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International Immunology, Vol. 14, No. 10, pp. 1135-1144, October 2002
© 2002 Japanese Society for Immunology

Trypanosoma cruzi down-regulates lipopolysaccharide-induced MHC class I on human dendritic cells and impairs antigen presentation to specific CD8+ T lymphocytes

Laurence Van Overtvelt1, Muriel Andrieu2, Valérie Verhasselt1, Francine Connan2, Jeannine Choppin2, Vincent Vercruysse1, Michel Goldman1, Anne Hosmalin2 and Bernard Vray1

1 Laboratoire d’Immunologie Expérimentale (CP 615), Faculté de Médecine, and Laboratoire de Parasitologie, Faculté des Sciences, Université Libre de Bruxelles, 808 route de Lennik, 1070 Brussels, Belgium 2 Laboratoire d’Immunologie des Pathologies Infectieuses et Tumorales, INSERM U445, Département d’Immunologie, ICGM, 27 rue du Faubourg St-Jacques, 75014 Paris, France

Correspondence to: B. Vray; E-mail: bvray{at}ulb.ac.be
Transmitting editor: G. Trinchieri


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Trypanosoma cruzi, the etiological agent of Chagas’ disease, may persist for many years in its mammalian host. This suggests escape from the immune response and particularly a suboptimal CD8+ T cell response, since these cells are involved in infection control. In this report, we show that T. cruzi inhibits the lipopolysaccharide (LPS)-induced up-regulation of MHC class I molecules at the surface of human dendritic cells (DC). To further investigate the functional consequences of this inhibition, a trypomastigote surface antigen-derived peptide (TSA-1514–522 peptide) was selected for its stable binding to HLA-A*0201 molecules and used to generate a primary T. cruzi-specific human CD8+ T cell line in vitro. We observed that DC infected with T. cruzi or treated with T. cruzi-conditioned medium (TCM) had a weaker capacity to present this peptide to the specific CD8+ T cell line as shown in an IFN-{gamma} ELISPOT assay. Interestingly, T. cruzi or TCM also reduced the antigen presentation capacity of DC to CD8+ T cell lines specific for the influenza virus M58–66 or HIV RT476–484 epitopes. This dysfunction appears to be linked essentially to reduced MHC class I molecule expression since the stimulation of the RT476–484 peptide-specific CD8+ T cell line was shown to depend mainly on the MHC class I–TCR interaction and not on the co-stimulatory signals which, however, were also inhibited by T. cruzi. This impairment of DC function may represent a novel mechanism reducing in vivo the host’s ability to combat efficiently T. cruzi infection.

Keywords: cytotoxicity, dendritic cell maturation, immune escape, IFN-{gamma} ELISPOT, trypomastigote surface antigen


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Trypanosoma cruzi is a protozoan parasite that causes Chagas’ disease, a disease affecting 18 million people in Latin America (1,2). This parasite lives free within the cytoplasm of infected host cells (3), suggesting that parasite antigens may be processed and presented on MHC class I molecules for recognition by CD8+ T cells (4). Furthermore, the parasitism of cells such as cardiomyocytes and smooth muscle cells, which generally express MHC class I but not MHC class II molecules, suggests that immune clearance may require an effective CD8+ T cell response. Indeed, in murine models of infection, it has been shown that CD8+ T lymphocytes play a crucial role in the control of the T. cruzi infection. They dominate inflammatory foci in parasitized tissues (5–7) and CD8+ T lymphocyte-depleted mice exhibit an uncontrolled parasite replication followed by death (8–11). The CD8+ T lymphocytes protect the host against T. cruzi probably through their cytolytic activity (12–17), and their production of IFN-{gamma} and tumor necrosis factor (TNF)-{alpha}, two pro-inflammatory cytokines known to be involved in macrophage activation and infection control (12,18–20). Interestingly, the presence of parasite-specific CD8+ T lymphocytes has also recently been identified in chagasic patients (21). Despite the demonstrated role of CD8+ T cells in the defense against T. cruzi, the parasite persists for many years in the mammalian host, suggesting a suboptimal CD8+ T lymphocyte response in addition to other escape mechanisms (22,23).

Stimulation of CD8+ T lymphocytes is mainly mediated by dendritic cells (DC) (13,24). DC are indeed the only antigen-presenting cells (APC) capable of priming naive T cells and the most potent APC to stimulate MHC class I- and II-restricted antigen-specific T cell responses both in vivo and in vitro. Major factors involved in their superior immuno-stimulatory capacity are their higher expression of MHC and co-stimulatory molecules, such as CD40, CD80 and CD86, as well as their higher ability to produce various cytokines such as IL-12. We have previously shown that human DC can be infected in vitro by T. cruzi (25). We may thus hypothesize that T. cruzi has evolved mechanisms which could inhibit parasite antigen presentation by human DC. Indeed, we observed that, in infected human DC, the parasite inhibits the basal production of IL-12 and TNF-{alpha}. Most interestingly, lipopolysaccharide (LPS)-induced DC maturation was profoundly impaired by T. cruzi infection, resulting in a reduced secretion of IL-12, TNF-{alpha} and IL-6 released normally at high levels by LPS-activated DC. In addition, up-regulation of the HLA-DR and CD40 molecules was significantly reduced. The same effects were induced when incubating human DC in the presence of the supernatant of a parasite suspension [termed T. cruzi-conditioned medium (TCM)] (25).

