Hanns F. Löhr, MD I Department of Internal Medicine Johannes Gutenberg-University Langenbeckstrasse 155131 Mainz, Germany. E-mail: firstname.lastname@example.org
Insufficient stimulatory capacities of autologous dendritic cells (DC) may contribute in part to impaired T cell stimulation and therefore viral persistence in patients with chronic hepatitis B virus (HBV) infection. In order to characterize the antigen presenting functions of DC from chronic HBV carriers and controls antigen specific T cell responses were analysed. CD34+ peripheral blood progenitor cells were differentiated to immature DC in the presence of GM-CSF, IL-6/IL-6R fusion protein and stem cell factor. Proliferative CD4+ T cell responses and specific cytokine release were analysed in co-cultures of DC pulsed with HBV surface and core antigens or tetanus toxoid and autologous CD4+ T cells. Cultured under identical conditions DC from chronic HBV carriers, individuals with acute resolved hepatitis B and healthy controls expressed similar phenotypical markers but chronic HBV carriers showed less frequent and weaker HBV antigen specific proliferative T helper cell responses and secreted less interferon-γ while responses to the tetanus toxoid control antigen was not affected. Preincubation with recombinant IL-12 enhanced the HBV specific immune reactivities in chronic HBV patients and controls. In conclusion, the weak antiviral immune responses observed in chronic hepatitis B may result in part from insufficient T cell stimulating capacities of DC. Immunostimulation by IL-12 restored the HBV antigen specific T cell responses and could have some therapeutical benefit to overcome viral persistence.
The reasons for viral persistence that develops in 5–10% of patients infected with the hepatitis B virus (HBV) are still un-known. The induction of potent immune responses seems to be decisive for whether a host can regulate and control noncytopathic infections like HBV or not [1,2]. In HBV transgenic mice the adoptive transfer of class I-restricted cytotoxic T cells (CTL) specific to the HBV surface antigen (HBsAg) resulted in acute immune mediated hepatitis therefore it appears that CTL are capable of destroying virus infected liver cells and thus may contribute to viral clearance . Antiviral CTL and HBV core antigen (HBcAg) specific class II restricted T helper (Th) cell responses correlate with viral elimination in patients with acute, self-limited hepatitis B. In addition, there is evidence that cytokines, like interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) are crucial for the noncytolytic inhibition of viral replication observed in the early phase of HBV infection before liver injury occurs [4–8]. This might indicate that the early and strong induction of IFN-γ secreting HBV specific Th1 cells might be critical for the clinical outcome by means of cytokine release, CTL activation as well as neutralizing anti-HBs antibody production [9,10].
HBV specific CTL as well as Th1 cells have also been demonstrated in blood and liver tissue of patients with chronic hepatitis B, but compared to patients with acute self-limited HBV infection the cellular immune reactivities are weak [7,10–12]. Although associations of immunogenetical, viral and environmental factors with chronic HBV infection have been described, their role in the immunopathogenesis of viral persistence is unknown [13–15]. We demonstrated recently that the therapeutic intradermal vaccination of patients with chronic hepatitis B with HBV surface antigen (HBsAg) resulted in lower specific Th1 cell and CTL responses than in healthy individuals [16,17]. These data suggested that impaired antigen processing or presentation by skin dendritic cells (DC), the most potent ‘professional’ antigen presenting cells [18–20], might be the cause of reduced antiviral T cell responses. To test this hypothesis we generated DC from peripheral blood progenitor cells of patients with chronic hepatitis B, individuals with acute resolved hepatitis B and healthy HBV seronega- tive controls and analysed the vigor and the cytokine release of antigen specific T helper cell responses.
MATERIALS AND METHODS
Dendritic cells and autologous T cells were prepared from 19 patients with chronic HBV infection characterized by persisting serum HBsAg and anti-HBc antibodies. Twenty-four healthy blood donors without the serological signs of HBV infection and four anti-HBs antibody and anti-HBc antibody positive blood donors with acute resolved HBV infection served as controls. Individuals with acute resolved hepatitis B were characterized by normal alanine aminotransferase (ALT) levels and absent HBV-DNA in the serum. Two chronic HBV patients had histologically confirmed liver cirrhosis and four patients had chronic inactive hepatitis B with negative HBV-DNA and normal ALT levels. Seven patients with chronic hepatitis B had been previously treated with IFN-α but all patients were untreated for at least six months before being enrolled in the study. Coinfections with the hepatitis C virus was serologically confirmed in one patient with chronic hepatitis B. No study participant was coinfected with the delta virus or human immunodeficiency virus.
