Immune effectors required for hepatitis B virus clearance

Abstract

To better define the mechanism(s) likely responsible for viral clearance during hepatitis B virus (HBV) infection, viral clearance was studied in a panel of immunodeficient mouse strains that were hydrodynamically transfected with a plasmid containing a replication-competent copy of the HBV genome. Neither B cells nor perforin were required to clear the viral DNA transcriptional template from the liver. In contrast, the template persisted for at least 60 days at high levels in NOD/Scid mice and at lower levels in the absence of CD4⁺ and CD8⁺ T cells, NK cells, Fas, IFN-gamma (IFN-γ), IFN-alpha/beta receptor (IFN-α/βR1), and TNF receptor 1 (TNFR1), indicating that each of these effectors was required to eliminate the transcriptional template from the liver. Interestingly, viral replication was ultimately terminated in all lineages except the NOD/Scid mice, suggesting the existence of redundant pathways that inhibit HBV replication. Finally, induction of a CD8⁺ T cell response in these animals depended on the presence of CD4⁺ T cells. These results are consistent with a model in which CD4⁺ T cells serve as master regulators of the adaptive immune response to HBV; CD8⁺ T cells are the key cellular effectors mediating HBV clearance from the liver, apparently by a Fas-dependent, perforin-independent process in which NK cells, IFN-γ, TNFR1, and IFN-α/βR play supporting roles. These results provide insight into the complexity of the systems involved in HBV clearance, and they suggest unique directions for analysis of the mechanism(s) responsible for HBV persistence.

Hepatitis B virus (HBV) is a noncytopathic human hepadnavirus that causes acute and chronic hepatitis and hepatocellular carcinoma (1). Approximately 350 million people worldwide are chronically infected by HBV, which greatly increases the risk of hepatocellular carcinoma (HCC) and causes more than 1 million deaths annually (2). Because HBV is not infectious in small animal or tissue culture models, systematic examination of the host–virus interactions during HBV infection has been difficult. The full spectrum of immunological requirements for HBV clearance is not completely defined.

Our current understanding is based to a large extent on comparison of the immune responses mounted against HBV in patients who clear and who fail to clear HBV (3), experiments in which potential effectors of clearance (e.g., HBsAg-specific CD8⁺ T cells, recombinant IFN-γ and TNF-α) have been adoptively transferred to or induced in HBV transgenic mice (4–8), and a limited number of experiments conducted in HBV-infected chimpanzees (9, 10, 18). Collectively, these studies have led to the current model for clearance of acute HBV infection, namely (i) that viral clearance during HBV infection is associated with entry of CD8⁺ T cells into the liver, the production of IFN-γ, and the induction of inflammatory liver disease (reviewed in ref. 3); (ii) that IFN-γ production, viral clearance, and liver disease are all impaired in the absence of CD8⁺ T cells (11); and (iii) that noncytolytic inhibition of HBV replication occurs during HBV infection (9) and is associated with IFN-γ expression in the liver (9, 10). Because viral clearance is itself a multifaceted process, dissection of the effectors responsible for control of viral replication and gene expression versus those required for elimination of the replicative template has been especially difficult because, until now, it has not been possible to take a reductive approach to identify the specific cellular and molecular effectors that are necessary for clearance of HBV.

We previously developed a model in which hydrodynamic injection of a naked plasmid DNA encoding a supergenomic HBV1.3-length transgene into inbred mice initiates high-titer HBV replication in the liver that is rapidly terminated if the mice are immunocompetent but persists indefinitely if they are globally immunodeficient (i.e., NOD/Scid) (12). Because of the power of mouse genetics, this model affords the unique opportunity to identify and characterize the immunological events that are required for HBV clearance. In the current study, we used this model to examine the clearance of intrahepatic HBV from hydrodynamically transfected mice that are genetically deficient in a variety of cellular (CD8⁺ T, CD4⁺ T, NK, and B cell) and molecular (IFN-γ, IFN-α/β receptor-1, TNF-α receptor-1, perforin, Fas) effectors that are assumed to be important for the clearance of HBV and other intracellular pathogens.

