Ssrna40 lyovec

The following TLR ligands (InvivoGen, each 1 µg/mL) were added into wells of 24-well plate containing 1-2 x 10⁶ PBMCs: Poly(I:C) HMW (TLR3), LPS-EK Ultrapure (lipopolysaccharide, TLR4), Imiquimod (IMQ) (R837, TLR7), Resiquimod (R848, TLR7/8), TL8-506 (TLR8), ssRNA40/LyoVec (TLR8) and CpG-ODN (TLR9). The concentration of 1 µg/mL is within the range of manufacturer recommendation and induced maximum cytokine response (not shown) (10 (link), 12 (link)). Golgi plug (1 µl/mL, BD Biosciences) was added to block the cytokines release and cell culture continued for 18h. For some experiments, PBMCs were stimulated with agonists alone or in combination with recombinant human HBsAg subtype adw (10 µg/mL, Fitzgerld) (15 (link)) and PepMix HBV (LEP) Ultra (2 µg/mL, JPT) and cultured for 5 days at 37°C incubator. In experiments for transcription factor induction, cells were re-stimulated with Phorbol-12-myristat-13-acetate (PMA, 50ng/mL) and Ionomycin (Ion, 1 µg/mL) on day 4.

In total, 50,000 cells/well were plated in a tissue-culture treated 96-well plate (Costar, No. 3596) in unsupplemented DMEM. Cells were then treated with 100 μM R848 (InvivoGen, No. tlrl-r848), 5 μg/mL ssRNA40/LyoVec (InvivoGen, No. tlrl-lrna40), and/or 5 μg/mL ORN06/LyoVec (InvivoGen, No. tlrl-orn6) and the indicated concentrations of the compounds. Cells were incubated overnight at 37 °C in a humidified atmosphere of 5% CO₂ and assayed for NF-κB signaling. Cell media was assayed with Quanti-Blue (InvivoGen, No. rep-qbs3) per the manufacturer’s recommendations. Specificity test in HEK-Blue hTLRs cells for CU-CPD107 using TLR-specific ligands to selectively activate the corresponding TLRs: 100 ng/mL of Pam₃CSK₄, 100 ng/mL of Pam₂CSK₄, 5 μg/mL of polyriboinosinic: polyribocytidylic acid [poly(I:C)], 20 ng/mL of LPS (lipopolysaccharide), 50 ng/mL of Flagellin, 2.9 µM of R848, 22.8 µM of R848, and 0.5 μM of ODN2006 were used to selectively activate hTLR1/2, hTLR2/6, hTLR3, hTLR4, hTLR5, hTLR7, hTLR8, and hTLR9 cells, respectively. The SEAP reporter is constructed as a IFN-β promoter fused to five NF-κB and AP-1-binding sites. SEAP was quantified by measuring absorbance at 620 nm. Data were normalized with ligand + DMSO as 100% activity and untreated cells as 0. Each data point represents the average and standard deviation of at least three biological replicates.

Poly(I:C) High Molecular Weight (HMW), Poly(A:U) HMW, Poly(I:C) HMW LyoVec, pppRNA LyoVec, LPS, 2'3'cGAMP, CL264, ODN2006, TL8-506, ssRNA40 LyoVec, and Bafilomycin A1 were purchased from InvivoGen. Human TNFα was purchased from PeproTech (NJ, USA). Universal IFNα was purchased from PBL Assay Science (NJ, USA). Z-FA-FMK was purchased from Sigma-Aldrich (MO, USA). Z-VAD-FMK was purchased from R&D Systems (MN, USA). RNAse III was purchased from Thermo Fischer. Human serums were purchased from NeoBiotech (France).
Synthesis of dsRNAs was performed by different manufacturers: IDT (IA, USA), Horizon Discovery (UK), or NittoAVECIA (CA, USA). A 45 grams batch of TL-532 was synthesized by NittoAVECIA under non-GMP standards. Lyophilized powders were resuspended using apyrogenic, nuclease-free, sterile NaCl 0.9% (InvivoGen). All dsRNAs were used without transfection reagents unless otherwise stated. NaCl 0.9% was used as the Mock condition for all experiments unless stated differently in the text.

To examine TLR8-mediated differentiation of T_FH cells, CD14⁺ monocytes and naïve CD4⁺ T cells were isolated from autologous human PBMCs of CHB patients (n=4) by negative selection (Miltenyi Biotec). The following co-culture experiments were adopted from previously published methods (12 (link)). First, purified CD14⁺ monocytes (1 x 10⁶ cells/mL) were left untreated or treated with TLR8-specific agonists ssRNA40/Lyovec (1 µg/mL, InvivoGen) or TL8-506 (1 µg/mL, InvivoGen) for 1.5 h. Isolated autologous naïve CD4⁺ T cells were directly added into the culture at a ratio of 2:1 (monocyte:CD4⁺) along with SEB (1 µg/mL, Toxins Technology). After 6 days, cells were either processed for T_FH cell sorting for further downstream study or re-stimulated overnight (O/N) with PMA (phorbol-12-myristat-13-acetate, 50 ng/mL, Sigma) and ionomycin (Ion 1 µg/mL, Sigma) as positive control to effectively activate T_FH cells. After 3-4h, Golgi plug (1 µl/mL, Invitrogen) was added and culture continued for additional 18h to investigate T_FH cell proliferation and differentiation related markers by flow cytometry. The culture supernatants were recovered and tested for IL-12p40, IL-18 and IL-21 cytokine quantifications by Luminex multiplex immunoassay.