Pegylated interferon a-2b up-regulates specific CD8+ T cells in patients with chronic hepatitis B

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http://www.wjgnet.com/1007-9327/16/6145.asp

ISSN 1007-9327 CN 14-1219/R World J Gastroenterol 2010 December 28; 16(48):

6145-6150

BRIEF ARTICLE

Pegylated interferon a-2b up-regulates specific CD8+ T cells in patients with

chronic hepatitis B

Ji Chen, Yan Wang, Xue-Jie Wu, Jun Li, Feng-Qin Hou, Gui-Qiang Wang

Telephone: +86-10-66551122 Fax: +86-10-66551680

Received: July 7, 2010   Revised: September 26, 2010

Accepted: October 3, 2010

Published online: December 28, 2010

Abstract

AIM: To investigate the effect of pegylated interferon (IFN) a-2b on specific

CD8+ T lymphocytes in patients with chronic hepatitis B (CHB).

METHODS: Twenty-one patients with CHB were treated with pegylated IFN a-2b.

Periphery blood mononuclear cells were isolated from fresh heparinized blood by

Ficoll-Hypaque density gradient centrifugation (density: 1.077 g/L, Pharmingen)

at weeks 0, 4, 8, 12, and 24, respectively. Frequency of circulating hepatitis B

virus (HBV) epitope-specific CD8 T cells was detected by flow cytometry.

Cytokines were detected by cytometric bead assay.

RESULTS: The frequency of circulating HBV core or env-specific CD8 T cells was

higher (P < 0.05), the number of HBV core specific CD8 T cells was greater at

week 24 (P < 0.05), the level of Th1-type cytokines [interleukin (IL)-12, tumor

necrosis factor-a, and IFN-g] was higher, while that of Th2-type cytokines

(IL-4, IL-6, and IL-10) was lower in responders than in non-responders (P <

0.05) after pegylated IFN a-2b treatment. The IL-6 level was correlated with HBV

DNA (r = 0.597, P = 0.04), while the inducible protein-10 (IP-10) level was

correlated with serum alanine aminotransferase (ALT) (r = 0.545, P = 0.005). The

IP-10 level at week 8 after pegylated IFN a-2b treatment could predict the

normalization of ALT in CHB patients (positive predict value = 56%, negative

predict value = 92%).

CONCLUSION: Pegylated IFN a-2b can enhance the immune response of CHB patients

by increasing the frequency of HBV specific CD8+ T cells and regulating the

Th1/Th2 cytokines.

© 2010 Baishideng. All rights reserved.

Peer reviewer: Dr. Jeff Butterworth, MB, FRCP, Department of Gastroenterology,

Shrewsbury and Telford Hospital NHS Trust, Mytton Oak Road, Shrewsbury,

Shropshire, SY3 8XQ, United Kingdom

Chen J, Wang Y, Wu XJ, Li J, Hou FQ, Wang GQ. Pegylated interferon a-2b

up-regulates specific CD8+ T cells in patients with chronic hepatitis B. World J

Gastroenterol 2010; 16(48): 6145-6150 Available from: URL:

http://www.wjgnet.com/1007-9327/full/v16/i48/6145.htm DOI:

http://dx.doi.org/10.3748/wjg.v16.i48.6145

INTRODUCTION

More than two billion people have been infected with hepatitis B virus (HBV) and

chronic HBV infection affects about 400 million people worldwide[1,2]. Chronic

hepatitis B (CHB) is a chronic inflammatory liver disease, which can progress to

end-stage liver diseases, such as cirrhosis and hepatocellular carcinoma.

Adaptive immunity plays a central role in the pathogenesis of chronic HBV

infection, and it is crucial to understanding the behavior of T cell response

for the design of effective strategies for the control of HBV infection[3-5].