Results and Discussion

Transfection Efficiency in Wild-Type and Knockout Mice.

To monitor transfection efficiency, HBV surface antigen (HBsAg) was measured in the blood of hydrodynamically transfected mice by quantitative ELISA (Fig. 1). Peak HBs antigenemia was achieved in all strains on day 4 after transfection, ranging between 1.1 and 6.1 μg/mL, with a mean of 3.3 ± 3.7 μg/mL for all mice (n = 297). The small differences in HBs antigenemia were insignificant given the abundance of HBV surface antigen (HBsAg) (>1 μg/mL) in all strains. An independent measure of transfection efficiency was obtained by quantifying the percentage of hepatocytes that expressed the HBV core antigen (HBcAg), which also peaked on day 4 after transfection. This number was similar in all strains, with a mean for all mice of 4.7 ± 2.7% (n = 47; Fig. 1). The comparable transfection efficiencies observed in this panel support the notion that the strains used in this study did not differ significantly in their intrinsic capacity to support HBV gene expression and replication.

Fig. 1.

Viral gene expression in wild-type and knockout mice. Serum HBsAg levels (open bars) were quantified by enzyme-linked immunosorbant assay (ELISA). Data represent averages (n ≥ 3). The average number of HBcAg⁺ hepatocytes in the liver (filled bars) was quantified by counting at least 100 grids per liver (×20 magnification) for at least three animals for each strain. All strains examined had comparable levels of serum HBsAg and HBcAg⁺ hepatocytes on day 4.

Effectors Required for Elimination of HBV from the Liver.

To identify immune effectors that eliminate HBV from the liver in this model, we monitored the abundance of the input plasmid (Fig. 2A) and HBV replicative DNA intermediates (Fig. 2B) in the liver as a function of time by real-time PCR. Separate real-time PCR assays were used to quantitate HBV and input plasmid DNA, respectively. The ratio of HBV DNA to input plasmid DNA was calculated for each sample, and a ratio of 2 or more was taken as evidence of viral replication. In addition, the frequency of HBcAg-positive hepatocytes as well as the cellular distribution of HBcAg (i.e., nuclear and cytoplasmic HBcAg) was monitored by immunohistochemical staining of liver sections isolated from individual mice of each strain (Figs. 2C and 3). Cytoplasmic HBcAg (cHBcAg) is an indicator of HBV replication (13–15), whereas nuclear HBcAg (nHBcAg) is a highly stable species that persists in the absence of HBV replication until cells are either killed or stimulated to divide (16, 17).

Fig. 2.

Control of HBV replication and clearance of the transcriptional template in wild-type and knockout mice. (A) Clearance of the transcriptional template was monitored by real-time PCR assay using probes that amplify a sequence on the backbone of the input plasmid. (B) Replication of the viral genome was monitored by real-time PCR assays using two primer sets specific for HBV and the input plasmid. Ratios of HBV:input plasmid greater than 2 indicated the presence of replicative DNA intermediates. (C) HBcAg-positive hepatocytes were quantified by counting at least 100 grids per liver (×20 magnification) for at least three animals for each strain. *, data were not collected at this time point.

Fig. 3.

Frequency and localization of HBcAg in wild-type and knockout mice. Representative sections stained for the presence of HBcAg are presented. Images were collected at ×40 magnification. (A) C57BL/6, day 4. All strains exhibited similar frequency and distribution of HBcAg on day 4. (B) CD8 knockout, day 20. CD4-, TNFR1-, and Fas-deficient animals exhibited similar frequency and distribution of HBcAg at this time point. (C) Fas knockout, day 60. CD4-, TNFR1-, and Fas-deficient animals exhibited similar frequency and distribution of HBcAg at this time point. (D) NOD/Scid, day 60. C57BL/6 mice were representative of the frequency and distribution of HBcAg staining observed in all strains examined on day 4. By day 20, C57BL/6 mice and mice deficient in B cells, perforin, or IFN-α/βR had no detectable HBcAg. In contrast, NOD/Scid animals and animals deficient in CD8, CD4, NK cells, IFN-γ, TNFR1, or Fas exhibited HBcAg in both the nucleus and cytoplasm of hepatocytes on day 20. IFN-γ- and NK cell-deficient animals cleared HBcAg by day 60 whereas animals deficient in CD8, CD4, Fas, or TNFR1 exhibited nuclear HBcAg at comparable frequencies between days 30 and 60. NOD/Scid animals exhibited hepatocytes with both nuclear and cytoplasmic HBcAg at stable levels between days 30 and 60.

