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Keywords:

  • human alveolar echinococcosis;
  • immunotherapy;
  • dendritic cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Dendritic cells (DC) are sentinels of immunity. We determined their role in the induction of immunity against alveolar echinococcosis, caused by the larval stage of the cestode Echinococcus multilocularis. Furthermore, we evaluated if unfractionated protein from E. multilocularis (Em-Ag) can be used as loading agent for DC (comparable to unfractionated tumour proteins) in order to generate antiparasitic cytotoxic T lymphocyte (CTL). Interestingly, immature DC did not mature in the presence of 1 µg/ml Em-Ag as analysed by FACS and mixed leucocyte reactions. Yet, their capacity to take up dextran was markedly reduced. Further maturation of immature Em-Ag pulsed DC could be induced by proinflammatory cytokines. These mature DC were slightly better inducers of T cell proliferation when compared with unpulsed mature DC. Importantly, by repetetive stimulation of autologous CD8+ lymphocytes with the Em-Ag pulsed mature DC, we were able to generate specifically proliferating CTL lines. Thus, immunotherapy with ex vivo generated Em-Ag pulsed DC might be of benefit for patients inheriting this incurable disease.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Metacestodes of the fox tapeworm Echinococcus multilocularis (Em) can proliferate in the liver of accidentally infected humans and cause a disease which is termed alveolar echinococcosis. Alveolar echinococcosis has a low incidence in Europe (approximately 0·02–1·4 new cases per year per 100 000 inhabitants) (1), but the severity of the disease and the relative inefficiency of available chemotherapeutics gives rise to the need for alternatives. Although single T cells that react specifically with unfractionated protein from E. multilocularis (Em-Ag) can be detected in patients (2), the lack of an effective immune response leads to the continuous growth of the lesion. This resembles the situation in tumours, where cytotoxic T lymphocyte (CTL) specific for tumour associated antigens are present but of weak activity (3,4).

Crude E. multilocularis antigen (Em-Ag) contains an undefined mixture of proteins of metacestode cells and products. It has antigenic but no mitogenic capacities. In the study presented here, we evaluated the effect of Em-Ag on ex vivo generated dendritic cells (DC), which are known to be potent inducers of immunity with the unique capacity to prime naive T-cells (5). First, we wanted to analyse the effect of Em-Ag on immature DC, which are present in most tissues and have the capacity to take up antigen efficiently (6). After activation by various stimuli such as bacterial components (e.g. LPS), inflammatory cytokines (TNF-α and IL-1), exposure to Leishmania major amastigotes (7) or CD40L (8), DC mature and migrate into lymph nodes to prime T cells (6). Capture of Ag, maturation and migration of DC are key events in the induction of immunity. We thus wanted to evaluate whether the parasitic proteins are capable of influencing Ag uptake and whether their presence induces maturation in immature DC. Second, we wanted to determine, whether the artificial maturation of DC by proinflammatory cytokines and prostaglandin (9) can be inhibited by the parasitic proteins. Third, we wanted to evaluate whether DC pulsed with Em-Ag might serve as a cellular vaccination for alveolar echinococcosis patients as described for tumour patients (5). The recent utilization of unfractionated proteins derived from poorly immunogenic tumour cells (10,11), or bacteria (12), to pulse DC has sparked our interest in this therapeutic approach.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Antibodies and reagents

The following monoclonal antibodies (mAbs) were used. FITC-labelled murine CD80 (BB1), CD40 (5C3), HLA-DR (G46-6) HLA-ABC (G46-2.6) mAb were purchased from Pharmingen (Hamburg, Germany), CD8 (DK25) mAb from Dako (Hamburg, Germany). PE-conjugated murine CD86 (IT2·2) mAb from Pharmingen, CD83 (Hb15a) mAb from Immunotech (Marseille, France), CD4 (MT310) and CD16 (DJ130c) mAb from Dako, CD14 (MO-P9), CD19 (Leu-12), and CD3 (SK7) mAb from Becton Dickinson (Heidelberg, Germany). Purified control IgG1-PE was purchased from Dako and IgG2b-PE, IgG1-FITC and IgG2b-FITC mAb from Pharmingen.

