Trypanosoma cruzi down-regulateslipopolysaccharide-induced MHC class I onhuman dendritic cells and impairs antigenpresentation to speci®c CD8+ T lymphocytes

Laurence Van Overtvelt1, Muriel Andrieu2, ValeÂrie Verhasselt1, Francine Connan2,Jeannine Choppin2, Vincent Vercruysse1, Michel Goldman1, Anne Hosmalin2 andBernard Vray1

1Laboratoire d'Immunologie ExpeÂrimentale (CP 615), Faculte de MeÂdecine, and Laboratoire deParasitologie, Faculte des Sciences, Universite Libre de Bruxelles, 808 route de Lennik, 1070 Brussels,Belgium2Laboratoire d'Immunologie des Pathologies Infectieuses et Tumorales, INSERM U445,DeÂpartement d'Immunologie, ICGM, 27 rue du Faubourg St-Jacques, 75014 Paris, France

Keywords: cytotoxicity, dendritic cell maturation, immune escape, IFN-g ELISPOT, trypomastigote surfaceantigen

Abstract

Trypanosoma cruzi, the etiological agent of Chagas' disease, may persist for many years in itsmammalian host. This suggests escape from the immune response and particularly a suboptimalCD8+ T cell response, since these cells are involved in infection control. In this report, we showthat T. cruzi inhibits the lipopolysaccharide (LPS)-induced up-regulation of MHC class I moleculesat the surface of human dendritic cells (DC). To further investigate the functional consequences ofthis inhibition, a trypomastigote surface antigen-derived peptide (TSA-1514±522 peptide) wasselected for its stable binding to HLA-A*0201 molecules and used to generate a primary T. cruzi-speci®c human CD8+ T cell line in vitro. We observed that DC infected with T. cruzi or treated withT. cruzi-conditioned medium (TCM) had a weaker capacity to present this peptide to the speci®cCD8+ T cell line as shown in an IFN-g ELISPOT assay. Interestingly, T. cruzi or TCM also reducedthe antigen presentation capacity of DC to CD8+ T cell lines speci®c for the in¯uenza virus M58±66

or HIV RT476±484 epitopes. This dysfunction appears to be linked essentially to reduced MHC class Imolecule expression since the stimulation of the RT476±484 peptide-speci®c CD8+ T cell line wasshown to depend mainly on the MHC class I±TCR interaction and not on the co-stimulatorysignals which, however, were also inhibited by T. cruzi. This impairment of DC function mayrepresent a novel mechanism reducing in vivo the host's ability to combat ef®ciently T. cruziinfection.

Introduction

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 in¯ammatory 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-g and tumor necrosis factor (TNF)-a, two

Correspondence to: B. Vray; E-mail: bvray@ulb.ac.be

Transmitting editor: G. Trinchieri Received 3 January 2002, accepted 3 July 2002

International Immunology, Vol. 14, No. 10, pp. 1135±1144 ã 2002 The Japanese Society for Immunology

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pro-in¯ammatory cytokines known to be involved inmacrophage activation and infection control (12,18±20).Interestingly, the presence of parasite-speci®c CD8+ Tlymphocytes has also recently been identi®ed in chagasicpatients (21). Despite the demonstrated role of CD8+ T cells inthe defense against T. cruzi, the parasite persists for manyyears in the mammalian host, suggesting a suboptimal CD8+ Tlymphocyte response in addition to other escape mechanisms(22,23).

Stimulation of CD8+ T lymphocytes is mainly mediated bydendritic cells (DC) (13,24). DC are indeed the only antigen-presenting cells (APC) capable of priming naive T cells andthe most potent APC to stimulate MHC class I- and II-restrictedantigen-speci®c T cell responses both in vivo and in vitro.Major factors involved in their superior immuno-stimulatorycapacity are their higher expression of MHC and co-stimulatory molecules, such as CD40, CD80 and CD86, aswell as their higher ability to produce various cytokines suchas IL-12. We have previously shown that human DC can beinfected in vitro by T. cruzi (25). We may thus hypothesize thatT. cruzi has evolved mechanisms which could inhibit parasiteantigen presentation by human DC. Indeed, we observed that,in infected human DC, the parasite inhibits the basal produc-tion of IL-12 and TNF-a. Most interestingly, lipopolysaccharide(LPS)-induced DC maturation was profoundly impaired byT. cruzi infection, resulting in a reduced secretion of IL-12,TNF-a and IL-6 released normally at high levels by LPS-activated DC. In addition, up-regulation of the HLA-DR andCD40 molecules was signi®cantly reduced. The same effectswere induced when incubating human DC in the presence ofthe supernatant of a parasite suspension [termed T. cruzi-conditioned medium (TCM)] (25).

