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The Journal of Immunology, 2003, 170: 1183-1190.
Copyright © 2003 by The American Association of Immunologists

Tumor Eradication by Hepatitis B Virus X Antigen-Specific CD8+ T Cells in Xenografted Nude Mice

Eunyoung Chun, Jihyun Lee, Hong Seok Cheong and Ki-Young Lee1

Division of Virology and Immunology, Mogam Biotech. Institute, Koosung-myun, Yongin-city Kyonggi-do, South Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously reported several CTL epitopes derived from the hepatitis B viral X Ag (HBx). In this study, we evaluated whether HBx-specific CTLs can be effectively used in adoptive cancer immunotherapy. To validate the possibility, four peptides containing a HLA-A2.1-restricted binding consensus motif were identified from the HBx protein and tested for their ability to activate CTL from PBMCs isolated from chronic carriers of HBV (n = 12). We selected two highly potent epitopes, HBx 52–60 (HLSLRGLFV) and HBx 115–123 (CLFKDWEEL), that are capable of inducing Ag-specific cytotoxic T cells in patient PBMCs. For adoptive immunotherapy using HBx-specific CTLs, we generated CTL clones restricted to the HBx 52–60 or HBx 115–123 peptide using a limiting dilution technique. LC-46, an HBx 52–60-specific clone, is CD62L-CD69+CD45RO+CD45RA-CD25dim and is stained by IFN-{gamma} (~92%), IL-2 (30%), and TNF-{alpha} (56%), but not by IL-5, IL-10, IL-12, or TNF-{beta}, indicating that the cells are fully activated T cytotoxic 1-type cells. When LC-46 cells were adoptively transferred into xenografted nude mice bearing human hepatomas expressing HLA-A2.1 molecules and intracellular HBx proteins, the tumors were eradicated. Taken together, our data provide solid evidence for the feasibility of adoptive immunotherapy with HBx-sensitized CTLs in hepatitis disease, including hepatocellular carcinoma (HCC).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hepatitis B viruses (HBV)2 are small hepatotropic pararetroviruses that replicate by reverse transcription and establish persistent liver infection in humans and other animals (1, 2). HBV are strongly associated with the development of primary liver cancer (hepatocellular carcinoma (HCC)), which is one of the most prevalent forms of human cancer worldwide (1). The factors that potentially induce HCC from chronic HBV infection are still unclear, but many researchers have suggested that the hepatitis B virus X (HBx) protein may be one of these factors (3, 4). HBx is a multifunctional protein with a number of reported activities. HBx might participate in viral pathogenesis as a transactivator of viral promoters and in carcinogenesis as a transactivator of cellular promoters, including those of genes involved in cell growth regulation such as c-fos, c-jun, c-myc, and epidermal growth factor receptor (4, 5, 6, 7, 8, 9, 10).

CTL play a key role in the control of HBV infection, and it is generally thought that they do so by destroying infected cells through cellular immune responses (11, 12, 13, 14). Because CTLs recognize complexes of antigenic peptides and MHC, considerable effort is currently ongoing to develop epitope-based vaccines to stimulate HBV-specific CTL responses (14, 15). Many different CTL epitopes derived from hepatitis B surface Ag (HBsAg), hepatitis B core Ag, and hepatitis B polymerase Ag have been reported, and some of them have shown significant results in both animal models and patients with human acute hepatitis B (11, 12, 13, 14, 15, 16). When HBsAg-specific CTLs are adoptively transferred into HBV transgenic mice, HBV gene expression and replication are abolished by the secretion of IFN-{gamma} and TNF-{alpha} after Ag recognition (17, 18). These results strongly suggest that virus-specific CTLs might be involved in the clearance of virus infection either directly or indirectly.

In this study, we investigated the role of the HBx protein as a potent HBV Ag for developing immunotherapy against both chronic hepatitis and hepatocellular carcinoma. We identified four HLA-A2-restricted CTL epitopes derived from HBx protein. Two epitope peptides, HBx 52–60 (HLSLRGLFV) and HBx 115–123 (CLFKDWEEL), strongly induced specific CTLs in blood taken from patients with chronic HBV. Notably, when an HBx 52–60-specific clone (LC-46) was adoptively transferred into nude mice xenografted with HCC, the tumors were eradicated. Taken together, these results demonstrate for the first time that adoptive immunotherapy using HBx-specific CTLs may be a promising approach for the treatment of hepatitis B viral diseases, including HCC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and mice