Given our previous results and the importance of the CD8+ T lymphocyte-mediated response in T. cruzi infection, it was important to study the functional consequences of T. cruzi infection or TCM treatment on the capacity of human DC to present antigens and to stimulate specific CD8+ T lymphocytes.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Culture medium and reagents
The culture medium for the DC consisted of RPMI 1640 (Biowhittaker, Verviers, Belgium) supplemented with L-glutamine (2 mM), gentamicin (20 µg/ml), 2-mercaptoethanol (50 µM), 1% non-essential amino acids (Gibco, Grand Island, NY) and 10% heat-inactivated FCS (Biowhittaker). Recombinant IL-4 was kindly provided by Schering-Plough (Kenilworth, NJ). Recombinant granulocyte macrophage colony stimulating factor (GM-CSF) was obtained from Novartis (Basel, Switzerland). LPS from Escherichia coli (0128:B12), and endotoxin-free PBS and BSA were purchased from Sigma (St Louis, MO). The culture medium for the CD8+ T cell lines consisted of RPMI 1640–glutamax supplemented with penicillin (50 U/ml), streptomycin (50 µg/ml), 1% non-essential amino acids, sodium pyruvate (1 mM), HEPES buffer (10 mM) (all from Life Technologies, Courbevoie, France) and 10% heat-inactivated human AB serum (Valbiotech, Paris, France).

T. cruzi trypomastigotes and TCM
T. cruzi trypomastigotes (Tehuantepec strain, Mexico) were maintained by weekly i.p. inoculations to BALB/c mice (6–8 weeks old) purchased from Bantin & Kingman Universal (Hull, UK) and maintained in our animal facilities on standard laboratory chow. To obtain large quantities of parasites, trypomastigotes (2.5 x 105 parasites/rat) were inoculated into irradiated (7 Gy X-ray) F344 Fischer rats (Iffa Credo, Brussels, Belgium). Trypomastigotes were obtained from the blood (containing 10 U heparin/ml) of infected rats by ion-exchange chromatography on DEAE–cellulose (Whatman DE52) equilibrated with phosphate saline glucose buffer at pH 7.4 (PBS glucose). Trypomastigotes were centrifuged (15 min, 1800 g, 4°C) and resuspended in endotoxin-free PBS (26).

TCM was prepared according to the method described by Kierszenbaum et al. (27) to obtain trypanosomal immunosuppressive factor. Briefly, suspensions of T. cruzi (2 x 107 trypomastigotes/ml in RPMI 1640 medium) were incubated at 37°C and 5% CO2 for 24 h. The parasites were then removed by filtration through a sterile 0.22-µm pore filter (Millipore, Bedford, MA). This TCM was aliquoted and stored at –20°C until used. When necessary, it was diluted in culture medium to obtain a final concentration of 75%.

Generation of human monocyte-derived DC
Human DC were generated from peripheral blood mononuclear cells (PBMC) as described (28). Briefly, PBMC from HLA-A*0201 healthy volunteers were isolated by density centrifugation of heparinized blood on Lymphoprep (Nycomed, Oslo, Norway), resuspended in culture medium and allowed to adhere onto six-well plates at 1.5 x 107/well. After 2 h at 37°C, the non-adherent cells were removed and the adherent cells were cultured in 3 ml of medium containing GM-CSF (800 U/ml) and IL-4 (500 U/ml). Every second day, GM-CSF and IL-4 were added. After 7 days of culture, non-adherent cells corresponding to the DC-enriched fraction were harvested, washed and used as APC. As previously reported (29), the DC-enriched fraction obtained according to this protocol routinely contained >95% of DC as assessed by morphology and flow cytometry analysis. DC were cultured in 24-well plates at 5 x 105 DC/ml with trypomastigotes at a parasite:cell ratio of 30:1. After 24 h, 45–90% of the cells were infected.

Flow cytometry analysis
The effect of T. cruzi on MHC class I surface expression was quantified by using DC incubated with LPS (100 ng/ml) with or without trypomastigotes (parasite:cell ratio, 30:1) or TCM (75%). DC, incubated in the presence of medium alone, were used as control. After 24 h, 1 x 105 DC were harvested and washed in PBS supplemented with 0.5% BSA, 10 mM NaN3 and incubated for 30 min at 4°C with FITC-conjugated anti-MHC class I (HLA-A, -B and -C) mouse IgG2a mAb (B9.12.1; Immunotech, Marseilles, France) or a corresponding isotype-matched control. Then, the DC were fixed with 1% paraformaldehyde before flow cytometry analysis (FACSCalibur; Becton Dickinson, Mountain View, CA).