Given our previous results and the importance of the CD8+ Tlymphocyte-mediated response in T. cruzi infection, it wasimportant to study the functional consequences of T. cruziinfection or TCM treatment on the capacity of human DC topresent antigens and to stimulate speci®c CD8+ T lympho-cytes.

Methods

Culture medium and reagents

The culture medium for the DC consisted of RPMI 1640(Biowhittaker, Verviers, Belgium) supplemented with L-gluta-mine (2 mM), gentamicin (20 mg/ml), 2-mercaptoethanol(50 mM), 1% non-essential amino acids (Gibco, GrandIsland, NY) and 10% heat-inactivated FCS (Biowhittaker).Recombinant IL-4 was kindly provided by Schering-Plough(Kenilworth, NJ). Recombinant granulocyte macrophagecolony stimulating factor (GM-CSF) was obtained fromNovartis (Basel, Switzerland). LPS from Escherichia coli(0128:B12), and endotoxin-free PBS and BSA were purchasedfrom Sigma (St Louis, MO). The culture medium for the CD8+ Tcell lines consisted of RPMI 1640±glutamax supplementedwith penicillin (50 U/ml), streptomycin (50 mg/ml), 1% non-essential amino acids, sodium pyruvate (1 mM), HEPES buffer(10 mM) (all from Life Technologies, Courbevoie, France) and10% heat-inactivated human AB serum (Valbiotech, Paris,France).

T. cruzi trypomastigotes and TCM

T. cruzi trypomastigotes (Tehuantepec strain, Mexico) weremaintained by weekly i.p. inoculations to BALB/c mice (6±8weeks old) purchased from Bantin & Kingman Universal (Hull,UK) and maintained in our animal facilities on standardlaboratory chow. To obtain large quantities of parasites,trypomastigotes (2.5 3 105 parasites/rat) were inoculatedinto irradiated (7 Gy X-ray) F344 Fischer rats (Iffa Credo,Brussels, Belgium). Trypomastigotes were obtained from theblood (containing 10 U heparin/ml) of infected rats by ion-exchange chromatography on DEAE±cellulose (WhatmanDE52) equilibrated with phosphate saline glucose buffer atpH 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 byKierszenbaum et al. (27) to obtain trypanosomal immuno-suppressive factor. Brie¯y, suspensions of T. cruzi (2 3 107

trypomastigotes/ml in RPMI 1640 medium) were incubated at37°C and 5% CO2 for 24 h. The parasites were then removedby ®ltration through a sterile 0.22-mm pore ®lter (Millipore,Bedford, MA). This TCM was aliquoted and stored at ±20°Cuntil used. When necessary, it was diluted in culture medium toobtain a ®nal concentration of 75%.

Generation of human monocyte-derived DC

Human DC were generated from peripheral blood mono-nuclear cells (PBMC) as described (28). Brie¯y, PBMC fromHLA-A*0201 healthy volunteers were isolated by densitycentrifugation of heparinized blood on Lymphoprep(Nycomed, Oslo, Norway), resuspended in culture mediumand allowed to adhere onto six-well plates at 1.5 3 107/well.After 2 h at 37°C, the non-adherent cells were removed andthe adherent cells were cultured in 3 ml of medium containingGM-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 fractionwere harvested, washed and used as APC. As previouslyreported (29), the DC-enriched fraction obtained according tothis protocol routinely contained >95% of DC as assessed bymorphology and ¯ow cytometry analysis. DC were cultured in24-well plates at 5 3 105 DC/ml with trypomastigotes at aparasite:cell ratio of 30:1. After 24 h, 45±90% of the cells wereinfected.

Flow cytometry analysis

The effect of T. cruzi on MHC class I surface expression wasquanti®ed by using DC incubated with LPS (100 ng/ml) with orwithout trypomastigotes (parasite:cell ratio, 30:1) or TCM(75%). DC, incubated in the presence of medium alone, wereused as control. After 24 h, 1 3 105 DC were harvested andwashed 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 ®xed with 1% para-formaldehyde before ¯ow cytometry analysis (FACSCalibur;Becton Dickinson, Mountain View, CA).