C57BL/6-based nude mice were purchased from Charles River Laboratories (Wilmington, MA). T2 cells were kindly provided by Y. Yang (The Scripps Research Institute, La Jolla, CA). T2 cells are MHC class I assembly deficient and, accordingly, express reduced amounts of HLA-A2, and no HLA-B5, on the cell surface (19). The T2 cell line was grown in RPMI 1640 medium (Life Technologies, Bethesda, MD) supplemented with 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO), 10% FBS (Life Technologies), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), and 0.1 mM HEPES (Sigma-Aldrich). The hybridoma cell line, BB7.2 (anti-HLA-A2), was purchased from American Type Culture Collection (Manassas, VA). The BB7.2 cells were maintained in RPMI medium supplemented with 10% FBS, and the culture supernatant was used for detection of cell surface Ags by a flow cytometer (BD Biosciences, San Jose, CA). The SNU-398, SNU-17, and Hep G2 cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea) (20), and were maintained in DMEM (Flow Laboratories, McLean, VA) supplemented with 10% FBS/glutamine/antibiotics in a humidified atmosphere of 5% CO2 at 37°C.

Synthetic peptides

Peptides were synthesized by a solid-phase method using the Fmoc-based protocol on an automated synthesizer (model 430A; Applied Biosystems, Foster City, CA). The crude products were purified on a reverse-phase preparative HPLC column (Vydac, Hesperia, CA). The homogeneity of the final products was assessed by analytical HPLC. Peptides were characterized by an amino acid analysis system (Hewlett Packard, Wilmington, DE) and electrospray mass spectrometry on a Platform II from VG (Manchester, U.K.). Pure peptide fractions were lyophilized and dissolved at 1 mg/ml in PBS.

HLA-A2 stabilization assay

Peptide binding to HLA-A2 molecules was measured using the T2 mutant cell line according to a protocol described previously (21). T2 cells (5 x 105/well) were incubated overnight with different concentrations of peptide in 96-well plates with culture medium (RPMI 1640 containing 10% FBS, 4 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin). The next day, cells were washed twice with cold PBS containing 2% FBS and incubated for 30 min at 4°C with anti-HLA-A2 mAb (BB7.2, 100 µl from culture supernatant) and 1/100 dilution of FITC-labeled goat anti-mouse IgG (Sigma-Aldrich). Cells were washed twice after each incubation, and HLA-A2 expression was measured by flow cytometry on a FACSCalibur (BD Biosciences). HLA-A2 expression (which is a measure of peptide binding to HLA-A2) was quantified as mean fluorescence intensity. Background fluorescence without BB7.2 AB was subtracted for each individual value.

Chronic HBV patient and healthy donor PBMCs

PBMCs from healthy donors and patients chronically infected with HBV were supplied by the Korea Red Cross (Seoul, Korea). The 12 patients included in this study were HLA-A2.1+. The diagnosis of chronic hepatitis B was based on standard diagnostic criteria performed by the Green Cross Reference Lab (GCRL, Green Cross, Seoul, Korea). Diagnostic parameters included clinical and biochemical evidence of liver cell injury (alanine aminotransferase and {alpha}-fetoprotein levels), together with serologic evidence of chronic infection (presence of HBsAg and HBV envelop Ag) in the absence of serologic evidence of hepatitis C virus infection. All patients were serologically negative for Abs to HIV (Table II).


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  		Table II. Analysis of HBV-infected patient for disease progression statusa

 
Preparation of effector cells and generation of HBx 52–60- and HBx 115–123-specific CTLs from patient PBMCs