As shown in Fig. 2A, input plasmid disappeared from wild-type and B cell-deficient animals between 10 and 20 days after transfection and between days 20 and 30 in perforin-deficient animals, suggesting that B cells and, surprisingly, perforin are not essential for clearance of the HBV transcriptional template from the liver in this model. Interestingly, input plasmid persisted through day 60 in all of the remaining strains (IFN-α/βR^−/−, IFN-γ^−/−, CD4⁺ T cell^−/−, CD8⁺ T cell^−/−, TNFR1^−/−, Fas^−/−, and NOD/Scid), suggesting that each of these effectors is required to clear the template DNA from the liver. Clearance of HBcAg-positive hepatocytes from the liver (Fig. 2C) in the different mouse strains occurred with kinetics that closely paralleled the clearance of input plasmid DNA (Fig. 2A), except IFN-α/βR^−/−, NK cell^−/−, and IFN-γ^−/− strains in which HBcAg-positive hepatocytes were cleared by day 40 despite the presence of input plasmid DNA. This small difference presumably reflects differential sensitivities between the two assays although we cannot exclude the possibility that input plasmid DNA persisted in the absence of HBcAg expression in these strains.

As shown in Fig. 2B, wild-type mice and mice deficient in B cells, NK cells, perforin, and IFN-α/βR controlled HBV replication with similar kinetics, i.e., between days 10 and 20 after transfection even if they had not eliminated the input HBV DNA plasmid (Fig. 2A) suggesting that noncytolytic mechanisms that suppress HBV replication are operative in this model and that B cells, NK cells, perforin, and IFN-α/βR do not contribute to that process. In contrast, HBV replication was detected for more than 30 days in the IFN-γ^−/−, CD4^−/−, CD8^−/−, TNFR1^−/−, Fas-deficient, and NOD/Scid strains (Fig. 2B), suggesting that each of these effectors is required to suppress HBV replication in this model. Multiple effectors are apparently absent in the NOD/Scid strain, as evidenced by the persistence of both input plasmid and HBV-replicative DNA in these animals at much higher levels than in mice lacking the individual effectors. Consistent with these data, nucleo-cytoplasmic HBcAg-positive hepatocytes were detectable in the IFN-γ^−/−, CD4^−/−, CD8^−/−, TNFR1^−/−, Fas^−/−, and NOD/Scid strains up to day 20 (Figs. 2C and 3B), after which HBcAg was strictly nuclear in all strains except NOD/Scid which continued to display nucleo-cytoplasmic HBcAg on day 60 (Fig. 3 C and D). Collectively, these results suggest that multiple effectors are absent in NOD/Scid mice, each of which is required to eliminate the transcriptional template and suppress HBV replication.

Association of the CD8⁺ T Cell Response with Clearance of HBcAg and the HBV Transcriptional Template.

Previous studies of HBV clearance in infected patients (3) and chimpanzees (9, 10, 11, 18) have demonstrated that CD4⁺ T cells and CD8⁺ T cells are required for elimination of HBV from the liver during natural infection. An essential role for Fas and TNFR1 in the clearance of HBV is less obvious. In our experiments, the failure of the CD4-deficient mice to clear intrahepatic HBV could reflect a direct or an indirect role for CD4⁺ T cells in the destruction of HBV⁺ hepatoctyes. Similarly, molecular effectors such as Fas and IFN-γ may act directly against HBV or indirectly through their modulation of the CD8⁺ T cell response. To investigate this possibility, we examined the CD8⁺ T cell response in wild-type C57BL/6 animals as well as animals that were deficient in CD4⁺ T cells, Fas, and IFN-γ.

