M Arasli, et al. www.hepato-gastroenterology.org DOI 10.5754/hge11580 2012; 59(115): Ahead of print.

Hepato-Gastroenterology

Liver, Original Open Access, Ahead of Print

Peripheral Blood Lymphocyte Dynamics and Viral Kinetics in Patients with Chronic Active Hepatitis B Virus

Infection Treated By Tenofovir

Mehmet Arasli1, Yucel Ustundag2, Basak Delikanli2, Ferda Harmandar2 and Cagatay Buyukuysal3

1Department of Immunology, 2Department of Internal Medicine and Gastroenterology, and 3Department of Biostatistics, Zonguldak Karaelmas University School of Medicine, Kozlu-Zonguldak, Turkey

Corresponding author: Yucel Ustundag, M.D., Department of Gastroenterology, Zonguldak Karaelmas Uni-versity School of Medicine, Kozlu-Zonguldak, Turkey; Tel.: +905359543556; E-mail: yucel_u@yahoo.com

KEY WORDS: Tenofovir; Antiviral action; Peripheral blood lymphocytes dynamics.

ABBREVIATIONS: Hepatitis B Virus (HBV); Interferon Gamma (IFN-γ); Interleukin-2 (IL-2); Interna-tional Units (IU); Natural Killer Cell (NK); Natural Killer T Cell (NKT); Natural Regulatory T Cells (nTregs); Simian Immunodeficiency Virus (SIV); T Helper1 (TH1); T Helper2 (TH2); T Helper17 (TH17); Transforming Growth Factor Beta (TGF-β).

ABSTRACT

Background/Aims: We investigated serum viral kinetics and peripheral blood lymphocyte dynamics in chronic hepatitis B patients during the first year of tenofovir therapy. Methodology: Fifteen patients, naive to any kind of previous antiviral therapy, were included in this study. The patients received tenofovir daily 245mg for 48 weeks. Fifteen age and gender compatible healthy subjects were enrolled as the control group. Clinical, biochemical, immunological and virological parameters were assessed at baseline, then at the first, third, sixth and twelfth months. Results: CD4+CD25+FOXP3+ nTregs percentages were significantly higher in the study group than that of healthy controls, CD4+CD28+ and CD4+CD38+ T cell percentages were signifi-cantly lower in the study group than those of control group (p<0.001). Twelve cases had undetectable HBV DNA levels after the one year therapy. We determined that there was an increase of the CD28+co-stimulator molecule on both the CD4+ and CD8+ T cells while a decrease of the CD8+CD38+ T cells, CD4+CD38+ T cells and CD4+CD25+FOXP3+ nTregs, in patients with tenofovir treatment, but only CD4+CD25+FOXP3+ nTregs were statistically significant. Conclusions: We found that both viral load and CD4+CD25+FOXP3+ nTreg percentages decreased significantly in patients with chronic hepatitis B virus infection during 1 year course of tenofovir treatment.

INTRODUCTION

Tenofovir is a highly potent antiviral agent effective against replication of DNA viruses such

as hepatitis B virus (HBV) and retroviruses, including human immunodeficiency virus. In

addition to its strong direct antiviral action, animal studies on simian immunodeficiency virus

(SIV) infected macaque monkeys clearly demonstrated that tenofovir induces strong CD8 cell

mediated immune responses that complement its antiviral potency (1). Furthermore, in vitro

and in vivo studies on murine models, demonstrated that tenofovir administration enhanced

natural killer (NK) and NKT cell activity and secretion of cytokines and chemokines (2-6).

Thus, it is possible that tenofovir may have immunopreserving or immunomodulatory effects

for HBV infected patients that are independent of its direct antiviral actions.

The course of chronic HBV infection is determined by the immune responses of host and the

viral factors (7-9). The viral load has been suggested to have ominous effects on the

components of the immune response (7,10). In line with this proposal, there is one recently

published report indicating good correlation of circulating T regulatory cells with viral load

reduction in chronic HBV treated patients with tenofovir. In this study, the authors indicated

that decreased CD8+ T cells and increased T regulatory activity may be inhibitory for HBV

specific T cell responses and this could be a part of mechanisms leading to T cell anergy or

exhaustion seen in chronic HBV infection (11). Moreover, innate lymphocytes such as

NK/NKT cells also take some roles in this failed host-virus interaction favoring viral survival.