Flow cytometric analysis

Cultured cells were washed, suspended at 3 × 105 in 50 µl of cold PBS, 0·1% sodium azide, 10 mg/ml BSA, and 200 µg/ml mouse IgG (Sigma, Fluka Biochemical, Buchs, Switzerland), and incubated for 10 min on ice. Subsequent staining with labelled mAbs or appropriate isotypic controls was performed for 30 min. Cells were then washed, and resuspended in 300 µl of cold PBS, 1% human serum albumin containing 10 µg/ml 7-amino-actinomycin D (7-AAD) (Sigma). Stained cells were analysed for three-colour immunofluorescence with a FACScalibur cell analyser (Becton Dickinson). Cell debris was eliminated from the analysis using a gate on forward and side scatter. 7-AAD staining was used to determine viable cells and to exclude cell debris (life gate). A minimum of 10 000 viable cells was analysed for each sample. Results were processed using Cellquest software (Becton Dickinson).

FITC-dextran labelling

Cells were incubated with FITC-dextran (0·1 mg/ml), either at 4°C (internalization control) or at 37°C for 1 h. DC were then washed twice with a cold buffer consisting of phosphate-buffered saline, 0·1% sodium azide, and 10 mg/ml bovine serum albumin.

DC generation from buffy coats

Buffy coats of healthy donors were obtained according to institutional guidelines. PBMC were prepared using Ficoll-Paque (Pharmacia, Uppsala, Sweden). PBMC were resuspended (15 × 106cells/well) in 6 well-plates (Nunc, Roskilde, Denmark) and incubated for 1 h at 37°C. Nonadherent cells were removed and the remaining cells were fed with 3 ml of X-VIVO 15 medium (Bio-Whittaker, Walkersville, MD, USA) containing 1% of heat-inactivated autologous plasma, 1000 IU GM-CSF/ml, and 1000 IU IL-4/ml (Stratagene, Hanover, Germany). Cells were refed with 0·5 ml of fresh medium containing 1000 IU GM-CSF and 1000 IU IL-4 per ml at days 2, 4 and 6. At day 7, the nonadherent cells were transferred to a new well with fresh medium. DC maturation was induced with a cocktail of cytokines as recently published (9). The following cytokines were added: IL-4, 1000 U/ml; IL-1β, 10 ng/ml; IL-6, 1000 U/ml (all from Stratagene), GM-CSF, 1000 U/ml (Leukomax, Novartis, Basel, Switzerland, kindly provided by Dr P.-Y. Dietrich, University of Geneva, Switzerland), prostaglandin (PGE)2, 1 µg/ml (Prostin, Pharmacia); TNF-α, 10 ng/ml (kindly provided by Dr J.-M. Dayer, University of Geneva, Switzerland). Cells were harvested after 2 days and used for flow cytometric analysis and/or culture with T cells. To have fresh autologous DC at each time point of restimulation of cultured CD8+ T cells, PBMC were frozen in 10% DMSO (Fluka Biochemical) and 90% human serum albumin (Blutspendedienst SRK, Bern, Switzerland).

Antigenic stimulants

The crude E. multilocularis antigen (Em-Ag) was kindly provided by T. Romig, Hohenheim. It was gained by using the same technics as recently described (13). Briefly, metacestodes from E. multilocularis were obtained from Meriones unguiculatus after artificial infection. The parasitic material was pressed through a metal sieve, homogenized and sonicated. The supernatant was harvested after centrifugation and the protein content was determined by the bicinchonic assay as indicated by the manufacturer (Pierce, Rockford, IL, USA). Final concentration in our trials was 1 µg/ml, a concentration that proved to be effective for lymphocyte stimulation. The antigen has no mitogenic properties.

Mixed leukocyte reaction

Allogenic T cells were obtained from buffy coats of healthy adults after Ficoll-Paque gradient centrifugation, adherence to plastic for 1 h at 37°C, and passage over a nylon wool column (Biotest, Dreieich, Germany). 1 × 105 allogenic T cells were cultured in the presence of graded numbers of irradiated (3000 rad, 137Cs source) DC. Cells were cultured for 4–5 days in round-bottomed 96-well plates in 200 µl of RPMI 1640 medium supplemented with l-glutamine, penicillin and containing 5% of heat-inactivated human AB+ Serum (Blood Transfusion Center, Annemasse, France). Tritiated thymidine (3H-TdR) (1 µCi/well, New England Nuclear, Boston, MA, USA) was added for the last 8–12 h of culture. 3H-TdR incorporation was measured in a liquid scintillation counter (Pharmacia, Wallace, Sweden). All conditions were set up in triplicate.

