Hepatitis B virus-induced defect of monocyte-derived dendritic cells leads to impaired T helper type 1 response in vitro: mechanisms for viral immune escape

Abstract

Dendritic cells (DC) are the most potent antigen-presenting cells and play a central role in the induction of antiviral immune responses. Recently, we have shown that monocyte-derived DC (MoDC) from patients with chronic hepatitis B virus (HBV) infection are functionally impaired. In our present study MoDC from healthy subjects were propagated in vitro and inoculated with HBV particles to investigate the precise mechanisms that underly MoDC dysfunction. T-cell proliferation assays revealed an impaired allostimulatory capacity of HBV-inoculated MoDC (HBV-MoDC) as well as a lower potential of stimulating autologous T cells against a recall antigen in comparison to control-MoDC. Interleukin-2, tumour necrosis factor-α and interferon-γ production by T cells in proliferation assays with HBV-MoDC was significantly lower than with control-MoDC and correlated with lower IL-12 production in HBV-MoDC cultures. The presence of the nucleoside analogue lamivudine (3TC), an inhibitor of HBV replication, restored impaired allostimulatory function of HBV-MoDC and up-regulated major histocompatibility complex class II expression. These results show that HBV infection compromises the antigen-presenting function of MoDC with concomitant impairment of T helper cell type 1 responses. This may play an important role for viral immune escape leading to chronic HBV infection. However, 3TC treatment can overcome HBV-MoDC-related T-cell hyporeactivity and this underscores its important role in enhanced immune responses to HBV.

Introduction

Persistent hepatitis B virus (HBV) infection affects more than 300 million individuals world-wide and represents a major public-health concern because of its propensity to progress to liver cirrhosis and hepatocellular carcinoma. Recovery from acute HBV infection requires both strong humoral and cellular immune responses. Control of HBV infection is associated with strong, polyclonal, multispecific CD8⁺ cytotoxic T lymphocyte and CD4⁺ T-cell-mediated responses to viral proteins.^1,2 Recent publications suggest that high viral load contributes to persistent T-cell hyporesponsiveness,^3,4 thus inhibition of viral replication has been assumed to be essential for recovery of T-cell reactivity.⁴

Dendritic cells (DC) are uniquely well-equipped in antigen-presenting function and act as key players in initiating T-lymphocyte activation against viral agents. An increasing number of viruses, such as hepatitis C virus (HCV),⁵ human immunodeficiency virus,⁶ vaccinia virus,⁷ herpes simplex virus⁸ and measles virus,⁹ may suppress antiviral immune responses by inducing functional paralysis of DC. The precise role of DC in HBV infection has not yet been determined. We have shown recently that monocyte-derived DC (MoDC) from patients with chronic HBV infection are functionally impaired.¹⁰ Treichel et al.^11,12 have found that asialoglycoprotein receptor (ASGPR), expressed on hepatocytes, mediates endocytosis of HBV particles. It has been suggested that the entrance of HBV virions may be the result of attachment of the viral pre-S1-related envelope to ASGPR. Recently Valladeau et al.¹³ showed that immature MoDC express ASGPR for receptor-mediated endocytosis. Based on these observations we established an in vitro model where immature MoDC were pulsed with HBV particles to elucidate the influence of the virus on MoDC properties.

Nucleoside analogues, such as lamivudine [(-)-β-l-2′,3′-dideoxy-3′-thiacytidine (3TC)] are potent and selective inhibitors of HBV replication.¹⁴ 3TC specifically inhibits the hepadnaviral DNA polymerase by competing with the corresponding dNTPs for incorporation into nascent DNA and by acting as a chain terminator after incorporation.¹⁵ It appears to be transported into the cell via pyrimidine nucleoside transporters and is activated enzymatically by different sets of cellular enzymes. 3TC has no stimulatory action on T cells.¹⁶

The influence of 3TC on MoDC has not yet been investigated. We therefore exposed HBV-inoculated MoDC (HBV-MoDC) in vitro to 3TC and observed the effect of 3TC on MoDC phenotype and function.

Materials and methods

Subjects

Peripheral blood was obtained from 19 female and 18 male healthy volunteers after obtaining written informed consent. The mean age of these subjects was 37 years (± 14·7 SD). Twenty-one of the healthy volunteers were vaccinated against HBV and had protective antibodies against hepatitis B surface antigen (anti-HBs), whereas the remaining 16 subjects were naïve. Healthy subjects who had a history of recovery from acute HBV infection or volunteers who suffered from chronic HBV infection were excluded.