To investigate whether the effect of T. cruzi on the MHC class I expression was due to intracellular infection and/or to parasite-derived molecules, trypomastigotes were stained with a cellular fluorescein dye. This allowed us to gate infected cells by flow cytometry as amastigote-containing cells were positive in FL1. For this, trypomastigotes (50 x 106/ml) were incubated for 15 min in 5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester (CFSE, 10 mM final concentration in PBS; Molecular Probes, Eugene, OR) (30). After washing 3 times in PBS, CFSE-stained trypomastigotes were added at different parasite:cell ratios to DC (10:1, 20:1 and 30:1). After 24 h, the DC were harvested, washed and treated for flow cytometry as described above to quantify MHC class I except that a phycoerythrin-conjugated anti-MHC class I (HLA-A, -B and -C) mouse IgG1 mAb (555.553; PharMingen) was used.

A sample (2 x 105 cells) of the DC suspension was centrifuged (Cytospin; Shandon Elliott, Pittsburgh, PA; 5 min, 400 g), fixed with methanol and then stained with Giemsa stain. The percentage of infected DC and the mean number of amastigotes per infected DC were recorded after microscopic examination of at least 300 cells.

Synthetic peptides
Synthetic peptides, derived from three types of antigens, were tested to study the APC function of T. cruzi-infected DC or TCM-treated DC. (i) The HLA-A*0201-restricted TSA-1514–522 (FVDYNFTIV) and TSA-189–97 (KLFPEVIDL) peptides, derived from the trypomastigote surface antigen TSA-1 (21). (ii) The HLA-A*0201-restricted influenza virus matrix M58–66 (GIL GFVFTL) peptide and the HLA-B27-restricted influenza virus nucleoprotein NP383–391 (SRYWAIRTR) peptide. (iii) The HLA-A*0201 restricted-HIV-1 LAI reverse transcriptase RT476–484 (ILKEPVHGV) peptide. The HLA-A*0201-restricted HTLV-1 Tax11–19 (LLFGYPVYV) peptide was used as a negative control. All these peptides were synthesized by Neosystem (Strasbourg, France) and were >80% pure as indicated by HPLC analysis. They were dissolved at 1 mM in 10% DMSO in water, aliquoted and frozen. The final concentration of DMSO in cell suspensions never exceeded 0.1%.

Binding of TSA-1 peptides to purified HLA-A*0201 molecules and stability of the formed complexes
The capacity of TSA-1514–522 and TSA-189–97 peptides to bind to HLA-A*0201 molecules and the stability of HLA–peptide complexes formed were tested using purified HLA-A*0201 molecules as previously reported (31,32). Peptides from influenza virus, M58–66 and NP383–391, were used as positive and negative control respectively.

Generation of human CD8+ T cell lines
TSA-1514–522-specific CD8+ T cell line was generated as previously described for the Melan-A/MART-specific CD8+ cytotoxic lymphocytes (33). Briefly, unfractionated PBMC from an uninfected HLA-A*0201 donor (4 x 106 cells/well) were seeded in 24-well plates with tetanus toxoid (1 µg/ml, for helper effect) and TSA-1514–522 peptide (1 µg/ml) in 2 ml of culture medium. IL-7 was added on day 3 (20 U/ml; Boehringer, Meylan, France). Given the very low frequency of peptide-specific T cells in unprimed donors, several cultures were performed in parallel. For each culture, replicates were treated independently and re-stimulated with irradiated peptide-pulsed autologous PBMC on day 7. For this, PBMC (10 x 106/ml) were pulsed with TSA-1514–522 (50 µg/ml for 4 h) and diluted to 106/ml. Then, 1 ml of each replicate supernatant was removed and replaced with 1 ml of complete medium containing pulsed PBMC (106 cells). One day later, 1 ml of each replicate was again removed and replaced with complete medium containing IL-2 (10 U/ml, Boehringer) and IL-7 (20 U/ml). This step was repeated 2 days later and then every week. After three successive re-stimulations, a significant cytolytic activity was detected in four of 10 wells (>=15% of peptide-specific 51Cr release at an effector cell:target cell ratio of 30:1). IL-2 was then added twice a week to a final concentration of 50 U/ml. A CD8+ T cell line was finally obtained after four to five stimulations.

HIV-1 LAI RT476–484 and influenza virus M58–66-specific CD8+ T cell lines were generated respectively from PBMC of HIV-seropositive HLA-A*0201 individuals or uninfected HLA-A*0201 donors, as previously described (31). Briefly, cells were cultured in culture medium and stimulated weekly with autologous PBMC or HLA-A2*0201-matched B lymphoblastoid cell lines preincubated with 1 µg/ml of the relevant peptide and irradiated. They were maintained between 0.7 and 1 x 106 cells/ml and fed with IL-2 (10 U/ml) twice a week, and used after 2 or 3 weeks of culture.

