Background: Virologic breakthrough (VBT) may occur in chronic hepatitis B (CHB) patients after switching from nucleos(t)ide analogues (NAs) to pegylated interferon alpha (Peg-IFN-alpha). This study aimed to characterize the clinical and immunological features of VBT.& nbsp;Methods: In NAs-treated patients switching to Peg-IFN-alpha, innate and adaptive immune cell proportions were examined in peripheral blood and liver biopsy specimens. In vitro effect of IFN-alpha on the expressions of toll-like receptors 2 (TLR2) and programmed cell death ligand 1 (PDL1) on monocytes, programmed cell death 1 (PD1) on CD8(+)T cells was examined. Peripheral blood mononuclear cells (PBMCs) were treated with TLR2 agonist and/or PDL1 blockade to evaluate their effect on HBV replication.& nbsp;Results: 33 of 166 patients switching to Peg-IFN-alpha experienced VBT after NA cessation, with majority being hepatitis B e antigen (HBeAg) positive or having higher hepatitis B core-related antigen (HBcrAg) levels. Patients with VBT exhibited lower proportions of TLR2(+)monocyte and increased PD1(+)HBV-specific CD8(+)T cell during the early phase of Peg-IFN-alpha therapy after NA cessation in peripheral blood, as well as fewer TLR2(+)CD68(+)macrophages but more PDL1(+)CD68(+)macrophages and PD1(+)CD8(+)T cells in liver tissues. Simultaneous use of TLR2 agonist and PDL1 blockage ex vivo suppressed HBV replication by promoting cytokines production and CD8(+)T cells cytotoxicity. Upon in vitro IFN-alpha stimulation, PDL1(+)monocytes and PD1(+)CD8(+)T cells were upregulated, whereas TLR2(+)monocytes were not increased in PBMC isolated from HBeAg-positive patients, or those with high HBcrAg titers.& nbsp;Conclusions: In NAs-treated patients, lower TLR2(+)monocyte and increased PD1(+)HBV-specific CD8(+)T cell proportions potentially contribute to VBT after switching to Peg-IFN-alpha therapy. This insufficient immunity may be associated with the HBeAg status and HBcrAg levels.