Bioassay for TNF quantification

A subclone (WEHI 1.14) of the TNF-sensitive WEHI 164 clone was used as described (14) with the following modifications. Fifty µl of graded dilutions from culture supernatant were added to 50 µl (2 × 104) WEHI 1.14 cells in flat-bottomed 96-well plates (Nunc) in duplicates and incubated for 24 h at 37°C. Twenty µl of MTS (333 µl/ml) (Promega, Madison, WI, USA) and 1 µl of the electron coupling reagent PMS (25 µm) (Sigma) were subsequently added to each well. After 2 h of incubation the resulting intensity of the colouration was measured at 490 nm in a Thermomax microplatereader (Molecular Devices, Menlo Park, CA, USA) and analysed using Softmax software from the same company. Recombinant human TNF-α (kindly provided by Dr J.-M. Dayer, University of Geneva, Switzerland) was used as a standard. The sensitivity of this assay was 0·1 pg/ml. This assay does not distinguish TNF-α from lymphotoxin. Thus, the resulting activity is referred to as TNF.

Purification of CD8+ T cells

Autologous CD8+ T cells were isolated using magnetic beads-conjugated mouse antihuman CD8+ mAb (Miltenyi, Bergisch Gladbach, Germany), a MACS column for positive selection (VS) and a vario-MACS magnet according to the manufacturer's instructions. The purified cells contained 96% to 99% CD8+ cells as assessed by flow cytometry.

CTL generation and restimulation

Mature DC were generated as described. At day 9, 1·5 × 106 autologous CD8+ cells were added to 5 × 104 DC and cultured in 1·5 ml per well X-VIVO 15 medium supplemented with 1% autologous plasma in 24-well plates. IL-2 was added in a concentration of 40 IU per ml at days 1, 4 and 7. Nine days after the first culture, CD8+ cells were harvested, counted and recultured under identical conditions using fresh generated autologous DC.

Proliferation of autologous T cells

To determine proliferation in autologous CD8+ cells induced by DC pulsed or not with crude Em-Ag, we cultured CTL generated after three restimulations in triplicates. CD8+ cells (5 × 104) were incubated with Em-Ag pulsed or unpulsed DC. IL-2 and 10 µg/ml Em-Ag were added if indicated. 3H-TdR (1 µCi/well) was added for 8 h after 3–4 days. 3H-TdR incorporation was assessed by using a liquid scintillation counter.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Effect of crude Em-Ag on immature DC

To investigate the effect of crude Em-Ag on DC differentiation from monocytic precursors, we cultured the adherent cell fraction of PBMC in the presence of GM-CSF, IL-4 and/or 1 µg/ml crude Em-Ag. After 7 days in culture, nonadherent cells were analysed. Flow cytometric analysis of the relevant surface markers known to be upregulated upon DC maturation revealed an unaltered expression profile of CD83, CD86, CD80, CD40, MHC class I and II in DC cultured in the presence of Em-Ag as compared to control DC (data not shown). Furthermore, DC cultured in the presence of Em-Ag for 2 additional days induced T cell proliferation comparable to immature control DC, whereas DC matured with proinflammatory cytokines were powerful in eliciting T cell proliferation (Figure 1). We compared the amount of autologous TNF in the supernatant of day 7 DC cultured for 24 h in the presence of 100 IU TNF-α and after washing for additional 24 h in absence of exogenous TNF-α, necrotic melanoma cells (which provide a medium intense maturation stimulus (15), 1 µg/ml Em-Ag or in the absence of these compounds. Em-Ag induced very little amounts of TNF, while TNF-α or necrotic melanoma cells induced the production of high amounts of TNF (Figure 2). To evaluate the influence of Em-Ag on dendritic cell receptor mediated endocytosis, we performed FITC-DX uptake assays with immature DC generated in the presence or absence of 1 µg/ml of Em-Ag. Surprisingly, the presence of Em-Ag during the 7 days of culture reduced the capacity of DC to take up the FITC-DX at 37°C (Figure 3). No uptake was detected at 4°C, indicating that the uptake is an active process and not a passive diffusion of labelled molecules. In four independent experiments, we found the level of FITC-DX uptake by DC cultured in the presence of Em-Ag to be 35·4% (SD: 1·9%) of the uptake seen in control DC.

image

Figure 1. Unaltered allostimulatory capacity of immature DC cultured in the presence of Em-Ag. Immature DC in presence (▴) or absence (●) of Em-Ag were cultured in GM-CSF and IL-4 for 2 days and subsequently used to stimulate allogeneic T cells. T cell proliferation was measured by 3H thymidine incorporation after 5 days. Immature DC cultured with TNF-α, IL-1β, IL-6 and PGE2 were used as control (▪). The data represent mean of triplicates ± SD. The figure is representative of five experiments with similar results. In all experiments, the proliferation of T cells without DC (▿) was below 600 c.p.m.