Supporting this hypothesis, a recently published study showed specific defect in CD56+ NK

cell activation and the reduced capacity to produce antiviral and Th1 skewing cytokines. In

this study, entecavir induced viral load reduction was proved to restore NK cell cytokine

producing capacity (12).

One of the best ways to understand immune evasion mechanisms of chronic HBV infection is

to scrutinize lymphocyte kinetics and viral dynamics during a successful antiviral therapy. We

believe that there is an obvious need of more confirmatory data in the pathogenesis of HBV

persistence despite host’s defense mechanims and thus, we investigated the peripheral blood

lymphocyte dynamics; CD3+ (total) T cells, CD4+ (Helper) T cells, CD8+

(Cytotoxic/Suppressor) T cells, CD4+CD28+ (co-stimulator positive Helper) T cells,

CD8+CD28+ (Cytotoxic) T cells, CD4+CD25+FOXP3+ natural regulatory T cells (nTregs),

CD8+CD38+ (activated CD8+) T cells, CD4+CD38+ (activated CD4+) T cells, CD3-

CD16+CD56+ NK (Natural Killer) cells and CD3+CD16+CD56+ NKT (Natural Killer T) cells,

CD19+ (total) B cells, CD23+CD19+ (activated) B cells; the CD4/CD8 ratio; and viral kinetics

during the course of tenofovir treatment in HBV monoinfected patients to see if there is any

contribution of antiviral immune responses to the success of tenofovir induced viral

inhibition.

METHODOLOGY

Patients

Fifteen patients (6 male, 9 female), naive to any kind of previous antiviral therapy, were

prospectively included in the study between the period of November 2008 and November

2010. We also included 15 healthy controls with a similar age and gender distribution with the

study group. The study protocol was designed according to guidelines of the Helsinki

declaration and it is approved by the local ethics committee of Zonguldak Karaelmas

University hospital. All the subjects in this study had hepatitis B surface antigen positivity for

at least 12 months. Hepatitis B e antigen was negative in 12 and positive in 3 of the cases. All

of the subjects enrolled in the study had positive serum tests for HBV-DNA with a viral load

of more than 1x104IU/mL for at least 2 occasions 3 months apart before entry to the study and

aminotransferase levels higher than 2 times the upper limit of normal range for a full year

prior to the study period. Liver biopsy sampling in all cases was routinely performed and

showed chronic active inflammation with fibrosis scores of ≥2 and necroinflammation scores

of ≥8 (Table 1).

The exclusion criteria included immunosuppressive or antiviral therapy within 1 year before

entry, the existence of a concurrent anti-hepatitis C virus, anti-hepatitis delta virus and/or anti-

human immunodeficiency virus positivity, liver cirrhosis, concomitant liver diseases other

than chronic hepatitis B virus infection, pregnancy, malignancy and systemic medical

illnesses like chronic renal failure, severe cardiopulmonary diseases or diseases which need

regular drug intake. We obtained informed consent from each patient.

Design

Single cohort with an interventional protocol was used as a study design in this investigation.

All of our cases received tenofovir at a daily dose of 245mg for 48 weeks. Biochemical,

immunological and virological parameters were assessed at baseline, then at the first, third,

sixth and twelfth month.

Serology and liver function tests

Biochemical investigations including serum alanine aminotransferase (ALT), aspartate

aminotransferase (AST) and bilirubin levels (Bil) were tested with routine automated

techniques. Enzyme linked immunofluorescence assay was used to detect hepatitis B markers

(HBsAg, HBsAb, HBeAg, HBeAb, HBcAg, HBcAb). Experimental methods were performed

according to the guidelines described in the reagent kit.

HBV-DNA quantification

Serum HBV-DNA load was measured by real time polymerase chain reaction using COBAS

Taqman system with a lowest detection of approximately 200 viral genome copies per mL.

The procedure was completed in accordance with the reagent kit (COBAS AmpliPrep, HBV

test, v2.0) package insert. The primer was present in the kit, the reaction volume was 40µL

and the reaction conditions were 37°C for 5 min, 94°C for 1 min then 40 cycles at 95°C for 5

sec and 60°C for 30 sec.

