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Journal of Virology, May 2005, p. 5568-5576, Vol. 79, No. 9
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.9.5568-5576.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

H-2 Kd-Restricted Hepatitis B Virus-Derived Epitope Whose Specific CD8+ T Lymphocytes Can Produce Gamma Interferon without Cytotoxicity

An Chen, Li Wang, Jingbo Zhang, Liyun Zou, Zhengcai Jia, Wei Zhou, Ying Wan, and Yuzhang Wu*

Institute of Immunology of PLA, Third Military Medical University, Chongqing, People's Republic of China

Received 9 August 2004/ Accepted 21 December 2004


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ABSTRACT
 
It is necessary to evaluate the cytokine secretion status of CD8+ T lymphocytes and elucidate the factors influencing cytokine secretion, because the secretion of cytokines is also an important feature of CD8+ T lymphocytes, and the cytokines usually play critical roles in the outcome of diseases. We showed here that peptide AYRPPNAPI, derived from the core antigen of hepatitis B virus (HBV), could bind to H-2 Kd and induce primed splenocytes from HBcAg expression plasmid-immunized mice to produce gamma interferon (IFN-{gamma}) in H-2 Kd- and CD8-dependent manners instead of in a CD4-dependent manner. The induced cells were mainly CD3 and CD8 positive but had no cytotoxic effect on the corresponding target cells. When administered into HBV transgenic mice, these cells can decrease the serum HBV load without causing liver damage. These results suggest that this peptide is a special kind of CD8+ T-cell epitope, for which specific CD8+ T cells can produce IFN-{gamma} when antigenic stimulation is encountered but which have no cytotoxic effect on the corresponding target cells both in vitro and in HBV transgenic mice. This phenomenon indicates initially that the functional mechanisms of CD8+ T cells can be determined by their epitope specificity, which may be associated with the development of epitope-based immunotherapeutic approaches for infectious diseases and tumors.


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INTRODUCTION
 
CD8+ T lymphocytes play a critical role in the immune response to various chronic and acute viral pathogens in humans and in other animals (4, 26, 38, 39). Vaccines or other reagents that can induce or enhance CD8+ T-cell responses offer hopes of curing viral infections (20, 21, 48). Identification of major histocompatibility complex class I (MHC-I)-restricted CD8+ T-cell epitopes is the basis for designing vaccines or other reagents concerned (35). The widely applied standard for this kind of epitope is that its specific CD8+ T lymphocytes are cytotoxic in vitro and/or in vivo; that is to say, the specific CD8+ T lymphocytes of the epitope can kill specific target cells expressing appropriate epitope peptides bound to the related MHC-I molecules (3, 32).

It is well known that the production of some kinds of cytokines, such as interferon (IFN) and tumor necrosis factor (TNF), is also the typical feature of CD8+ T lymphocytes (13). The cytokines produced by CD8+ T lymphocytes are usually the key factors through which CD8+ T lymphocytes exert their effects (10, 22, 37). It is necessary to evaluate the ability of epitope-specific CD8+ T lymphocytes to produce cytokines during the identification of MHC-I-restricted CD8+ T-cell epitopes.

Previous research with hepatitis B virus (HBV) infection has identified several HBV antigen-derived MHC-I-restricted cytotoxic T lymphocyte (CTL) epitopes, including human HLA-I- and murine H-2-restricted epitopes (7, 18, 34). All of these epitopes were first identified via cytotoxicity assays, that is to say, these epitope-specific CTLs can kill the target cells that present or are pulsed with the corresponding epitope peptide (15, 25). In the pathogenesis of hepatitis B, these epitope-specific CTLs kill the infected hepatocytes to eliminate HBV inside them, causing liver injury, of course, at the same time. Some of these CTLs can simultaneously inhibit the replication of HBV via the IFN-{gamma}/TNF-{alpha} pathway (noncytotoxic cure), because they have been proved to produce IFN-{gamma} after stimulation by antigen peptide. It is well recognized that curing HBV infection depends more on cytokines than on cytotoxicity: the cytokines secreted by CTLs play a more important role than the cytotoxicity of CTLs in eliminating HBV infection (9, 10, 12).

The core antigen of HBV (HBcAg) is a structural protein of which specific CTLs can be easily detected in individuals infected by HBV (7). A. Kuhrober and his colleagues (18) reported that HBcAg87-95 was an H-2 Kd-restricted CTL epitope and that its specific CTLs could kill P815 cells expressing this antigen. This is the only H-2 Kd-restricted epitope within HBcAg identified until now. We want to know whether this epitope-specific CTL produces cytokines like IFN-{gamma} after stimulation with antigen and whether there are other Kd-restricted CTL epitopes within this antigen beside HBcAg87-95.

In the present study, we predicted the murine H-2 Kd-restricted T-cell epitopes in HBcAg and analyzed the affinity of the three candidate epitope peptides binding to H-2 Kd. The splenocytes from HBcAg expression plasmid-immunized BALB/c mice were incubated with the peptide and interleukin-2 (IL-2) in vitro. The function of the stimulated splenocytes was measured in vitro and in vivo. We found that the epitope HBcAg87-95-specific CD8+ T cells could produce IFN-{gamma}. We also found that peptide HbcAg131-139 was an H-2 Kd-restricted CD8+ T-lymphocyte epitope and that its specific CD8+ T lymphocytes could produce IFN-{gamma} but had no cytotoxicity either in vitro or in HBV transgenic mice.