PBMCs from chronic HBV patients who were positive for the HLA-A2 molecule were separated by centrifugation on Ficoll-Hypaque (Sigma-Aldrich) and washed twice with RPMI 1640 medium without serum. After lysis of the erythrocytes, the cells were resuspended in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, and 10% (v/v) human AB serum, and placed in a six-well plate at 2.5 x 107 cells/well. The cells were stimulated for 2 wk with four HBx-derived peptides (HBx 15–23, HBx 52–60, HBx 92–100, and HBx 115–123, respectively). Briefly, the peptides were added at 10 µg/ml during the first week of stimulation. On days 3 and 10, 2.5 ml of RPMI 1640 with 10% (v/v) human AB serum and rIL-2 at 20 U/ml was added to each well. On day 7, the cultures were restimulated with peptide (10 µg/ml), human rIL-2 (rhIL-2; 20 U/ml) (Boehringer Mannheim, Mannheim, Germany), and gamma-irradiated (3000 rad) allogenic HLA-A2-positive feeder cells. On day 14, the cells were tested for the ability to lyse 51Cr-labeled T2 cells that had been incubated for 4 h with synthetic peptides at 10 µg/ml in a 4-h 51Cr release assay. For the generation of HBx-specific cloned CD8+ T cells, PBMCs derived from patients 1 and 11 (as shown in Fig. 2) were stimulated for 2 wk, as described above. After 14 days, CD8+ and CD4+ T cells were purified with anti-CD8 and anti-CD4 Abs conjugated to magnetic beads (Miltenyi Biotec, Bergish Gladach, Germany). CD4+ T cells were used in the CTL assay, as described in Fig. 3A. Purified CD8+ T cells were restimulated for a further 2 wk with HBx 52–60 and HBx 115–123 peptides. The peptides were added at 10 µg/ml during the first week of stimulation. On days 17 and 24, 2.5 ml of RPMI 1640 with 10% human AB serum and hIL-2 at 20 U/ml was added to each well. On day 21, the cells were harvested and plated into 96-well plates (1 cell/well) by a limiting dilution technique. The cells were restimulated with peptide (10 µg/ml), hIL-2 (20 U/ml), and gamma-irradiated (3000 rad) allogenic HLA-A2-positive feeder cells. The cultures were refed every 3 days by replacing 50% of the volume in each well with fresh culture medium. After 12–24 days, wells were scored visually for growing cells. Proliferating cell clusters were chosen for further expansion only when the frequency of positive wells at a given cell dilution was less than 20%. This cloning procedure was repeated four times, until CD8+CTL clones showed stable proliferation and continuous cytotoxicity specific for the HBx 52–60 and HBx 115–123 peptides. CTL clones were maintained by restimulation every 2 wk.



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  		FIGURE 2. Epitope-specific CTL response in PBMCs from patients with chronic HBV. A 51Cr release assay was performed on PBMCs derived from chronic HBV patients, as described in Table II. The peptides, HBx 15–23, HBx 52–60, HBx 92–100, and HBx 115–123, respectively, were added at 10 µg/ml during the first week of stimulation. On days 3 and 10, 1 ml of RPMI 1640 with 10% (v/v) human AB serum and hIL-2 at 20 U/ml was added to each well. On day 7, the cultures were restimulated with peptide (10 µg/ml), hIL-2 (20 U/ml), and gamma-irradiated (3000 rad) allogenic HLA-A2-positive feeder cells. On day 14, the cells were tested for the ability to lyse 51Cr-labeled T2 cells that had been incubated for 4 h with synthetic peptides at 10 µg/ml in a 4-h 51Cr release assay at E:T ratios of 10:1. All assays were performed in triplicate, and representative data are shown. The mean percent cytotoxicity ± SE values of results of triplicate experiments are presented. Spontaneous lysis was <25% in all assays.

 


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  		FIGURE 3. HBx 52–60 (A, B, and C)- and HBx 115–123 (D, E and F)-specific cytotoxic activity mediated by CD8+ T cells derived from patient PBMCs. A and D, PBMCs derived from patient 1 (A) and patient 11 (D) were stimulated with HBx 52–60 and HBx 115–123 peptide for 2 wk, as described in Materials and Methods. CD4+ and CD8+ T cells from the cultures were purified with anti-CD4-- and anti-CD8-conjugated magnetic beads. 51Cr release assays were performed at different E:T ratios, as described in Materials and Methods. •, T2 target pulsed without peptide, and {circ}, T2 target pulsed with HBx 52–60 peptide (A) and HBx 115–123 peptide (D), represent total cultured cells after culture for 2 wk. {blacktriangleup}, T2 target pulsed without peptide, and {triangleup}, T2 target pulsed with HBx 52–60 peptide (A) and HBx 115–123 peptide (D), represent purified CD8+ T cells. {blacksquare}, T2 target pulsed without peptide, and {square}, T2 target pulsed with HBx 115–123 peptide, represent purified CD8+ T cells. B, C, E, and F, Two clones, LC-46 (B and C) and LC-23 (E and F), were generated from patients 1 and 11, respectively, as described in Materials and Methods. Specific activities were tested with respect to both Ag specificity (B and E) and dependency (C and F). •, T2 target pulsed without peptide as a negative control; {circ}, T2 target pulsed with HBx 52–60 peptide (C) and HBx 115–123 peptide (F), respectively; {blacktriangleup}, T2 target pulsed with HPV E7 11–20 peptide as a control.