Flow cytometric analysis of peripheral blood lymphocytes

Peripheral blood lymphocyte phenotypes were analyzed following the immunofluorescense

procedure recommended by Beckman Coulter. Briefly, peripheral blood samples were

incubated with 10-20µL FITC, PE and PC5 labeled mouse anti-human monoclonal antibodies

in the dark for 15 minutes at room temperature. Erythrocytes were lysed in ImmunoPrep

solutions maintained for Coulter TQ Prep (Beckman Coulter, Miami, Fl, USA) according to

the manufacturer's instructions. For each analysis, Beckman Coulter Cytomics FC500 five-

color flow cytometry equipped with 488nm Argon Ion laser was used and 30000 cells were

collected. The mouse anti-human monoclonal antibodies specific for CD45, CD14, CD3,

CD4, CD8, CD19, CD16+56+, CD23, CD25, CD28, CD38 and isotopic controls were

purchased from Beckman Coulter (Immunotech, Marseille, France). FoxP3-PE (Clone

236A/E7) moAb were purchased from eBioscience, Inc. (SanDiego, CA, USA). We used

IntraPrep reagent (Beckman Coulter) as fixative and permeabilizing agent. Procedure was

applied according to the manufacturer's instructions. Briefly, for CD4-FITC/CD25-

PC5/FoxP3-PE staining, peripheral blood mononuclear cells obtained from the study subjects

and control cases were used. Following the CD4 and CD25 surface staining, fixation and

permeabilization steps and intracytoplasmic FoxP3 staining were performed. Two hundred

thousand cells were counted in flow cytometry. Kaluza (version 1.0) flow cytometry analysis

software (Beckman Coulter, Miami, Fl, USA) was used to assess the phenotype analysis of

lymphocytes (Figure 1a-d).

Statistical analyses

For statistical analysis, we used SPSS for Windows, Release 18, programme on an individual

computer (SPSS, IL Chicago, USA). All continuous data were expressed as mean±standard

deviation if normally distributed or as medians if not normally distributed and were compared

using Wilcoxon test. For comparison of the results, a value of p<0.05 was considered

statistically significant. Descriptive statistics was also done to examine the age, gender, HBV-

DNA, hepatitis e antigen status, liver enzymes and total bilirubin.

RESULTS

At the beginning, 14% of our cases had serum levels of HBV-DNA over 107 copies per mL

and 12 (80%) cases had undetectable HBV-DNA levels after sixth months of therapy which

remained negative in all these cases at the end of 1 year treatment (Figure 2). The baseline

percentages of CD3+ (total T cells), CD4+ (helper T cells), CD8+ (cytotoxic/suppressor T

cells), CD19+ (total B cells), CD19+CD23+ (activated B cells), CD8+CD38+ (activated CD8 T

cells), CD8+CD28+ (cytotoxic T cells), CD3-CD16+CD56+ (NK cells), CD3+CD16+CD56+

(NKT cells) and CD4/CD8 ratio, were not different between the study and control groups.

The most striking findings in this study were the differences in the percentages of

CD4+CD25+FOXP3+ T regulatory cells, co-stimulator positive CD4+CD28+ helper T cells

and activated CD4+CD38+ helper T cells between the study and the healthy control groups

(respectively for nTregs 3.50±2.50 vs. 1.97±1.33, p=0.012, for CD4+CD28+ helper T cells

77.60±22.75 vs. 89.65±10.57, p=0.03, for CD4+CD38+ Helper T cells 10.02±3.74 vs.

19.89±6.66, p=0.0001) (Table 2). CD4+CD25+FOXP3+ natural T regulatory cell percentages

were significantly higher in the study group than that of healthy controls; CD4+CD28+ co-

stimulator positive helper and CD4+CD38+ activated helper T cell percentages were

significantly lower in the study group than those of the control group. We also determined

that there was an increase of CD28+ co-stimulator molecule on both CD4+ (10/15 patients,

67%) and CD8+ T cells (11/15 patients, 73%), a decrease of CD38 activation molecule on

both CD4+ (10/15 patients, 67%) and CD8+ T cells (11/15 patients, 73%) in the study group

during the course of tenefovir treatment. But none of them was statistically significant. There

was also no difference in the percentages of CD3+ (total T cells), CD4+ (helper T cells),

CD19+ (total B cells), CD19+CD23+ (activated B cells), CD8+CD38+ (activated CD8 T cells),

CD8+CD28+ (cytotoxic T cells), CD3-CD16+CD56+ (NK cells), CD3+CD16+CD56+ (NKT

cells) and CD4/CD8 ratio, at the beginning and throughout the study period (Figures 3 and 4).