HLA-typing

Genomic DNA from whole blood was extracted using a NucleoSpin® Blood Kit (Macherey-Nagel, Düren, Germany). Polymerase chain reaction with sequence-specific primers (PCR-SSP; Institute of Immunology, Department of Transplantation, University of Heidelberg, Germany) was used to amplify human leucocyte antigen (HLA)-DRB1 and -DQB1 as described by Ollerup et al.^17,18 For subtyping of HLA-DRB1 genes allele-specific primer mixes were used (Dynal, Hamburg, Germany).

Cytokines

All cytokines were recombinant human proteins. Final concentrations were interleukin-4 (IL-4), IL-6 (R&D Systems, Wiesbaden-Norderstedt, Germany) and granulocyte–macrophage colony-stimulating factor (GM-CSF; Sigma-Aldrich, Taufkirchen, Germany), 1000 IU/ml each; IL-2 (R&D Systems), 20 IU/ml; tumour necrosis factor-α (TNF-α; R&D Systems), 10 ng/ml; IL-1β, 10 ng/ml and prostaglandin E₂ (PGE₂; Sigma-Aldrich), 1 μg/ml.

Isolation of HBV particles

HBV particles were generated, using sucrose gradient ultracentrifugation, from the serum of highly viraemic patients who were chronically infected with wild-type strains of HBV.¹⁹ The isolation procedure was performed as described by Gerken et al.²⁰ Presence of viral particles was confirmed by transmission electron microscopy (TEM) as described previously¹⁰ and by PCR using HBV-specific primers. Primer MD16 (position 169–186) 5′-GTCCTAGGAATCCTGATG and primer MD19 (position 2718–2737) 5′-GGGTCACCATATTCTTGGGA were used complementary to the surface gene region.

Detection of the precore gene region was performed as described by Wang et al.²¹ with minor modifications.

Generation of MoDC from peripheral blood mononuclear cells (PBMC)

Isolation of PBMC from 37 healthy subjects was performed by Ficoll-separation (Biochrom AG Seromed, Berlin, Germany) as desribed previously.¹⁰ Briefly, PBMC were washed and resuspended at 4 × 10⁶ cells/ml and cultured in serum-free AIM-V medium containing l-glutamine, streptomycin sulphate (50 μg/ml), and gentamicin sulphate (10 μg/ml). After incubation for 1·5 hr at 37°, non-adherent cells were removed by gentle rinsing with RPMI-1640 (Bio Whittaker, Walkersville, MD). Adherent cells were cultured in AIM-V with recombinant GM-CSF and IL-4 for 6 days at 37° in 5% CO₂.

Blood was taken twice from nine of the 37 subjects. In this subgroup isolation of monocytes from PBMC was performed by magnetic depletion of T cells, natural killer cells, B cells, and basophils using a monocyte isolation kit (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany), resulting in > 95% purity of CD14⁺ cells. The highly pure monocyte populations were cultured as described above.

At day 0 of culture, cells were inoculated with viral particles (7·5 million HBV particles/ml culture medium). HBV-MoDC and non-HBV-pulsed control-MoDC were exposed to 3TC (kind gift from Glaxo Wellcome, Research Triangle Parc, NC) continuously from day 1 postplating, at a concentration of 0·25, 0·5, 1, 2, 4, 8, or 16 mm in order to investigate the influence of 3TC at different concentrations on MoDC. Non-3TC-treated MoDC/HBV-MoDC from the same healthy volunteers, propagated in parallel, served as controls. After 6 days of culture, cells were harvested. In eight healthy volunteers only a part of HBV-MoDC and control-MoDC culture plates were harvested on day 6 and the remaining wells were maintained for a further 48 hr with a cytokine cocktail comprising TNF-α, IL-1β, IL-6 and PGE₂ to promote maturation.

Intracellular detection of virus

HBV-MoDC were collected after 6 days of culture, and DNA extraction was performed using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). DNA from HBV-MoDC was analysed with different sets of oligonucleotide primers (Roth, Karlsruhe, Germany) as described above. Detection of viral particles intracellularly was assessed by TEM.¹⁰

Detection of apoptosis in MoDC cultures

Viability of HBV-MoDC and control-MoDC was assessed on day 6 of culture. Cells were stained with fluorescein isothiocyanate (FITC) -conjugated annexin V and propidium iodide (PI) using the annexin V–FITC apoptosis detection kit (BD PharMingen, Heidelberg, Germany). Flow cytometry was performed using a FACSscan Flow Cytometer (BD PharMingen).