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MATERIALS AND METHODS
 
Epitope prediction and peptide preparation. Two predictive algorithms—SYFPEITHI(http://www.uni-tuebingen.de/uni/kxi/) (31) and the Bioinformatics and Molecular Analysis Section(BIMAS) MHC Peptide Binding Predictions program (http://bimas.dcrt.nih.gov/molbio/hla_bind) (30)—were used to predict putative H-2 Kd-restricted T-cell epitopes from within the amino acid sequences of HBcAg (subtype ayw). The three highest-scored 9- or 10-amino-acid candidate epitope peptides in both algorithms were selected as candidates for further identification. These three peptides and the previously identified CTL epitope peptide HBcAg87-95 (18) were chemically synthesized via a solid-phase 9-fluorenylmethoxy carbonyl program and were purified by high-performance liquid chromatography. The purity and molecular weight of the peptide were determined by high-performance liquid chromatography-mass spectrometry, as described previously (49).

Peptide-MHC binding affinity assay. To analyze the affinity of candidate epitope peptides binding to MHC molecules, the ability of synthetic peptide to stabilize Kd molecules on the surface of the transporter associated with antigen processing-deficient and transfected RMA-S/Kd cell line (27) (a gift from J. R. Bennink, National Institutes of Health) was measured by indirect immunofluorescence by the method introduced by A. Mullbacher et al. (28) and B. Tirosh et al. (40). Briefly, RMA-S/Kd cells were incubated with peptide at a series of concentrations in serum-free RPMI 1640 culture overnight and stained with the anti-Kd monoclonal antibody and fluorescence-labeled secondary antibody for a flow cytometric assay. For the detection of affinity, the culture supernatant of HB159 (American Type Culture Collection) was used as the primary antibody, and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G (IgG) (BD-Pharmingen, San Diego, Calif.) was used as the secondary antibody. The affinity is expressed as FI (fluorescence intensity), which was calculated as (MFI of sample – MFI of background)/MFI of background. In this formula, MFI of background is the value without peptide incubation. Samples were measured in triplicate, and the mean FIs were calculated.

Construction of HBcAg expression plasmid and establishment of transfected cell line P815C. The HBcAg-encoding DNA sequence was amplified by PCR from plasmid HBV1.3 ayw (47), kindly provided by F. V. Chisari (Scripps Institute, La Jolla, Calif.). The amplified sequence was subcloned into the vector pcDNA3.1+ (Invitrogen, Groningen, The Netherlands) for stable mammalian expression. The constructed plasmid pcHBc3.1+ was identified by restriction enzyme digestion, gel electrophoresis, and DNA sequencing. The plasmid pcHBc3.1+ was linearized and transfected into P815 cell line via electroporation as described by Chiasri et al. (15). The established cell line P815C that stably expressed HBcAg was subcloned by limited dilution after being screened with G418. The corresponding antigen expressed by the cloned P815C cells was identified by flow cytometry with rabbit anti-HBcAg antibody and FITC-labeled goat anti-rabbit IgG. More than 95% of the tested cells were FITC positive.

Immunization of mice with plasmid and preparation of splenocytes. BALB/c mice (male, 8 to 10 weeks old, from the Center of Experimental Animals, Medical Academy of China, Beijing, China) were immunized with plasmid pcHBc3.1+ as described by Chisari et al. (15). Briefly, plasmid DNA (50 µg) was injected into the tibialis anterior muscles (100 µg/mouse). Two weeks after the first DNA injection, mice were administered a booster injection, with the same dosage as in the first one. Ten days after the second injection, the mice were sacrificed, and the separated splenocytes were incubated with irradiated and synthetic peptide-pulsed P815 cells in RPMI 1640 plus 10% fetal calf serum (FCS).

Enzyme-linked immunospot (ELISPOT) assay. The ELISPOT assay (Diaclone, Besan, France), as described previously (2, 29), was employed to assess whether the synthetic peptides could stimulate the immunized murine splenocytes to produce IFN-{gamma}, a classical cytokine commonly produced by CTLs. Briefly, the splenocytes were collected after incubation for 1 or 3 weeks; 5 x 104 or 104 splenocytes in 50 µl of culture medium were put into the culture wells of 96-well microtiter plates. Before this, the same numbers of P815 cells were pulsed with peptide at a concentration of 30 µg/ml in 50 µl of culture medium and seeded as stimulator cells in the same culture wells. The following experiments were conducted according to the kit instructions. Aluminum foil was used to wrap the plate to reduce the background and improve well-to-well reproducibility of staining in this assay (16). The number of spots was counted with a light microscope, and the results were expressed as the number of spot-forming cells (SFC) per 106 or 104 splenocytes. In some of the experiments, monoclonal antibodies against H-2 Kd, murine CD4, or murine CD8 (eBioScience, San Diego, Calif.) were added into the culture system at a concentration of 10 or 20 mg/liter to clarify the roles of H-2 Kd, CD4, and CD8 in IFN-{gamma} production.