Expectedly, CD4+CD25+FOXP3+ regulatory T cells were found to decrease significantly

during the study period in all of our cases (baseline 3.5±2.49, 1st month 3.2±2.5, 3rd month

2.0±1.84, 6th month 0.9±1.4, 12th month 1.07±1.32) (Figure 5). While HBV-DNA levels were

negative after 6 months of tenofovir treatment in all cases, three patients (20%) had developed

breakthrough infection with increased HBV-DNA levels (range; 200-180,000IU) due to

compliance problems. No drug resistance was detected during the study period in these cases

with increased HBV-DNA levels after 6 months of treatment.

DISCUSSION

Tenofovir is a highly potent antiviral agent effective against replication of DNA viruses such

as hepatitis B virus (HBV) and retroviruses including the human immunodeficiency virus.

Resistance to tenofovir has not been described thus far in HBV mono-infected patients with

lamivudine pre-treatment. In our study, we investigated the decrement of HBV-DNA and

the changes of peripheral blood lymphocyte profile in 15 patients with chronic hepatitis B

virus infection during a 1-year course of tenofovir treatment. We showed high levels of

virological and biochemical remission with tenofovir treatment both in ‘e’ antigen positive

and negative patients. At the beginning of the study, 14% of our cases had serum levels of

HBV-DNA over 107 copies/mL and 12 (80%) cases had undetectable HBV-DNA levels after

sixth months of therapy, which remained negative in all of these cases at the end of 1 year of

treatment. While HBV-DNA levels were negative after 6 months of tenofovir treatment, 3

patients (20%) had developed increased HBV-DNA levels (range 200 IU-180 000 IU) due to

compliance problems. Meanwhile, flow cytometry findings indicated that there was no

statistically significant difference on the percentages of CD3+, CD4+, CD8+, CD19+,

CD19+CD23+, CD4+CD38+, CD8+CD38+, CD4+CD28+, CD8+CD28+, CD3-CD16+CD56+,

CD3+CD16+CD56+ and CD4/CD8 ratio, at the beginning and throughout the study period.

Although there were an increase of CD28 co-stimulator molecule on both CD4+ and CD8+ T

cells and a decrease of CD38 activation molecule on both CD4+ and CD8+ T cells, none of

these results was statistically significant. Thus far, during a long period, CD38 positivity on

CD8+ T cells have been used as an indicator of treatment on HIV positive patients. Actually,

the decreasing percentage of CD8+CD38+ T cells has been used for monitoring the viral load

to evaluate the effectiveness of antiretroviral therapy (ART) in HIV-infected patients (13-15).

In different publications, CD8+CD38+ lymphocytes percentages and MFI (mean fluorescence

intensity) values correlate with viral load and could also discriminate patients with a good

virological response from those with a poor virological response in HIV-infected patients (16-

18). We were expecting a similarly decreasing circumstance of CD8+CD38+ lymphocyte

percentages in chronic hepatitis B patients with tenofovir therapy. However, our CD8+CD38+

lymphocyte results were not statistically significant. We feel that a larger group of patients is

needed to assess our results.

Compared with healthy controls, our highly viremic cases with chronic hepatitis B infection

showed higher baseline level of CD4+CD25+FOXP3+ cell percentages and lower baseline

levels of CD4+CD28+ (co-stimulator positive) and CD4+CD38+ (activated) helper T cells. This

finding can be supportive data emphasising a potential role of this T cell profile in the

persistence of hepatitis B infection. CD4+CD25+FOXP3+ cells were supposed to be nTregs

(8). nTregs take role in suppressing the different parts of the immune system, (dendritic cells,

mast cells, basophils, eosinophils and different effector cells of CD8+, TH1, TH2 and TH17)

(19,20). It has been reported that CD4+CD25+FOXP3+ nTregs secrete interleukin 10 and

transforming growth factor beta (TGF-β) both of which have inhibitory effects on

proliferation, cytokine-production (IFN-γ, IL-2) and cytolytic activity of naive and antigen

specific CD4+ and CD8+ cells (21,22). These cells are responsible for suppressing the

immunity to both self reactive and foreign antigens like viruses. Studies with several viruses

such as HCV, retroviruses and herpes viruses demonstrated that CD4+CD25+FOXP3+ nTregs

may affect the magnitude of immunity and the outcome of infection (23). nTregs may also

play an important role in the suppression of antiviral T cell responses in the acute and chronic

phase of HBV infection and thus may help the virus to establish viral persistence (22). In one

study, circulating CD4+CD25+FOXP3+ nTregs were detected in a higher percentage of

patients with chronic hepatitis B virus infection compared to healthy controls and individuals

with a resolved HBV infection. nTregs contribute to the impaired immune response in

patients with chronic hepatitis B virus infection (24).