The study has been approved by a local ethical committee and all study participants gave their informed consent to the experiments.
Generation of dendritic cells from CD34+ progenitor cells
CD34+ haematopoietic progenitor cells were isolated using immunomagnetic beads (MACS, Milteny, Bergisch Gladbach, Germany) from 100 ml heparinized peripheral blood of patients with chronic hepatitis B or healthy controls. The purity of re-covered CD34+ cells was routinely determined as 85–95% by flow cytometry. The CD34+ cells were cultured at 106 cells/well for 14–16 days under serum free conditions with medium (X-VIVO 15, Biowhittaker Inc., Walkersville, USA) supplemented with L-glutamine (300 μg/ml). In addition, the growth medium con-tained 100 ng/ml stem cell factor (SCF; Strathmann Biotech, Hannover, Germany), 100 ng/ml human granulocyte-macrophage colony stimulating factor (GM-CSF, Leukomax, Novartis, Nürnberg, Germany) and 10 ng/ml IL-6/IL-6R fusion protein (hyper-IL-6; 13).
Flow cytometry analysis
Flow cytometry was used to analyse the purity of progenitor cells and T cells after isolation with immunomagnetic beads. Briefly, 5 × 104 cells were washed and incubated with FITC-conjugated monoclonal antibodies (anti-CD34 or anti-CD4, Pharmingen BD, Heidelberg, Germany) for 45 min on ice. FITC- or PE-conjugated IgG isotype-matched antibodies (Dianova, Hamburg, Germany) were used as controls. Fluorescence analyses were performed on a FACScan using the Cell Quest software (B & D, Heidelberg, Germany).
For the analysis of differentiation markers 5 × 105 DC were incubated with 5 μl FITC-conjugated anti-CD1a, anti-CD14 and anti-HLA-DR (Dianova), anti-CD80 (Pharmingen), anti-CD86 (Diaclone Hoelzel, Köln, Germany) or PE-conjugated anti-IL-12RβI antibodies.
Antigens and synthetic peptides
Recombinant major HBsAg (adw subtype) and HBcAg were purchased from Diasorin (Düsseldorf, Germany). Tetanus toxoid (TT) used as irrelevant control antigen was a gift from Dr Blachkolb (Chiron Behring, Marburg, Germany).
To test the T cell stimulating capacities 5 × 105 DC were incubated for four hours together with medium or individual antigens at a concentration of 20 μg/ml. The DC were irradiated at 3000 rad, washed and brought in triplicates to 96-round bottom well-microtiter plates (Greiner, Frickenhausen, Germany) at a concentration of 1 × 104/well together with 1 × 105 isolated autolo-gous CD4+ T cells. Anti-CD3/anti-CD28-stimulated (1 μg/ml, orthoclone-3; anti-LeuTM28 Pure, Becton & Dickinson, San Jose, USA) cells served as positive and unstimulated cells as negative controls. As an additional control autologous CD14+ blood monocytes preincubated as described above were used to stimulate CD4+ T cells. After six days of incubation at 37°C in a humidified atmosphere the plates were pulsed with 0·25 μCi/well 3H-thymidine and 20 h later the incorporated radioactivity was measured by liquid scintillation technique (Beta-Plate, LKB Wallac). Data are presented as mean stimulation indices (SI) and standard errors of the means (±SEM) of triplicates calculated from the ratios of incorporated radioactivities (counts per minute, cpm) of T cell cultures in the presence or absence of antigen. SI values higher than three were considered positive.
Cytokine release assay
To analyse the cytokine release 50 μl of the supernatants from medium, HBsAg, HBcAg or TT stimulated T cell and DC cultures were harvested after 24 h for IL-10 and after 48 h for IFN-γ analysis. All supernatants were stored immediately at −20°C until tested. IL-10 and IFN-γ concentrations were measured using commercially available ELISA kits with a detection limit of 1·2 or 2·0 pg/ml, respectively, according to the manufacturers instructions (Pelikline CompactTM, Central Laboratory, Amsterdam, the Netherlands).
In selected experiments it was analysed whether the T cell hyporesponsiveness could be overcome by the addition of interleukin-12 (IL-12). DC preparations were incubated for the last two days of differentiation (day 14–16) with recombinant IL-12 in two concentrations (100 and 1000 pg/ml) together with HBcAg (20 μg/ml). The DC were washed extensively to remove IL-12 and other cytokines from the cell cultures before the T cells were added and the proliferation assay was performed.