Cytolytic activity assay
The cytolytic activity of the CD8+ T cell lines was assessed by a standard 51Cr-release test, 5–7 days following re-stimulation. The target cells [1–2 x 106 HLA-A2*0201 T/B hybrid cell line named T1 cells (34) or Epstein–Barr virus-transformed B cells] were labeled with 100 µCi 51Cr (10 mCi/ml, Dupont/NEN, Boston, MA) and pulsed with the relevant peptide (TSA- 1514–522; 5 µg/ml). For dose–response analysis, the target cells were incubated with peptide concentrations ranging from 10–15 to 10–5 M. After 1.5 h, target cells were washed twice with 0.9% NaCl containing 5% FCS and distributed in V-bottom 96-well plates (5 x 103 cells/well). Effector cells were added at different E:T ratios in a final volume of 0.2 ml of culture medium and incubated at 37°C for 4 h. The culture supernatants were harvested and 51Cr release was measured in a {gamma}-counter. In each test, non-pulsed target cells served as controls. Spontaneous 51Cr release was measured in the supernatant of target cells incubated in the absence of CD8+ T lymphocytes. The lysis of target cells gave the total 51Cr incorporated. Spontaneous 51Cr release was <25% of total 51Cr incorporated in all assays (31). The percentage of cell lysis was determined as follows: 100 x [(experimental release – spontaneous release)/ (Total 51Cr incorporated – spontaneous release)]

ELISPOT assay for single-cell IFN-{gamma} release
The IFN-{gamma} ELISPOT assay was performed as previously described (31). Ninety-six-well nitrocellulose plates (MultiScreen HA; Millipore) were coated with 15 µg/ml capture mouse anti-human IFN-{gamma} mAb 1-D1K (Mabtech, Stockholm, Sweden) in PBS overnight at 4°C. Wells were washed with PBS and saturated with RPMI/10% human AB serum. Then, the CD8+ T cell lines were seeded at serial dilutions (1–50 x 103 cells/well) overnight, in triplicate, with 104 stimulating DC/well. The plates were then washed in PBS and incubated with 1 µg/ml of the biotinylated anti-IFN-{gamma} mAb 7-B6-1 biotin (Mabtech) for 2 h at 37°C. The plates were washed several times with PBS/Tween 20 (0.05%) and the alkaline phosphatase-labeled Extravidin (1:6000 dilution; Sigma) was added for 1 h at 37°C. The plates were washed again with PBS/Tween 20 (0.05%) and revealed with a chromogenic alkaline phosphatase-conjugated substrate (Bio-Rad, Ivry sur Seine, France). After 30 min, the plates were washed under running tap water and air dried overnight. Spots were visualized through a stereomicroscope (MZ6, magnification x40; Leica, Heerbrugg, Switzerland,). Only large spots with fuzzy borders were scored as IFN-{gamma} spot-forming cells (SFC). Responses were considered as significant if (i) a minimum of five SFC were present per well, (ii) this number was at least 2-fold that obtained with the negative control at the cell concentration used, (iii) the same result was obtained using at least two different effector cell numbers and (iv) the number of spots obtained was proportional to the number of plated cells. As a negative control, DC were pulsed with the HLA-A*0201-restricted HTLV-1 Tax11–19 peptide. The positive control consisted of 500 effector cells plated with 50 ng/ml phorbol myristate acetate and 500 ng/ml ionomycin.

Using data that are means of triplicates, the percentage of inhibition of IFN-{gamma} SFC was calculated as follows: 100 x [(SFC with DC) – (SFC with T. cruzi-infected or with TCM-treated DC)/(SFC with DC)].