In our study, we found that both viral load and CD4+CD25+FOXP3+ nTreg percentages

decreased significantly in patients with chronic hepatitis B virus infection during 1 year

course of tenofovir treatment (baseline 3.5±2.49, 1st month 3.2±2.5, 3rd month 2.0±1.84, 6th

month 0.9±1.4, 12th month 1.07±1.32). Our findings were consistent with another study in

which circulating nTregs were reported to be declining in parallel with HBV-DNA level (25).

This finding was also demonstrated in a recent study which investigated the relationship

between antigen stimulated nTregs expression and their function with HBV viral load in 35

patients who received tenofovir treatment for chronic HBV infection (11). The authors

demonstrated that e antigen negative cases had higher basal CD4+CD25+FOXP3+ nTregs than

healthy controls and e antigen positive cases (11). In this study, significant reduction in

CD4+CD25+FOXP3+ nTregs was observed in week 12, during the tenofovir treatment. They

also found that baseline CD8 percentages were lower on both groups of chronic hepatitis B

patients and healthy controls. However, following the 24 weeks of tenofovir therapy, there

was a modest increase in CD8+ T cell percentages in eAg positive patients and a significant

increase in CD8+ T cell percentages in eAg negative patients. Differently from this study,

most of the patients in our study group consisted of e antigen negative cases. Moreover, in

our study, CD8+ T cell percentages of both healthy and study groups were not different and

also CD8 T cell percentages did not change during the course of tenofovir treatment.

However, we found that peripheral blood nTregs percentages in our study subjects were

higher than that of healthy controls.

It is already known that high viral load can influence the functionality of CD8 T cell function

(7,26) and lamivudin treatment enhances HBV specific T cell effector function (27). Possibly,

active viremia can result in the induction of T regulatory cells which may be reversed on

therapeutic virus suppression. This may lead to enhanced antiviral effector T cell response.

Thus, we can not omit the possibility that virus specific T cells can restore their function after

viral clearance with tenofovir treatment. Indeed, one report from the literature supported the

importance of functional effects of tenofovir on the cellular and native immune system. This

report was an animal study which showed the potential immunomodulatory actions of

tenofovir in macaques infected with SIV, such as in the induction of strong CD8 cell mediated

immune responses, the enhancement of NK and NKT cell activity and the secretion of

cytokines and chemokines (1).

If we concentrate on chronic hepatitis B virus infection, we know that natural killer (NK) and

natural killer T (NKT) cells, which are representative of the innate immune system, have also

failed in early stages of HBV infection (8,28). However, in our study we could not find any

difference in the pretreatment percentages of NK and NKT cells between the study groups

and healthy controls. Moreover, there was no change in the percentages of these cells

throughout the study period. Similar to the results of our study, a recent investigation on this

issue clearly showed that total NK cell percentages remain similar to controls throughout the

6 month treatment period (12). However, the investigators found that the cytotoxic capacity of

NK cells is retained and 6 month treatment with entecavir induced enhancement of NK cell

activation and interferon gamma production in parallel with the level of HBV-DNA viral load

(12).

In contrast to the strong and multispecific T cell responses observed during acute self-limited

HBV infection, patients with chronic hepatitis B tend to have weak and narrowly focused

immune responses that are thought to play an important role in ongoing viral replication and

persistence of viral infection (7,10). The cause of paralysed, weak and undetectable virus

specific T cell responses is not yet known. A high viral load may be an important factor that

contributes to T cell failure. It has been suggested that this T cell anergy or dysfunction can be

induced by the virus itself (7,28,29-31). Thus, it is to be expected that antiviral treatments can

possibly correct transient or persistent reconstitution of virus-induced impaired cellular

immune system. Indeed, several studies with antivirals including lamivudine, adefovir and

entecavir have provided evidence along this line (32-35). For example, the studies with

lamivudine have shown us that inhibition of viral replication transiently restores T cell hypo

responsiveness in Hbe antigen positive patients (32,36,37). Lamivudine induces cytotoxic T

lymphocytes, which are suggested to be contributory to its antiviral effects (38). However,

with a prolonged treatment period using this drug, the peripheral CD4 positive T cells decline

to pre-treatment levels. The transient nature of this immune reconstitution was associated with

lamivudine resistance which commonly develops during its prolonged therapy period. Some

other authors reported that adefovir induced inhibition of viral replication transiently leads to

an increase in HBV specific CD4+ T cells, but no increase in HBV specific CD8+ T cells in

