Although several reports have demonstrated that HBV control does not require massive destruction of infected hepatocytes 1835
, inhibition of HBV replication in humans has always been linked to immune-mediated events leading to liver inflammation. Thus, subjects with persistent HBV infection but without signs of liver damage have generally been considered to lack an active cellular response to HBV 3637
. We demonstrate the presence of functionally active HBV-specific CD8 cells in the large majority of chronic HBV-infected patients lacking evidence of liver damage but controlling HBV replication. Furthermore, HBV-specific CD8 cells are not only present in the circulation but are within the liver in this group of patients, demonstrating that peripheral tolerance and T cell exhaustion of HBV-specific CD8 cells do not occur in this patient population.
HCV-specific CTLs are also present in the normal liver of chimpanzees 1 yr after resolution of acute HCV infection 38
. Furthermore, our findings have parallels with a murine model of influenza virus infection in which efficient virus control is strictly dependent on the kinetics and distribution of the virus-specific CD8 response, but is not associated with pathological damage induced by the immune response 3940
. The pattern of the HBV-specific CD8 response is also similar to that seen in other human persistent virus infections, such as EBV 33
and HTLV-I 4142
, in which a powerful CTL response can play an important role in limiting virus replication without causing inflammatory disease.
Our data do not establish whether HBV-specific CD8 cells can control virus replication through the secretion of cytokines alone or whether direct lysis of infected cells is also involved. The sparsely scattered pattern of CD8+
cells within the liver parenchyma of patients with low level viral replication suggests that secreted cytokines may be playing a major role in antiviral control. The recent demonstration of the efficacy of IFN-γ in activating a pathway of intracellular virus inactivation in the hepatocytes reinforces this interpretation 3543
. However, some degree of direct hepatocyte lysis caused by HBV-specific CD8 cells may exist, which is not detectable by serum liver enzyme measurement.
Patients with evidence of liver inflammation and a high level of HBV replication show a different pattern of distribution of HBV-specific CD8 cells. The frequency of these cells is not above the background level in the circulation of the great majority of the subjects tested. However, HBV-specific CD8 cells are not completely deleted, as they are detectable in the liver compartment. Although HLA–peptide tetramers have the advantage of allowing direct and reproducible quantification of HBV-specific CD8 cells 25
, they also have potential limitations. The detection of virus-specific CD8 cells is dependent on the expression of TCRs on their cell surface. Therefore, one interpretation of the absence of tetramer-binding cells could be TCR downregulation in the presence of high levels of Ag 21
. However, the fact that we can detect HBV-specific CD8 cells in the liver, the site of maximal HBV replication, implies that TCR downregulation does not completely abrogate their detection, and that the absence of these cells in the periphery is due to a genuine compartmentalization.
Another limitation of the tetramer technology is that it can only be applied to the study of defined epitopes, and cannot quantify the entire spectrum of CD8+
cells specific for HBV. We therefore cannot rule out the possibility that HBV-specific CD8 responses in chronic patients could be directed against epitopes other than those covered by the HBV tetramers used in this study. However, these tetramers have been synthesized because they cover the most frequent CTL epitopes found in acutely and chronically infected patients after screening with multiple peptides 6789
. Furthermore, CD8 cells specific for core 18–27 appear to be numerically dominant in the immunoprotective response associated with the control of acute infection 25
In the context of this dominant CD8 response, we found that frequencies of intrahepatic Tc 18–27+
CD8 cells are lower in the majority of patients with high virus load than in patients controlling the virus. However, the total number of intrahepatic Tc 18–27+
CD8 cells is likely to be of the same magnitude in the two groups, because the higher CD8 infiltration in the group of patients with liver inflammation could compensate for the lower frequency of tetramer-specific CD8 cells. If the quantity of core 18–27-specific cells is not the variable determining the liver pathology, hepatocyte damage may not be primarily due to lysis by HLA class I–restricted HBV-specific CD8 cells, but might be the consequence of the large infiltrate of T cells. It is tempting to speculate that this infiltration may be largely nonvirus specific 44
. Such recruitment of nonantigen-specific CD8 cells mediated by IFN-γ has been demonstrated in a transgenic mouse model of fulminant hepatitis 45
and in the setting of poor viral control in a mouse model of influenza infection 39
Support for the concept that the CD8 liver infiltrate may have a large nonvirus-specific component comes from several studies. In chronic hepatitis C, the frequency of liver-infiltrating HCV-specific CD8 cells is very low 46
, suggesting that the bulk of intrahepatic CD8 cells are nonantigen specific. In a transgenic mouse model of fulminant hepatitis, liver damage is only seen when infiltration of nonantigen-specific CD8 cells follows the Ag-specific component 45
. Furthermore, in recent results from chimpanzees infected with HBV, liver damage occurs concomitant with massive infiltration of CD8 cells 18
. This sequestration occurs after clearance of most of the HBV, and is therefore unlikely to be composed primarily of HBV-specific CD8 cells.