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image

Figure 2. Em-Ag induces a slight TNF production in immature DC. Bioactive TNF was determined in the 48 h supernatant of immature DC incubated for 2 days with 1 µg/ml Em-Ag. Untreated immature DC and DC stimulated with 100 U TNF-α and necrotic melanoma cell material served as a control. One representative experiment out of four is shown. The data represent mean of triplicates ± SD.

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image

Figure 3. Internalization of FITC-dextran by immature DC. Day 7 immature DC cultured in the absence (left) or presence (right) of Em-Ag were incubated with FITC-dextran for 1 h at 4°C (thin lines) or 37°C (bold lines). One representative experiment out of four is shown.

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Effect of Em-Ag on mature DC

To analyse the effect of Em-Ag on the maturation of DC, we induced maturation of immature day 7 DC using the proinflammatory cytokines indicated above and PGE2 in the presence or absence of 1 µg/ml crude Em-Ag. After 2 days of maturation, we analysed the surface expression of relevant molecules on DC. In three independent experiments, we found no significant difference in the percent expression or the average mean fluorescent intensity (MFI) of all parameter analysed (Table 1). The percentage of the surface expression of CD83 served as indicator for DC purity, which was approximately 80%. To test the functional capacity of DC matured in the presence or absence of Em-Ag, we performed allogenic mixed leucocyte reactions (MLR) experiments. We found an increased allostimulatory capacity of DC cultured in the continuous presence of Em-Ag (Figure 4). These experiments taken together indicate that Em-Ag does not inhibit the maturation of immature DC when maturation stimuli are present in abundance.

Table 1.  FACS profile of mature DC generated in the presence or absence of 1 µg/ml Em-Ag
 Mature DC1 mg/ml Em-Ag
Medium %Range %MFI (SD)Medium %Range %MFI (SD)
CD8383·072–9046 (6)84·069–9447 (15)
MHC-II99·099176 (4)99·399–99·4270 (115)
CD86/MHC-II90·082–95107 (61)88·782–95102 (62)
CD80/CD8345·035–5825 (10)58·026–8528 (15)
CD40/CD8353·329–8121 (5)74·052–8826 (12)
MHC-I/CD8392·589–9667 (35)90·087–9365 (42)
image

Figure 4. Higher capacity of Em-Ag pulsed DC to induce allostimulatory proliferation. Immature DC pulsed with Em-Ag (●) or not pulsed (▪) and consecutively cultured for 2 days in the presence of the maturation inducing cytokines TNF-α, IL-1β, IL-6 and PGE2. T cell proliferation was measured as 3H thymidine incorporation. The data represent mean of triplicates ± SD. The figure is representative of five experiments with similar results. In all experiments, the proliferation of T cells without DC (▿) was below 600 c.p.m.

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DC pulsed with crude Em-Ag induce autologous T-cell proliferation

In order to evaluate if mature DC pulsed with crude Em-Ag are capable of inducing autologous CD8+ T cell proliferation we cultured purified CD8+ T cells with mature DC pulsed or not pulsed with Em-Ag. After 3–4 rounds of restimulation with autologous DC, the proliferation of the T cell lines in presence of pulsed or unpulsed DC was measured (3H-TdR incorporation). Em-Ag pulsed DC only induced the proliferation in CD8+ T cell lines that were generated in the presence of Em-Ag pulsed DC. Furthermore, the addition of high concentrations of Em-Ag (10 µg/ml) did not inhibit the proliferation (Figure 5). Neither pulsed nor unpulsed DC induced a comparable proliferation in T cell lines generated with unpulsed DC. These data demonstrate an Em-Ag dependent proliferation of CD8+ T cell lines generated with Em-Ag pulsed autologous DC.

image

Figure 5. Em-Ag pulsed DC induce autologous CTL proliferation. After three rounds of restimulation with Em-Ag, pulsed or unpulsed DC, CD8+ CTL lines were cultured with pulsed or unpulsed DC. 10 µg of crude Em-Ag was added to the proliferation assays if indicated. 3H thymidine incorporation was measured in triplicate. Data represent the mean ± SD. Three experiments were performed with similar results.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Little is known about the complex interactions of E. multilocularis with the human immune system. The ability of E. multilocularis to persist in an immunocompetent host, and thus to successfully avoid the host immune system, can potentially be ascribed to two mechanisms. First, the parasitic lesion may manifest low immunogenicity due to an inert capsule (the outer, acellular, so-called laminated layer) or, alternatively, interact actively with the antigen presentation by professional APC (16). Second, the efferent arm of the immune response might be blocked, which means that specific T cells could be deactivated in the presence of the lesion itself or products produced by the parasite as suggested by others (17). To gain further insights in the mechanisms involved and to evaluate the possibility of an immunotherapy using ex vivo generated and protein pulsed autologous DC, we performed this study.