HBe antigen positive cases (33,34). Another study, again on HBe antigen positive patients,

showed that the inhibition of viral replication by adefovir reduces regulatory T cells and

enhances T cell immune responses in Hbe antigen positive chronic hepatitis B (33). A further

study on a large sample size of HBe antigen positive cases with a high viral load showed that

powerful inhibition of viral replication by entecavir partially restored impaired T cell

responsiveness, which lasted throughout the study period (4-48 weeks). Since there is no

known direct effect of entecavir, adefovir or lamivudine on T cell population, it is most

probably true that the T cell failure is associated with the degree of viral replication.

As far as we know, all of these studies were performed on HBe antigen positive cases. We

also know that neonatal tolerance to the ‘e’ antigen induces a lack of antiviral response in the

newborn following mother to infant transmission of HBV (39-40). Thus, the soluble Hbe

antigen has been suggested to contribute to the T cell hypo responsiveness by down regulating

HLA class II restricted T cell responses (41). Immunization of mice with the HBe antigen can

favour the production of T helper 2 cytokines which in turn cause suppression of T helper 1

production and deletion of T helper 1 cells by Fas-FasL mediated mechanisms (42). T helper

1 cells play a part in non-cytolytic control of viral replication and a dominant T helper 2

cytokine profile contributes to viral persistence. In our study, most of the patients were ‘e’

antigen negative and only 3 patients had ‘e’ antigen positivity. Although we had the same

results in ‘e’ antigen negative and ‘e’ antigen positive cases, ‘e’ antigen negativity and low

pre-treatment HBV-DNA levels could have been contributory to our findings. Possibly, a

dramatic decline in HBV-DNA levels in the ‘e’ antigen positive cases reflects a simultaneous

decrease in ‘e’ antigen levels and this may be responsible for the loss of T cell tolerance or

paralysis during the antiviral drug treatment of chronically infected patients with HBV.

In conclusion, persistent HBV infection in our patients showed a peripheral blood lymphocyte

profile in favor of nTregs over the co-stimulatory helper T cells. We also found that adequate

and prolonged HBV-DNA suppression with tenofovir treatment does not make significant

quantitative changes in the peripheral blood B, CD4+, CD8+ T lymphocyte cell percentages,

CD4/CD8 ratio or NK and NKT cells. However, the percentages of T regulatory cells

significantly decreased during HBV-DNA clearance with tenofovir treatment. This may have

prominent effects on the dynamic balance between HBV and the immune system through

potentiating antiviral effector T cell functions. Thus further studies are needed investigating

functional changes in the cell mediated immune responses during antiviral induced HBV-

DNA suppression. Furthermore, the presence or absence of the ‘e’ antigen has different

immunological effects on the T cell profile and T cell functions and therefore we need a larger

sample size of ‘e’ antigen negative and ‘e’ antigen positive cases on tenofovir treatment to

draw firm conclusions regarding this interesting subject.

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FIGURE 1. Flow cytometric CD4+CD25+FOXP3+ Regulatory T cell analysis. (a) In CD4-SS histogram 200,000 PBMCs were collected and CD4+ T helper lymphocytes were gated. In the first column, CD4+CD25+High cells were gated. The percentage value shows the amount of CD4+CD25+High cells in the CD4+ T helper cells group. This group of cells was significantly expressing intra-cytoplasmic FOXP3 transcription factor (in second column). The horizontally located histograms show CD4+CD25+FOXP3+ Regulatory T cells respectively; (b) in healthy control group; (c) in a chronically infected hepatitis B patient before the treatment; and, (d) after the treatment. Primarily, PBMCs were stained with anti-human moAbs CD4-FITC and CD25-PC5, after the fixation and the permeabilization steps, intra-cytoplasmic FoxP3-PE staining were performed. In a different tube, to exclude the non-specific binding, CD4-FITC, IgG1-PE and IgG1-PC5 isotypic controls were studied.