Different studies in chronic and early acute phases of HBV infection suggested

that the functional impairment of HBV-specific cell-mediated immune response

plays an important role in HBV persistence[6-14]. Moreover, recent studies

showed that both positive and negative signals regulate the antigen-specific T

cell function and are important for the better outcome of patients with HBV

infections[15-17].

Pegylated interferon (IFN) a-2b can modulate and reduce antiviral function

of CHB patients by enhancing their immune responses. However, the exact effect

of pegylated IFN a-2b on the immune responses of patients with HBV infections

remains unclear. The present study was designed to investigate the effect of

pegylated IFN a-2b on HBV specific CD8+ T cells and secretion of cytokines in

CHB patients.

MATERIALS AND METHODS

Patients and study design

Twenty-one consecutive CHB patients (17 males and 4 females) at the age of 20-39

years (mean 25 years), admitted to our hospital from January 2008 to May 2009

were included in this study. Diagnosis of HBV infection was established as

previously described[18]. Clinical data and characteristics of the patients are

summarized in Table 1. The patients were treated with pegylated IFN a-2b

(PegIntron from Schering-Plough), at the dose of 0.5-1 mg/kg of body weight,

once a week for 24 wk. Clinical and laboratory data about the patients were

detected before treatment, or at weeks 4, 8, 12, and 24 after treatment.

Patients co-infected with HBV and HCV or with detectable antibodies against

hepatitis delta virus or against human immunodeficiency virus were excluded, as

were those with other causes of liver disease, including alcohol abuse. No

patient had decompensated liver disease (evidence or history of ascites,

variceal bleeding, hepatic encephalopathy or jaundice).

Isolation of peripheral blood monouclear cells

Peripheral blood monouclear cells (PBMC) were isolated from fresh heparinized

blood by Ficoll-Hypaque density gradient centrifugation (density: 1.077 g/L,

Pharmingen). Blood was two-fold diluted with RPMI 1640 medium containing 300

mg/mL L-glutamin, 100 U/mL penicillin, 100 mg/mL streptomycin and 10% fetal calf

serum, then added into the isovolumic Ficoll, centrifuged for 400 × g at 21℃

for 35 min. The cells were washed twice with phosphate buffered saline (PBS).

Human leukocyte antigen-A2 typing

One hundred microliters of fresh heparinized blood (100) was incubated with

human leukocyte antigen-A2 primary antibody for 30 min. Erythrocytes were lysed

with an erythrocyte lysate at 37℃, washed with PBS, and then incubated with

secondary antibody, washed again and analyzed on Becton Dickinson FACS (Becton

Dickinson, USA).

Analysis of HBV epitope-specific CD8+ T cells

Frequency of HBV epitope-specific CD8 T cells was detected by flow cytometry

after incubated with HBV core18-27 tetramers (ProImmune, Oxford, UK) and HBV env

335-343 pentamers (ProImmune, Oxford, UK). Freshly isolated PBMC were incubated

with PE-labeled tetramer or pentamer in PBS (10% FCS) for 15 min at 37℃,

washed once with PBS (1% FCS) and then incubated on ice for 30 min with

FITC-anti-CD8 (ProImmune, Oxford, UK), washed twice with PBS, adjusted to 1 ×

106 cells/vial, and fixed in 2% paraformaldehyde for analysis. About 1 × 106

PBMC were harvested and analyzed within the CD8 gate on Becton Dickinson FACS

using the CELLQuestâ„¢ software.

Secretion of cytokines

Serum levels of interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN-g and

inducible protein-10 (IP-10) in CHB patients were measured by cytometric bead

assay (BD, USA) according to its manufacturer’s instructions.

Serological assessment

Fasting serum levels of liver enzymes [alanine aminotransferase (ALT), aspartate

aminotransferase] were measured with a Hitachi-7180 automatic biochemistry

analyzer (Hitachi Inc., Japan) following the standard laboratory methods. HBV

DNA was detected by real time polymerase chain reaction (Amplicor, Roche).

Statistical analysis

All data were analyzed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA).