To investigate the role of co-stimulatory molecules, IL-12 and MHC class I molecules for the stimulation of the RT476–484-specific CD8+ T cell line, the following mAb were added at 5 µg/ml to the DC/CD8+ T lymphocytes co-culture: human CD154 (CD40L) muCD8 fusion protein and human CD152 (CTLA-4) muIg fusion protein (IgG2a) from Ancell Corp. (Bayport, MN); mAb anti-human IL-12 (IgG1) and mAb anti-human MHC class I (W6/32, IgG1) from R & D System (Minneapolis, MN).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
T. cruzi infection and TCM treatment of human DC inhibit LPS-induced MHC class I up-regulation
As MHC class I molecule expression is central for CD8+ T lymphocyte stimulation, we first tested the effects of T. cruzi on MHC class I molecule expression of human DC. T. cruzi infection had no effect on their basal expression of MHC class I molecules (data not shown). This is in line with our previous results showing that T. cruzi had no effect on the basal phenotype of human DC (25). In contrast, when using DC in the presence of LPS—as a maturation agent (35)—the up-regulation of MHC class I molecules was significantly inhibited by T. cruzi infection (Fig. 1A). Indeed, we observed that the addition of T. cruzi trypomastigotes to DC inhibited in a dose-dependent manner the LPS-induced up-regulation of MHC Class I. A maximal inhibiting effect was observed at a 30:1 parasite:cell ratio (Table 1). At this optimal ratio, we did not observe any cell cytotoxicity, and the percentage of infected DC and the mean number of amastigotes per infected DC were 70% and 2.6 respectively, in accordance with previous results (25). In order to determine whether the down-regulation of MHC class I molecules was due to intracellular infection and/or was mediated by products released by T. cruzi in the culture medium, LPS-treated DC were incubated with TCM. As illustrated in Fig. 1(B), TCM reproduced the inhibitory effects of live parasites on MHC class I expression.



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  		Fig. 1. T. cruzi infection and TCM treatment of DC inhibit LPS-induced MHC-I up-regulation. DC were incubated with LPS (100 ng/ml) with or without trypomastigotes (parasite:cell ratio, 30:1) (A) or TCM (75%) (B). DC, incubated in the presence of medium alone, were used as control. After 24 h, DC were harvested, washed and the expression level of MHC class I was measured by flow cytometry analysis. Thick lines represent FACS profiles obtained for DC incubated with medium alone. Thin lines represent FACS profiles obtained for DC incubated with LPS alone. Dotted lines represent FACS profiles obtained for DC incubated with LPS in the presence of trypomastigotes (A) or TCM (B). The solid histograms represent FACS profiles after staining with the corresponding isotype-matched control mAb. Results of one representative experiment out of eight on different blood donors are shown.

 

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  		Table 1. T. cruzi infection inhibits the LPS-induced up-regulation of MHC class I molecules on DC
 
To further confirm this observation, we analyzed the expression of MHC class I molecules on infected and uninfected DC. Therefore, T. cruzi were stained with CFSE, a cellular fluorescein dye (30), washed and then added to DC. This allowed us to detect infected DC by flow cytometry (Fig. 2). The expression of MHC class I was then assessed using a phycoerythrin–mAb anti-MHC class I molecule, and its expression was analyzed on infected and uninfected DC. As shown in Table 1, infected DC presented a reduced expression of MHC class I molecules. In addition, DC that did not contain amastigotes also presented a reduced expression of MHC class I molecules to the same extent, suggesting that intracellular infection is not necessary for the observed effects. This observation confirms results obtained with LPS-treated DC incubated with TCM and indicates that not only intracellular infection but also parasite-derived molecules can exert such an inhibitory effect on the expression of MHC class I molecules.



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  		Fig. 2. Fluorescence of infected DC using CFSE-stained T. cruzi. T. cruzi trypomastigotes were stained with CFSE and then added to DC for 24 h. Fluorescence of control DC and DC co-cultured with CFSE-stained T. cruzi at different parasite:cell ratios (10:1, 20:1 and 30:1) was analyzed by flow cytometry. Numbers refer to the percentages of infected cells. Data of one representative experiment of three are shown.

 
Generation and characterization of primary T. cruzi-specific CD8+ T lymphocytes
To evaluate the functional consequences of MHC class I down-regulation on LPS-treated DC by T. cruzi, the capacity of T. cruzi-infected DC or TCM-treated DC to stimulate a parasite-specific CD8+ T cell line was tested. Therefore, we first generated in vitro a primary T. cruzi-specific CD8+ T cell line using a peptide stimulation strategy (33). We selected two peptides from the trypomastigote surface antigen TSA-1 (TSA-1514–522 and TSA-189–97) containing a HLA-A*0201 binding motif and identified as HLA-A*0201-restricted CTL epitopes in T. cruzi-infected patients (21). As a prerequisite for optimal peptide presentation, they were first tested for their capacity to form a sufficient amount of MHC class I–peptide complexes and for the good stability of these formed complexes using an ELISA-based binding assay measuring peptide binding to purified HLA-A*0201 molecules (Fig. 2A and B) (32). The TSA-1514–522 peptide, which showed the highest level of binding at 10–6 M and the highest HLA-A2 complex stability (>50% of non-dissociated complexes after 24 h at 37°C) (36), was selected to generate a primary T. cruzi-specific CD8+ T cell line.