FIGURE 2. We found that 80% cases had undetectable HBV DNA levels after sixth months of therapy which remained negative in all these cases at the end of 1 year treatment.

FIGURE 3. There was no significantly difference on the CD4/CD8 ratio and the percentages of CD3+, CD4+, CD8+ T cells during the study period in all of our cases.

FIGURE 4. We determined that there was no difference on percentages of CD3-

CD16+CD56+ (NK) and CD3+CD16+CD56+ (NKT) cells at the beginning and throughout the study period in all of our cases.

FIGURE 5. CD4+CD25+FOXP3+ nTregs were found to decrease significantly during the study period in all of our cases.

TABLE 1. Patient characteristics at entry into the study.

Characteristics Patients (n=15)

Gender (male/female) 6/9 Mean age (in years) Male Female

50.3±9.2 48.8±14

Serum ALT levels (IU/L) 93.2±56 Serum AST levels (IU/L) 67.5±36.7 Serum total bilirubin level (mg/dL) 2.1± 0.7 Serum HBV DNA (copy/mL) >107 copies per mL <107 copies per mL

40% (6/15) 60% (9/15)

Liver histopathology-stage Stage 2 fibrosis Stage 3 fibrosis Stage 4-5 fibrosis

53% (8/15) 20% (3/15) 26% (4/15)

Hbe-Ag positivity 20% (3/15)

TABLE 2. Viral, biochemical and immunological chracteristics of the control and the study groups.

Treatment response over (months) Healthy

controls Study Group Baseline 1st month 3rd month 6th month 12th month

HBV DNA negation 0

3 (20%) 11 (73%) 12 (80%) 12 (80%)

HBe antigen negation ALT normalization 0

4 (26.6%) 8 (60%) 13

(86.6%) 14 (93.3%)

T lymphocyte subgroups (%) CD3+ T lymphocytes CD4+ T lymphocytes CD8+ T lymphocytes CD4+/CD8+lymphocyte CD4+CD25+T lymphocyte* CD19+ lymphocytes NK cells NKT cells CD8+CD28+lymphocyte CD8+CD38+lymphocyte CD4+CD28+lymphocyte * CD4+CD38+lymphocyte* CD19+CD23+lymphocyte

67,8±4.2 45.5±4.2 26.4±2.2 1.7±0.2 1.9±1.3* 12±3 11,2±4,3 3.2±1.9 42.7±20.3 11.4±4.4 89.6±10.5 19.8±6.6 41.3±8.8

68.9±8.3 46.1±8.9 28.2±10.2 1.9±1.1 3.5±2.4 12.1±3.4 12.4±6.4 4±2.9 35.9±16.2 7.1±3.6 77.6±22.7 10±3.7 38.2±15.9

68.8±8.8 45.2±7.6 28.6±9.8 1.8±0.9 3.2±2.5 11.3±3.4 11.7±7.1 4.3±2.9 38.8±11.9 6±3.1 82.6±23.4 9.2±3.4 38.1±16.5

67.7±6.8 44.5±7.7 28.6±8.7 1.7±0.7 2±1.8 11.3±3.7 11.8±6.9 5.2±2.9 41±13.8 5.4±2.4 83.3±25 8.8±3.8 37.3±17

69.1±7.7 45.5±6.7 25.6±7.4 1.9±0.8 0.9±1.4 12±3.7 10.8±6.6 4.2±2.4 39.5±13.3 5.1±3 80.9±23.9 8.3±3.5 37.1±17.1

69.3±8.3 45.9±6.7 26.7±8.2 1.9±0.9 1±1.3 11.5±4 10.9±6.7 4.7±2.5 49±16.4 5.6±3 84.6±24.3 7.8±4.2 37.7±18.4

Biochemical measurements Serum ALT level (IU/L) Serum AST level (IU/L) Total serum bilirubin level (mg/dL)

26±12 21±11 1.0±0.3

93.2±56 67.6±36.7 2.1±0.7

54.6±27.8 47.8±21.2 1.9±0.5

43.2±23 43.1±22.3 1.5±0,.5

31.7±11.7 36.7±18.8 1.2±0.4

30.2±13.2 36.2±20.7 1.2±0.3