Endocytosis assay with dextran

For quantification of endocytosis 1 mg/ml of FITC-dextran (mol. wt 40000) (Sigma-Aldrich) was added to 2 × 10⁵ HBV-MoDC and control-MoDC for 30 min at 37°. As a control, MoDC were stained with FITC-dextran and stored for 30 min at 4°. After incubation cells were washed three times and subjected to FACS analysis.

Quantification of spontaneous IL-12 release in MoDC cultures

Supernatants from MoDC cultures from 32 subjects were collected on day 5 and supernatants from the cytokine-cocktail-stimulated MoDC cultures (n = 8) were collected on day 8. Release of the biologically active IL-12p70 heterodimer was detected using an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, detection limit 5 pg/ml) and following the manufacturer's protocol.

Quantification of IL-12 expression in MoDC by intracellular cytokine staining

Two-colour flow intracellular IL-12 staining of CD11c(+) HBV-MoDC and CD11c(+) control-MoDC with FITC-anti-IL-12 (p40/p70) monoclonal antibodies (mAb) was performed using a Cytofix/Cytoperm Plus™ Kit with GolgiPlug™ (BD PharMingen). Stained cells were subjected to FACS analysis.

Detection of toxicity of 3TC on MoDC cultures

MoDC cultures treated with different 3TC concentrations (see above) were harvested and cells were stained with FITC-conjugated annexin V and PI. Staining and analysis were performed as described above. Untreated MoDC served as controls.

Phenotypic analysis of cultured MoDC

A panel of fluorochrome-labelled mAb (BD PharMingen) was chosen for staining of 3TC (0·5 mm) -treated and untreated HBV-MoDC. The corresponding MoDC-controls were stained in parallel. FITC-labelled mouse anti-human CD3, CD14, CD40, CD1a, CD80 and CD86 mAb, and phycoerythrin-conjugated mouse anti-human HLA-DR, CD11c, CD19, and CD83 mAb were used. Staining was performed as described previously.¹⁰

Allogenic T-cell stimulatory activity of HBV-MoDC and controls with and without 3TC treatment.

Mixed lymphocyte reaction (MLR) was performed to compare the allostimulatory capacity of control-MoDC and HBV-MoDC from 37 healthy volunteers. Furthermore, MLR was set up in parallel with 3TC-pulsed control-MoDC and 3TC-pulsed HBV-MoDC as stimulators. PBMC as allogeneic responder cells from healthy volunteers were isolated from peripheral blood by density gradient centrifugation using Ficoll–Hypaque. MLR was performed as described previously.¹⁰

PBMC from 10 healthy volunteers were exposed to HBV for 6 days in vitro. Subsequently, MLR was performed with irradiated control-MoDC as stimulator cells and HBV-pretreated PBMC to investigate the influence of HBV on responder PBMC. Control MLR experiments were performed with non-virus-inoculated PBMC from the same subjects.

T helper cell type 1 (Th1)/Th2 cytokine quantification

Supernatants from MLR from 30 subjects were collected on day 5 for cytokine quantification. Cytokines were measured by flow cytometry using the ‘Human Th1/Th2 Cytokine Cytometric Bead Array (CBA) Kit’ (BD PharMingen). The assay sensitivities for IL-2, IL-4, IL-5, IL-10, TNF-α and interferon-γ (IFN-γ) are 6·6, 6·5, 2·8, 4·7, 4·3 and 15·6 pg/ml, respectively.

Generation of autologous antigen-specific T cells

MoDC from healthy subjects who were vaccinated with a commercially available tetanus toxoid (TT) were generated and inoculated with viral particles as described above. Autologous HBV-MoDC and control-MoDC cultures were pulsed with TT (RIVM, Bilthoven, the Netherlands) at 7·5 fast-fatigable units (FF)/ml final concentration at day 1 of culture. Unpulsed HBV-MoDC and control-MoDC cultures from the same healthy volunteers, propagated in parallel, served as controls. MoDC (4 × 10⁴ cells/well) were irradiated (3000 rads) and incubated with autologous responder PBMC (2 × 10⁵ cells/well) as co-cultures supplemented with 2 mm l-glutamine, 2 × 10⁻⁵ m 2-mercaptoethanol and IL-2 (20 IU/ml) at various stimulator : responder ratios. In addition, PBMC (2 × 10⁵ cells/well) were plated in round-bottomed wells with AIM-V medium supplemented with 2 mm l-glutamine, 2 × 10⁻⁵ m 2-mercaptoethanol, IL-2 (20 IU/ml) and phytohaemagglutinin (1% v/v, Sigma-Aldrich) for total T-cell stimulation.

