In this report, we demonstrate that intrahepatic expression of HCV core impairs βgal-specific CD8+ T cells, resulting in failed clearance of Ad-LacZ infection. The functional analysis of βgal-specific CD8+ T cells revealed that the expression of HCV core by hepatocytes alters the magnitude of the βgal-specific CD8+ T cell response and suppresses both CD8+ T cell effector cytokine production (i.e., IFN-γ and TNF-α) and cytolytic potential. In addition, high levels of PD-1 expression by intrahepatic βgal-specific CD8+ T cells and B7-H1 expression by liver DCs were sustained in core(+) mice as compared with that in core(−) mice. Suppression of CD8+ T cell responses was limited to the liver compartment where core is expressed and was not observed in the spleen. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway rescued exhausted CD8+ T cells in core(+) mice and led to successful clearance of Ad-LacZ infection.
Chronic HCV infection in humans is characterized by CD8+
T cell exhaustion and dysfunction (37
). Recently, it has been reported that increased expression of PD-1 is associated with the impaired HCV-specific CD8+
T lymphocytes observed in chronic HCV patients (11
). However, the underlying mechanism(s) for HCV-mediated impaired CD8+
T cell responses has yet to be determined. Based on our finding of impaired intrahepatic CD8+
T cell responses in core(+) mice, it is possible that HCV core-induced T cell dysfunction may contribute one of the viral factors that causes impaired CD8+
T cell responses as seen in chronic HCV patients. From our study we found that splenic CD8+
T cell responses are not altered in core(+) mice. One possible explanation is that HCV core-expressing hepatocytes may not inhibit the CD8+
T cell response at the induction phase. Instead, the inhibition may rather occur at the point where CD8+
T cells enter the livers of core(+) mice and either undergo apoptosis or become defective in further proliferation/effector activity. That is, it is likely that HCV core-expressing hepatocytes alter the liver inflammatory conditions, thereby inhibiting CD8+
T cell responses either directly or indirectly by DC dysregulation. Furthermore, we have observed increased caspase-3 staining by intrahepatic CD8+
T cells in core(+) mice during Ad-LacZ infection, indicating enhanced apoptosis of T cells as a potential mechanism for the observed dysregulation of T cell responses in the livers of core(+) mice (data not shown).
Based on our findings that impaired CD8+
T cell responses correlate with sustained PD-1 expression in core(+) mice, it is likely that the PD-1/B7-H1 pathway plays a major inhibitory role in our HCV core transgenic model. The role of PD-1/B7-H1 as a mechanism for liver tolerance has been well established. Indeed, PD-1 expression by T cells has been shown to inhibit intrahepatic antiviral immune responses at the effector phase (20
). The initial induction of PD-1 by CD8+
T cells in both core(+) and core(−) mice at the early stage of viral infection provides further evidence that PD-1 plays an important role in controlling intrahepatic T cell responses. However, at later time points the expression of PD-1 by β
gal-specific intrahepatic CD8+
T cells remains significantly elevated in core(+) mice whereas it decreases appreciably in core(−) mice. Our studies suggest that enhanced expression of PD-1 and B7-H1 in core(+) mice following Ad-LacZ infection modulates both antiviral immunity and effector CD8+
T cell deletion. The loss of PD-1 expression by CD8+
T cells in core(−) mice at later time points during Ad-LacZ infection is likely due to successful viral control, leading to clearance of Ag from the liver. The sustained PD-1 expression in core(+) mice is due to higher levels of B7-H1 expression by liver DCs in core(+) mice and an inability to rapidly clear viral Ag. More so, we believe that enhanced expression of PD-1 on β
T cells is due to a partially exhausted phenotype exhibited by the T cells in an environment characterized by Ag persistence.