Phenotypic analysis showed that this cell line comprised 90% CD8+ and no CD4+ lymphocytes (data not shown). It had a specific cytotoxic activity against TSA-1514–522 peptide-pulsed target cells (Fig. 3C). MHC class I restriction was attested by absence of lysis on a non-HLA-A*0201 lymphoblastoid B cell line pulsed with the TSA-1514–522 peptide (Fig. 3C) and by intracellular cytokine detection using flow cytometry, where only CD3+CD8+ lymphocytes produced IFN-{gamma} specifically in response to the TSA-1514–522 peptide (data not shown). The CD8+ T cell line recognized the TSA-1514–522 peptide in a dose-related fashion and the concentration leading to half-maximal lysis was as low as 10–11 to 10–12 M (Fig. 3D). This cell line was maintained in long-term culture for >3 months. These data indicated that precursor CD8+ T lymphocytes against T. cruzi antigen are present in healthy individuals and can be detected by repeated peptide stimulations in vitro.



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  		Fig. 3. Generation and characterization of primary T. cruzi-specific CD8+ T lymphocytes. (A and B) Binding of TSA-1514–522 and TSA-189–97 peptides to purified HLA-A*0201 molecules and stability of complexes formed. (A) Binding of different concentrations of peptides TSA-1514–522 (filled squares) and TSA-189–97 (filled triangles) to purified HLA-A*0201 molecules was measured in an ELISA. These results are compared with those obtained with a positive control peptide (the HLA-A2*0201-restricted influenza virus M58–66 peptide; open triangles) and a negative control peptide (the HLA-B27-restricted influenza virus NP383–391 peptide; open diamonds). (B) The stability of the re-natured complexes at 37°C was assessed over time. The results are expressed in arbitrary fluorescence units and are representative of two independent experiments. (C and D) Specific cytotoxicity of the CD8+ T cell line in response to TSA-1514–522 peptide. (C) Cytotoxic activity of the TSA-1514–522-specific CD8+ T cell line evaluated after five stimulations. Lysis of the targets, including T1 cells pulsed with TSA-1514–522 (filled squares) or unpulsed (open squares) and non-HLA-A*0201 Epstein–Barr virus-transformed cells pulsed with TSA-1514–522 (filled triangles) or unpulsed (open triangles), was evaluated in a 4 h 51Cr-release assay. (D) Titration of TSA-1514–522 peptide-specific recognition. T1 cells were pulsed with the peptide TSA-1514–522 at the indicated concentrations and incubated at an E:T ratio of 15:1 with the CD8+ cell line obtained after six stimulations. 51Cr release was measured after 4 h. The results shown are representative of two independent experiments.

 
T. cruzi impairs the ability of human DC to stimulate specific CD8+ T cell lines
Using the TSA-1514–522-specific CD8+ T cell line in an IFN-{gamma} ELISPOT assay, we investigated the capacity of T. cruzi-infected DC or TCM-treated DC to present the TSA-1514–522 peptide. TSA-1514–522-pulsed DC were able to induce a specific IFN-{gamma} production by the CD8+ T cell line. By contrast, when DC were infected with T. cruzi or treated with TCM, the specific IFN-{gamma} release was inhibited up to 44 and 59% respectively (Fig. 4A and D). The TSA-1514–52-specific-CD8+ T cell line did not produce IFN-{gamma} in response to the infected DC nor lyse infected DC except when exogenous TSA-1514–522 peptide was added to DC (data not shown). Therefore, the epitope was not presented by DC following infection.



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  		Fig. 4. T. cruzi alters antigen presentation of human DC to antigen-specific CD8+ T cell lines. HLA-A2*0201-matched DC were incubated in medium containing LPS (100 ng/ml) alone or with trypomastigotes (‘infected’, parasite:cell ratio 30:1, A–C) or TCM (‘TCM’, 75%, D–F). After 24 h, DC were washed and pulsed with the control Tax11–19 peptide, or the relevant TSA-1514–522 (A and D), M58–66 (B and E) or RT476–484 (C and F) peptides (1 µg/ml) for 2 h at 37°C. Then, DC were washed again before being added to CD8+ T cell lines specific for T. cruzi (TSA-1514–522 peptide), influenza virus (M58–66 peptide) or HIV (RT476–484 peptide) for an IFN-{gamma} ELISPOT assay. The number of IFN-{gamma} SFC per well was counted. Data are mean values of SFC ± SEM from triplicate wells with three (or two) different effector cells numbers per well and are representative of one experiment out of n independent experiments (n = 4 in A, C, D and F; n = 6 in B; and n = 3 in E). The percentages of inhibition were calculated as described in Methods.

 
To verify whether the inhibition of the ability of DC to stimulate CD8+ T lymphocytes was general or specific for the TSA-1514–522 peptide, we investigated the capacity of T. cruzi-infected DC or TCM-treated DC to present the influenza virus M58–66 peptide or the HIV-1 RT476–484 peptide to specific CD8+ T cell lines. While M58–66-pulsed DC induced specific IFN-{gamma} production by the CD8+ T cell line, this production was decreased up to 86% following infection with the parasite and 45% following treatment with TCM (Fig. 4B and E). Presentation of the HIV-1 RT476–484 peptide to specific CD8+ T cell lines generated from PBMC of HIV-seropositive HLA-A*0201+ donors was also inhibited up to 39% by infection with the parasite and 69% by treatment with TCM (Fig. 4C and F). Inhibition was maximal at the lower effector:stimulation ratios. Similar results were obtained with T. cruzi-infected DC and TCM-treated DC from different HLA-A*0201 donors (Table 1). These results show that T. cruzi can alter the antigen presentation function of DC to CD8+ T cell lines, not only those specific for one of their own antigen but also of unrelated antigens.