 
51Cr release assay

CTL function was measured with a 51Cr release assay, as described previously. Briefly, peptide-pulsed target cells were prepared by incubating T2 cells with synthetic peptides (10 µg/ml) for 4 h in a CO2 incubator and washed extensively with PBS buffer to eliminate unbound peptides. Subsequently, the cells were incubated with 100 µCi 51Cr for 4 h at 37°C. They were brought into contact with each other by centrifugation for 2 min and incubated for 4 h in 96-well round-bottom plates. The specific lysis was calculated as follows: (experimental release - spontaneous release)/(100% release - spontaneous release) x 100. All assays were performed in triplicate. Negative controls included target cells pulsed with an irrelevant peptide (human papillomavirus (HPV) E7 11–20 peptide) (22) or unpulsed target cells. Spontaneous lysis was <25% in all assays.

Intracellular cytokine staining

For the analysis of intracellular cytokines, Abs against hIL-2 (MQ1-17H12), IL-5 (JES1-39D10), IL-6 (MQ2-6A3), IL-10 (JES3-19F1), IL-12 (C11.5), TNF-{alpha} (MAB11), TNF-{beta} (359-81-11), and IFN-{gamma} (B27) were purchased from BD PharMingen (San Diego, CA). The cells were incubated for 5 h at 37°C in RPMI containing 10% FBS and 2 µg/ml of brefeldin A (Sigma-Aldrich). The cells were then washed and stained with FITC-conjugated anti-CD8 Ab for 30 min on ice. After induction, the cells were washed in PBS containing 3% FBS and 0.1% NaN3 and fixed with 4% formaldehyde in PBS for 20 min. After washing, the cells were permeabilized with 0.5% saponin (Sigma-Aldrich) in PBS for 10 min, centrifuged, and resuspended in 50 µl of the same solution. The cells were then stained with PE-conjugated anti-IL-2, anti-IL-5, anti-IL-6, anti-IL-10, anti-TNF-{alpha}, anti-TNF-{beta}, or anti-IFN-{gamma} Abs, respectively, for 30 min. After incubation, the cells were washed in PBS containing 3% FBS and 0.1% NaN3 and analyzed with a FACSCalibur apparatus (BD Biosciences). The results were processed under the CellQuest software and represented as dot plots.

Surface marker analysis

Cells were washed three times with PBS containing 1% BSA, counted, and distributed into staining tubes (1 x 106 cells/tube). Cells were stained with an FITC-conjugated anti-CD8 Ab (RPA-T8) and PE-conjugated Abs against human CD4 (RPA-T4), CD25 (M-A251), CD62 (Dreg 56), CD69 (FN-50), CD45RA (HI100), or CD45RO (UCHL1). All conjugated Abs were purchased from BD PharMingen. The cells were incubated for 40 min on ice, washed three times with PBS buffer, and analyzed with a FACSCalibur apparatus (BD Biosciences). For HLA-A2.1 typing in SNU-398, 1 x 106 cells were incubated with BB7.2 (100 µl) or mouse serum (100 µl; diluted 1/1000) for 40 min on ice. The cells were washed three times with PBS, further incubated with FITC-labeled goat anti-mouse IgG (100 µl; diluted 1/100; Sigma-Aldrich) for 40 min on ice, and then washed three times with PBS and analyzed with a FACSCalibur apparatus.

Western blotting of the HBx protein

SNU-398, SNU-17, and Hep G2 cells were seeded into six-well plates and grown until confluent. Cells were harvested and lysed in modified protein lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM DTT, 1% Nonidet P-40, 0.3% deoxycholate, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 0.5 mM PMSF). The lysates were separated by SDS-PAGE and electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was blocked with 5% nonfat dry milk in TBST buffer (20 mM Tri-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C with an anti-HBx Ab (3F6-G10 clone) (Serotec, Oxford, U.K.). Subsequently, the membrane was washed with TBST buffer and incubated with an appropriate secondary Ab. The protein bands were visualized using ECL kits (Amersham, Buckinghamshire, U.K.).