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To investigate whether the effect of T. cruzi on the MHCclass I expression was due to intracellular infection and/or toparasite-derived molecules, trypomastigotes were stainedwith a cellular ¯uorescein dye. This allowed us to gate infectedcells by ¯ow cytometry as amastigote-containing cells werepositive in FL1. For this, trypomastigotes (50 3 106/ml) wereincubated for 15 min in 5-(and 6-)-carboxy¯uorescein di-acetate succinimidyl ester (CFSE, 10 mM ®nal concentration inPBS; Molecular Probes, Eugene, OR) (30). After washing 3times in PBS, CFSE-stained trypomastigotes were added atdifferent parasite:cell ratios to DC (10:1, 20:1 and 30:1). After24 h, the DC were harvested, washed and treated for ¯owcytometry as described above to quantify MHC class I exceptthat a phycoerythrin-conjugated anti-MHC class I (HLA-A, -Band -C) mouse IgG1 mAb (555.553; PharMingen) was used.

A sample (2 3 105 cells) of the DC suspension wascentrifuged (Cytospin; Shandon Elliott, Pittsburgh, PA; 5 min,400 g), ®xed with methanol and then stained with Giemsastain. The percentage of infected DC and the mean number ofamastigotes per infected DC were recorded after microscopicexamination of at least 300 cells.

Synthetic peptides

Synthetic peptides, derived from three types of antigens, weretested to study the APC function of T. cruzi-infected DC orTCM-treated DC. (i) The HLA-A*0201-restricted TSA-1514±522

(FVDYNFTIV) and TSA-189±97 (KLFPEVIDL) peptides, derivedfrom the trypomastigote surface antigen TSA-1 (21). (ii) TheHLA-A*0201-restricted in¯uenza virus matrix M58±66 (GIL-GFVFTL) peptide and the HLA-B27-restricted in¯uenza virusnucleoprotein 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-1Tax11±19 (LLFGYPVYV) peptide was used as a negativecontrol. All these peptides were synthesized by Neosystem(Strasbourg, France) and were >80% pure as indicated byHPLC analysis. They were dissolved at 1 mM in 10% DMSO inwater, aliquoted and frozen. The ®nal concentration of DMSOin cell suspensions never exceeded 0.1%.

Binding of TSA-1 peptides to puri®ed HLA-A*0201molecules and stability of the formed complexes

The capacity of TSA-1514±522 and TSA-189±97 peptides to bindto HLA-A*0201 molecules and the stability of HLA±peptidecomplexes formed were tested using puri®ed HLA-A*0201molecules as previously reported (31,32). Peptides fromin¯uenza virus, M58±66 and NP383±391, were used as positiveand negative control respectively.

Generation of human CD8+ T cell lines

TSA-1514±522-speci®c CD8+ T cell line was generated aspreviously described for the Melan-A/MART-speci®c CD8+

cytotoxic lymphocytes (33). Brie¯y, unfractionated PBMCfrom an uninfected HLA-A*0201 donor (4 3 106 cells/well)were seeded in 24-well plates with tetanus toxoid (1 mg/ml, forhelper effect) and TSA-1514±522 peptide (1 mg/ml) in 2 ml ofculture medium. IL-7 was added on day 3 (20 U/ml;Boehringer, Meylan, France). Given the very low frequencyof peptide-speci®c T cells in unprimed donors, severalcultures were performed in parallel. For each culture, replic-

ates were treated independently and re-stimulated withirradiated peptide-pulsed autologous PBMC on day 7. Forthis, PBMC (10 3 106/ml) were pulsed with TSA-1514±522

(50 mg/ml for 4 h) and diluted to 106/ml. Then, 1 ml of eachreplicate supernatant was removed and replaced with 1 ml ofcomplete medium containing pulsed PBMC (106 cells). Oneday later, 1 ml of each replicate was again removed andreplaced with complete medium containing IL-2 (10 U/ml,Boehringer) and IL-7 (20 U/ml). This step was repeated 2 dayslater and then every week. After three successive re-stimulations, a signi®cant cytolytic activity was detected infour of 10 wells (>15% of peptide-speci®c 51Cr release at aneffector cell:target cell ratio of 30:1). IL-2 was then addedtwice a week to a ®nal concentration of 50 U/ml. A CD8+ T cellline was ®nally obtained after four to ®ve stimulations.

