Interestingly, virtually all the intrahepatic COR93-specific CD8+ T cells in HBV transgenic mice strongly expressed the co-inhibitory molecule compound library PD-1 on day 1.5 ex vivo and remained so until day 28 (Figure 2E), while PD-1 expression was virtually absent in their counterparts in cVac infected nontransgenic animals (Figure 2J), suggesting that PD-1 signaling may have contributed to the functional impairment of intrahepatic COR93-specific CD8+ T cell responses in HBV transgenic mice. To determine if the dysfunctional T cell responses in the HBV transgenic liver reflect active suppression of functional differentiation by PD-1 signaling, the COR93-specific TCR transgene was crossed for two generations onto a MHC class I matched PD-1 deficient background (kindly provided by Dr.

Arlene Sharpe, Harvard Medical School) [31], yielding PD-1 deficient COR93-specific TCR transgenic animals. Equal numbers of PD-1 deficient and wild type COR93-specific na?ve CD8+ T cells were adoptively transferred into HBV-transgenic mice, and analyzed for expansion, IFN�� producing ability and Granzyme B (GrB) expression on day 7 after adoptive transfer. The results were correlated with the degree of liver damage and HBV gene expression monitored by serum alanine aminotransferase (ALT) activity and HBV gene Northern Blot (NB) analysis, respectively. As shown in Figure 3A, PD-1 deficient COR93-specific CD8+ T cells expanded much more vigorously in the liver than wild type COR93-specific CD8+ T cells, and a larger fraction of PD-1 deficient T cells expressed IFN�� and Granzyme B (Figure 3B and C) and they induced a more severe liver disease, monitored as serum alanine aminotransferase (ALT) activity (Figure 3D).

Furthermore, HBV gene expression was significantly reduced in the recipients of PD-1 deficient COR93-specific CD8+ T cells but not in the wild type T cell recipients (Figure 3E), reflecting the superior cytolytic and interferon gamma-producing activity of the PD-1 deficient cells. Collectively, these results indicate that PD-1 signaling suppresses the expansion and functional differentiation of HBV-specific CD8+ T cells after antigen recognition in the liver. Figure 3 PD-1 deficient COR93-specific CD8+ T cells develop cytolytic ability in the liver of HBV transgenic mice.

Intrahepatic priming of HBV-Specific na?ve T cells after adoptive transfer into HBV transgenic mice Because the hepatocytes in HBV transgenic mice replicate HBV at high level and release viral particles and subviral antigens into the circulation [19], HBV derived antigen could be presented to na?ve T cells either by the hepatocytes themselves or by professional antigen Drug_discovery presenting cells (pAPCs) that acquire virus particles and/or subviral antigens in the liver or in peripheral lymphoid organs.