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The Journal of Immunology, 2000, 164: 3741-3748.
Copyright © 2000 by The American Association of Immunologists

T Cell Clones Raised from Chronically Infected Healthy Humans by Stimulation with Toxoplasma gondii Excretory-Secretory Antigens Cross-React with Live Tachyzoites: Characterization of the Fine Antigenic Specificity of the Clones and Implications for Vaccine Development1

Ignazia Prigione2,*, Paola Facchetti*, Laurence Lecordier{dagger}, Didier Deslée{dagger}, Sabrina Chiesa*, Marie-France Cesbron-Delauw{dagger} and Vito Pistoia*

* Laboratorio di Oncologia, Istituto G. Gaslini, Genoa, Italy; and {dagger} Laboratoire des Mécanismes Moléculaires de la Pathogenèse des Sporozoaires, Institut Pasteur and Institut de Biologie de Lille, Lille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Excreted-secreted Ags (ESA) of Toxoplasma gondii (Tg) play an important role in the stimulation of the host immune system in both acute and chronic infections. To identify the parasite Ag(s) involved in the maintenance of T cell-mediated long term immunity, 40 ESA-specific T cell clones were derived from three chronically infected healthy subjects. All the clones were CD4+ and recognized both ESA and live tachyzoites in a HLA-DR-restricted manner. Conversely, CD4+ tachyzoite-specific T cell clones from the same subjects proliferated in response to ESA, pointing to shared immunodominant Ags between ESA and Tg tachyzoites. By T cell blot analysis using SDS-PAGE-fractionated parasite extracts, the following patterns of reactivity were detected. Of 25 clones, 6 recognized Tg fractions in the 24- to 28-kDa range and proliferated to purified GRA2, 5 reacted with Tg fractions in the 30- to 33-kDa range; and 4 of them proved to be specific for rSAg1. Although surface Ag (SAg1) is not a member of ESA, small amounts of this protein were present in ESA preparation by Western blot. Of 25 clones, 8 responded to Tg fractions in the 50- to 60-kDa range but not to the 55-kDa recombinant rhoptries-2 parasite Ag, and 6 did not react with any Tg fraction but proliferated in response to either ESA or total parasite extracts. In conclusion, CD4+ T cells specific for either ESA (GRA2) or SAg1 may be involved in the maintenance of long term immunity to Tg in healthy chronically infected individuals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Toxoplasma gondii (Tg)3 is an obligate intracellular protozoan parasite that infects all mammalian cells (1, 2). Human infection is generally asymptomatic and self-limiting in immunocompetent hosts. These individuals remain chronically infected, the parasites persisting encysted in brain and muscle, and develop life-long protective immunity against reinfection (1, 2). In contrast, in immunocompromised individuals, toxoplasmosis represents one of the major opportunistic infections (3, 4). It is most often due to reactivation of the latent infection and may result in toxoplasmic encephalitis (3, 4).

The central role of cell-mediated immunity in host defense against the acute infection as well as in the control of the chronic state is well recognized (5, 6, 7). Different cytokines (8, 9) and cell populations (10, 11, 12, 13, 14) are involved in the effector and regulatory phases of the immune response to the parasite. We and others have previously shown that Tg-specific CD4+ T cells generated in vitro from chronically infected healthy subjects display a predominant Th0/Th1 profile of cytokine production and efficiently lyse autologous Tg-infected APC (13, 14, 15, 16, 17, 18, 19).

In the search of parasite Ags involved in protective immunity, most of the work has been focused on surface Ags (SAgs) specifically expressed at the proliferative tachyzoite stage with a particular interest in SAg1 (20, 21, 22, 23, 24).

A key role in stimulation of the host immune system has also been documented for parasite excretory-secretory Ags (the so-called ESA), which are expressed at both the tachyzoite and encysted bradyzoite stages (25, 26). ESA represent the majority of the Tg circulating Ags in sera from hosts with acute Tg infection (27). Moreover, ESA secretion by the encysted form of Tg has been proposed as a mechanism that maintains long-lasting immunity to the parasite (26). It has been observed that ESA are highly immunogenic during both human (28) and experimental (29) infections, and their role in inducing protective immunity (either Ab-dependent or cell-mediated) has been demonstrated in different experimental models (29, 30, 31).

The major components of ESA are the GRA molecules that are stored within Tg dense core granules and secreted into the parasitophorous vacuole after parasite invasion (32, 33).