51Cr releasing assay. After 1 week of incubation, the splenocytes were collected and the standard 4-h 51Cr releasing assay was used to assess the cytotoxicity of the splenocytes. The transfected P815C cells which could stably express HBcAg or P815 cells pulsed with the corresponding peptide were used as the target cells. The detailed protocol and the cytotoxicity calculation were described previously (23, 46). The percentage of specific lysis was calculated as (experimental release – spontaneous release)/(maximal release minus spontaneous release) x 100%.

Flow cytometric determination of CD3 and CD8 expression on murine splenocytes. The expression of CD3 and CD8 on splenocytes before and after peptide stimulation for 1 week was measured, respectively, by flow cytometry (42, 44). Briefly, the collected splenocytes were incubated with PE-labeled anti-mouse CD8 monoclonal antibody and phycoerythrin (PE)-Cy5-labeled anti-mouse CD3 monoclonal antibody (eBioScience) for 30 min at 37°C, avoiding direct light. After being washed twice with fluorescence-activated cell sorter (FACS) buffer (0.5% bovine serum albumin and 0.05% sodium azide in phosphate-buffered saline [PBS]), the splenocytes were resuspended in 500 µl of PBS. The samples were analyzed with the FACSCalibur flow cytometer (BD Biosciences), and the FACS data were analyzed by using CELLQuest software (BD Biosciences).

Peptide-specific CD8+ lymphocytes and HBV transgenic mice. Peptide-specific CD8+ lymphocytes were derived from the splenocytes from plasmid pcHBc3.1+ immunized BALB/c mice as described previously (10, 15). The separated splenocytes were stimulated once a week with both peptide-pulsed irradiated P815 cells and murine IL-2 (supernatant of EL4 IL-2 cells). After 3 weeks of stimulation, the cells were collected, washed, resuspended in Hanks balanced salt solution containing 2% FCS, and injected intravenously into HBV transgenic mice. For each mouse, 107 splenocytes in a volume of 500 µl were injected. The HBV transgenic BALB/c mice (male, 6 to 8 weeks old) were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. The mice were purchased from Transgenic Engineering Lab, Infectious Disease Center, Guangzhou, China (11, 45). All HBV transgenic mice used in this study were identified as positive both for serum HBV surface antigen (HBsAg) and serum HBV e antigen (HBeAg). The serum HBV virus load in these mice was at 104 to 105 copies per ml of serum. HBV DNA, HBV mRNA, and HBcAg in hepatocytes of these animals were all positive by Southern blotting, Northern blotting, and immunohistochemistry, respectively. These animals had no cytopathological changes in the liver and had normal serum alanine aminotransferase (sALT) levels.

sALT and HBV DNA analysis. The hepatocyte injuries of CTL-injected HBV transgenic mice were monitored by measuring sALT levels with a commercially provided reagent. The serum virus load of HBV transgenic mice before and after cell injection was dynamically observed by quantitative PCR with the reagent provided by Fuxing Biological Company (Shanghai, China) according to the instructions for this reagent. The results were expressed as the number of HBV DNA copies per milliliter of serum (5, 24). To determine the factors responsible for the inhibition of HBV replication, each HBV transgenic mouse was injected with 250 µg of hamster anti-IFN-{gamma} monoclonal antibody (eBioScience) 24 h before the administration of CD8+ T cells in some of the experimental groups (10). The irrelevant antibody hamster IgG (eBioScience) was employed in the same manner as the negative control. The mice were sacrificed at certain time points after treatment with CD8+ T cells, and the serum HBV DNA load was measured as described above.

Immunohistochemical analysis for HBcAg expression. The intracellular expression of HBcAg was assessed by immunohistochemical analysis as described by Guidotti et al. (10). Briefly, the frozen sections from HBV transgenic mice were treated with 3% H2O2 diluted in formaldehyde for 10 min, washed with PBS, and then incubated with rabbit anti-HBcAg primary antibody (eBioScience) at a 1:50 dilution and at 37°C for 60 min. After the sections were washed with PBS, horseradish peroxidase-labeled secondary antibody goat anti-rabbit IgG (DAKO, Carpinteria, Calif.) was added for further incubation for 30 min at room temperature. After being washed again, sections were stained with diaminobenzidine substrate (DAKO) at room temperature for about 5 min and counterstained with hematoxylin. The positive cells confirmed by immunohistochemistry in 500 hepatocytes within the randomly selected five fields of each section (three sections from each mouse) were counted. The expression of HBcAg in each mouse was shown as the mean percentage of HBcAg positive cells in hepatocytes of three separately treated sections.