The number of intrahepatic HBcAg-specific CD8⁺ T cells in transfected mice was measured directly by ex vivo staining of intrahepatic lymphocytes with a soluble H-2K^b:Ig dimer complexed with a peptide corresponding to the MHC class I H-2b-restricted, HBV core-derived immunodominant CTL epitope (HBcAg93) (19). On day 14 after transfection, HBcAg93-specific CD8⁺ T cells were abundant in the liver and comprised >30% of the intrahepatic CD8⁺ T cells in C57BL/6 mice at this time point. To compare interstrain differences, the percentage of intrahepatic HBcAg93-specific CD8⁺ T cells was normalized to the percentage observed in wild-type C57BL/6 animals (Fig. 4). HBcAg93-specific CD8⁺ T cells were undetectable in the intrahepatic lymphocyte populations of the CD4-deficient animals on day 14 after transfection (Fig. 4). The absence of detectable HBcAg93-specific CD8⁺ T cells did not appear to be an artifact of the hydrodynamic transfection method because we were also unable to detect HBcAg93-specific CD8⁺ T cells in the spleen or liver of CD4-deficient mice subjected to an intramuscular DNA prime, intravenous HBV core vaccinia boost regimen that was shown to induce strong HBcAg93-specific CD8⁺ T cells responses (20). These results suggest that CD4⁺ T cell help is required to mount a CD8⁺ T cell response to HBV and suggest that CD8⁺ T cells are the primary cellular effector responsible for HBV clearance in this model.

Fig. 4.

The HBV HBcAg93-specific intrahepatic CD8⁺ T cell response. Intrahepatic lymphocytes were isolated from mice on day 14 after transfection and then stained ex vivo with FITC-anti-CD8 and the HBV HBcAg93-dimer followed by a PE-anti-mouse Ig secondary antibody. The percentage of intrahepatic CD8⁺ T cells specific for HBcAg93 was measured and averaged (n ≥ 3). Values plotted were expressed as a percentage of all CD8⁺ T cells normalized against C57BL/6.

To determine whether additional effectors mediate HBV clearance via an effect on the CD8⁺ T cell response, we also analyzed the frequency of HBcAg93-specific intrahepatic CD8⁺ T cells in additional knockout strains. For example, IFN-γ is known to stimulate development of CD8⁺ T cells (21, 22), and the absence of IFN-γ was associated with delayed elimination of the input plasmid (Fig. 2A), control of HBV replication (Fig. 2B), and clearance of HBcAg (Fig. 2C). Interestingly, IFN-γ-deficient animals produced nearly wild-type levels of HBcAg93-specific CD8⁺ T cells (Fig. 4), suggesting that the impact of IFN-γ deficiency on HBV clearance was not due to a defective CD8⁺ T cell response but rather, perhaps, due to defects in the effector function(s) of the CD8⁺ T cells induced.

Besides mediating the cytolytic activity of CD8⁺ T cells, Fas also regulates T cell homeostasis (23). Because Fas-deficient mice failed to clear the input transcriptional template from the liver in this model, we asked whether they were able to mount a CD8⁺ T cell response to HBV. Importantly, the frequency of HBcAg93-specific CD8⁺ T cells in the Fas knockout mice was comparable to that observed in wild-type C57BL/6 animals (Fig. 4), and, when determined by intracellular cytokine staining (20), the fraction of IFN-γ-positive CD8⁺ T cells in Fas knockout mice (73.8 ± 13.2%) and wild-type C57BL/6 animals (70.2 ± 5.6%) was also comparable, indicating that Fas is not required for induction of the CD8⁺ T cell response to HBV. On the other hand, the data shown in Fig. 2A suggest that the death pathway induced by Fas/FasL interactions is absolutely required for control of HBV in the hydrodynamic transfection model.