In this work, we have attempted to identify the immunodominant secreted Ag(s) involved in the maintenance of T cell-mediated immunological memory against the parasite in healthy subjects.

To this end, we have characterized a panel of T cell clones derived in vitro from chronically infected healthy donors and cross-reactive with both secreted Ags and live tachyzoites. Our results suggest that, among the different Ags, the GRA2-secreted Ag as well as the major tachyzoite SAg, SAg1, play a relevant role in the maintenance of T cell-mediated memory responses to Tg in chronically infected healthy humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Donors

Heparinized peripheral blood samples were obtained from three healthy donors with serological evidence of prior Tg infection.

Parasite

The RH strain of Tg maintained by repeated passages in Swiss CD1 mice was used throughout the experiments. Tachyzoites were isolated from murine peritoneal fluids, attenuated by serial passages in Vero cells, counted, and used for T cell stimulation (11).

Antibodies

Toxoplasma GRA proteins were detected using the following mAbs: TG17-43 (anti-GRA1), TG17-179 (anti-GRA2) and TG17-113 (anti-GRA5) (34). GRA6 was stained using a polyclonal anti-rGRA6 mouse antiserum (35). A polyclonal antiserum from rats immunized with the SAg1 48–67 MAP peptidic construct was used to detect SAg1 in denaturing conditions (36). The anti-rhoptries (ROP)-2 mAb 4A7 was kindly provided by Dr. Jean-François Dubremetz (Institut Pasteur, Lille, France).

Ag preparations

ESA preparation. Filtered RH strain tachyzoites (1.5 x 108) were incubated at 37°C for 3 h under mild incubation in test tubes containing 1.5 ml RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 10% (v/v) heat-inactivated FCS (Life Technologies). After centrifugation for 10 min at 1000 x g, the ESA-containing supernatants were filtered on a 0.22-µm pore size Millipore membrane (Millipore, Bedford, MA). After addition of 100 U/ml aprotinin (Sigma, St. Louis, MO), ESA preparation was stored at -70°C until used (29).

Purification of the GRA2 Ag. GRA2 was purified from Nonidet P-40-tachyzoite extracts by HPLC, and the purity was assessed by silver staining as previously described (31, 37).

Recombinant Ags. rGRA1 was produced in Escherichia coli as a hybrid protein fused to GST (38). N-terminal and C-terminal rGRA6 correspond to the two hydrophilic N and C terminus domains of GRA6 produced in E. coli as GST-fusions (L. Lecordier and M.-F. Cesbron-Delauw, unpublished observations); these rGST-fusion proteins were purified as reported (39). rGST protein was expressed and used as negative control in cell proliferation assays. The purity of all GST-fusions was assessed by Coomassie blue staining of gel electrophoresis, showing for each r protein a single band (data not shown). rROP2 was provided by Innogenetics, (Ghent, Belgium); ROP2-derived peptides (197–216, 501–524) were synthesized as reported (40). rSAg1 was kindly provided by Dr. E. Petersen, (Statens Seruminstitut, Copenhagen, Denmark) (41).

SDS-PAGE and immunoblotting

SDS-PAGE was performed on 13% polyacrylamide gels according to the procedure of Laemmli (43) using, respectively, ESA or a total tachyzoite extract under reducing conditions (15% ß-mercaptoethanol). Gels were transferred to nitrocellulose membranes (44) and blocked in 5% nonfat dry milk in PBS. Membranes were incubated with primary Abs and then with antispecies alkaline phosphatase conjugates (Sanofi Pasteur Diagnostic, Marnes-La-Coquette, France), both diluted in PBS-1% nonfat dry milk. The alkaline phosphatase activity was detected with the ProtoBlot nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate color development system (Promega, Madison, WI). For enhanced chemiluminescence (ECL), blots were incubated with peroxidase-conjugated second Abs (Jackson ImmunoResearch Laboratories, West Grove, PA), and signals were detected via the ECL system (Pierce, Rockford, IL).

