 3 h. Cells then underwent staining with BDCA‐2‐FITC to estimate pDCs surface marker expression and interferon‐α‐PE staining (MACS Miltenyi Biotec) after fixing with permeabilizing solution for intracellular antigen detection (Figure 2).

R837 (selective TLR7 agonist), R848 (TLR7 & TLR8 agonist) and TL8-052 (selective TLR8 agonist) were acquired from Invivogen and stored at −20°C. TLR-agonist R837 was used at a concentration of 5 μg/mL. R848 at 10 μg/mL and TL8-052 at 5 μg/mL.
Vesatolimod, Gardiquimod, Telratolimod, Selgantolimod and Motolimod were acquired from a commercial distributor (MedChemExpress) and stored as per the manufacturer’s instructions. Concentrations were used in increasing concentration to establish working concentrations as follows: TLR-agonist GS-9620/Vesatolimod, 0.1 μM, 1 μM, 3 μM, 10 μM, 20 μM, 30 μM, 60 μM (selective TLR7 agonist), Gardiquimod, 0.1 μM, 1 μM, 3 μM, 10 μM, 20 μM, 30 μM (selective TLR7 agonist), 3 M-052/Telratolimod, 0.1 μM, 1 μM, 3 μM, 10 μM, 20 μM, 30 μM (TLR7 & TLR8 agonist), GS-9688/Selgantolimod, 0.1 μM, 1 μM, 3 μM, 10 μM, 20 μM, 30 μM (selective TLR8 agonist), MTX-1337/Motolimod, 0.1 μM, 1 μM, 3 μM, 10 μM, 20 μM, 30 μM (selective TLR8 agonist).
PolyIC-lyovec (LMW) was acquired from Invivogen and dissolved in LAL-water as per the manufacturer’s instructions and used at a concentration of 2 μg/mL. Salmonella Lipopolysaccharide (LPS) was used as positive controls at a concentration of 10 ng/mL.

Human PBMC were obtained by density gradient centrifugation and human B-cells were negatively sorted from PBMC using EasySep Human B-cell Isolation Kit (StemCell) according to the manufacturer’s instructions. Purified B-lymphocytes were seeded at 1 × 10⁶ cells/ml in IMDM medium (Lonza) supplemented with 20% fetal bovine serum (Deutscher), 1% penicillin/streptomycin (Gibco) and stimulated for 4 days with 100 ng/ml human recombinant CD40L (Enzo Life Sciences) alone or with 50 ng/ml recombinant human IL-4 (Peprotech) or 2 μg/ml Gardiquimod (InvivoGen) or 2.5 μg/ml CpG oligodeoxynucleotide 2006 (InvivoGen) or 100 ng/ml IL-21 (R&D Systems) or 100ng/ml INFγ (R&D Systems) or 50 ng/ml Pam3CSK4 (InvivoGen) or 0.5 μg/ml goat anti-human kappa (Southern Biotech).

Total CD20⁺ B cells were plated at 100,000 cells/well in a 96-well-plate in RPMI 10% FBS and 2.5 μg/ml polyclonal F(ab’)₂ anti-human IgM (Jackson Immunoresearch), 1.0 μg/ml CpG ODN2006 (In vivogen) or 2 μg/ml Gardiquimod (In vivogen). Expression of surface activation markers CD69 and CD86 was analyzed on gated CD19⁺CD27^- IgD⁺ or IgD^- cells after 48 hours by flow cytometry. For functional analysis of IGHD variants flow cytometrically sorted CD19⁺CD27IgM⁺IgD⁺ and CD19⁺CD27^-IgM⁺IgD^- B cells were stimulated by anti-human IgM or anti-human IgD antibodies. Expression of CD69 was analyzed after 48 hours.