As a crucial step in mounting an immune response, immature DC take up Ag by phagocytosis (18), macropinocytosis (19) or receptor mediated endocytosis (20). For parasitic infections, especially the recently described IgE/Fcε dependent phagocytosis and also the signalling pathway might be of high interest (21). We found a reduced capacity of DC grown in presence of the Em-Ag to take up Dextran as a model Ag. This could be attributed to a nonspecific mechanism, i.e. a slight maturation that cannot be detected by simple analysis of maturation surface markers or functional assays. Furthermore, we cannot formally exclude the overloading of uptake mechanisms by the presence of Em-Ag. However, with regard to the low concentration of the Ag, it seems more likely that a protein of the Em-Ag fraction has the specific capacity to block the Ag uptake of immature DC. The mannose receptor responsible for the uptake of dextran recognizes the pattern of carbohydrates that are displayed on the surface or cell walls of infectious agents (22). Because the laminated layer of the alveolar echinococcosis lesion is composed of protein and nonprotein molecules, it might be possible that proteins of E. multilocularis inhibit the recognition or uptake of carbohydrates by the pattern recognition receptor of DC. In contrast to bacterial products such as LPS (19), the crude protein fraction of E. multilocularis does not induce DC maturation. This finding might support the view of alveolar echinococcosis as an inert lesion. In total, our findings indicate that immature DC, in contact with the parasitic lesions, phagocytose very little antigen and are not matured, and thus do not migrate and do not present Ag with sufficient costimulation, a situation which might induce anergy (23) or tolerance (24) of Ag specific T lymphocytes.

The Em-Ag does not actively block the maturation of DC, since ex vivo generated and matured Em-Ag pulsed DC display similar maturation features as control DC. This finding would allow Em-Ag pulsed mature DC to be used as a cellular vaccine in alveolar echinococcosis patients without the risk of inducing tolerance with partially matured DC. However, we cannot exclude the possibility that, in vivo, a nonprotein of the alveolar echinococcosis lesion can inhibit the maturation of DC.

T cells are believed to play an important role in the immunological control of Em infections (25), a finding highlighted by the occurrence of massive alveolar echinococcosis lesions in a HIV infected child (26) and by the enhanced metastasic formation in T cell depleted mice (27). Furthermore, we recently demonstrated the presence of antigen specific CD4+ and CD8+ T cells in alveolar echinococcosis patients (2). We thus evaluated whether Em-Ag pulsed DC are capable of stimulating autologous CD8+ T cells. Because repetitive restimulation with DC allows the generation of T cell lines even if the precursor frequency is extremely low (28), we were able to use PBMC of healthy donors for our experiments. Recently, a laminated layer-associated protein (29) has been described but not yet sequenced. Thus, no CTL epitope prediction with bioinformatic tools is available so far, which would allow the synthesis of peptides with determined HLA specific binding properties. As a consequence, it is difficult to demonstrate the specificity of CTL lines generated with Em-Ag pulsed APC. We tried to overcome this limitation by demonstrating the Em-Ag dependency of the T cell lines generated by repetitive stimulation. After 3–4 restimulations, CTL lines generated with pulsed DC only proliferated in the presence of pulsed DC, while neither pulsed nor unpulsed DC induced proliferation in T cell lines generated in the presence of unpulsed DC. These data might indirectly demonstrate the generation of T cell lines specific for peptide derived from crude Em protein. As in the allogenic MLRs, the presence of Em-Ag in high dosage did not influence the autologous lymphocytic proliferation. The modification of DC function by an E. multilocularis derived macrophage modifying factor demonstrated in a mouse model (30) was thus not observed in our experiments.

In conclusion, we have analysed the effect of Em-Ag on the afferent part of the immune response (inhibition of Ag-uptake by the mannose receptor and no induction of DC maturation) and on the efferent part (no inhibition of DC maturation and T cell proliferation). Furthermore, we have demonstrated the Em-Ag dependent proliferation of CD8+ T cell lines after restimulation with pulsed mature DC. We suggest that the utilization of Em-Ag pulsed DC in immunotherapeutic protocols, similar to currently applied tumour vaccination studies, might be of benefit for alveolar echinococcosis patients.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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