Statistical methods

The non-parametric Mann–Whitney Rank Sum Test with two-tailed P-values, or for paired samples, the Wilcoxon Signed Ranks Test, were used. A P-value of < 0·05 was considered statistically significant. Data from MLR and cytokine detection as well as from apoptosis and endocytosis assays are summarized with means ± SEM.

Results

Virus is present in HBV-MoDC from healthy volunteers

DNA encoding a highly conserved motif of the pre-core and surface region could be detected in HBV-MoDC cultures from all subjects (Fig. 1). Moreover, the presence of viral particle formation (Dane and core particles) in HBV-MoDC has been confirmed by TEM (data not shown).

Figure 1.

PCR was conducted on DNA from HBV-MoDC propagated in GM-CSF + IL-4. PCR was performed in all 37 healthy volunteers. This figure shows representative results of HBV-MoDC from 11 subjects. (a) All were positive for the surface region and (b) all were positive for the precore region. HBV-infected liver tissue and control-MoDC served as positive and negative controls, respectively.

Inoculation of MoDC with HBV does not induce cell death or apoptosis

After 6 days of culture in GM-CSF/IL-4-supplemented medium, the yield of viable MoDC inoculated with and without HBV was similar as determined by trypan blue dye exclusion analysis. MoDC deriving from highly enriched monocytes were 55–80% pure based on the expression of CD11c⁺, HLA-DR⁺, CD1a⁺, CD40⁺, CD80⁺, CD86⁺ cells and lacking expression of lineage markers for T cells (CD3), B cells (CD19) and monocytes (CD14). The percentage of intact viable cells (annexin V⁻ PI⁻) at day 6 of culture did not differ significantly in either group (P = 0·9, n = 10). The percentage of early apoptotic cells was 15·0% in the HBV-MoDC group as compared to 14·4% in the control group (P = 0·9). The percentage of cells that underwent apoptotic death or that died as a result of a necrotic pathway was 10·0% in HBV-MoDC cultures and 10·5% in the control group (P = 0·9).

Mannose receptor-mediated endocytosis is not affected by exposure of MoDC to HBV

The decrease in antigen uptake has been used to monitor the maturation of HBV-MoDC and controls. FACS analysis revealed that HBV-MoDC and control-MoDC internalize comparable amounts of FITC-dextran [mean fluorescence intensity (MFI): 99·5 ± 8·2 and 99·0 ± 10·6, P = 0·97, n = 7].

HBV-MoDC exhibit impaired spontaneous IL-12 release

Cytokine detection by ELISA showed that spontaneous IL-12 p70 release of HBV-MoDC is significantly lower as compared to control-MoDC (Fig. 2a). Similar results of IL-12 p70 levels have been obtained in the subgroup where MoDC cultures derived from highly purified monocytes (15·9 pg/ml ± 2·3 versus 27 pg/ml ± 4·9 in controls, P < 0·05).

Figure 2.

(a) Spontaneous release of IL-12 in GM-CSF/IL-4-supplemented HBV-MoDC cultures is significantly lower in comparison to that in control-MoDC cultures (n = 32) as assessed by ELISA. The results are expressed as mean values ± SEM. (b) MLR supernatants were assessed by FACS analysis for production of Th1 and Th2 cytokines. IL-2, IFN-γ and TNF-α secretion in MLR with HBV-MoDC were significantly lower as compared to controls. The results are expressed as mean values ± SEM, P-values are indicated in case of statistical significance.

These findings have also been confirmed by quantification of IL-12 as determined by intracellular cytokine staining. The percentage of HBV-MoDC producing intracellular IL-12 was significantly lower in comparison to controls (19·5% ± 4·2 versus 39·8% ± 5·8, n = 18, P < 0·05) as measured by flow cytometry. Similar to our previous observations¹⁰ significantly higher levels of IL-12p70 were secreted by MoDC in both groups after addition of the cytokine cocktail for induction of terminal MoDC maturation. The levels of IL-12p70 production between control-MoDC and HBV-MoDC were not significantly different after cytokine-induced maturation (968 pg/ml ± 62·6 versus 917 pg/ml ± 89·2, P = 0·65), indicating a certain potential of recovery of HBV-MoDC.

T-cell-induced production of IL-2, IFN-γ and TNF-α is reduced in MLR with HBV-MoDC as stimulators

The results of cytokine measurements by FACS analysis are shown in Fig. 2(b). IL-2, IFN-γ and TNF-α secretion were significantly lower in MLR elicited with HBV-MoDC as compared to those from control-MoDC. The concentrations of IL-10, IL-4 and IL-5 in MLR supernatants were comparable in both groups.