Descriptive baseline data were expressed as mean ± SD for continuous variables.

Differences between groups were assessed using Kruskal-Wallis H for continuous

variables. Spearman P test was performed for correlation analysis. The accuracy

of serum factors for predicting virologic response was assessed using the

receiver operating characteristic curve. P < 0.05 was considered statistically

significant.

RESULTS

Frequency of circulating HBV epitope-specific CD8 T cells in CHB patients after

pegylated IFN a-2b treatment

Circulating HBV epitope-specific CD8 T cells were detected 13 out of the 21 CHB

patients (Table 1). The frequency of HBV core 18-27 tetramers+/CD8+ T cells at

week 0 was 0.013 ± 0.002, which increased to 0.026 ± 0.015, 0.029 ± 0.019,

0.036 ± 0.025, and 0.045 ± 0.027, respectively, at weeks 4, 8, 12, and 24

after IFN a-2b treatment (Figure 1), with a significant difference between weeks

8 and 0, and between weeks 24 and 0 (P < 0.05). The frequency of HBV env 335-343

pentamers+/CD8+ T cells began to increase at week 8 with a significant

difference between weeks 24 and 0 (P < 0.05). No significant difference was

observed in frequency of HBV core and HBV env specific CD8 T cells.

To further analyze the effect of pegylated IFN a-2b on HBV-specific CD8 T cells,

13 patients were divided into responders (n = 7) and non-responders (n = 6).

Responders were defined as their ALT returned to its normal level and their HBV

DNA was decreased to over 2log, and/or their serum HBeAg was conversed. The

frequency of HBV core18-27 tetramers+/CD8+ T cells was 0.014 ± 0.011, 0.029 ±

0.022, 0.029 ± 0.021, 0.067 ± 0.029, and 0.05 ± 0.025, respectively, in

responders at weeks 0, 4, 8, 12 and 24 after treatment, which was higher than

that in non-responders (0.012 ± 0.007, 0.018 ± 0.009, 0.028 ± 0.019, 0.025 ±

0.021 and 0.030 ± 0.01, respectively). No significant difference was found in

frequency of HBV core specific CD8 T cells between responders and non-responders

at baseline, even at weeks 4, 8, and 12 after treatment (Figure 2), with a

significant difference observed at week 24 (P < 0.05, Figure 3). The frequency

of HBV env specific CD8 T cells was higher in responders than in non-responders

(P < 0.05, Figure 2).

Secretion of cytokines after pegylated IFN treatment and its correlation with

virologic responses

The serum levels of IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-a

IFN-g, IL-12, and IP-10 were measured at baseline, during the treatment and

follow-up. The serum IL-2 level was very low in CHB patients, which was almost

undetectable. The levels of Th1-type cytokines including IL-12, TNF-a and IFN-g

were increased while those of Th2-type cytokines including IL-4, IL-6 and IL-10

were decreased at week 48 after treatment (Figure 4). The baseline IP-10 level

was increased from week 4 and decreased from week 48 after treatment.

The baseline IL-6 level was correlated with HBV DNA in responders (r =

0.597, P <0.05) but not with HBV DNA in non-responders. IL-10 was correlated

with IL-6 (r = 0.762, P = 0.002), and IL-12 was correlated IFN-g (r = 0.485, P =

0.026).

The IP-10 level was closely correlated with the serum ALT level not only in

responders but also in non-responders (r = 0.545, P = 0.005, Figure 5),

indicating that IP-10 level fluctuates with serum ALT level. The baseline IP-10

level was lower in patients with their ALT < 40 U/L than in those with their ALT

> 40 U/L.

Predictability of IP-10

To determine whether IP-10 can predict the normalization of ALT (< 40 U/L) after

peg-IFN a-2b treatment, receiver operating characteristic curve was plotted for

IP-10. The IP-10 level at week 8 after treatment was predictable. The area under

the curve was 0.741 (P = 0.065). A cutoff value of 437.78 was chosen.

