Figure 1. CHB patient-derived PBMCs had impaired TLR9-mediated IFN-α production.

(A) Comparison of the ability of healthy donors and CHB patients to produce IFN-α. PBMCs (2×10⁶ cells) obtained from healthy donors (n = 20) and treatment-naïve CHB patients (n = 30) were stimulated with CpG ODN2216 (TLR9 ligand, 3 µM), GardiQuimod (TLR7 ligand 1 µg/ml) or NDV (3.12 HA units/ml) for 48 hours Cell culture supernatants were collected to measure IFN-α production. (B) PBMCs (2×10⁶ cells) from patients or healthy donors were stimulated with 3 µM CpG ODN2216 for 3 hours and brefeldin A were added to the culture supernatant while stimulation. The cells were then collected and stained with BDCA-2 and IFN-α. The IFN-α producing pDCs were then analysed. (C) Comparison of the frequency of pDCs in healthy donors and CHB patients. PBMCs from healthy donors (n = 10) and treatment-naïve CHB patients (n = 12) were surface stained with CD123-FITC and BDCA-2-APC to identify the pDC population by flow cytometry. The pDC frequency was then calculated for each healthy donor and patient. (D) Comparison of the functional pDCs ratio in healthy donors and CHB patients. PBMCs obtained from healthy donors (n = 10) and treatment-naïve CHB patients (n = 12) were stimulated with CpG ODN2216; then, the PBMCs were surface stained with CD123-PE and HLA-DR-PerCP to identify the pDC population. The cells were then fixed, and intracellular IFN-α staining was performed to identify the IFN-α producing cells. Dotted numbers indicate the percentage of IFN-α-positive cells.