Preparation of Tg tachyzoite Ag-bearing nitrocellulose particles

Ag-bearing particles were prepared as previously described (42). After SDS-PAGE and electrotransfer (see above), the 110-mm-wide nitrocellulose sheet was washed in PBS for 30 min at room temperature. The mw standards and the overall material distribution were both visualized by Ponceau red staining of the nitrocellulose sheet. Distribution of the Ags was analyzed by Western immunoblotting of vertical strips excised at the edges of the nitrocellulose sheet using the above mentioned Abs. The 60-mm-wide membrane containing the blotted material was then divided vertically into 3-mm-high horizontal strips. Positive control was prepared by dotting 25 µg of parasite extract onto a 20-mm2 nitrocellulose circle. Negative control was a similar nitrocellulose circle not loaded with parasite Ags. Each horizontal strip and circle was transferred to a sterile tube, dissolved in 1 ml DMSO (Merck, Rahway, NJ), and incubated for 1 h to ensure sterility. Ag-bearing nitrocellulose particles were precipitated by adding an equal volume of carbonate-bicarbonate buffer (50 mM, pH 9.5), dropwise with vigorous vortexing. The particles were washed twice with HBSS (Life Technologies), finally resuspended in 1.5 ml culture medium and stored at -20°C.

Cell culture and cloning

Peripheral blood mononuclear cells (MNC) were isolated by centrifugation on Ficoll-Hypaque density gradients and resuspended at the concentration of 1 x 106 cells/ml in RPMI 1640 (HyClone Laboratories, Logan, UT) supplemented with L-glutamine, penicillin-streptomycin, nonessential amino acids (BioWhittaker, Walkersville, MD), and 10% pooled human sera obtained from Tg-seronegative donors. MNC were subsequently cultured for 7 days in 24-well plates with or without 5 x 104 tachyzoites/ml or ESA preparation at 1:20 final dilution (17). Low density lymphoid blasts were then purified on a Percoll (Pharmacia, Uppsala, Sweden) density gradient and cloned by limiting dilution immediately after isolation (17). The cloning procedure was performed as follows. Percoll-enriched blasts were seeded in 96-well U-bottom plates at a concentration of 0.5 cell/well in 0.2 ml complete medium supplemented with 20 U/ml rIL2 (Chiron Therapeutics, Emeryville, CA) in the presence of 1 µg/ml PHA-P (Murex Biotech, Dartford, U.K.) and 105 {gamma}-irradiated (60 Gy) allogeneic MNC (17). Alternatively, blasts obtained from MNC stimulated with tachyzoites or ESA were cloned with the same procedure but in the presence of autologous irradiated MNC and live tachyzoites (2500/well) or ESA preparation (1:20 final dilution), respectively. Cells were fed weekly with fresh medium containing 50 U/ml rIL2 and proliferating microcultures were expanded in rIL2-containing medium.

Cell proliferation assays

To test Ag specificity of T cell clones, 2 x 104 blasts were incubated in duplicate with the appropriate Ag in the presence of 5 x 104 autologous irradiated (60 Gy) MNC as APC in 96-well flat-bottom plates in a total volume of 0.2 ml for 72 h (17). The different Ags were used at the following concentrations: Tg tachyzoites at 3500 parasites/well; ESA preparation at a 1:20 (v/v) final dilution; purified GRA2, rGRA Ags, rROP2, and rSAg1 at 1 µg/ml; ROP2 peptides at 0.5–5 µg/ml; and the Ag-bearing particles at a 1: 10 (v/v) final dilution. Cells were pulsed with 0.5 µCi/well [3H]thymidine (ICN Biomedicals, Costa Mesa, CA) for the last 18 h of culture and harvested. The cell-associated radioactivity was determined by liquid scintillation counting.

The capacity of anti-MHC products mAb to inhibit the tachyzoite/Ag-induced proliferation of T cell clones was investigated as reported (11). In brief, T cell blasts were cultured with autologous irradiated (60 Gy) MNC and tachyzoites or Ag in the presence or absence of various dilutions of anti-HLA-DR or anti-HLA-ABC mAb. Cell proliferation was assessed after 72 h.

To investigate the pattern of cytokine production by Tg- or ESA-specific T cell clones, T cell blasts were washed twice, resuspended at the concentration of 1 x 106 cells/ml in complete medium, and cultured for 48 h with 1 µg/ml PHA-P plus 5 ng/ml PMA (Sigma) (17). Controls were T cell clones cultured in complete medium for 48 h without stimuli. Supernatants were collected and stored at -80°C until tested. IL-4 and IFN-{gamma} were assayed using ELISA kits from Amersham (Little Chalfont, U.K.) and Medgenix (Fleurus, Belgium), respectively.