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RESULTS
 
Prediction of putative CTL epitopes restricted with H-2 Kd. To predict the putative H-2 Kd-restricted CTL epitopes in HBcAg, two programs (BIMAS and SYFPEITHI), were used to scan the complete amino acid sequence of this antigen. The previously identified CTL epitope peptide HBcAg87-95 (SYVNTNMGL) was the highest-scored peptide in both programs, and the next three highest-scored 9-amino-acid peptides or 10-amino-acid peptides were chosen as candidates for further identification (Table 1). These four peptides were chemically synthesized, purified, and identified. The molecular weight of each peptide determined by mass spectrometry assay was similar to its theoretical molecular weight, and the purities of these peptides were all >95% (data not shown).


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  		TABLE 1. Results of prediction of potential CTL epitope, restricted with H-2 Kd

Peptide HbcAg131-139 binds to H-2 Kd efficiently. The affinity of candidate peptide binding to H-2 Kd was measured with transporter associated with antigen processing-deficient RMA-S/Kd cells that were stably transfected with H-2 Kd, for the Kd-restricted epitope peptide could bind to Kd molecule and enhance the expression of Kd on the surface of RMA-S/Kd when exposed to exogenous Kd binding peptide. The enhanced expression of Kd could be determined as the enhanced intensity of fluorescence by FACS assay. The previously identified Kd-restricted CTL epitope peptide (SYVNTNMGL) derived from HBcAg87-95 and the unrelated HLA-A2-restricted epitope peptide (FLPSDFFPSV) derived from HBcAg18-27 were used as the positive and negative controls, respectively. As shown in Fig. 1, HBcAg131-139 could bind to Kd molecules on the surface of RMA-S/Kd, and the affinity of this peptide was lower than that of peptide HBcAg87-95 but significantly higher than that of HBcAg18-27. Within the concentration range tested, the intensity of fluorescence, i.e., the Kd bound to this peptide, increased with the increasing concentrations of peptide HBcAg131-139 incubated with RMA-S/Kd cells. The other two candidates, HBcAg117-126 and HBcAg131-140, and the negative control peptide, HBcAg18-27, could not significantly enhance Kd expression on RMA-S/Kd cells.



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  		FIG. 1. Peptide HBcAg131-139 binding to H-2 Kd RMA-S/Kd cells were incubated with peptide at a series of concentrations in serum-free culture medium overnight, and the Kd amount stabilized on the surface of RMA-S/Kd by peptides was determined by flow cytometry. The FI value was calculated as (MFI of sample – MFI of background)/MFI of sample. The values in each point represent the means of triplicate experiments.

IFN-{gamma} production by primed murine splenocytes induced by peptide HBcAg131-139. The splenocytes of pcHBc3.1+ plasmid-immunized mice were separated routinely and were incubated with irradiated P815 cells pulsed with peptide at a concentration of 30 µg/ml. The culture supernatant of EL-4 (Academy of China Cell Bank, Shanghai, China) was used as a source of IL-2 at the volume proportion of 2.5% in RPMI-1640 plus 10% FCS. Peptide HBcAg18-27 was used as the negative control. One week later, the splenocytes were collected, and IFN-{gamma} production by these cells was determined by ELISPOT assay as described in Materials and Methods. As shown in Fig. 2A, splenocytes from immunized mice could produce IFN-{gamma} after stimulation with peptide HBcAg131-139 or peptide HBcAg87-95 in vitro. The rate of IFN-{gamma}-producing cells was about 2,000 per 106 splenocytes after incubation with HBcAg131-139, almost reaching the level of splenocytes stimulated with HBcAg87-95, which had been identified as a Kd-restricted CTL epitope. These results were consistent with those obtained by enzyme-linked immunosorbent assay with the culture supernatant as a sample (data not shown). There were few splenocytes secreting IFN-{gamma} after stimulation with other three peptides, including the other two candidates.



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  		FIG. 2. ELISPOT assay for determination of IFN-{gamma} secreted by primed splenocytes stimulated with peptides. The splenocytes from pcHBc3.1+ plasmid-immunized mice were separated routinely and were incubated with peptide-pulsed irradiated P815 cells and IL-2 in RPMI 1640 plus 10% FCS for 1 week (A and B) or 3 weeks (C). The splenocytes were collected, and the ELISPOT assay was used to determine IFN-{gamma} production by these cells. (A) The primed splenocytes were incubated with each peptide; HBcAg18-27 was used as the negative control. (B) The effect of monoclonal antibodies against H-2 Kd, CD4, and CD8 on the production of IFN-{gamma} by primed splenocytes stimulated with peptide HBcAg131-139. (C) The frequency of IFN-{gamma}-producing cells in the total number of splenocytes tested after stimulation for 3 weeks in vitro with each peptide.

To clarify the relationship between the secretion of IFN-{gamma} by splenocytes stimulated with HBcAg131-139 and H-2 Kd, CD4, and CD8, respectively, monoclonal antibodies against H-2 Kd, murine CD4, or murine CD8 were added into the culture wells at a concentration of 10 or 20 mg/liter during the ELISPOT assay. As shown in Fig. 2B, both anti-Kd monoclonal antibody and anti-CD8 monoclonal antibody inhibited the stimulated splenocytes to produce IFN-{gamma}: the number of IFN-{gamma} SFC was reduced significantly by both antibodies in a dose-dependent manner. On the contrary, anti-CD4 monoclonal antibody had no obvious effects on the secretion of IFN-{gamma}, even at higher concentrations. The results indicated that the secretion of IFN-{gamma} by splenocytes was H-2 Kd and CD8 dependent, but not CD4 dependent.