Conclusion.

This examination of HBV clearance in a panel of immunodeficient mouse strains was undertaken to characterize the mechanism(s) of viral clearance from the liver in an in vivo model of HBV replication. Hepatic clearance of the input plasmid that, like the viral closed circular DNA (cccDNA) (24, 25), serves as the viral transcriptional template was found to require many effectors, including CD8⁺ T cells, CD4⁺ T cells, NK cells, Fas, TNFR1, IFN-α/βR, and IFN-γ. The fact that the transcriptional template persists despite the absence of all these effectors suggests that they impact the same pathway, presumably the induction and/or the effector functions of the HBV-specific CD8⁺ T cell response (see below). Our finding that HBcAg93-specific CD8⁺ T cells do not develop in CD4-deficient animals illustrates this point. Indeed, the results are consistent with a model in which CD8⁺ T cells are the key cellular effectors mediating HBV clearance from the liver, and in which CD4⁺ T cells, NK cells, Fas, IFN-γ, IFN-α/βR, and TNFR1 contribute to the induction of the antiviral CD8⁺ T cell response and elimination of HBV-positive hepatocytes. Surprisingly, although clearance of this plasmid, like clearance of cccDNA, is likely to be a cytodestructive process, it did not require perforin, the major cytolytic mediator in CD8⁺ T cells (26). Because the input plasmid persisted for the duration of the study in Fas- and TNFR1-deficient animals, the data suggest that, unexpectedly, one or both of those pathways may play a role in elimination of the natural cccDNA transcriptional template during natural HBV infection in man.

The observation that clearance of the HBV transcriptional template from the liver required Fas was not due to defective induction of the CD8⁺ T cell response to HBV in the Fas knockout mice, in contrast to the CD4-deficient mice, because HBcAg-specific CD8⁺ T cells were induced at comparable freqencies in Fas-deficient and wild-type C57BL/6 animals. This finding may suggest that Fas mediates the cytolytic effector function(s) of the HBV-specific CD8⁺ T cells in this model and that perforin does not. Previous experiments suggested that both FasL and perforin must be delivered simultaneously by cytotoxic T lymphocytes (CTLs) to kill HBsAg-positive hepatocytes in vivo (5). Notably, however, those experiments involved the adoptive transfer of in vitro-passaged, terminally differentiated CTL clones, which may differ significantly from the primary T cell response induced in this in vivo HBV transfection model. Viral clearance in this model may use mechanism(s) more akin to the clearance of replication-deficient adenovirus in vivo, which requires Fas- and TNFR1-mediated pathways of cytolysis but is independent of perforin- and granule protease-dependent cytotoxicity mechanisms (27).

Clearance of HBV replicative intermediates also required multiple effectors, but fewer than were required for clearance of the transcriptional template. Whereas the replicative intermediates disappeared, as expected, coincident with loss of the viral transcriptional template in several of the strains, their rapid disappearance in the face of persistent template in IFN-α/βR-deficient and NK cell-deficient mice implies that they were eliminated noncytolytically by other effectors. Their delayed but eventual disappearance in CD4- CD8-, TNFR1-, and Fas-deficient strains that failed to clear the transcriptional template reinforces that interpretation. The fact that HBV replication eventually stopped in all of the other lineages indicates that redundant pathways must exist to produce the antiviral signal(s) that ultimately suppress it.

Collectively, these results validate the use of the hydrodynamic transfection model to deconvolute the cellular and molecular mechanisms responsible for clearance of HBV. The results are consistent with a model in which CD4⁺ T cells serve as master regulators of the adaptive immune response to HBV; CD8⁺ T cells are the key cellular effectors mediating HBV clearance from the liver, apparently by a Fas-dependent, perforin-independent process in which NK cells, IFN-γ, TNFR1, and IFN-α/βR play supporting roles. The outcome of natural HBV infections is a product of a complex mixture of variables, including differences deriving from both host and virus as well as extrinsic factors. The HBV transfection model represents one, well-controlled experimental scenario that mimics acute HBV infection by permitting observation of HBV clearance in immunologically naïve hosts but that more closely resembles chronic HBV in the relatively low frequency of HBV-positive hepatocytes in the liver. We note that future examination of HBV clearance under conditions that mimic other aspects of acute and chronic HBV are necessary to identify effectors universally required for clearance of all HBV versus those that are conditionally required. The data presented herein provide insight into the complexity of the systems involved in HBV clearance, and they suggest unique directions for further analysis of the mechanism(s) responsible for HBV clearance and persistence.