HCC xenografts and adoptive transfer

For tumor implants, SNU-398 cells (10 x 106) in 100–150 µl of PBS were injected s.c. into the lower dorsal region of female nude mice 7–9 wk of age. The tumors were treated on day 8 when they had reached an average size of 300 mm3. In brief, 50 µl of suspension containing HBx 52–60-specific CTLs (5 x 106 cells) or 50 µl PBS alone was injected i.v. into xenografted mice. The individual treatment groups consisted of five to six mice. Tumor volume (mm3) was calculated by the formula (length (mm) x shortest width (mm) x longest width (mm)), in which length was the longest axis. For monitoring transferred T cells in tumor-bearing mice, ~200 µl blood was obtained from each mouse by tail bleeding. The cells were stained with an anti-HLA-A2.1-specific Ab (BB7.2) to discriminate transferred T cells from mouse PBMC cells and were analyzed with flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selection of HBx-derived epitopes bound by HLA-A2 molecules

To identify HLA-A2 epitopes, the HBx amino acid sequence from the adw type of HBV was obtained from the National Center for Biotechnology Information data base. Using the BIMAS computer program (National Institutes of Health), six CTL epitopes were selected based on the binding scores generated by the program (Table I) (23). The selected epitopes were then tested for their binding affinity to HLA-A2 molecules using the human Ag processing-defective cell line T2. Although T2 cells express very few HLA-A2 molecules in normal culture conditions, they express the molecules at a much higher level when allowed to bind with appropriate peptides that stabilize the HLA-A2 molecules. Thus, up-regulation of peptide-induced HLA-A2 expression in T2 cells can be regarded as an indication of the presence of HLA-A2-restricted epitopes (24, 25). To determine the optimal concentration for binding affinity, the amount of HLA-A2 expressed on the T2 cell surface was quantified by staining the cells with the HLA-A2-specific Ab BB7.2 after the addition of peptides at different concentrations.


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  		Table I. HLA-A2-restricted epitopes derived from HBx protein

 
Among the six different peptides tested, four peptides, HBx 15–23, HBx 52–60, HBx 92–100, and HBx 115–123, showed significant binding affinities to HLA-A2 (Fig. 1). Surface HLA-A2 expression on the T2 cells reached a plateau at a peptide concentration of 10 µg/ml, indicating that this concentration is sufficient for maximal surface expression of HLA-A2 molecules. HBx 52–60 exhibited a relatively higher affinity than any of the other peptides tested. HBx 115–123 was found to be a moderate binder, whereas HBx 15–23 and HBx 92–100 were found to be relatively weak binders (Fig. 1). HBx 36–44 and HBx 102–110 did not show significant binding in our experimental setting.



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  		FIGURE 1. Determination of the binding affinity of synthetic peptides derived from the HBx protein to HLA-A2.1 molecules. After incubation with the peptide at different concentrations as described in Materials and Methods, elevation of HLA-A2.1 expression on the T2 cell surface was detected by measuring the mean fluorescence intensities with flow cytometry using the BB7.2 Ab. The data shown are representative of three independent experiments ({circ}, HBx 15–23; •, HBx 36–44; {blacktriangleup}, HBx 52–60; {triangleup}, HBx 92–100; {blacksquare}, HBx 102–110; {square}, HBx 115–123).

 
CTL response to HLA-A.2-restricted HBx epitopes in HBV chronic carriers

Because the binding of a peptide to class I MHC molecules does not necessarily mean that they are able to be recognized by and to induce MHC-restricted CTL, we examined whether these peptides could generate peptide-specific CTLs in patients with chronic HBV infection (Table II). PBMCs from HLA-A2-positive patients were stimulated for 2 wk with the four HBx-derived peptides that showed significant binding affinities to HLA-A2 molecules (Fig. 1B).

The HBx 115–123 (CLFKDWEEL) peptide, which was shown to bind to HLA-A2 with moderate affinity (Fig. 1B), elicited strong CTL activity in PMBCs from 3 of 12 patients (patients 1, 3, and 11) and a weak CTL response in PMBCs from 3 of 12 patients (patients 4, 5, and 8) (Fig. 2). The HBx 52–60 (HLSLRGLFV) peptide, which exhibited high affinity to HLA-A2, elicited strong CTL activity in PMBCs from 5 of 12 patients (patients 1, 3, 5, 8, and 11) and a weak CTL response in PMBCs from 3 of 12 patients (patients 2, 4, and 10) (Fig. 2). Interestingly, in PMBCs from patients 1, 2, 3, 4, 5, and 8, two of four peptides, HBx 52–60 and HBx 115–123, elicited significant CTL activity (Fig. 2).