Allostimulatory function of HBV-MoDC is significantly reduced as compared to controls

HBV-MoDC were propagated from highly enriched monocytes of 37 healthy volunteers in response to GM-CSF and IL-4 and overall results showed a significantly lower T-cell stimulatory activity as compared to control-MoDC cultures from the same subjects (Fig. 3). This was statistically significant at stimulator : responder ratios of 1 : 10 and 1 : 20. Consistent MLR results have been obtained in the subgroup with stimulator MoDC derived from highly purified monocytes (mean counts in MLR with control-MoDC and HBV-MoDC: 134 704 counts per minute (c.p.m.) ± 14 687 and 77 362 c.p.m. ± 10745; 112 985 c.p.m. ± 16 689 and 65 299 c.p.m. ± 13 575; and 99 464 c.p.m. ± 15 286 and 49 302 c.p.m. ± 12 512, respectively, at stimulator : responder ratios of 1 : 10, 1 : 20 and 1 : 40). Results in the subgroup were statistically significant even at the lowest stimulator : responder ratio (P < 0·05). MLR results revealed a strong correlation between reduced T-cell stimulatory capacity and low IL-12 as well as reduced Th1 cytokine levels (Table 1). These results were consistent in repeated experiments. The T-cell response in MLR with HBV-pulsed responder PBMC from healthy volunteers and unpulsed allogeneic MoDC as stimulators was comparable with that detected in MLR with non-HBV-exposed control-PBMC (P = 0·89, P = 0·97 and P = 0·20 at stimulator : responder ratios of 1 : 10, 1 : 20 and 1 : 40, respectively).

Figure 3.

Allogeneic T-cell proliferation induced by HBV-MoDC was measured in MLR. HBV-MoDC exhibit significantly impaired allostimulatory capacity compared to control-MoDC. Results were significant at stimulator : responder ratios of 1 : 10 (P < 0·001) and 1 : 20 (P < 0·005). The results are expressed as mean c.p.m. ± SEM from 37 subjects.

Table 1.

Correlation between MLR activity and cytokine milieu induced by HBV-MoDC

MLR activity† (%)	IL-12 (pg/ml)‡ (mean ± SEM)	IFN-γ (pg/ml)§ (mean ± SEM)	TNF-α (pg/ml)§ (mean ± SEM)	IL-2 (pg/ml)§ (mean ± SEM)
25–59% (n = 13)	11 ± 4 (44%)	2914 ± 361 (57%)	84 ± 19 (63%)	64 ± 21 (63%)
60–98% (n = 17)	12 ± 2 (48%)	3762 ± 328 (73%)	109 ± 10 (82%)	75 ± 22 (74%)
100% (n = 30)*	25 ± 5 (100%)*	5121 ± 490 (100%)*	133 ± 17 (100%)*	102 ± 24 (100%)*

^†T-cell allostimulatory capacity of HBV-MoDC in comparison to controls(*) cultured in parallel.

^‡IL-12 production of HBV-MoDC in comparison to controls (*).

^§Cytokine production in MLR induced by HBV-MoDC in comparison to controls (*).

HBV-MoDC reveal a lower potential of stimulating autologous T cells against TT

Stimulatory responses against autologous recall antigen-specific T cells were significantly lower in the HBV-MoDC group as compared to control MoDC (Fig. 4). T-cell responses from vaccinated (n = 8) and from naïve subjects (n = 7) were not significantly different.

Figure 4.

PBMC responses against a recall antigen in proliferation assays with autologous HBV-MoDC or control-MoDC which were pulsed with TT. Unpulsed autologous HBV-MoDC and control-MoDC from the same healthy volunteers served as controls. PBMC were activated with PHA for maximal T-cell stimulation. T-cell responses induced by HBV-MoDC were significantly lower as compared to the control group (n = 15). Bars indicate [³H]thymidine incorporation (mean c.p.m. ± SEM) into autologous PBMC.

HLA-DR expression is significantly increased on 3TC-treated HBV-MoDC as compared to untreated controls

3TC-treated and untreated HBV-MoDC and controls that were not exposed to virus were subjected to immunophenotypic analysis by flow cytometry. MoDC cultured in GM-CSF/IL-4 displayed the phenotype of differentiating but not yet mature cells with low MFI of CD83 and CD80, moderate expression of CD86 and CD40. In the non-3TC-exposed MoDC cultures the MFI of CD80 was significantly lower in HBV-MoDC as compared to control-MoDC (Table 2). The MFI of HLA-DR was significantly higher in HBV-MoDC exposed to 0·5 mm 3TC as compared to untreated HBV-MoDC (Table 2). A similar trend for HLA-DR expression could be detected in the control group under drug exposure. The MFI of CD86, CD40, CD11c, CD1a and CD83 was not significantly different in the various experimental groups. As a result of the gating strategy used to evaluate the expression of HLA-DR and co-stimulatory molecules on MoDC no differences were observed between MoDC which derived from highly enriched monocytes and those deriving from purified monocytes.