HIV-1 LAI RT476±484 and in¯uenza virus M58±66-speci®cCD8+ T cell lines were generated respectively from PBMC ofHIV-seropositive HLA-A*0201 individuals or uninfected HLA-A*0201 donors, as previously described (31). Brie¯y, cellswere cultured in culture medium and stimulated weekly withautologous PBMC or HLA-A2*0201-matched B lympho-blastoid cell lines preincubated with 1 mg/ml of the relevantpeptide and irradiated. They were maintained between 0.7and 1 3 106 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 astandard 51Cr-release test, 5±7 days following re-stimulation.The target cells [1±2 3 106 HLA-A2*0201 T/B hybrid cell linenamed T1 cells (34) or Epstein±Barr virus-transformed B cells]were labeled with 100 mCi 51Cr (10 mCi/ml, Dupont/NEN,Boston, MA) and pulsed with the relevant peptide (TSA-1514±522; 5 mg/ml). For dose±response analysis, the target cellswere incubated with peptide concentrations ranging from10±15 to 10±5 M. After 1.5 h, target cells were washed twice with0.9% NaCl containing 5% FCS and distributed in V-bottom96-well plates (5 3 103 cells/well). Effector cells were added atdifferent E:T ratios in a ®nal volume of 0.2 ml of culture mediumand incubated at 37°C for 4 h. The culture supernatants wereharvested and 51Cr release was measured in a g-counter. Ineach test, non-pulsed target cells served as controls.Spontaneous 51Cr release was measured in the supernatantof target cells incubated in the absence of CD8+ T lympho-cytes. The lysis of target cells gave the total 51Cr incorporated.Spontaneous 51Cr release was <25% of total 51Cr incorporatedin all assays (31). The percentage of cell lysis was determinedas follows: 100 3 [(experimental release ± spontaneousrelease)/ (Total 51Cr incorporated ± spontaneous release)]

ELISPOT assay for single-cell IFN-g release

The IFN-g ELISPOT assay was performed as previouslydescribed (31). Ninety-six-well nitrocellulose plates(MultiScreen HA; Millipore) were coated with 15 mg/ml capturemouse anti-human IFN-g mAb 1-D1K (Mabtech, Stockholm,Sweden) in PBS overnight at 4°C. Wells were washed with PBSand saturated with RPMI/10% human AB serum. Then, theCD8+ T cell lines were seeded at serial dilutions (1±50 3 103

cells/well) overnight, in triplicate, with 104 stimulating DC/well.The plates were then washed in PBS and incubated with

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1 mg/ml of the biotinylated anti-IFN-g mAb 7-B6-1 biotin(Mabtech) for 2 h at 37°C. The plates were washed severaltimes with PBS/Tween 20 (0.05%) and the alkaline phos-phatase-labeled Extravidin (1:6000 dilution; Sigma) wasadded for 1 h at 37°C. The plates were washed again withPBS/Tween 20 (0.05%) and revealed with a chromogenicalkaline phosphatase-conjugated substrate (Bio-Rad, Ivry surSeine, France). After 30 min, the plates were washed underrunning tap water and air dried overnight. Spots werevisualized through a stereomicroscope (MZ6, magni®cation340; Leica, Heerbrugg, Switzerland,). Only large spots withfuzzy borders were scored as IFN-g spot-forming cells (SFC).Responses were considered as signi®cant if (i) a minimum of®ve SFC were present per well, (ii) this number was at least2-fold that obtained with the negative control at the cellconcentration used, (iii) the same result was obtained using atleast two different effector cell numbers and (iv) the number ofspots 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 controlconsisted of 500 effector cells plated with 50 ng/ml phorbolmyristate acetate and 500 ng/ml ionomycin.

Using data that are means of triplicates, the percentage ofinhibition of IFN-g SFC was calculated as follows: 100 3 [(SFCwith DC) ± (SFC with T. cruzi-infected or with TCM-treated DC)/(SFC with DC)].

To investigate the role of co-stimulatory molecules, IL-12and MHC class I molecules for the stimulation of the RT476±484-speci®c CD8+ T cell line, the following mAb were added at 5mg/ml to the DC/CD8+ T lymphocytes co-culture: humanCD154 (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

T. cruzi infection and TCM treatment of human DC inhibitLPS-induced MHC class I up-regulation

As MHC class I molecule expression is central for CD8+ Tlymphocyte stimulation, we ®rst tested the effects of T. cruzi onMHC class I molecule expression of human DC. T. cruziinfection had no effect on their basal expression of MHC classI molecules (data not shown). This is in line with our previousresults showing that T. cruzi had no effect on the basalphenotype of human DC (25). In contrast, when using DC inthe presence of LPSÐas a maturation agent (35)Ðthe up-regulation of MHC class I molecules was signi®cantly inhibitedby T. cruzi infection (Fig. 1A). Indeed, we observed that theaddition of T. cruzi trypomastigotes to DC inhibited in a dose-dependent manner the LPS-induced up-regulation of MHCClass I. A maximal inhibiting effect was observed at a 30:1parasite:cell ratio (Table 1). At this optimal ratio, we did notobserve any cell cytotoxicity, and the percentage of infectedDC and the mean number of amastigotes per infected DCwere 70% and 2.6 respectively, in accordance with previousresults (25). In order to determine whether the down-regulationof MHC class I molecules was due to intracellular infection

and/or was mediated by products released by T. cruzi in theculture medium, LPS-treated DC were incubated with TCM. Asillustrated in Fig. 1(B), TCM reproduced the inhibitory effectsof live parasites on MHC class I expression.