After stimulation once a week with epitope peptide for 3 weeks, the IFN-{gamma}-producing cells were measured by ELISPOT assay. As shown in Fig. 2C, the number of IFN-{gamma}-producing cells increased greatly after stimulation with peptide HBcAg87-95 and peptide HBcAg131-139 for three cycles. The rate of the IFN-{gamma}-producing cells was about 2,400 and 2,500 per 104 splenocytes after incubation with HBcAg131-139 and HBcAg87-95, respectively. There were still rare splenocytes secreting IFN-{gamma} after stimulation with other three peptides.

Cytotoxicity of peptide-stimulated primed mouse splenocytes in vitro. The cytotoxicity of the splenocytes was determined by a standard 51Cr-releasing assay after 1-week incubation in vitro under the same conditions as those for the ELISPOT assay. The transfected P815C cells which could stably express HBcAg and P815 cells pulsed with the corresponding peptide (30 µmol/liter) were used as the target cells. Peptides HBcAg87-95 and HBcAg18-27 were used as positive and negative controls, respectively. The results showed that splenocytes incubated with peptide HBcAg131-139 could hardly kill P815C and that the cytotoxicity of these splenocytes could not be distinguished from that of the negative control. However, in the positive control, splenocytes incubated with HBcAg87-95 could kill the target P815C cells, and the percentage of specific lysis reached 60% at the ratio of indicated effector cells to target cells (E:T ratio) of 100:1 (Fig. 3A). Similarly, splenocytes incubated with HBcAg87-95 could kill P815C cells and also P815 cells pulsed with the corresponding peptide, and the percentage of specific lysis of the latter cells was higher than that of the former cells as the target cells (80% at an E:T ratio of 100:1). On the contrary, splenocytes stimulated with HBcAg131-139 still could not kill P815 cells pulsed with the corresponding peptide (Fig. 3B). When the cytotoxicity was measured after stimulation for 3 weeks with peptide HBcAg131-139-pulsed irradiated P815 cells and IL-2, the specific killing efficiency on the corresponding target cells still could not be distinguished from that of the negative control (data not shown). In short, the splenocytes incubated with peptide HBcAg131-139 had no cytotoxicity, although they could produce IFN-{gamma} when encountering antigen peptide in vitro.



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  		FIG. 3. Cytotoxicity of peptide-stimulated primed splenocytes. Splenocytes of pcHBc3.1+ plasmid-immunized mice were separated routinely and incubated with peptide at a concentration of 30 µmol/liter and IL-2 in RPMI 1640 with 10% FCS for 1 week. A standard 4-h 51Cr releasing assay was used to determine the cytotoxicity of these cells. The percentage of specific lysis was calculated as (experimental release – spontaneous release)/(maximal release – spontaneous release) x 100%. The lysis values shown as the E:T ratio represent the means of triplicate experiments. (A) The transfected P815C cells that stably express HBcAg were used as target cells. (B) P815 cells pulsed with the corresponding peptide at the concentration of 30 µmol/liter were used as target cells.

Expression of CD3 and CD8 on peptide HBcAg131-139-stimulated murine splenocytes. The phenotype of splenocytes from plasmid pcHBc3.1+-immunized mice were detected by FACS before and after stimulation for 1 week. The same splenocytes incubated with peptide HBcAg18-27 and IL-2 for the same period of time were used as the negative control. The results are shown in Fig. 4. Before stimulation, only about 22.6% of the splenocytes from immunized mice were CD3+ CD8+ (Fig. 4A), and the percentage of CD3+ CD8+ cells reached 65.4% after stimulation with peptides HBcAg131-139 and IL-2 for 1 week (Fig. 4B). In the negative control, after stimulation with peptide HBcAg18-27 and IL-2 for 1 week, the percentage of CD3+CD8+ cell remained almost unchanged (26.2%) (Fig. 4C). These results indicated that peptide HBcAg131-139 could promote the proliferation of CD8+ T cells; in other words, HBcA131-139-specific T cells were CD8 positive.



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  		FIG. 4. CD3 and CD8 expression of splenocytes before and after peptide stimulation for 1 week. The expression of CD3 and CD8 by peptide-stimulated splenocytes was measured by flow cytometry. The splenocytes were stained simultaneously with PE-labeled anti-murine CD8 monoclonal antibody and PE-Cy5-labeled anti-murine CD3 monoclonal antibody. The representative results of the FACS assay are shown. The numbers in each figure are the percentage of CD3+CD8+ cells in the splenocytes tested. (A) Splenocytes from immunized mice before any stimulation. (B) Splenocytes from immunized mice stimulated with peptide HBcAg131-139 at a concentration of 30 µmol/liter and IL-2 for 1 week. (C) Splenocytes from immunized mice incubated with peptide HBcAg18-27 and IL-2 for 1 week.