Generation and HLA-A2 restriction analysis of the HBx 52–60- and HBx 115–123-specific CTL lines

The HBx 52–60 (HLSLRGLFV) and HBx 115–123 (CLFKDWEEL) peptides induced CTL activity with the highest frequency among the four peptides in tested patients (Fig. 2). To identify CD8+ T cell-mediated killing against the peptide, PBMCs derived from patients 1 and 11 were stimulated with HBx 52–60 and HBx 115–123, respectively, for 14 days. The cultured cells were separated into three populations, unseparated cells (Fig. 3, A and D, circles), purified CD8+ T cells (Fig. 3, A and D, triangles), and purified CD4+ T cells (Fig. 3, A and D, squares), using magnetic beads, and a 51Cr release assay was performed. Specific cytotoxic activities were observed in both unseparated cells and purified CD8+ cells, but not in purified CD4+ T cells, suggesting that these cytotoxic responses are HLA-A2 restricted and CD8+ T cell-mediated killing. Based on this observation, patient 1’s HBx 52–60-specific cell line and patient 11’s HBx 115–123-specific cell line were selected for cloning.

LC-46 (Fig. 3B), derived from patient 1, and LC-23 (Fig. 3E), derived from patient 11, were generated by a limiting dilution technique described in Materials and Methods. Specific activities were tested against T2 pulsed with HBx 52–60 (Fig. 3B, {circ}), T2 pulsed with HBx 115–123 (Fig. 3E, {circ}), T2 pulsed with HPV E7 11–20 as control peptide (Fig. 3, B and E, {blacktriangleup}), and T2 pulsed without peptide (Fig. 3, B and E, •). It is previously reported that, in HPV-infected patient, CTL capable of recognizing HPV E7 11–20 peptide that has moderate affinity with HLA-A2 molecules is strongly detected in the tetramer-binding assay (26). In our study, the peptide was used as an irrelevant control to validate the specificity of the T clones.

As expected, the LC-46 and LC-23 clones were found to be Ag specific against HBx 52–60 and HBx 115–123, respectively. Furthermore, as indicated in Fig. 3, C and F, their specific killing was shown to be dependent on the amount of prepulsed peptides.

Characterization of HBx-specific cloned CD8+ T cells

To ascertain whether the cloned CD8+ T cells were activated, we performed surface marker analysis with seven different mAbs, against CD25, CD45RO, CD45RA, CD62, and CD69. It has been well defined that, in activated or effector CD8+ T cells, the CD25, CD45RO, and CD69 molecules are strongly increased, whereas CD45RA and CD62 molecules are significantly down-regulated as compared with naive CD8+ T cells (27, 28). To evaluate the expression levels of these molecules on LC-46 cells cloned by the HBx 52–60 peptide, PBMCs derived from a healthy donor were stained for these molecules (Fig. 4). Consistent with previous reports, the CD25, CD45RO, and CD69 molecules were strongly increased in LC-46 cells, whereas no significant increase of CD45RA and CD62 was observed (Fig. 4B) compared with that of the healthy donor (Fig. 4A). Similar results were also observed with the staining of LC-23 T cells (data not shown), indicating that the cloned T cells tested in this study might be activated phenotypically.



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  		FIGURE 4. Analysis of surface markers on the LC-46 clone (HBx 115–123 peptide specific). The LC-46 clone was stained with an FITC-conjugated anti-CD8 Ab and PE-conjugated Abs against human CD4, CD25, CD62, CD69, CD45RA, or CD45RO. Expression levels of different activated markers were analyzed with a FACSCalibur apparatus, and the data were processed with CellQuest software and are represented by dot plots. The upper (A) and lower panels (B) represent the results from the total PBMCs derived from a healthy donor and the LC-46 clone, respectively.

 
We next analyzed the intracellular cytokine profile of the cells to further identify their functional pattern. The cells were stained with an FITC-conjugated anti-CD8 Ab and PE-conjugated Abs against IL-2, IL-5, IL-6, IL-10, IL-12, TNF-{alpha}, TNF-{beta}, or IFN-{gamma}. It has been reported that CD8+ T cells can be classified into two types according to their cytokine pattern: T cytotoxic (Tc) 1 (IFN-{gamma}, IL-2, and TNF producing) and Tc 2 (IL-4, IL-5, IL-6, IL-10, and IL-13 producing) (29, 30). As shown in Fig. 5, nearly 30 and 56% of the CD8+ T cells produced IL-2 and TNF-{alpha}, and nearly 92% of them produced IFN-{gamma}. We did not find any difference in the percentage of Tc 2-type cytokines (IL-5, IL-6, and IL-10) (Fig. 5).