To further con®rm this observation, we analyzed theexpression of MHC class I molecules on infected anduninfected DC. Therefore, T. cruzi were stained with CFSE, acellular ¯uorescein dye (30), washed and then added to DC.This allowed us to detect infected DC by ¯ow cytometry(Fig. 2). The expression of MHC class I was then assessedusing a phycoerythrin±mAb anti-MHC class I molecule, and itsexpression was analyzed on infected and uninfected DC. Asshown in Table 1, infected DC presented a reduced expres-sion of MHC class I molecules. In addition, DC that did not

Fig. 1. T. cruzi infection and TCM treatment of DC inhibitLPS-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 mediumalone, were used as control. After 24 h, DC were harvested, washedand the expression level of MHC class I was measured by ¯owcytometry analysis. Thick lines represent FACS pro®les obtained forDC incubated with medium alone. Thin lines represent FACS pro®lesobtained for DC incubated with LPS alone. Dotted lines representFACS pro®les obtained for DC incubated with LPS in the presenceof trypomastigotes (A) or TCM (B). The solid histograms representFACS pro®les after staining with the corresponding isotype-matchedcontrol mAb. Results of one representative experiment out of eighton different blood donors are shown.

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contain amastigotes also presented a reduced expression ofMHC class I molecules to the same extent, suggesting thatintracellular infection is not necessary for the observed effects.This observation con®rms results obtained with LPS-treatedDC incubated with TCM and indicates that not only intra-cellular infection but also parasite-derived molecules canexert such an inhibitory effect on the expression of MHC class Imolecules.

Generation and characterization of primary T. cruzi-speci®cCD8+ T lymphocytes

To evaluate the functional consequences of MHC class Idown-regulation on LPS-treated DC by T. cruzi, the capacity ofT. cruzi-infected DC or TCM-treated DC to stimulate aparasite-speci®c CD8+ T cell line was tested. Therefore, we®rst generated in vitro a primary T. cruzi-speci®c CD8+ T cellline using a peptide stimulation strategy (33). We selected twopeptides from the trypomastigote surface antigen TSA-1 (TSA-1514±522 and TSA-189±97) containing a HLA-A*0201 bindingmotif and identi®ed as HLA-A*0201-restricted CTL epitopes inT. cruzi-infected patients (21). As a prerequisite for optimalpeptide presentation, they were ®rst tested for their capacity toform a suf®cient amount of MHC class I±peptide complexesand for the good stability of these formed complexes using an

ELISA-based binding assay measuring peptide binding to

puri®ed HLA-A*0201 molecules (Fig. 2A and B) (32). TheTSA-1514±522 peptide, which showed the highest level ofbinding 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-speci®c CD8+ Tcell line.

Phenotypic analysis showed that this cell line comprised

90% CD8+ and no CD4+ lymphocytes (data not shown). It hada speci®c cytotoxic activity against TSA-1514±522 peptide-pulsed target cells (Fig. 3C). MHC class I restriction wasattested by absence of lysis on a non-HLA-A*0201 lympho-

blastoid B cell line pulsed with the TSA-1514±522 peptide(Fig. 3C) and by intracellular cytokine detection using ¯owcytometry, where only CD3+CD8+ lymphocytes producedIFN-g speci®cally in response to the TSA-1514±522 peptide

(data not shown). The CD8+ T cell line recognized theTSA-1514±522 peptide in a dose-related fashion and theconcentration leading to half-maximal lysis was as low as10±11 to 10±12 M (Fig. 3D). This cell line was maintained in long-

term culture for >3 months. These data indicated thatprecursor CD8+ T lymphocytes against T. cruzi antigen arepresent in healthy individuals and can be detected byrepeated peptide stimulations in vitro.

Fig. 2. Fluorescence of infected DC using CFSE-stained T. cruzi. T. cruzi trypomastigotes were stained with CFSE and then added to DC for24 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) wasanalyzed by ¯ow cytometry. Numbers refer to the percentages of infected cells. Data of one representative experiment of three are shown.