Dendritic cell culturing
Samples of 100 ml heparinized blood were collected from 19 patients chronically infected with the hepatitis B virus (HBsAg and anti-HBc positive), four controls with acute resolved HBV infection (anti-HBs and anti-HBc positive) and 24 healthy blood donors (HBsAg and anti-HBc negative). CD34+ pro-genitor cells were isolated using immunomagnetic beads from peripheral blood mononuclear cells (PBMC; mean recovery 0·1–0·2%). Culturing the progenitor cells in the presence of GM-CSF, hyper-IL-6 and stem cell factor for 14–16 days resulted in the differentiation to proliferating immature DC. Flow cytometric analysis revealed that these DC expressed CD1a (30–90%), CD86 (29–98%) and HLA-DR (28–72%) surface markers but had lost the stem cell marker CD34 (Fig. 1). No differences were detected between DC derived from chronic HBV patients and controls. The preparations contained virtually no T cells or monocytes as verified by the absence of CD3 or CD14 staining (data not shown).
T cell stimulation by antigen-pulsed autologous dendritic cells
In order to analyse the T cell stimulating capacities, DC pulsed with various concentrations of HBsAg, HBcAg or TT were cultured together with autologous CD4+ T cells derived from frozen PBMC samples. In selected experiments the inhibition of pro- liferative T cell responses by preincubation with anti-HLA DR antibodies (L243, ATCC) indicated class II-restricted specific T cell stimulation (Fig. 2).
In response to HBcAg only 8 of 19 (42%) chronic HBV carriers showed positive T cell stimulation indices (SI > 3·0) as compared to 17 of 24 (71%) healthy controls and three of the four individuals with acute resolved HBV infection (75%). Furthermore, the vigors of the HBcAg specific T cell responses were lower in patients with chronic HBV infection compared to healthy HBV naïve controls (SI 3·7 ± 1·1 versus 10·2 ± 3·6; P < 0·05) and individuals with resolved hepatitis B (SI 6·4 ± 2·0; P = n.s. Fig. 3).
Similar results were obtained after HBsAg specific stimulation, although specific proliferative T cell responses were less frequently observed in all study groups. In detail, only two of 11 tested chronic HBV carriers (18%), two tested individuals with resolved HBV infection (100%) and six of 12 tested healthy HBV seronegative controls (50%) showed relevant T cell reactions (SI > 3). Again, the vigors of the HBsAg specific T cell responses were lower in patients with chronic HBV infection than in healthy seronegative controls (SI 1·5 ± 0·3 versus 14·1 ± 6·5; P < 0·05) and individuals with acute resolved HBV infection (SI 9·5 ± 2·5; P < 0·001; Fig. 3).
However, strong T cell proliferation was observed when DC were pulsed with tetanus toxoid in all but one of the 17 tested HBV carriers as well as in seven tested HBV naïve healthy controls and two individuals with acute resolved HBV infection. Furthermore, the vigors of the T cell stimulation were not sig- nificantly different between HBV naïve healthy controls and chronic HBV carriers (SI: 35·6 ± 13·4 versus 29·3 ± 7·6, P = n.s.) or individuals with acute resolved HBV infection (SI: 35·6 ± 13·4 versus 18·4 ± 8·9; P = n.s.; Fig. 3).
In control experiments of CD14+ monocytes used as anti- genpresenting cells and autologous CD4+ T cells the majority of chronic HBV patients and seronegative controls showed TT- specific T cell stimulation while 5 chronic HBV patients showed weak reaction to HBcAg (data not shown).
In vitro cytokine release of DC-stimulated CD4+ T cells
The functional capacities of T helper cells stimulated by auto-logous DC were analysed by their cytokine release in response to HBV and TT antigens. Upon HBcAg stimulation CD4+ T cells from 19 tested chronic HBV carriers showed a reduced IFN-γ secretion compared to 14 tested healthy controls (110 ± 55 pg/ml versus 585 ± 282 pg/ml; P = 0·05) but similar levels of IL-10 production (210 ± 53 versus 251 ± 94 pg/ml; P = n.s.). Furthermore, the two tested individuals with acute resolved HBV infection secreted more IFN-γ (521 ± 264 pg/ml; P > 0·02) and IL-10 (1055 ± 119 pg/ml, P = 0·0008) than chronic HBV carriers (Fig. 4).
