As expected, cVac infection per se did not induce liver disease (Figure 9D,; red bar), nor did it suppress HBV gene expression (Figure 9E), Pacritinib suggesting that the induction of liver disease and the suppression of HBV gene expression in cVac infected HBV transgenic mice after COR93-specific CD8+ T cell adoptive transfer were mediated by the T cells. These results suggest that functional differentiation of HBV-specific CD8+ T cells in the HBV transgenic mouse liver is sufficiently restored in the context of a systemic virus infection to both cause hepatitis and inhibit viral gene expression. Activation of professional antigen presenting (pAPC) cells is believed to be essential for the induction of functional CD8+ T cell responses after virus infections, and several studies have demonstrated that ligation of CD40 induces pAPC activation, resulting in the induction of CD8+ T cell responses [40]�C[42].

To examine whether CD40 activation could induce functional differentiation of HBV-specific CD8+ T cells in this model, we adoptively transferred na?ve COR93-specfic CD8+ T cells into HBV transgenic mice and nontransgenic controls that had been injected intravenously either with 100 ��g/mouse of an agonistic anti-CD40 antibody (��CD40) [43], [44] or with saline (NaCl) 16 hours before transfer. Seven days later, the mice were sacrificed, and intrahepatic COR93-specific CD8+ T cells were analyzed for expansion, IFN�� producing ability and Granzyme B (GrB) expression. The results were correlated with the degree of liver damage and HBV gene expression monitored by serum ALT activity and Northern Blot analysis, respectively.

As shown in Figure 10A, by day 7, ��CD40 treatment increased the intrahepatic expansion of COR93-specific CD8+ T cells in HBV transgenic mice by 5 fold compared to the saline treated transgenic animals. Furthermore, by day 7, approximately 40% of intrahepatic COR93-specific CD8+ T cells in the ��CD40 treated animals produced IFN�� in response to 5 hours in vitro peptide stimulation (Figure 10B), and almost all the COR93-specific CD8+ T cells expressed GrB directly ex vivo (Figure 10C), contrasting strikingly to their counterparts in the saline treated animals. Interestingly the induction of T cell effector functions coincided with PD-1 downregulation in intrahepatic COR93-specific CD8+ T cells (Figure 10D), suggesting that activation of CD40 signaling counteracted the PD-1 mediated negative signaling.

In contrast to these observations, neither the expansion nor the functional differentiation of the COR93-specific CD8+ T cells were enhanced in ��CD40 treated nontransgenic recipients (not shown). Importantly, ��CD40 treated HBV transgenic recipients displayed higher serum ALT activity (Figure 10E) and very strong suppression of intrahepatic Batimastat HBV gene expression (Figure 10F), after adoptive transfer of na?ve COR93-specific CD8+ T cells compared to saline treated animals.