Table 1. T. cruzi infection inhibits the LPS-induced up-regulation of MHC class I molecules on DC

MHC class I expression by DC (median ¯uorescence intensity)

UntreatedDC

LPS-treatedDC

T. cruzi-infected andLPS-treated DC (10:1)a

T. cruzi-infected andLPS-treated DC (20:1)a

T. cruzi-infected andLPS-treated DC (30:1)a

All DC 17 33 29 24 20Infected DC 31 24 20Uninfected DC 28 22 19

DC were incubated for 24 h with (or without) LPS and graded ratios of CFSE-stained T. cruzi. The expression of MHC class I molecules wasassessed by ¯ow cytometry using a phycoerythrin-conjugated anti-MHC class I molecule mAb. The analysis was gated on all the DC orselectively on the infected DC (cells positive in FL1, see Fig. 2) and on the uninfected DC (cells negative in FL1, see Fig. 2).

aParasite:cell ratio. Data of one representative experiment of three are shown.

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T. cruzi impairs the ability of human DC to stimulate speci®cCD8+ T cell lines

Using the TSA-1514±522-speci®c CD8+ T cell line in an IFN-gELISPOT 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 aspeci®c IFN-g production by the CD8+ T cell line. By contrast,when DC were infected with T. cruzi or treated with TCM, thespeci®c IFN-g release was inhibited up to 44 and 59%respectively (Fig. 4A and D). The TSA-1514±52-speci®c-CD8+

T cell line did not produce IFN-g in response to the infected DCnor lyse infected DC except when exogenous TSA-1514±522

peptide was added to DC (data not shown). Therefore, theepitope was not presented by DC following infection.

To verify whether the inhibition of the ability of DC tostimulate CD8+ T lymphocytes was general or speci®c for theTSA-1514±522 peptide, we investigated the capacity of T. cruzi-infected DC or TCM-treated DC to present the in¯uenza virusM58±66 peptide or the HIV-1 RT476±484 peptide to speci®c CD8+

T cell lines. While M58±66-pulsed DC induced speci®c IFN-gproduction by the CD8+ T cell line, this production wasdecreased up to 86% following infection with the parasite and45% following treatment with TCM (Fig. 4B and E).Presentation of the HIV-1 RT476±484 peptide to speci®c CD8+

T cell lines generated from PBMC of HIV-seropositive HLA-A*0201+ donors was also inhibited up to 39% by infection withthe 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 andTCM-treated DC from different HLA-A*0201 donors (Table 1).These results show that T. cruzi can alter the antigenpresentation function of DC to CD8+ T cell lines, not onlythose speci®c for one of their own antigen but also of unrelatedantigens.

Role of co-stimulation

To stimulate primary T cells ef®ciently during DC-mediatedantigen presentation, co-stimulatory signals through CD28±CD80/86 and CD40L±CD40 interactions are required in

Fig. 3. Generation and characterization of primary T. cruzi-speci®c CD8+ T lymphocytes. (A and B) Binding of TSA-1514±522 and TSA-189±97

peptides to puri®ed HLA-A*0201 molecules and stability of complexes formed. (A) Binding of different concentrations of peptides TSA-1514±522

(®lled squares) and TSA-189±97 (®lled triangles) to puri®ed HLA-A*0201 molecules was measured in an ELISA. These results are comparedwith those obtained with a positive control peptide (the HLA-A2*0201-restricted in¯uenza virus M58±66 peptide; open triangles) and a negativecontrol peptide (the HLA-B27-restricted in¯uenza virus NP383±391 peptide; open diamonds). (B) The stability of the re-natured complexes at37°C was assessed over time. The results are expressed in arbitrary ¯uorescence units and are representative of two independentexperiments. (C and D) Speci®c cytotoxicity of the CD8+ T cell line in response to TSA-1514±522 peptide. (C) Cytotoxic activity of theTSA-1514±522-speci®c CD8+ T cell line evaluated after ®ve stimulations. Lysis of the targets, including T1 cells pulsed with TSA-1514±522 (®lledsquares) or unpulsed (open squares) and non-HLA-A*0201 Epstein±Barr virus-transformed cells pulsed with TSA-1514±522 (®lled triangles) orunpulsed (open triangles), was evaluated in a 4 h 51Cr-release assay. (D) Titration of TSA-1514±522 peptide-speci®c recognition. T1 cells werepulsed 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 obtainedafter six stimulations. 51Cr release was measured after 4 h. The results shown are representative of two independent experiments.

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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-speci®c 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 presen-

tation of RT476±484 peptide to the CD8+ T cell line had no effect.