Materials and Methods

Animal Studies.

The following mouse strains were used in this study. C57BL/6 mice and B10.D2 mice were obtained from the rodent breeding colony of The Scripps Research Institute, which is derived from breeders obtained from the Jackson Laboratory (Bar Harbor, ME). CB17-NOD/LtSzPrkdc^scid/J (NOD/Scid) (28), C57BL/6J-Lyst^bg- (NK^−/−) (29), and B6.129-Fas^tmlOsa (Fas^−/−) (37) mice were obtained directly from the Jackson Laboratory. Breeding pairs of strains B6.129S2-CD8a^tm1Mak (CD8^−/−) (31), B6.129S7-Ifng^tm1Ts (IFN-γ^−/−) (32), C57BL/6-Pfp^tmSdzs (perforin^−/−) (33), B6 × 129-CD4^tm1Mak (CD4^−/−) (34), B6.129-Igh-6^tm1Cgn (B cell^−/−) (35), and B6.129-Tnfrsf1a^tm1Mak (TNFR1^−/−) (36) were obtained from Michael Oldstone (The Scripps Research Institute). Breeding pairs of B6;129-IFNAR1^tm1Agt (IFN-α/βR^−/−) N9 mice (37) mice were donated by Jonathan Sprent (The Scripps Research Institute). Animals were treated according to the National Institutes of Health Guidelines for Animal Care and the Guidelines of The Scripps Research Institute. Mice were used at 6–10 weeks of age, except for the B6.129-Fas^tmlOsa mice, which were 7–12 weeks of age at the time of injection.

Hydrodynamic Transfection of Mice with HBV1.3 Plasmid.

A total of 13.5 μg of pT-MCS-HBV1.3 and 4.5 μg of the Sleeping Beauty transposase expression plasmid pCMV-SB were injected into the tail vein of 6- to 10-week-old mice in a volume of saline equivalent to 8% of the body mass of the mouse (e.g., 1.6 mL for mouse of 20 g) (12, 38). The total volume was delivered within 3–8 seconds. Cohorts were defined by matching mice on the basis of serum alanine aminotransferase activity and serum HBsAg values on day 1 after injection of HBV1.3 DNA. The percentage of HBcAg-positive hepatocytes in the liver serves as a lower limit estimate of transfection efficiency in these experiments and ranged on average between 1 and 10%.

Quantitation of HBV DNA and Input DNA by Real-Time PCR.

Total genomic DNA was purified from the liver as described in ref. 39. HBV DNA and input vector copy numbers were determined by real-time PCR using 0.5 μg of total genomic liver DNA in 50-μL reactions performed in triplicate. HBV DNA was amplified by using primers HBV469U (5′-CCCGTTTGTCCTCTAATTCC-3′) and HBV569L (5′-GTCCGAAGGTTTGGTACAGC-3′); input vector was amplified by using primers pTHBV-5585U20 (5′-CCAGTCGGGAAACCTGTCGT-3′) and pTHBV-5675L20 (5′-GCAGCGAGTCAGTGAGCGAG-3′), which anneal to sequences within the Sleeping Beauty transposase IR sequences in the plasmid backbone of vector pT-MCS-HBV1.3. Reactions were performed in Sybr green reaction mix (Applied Biosystems, Foster City, CA). Cycling parameters were (i) 1 cycle: 95 °C, 5 min; (ii) 40 cycles: 95 °C, 30 seconds; 60 °C, 60 seconds; and (iii) 1 cycle: 72 °C, 10 min on a Bio-Rad iCycler iQ Real Time PCR Detection System (Bio-Rad, Hercules, CA). Plasmid pT-HBV1.3 diluted with 0.5 ng/μL human genomic DNA to 10⁷, 10⁵, 10³, and 10¹ copies per reaction was used as a standard. All samples were analyzed in triplicate. The lower limit of accurate detection in this assay was 2.5 × 10² genome equivalents per reaction, or 2.5 × 10³ copies per 10⁶ cells, assuming 5 pg of genomic DNA per hepatocyte. A >2-fold excess of HBV over input plasmid was interpreted as evidence that active replication was ongoing in the liver in that sample.