Following stimulation with HBsAg the weak IFN-γ and IL-10 secretion by T cells was not significantly different in 12 tested chronic HBV carriers, nine tested HBV naïve controls and the two tested individuals with acute resolved HBV infection (IFN-γ: 28± 9 versus 69 ± 39 versus 18 ± 1 pg/ml; P = n.s.; IL-10: 21± 5 versus 67 ± 30 versus 14 ± 3 pg/ml; P = n.s.; Fig. 4).
The stimulation of T cells with the TT control antigen, however, led to strong IFN-γ production in chronic HBV carriers, patients with resolved HBV infection (1125 ± 349 pg/ml versus 2349 ± 1154 pg/ml; P = n.s.) and healthy HBV seronegative controls (1211 ± 565 pg/ml; P = n.s.). In contrast to HBcAg the TT induced mean IL-10 production was rather weak in chronic HBV patients (44 ± 14 pg/ml), in resolved HBV infection (174 pg/ml) and HBV naïve healthy controls (64 ± 23 pg/ml) (Fig. 4).
Ratios for IFN-γ and IL-10 production were calculated to evaluate Th1/Th2 cytokine profiles. In contrast to Th1 cell induction in healthy HBV naïve controls chronic HBV carriers as well as individuals with acute resolved HBV infection showed pre- ferential Th0/Th2 cytokine production upon stimulation with HBcAg (Table 1).
Table 1. Ratio of interferon-γ and interleukin-10 production by DC-stimulated T cells
Ratio of mean IFN-γ and IL-10 production representing the Th1/Th2 differentiation of stimulated T cells. CD4+ T cells were stimulated with autologous antigen pulsed dendritic cells and cytokines were analysed in the cell culture supernatants. TT, tetanus toxoid; HBcAg, HBV core antigen; HBsAg, HBV surface antigen. HC, HBV naïve healthy controls; CHB, patients with chronic hepatitis B; RHB, patients with resolved HBV infection.
Enhancement of antigen-specific T cell responses by interleukin-12
Insufficient autocrine interleukin-12 (IL-12) release could contribute to reduced DC activation and decreased IFN-γ production by T cells thus leading to viral persistence. The flow cytometric analysis revealed that the CD1a+DC from HBV patients and controls expressed IL-12RβI molecules on their surface (data not shown).
The DC preincubation for 2 days with 100 or 1000 pg/ml recombinant human IL-12 (generously provided by M. Gatley, Nutley, USA) resulted in two-fold higher proliferative HBcAg specific T cell responses in two tested HBV seronegative healthy controls as well as in four of five tested chronic HBV carriers demonstrating the reversible nature of insufficient T cell stimulation by autologous DC (Fig. 5).
The vigour and cytokine release of antiviral T helper cell re-sponses may determine whether an acute HBV infection will be controlled or persist in infected humans [1,5]. Antigen processing and presentation by DC, the most potent professional antigen presenting cells, represents a critical step in the early phase of HBV infection since DC are responsible for the induction of antiviral T cell responses [19,21]. In this study, DC derived from CD34+ peripheral blood progenitor cells were used to induce HBV antigen specific T cell responses because it has been de-monstrated that they induce stronger primary and memory T cell responses than DC preparations derived from monocytes [22–24]. GM-CSF and stem cell factor were found essential for the dif- ferentiation of expanded haematopoietic progenitor cells into immature DC. The addition of hyper-IL-6, a designer cytokine molecule of covalently linked human IL-6 and soluble IL-6 receptor, drives pluripotent stem cells to develop into immature DC capable to process soluble protein antigens [25,26]. Here, we show that these DC expressed identical surface markers, i.e. CD1a, CD86 and HLA-DR molecules as Langerhans cells generated by different protocols [25,27–29] and therefore they can be considered as functional antigen presenting DC. Another advantage of hyper-IL-6 conditioned stem cell derived DC is that these strongly proliferate and can easily be expanded in vitro under serum free conditions. Thus, such progenitor cell derived DC have the po-tential to be used in the future for adoptive transfer therapies [27,30–32].