Currently, it remains unclear how intrahepatic expression of HCV core suppresses CD8+ T cell responses during Ad-LacZ infection. It is possible that HCV core is secreted from hepatocytes in core(+) mice and that extracellular core protein exerts an immunomodulatory function. However, we were unable to detect secreted HCV core by using ELISA on our core(+)-infected mice (data not shown). This could be due to a detection limit with our core ELISA. However, we would rather believe that the expression of HCV core alone (not as polyproteins including the E1 and E2 glycoproteins) might not allow for an appreciable secretion of HCV core in core(+) mice. Thus, it is likely that intracellular core protein plays a role in the impaired antiviral activity of CD8+ T cells as observed in core(+) mice.
Based on the importance of innate immunity linking to adaptive immunity, it is tempting to speculate on the potential mechanism of HCV core-mediated suppression of antiviral CD8+
T cell responses. First, HCV core has been shown to alter type 1 IFN responses in vitro (39
). Type 1 IFNs induce DC maturation and NK cell activation. Furthermore, the production of type 1 IFNs is believed to be critical to overcome baseline tolerance in the liver and thus HCV core-mediated alteration of type 1 IFNs in our murine model may explain the observed dysregulation of CD8+
T cell responses. Second, HCV core expression by hepatocytes has been shown to impair NK-mediated activation of DC cells in vitro (42
). Indeed, a human cell line expressing HCV core has been reported to up-regulate HLA-E, a ligand for NK inhibitory receptors. Recently, the immunoregulatory role for NK cells has emerged such that NK cells can influence adaptive immune responses by their ability to secrete immunomodulatory cytokines and chemokines. It is possible that altered expression of NK inhibitory ligands in core(+) mice impairs the induction of effector immune responses. Third, IL-10 has been shown to be a key mediator in the suppression of T cell responses during persistent viral infection (44
). Interestingly, anti-IL-10R treatment during chronic lymphocytic choriomeningitis virus infection was shown to enhance antiviral T cell responses and reduce expression of PD-1 by T cells (46
). It is possible that HCV core-expressing hepatocytes alter DC activation and function and lead to enhanced IL-10 production. As a result of DC dysfunction, core(+) mice might not generate effective CD8+
T cell responses.
Nevertheless, our studies demonstrate that in vivo treatment with anti-B7-H1 blocking Ab can restore effective antiviral intra-hepatic T cell responses in core(+) mice. Importantly, blockade of this pathway enhances viral clearance by releasing HCV core-mediated immune suppression. At this point, it is not clear if the increased numbers of virus-specific T cells observed in the B7-H1 blockade group is due to decreased deletion of activated T cells in the liver. We have shown thus far that the proliferation marker Ki67 is expressed at higher levels in core(+) mice that received anti-B7-H1 blocking Ab. Importantly, we have also observed a decreased TUNEL-positive signal in the livers of core(+) mice treated with B7-H1 blockade (J. R. Lukens, unpublished observations). These results suggest that the observed increase in the frequency of βgal-specific T cells in the anti-B7-H1 treatment group is due to both decreased deletion of CD8+ T cells and enhanced proliferation of virus-specific CD8+ T cells. Interestingly, blockade of the PD-1 pathway might affect the ability of activated CD8+ T cells to infiltrate into the lobular region where virus-infected hepatocytes reside. Furthermore, anti-B7-H1 blockade treatment may be a potential therapeutic treatment strategy to treat chronic HCV patients. The HCV core transgenic model described in this report will be useful for testing the efficacy of therapeutic agents to restore antiviral CD8+ T cell activity as well as their effector function on viral clearance.
In summary, we report that the expression of HCV core in the liver compartment of mice impairs adenovirus clearance and CD8+ T cell responses. The suppressed CD8+ effector T cell responses observed in core(+) mice coincides with an elevated expression of PD-1 by virus-specific CD8+ T cells and B7-H1 by liver DCs. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway rescues viral clearance, CD8+ T cell effector cytokine production, and granzyme B expression in core(+) mice. These findings implicate a potential therapeutic strategy to manipulate the PD-1/B7-H1-negative signaling pathway to restore effector T cell responses in chronic HCV patients.