Effects of epitope-specific CTLs on HBV transgenic mice in vivo. To investigate the effects of epitope-specific CTLs in vivo, the splenocytes from plasmid pcHBc3.1+-immunized BALB/c mice were incubated with the peptide-pulsed irradiated P815 cells and IL-2 as described in Materials and Methods. After 3 weeks of stimulation, the splenocytes were collected, and about 85% of them were identified as CD8 positive. Stimulated splenocytes at a concentration of 107 were injected intravenously into HBV transgenic mice that were commonly used as the model for chronic asymptomatic HBV carriers. The splenocytes stimulated with peptide (HBcAg18-27)-pulsed, irradiated P815 cells for 3 weeks were used as the negative control. At different time points after cell injection, the murine sALT level was determined to evaluate the hepatocyte injuries caused by the cells. As shown in Fig. 5A, peptide HBcAg87-95-specific CD8+ T cells produced a mild and transient liver injury. The sALT level began to rise within 1 day after the administration of T cells, peaked at day 3 (309 U/liter), and then returned to baseline within 1 week. On the contrary, the peptide HBcAg131-139-specific CD8+ T cells and negative control splenocytes rarely caused liver injury, as the sALT level of the HBV transgenic mice did not rise obviously after cell administration.



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  		FIG. 5. Effects of HBV-derived peptide-specific CTLs in HBV transgenic mice. Epitope peptide-specific CD8+ T cells (107) were injected intravenously into both HBsAg- and HBeAg-positive HBV transgenic mice. The peptide HBcAg87-95- and peptide HBcAg18-27-stimulated splenocytes were used as the positive and negative controls, respectively. The effects of CTLs in HBV transgenic mice before and at different time points after the cell injection were determined. The results represent the mean results from four mice with matched age, sex, and HBsAg level. (A) The sALT level before and after cell injec-tion. (B) The serum virus loads (copies of HBV DNA per ml of serum) before and after cell injection, detected by quantitative PCR. (C) The modulation of anti-IFN-{gamma} monoclonal antibody of the HBV replication inhibition caused by transferred CD8+ T cells. The hamster anti-IFN-{gamma} monoclonal antibody was injected at 24 h before T cells were transferred, and the serum HBV DNA load of HBV transgenic mice was measured at some time points after cells were transferred. The irrelevant hamster IgG was used as a control. (D) The effects of CD8+ T cells on HBcAg expression in the livers of transgenic mice. The frozen liver sections were prepared after the administration of T cells. HBcAg levels were determined by immunohistochemistry, and the mean percentage of HBcAg-positive cells in all cells counted from four mice is shown.

The serum HBV load of HBV transgenic mice before and after cell injection was determined by quantitative PCR to assess the curative effect of HBV-derived epitope-specific CD8+ T cells in HBV transgenic mice. Both peptide HBcAg87-95 and peptide HBcAg131-139-specific CD8+ T cells could significantly inhibit the replication of HBV in HBV transgenic mice. The virus load in serum of HBV transgenic mice declined significantly and was lower than the detection limit on day 5 after cell administration. The detection limitation for the assay was 100 copies per ml of serum, and the DNA load lower than this level was employed as the negative control. According to the results, we may conclude that the HBV DNA load of HBV transgenic mice decreased to <100 copies per ml of serum on day 5 after the transfer of peptide HBcAg87-95-induced CD8+ T cells and peptide HBcAg131-139-induced CD8+ T cells. The negative control cells had no obvious inhibitory effect on HBV replication in HBV transgenic mice (Fig. 5B).

When the anti-IFN-{gamma} monoclonal antibodies were administered before CD8+ T cells were transferred, the inhibition of hepatic HBV replication induced by CD8+ T cells after stimulation with both peptide HBcAg87-95 and HBcAg131-139 was partially but significantly blocked (Fig. 5C). The control antibody (irrelevant hamster IgG) had no effects on the decrease in HBV DNA caused by the injected cells. This result is consistent with previous findings that the HBV replication inhibition caused by CTLs was partially mediated by IFN-{gamma} (9) and suggests that CD8+ T cells induced by both peptides could produce IFN-{gamma} in HBV transgenic mice.

The expression of HBcAg in hepatocytes of HBV transgenic mice could also be inhibited by CD8+ T cells stimulated by both peptides HBcAg8795 and HBcAg131-139. We observed four HBV transgenic mice with matched age, sex, and serum HBsAg levels in each group; the mean results are shown in Fig. 5D. The percentage of HBcAg-positive cells in the counted hepatocytes in HBV transgenic mice was about 75%, which then decreased to <10% on day 7 after the administration of CD8+ T cells induced by both peptides, HBcAg87-95 and HBcAg131-139. This suggested that CD8+ T cells stimulated by both peptides could significantly inhibit HBcAg expression in the livers of HBV transgenic mice.

Briefly, specific CD8+ T cells induced by peptide HBcAg87-95 could inhibit HBV replication and HBcAg expression and simultaneously kill hepatocytes. However, specific CD8+ T cells induced by peptide HBcAg131-139 could inhibit HBV replication and HBcAg expression but did not cause injuries to the liver without elevation of sALT levels in HBV transgenic mice.


