Early time point of anti-TNF-α delivery is critical for HBV clearance. BALB/c mice were intraperitoneally administered a single injection of etanercept (250 µg) at different time points after pAAV/HBV1.2 hydrodynamic injection. (a) Differences in titers of serum HBsAg (left) and serum HBV viral loads (right) of HBsAg-positive mice with a single injection of etanercept at different time points were quantified. *P<0.05; **P<0.01, Student's t-test, compared with PBS group. (b) Intrahepatic lymphocytes from mice receiving a single etanercept injection at different time points were isolated, and the PD-1 and CD127 expression of liver-infiltrating CD8+ lymphocytes was analyzed by flow cytometry. *P<0.05; **P<0.01, Student's t-test. The data are representative of at least three independent experiments in each group. HBsAg, HBV surface antigen; HBV, hepatitis B virus; PBS, phosphate-buffered saline; PD-1, programmed cell death-1; TNF, tumor necrosis factor.