Immunophenotypic studies

Immunophenotypic analyses of Tg- or ESA-specific T cell clones were performed by direct immunofluorescence using FITC-conjugated mAbs (CD3, CD4, CD8) from Becton Dickinson (Mountain View, CA). Cells were incubated for 30 min on ice with saturating amounts of mAb, washed twice, and analyzed by a FACScan flow cytometer (Becton Dickinson) as reported (17).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment and characterization of ESA-specific T cell clones

In preliminary experiments, peripheral blood MNC from six healthy donors, three seropositive and three seronegative for Tg infection, were tested for proliferative response to a Tg ESA preparation. After 7 days of culture, [3H]thymidine incorporation was detected in MNC from seropositive subjects only (data not shown). Furthermore, the two ESA preparations used throughout all of the experiments did not stimulate the proliferation of PHA-induced CD4+ T cell clones.

Next, MNC from Tg-seropositive donors were incubated for 7 days with ESA preparation and subsequently enriched for lymphoid blasts by a Percoll gradient. T cell clones were then generated from cultured blasts under limiting dilution conditions in the presence of feeder cells, ESA or PHA, and rIL2. Forty ESA-specific, CD4+ T cell clones were collectively obtained from the three donors. All of the clones proliferated in response to both ESA preparation and live Tg tachyzoites (Table IGo).


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  		Table I. Proliferative responses of CD4+ T cell clones raised by ESA stimulation to ESA and Tg tachyzoites1

 
To demonstrate the HLA-restricted nature of ESA-specific T cell clones, three representative clones (clones 17, 22 and 26; see Table IGo) were incubated with ESA preparation or live Tg tachyzoites in the presence of an anti-HLA-DR mAb or of an isotype-matched anti-HLA class I mAb. As shown in Fig. 1Go, proliferation of the three T cell clones in response to ESA or tachyzoites was virtually abrogated by treatment with the anti-HLA-DR, but not with the anti-HLA class I mAb.



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  		FIGURE 1. Anti-HLA-DR mAb inhibits the proliferation of ESA-specific T cell clones to ESA and Tg tachyzoites. Blasts (2 x 104) were cultured for 72 h with 5 x 104 irradiated autologous MNC in the presence of medium alone (CTR), ESA, or tachyzoites. The proliferative responses to ESA and tachyzoites were determined in the presence or absence of anti-HLA-DR or anti-HLA class I mAbs, both of the IgG2a subclass. Cell proliferation was assessed by [3H]thymidine incorporation. Results were obtained with three representative CD4+ ESA-specific T cell clones.

 
In subsequent experiments, 20 additional CD4+ T cell clones were raised by stimulation with Tg tachyzoites and tested for proliferative responses to the ESA preparation. All the Tg tachyzoite-specific clones incorporated [3H]thymidine on incubation with ESA; Fig. 2Go shows the results obtained with 10 representative cell clones. When an anti-HLA-DR mAb was added to the cultures, T cell proliferation was abolished (data not shown).



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  		FIGURE 2. Tg tachyzoite-specific T cell clones proliferate in response to ESA preparations. Cells (2 x 104) cells from Tg-specific T cell clones were cultured with 5 x 104 irradiated autologous MNC in the presence of medium alone (CTR), ESA preparation, or live tachyzoites. Proliferation was assessed 3 days later by [3H]thymidine incorporation.

 
These results, obtained collectively by the study of 60 Tg-specific T cell clones, provide compelling evidence for the cross-recognition of ESA and Tg .

Response of ESA-specific T cell clones to fractionated Tg Ags.

In a subsequent series of experiments, the fine antigenic specificity of ESA-specific T cell clones was investigated. To this end, T cell clones raised by ESA stimulation were tested for reactivity to Tg antigenic fractions by T cell blot analysis, whereby T lymphocytes are challenged with Ags fixed onto solid particles (42).

To this end, a total extract of Tg was fractionated by SDS-PAGE and transferred to nitrocellulose; horizontal strips of the membrane converted into Ag-bearing particles were then used to stimulate T cell clones in the presence of irradiated feeder cells.