Correspondingly, the positive and negative predictive value was 56% and 92%,

respectively (Table 2).

DISCUSSION

HBV has a high propensity to persist and several strategies have been developed

for control of its evading from T cell responses, including the direct

inhibitory effect of viral proteins on T cell responses and the emergence of

escape mutations[19-21]. Moreover, HBV infection is more common in immune

deficient individuals, such as infants, patients with cancer and those treated

with steroid hormone, thereby can interfere with viral clearance by the innate

immune system[22,23]. Inefficient innate responses and rapid spread of HBV may

in turn delay and impair adaptive responses because of inefficient promotion of

T cell priming by innate immunity and through T cell exhaustion induced by a

rapidly increased viral load. However, the actual impact of exhaustion by

persistent exposure to high antigen concentrations on virus persistence has only

been partially defined.

Furthermore, two kinds of drugs (nucleoside analogs and IFN) are usually

used in antiviral treatment of CHB patients. IFN is involved in numerous immune

interactions during viral infection, as an inducer, regulator, and effector of

both innate and adaptive antiviral systems. IFN-a and beta are produced rapidly

due to viral factors, such as envelope glycoprotein, CpG DNA or dsRNA, and

interact with cellular pattern-recognition receptors, such as mannose receptors,

toll-like receptors, and cytosolic receptors[24]. In addition, IFN modulates

both innate and adaptive immunity, ultimately resulting in an enhanced antiviral

effector function.

In the present study, the frequency of HBV epitope-specific CD8+ T cells in

peripheral blood was persistently increased at weeks 4, 8, 12 and 24 after

peg-IFN a-2a treatment, while the number of HBV epitope-specific CD8 T cells in

HBV core 18-27 tetramers and HBV env 335-343 pentamers was greater in responders

than in non-responders after pegylated IFN a-2b treatment, suggesting that the

therapeutic effect of pegylated IFN a-2b on HBV infection may be attributed to

the elevated HBV-specific CD8 T cells, and that the immune response mediated by

HBV-specific cells plays an important role in control of HBV. However, the

frequency of HBV core 18-27 tetramers+/CD8+ T cells was higher than that of HBV

env 335-343 pentamers+/CD8+ T cells after pegylated IFN treatment, suggesting

that the HBV core epitope plays a more critical role in induction of a stronger

immune response to HBV infection than to HBV env epitope. Pegylated IFN a-2b

could enhance specific immune response of CHB patients. Further study should be

performed with a large sample size.

Cytokines play an important role in immune modulation. Clearance of HBV

infection is mediated by a strong polyclonal cellular response of both CTL and

Th1 cells. Chronic HBV infection is caused mainly by an increased response of

Th2 cells and impaired production of type 1 cytokines. IL-10, a Th2-type

cytokine secreted by T-cells, activated B cells and monocytes, is a powerful

inhibitor of Th1 activation and suppresses cell-mediated immunity in mice and

humans[25,26]. Of the detected cytokines, Th2-type cytokines such as IL-4, IL-6

and IL-10, were altered conspicuously. After treatment, the level of Th2-type

cytokines (IL-4 and IL-10) was down-regulated, thus confirming the immune

recover potential of pegylated IFN a-2b, the level of IL-12 which can promote

the differentiation of Th1-type cytokines was low, and the production of

Th1-type cytokines was increased, indicating that the immune function of

pegylated IFN a-2b can be achieved by regulating the balance of Th1/Th2

cytokines.

IL-6 is a multifunctional cytokine with both differentiation and

growth-promoting effects for a variety of target cells. IL-6 is generally

considered an important cytokine in the network of cytokines that regulate

immune reactions and acute phase responses[27]. It was reported that IL-6 is

correlated with liver fibrosis/cirrhosis[28] and is a cell attachment site for

HBV[29]. In the present study, the IL-6 level was correlated with HBV DNA plasma

only in responders.