Using autologous DC as antigen-presenting cells, T cells from patients with chronic hepatitis B showed less frequent and less vigorous class II restricted proliferative responses to HBcAg and HBsAg in vitro than T cells from individuals with acute resolved HBV infection and from healthy HBV seronegative controls whereas vigorous TT specific T helper cell responses were observed in all patient groups [18,33]. In control experiments using autologous monocytes as antigen-presenting cells weak HBcAg specific T cell responses were observed in some chronic HBV carriers but not in HBV naïve healthy controls. Therefore, it seems likely that the DC preparations used in this study were able to induce strong primary immune responses in healthy controls whereas in patients with ongoing HBV infection the induction of specific T cell responses was impaired. Similar results were obtained using a different protocol with monocyte derived DC from patients with chronic hepatitis B and healthy controls (Böcher et al. unpublished observation).
To analyse whether antigen presentation by autologous DC may result in the stimulation of functionally different T cell subsets the cytokine release of antigen-stimulated T cells was studied in chronic HBV patients and controls. T cells from pa-tients with chronic hepatitis B secreted lower amounts of IFN-γ upon stimulation with HBcAg pulsed DC compared to the HBV seronegative healthy controls and individuals with acute resolved hepatitis B. T cells from either chronic HBV patients or the con-trol groups stimulated with HBsAg pulsed DC showed very low IFN-γ concentrations in their supernatants whereas TT stimulation led to strong IFN-γ secretion in all study groups. Irrespectively to the antigen the concentrations of the Th2 marker cytokine IL-10 was low in the T cell supernatants of chronic HBV carriers, individuals with acute resolved hepatitis B and HBV seronegative blood donors. Thus, from the calculated ratios of IFN-γ and IL-10 production it may be indicated that HBcAg pulsed DC induced a preferential Th1/Th0 cell differentiation in healthy HBV naïve controls and such with acute resolved hepatitis B but a Th2-like cytokine pattern in chronic HBV carriers. T cell stimulation with HBsAg induced a weak Th0- like cytokine response and the TT control antigen specific T cell stimulation resulted in strong Th1 cellular responses in all patient groups.
It was suggested that a decreased T cell response against HBcAg may contribute to viral persistence in chronic HBV infection . Whether putative HBV infection of the DC, that may persist at low levels even after HBs/anti-HBs seroconversion , interferes with HBV specific antigen processing and presentation pathways has to be investigated in future experiments. The induction of HBV specific Th2 cell responses could result in T cell exhaustion or suppression and therefore favour development of chronicity while strong Th1 responses are associated with viral elimination [32,35]. Thus, the low IFN-γ/IL-10 ratios in response to HBcAg might indicate that virus specific T cell responses in chronic hepatitis B are dysregulated by impaired DC priming [36–38]. Once again, these results could be confirmed by our group with the use of a different protocol (W. Böcher et al. unpublished observations). Thus, strong evidence is given that defective DC priming could contribute to viral persistence in chronic hepatitis B.
Human DC secrete IL-12 and require IL-12 inducing factors to stimulate Th1 cells and CTL [39–41]. In this study the recent observation of IL-12 RβI expression on DC surfaces was confirmed arguing that IL-12 might act directly on DC to induce their functional activation . The preincubation of DC from chronic HBV carriers and controls with two concentrations of recombinant human IL-12 resulted in a two-fold increase of proliferative T cell responsiveness indicating that insufficient IL-12 secretion by the DC could contribute to the weak HBV-specific T cell reactivities observed in patients with chronic hepatitis B [43–45].
In conclusion, autologous DC pulsed with recombinant HBV antigens induced less frequent and weaker proliferative T cell responses in chronic HBV carriers than in healthy HBV seronegative controls and individuals with acute resolved hepatitis B. Compared to the control groups the T cells from patients with chronic HBV infection secreted less IFN-γ after stimulation with HBcAg pulsed DC. These results may indicate that defective DC priming could contribute to partial T cell anergy and weak cellular immune responses observed in chronic hepatitis B. Impaired HBV specific antigen presentation or processing pathways are one hypothesis which will be analysed in future. The addition of recombinant IL-12 in vitro, however, could restore the HBV specific T cell hyporesponsiveness in some patients. The data presented here could be of critical importance for the use of IL-12 as adjuvant in combination with future therapeutic vaccine strategies or the therapeutic transfer of in vitro stimulated dendritic cells to overcome viral persistence.
This study has been approved by a local ethical committee. All patients and controls included in this study gave their informed consent to the experiments in accordance with guidelines of the Helsinki Declaration. None of the authors had commercial interests or other conflict of interests. This paper was supported by grants of the Deutsche Forschungsgemeinschaft (SFB 490-A4 and 548-B3).