Role of co-stimulation
To stimulate primary T cells efficiently during DC-mediated antigen presentation, co-stimulatory signals through CD28–CD80/86 and CD40L–CD40 interactions are required in addition to the TCR engagement by MHC class I–peptide complexes. The role of the CD28–CD80/86 and CD40L–CD40 pathways was thus investigated in the RT476–484-specific CD8+ T cell line using the CD154 (CTLA-4) and the CD152 (CD40L) fusion protein respectively. Anti-MHC class I and anti-IL-12 antibodies were also used. Interestingly, addition of soluble CD154 or CD152 or an anti-IL-12 antibody during the presentation of RT476–484 peptide to the CD8+ T cell line had no effect. In contrast, addition of the blocking anti-MHC class I antibody decreased by 60% IFN-{gamma} production by this CD8+ T cell line (Fig. 5). Moreover, control experiments showed that the same concentrations of soluble CD154, CD152 and anti-IL-12 were effective at abrogating the primary response of alloreactive T cells in a MLR (data not shown) (37). These results indicate that co-stimulation through the CD28–CD80/CD86 and CD40L–CD40 pathways is not required for the RT476–484 line, which is mainly dependent on recognition of the MHC–peptide complex. Therefore, in this case, even though T. cruzi inhibits CD40 up-regulation and IL-12 production by LPS-stimulated DC (25), the decreased expression of MHC class I molecules seems to be responsible for CD8+ T cell response inhibition.



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  		Fig. 5. Role of the co-stimulatory molecules. HLA-A2*0201-matched DC were incubated in medium containing LPS (100 ng/ml). After 24 h, DC were washed and pulsed with the control Tax11–19 peptide or the RT476–484 peptide (1 µg/ml) for 2 h at 37°C. Then, DC were washed again before being cultured with the RT476–484-specific CD8+ T cell line for an IFN-{gamma} ELISPOT assay in the presence or absence of 5 µg/ml of either anti-human IL-12 antibody, human CD152 (CTLA-4) and human CD154 (CD40L) fusion protein, and anti-human MHC-I antibody or corresponding isotype control. The number of IFN-{gamma} SFC per well was counted. Three different effector cell concentrations were tested. Data are mean values of SFC ± SEM from triplicate wells with 3000 effector cells. The mean values of SFC from triplicate wells with control samples (DC pulsed with 1 µg/ml control Tax11–19 peptide) was subtracted from the experimental values and was <10%. The results shown are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The data presented here show that T. cruzi inhibits LPS-induced MHC class I up-regulation on the surface of human DC. This inhibition is mediated by soluble factor(s) released by the parasite itself since TCM, added to the DC culture medium, exerts the same effect as intracellular T. cruzi infection. These data are reinforced by the fact that when DC are co-cultured with T. cruzi, both infected and non-infected DC show a down-regulation of MHC class I expression. So that, in vitro, in addition to a direct effect of the intracellular parasites (amastigotes) on MHC class I expression, a similar effect is also observed through the presence of free trypomastigotes present in the cell suspension and releasing parasite-derived molecules similar to TCM. One can hypothesize that, in vivo, the parasites release continuously TCM that acts in a systemic way by diffusing in all the host’s body.

The functional consequence of parasite–DC interactions seems to be that T. cruzi impairs the ability of human DC to present MHC class I-restricted peptides to specific CD8+ T cell lines. The interference with MHC class I antigen presentation was observed not only with a parasite-derived antigen (TSA-1514–522 peptide), but also with two unrelated antigens (M58–66 and RT476–484 peptides). Starting from these data, it would be interesting to look at the potential exacerbation of co-infections such as HIV in chagasic patients (37). In particular, DC from chagasic patients may be deficient in eliciting novel CD8+ T lymphocytes since DC are the only APC that can induce primary T cell responses.

We have previously shown that T. cruzi also inhibits co-stimulatory molecules up-regulation during LPS-induced DC maturation (25). It is difficult to completely rule out the possibility that the effect observed could be due to small changes in the level of production or expression of cytokines and/or co-stimulatory molecules. However, the reduced ability of T. cruzi-infected DC or TCM-treated DC to stimulate CD8+ T cell lines seems to be linked mainly to reduced MHC class I molecule expression since the stimulation of at least one CD8+ T cell line (the RT476–484 peptide-specific CD8+ T cell line) depends mainly on the interaction between MHC class I–TCR, but not on the co-stimulatory signals. This inhibition of cell surface MHC class I expression may contribute to the inability of T. cruzi-specific CD8+ T cells to efficiently eradicate the parasites in infected individuals and suggests the existence of a novel escape mechanism. This should ideally be tested using functional analysis of DC harvested from chagasic patients.