Table 2.

Phenotypic characteristics of HBV-MoDC and control-MoDC cultured in GM-CSF and IL-4

		MFI (mean ± SEM)
Antigen		Lamivudine(–)	Lamivudine (+)
CD80	HBV-MoDC	13·1 ± 0·9*	16·1 ± 1·4
	Controls	16·8 ± 2·0	17·8 ± 1·8
CD86	HBV-MoDC	43·2 ± 3·9	50·3 ± 6·0
	Controls	44·8 ± 3·8	44·8 ± 5·6
HLA-DR	HBV-MoDC	212·1 ± 27·7	346·8 ± 53·9**
	Controls	245·1 ± 32·6	344·0 ± 57·0
CD40	HBV-MoDC	58·0 ± 5·1	58·6 ± 7·9
	Controls	55·6 ± 4·5	59·3 ± 7·1
CD83	HBV-MoDC	10·0 ± 0·8	9·4 ± 0·9
	Controls	10·0 ± 0·6	8·4 ± 0·5

^*P < 0·05 versus controls [lamivudine(–)].

^**P < 0·05 versus HBV-MoDC [lamivudine(–)].

Allostimulatory capacity of HBV-MoDC is significantly enhanced by treatment with 3TC

Treatment of HBV-MoDC with 0·25 and 0·5 mm of 3TC for 6 days markedly enhanced their allostimulatory function as compared to untreated HBV-MoDC (P < 0·05) and approached levels that were similar to those of controls (Fig. 5). Consistent results have been obtained in the subgroup that derived from monocytes with prior depletion of contaminating cells [mean counts in MLR with 3TC-exposed (0·5 mm) HBV-MoDC: 130 885 c.p.m. ± 13 750, as compared to 77 362 c.p.m. ± 10 745 in untreated HBV-MoDC at a stimulator : responder ratio of 1 : 10, P < 0·05).

Figure 5.

Allogeneic T-cell proliferation induced by 3TC-treated HBV-MoDC and controls was measured in MLR from 27 subjects. HBV-MoDC pulsed with 0·25 and 0·5 mm 3TC exhibited significantly enhanced allostimulatory capacity compared to untreated HBV-MoDC (P < 0·05). Results (mean ± SEM) of [³H]thymidine incorporation are shown at a stimulator : responder ratio of 1 : 10 and are expressed as a percentage of MLR activity induced by untreated control-MoDC that are represented in the first bar as 100%.

Treatment of control-MoDC with higher concentrations of 3TC (2 and 4 mm) led to a decrease in the proliferative response, a phenomenon that might be because of increased cytotoxicity of 3TC in higher concentrations. This might also explain the observation that the extent of improvement of HBV-MoDC function could not be observed in cultures exposed to higher 3TC concentrations, such as 2 and 4 mm. Moreover, we did not observe an increase of IL-12 secretion induced by 3TC treatment at any concentration (data not shown).

Cytotoxic effects of 3TC on MoDC cultures

In MoDC cultures the majority of cells were viable, as indicated by low staining by both annexin V and PI. Results from MoDC experiments exposed to various 3TC concentrations are expressed as the ratio between the percentage of early apoptotic cells in 3TC-treated MoDC cultures as compared to untreated controls (Fig. 6a). When cells were treated with 3TC (at concentrations of 0·25, 0·5, 1, 2, 4, 8 and 16 mm) the percentage of early apoptotic cells in MoDC increased with augmentation of the drug dose. Comparison of the results from days 2 and 4 demonstrates that induction of early apoptosis is enhanced by prolonging drug exposure time. Percentage of late apoptotic or dead cells is markedly increased at higher 3TC concentrations on day 2 of MoDC culture (Fig. 6b). Multiple experiments demonstrated that MoDC cultures exposed continuously to 8 mm of 3TC for 4 days revealed approximately a 35% reduction of cell viability, whereas cell viability was reduced approximately to 50% at a concentration of 16 mm.

Figure 6.

(a) Apoptosis index at different 3TC concentrations represents the ratio between the percentage of cells that underwent early apoptosis in 3TC-treated MoDC cultures (n = 10) and that of untreated control-MoDC (n = 10). Results show that 3TC increases percentage of cells that undergo early apoptosis as compared to unpulsed controls. The influence of 3TC on MoDC cultures is dose dependent, as shown in the graphs.(b) The ratio between 3TC-treated late apoptotic or dead cells and untreated controls is similar in cultures treated with 0·25, 0·5, 1, 2 and 4 mm 3TC. Induction of late apoptosis or cell death is markedly increased in MoDC cultures treated with 8 mm and 16 mm 3TC at day 2 after infection.