The data also show that viral replication does not depend on the quantity of Tc 18–27+
CD8 cells. We found that completely different levels of HBV replication can coexist with slightly different numbers in the circulation and with comparable numbers of intrahepatic HBV-specific CD8 cells. Escape of CTL recognition due to mutations within the epitope is not the explanation of this finding. The presence of similar numbers of virus-specific CD8 cells despite large differences in viral load seems counterintuitive if CTLs have an important role in viral control. However, this conundrum has also been observed in HTLV-1 4147
and fits a mathematical model where steady-state CTL numbers do not correlate with virus load 30
. This model suggests instead that the primary factor controlling viral load is CTL responsiveness, which denotes the rate at which virus-specific CD8 cells expand and exert antiviral activity. Since efficient CTL responsiveness will result in a lowering of the viral load, virus-specific CD8 numbers would fall with the lower antigenic stimulus. Thus, at equilibrium, there may be little discernible difference in actual CTL abundance between patients with high and low levels of HBV replication.
Our data from the two groups of HBV patients are consistent with this model in that the most striking difference between them is their CTL responsiveness rather than actual numbers at equilibrium. Patients controlling the virus demonstrate circulating HBV-specific CD8 cells expressing the phenotype of Ag-experienced resting cells. These cells exhibit efficient proliferation after reencounter with the viral Ag, and can exert antiviral effector functions after such expansion, demonstrating that patients without liver inflammation and with low viral load are characterized by the potential to rapidly mount strong CD8 effector mechanisms. This reservoir of circulating HBV-specific CD8 cells able to expand after recognition of the specific virus sequence is not detectable in patients unable to control the virus. This prevents phenotypic and functional comparison of equivalent populations of HBV-specific CD8 between the two groups, which might reveal possible mechanisms contributing to chronicity. Whether these different outcomes of chronic infection result from differences in the efficiency, kinetics 48
, and distribution 40
of the antiviral CD8 response, differences in CD4 T cell help 49
, or differences in the size or fitness of the initial virus inoculum 2139
remains to be determined. However, recent studies (Boni, C., manuscript in preparation) in HBeAg+
chronic patients undergoing lamivudine treatment have shown that a reduction in viral load allows repopulation with functionally active HBV-specific CD8 cells. This reconstitution of a circulating reservoir of HBV-specific CTLs, combined with the absence of hepatic inflammation mediated by these cells when viral load is low, supports an immunotherapeutic approach designed to boost HBV-specific CD8 responses.
In conclusion, measurement of circulating and intrahepatic HBV-specific CD8 cells in patients with differing viral load and liver pathology has provided new insights into the pathogenesis of HBV infection. Our data show an active HBV-specific CD8 response in patients controlling HBV replication. The presence of liver-infiltrating HBV-specific CD8 cells in the absence of liver inflammation suggests that control of HBV replication and liver damage may be independent events in these patients. Since comparable quantities of core 18–27-specific CD8 cells are demonstrable in the liver with a variable extent of damage, it is plausible that hepatocyte lysis might be the consequence of the dense infiltrate of nonantigen-specific T cells.