IP-10, a chemotactic CXC chemokine of 77 aa in its mature form[30,31], can

be produced by a variety of cells, including hepatocytes[32,33]. The correlation

between IP-10 levels and necroinflammatory activity, as well as the high and low

IP-10 levels before and after pegylated IFN a-2b treatment, may imply that IP-10

plays a role in the natural pathogenesis of HBV-induced liver damage[34]. It was

reported that the baseline IP-10 level can predictive the response of CHB

patients to HCV treatment, and is correlated with liver inflammation and

fibrosis[35,36]. In this study, the baseline IP-10 level in CHB patients could

predict the normalization of ALT after pegylated IFN a-2b treatment.

In conclusion, given the importance of protective T cell responses in

control of HBV, the correlation between immunomodulatory molecules and pegylated

IFN a-2b treatment in restoration of the immune responses of antiviral T cells

are highly desirable. Pegylated IFN a-2b therapy can enhance the immune response

of CHB patients by influencing the production of cytokines. IP-10 can

potentially predict the normalization of ALT, which is correlated with liver

damage. Further study is needed with a large sample size.

ACKNOWLEDGMENTS

The authors thank Dr. Ming Yu and Hong-Li Xi for their technical support help in

this study.

COMMENTS

Background

More than two billion people have been infected with hepatitis B virus (HBV) and

chronic HBV infection affects about 400 million people worldwide. Two kinds of

drugs [nucleoside analogs and interferon (IFN)] are mainly used in treatment of

chronic hepatitis B (CHB) patients. IFN is involved in numerous immune

interactions as an inducer, regulator, and effector in treatment of viral

infections. Cytokines play an important role in immune modulation. Clearance of

HBV infection is mediated by a strong polyclonal cellular response of both CTL

and Th1 cells. Chronic HBV infection is caused mainly by an increased response

of Th2 cells and impaired production of type 1 cytokines. Inducible protein 10

(IP-10) is a chemotactic CXC chemokine of 77 aa in its mature form.

Research frontiers

IFN-a and b are produced rapidly due to viral factors, such as envelope

glycoproteins, CpG DNA or dsRNA, and interact with cellular pattern-recognition

receptors, such as mannose receptors, toll-like receptors, and cytosolic

receptors. IP-10 can be produced by a variety of cells, including hepatocytes.

The results of this study show that the baseline IP-10 level can predict the

response of patients with HBV infection to its treatment with pegylated IFN

a-2b.

Innovations and breakthroughs

The present study demonstrated the correlation between pegylated IFN a-2b

treatment and HBV-specific T lymphocytes. In addition, the effect of pegylated

IFN a-2b on HBV infection could be achieved by balancing the production of

Th1/Th2 cytokines and IP-10 could predict the outcome of patients with HBV

infection after pegylated IFN a-2b treatment.

Applications

In this study, pegylated IFN a-2b could up-regulate HBV epitope specific CD8+ T

cells. The specific cellular immune response could control HBV. IP-10 serum

level could predict the outcome of patients with HBV infection after pegylated

IFN a-2b treatment, thus providing a new index for the treatment of HBV

infection. Pegylated IFN a-2b may be used as a novel strategy for the treatment

of HBV infection by regulating the cytokines.

Terminology

Human leukocyte antigen (HLA) typing is a method to define the HLA+ and HLA+

blood for studied subjects. Flow cytometry is used to define the HBV epitope

specific CD8+ T lymphocytes. Cytometric bead assay is a new technique for

detecting serum concentration of cytokines.

Peer review

This is a very interesting study, showing that pegylated IFN a-2b therapy can

increase the frequency of specific CD8+ T lymphocytes in CHB patients. This may

contribute to the better control of HBV replication and to the recovery of CHB

patients, thus having a promise for therapeutic interventions. The experiments

support the claim of the authors.

REFERENCES<CUT>