Discussion

The precise mechanism of how viral infections evade the immune response and lead to chronic infection still requires elucidation. Viruses have developed a broad range of mechanisms to escape the host immune response.^22,23 Recently, it has become evident that many viral immune escape mechanisms specifically target DC function.^5–9 Previous results from our studies in patients with chronic HBV infection have shown that DC are susceptible to HBV and that the presence of intracellular HBV particles is associated with impaired allostimulatory function of MoDC.¹⁰

Although the HBV in vitro model established in this study represents an unphysiological situation and does not fully reflect the overall in vivo situation, it serves as an appropriate tool to elucidate further the influence of HBV on antigen-presenting cells under ‘standardized’ conditions. HBV is a non-cytopathic virus, thus comparable percentages of dead and apoptotic cells have been observed in HBV-inoculated MoDC and controls. Inoculation of highly enriched MoDC cultures with HBV particles correlated with poor stimulation of T cells in MLR experiments. HBV-pulsed and unpulsed responder PBMC from healthy volunteers showed a comparable proliferative response in MLR with normal control-MoDC. These results suggest that low MLR activity is not the result of a functional defect or of the anergy of responder PBMC but rather of a defect of allostimulatory capacity of HBV-MoDC. This is supported by data from experiments in a subgroup where MLR was performed with highly purified populations of MoDC. Results in this subgroup were consistent with our findings in MLR experiments with highly enriched MoDC and showed an even more pronounced impaired MLR activity at the lowest stimulator : responder ratio. Furthermore, we found that HBV-MoDC induce a lower T-cell response towards TT as recall antigen in the autologous setting, indicating that a generalized altered function of antigen-presenting capacity may occur during HBV infection. Kanto et al.²⁴ did not find a reduced T-cell response towards a recall antigen during HCV infection, suggesting not a reduced immunodeficiency state but rather a distinct viral mechanism of immune evasion. Although generalized immunosuppression is not a clinical characteristic of chronic viral hepatitis it has been reported recently that patients with chronic HCV infection reveal a surprisingly high rate of primary non-response to standard HBV and hepatitis A virus (HAV) vaccines.^25,26 Analysing and subdividing MLR data from HBV-MoDC cultures in healthy volunteers who were vaccinated against HBV and showed protective anti-HBs serum titres to those who were naïve we did not find a significant difference between the MLR results from both groups. In our study we observed a wide diversity in the strength of functional alteration of HBV-MoDC derived from 37 healthy volunteers. Markedly reduced T-cell stimulatory capacity corresponded to low IL-12 production in HBV-MoDC cultures and low IFN-γ, TNF-α and IL-2 secretion in MLR. Moreover, in five cases the proliferative response of T cells did not increase in the presence of increasing concentrations of stimulator HBV-MoDC. Higher allostimulatory activities in the experimental group correlated with higher IL-12 and Th1 cytokine secretion, although not to the extent observed in controls. This is in accordance with a recent report by Lohr et al.²⁷ where a reduced T helper cell induction by autologous dendritic cells in patients with chronic HBV infection could be restored by exogenous IL-12. The importance of Th1 cells for virus clearance has been demonstrated in patients with HBV infection as well as in animal models.^28–30 However, the role of IL-12 in viral infections remains controversial and some earlier reports indicate that IL-12 may not be necessary for induction of Th1 response in viral infection.^31,32

Ferrari et al.³³ have shown that eradication of HBV infection in self-limited acute hepatitis is associated with a strong HLA-class-II-restricted, CD4-mediated response to HBV antigens. As CD4 T-cell-mediated responses critically depend on production of Th1 cytokines such as IFN-γ and TNF-α, imbalance of Th1 and Th2 appears to be important in chronic viral infections.^28,34,35 Studies in the transgenic mouse model³⁶ as well as in the chimpanzee model³⁷ have shown that most viral DNA is eliminated from the cytoplasm of hepatocytes by a non-cytolytic antiviral effect of IFN-γ and TNF-α released within the liver. IFN-γ and TNF-α have been reported to suppress replication of HBV in vitro³⁸ and in vivo.³⁹ TNF-α represents the major cytokine involved in acute HBV-induced liver disease due to T-cell activation.⁴⁰ This is supported by the data of Hohler et al.⁴¹ showing that defective virus clearance in patients with chronic HBV infection is associated with TNF-α promoter polymorphism.