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DISCUSSION
 
In this study, we identified the HBcAg-derived epitope, HBcAg131-139 (AYRPPNAPI), which had high affinity to H-2 Kd molecules and could induce splenocytes from HBcAg expression plasmid-immunized BALB/c mice to produce IFN-{gamma}. This cytokine secretion was H-2 Kd and CD8 dependent, but not CD4 dependent, which indicated that the secretion of IFN-{gamma} was H-2 Kd restricted and that the effector cells were CD8 positive but not CD4 positive. A 51Cr releasing assay revealed that these effector cells did not kill the specific target cells, transfected cell line P815C, or peptide-pulsed P815 cells. After stimulation with peptide, most of the primed splenocytes were CD8 positive. These stimulated splenocytes were then injected intravenously into HBV transgenic mice, the widely applied models of chronic asymptomatic HBV carriers (6, 10, 11, 36, 45). CTLs specific to this peptide identified by us significantly decreased the serum virus load and HBcAg expression but caused no injuries to the liver of HBV transgenic mice.

The results indicated that the absence of cytotoxicity does not attribute to the failure of antigen processing or epitope presentation by P815C cells, because these splenocytes could not kill the P815 cells pulsed with the corresponding peptide either, in which the epitope would be well represented. It seems that the absence of cytotoxicity in vitro is not due to the fact that the effector cells were too few, resulting in a low E:T ratio, although there were only about 0.2% splenocytes producing IFN-{gamma}. The reasons may include the folllowing. (i) The peptide HBcAg87-95-induced splenocytes could kill the corresponding target cells, although the frequency of IFN-{gamma}-producing cells was similar to that of peptide HBcAg131-139-induced splenocytes (about 2,000 per 1 million splenocytes). (ii) In a study by Wang et al. (41), the frequency of IFN-{gamma}-producing cells was only about 500 per million T cells, but the specific killing efficiency of the target cells was about 50% at an E:T ratio of 50:1, so we think this ratio of effector to target cells may not interfere with the detection of cytotoxicity. (iii) More importantly, we found that most of the peptide HBcAg131-139-induced murine splenocytes were CD8+ cells (about 85%) after 3 weeks and three cycles of stimulation in vitro. At that time, the frequency of IFN-{gamma}-producing cells in the tested splenocytes was about 24% (about 2,400 SFC when 104 induced cells were used for determination, close to that provided by Kasahara et al. (17), but no obvious changes in the specific killing efficiency were found in the corresponding target cells (data not shown).

In HBV transgenic mice, the liver injury is caused mainly by cytotoxicity, and the inhibition of HBV replication is attributed mainly to cytokines produced by the injected cells (1, 10). The results from our studies indicated that splenocytes stimulated with peptide HBcAg131-139 produced cytokines without cytotoxicity when encountering antigen in vivo. On the contrary, CTLs specific to the previously identified H-2 Kd-restricted epitope, HBcAg87-95, could produce IFN-{gamma}, kill the specific target cells, and lower the serum virus load, as well as kill hepatocytes and elevate sALT levels, when injected into HBV transgenic mice. The anti-IFN-{gamma} antibody could partially but significantly abolish the inhibition of HBV DNA replication by CD8+ T cells induced by both peptides, HBcAg87-95 and HBcAg131-139. This result was consistent with the viewpoint that the HBV replication inhibition is mainly mediated by IFN-{gamma} and suggested that the peptide HBcAg131-139-induced CD8+ T cells could produce IFN-{gamma} in HBV transgenic mice.

Although its specific T cells have no cytotoxicity both in vitro and in vivo, we propose that the peptide HBcAg131-139 is an H-2 Kd-restricted CD8+ T lymphocyte epitope. The reasons may include the following. (i) The pathway through which we identified this epitope was the classical and widely used method for identifying MHC-I-restricted CD8+ T lymphocyte epitopes (33). (ii) This peptide could bind to the H-2 Kd molecule efficiently and in a dose-dependent manner. (iii) This peptide could induce primed splenocytes to produce IFN-{gamma}, a typical type I cytokine commonly produced by CD8+ T lymphocytes. The secretion of IFN-{gamma} and other related cytokines is the important feature of CD8+ T lymphocytes. (iv) The production of IFN-{gamma} by these peptide-stimulated splenocytes could be blocked by anti-H-2 Kd and an anti-CD8 monoclonal antibody but not by an anti-CD4 monoclonal antibody, which indicated that this IFN-{gamma} secretion is H-2 Kd and CD8-dependent, but not CD4 dependent. (v) Most of the primed murine splenocytes were CD3 and CD8 positive after stimulation with this peptide, but this shift was not observed when stimulated with the negative control peptide HBcAg18-27 and IL-2.

The compartmentalization of cytokine secretion function and cytotoxicity in CD8+ T cells has been previously described in human hepatitis C virus (HCV) infection by Lancaster and his colleagues (19). The authors found that there were noncytolytic IFN-{gamma}-producing HCV-specific CD8+ T cells in the peripheral blood of chronic HCV infection patients; in other words, this kind of HCV-specific CD8+ T cell could produce IFN-{gamma} but had no cytotoxicity. Unfortunately, the authors did not clarify or emphasize the epitope specificity of these CD8+ T cells. Our results lend further credence to the occurrence of this phenomenon. Moreover, to our knowledge, this is the first time this kind of epitope has been described, and no one has previously reported the identification of a CD8+ T-cell epitope for which specific CD8+ T cells have such special properties.