Fig. 4. T. cruzi alters antigen presentation of human DC to antigen-speci®c CD8+ T cell lines. HLA-A2*0201-matched DC were incubated inmedium containing LPS (100 ng/ml) alone or with trypomastigotes (`infected', parasite:cell ratio 30:1, A±C) or TCM (`TCM', 75%, D±F). After24 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 (Cand F) peptides (1 mg/ml) for 2 h at 37°C. Then, DC were washed again before being added to CD8+ T cell lines speci®c for T. cruzi (TSA-1514±522 peptide), in¯uenza virus (M58±66 peptide) or HIV (RT476±484 peptide) for an IFN-g ELISPOT assay. The number of IFN-g SFC per wellwas counted. Data are mean values of SFC 6 SEM from triplicate wells with three (or two) different effector cells numbers per well and arerepresentative 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 ofinhibition were calculated as described in Methods.

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In contrast, addition of the blocking anti-MHC class I antibodydecreased by 60% IFN-g production by this CD8+ T cell line(Fig. 5). Moreover, control experiments showed that the sameconcentrations of soluble CD154, CD152 and anti-IL-12 wereeffective at abrogating the primary response of alloreactive Tcells in a MLR (data not shown) (37). These results indicatethat co-stimulation through the CD28±CD80/CD86 andCD40L±CD40 pathways is not required for the RT476±484 line,which is mainly dependent on recognition of the MHC±peptidecomplex. Therefore, in this case, even though T. cruzi inhibitsCD40 up-regulation and IL-12 production by LPS-stimulatedDC (25), the decreased expression of MHC class I moleculesseems to be responsible for CD8+ T cell response inhibition.

Discussion

The data presented here show that T. cruzi inhibits LPS-induced MHC class I up-regulation on the surface of humanDC. This inhibition is mediated by soluble factor(s) released bythe parasite itself since TCM, added to the DC culture medium,exerts the same effect as intracellular T. cruzi infection. Thesedata are reinforced by the fact that when DC are co-culturedwith T. cruzi, both infected and non-infected DC show a down-regulation of MHC class I expression. So that, in vitro, inaddition to a direct effect of the intracellular parasites(amastigotes) on MHC class I expression, a similar effect isalso observed through the presence of free trypomastigotespresent in the cell suspension and releasing parasite-derivedmolecules similar to TCM. One can hypothesize that, in vivo,the parasites release continuously TCM that acts in a systemicway by diffusing in all the host's body.

The functional consequence of parasite±DC interactionsseems to be that T. cruzi impairs the ability of human DC to

present MHC class I-restricted peptides to speci®c CD8+ Tcell lines. The interference with MHC class I antigen presen-tation 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, itwould 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 de®cient in eliciting novelCD8+ T lymphocytes since DC are the only APC that caninduce primary T cell responses.

We have previously shown that T. cruzi also inhibits co-stimulatory molecules up-regulation during LPS-induced DCmaturation (25). It is dif®cult to completely rule out thepossibility that the effect observed could be due to smallchanges in the level of production or expression of cytokinesand/or co-stimulatory molecules. However, the reduced abilityof T. cruzi-infected DC or TCM-treated DC to stimulate CD8+ Tcell lines seems to be linked mainly to reduced MHC class Imolecule expression since the stimulation of at least one CD8+

T cell line (the RT476±484 peptide-speci®c CD8+ T cell line)depends mainly on the interaction between MHC class I±TCR,but not on the co-stimulatory signals. This inhibition of cellsurface MHC class I expression may contribute to the inabilityof T. cruzi-speci®c CD8+ T cells to ef®ciently eradicate theparasites in infected individuals and suggests the existence ofa novel escape mechanism. This should ideally be testedusing functional analysis of DC harvested from chagasicpatients.

To our knowledge, this is the ®rst report indicating a reducedexpression of MHC class I molecules on human DC by aprotozoan parasite (T. cruzi) and its functional consequences.Down-regulation of MHC class I molecules on T. cruzi-infectedDC may decrease the protective effect of speci®c CD8+ Tlymphocytes. In contrast, such a down-regulation of MHCclass I molecules was not observed in T. cruzi-infected mousemacrophages so that no impairment of antigen presentation tothe speci®c CD8+ T cell line was observed (39). On the otherhand, an up-regulation of MHC class I expression on the J774macrophage cell line and on ®broblasts was induced byT. cruzi, and was shown to be dependent on the presence ofIFN-a/b (40). Toxoplasma gondii, another obligate intracellularprotozoan parasite, is also known to impair the up-regulationof MHC class I expression on IFN-g-activated bone marrow-derived macrophages, but data on functional consequencesare lacking (41).