Levels of Th2 cytokines, such as IL-4 and IL-5 in MLR performed with HBV-MoDC did not differ significantly when compared to MLR with control-MoDC. Similar to our previous observations in HBV patients, we did not observe an IL-10-related inhibitory effect on T-cell activity. Taken together, there is evidence that the presence of HBV in MoDC leads to an impaired Th1 response.

Various authors describe a positive correlation between HLA-DR *07 haplotypes and increased susceptibility to viral infection.⁴² Others suggest that subjects with HLA-DRB1*1301 and 1302 alleles have shown protection from chronic HBV infection.⁴³ In this study, we did not find a correlation between the extent of reduced allostimulatory function and HLA type (data not shown).

Investigating further the underlying mechanisms of functional MoDC impairment we observed a trend towards lower HLA-DR expression on HBV-pulsed MoDC as compared to control-MoDC. HLA-DR represents the most important stimulatory determinant in MLR. Therefore, lower expression of HLA-DR may additionally contribute to reduced allostimulatory activity of MoDC. The influence of viral infection on major histocompatibility complex (MHC) class II expression has already been reported in the literature. For instance, low MHC class II expression on DC resulting in defective lymphocyte activation has been described in transgenic HBV mice.⁴⁴ Similar results have been described in human monocytes infected with measles virus⁴⁵ and more recently in DC infected with cytomegalovirus.⁴⁶ In accordance with our previous observations in patients chronically infected with HBV,¹⁰ in this in vitro study we observed a lower expression of CD80 on HBV-MoDC in comparison to controls. Viral interference with antigen presentation capacity has already been reported for many viruses⁴⁷ and may also represent a mechanism of how HBV evades the immune response.

Boni et al.⁴ reported that 3TC-related reduction of viral load can overcome T-cell hyporesponsiveness in patients with chronic HBV infection. Their study suggested, that HBV-related proteins interfere with the general function of the immune system. Viruses are able to block the presentation of endogenous antigens by antigen-presenting cells as a result of the expression of viral proteins interfering with antigen presentation (VIPRs).⁴⁷ In our study, we investigated whether HBV-related MoDC dysfunction can be restored by 3TC-induced inhibition of viral replication. To date, the antiviral effect of 3TC has been investigated in primary hepatocyte cultures⁴⁸ and various cell lines.⁴⁹ Our results indicate that 3TC does not exert a direct stimulatory effect on T cells but rather enhances allostimulatory capacity of HBV-MoDC that correlates with an increased MHC class II expression. It has been reported that VIPRs are capable of interfering with expression and regulation of MHC class II molecules.⁵⁰ Therefore, we are currently investigating the role of signal transduction cascades, such as the Jak/Stat pathway, to elucidate further mechanisms of HBV-related dysfunction and 3TC-mediated up-regulation of MHC class II. The observed effect of 3TC on HBV-MoDC is dose dependent and has been shown to be less pronounced by increasing the drug concentrations, a phenomenon that might be the result of increased cytotoxicity of 3TC in higher concentrations. Conclusively, there is evidence that 3TC does not only represent a potent inhibitor of viral replication but seems to possess additional immunomodulatory activity on MoDC. However, further studies are required to investigate the precise mechanisms of how 3TC restores HBV-induced MoDC dysfunction. Modulation of MoDC function directly in vivo or ex vivo may represent a promising approach to overcome T-cell hyporeactivity in chronically HBV-infected patients and may have important implications for DC-based immunotherapy of HBV infection.

Abbreviations

anti-HBs

antibodies against hepatitis B surface antigen

ASGPR

asialoglycoprotein receptor

DC

dendritic cells

GM-CSF

granulocyte–macrophage colony-stimulating factor

HAV

hepatitis A virus

HBV

hepatitis B virus

HBV-MoDC

HBV-inoculated MoDC

HCV

hepatitis C virus

IFN

interferon

IL

interleukin

mAb

monoclonal antibodies

MFI

mean fluorescence intensity

MHC

major histocompatibility complex

MoDC

monocyte-derived DC

MLR

mixed lymphocyte reaction

PGE₂

prostaglandin E₂

PBMC

peripheral blood mononuclear cells

PCR-SSP

polymerase chain reaction with sequence-specific primers

3TC

[-]-beta-L-2′,3′-dideoxy-3′-thiacytidine, lamivudine

Th1

T helper cell type 1

TNF-α

tumour necrosis factor-α

TEM

transmission electron microscopy

TT

tetanus toxoid

VIPRs

viral proteins interfering with antigen presentation