Activation of Human PBMCs by TLR Ligands

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.