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  		FIGURE 5. Analysis of the intracellular cytokine profile of the LC-46 clone (HBx 52–60-specific T cell). Cells were intracellularly stained with PE-conjugated Abs against human IL-2, IL-5, IL-6, IL-10, IL-12, IFN-{gamma}, TNF-{alpha}, or TNF-{beta} and with an FITC-conjugated anti-CD8 Ab, and then analyzed with a FACSCalibur apparatus. Data were processed with CellQuest software and are represented by dot plots.

 
Regression of tumors in xenografted nude mice

HBx is an essential viral protein that might be a cofactor in the development of hepatocellular carcinoma during chronic HBV infection (3, 4). It is generally thought that class I-restricted CD8+ CTL play a key role in the control of HBV infection (11, 12, 13, 14). Furthermore, it has been reported that, when the HBsAg-specific CTL is adoptively transferred to HBV transgenic mice, HBV gene expression and replication are abolished by the secretion of IFN-{gamma} and TNF-{alpha} after Ag recognition (17, 18). In this study, we tried to adoptively transfer HBx 52–60 (clone LC-46) peptide-specific CD8+ T cells, which are activated and able to express IFN-{gamma}, TNF-{alpha}, and IL-2 into xenografted nude mice. Before engrafting HCC into nude mice, we tested the expression of intracellular HBx protein and HLA-A2.1 molecules on the cell surface in the SNU-398 cell line. As shown in Fig. 6, the cells were HLA-A2.1 positive (Fig. 6A) and were shown to strongly express HBx protein (Fig. 6B). It is possible, therefore, that the HBx 52–60 peptide-specific CTL (clone LC-46), which is capable of producing IFN-{gamma} and TNF-{alpha}, may be able to regress HBx-expressed HCC in xenografted nude mice. To evaluate this possibility, we xenografted human hepatoma cells (SNU-398) into nude mice. When 1 x 107 SNU-398 cells were injected s.c., the nude mice bore tumors of ~300 mm3 on day 8. The mice were subsequently injected i.v. with 5 x 106 cells of LC-46 T cells, and the tumor size was measured. As shown in Fig. 6C, the tumor masses in adoptively transferred mice were reduced in size and were hardly detectable on day 14, and these mice stayed alive for more than 1 mo. Tumor mass regression was not observed in the mice injected with control purified CD8+ T cells derived from a healthy donor or with PBS alone; instead, the tumors grew rapidly until day 18 (Fig. 6C, open bar and filled bar, respectively). Interestingly, the transferred LC-46 clone was maintained for at least 2 wk (Fig. 6D, {circ}), whereas the purified CD8+ T cells derived from the healthy donor dramatically decreased over that time (Fig. 6D, {triangleup}). In Fig. 6, our data demonstrate that the HBx-derived epitope, HBx 52–60, endogenously processed in SNU-398, may be specifically recognized by the adoptively transferred LC-46 clone, after which cytotoxic events, including cytokine-mediated mechanisms (IFN-{alpha}/{beta}, IFN-{gamma}, or TNF-{alpha}), act synergistically on xenografted tumor cells.



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  		FIGURE 6. Eradication of tumors by an adoptively transferred HBx-specific CD8+ T clone. A, Expression of HLA-A2.1 molecules on SNU-398 cells. Dotted line, treated with secondary Ab alone as a control. B, Intracellular expression of HBx protein on SNU-398 cells. SNU-17, human cervical cancer cells as a control. Hep G2, human hepatoma cells as a negative control cell. C, Regression of tumors by the adoptively transferred LC-46 clone (HBx 52–60 peptide-specific CD8+ T cells) in xenografted nude mice. For the tumor implant, SNU-398 cells (10 x 106) in 100–150 µl of PBS were injected s.c. into the lower dorsal region of nude mice. After 8 days, when the tumor reached an average size of 300 mm3, as indicated in C (*), 50 µl of suspension containing the LC-46 clone (5 x 106 cells) (• line), control CD8+ T cells (5 x 106 cells) (open bar), and 50 µl PBS alone (filled bar) was injected i.v. into xenografted mice. Tumor volume (mm3) was calculated by the formula: length (mm) x shortest width (mm) x longest width (mm), in which length was the longest axis. Data from groups of five mice are expressed as mean ± SE (p < 0.01). The tumor-free mice were followed for >80 day. D, Monitoring adoptive transferred T cells in xenografted nude mice. After the LC-46 clone was injected into tumor-bearing nude mice, 200 µl blood was isolated via tail vein from each mouse, stained with anti-HLA-A2.1 Abs (BB7.2) to identify the transferred LC-46 clone in the blood sample, and analyzed with flow cytometry. {circ} and {triangleup}, Represent the percentages of transferred LC-46 cells and purified CD8+ T cells derived from a healthy donor, respectively. •, Represents PBS alone as a negative control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The HBx protein is known to be a promiscuous transactivator of many viral and cellular promoters in HBV-infected cells, including the promoters of genes involved in cell growth regulation, such as c-fos, c-jun, c-myc, and the epidermal growth factor receptor. In addition to these essential roles, the protein is thought to play a role in the development of HCC during chronic infection (4, 5, 6, 7, 8, 9, 10). In HBx transgenic mice, it has been observed that the development of HCC is critically related to the expression level of HBx protein and the expression of HBx increases the sensitivity to the tumorigenic effects of hepatocarcinogens 2- to 3-fold (31). Although the roles of HBx in viral infection and carcinoma have been well elucidated, its role as either an immunogen or cancer Ag in HBV infection is still unclear.