Twenty-five ESA-specific T cell clones were tested. No T cell clone proliferated on incubation with nitrocellulose particles devoid of Ag. Four patterns of reactivity were detected; Fig. 3Go shows one representative experiment with a single T cell clone for each pattern observed. Eight of 25 clones recognized Tg fractions corresponding to an approximate molecular mass of 50–60 kDa (Fig. 3GoAI) and containing the ROP2 Ag in immunoblotting assays, as shown in Fig. 3GoB. Five of 25 clones proliferated in response to fractions spanning an approximate molecular mass of 30–33 kDa (Fig. 3GoAII) and containing the GRA6 and SAg1 Ags, as assessed by immunoblotting (Fig. 3GoB). Six of 25 clones reacted with Tg fractions corresponding to an approximate molecular mass of 24–28 kDa (Fig. 3GoAIII). These fractions reacted with mAbs to GRA1 and GRA2 Ags in immunoblotting assays (Fig. 3GoB). Finally, 6 of 25 clones responded to the ESA preparation but not to any Tg fraction (Fig. 3GoAIV). The latter clones proliferated when challenged with live tachyzoites (see Table IGo) or with a positive control represented by total parasite extract blotted on nitrocellulose particles (data not shown).



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  		FIGURE 3. Proliferative response of ESA-specific T cell clones to fractionated Tg extracts (T cell blot analysis). A, Two x 104 blasts from ESA-specific T cell clones were cultured for 72 h with 5 x 104 irradiated autologous MNC in the presence of Ag-bearing nitrocellulose particles (1:10 final dilution) prepared as described in Materials and Methods. Strip numbers (1 to 20) refer to individual horizontal strips processed into Ag-bearing nitrocellulose particles. Their relative molecular masses can be deduced from the Western immunoblot shown in B. Proliferation was assessed by [3H]thymidine incorporation. Panels show the response of one representative T cell clone for each pattern of reactivity observed. B, Distribution analysis of the Ag on nitrocellulose strips by Western immunoblot of a total tachyzoite extract. The vertical strips were probed with the anti-GRA1 mAb Tg 17–43 (GRA1), the anti-GRA2 mAb Tg 17–179 (GRA2), the anti-GRA5 mAb Tg 17–113 (GRA5), anti-GRA6 mouse polyclonal Abs (GRA6), anti-SAg1 rat polyclonal Abs (SAg1), and the anti-ROP2 mAb 4A7 (ROP2). Horizontal lines correspond to the 3-mm-high horizontal strips that are processed into Ag-bearing nitrocellulose particles.

 
Reactivity of ESA-specific T cell clones to purified or recombinant Tg Ags

Next, the three groups of clones reactive to well-defined Tg fractions were tested for proliferative responses to the Ags detected by immunoblotting in the same fractions.

The six clones that recognized Ag(s) ranging from 24 to 28 kDa were incubated with rGRA1 or native GRA2 (Fig. 3GoB) in the presence of irradiated feeder cells. As shown in Fig. 4GoA, all of these clones proliferated in response to GRA2, but not to GRA1.



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  		FIGURE 4. ESA-specific T cell clones proliferate on challenge with GRA2 and SAg1 Ags. Blasts (2 x 104) from ESA-specific T cell clones were cultured with 5 x 104 irradiated autologous MNC in the presence of 1 µg/ml purified or recombinant Tg Ags. Proliferation was assessed 3 days later by [3H]thymidine incorporation. A, Responses to purified GRA2 or rGRA1 of six different clones reactive to 24- to 28-kDa Tg fractions. B, reactivity to rSAg1 or C-terminal and N-terminal rGRA6 of five clones that recognize 30- to 33-kDa Tg fractions. The figure shows the response to N-terminal rGRA6, but identical results were obtained with C-terminal rGRA6.

 
The five clones reactive with Tg fractions with a molecular mass of 30–33 kDa were challenged with C-terminal or N-terminal rGRA6 or with rSAg1 (see Fig. 3GoB). Fig. 4GoB shows that four of five clones responded to SAg1, but not to C-terminal or N-terminal rGRA6. The fifth clone from this group did not proliferate on incubation with any of the tested Ags.

The eight clones that responded to the 50- to 60-kDa Tg fractions were challenged with rROP2 (see Fig. 3GoB), but no cell proliferation was detected (data not shown). In additional experiments, the same clones were incubated with two ROP2 peptides that are recognized by T cells from most Tg-seropositive individuals (40). Again, no cell proliferation was observed. Finally, the same clones were tested against GRA2 in view of the hypothesis that GRA2 dimers, with a molecular mass that would fall into the 50- to 60-kDa range, could be target Ags. However, no [3H]thymidine incorporation was detected under these conditions (data not shown).

