In vitro culture of PBMCs with TLR7/8 ligands and inhibitors.

Plates for PBMCs culture were precoated with anti-IgM antibodies (Jackson ImmunoResearch, 109-005-129) at a concentration of 5 μg/mL in PBS at 4°C overnight. PBMCs from patients or controls were isolated and resuspended in complete 1640 medium (RPMI 1640 medium, 10% FBS, 100 μg/mL of penicillin and streptomycin, 2 mM l-glutamine, 100 mM sodium pyruvate, 1 × non-essential amino acid, 55 μM β-mercaptoethanol) (Gibco, Thermo Fisher Scientific). Cells were cultured at 37°C in a 5% CO₂ incubator at the density of 2.5 × 10⁵ cells per well in 96-well plate. The cells were stimulated with TLR7/8 ligand-R848 (Enzo Life Sciences, ALX-420-038-M005) for up to 12 days. After culture, cells were lysed for Western blot analysis, and culture supernatant was harvested for IgA1 ELISA and lectin binding assay. For blockade of TLR7 activation, a synthetic small compound (Zinc 4756232), reported to be a TLR7 inhibitor (78), was synthesized by Specs. TLR8-specific inhibitor CU-CTP8m (79) (CAS 125079-83-6) was synthesized by DC Chemicals. Loxoribine (InvivoGen, tlrl-lox), a selective TLR7 agonist, was also included in the culture system at a concentration of 0.5 mM to induce the synthesis of IgA1 and Gd-IgA1.