In this present study, we evaluated whether HBx-specific CTLs exist in chronic HBV patients, and if so, whether these cells are capable of recognizing HBx-derived epitopes and can be used in adoptive immunotherapy of HCC. In previous reports, anti-HBx Abs had been detected in the serum of patients with chronic HBV (32, 33, 34). These observations strongly suggest that HBx protein can be used as a target Ag for the treatment of HBV-related diseases, and also that CTL-restricted epitopes within HBxAg might be applied in epitope-based therapeutics. Recently, our group and Chung et al. (35, 36) reported that several HLA-A2.1-restricted CTL epitopes were identified in patients, but this information was insufficient to expand the epitope to immunotherapy targeting HCC.

In this study, we report, for the first time, that HBx 52–60- and HBx 115–123-specific T cell clones can be generated from the PMBCs of patients with chronic hepatitis. The cells appear to be activated (CD25dimCD45RO+CD45RA-CD62-CD69+) and restricted to the HLA-A2.1 molecule and the peptide with which they were pulsed, and appear to be Tc 1-type cells with respect to their cytokine profile (IFN-{gamma}, TNF-{alpha}, and IL-2 cytokines). Collectively, these data suggest that the HBx 52–60 and HBx 115–123 peptides may be processed endogenously in vivo, may be present on the cell surface with HLA-A2.1 molecules in HBV-infected cells, and may result in the generation of Ag-specific CTLs in humans. We also demonstrate that when HBx 52–60-specific CD8+ T cells were transferred to nude mice xenografted with human HCC, which is positive for HLA-A2 molecules and is able to express HBx protein, the tumor was eradicated. This provides evidence for the feasibility of adoptive immunotherapy with HBx-specific CTLs in HCC. We speculate that this result may arise in two ways: 1) tumor eradication may result from direct killing mediated by Ag-specific CD8+ T cells, and 2) IFN-{alpha}/{beta}-, IFN-{gamma}-, or TNF-{alpha}-mediated killing may occur because the cells are able to secrete these cytokines. Similar observations have been reported (37, 38, 39).

Recently, it has been reported that epitope-peptides derived from tumor-specific, tumor-associated Ag, or viral proteins can be applied as a potent antigenic form to induce antitumor or antiviral immune responses in therapeutic approaches such as DNA vaccine, therapeutic proteins including synthetic polypeptides, or dendritc cell therapy (40, 41, 42). The majority of the peptide-based approaches use peptides with defined epitopes for the stimulation of T cell, mostly CD8+ CTL, response. And thus, two epitopes, HBx 52–60 and HBx 115–123, evaluated in this study may be used as one of potent epitopes in treatment of hepatitis, including hepatocellular carcinoma induced by HBV when the epitope is applied to dendritic cell therapy, DNA vaccine, or protein-based vaccine.

In conclusion, HBx-derived epitopes are highly promising agents for the development of both epitope-based therapeutics and immunotherapy, including adoptive transferred method against hepatitis.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Ki-Young Lee, Division of Virology and Immunology, Mogam Biotech. Institute, 341 Pojung-ri, Koosung-myun, Yongin-city Kyonggi-do 449-910, South Korea. E-mail address: thylee{at}mogam.re.kr Back

2 Abbreviations used in this paper: HBV, hepatitis B virus; HBsAg, hepatitis B surface Ag; HBx, hepatitis B viral X Ag; HCC, hepatocellular carcinoma; hIL, human IL; HPV, human papillomavirus; Tc, T cytotoxic. Back

Received for publication September 19, 2002. Accepted for publication November 20, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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