In summary, 6 of 25 (24%) ESA-specific T cell clones recognized GRA2, 4 of 25 (16%) reacted with SAg1, 8 of 25 (32%) responded to an as yet unidentified 50- to 60-kDa Ag, and 6 of 25 (24%) did not recognize any Tg fraction. The last ESA-specific T cell clone (1 of 25; i.e., 4%) proliferated in response to 30- to 33-kDa Tg fractions but not to SAg1.

The above experiments indicated that a number of T cell clones were specific for SAg1, which, however, does not belong to the ESA family. Therefore, Western blot experiments were conducted to investigate whether or not some SAg1 protein was present in the ESA preparation used throughout this study. As shown in Fig. 5Go, a faint band of immunoreactive SAg1 was detected after prolonged exposure. In the same figure, it is shown that GRA1, GRA2, GRA5, and GRA6, which represent typical Tg Ags associated with the dense granules, were well represented in ESA preparation, whereas ROP2, as expected, was not found.



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  		FIGURE 5. Western immunoblot analysis of the ESA preparation electrophoresed in reducing conditions. The blots were probed with the anti-GRA1 mAb Tg 17–43 (GRA1), the anti-GRA2 mAb Tg 17–179 (GRA2), the anti-GRA5 mAb Tg 17–113 (GRA5), anti-GRA6 mouse polyclonal Abs (GRA6), anti-SAg1 rat polyclonal Abs (SAg1), and the anti-ROP2 mAb 4A7 (ROP2). They were developed by ECL. The major stained proteins are the four tested GRA Ags. After prolonged exposure, a weak band corresponding to SAg1 became detectable. No signal was found with the anti-ROP2 mAb.

 
These results lend support to the hypothesis that the presence of SAg1 in ESA preparation is likely attributable to shedding from the parasite surface membrane.

Cytokine production by ESA-specific T cell clones

ESA-specific T cell clones subdivided into four groups according to the patterns of Tg fraction reactivity were subsequently tested for cytokine production after PHA-PMA stimulation. These polyclonal activators are potent inducers of cytokine gene expression and have been previously shown to trigger cytokine secretion by Tg-specific T cell clones with patterns similar to those induced by Ag-specific stimulation (17). The cytokines tested were IL-4 and IFN-{gamma}, the production of which allows identification of the Th0, Th1, or Th2 orientation of helper T cell clones. Most of the clones produced both IL-4 and IFN-{gamma} irrespective of their antigenic specificity (Fig. 6Go), although, in accordance with previous studies, there was a consistent trend to hyperproduction of IFN-{gamma} (Fig. 6Go) (11, 17). Notably, four of the above clones were incubated with an insolubilized CD3 mAb before being tested for cytokine production. IL-4 and IFN-{gamma} were produced according to the same patterns observed on PHA-PMA stimulation (not shown).



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  		FIGURE 6. Production of IFN-{gamma} and IL-4 by ESA-specific T cell clones subdivided according to their antigenic reactivity. A, GRA2-reactive T cell clones; B, SAg1-specific T cell clones; C, clones reactive with 50- to 60-kDa fractions; D, Clones that do not recognize any tested Tg fraction although proliferating in response to Tg tachyzoites or total parasite extracts. Cloned T cells were stimulated with PHA and PMA for 48 h before their supernatants were harvested. Cytokines were detected by ELISA. Results are expressed as nanograms per ml.

 
Thus, the overall patterns of IL-4 and IFN-{gamma} production by ESA-specific T cell clones appear to be unrelated to their antigenic specificity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Healthy individuals chronically infected with Tg provide an useful model in which the mechanisms involved in long term immunity against the parasite and the Tg Ags recognized by memory T cells can be investigated. Among the Tg Ags characterized thus far, ESA are peculiar since, at least for most of them, expression is maintained during both the acute and the chronic stages of the parasite (25, 26). Thus, in principle, ESA could play a role in the persistent stimulation of cell-mediated immunity in chronically infected healthy subjects (26).

This hypothesis, originally formulated by Dessaint and Capron (26), was tested in this study by raising ESA-specific and tachyzoite-specific T cell clones from the peripheral blood of three healthy individuals who were seropositive for Tg infection.

