We next explored whether dual-vector therapy could restore CTL responses by detecting the percentages and activation status of liver-resident NK, CD4+ T, and CD8+ T cells in HBV+ mice. While no significant changes were detected in hepatic CD4+ T, NK and NKT cells (Supporting Fig. 6), the percentage and absolute number of hepatic CD8+ T cells and activating CD8+ T cells were markedly up-regulated by dual-vector administration (Fig. 5A,B). Furthermore, dual-vector treatment markedly augmented CTL function (e.g., CD107a+ and IFN-γ+ CD8+ T-cell percentages increased) in liver compared to ssRNA vector Ibrutinib nmr (Fig.

5C), along with down-regulation of PD-1 and up-regulation of CD28 (Fig. 5D), suggesting that dual therapy reverses CD8+ T-cell anergy by regulating the PD-1/CD28 axis. Moreover, the percentages of HBV-specific hepatic CD8+ T cells as well as HBV-specific CD107a+ and IFN-γ+ CD8+ T cells were also significantly enhanced by dual-vector treatment (Fig. 5E). In addition, we also observed the T-cell responses in spleen and peripheral blood and found similar results in line with the microenvironment. As shown in Supporting Fig. 7, the percentages and absolute numbers of CD8+ T cells

as well as activated CD8+ T cells in both spleen and peripheral blood increased after dual vector therapy, suggesting Small molecule library ic50 that the restoring of CD8+ T-cell response exerted by dual vector is systemic. To further explore the role of CD8+ T cells, we depleted them from HBV+ mice with an anti-CD8β mAb. CD8+ T cells, but not CD4+ T, are critical for dual-vector-mediated inhibition of HBV replication, as HBV DNA copies, HBx expression, and HBsAg levels

in serum became significantly higher, while serum IFN-γ levels declined, 上海皓元 after CD8+ T-cell depletion (Fig. 6A,B). NK cell-depletion also partly attenuated dual-vector-mediated inhibition of HBV, suggesting that NK cells might also participate in dual-vector-mediated HBV inhibition. But the role of CD8+ T cells was the most prominent. To determine the role of CD8+ T cells, we adoptively transferred splenic CD8+ T cells or CD8+ T-cell-depleted splenic lymphocytes from wild-type (WT) mice into HBV+ Rag-1−/− mice. Adoptively transferring CD8+ T cells but not non-CD8+ T cells or IFN-γ−/− CD8+ T-cells (e.g., CD8+ T cells from IFN-γ−/− mice) inhibited HBV replication (serum HBsAg) in HBV-persistent Rag-1−/− mice when dual vector treatment was used (Fig. 6C). Although dual vector promoted CD8+ T cell activation, HBsAg inhibition was markedly impaired in HBV-persistent IFN-γ−/− mice (the inhibitory rate is 85.16 ± 4.75% and 55.24 ± 10.43% in WT HBV+ and IFN-γ−/− HBV+ mice, respectively) (Fig. 6D). These results suggest that recovering anti-HBV adaptive immunity by reversing hepatocyte-intrinsic tolerance is at least partially dependent on functional rescue by IFN-γ-producing CD8+ T cells.