The inhibitory effect of a soluble factor from T. cruzi on LPS-mediated maturation of DC might be due to competition with aLPS receptor. It has indeed been indicated that GPI-anchoredmucin-like glycoproteins from T. cruzi signal macrophagesthrough receptors of the Toll family. However, the receptortriggered by these glycoproteins seemed distinct from TLR4which is triggered by LPS (42).

In line with our results, a reduced expression of MHC class Imolecules has been previously shown in viral infections. Inparticular, HIV-infected human DC express lower levels ofMHC class I molecules and this may explain the inability ofCD8+ T lymphocytes to be primed early enough to eliminatethe onset of infection in vivo (43). HSV infection is also knownto down-regulate MHC class I expression on human DCprobably as a result of the inhibitory effect of infected-cell

Fig. 5. Role of the co-stimulatory molecules. HLA-A2*0201-matchedDC were incubated in medium containing LPS (100 ng/ml). After24 h, DC were washed and pulsed with the control Tax11±19 peptideor the RT476±484 peptide (1 mg/ml) for 2 h at 37°C. Then, DC werewashed again before being cultured with the RT476±484-speci®cCD8+ T cell line for an IFN-g ELISPOT assay in the presence orabsence of 5 mg/ml of either anti-human IL-12 antibody, humanCD152 (CTLA-4) and human CD154 (CD40L) fusion protein, andanti-human MHC-I antibody or corresponding isotype control. Thenumber of IFN-g SFC per well was counted. Three different effectorcell concentrations were tested. Data are mean values of SFC 6SEM from triplicate wells with 3000 effector cells. The mean valuesof SFC from triplicate wells with control samples (DC pulsed with1 mg/ml control Tax11±19 peptide) was subtracted from theexperimental values and was <10%. The results shown arerepresentative of two independent experiments.

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protein (ICP)-47, an immediate/early protein, on TAP (44,45)and thereby to inhibit MHC class I-mediated peptide presen-tation (45,46). Similar observations have been reported withviruses (adenovirus, Epstein±Barr virus, and human andmouse cytomegalovirus) infecting other cell types. Thesehave been found to subvert MHC class I presentation by avariety of mechanisms, including interference with peptidetransport and entrapment of MHC class I (47±49).

Despite the fact that T. cruzi probably synthesizes the TSA-1antigen during infection, the TSA-1514±522-speci®c CD8+ T cellline did not recognize infected DC except when exogenousTSA-1514±522 peptide was added (data not shown). This mayre¯ect a requirement by some TCR for a higher threshold ofantigenic determinant density than that expressed by theparasite-infected target cells in this experiment. More likely,these results re¯ect the selection of low-af®nity CD8+ Tlymphocytes by the repetitive peptide stimulation techniquethat fails to lyse target cells expressing endogenous deter-minants which were shown to require higher peptide concen-trations to sensitize target cells.

The CD8+ T cell responses found in vivo, although inef®cientto sterilize parasite infection, are induced despite the down-regulation of MHC class I and of co-stimulation moleculesupon maturation of DC found here and in our previous work(25). They may be elicited only against highly expressedepitopes that allow them to reach the threshold level of MHCclass I±peptide density to induce CD8+ responses (50). Theymay also be induced in the lymphoid organs at sites distantfrom T. cruzi and its soluble products. Indeed, DC havedeveloped specialized and ef®cient cross-presentationmechanisms that may allow MHC class I-restricted presenta-tion of endocytosed parasites or apoptotic debris frominfected cells (51±54).

In conclusion, our data suggest that the inhibition of MHCclass I up-regulation on LPS-treated DC may be involved in thegeneration of a suboptimal CD8+ T lymphocyte response uponinfection with T. cruzi. In vivo, this could be a mechanismallowing the parasite to escape immune recognition and favorthe 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 supportedby grants from Centre de Recherche Interuniversitaire enVaccinologie, Fonds Emile Defay, la Banque Nationale de Belgique,the Agence Nationale de Recherches sur le SIDA (ANRS) andEnsemble Contre le SIDA (SIDACTION). L. V. O is the recipient of agrant from the Fonds pour la Formation aÁ la Recherche dans l'Industrieet dans l'Agriculture (FRIA).

Abbreviations

APC antigen-presenting cellCFSE 5-(and 6)-carboxy¯uorescein diacetate succinimidyl

esterDC dendritic cellGM-CSF granulocyte macrophage colony stimulating factorLPS lipopolysaccharidePBMC peripheral blood mononuclear cellSFC spot-forming cellTCM T. cruzi-conditioned medium

TNF tumor necrosis factorTSA-1 trypomastigote surface antigen-1

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