All of the clones obtained were CD4+ and HLA class II restricted independent of the stimuli used to raise them. In the majority of the studies in which human Tg- specific T cell clones were derived from healthy donors (15, 16, 17, 18, 19), a CD4+ immunophenotype was consistently detected. Nonetheless, some investigators have been successful in expanding in vitro CD8+ T cells cytotoxic to Tg-infected cells from blood of healthy seropositive donors (13, 14, 45). In acute toxoplasmosis, both CD4+ and CD8+ T cell clones could be easily grown from patients’ peripheral blood (46). The reasons for such differences between acute and chronic human Tg infections are unknown but may relate to the different immune effector mechanisms activated in vivo by the parasite (18).

The characterization of the Tg Ags recognized by ESA-specific T cell clones was conducted using three approaches: 1) ESA-specific T cell clones were challenged with live Tg tachyzoites and, conversely, tachyzoite specific-T cell clones were stimulated with ESA; 2) ESA-specific T cell clones were cultured with Tg tachyzoite fractions to define the molecular mass of target Ags and 3) the fine specificity of ESA-specific T cell clones was assessed by their incubation with purified or recombinant Tg Ags selected on the basis of the latter experiments.

As for the first point, all ESA-specific T cell clones proliferated when challenged with Tg tachyzoites, and the same result was obtained when the opposite strategy was used. These results suggest that ESA and live tachyzoites share similar immunodominant Ags. However, because both ESA and tachyzoites are complex antigenic systems the composition of which is only partially known, additional studies were performed to gain more insight into these issues.

The second approach, i.e., stimulation of ESA-specific T cell clones with fractionated parasite extracts, allowed the identification of three major patterns of reactivity against fractions of different molecular masses (24–28 kDa, 30–33 kDa, 50–60 kDa). A fourth group of clones proliferated on incubation with ESA, but not in the presence of any of the tested parasite fractions. The possibility that the latter clones were not ESA specific was ruled out by the following: 1) ESA-induced cell proliferation was abrogated by anti-HLA class II Abs; and 2) all of the clones from this group incorporated [3H]thymidine on challenge with Tg tachyzoites or total parasite extracts. Thus, failure of some ESA-specific T cell clones to react with fractionated parasite Ags may be due to the denaturing conditions of the SDS/PAGE used for Tg tachyzoite fractionation and/or to the presence in the ESA preparation of low molecular mass Ag(s) that were not retained in the polyacrylamide gel.

Finally, the fine specificity of two groups of ESA-specific T cell clones was determined after challenge with purified or recombinant Ags. These studies demonstrated that one group of ESA-specific T cell clones recognized the GRA2 Ag and another one the SAg1 Ag.

GRA2 belongs to a family of at least nine proteins associated with the so-called dense granules which are specialized secretory organelles found in all Apicomplexan parasites (33). After parasite host-cell invasion, the GRA proteins are secreted into the parasitophorous vacuole where most of them behave like membrane-associated proteins and like components of the cyst wall (33, 34). GRA proteins have been shown to be major soluble components of ESA preparations, obtained on serum-stimulated secretion of extracellular parasites (33, 47).

These Ags are actively secreted by the parasite and constitute the major circulating Ags detectable during the acute phase of the infection (27). ESA elicit both humoral and cellular immune responses in Tg-infected hosts (28, 29, 30). Furthermore, ESA immunization may confer a high level of protection to both mice and rats against congenital Toxoplasma infections (29, 30, 31). In the latter experimental models, such protective immunity could be afforded by animal immunization with purified GRA2 (31, 33).

SAg1 is a 30-kDa glycoprotein that together with SAG2 (22 kDa), p23, p35, and SAG3 (43 kDa) is a major component of the surface proteins of Tg tachyzoites (20). The five proteins share glycosylphosphatidylinositol structures for anchoring these molecules at the surface membrane. SAg1 was not supposed to be released from dense core granules after serum stimulation and, therefore, to be absent from ESA preparation (47). However, Western blot experiments showed that, under particular experimental conditions, a faint SAg1-specific band was demonstrable. These findings suggest that low amounts of SAg1 were released during ESA preparation owing to shedding from the surface of the parasite or to occasional parasite lysis.