CD8+ T lymphocyte epitope peptide binding to the MHC-I molecule with certain affinity is the foundation for presenting the epitope peptide as a peptide-MHC complex by antigen-presenting cells and to induce functional CD8+ T lymphocytes. We found that peptide HBcAg131-139 could bind to H-2 Kd, and the affinity of this peptide is lower than that of HBcA87-95. Kuhrober and colleagues (18) proved that HBcAg87-95-specific CD8+ T lymphocytes had cytotoxicity in vitro but did not provide proof of the IFN-{gamma} production of this type of CTL. In our study, we found that this type of CD8+ T lymphocyte could produce IFN-{gamma} as well. It is necessary for a CD8+ T lymphocyte epitope peptide to bind to the related MHC-I molecule at a certain level of affinity; there may be a threshold in the binding affinity for a peptide to be a T-cell epitope, but this does not mean that the higher the binding affinity, the more immunodominant epitope the peptide is. A peptide with high binding affinity may not necessarily be the CD8+ T lymphocyte epitope (14, 18, 28, 40). There are no more clues about the relationship between the binding affinity and the functional mechanisms of CD8+ T lymphocytes specific to this peptide. In this study, we found a type of specific CD8+ T lymphocyte that could produce IFN-{gamma} without cytotoxicity, and we also measured the corresponding peptide-MHC-I molecule binding affinity. Although Franco and colleagues (8) have shown that the priming and memory generation of antigen-specific CD8+ CTLs do not require help if the immunogen binds to MHC-I molecules with high affinity, we still cannot decide the accurate relationship between the affinity of epitope peptide binding to MHC-I molecules and the functional mechanisms of its specific CD8+ T lymphocyte. However, it would be very interesting to investigate the relationship in more experimental models.

Antigen-specific CD8+ T lymphocytes play key roles in eliminating pathogen infections, including HBV infection. In this model, different pathogen-specific CD8+ T lymphocytes may influence the outcomes of infection via the same or different mechanisms, so it is very important to clarify the exact mechanisms used by each kind of epitope-specific CD8+ T lymphocytes to control or eliminate the pathogens (13, 43). For example, in hepatitis B infection, these antigen-specific CD8+ T lymphocytes can kill the infected hepatocytes through their cytotoxicity to eliminate HBV, resulting in hepatitis at the same time. Some of the CD8+ T lymphocytes can inhibit the replication of HBV simultaneously through the IFN-{gamma}/TNF-{alpha} pathway (noncytotoxicity cure), and this mechanism can be used to inhibit HBV replication without hepatocyte damage. In this sense, the cytokines produced by CD8+ T lymphocytes are more important than the cytotoxicity of CD8+ T lymphocytes. In the present study, we found that HBcAg-derived peptide (HBcAg131-139) could induce primed splenocytes to produce IFN-{gamma} without cytotoxicity. This kind of CD8+ T lymphocyte may exert more important roles in the treatment of HBV infection. Our results indicate that there are many epitope-specific CD8+ T lymphocytes of which functional mechanisms are different from one another: some produce cytokines, some exert cytotoxicity, and some have both effects. Based on our results, we can propose preliminarily that the functional mechanisms are related, at least to some degree, to epitope specificity. In future work, we can select the proper epitopes according to the functional mechanisms of their specific CTLs and the pathogenesis of certain diseases to design vaccines or other reagents to cure infectious diseases and other diseases without injuries to the hosts.

Of course, HBV does not infect mice; we used mice as a model, through which we proved that each kind of epitope-specific CD8+ T lymphocyte can have different functional mechanisms and that the mechanisms of CTLs are related, at least to some degree, to their epitope specificity. Based on this result, if we find human HLA-I-restricted CD8+ T lymphocyte epitopes similar to those within antigens of HBV and other pathogens, we can use the proper epitope(s) to construct vaccines to induce CTL responses which can be used for the treatment of chronic hepatitis B without liver damage and of other infectious diseases without injury to the hosts.


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ACKNOWLEDGMENTS
 
This work was supported by the National Key Basic Research and Development Plan of China (973 Program Projects; no. 2001CB510001) and the Major Program of the National Natural Science Foundation of China (no. 11111111).

We thank F. V. Chisari for providing plasmid HBV1.3 ayw, J. R. Bennink for providing the RMA-S/Kd cell line, and L. Zhao, H. Fan, and Liu Haihong for help in manuscript preparation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Immunology, PLA, Third Military Medical University, Chongqing 400038, People's Republic of China. Phone: 86-23-68752680. Fax: 86-23-68752789. E-mail: yuzhangwu63{at}yahoo.com.cn. Back


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Journal of Virology, May 2005, p. 5568-5576, Vol. 79, No. 9
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.9.5568-5576.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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