To our knowledge, this is the first report indicating a reduced expression of MHC class I molecules on human DC by a protozoan parasite (T. cruzi) and its functional consequences. Down-regulation of MHC class I molecules on T. cruzi-infected DC may decrease the protective effect of specific CD8+ T lymphocytes. In contrast, such a down-regulation of MHC class I molecules was not observed in T. cruzi-infected mouse macrophages so that no impairment of antigen presentation to the specific CD8+ T cell line was observed (39). On the other hand, an up-regulation of MHC class I expression on the J774 macrophage cell line and on fibroblasts was induced by T. cruzi, and was shown to be dependent on the presence of IFN-{alpha}/ß (40). Toxoplasma gondii, another obligate intracellular protozoan parasite, is also known to impair the up-regulation of MHC class I expression on IFN-{gamma}-activated bone marrow-derived macrophages, but data on functional consequences are lacking (41).

The inhibitory effect of a soluble factor from T. cruzi on LPS-mediated maturation of DC might be due to competition with a LPS receptor. It has indeed been indicated that GPI-anchored mucin-like glycoproteins from T. cruzi signal macrophages through receptors of the Toll family. However, the receptor triggered by these glycoproteins seemed distinct from TLR4 which is triggered by LPS (42).

In line with our results, a reduced expression of MHC class I molecules has been previously shown in viral infections. In particular, HIV-infected human DC express lower levels of MHC class I molecules and this may explain the inability of CD8+ T lymphocytes to be primed early enough to eliminate the onset of infection in vivo (43). HSV infection is also known to down-regulate MHC class I expression on human DC probably as a result of the inhibitory effect of infected-cell protein (ICP)-47, an immediate/early protein, on TAP (44,45) and thereby to inhibit MHC class I-mediated peptide presentation (45,46). Similar observations have been reported with viruses (adenovirus, Epstein–Barr virus, and human and mouse cytomegalovirus) infecting other cell types. These have been found to subvert MHC class I presentation by a variety of mechanisms, including interference with peptide transport and entrapment of MHC class I (47–49).

Despite the fact that T. cruzi probably synthesizes the TSA-1 antigen during infection, the TSA-1514–522-specific CD8+ T cell line did not recognize infected DC except when exogenous TSA-1514–522 peptide was added (data not shown). This may reflect a requirement by some TCR for a higher threshold of antigenic determinant density than that expressed by the parasite-infected target cells in this experiment. More likely, these results reflect the selection of low-affinity CD8+ T lymphocytes by the repetitive peptide stimulation technique that fails to lyse target cells expressing endogenous determinants which were shown to require higher peptide concentrations to sensitize target cells.

The CD8+ T cell responses found in vivo, although inefficient to sterilize parasite infection, are induced despite the down-regulation of MHC class I and of co-stimulation molecules upon maturation of DC found here and in our previous work (25). They may be elicited only against highly expressed epitopes that allow them to reach the threshold level of MHC class I–peptide density to induce CD8+ responses (50). They may also be induced in the lymphoid organs at sites distant from T. cruzi and its soluble products. Indeed, DC have developed specialized and efficient cross-presentation mechanisms that may allow MHC class I-restricted presentation of endocytosed parasites or apoptotic debris from infected cells (51–54).

In conclusion, our data suggest that the inhibition of MHC class I up-regulation on LPS-treated DC may be involved in the generation of a suboptimal CD8+ T lymphocyte response upon infection with T. cruzi. In vivo, this could be a mechanism allowing the parasite to escape immune recognition and favor the establishment of persistent infection.


   Acknowledgements
 
The authors wish to thank J.-G. Guillet for constant support, J. F. Desoutter and S. Figueiredo for valuable technical assistance, and I. Mazza for help in preparing the manuscript. This work was supported by grants from Centre de Recherche Interuniversitaire en Vaccinologie, Fonds Emile Defay, la Banque Nationale de Belgique, the Agence Nationale de Recherches sur le SIDA (ANRS) and Ensemble Contre le SIDA (SIDACTION). L. V. O is the recipient of a grant from the Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture (FRIA).


   Abbreviations
 
APC—antigen-presenting cell

CFSE—5-(and 6)-carboxyfluorescein diacetate succinimidyl ester

DC—dendritic cell

GM-CSF—granulocyte macrophage colony stimulating factor

LPS—lipopolysaccharide

PBMC—peripheral blood mononuclear cell

SFC—spot-forming cell

TCM—T. cruzi-conditioned medium

TNF—tumor necrosis factor

TSA-1—trypomastigote surface antigen-1


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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