SAg1 is considered as one of the most immunogenic Ags of Tg due to its ability to elicit a vigorous Ab response (48, 49). Anti-SAg1 Abs are detected in both early and chronic phases of human Tg infection (48, 49). Finally, in animal models, SAg1 or derivative peptides (36) have been found to activate CD8-dependent or Ab-dependent protective responses against the parasite (22, 23, 24).

The last group of clones displayed consistent reactivity with Tg fractions in the molecular mass range from 50 to 60 kDa. A 56-kDa Tg Ag associated with the rhoptries-secretory organelles of Tg, and named ROP2 was previously identified as one of the Tg Ags capable of eliciting CD4+ T cell responses in humans (50). Even if ROP2 was not expected to belong to ESA preparations (33), we have investigated whether our 50- to 60-kDa fraction-reactive T cell clones proliferated on exposure to either rROP2 or synthetic peptides thereof (40). One of the latter peptides has been recently characterized as a major T cell epitope of ROP2 (40). No proliferation in response to rROP2 or its derivative peptides was detected. Because the ROP2-specific T cell clone reported in the literature was initially isolated on cell stimulation by a Tg-soluble Ag preparation (50), the lack of any response to ROP2 in our experiments is most probably due to differences in the antigenic preparations used to raise T cell clones.

Therefore, the molecular target(s) recognized by the 50- to 60-kDa group of T cell clones may represent a novel Ag(s) associated with ESA. In this connection, a recent study in mice has allowed the isolation of a CD4+ T cell clone reactive with a new ESA of the apparent molecular mass of 40 kDa (51).

Previous in vitro and in vivo studies have emphasized the role of type I cytokines, e.g., IFN-{gamma}, produced by Tg-specific T cells in the defense against the parasite (8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 52). In this study, we investigated whether the nature of the Ag recognized by ESA-specific T cell clones could influence their profiles of cytokine expression. These experiments demonstrated that this was not the case, because all of the clones displayed a Th0-type pattern of cytokine production with a high IFN-{gamma}:IL-4 ratio irrespective of their antigenic specificity.

This study demonstrates that: 1) Tg Ags, e.g., SAg1 and GRA2, play an important role in the long term stimulation of Tg-specific helper T cells; 2) both tachyzoite-specific, i.e., SAg1, and ESA expressed during the whole intermediate host life cycle of the parasite, i.e., GRA2, are involved in chronic stimulation of cell-mediated immunity against Tg; 3) other Ags that await further characterization may also play a role in the phenomenon. The finding that both SAg1 and GRA2 were target Tg Ags of T cell clones from healthy chronically infected subjects lends support to a model whereby memory T cell responses may be maintained throughout transient parasitemia taking place after the occasional rupture of tissue cysts. These waves of parasitemia would allow antigenic restimulation of both SAg1- and GRA2-specific helper T cell clones to occur.

Thus far, most of the efforts for Tg vaccine development have been addressed to Ags expressed by the parasite at the tachyzoite stage, although a relevant proportion of Tg-induced pathology is attributable to cysts containing bradyzoites or to disease reactivation from this "dormant stage" (53).

Attenuated Tg tachyzoite vaccines have been successfully employed for animal use, but vaccination with live organisms cannot be safely performed in humans. Thus, the approach to a human Tg vaccine must be based on the use of recombinant Ags or synthetic peptides, that, ideally, should protect the host from all the life cycle stages of the parasite (53). Our data showing that CD4+ Tg-specific T cell clones from chronically infected healthy donors react with both SAg1 and GRA2 support the hypothesis that a combination of these Ags or of appropriate derivative peptides represent suitable candidates for vaccine development in humans.


   Acknowledgments
 
We thank the blood donors for their cooperation, Dr. E. Petersen for providing rSAg1, and Eliana Campochiaro for secretarial assistance.


   Footnotes
 
1 This work was supported in part by Ministero della Sanità, Progetti di Ricerca Corrente. Back

2 Address correspondence and reprint requests to Dr. Ignazia Prigione, Laboratorio di Oncologia, Istituto G. Gaslini, Largo G. Gaslini 5, 16147 Genoa, Italy. Back

3 Abbreviations used in this paper: Tg, Toxoplasma gondii; ESA, excreted-secreted Ags; GRA, dense granule proteins; SAg, surface Ag; MNC, mononuclear cells; ROP, rhoptries; ECL, enhanced chemiluminescence. Back

Received for publication June 29, 1999. Accepted for publication January 21, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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