Immunohistochemical Staining for HBcAg.

HBV core protein was visualized by immunohistochemical staining of tissues fixed in zinc-buffered formalin using anti-core polyclonal rabbit antibody (DAKO, Carpinteria, CA) exactly as described in ref. 39. The average percentage of core positive cells at each time point for a given mouse strain was determined from ≥3 livers per time point per strain, counting ≥100 independent fields per liver at ×20 magnification, with an average of 323 ± 29 hepatocytes counted per field (n = 297 fields).

HBV Surface Antigen Antigenemia.

Serum concentrations of HBsAg were quantitated by sandwich ELISA using HBsAg standards and antibodies provided in a commercial ELISA kit (Abbott Laboratories, Chicago, IL) to generate a calibration curve in which the dynamic range of accurate measurement was between 3 ng/mL and 80 μg/mL.

Lymphomononuclear Cell Preparation.

Single cell suspensions were prepared from the liver as previously described in refs. 7 and 40. Briefly, livers were first perfused with 10 mL of PBS (Invitrogen, Carlsbad, CA) via the portal vein to remove circulating lymphocytes and pressed through a 70-μm Cell Strainer (Becton Dickinson, Franklin Lakes, NJ). Total liver cells were digested with 10 mL of RPMI medium 1640 (Invitrogen), containing 0.02% (wt/vol) collagenase IV (Sigma, St. Louis) and 0.002% (wt/vol) DNase I (Sigma), for 40 min at 37 °C. Cells were washed with RPMI medium 1640 and then underlaid with one of the following: 24% (wt/vol) metrizamide (Sigma) in PBS, 55% Percoll (Amersham Biosciences, Piscataway, NJ) in PBS, or 10% Percoll in Histopaque 1083 (Sigma). After centrifugation for 20 min at 1500 × g, intrahepatic lymphocytes (IHLs) were isolated at the interface. Red blood cells were lysed by ACK lysis buffer (0.15 M NH₄Cl, 10.0 mM KHCO₃, and 0.1 mM Na₂EDTA, pH 7.2) and then the remaining lymphmononuclear cells were washed twice with RPMI medium 1640 and analyzed.

Ex Vivo Lymphocyte Analysis.

A recombinant soluble dimeric H-2K^b:Ig Fusion Protein (BD/PharMingen, San Diego) was complexed with the HBV core antigen 93–100 peptide (HBcAg93, MGLKFRQL) according to the manufacturer’s instructions to produce the HBcAg93-dimer. Briefly, the dimeric H-2K^b:Ig fusion protein was incubated with the HBcAg93 peptide at a 10-fold molar excess for 12 h at 37 °C. Lymphmononuclear cells isolated from the liver were stained with the HBcAg93 dimer and FITC-conjugated anti-mouse CD8 and APC-conjugated anti-mouse CD3 or APC-conjugated anti-mouse CD45 (BD/PharMingen) for 1 h on ice. The H-2K^b:Ig dimer without peptide (“unloaded dimer”) and an H-2K^b:Ig loaded with a betagalactosidase-derived peptide (DAPIYTNV) (Bgal-dimer) were used as negative controls. Cytometric data were acquired by using a FACSCalibur flow cytometer (Becton Dickinson), and data were analyzed by using CELLQuest software (Becton Dickinson).