Intracellular staining was performed using fixation and permeabilization buffers (eBioscience) according to the manufacturer’s instructions. Flow cytometry was performed using FACSCalibur and data were analyzed with CellQuest software (BD Biosciences). Cell depletion mAbs were purified from 2.43 (α-CD8β), GK1.5 (α-CD4), and PK136 (α-NK1.1) hybridoma cell lines. To deplete cells, mice were injected intraperitoneally with 1 mg of mAb. Three days later the dual vector was administered intravenously. Splenic lymphocytes
were separated from WT C57BL/6 or IFN-γ−/− mice. buy RXDX-106 Lymphocytes were incubated with microbeads directly conjugated to antimouse CD8 Ab (10 μL /107 cells) at 4°C for 20 minutes. Labeled cells were removed on MACS columns in a magnetic field. After washing twice with PBE solution, the column was removed from the magnet and flushed with PBE. After washing with PBS, CD8+ T cells were sorted and purity was analyzed by fluorescent-activated cell sorter FACS (>90%). The sorted splenic CD8+ T cells from WT C57BL/6, IFN-γ−/− or IFNAR−/− mouse, or CD8-depleted splenic lymphocytes, were transferred intravenously into recipient Rag 1−/− HBV carrier mice (5 × 106 cells/recipient). Plasmid construction, lentiviral packaging, reverse transcription, and
real-time PCR analysis, western blot, enzyme-linked immunosorbent assay (ELISA) assay, selleck screening library and immunohistochemistry are included in the Supporting Information. Statistical analysis was performed using a paired Student’s t test. P < 0.05 was considered statistically significant. HBV inhibits TLRs or other PRR-mediated innate immune responses4, 5 by suppressing the host antiviral type I IFN signal pathway, leading to cell-intrinsic immunotolerance. To explore this cell-intrinsic immunotolerance, we first evaluated expression of type I IFNs, IFN-inducible genes (ISG15 and MxA), and immunosuppressive cytokines (TGF-β and interleukin [IL]-10) in HBV-persistent HepG2.2.15 上海皓元医药股份有限公司 cells. We found that IFN-α, IFN-β, ISG15, and MxA expression was significantly lower, while TGF-β and IL-10 was higher, in HepG2.2.15 cells than in control HepG2 cells (Supporting Fig. 1A). We also evaluated
gene expression in human primary HCC cells harvested from HBV+ CHB patients and found similar results (Supporting Fig. 1B). To further confirm this in vivo, we established HBV-persistent mice by hydrodynamic injection of pAAV/HBV1.2 plasmid into C57BL/6 mice (Supporting Fig. 2A-D). Four weeks later, a time when HBV-carrier established, liver tissue exhibited high HBsAg expression without liver injury (Supporting Fig. 2B). The higher levels of HBsAg and HBV-DNA in serum can persist for at least 6 months (Supporting Fig. 2C,D) with no nonspecific inflammatory and liver injury, suggesting that the HBV-persistent mice had been successfully established. Mice with serum HBsAg levels >500 ng/mL were defined as HBV-persistent mice (HBV+), and were inoculated with HBV vaccine (rHBs/CFA).