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* Institut für Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Mannheim, Universität Heidelberg, Mannheim, Germany;
Abteilung für Neuropathologie, Klinikum der Universität zu Köln, Köln, Germany;
Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London, United Kingdom; and
Institut für Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactosidase (-gal) and studied whether 1) secreted or cytoplasmic Ags and 2) tachyzoites or bradyzoites, which persist intracerebrally, induce CD8 T cells. We monitored the frequencies and kinetics of -gal-specific CD8 T cells in infected mice by MHC class I tetramer staining. Upon oral infection of B6C (H-2bxd) mice, only -gal-secreting tachyzoites induced -gal-specific CD8 T cells. However, upon secondary infection of mice that had received a primary infection with tachyzoites secreting -gal, -gal-secreting tachyzoites and bradyzoites transiently increased the frequency of intracerebral -gal-specific CD8 T cells. Frequencies of splenic and cerebral -gal-specific CD8 T cells peaked at day 23 after infection, thereafter persisting at high levels in the brain but declining in the spleen. Splenic and cerebral -gal-specific CD8 T cells produced IFN-
and were cytolytic upon specific restimulation. Thus, compartmentalization and stage specificity of an Ag determine the induction of CD8 T cells in toxoplasmosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Control of T. gondii is critically dependent on CD4 and CD8 T cells, which both produce protective IFN- (3, 4, 5). In addition, perforin-mediated killing of T. gondii-infected cells by CD8 T cells contributes to parasite control in chronic Toxoplasma encephalitis (TE), during which CD8 T cells represent 
30–50% of all inflammatory leukocytes (6, 7). The crucial role of CD8 T cells in the control of T. gondii is further evidenced by immunogenetic studies in mice, which revealed that the MHC class I Ld molecule confers protection against toxoplasmosis, although immunodominant MHC class I-restricted epitopes of T. gondii have not yet been identified (8, 9). Impairment of T cell immunity may lead to a lethal reactivation of intracerebrally persisting cysts as observed in AIDS patients.
At present it is unknown which stages of the parasite, i.e., the tachyzoite and/or bradyzoite, are recognized by CD8 T cells. Moreover, it is yet unresolved whether both secreted and cytoplasmic Ag of T. gondii induce a CD8 T cell response, although there is clear evidence that at least the tachyzoite-specific SAG1 protein, which is released from the cell surface of T. gondii, induces T. gondii-specific CD8 T cell responses (10). Previously, a systematic analysis on the role of Ag compartmentalization for the induction of CD8 T cells was successfully performed by the expression of a heterologous Ag under various experimental conditions in bacterial pathogens as well as in the protozoal kinetoplast Trypanosoma cruzi (11, 12, 13). These studies revealed that the compartmentalization of pathogen-derived Ag plays a decisive role for the resulting CD8 T cell response and that depending on the pathogen secreted, both secreted and cytoplasmic Ag elicit Ag-specific CD8 T cell responses. However, such a systematic analysis has not been performed in toxoplasmosis.
To systematically analyze how the compartmentalization and stage specificity of an Ag determine the induction of a parasite-specific CD8 T cell response and to dissect in detail the kinetic and hierarchy of the CD8 T cell response with peptide-specific precision, we expressed the model Ag Escherichia coli -galactosidase (-gal) that contains defined CD8 T cell epitopes either as a secretory or cytoplasmic Ag in tachyzoites under the control of the tachyzoite-specific SAG1 promoter as well as a secretory Ag in bradyzoites under the control of the bradyzoite-specific SAG4 promoter of T. gondii. Analysis of mice infected with cysts of the various recombinant T. gondii parasites demonstrated that tachyzoites secreting -gal induced a long-lasting dominant CD8 T cell response against the Ld-restricted -gal876–884 epitope and a short-lived subdominant CD8 T cell response against the Kb-restricted -gal497–504 epitope, but they did not induce a CD8 T cell response against the Kb-restricted -gal96–103 epitope. Cerebral Ld-restricted, -gal876–884-specific, IFN--producing, and cytotoxic CD8 T cells persisted at high frequency in chronic TE, indicating that control of persisting T. gondii is at least partially dependent on these CD8 T cells. These findings provide novel insights into the potential role of stage-specific expression and compartmentalization of Ags in the induction of a T. gondii-specific CD8 T cell response and may have important implications for the design of T. gondii-based vaccines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tachyzoites of T. gondii (Prugniaud strain) were propagated in human foreskin fibroblast (HFF) monolayers grown in DMEM (PAA Laboratories, Cölbe, Germany) containing 3.7 g/L sodium bicarbonate (Sigma-Aldrich, Deisenhofen, Germany), 10 mM HEPES (Sigma-Aldrich), 1 mM L-glutamine (Sigma-Aldrich), 10% FCS (PAA Laboratories), and 10 µg/ml gentamicin (Sigma-Aldrich). The hypoxanthine-xanthine-guanine-phosporbosyl-transferase (HXGPRT) gene-deprived T. gondii PruHX- was generated by double homologous recombination using the plasmid pRH
HXGPRT and 6-thioxanthine selection (14, 15).
Construction of expression vectors
To express cytoplasmic -gal under the control of the sag1 promoter, the vector pS1LACZ-HX was constructed by amplifying the E. coli lacZ gene with PCR primers 5'-GCC ATG CAT ATG GAG AAG TTC CTA TTC CTA TTC CGA AG-3' (sense) and 5'-TGA TTA ATT AAG GAG ATC TTT TTT GAC ACC AGA CCA ACT GGT A-3' (antisense) using the plasmid pSAG1-Gal (16) as template DNA. PCR was conducted using AmpliTaq DNA polymerase (PerkinElmer, Dreieich, Germany). The resulting PCR product was introduced with NsiI and PacI restriction enzyme sites at its 5' and 3' ends, respectively, and was subsequently cloned into the NsiI and PacI-digested plasmid, pHXGPRTLoxSLacZ (17).
To express secretory -gal under the control of the sag4 promoter, the vector pS4spLACZ-HX was constructed by amplifying the lacZ gene fused to the signal peptide of sag1 from the plasmid pSAG-gal with the PCR primers, 5'-AAA CTG CAG TTT CCG AAG GCA GTG AGA CGC-3' (sense) and 5'-TGA TTA ATT AAG GAG ATC TTT TTT GAC ACC AGA CCA ACT GGT A-3' (antisense). The resulting PCR fragment was introduced with a PstI and a PacI site at the 5'and 3' ends, respectively, and was cloned into the NsiI and PacI-digested plasmid, psag4GFPsag4-HX (D. Soldati, unpublished data). All restriction enzymes were supplied by New England Biolabs (Frankfurt, Germany).
DNA sequences of all lacZ-containing plasmids used in this study were determined (MWG Biotech, Ebersberg, Germany) within the regions of the three known CD8 T cell epitopes (18, 19, 20).
Parasite transfection and drug selection using HXGPRT as a selectable marker gene
Parasites PruHX- were harvested from freshly lysed cultures of T. gondii-infected HFF cells and transfected by electroporation as described previously (21). T. gondii mutants PruS1LACZ and PruS4SPLACZ were generated by transfecting the parental PruHX- with the expression plasmids pS1LACZ-HX and pS4spLACZ-HX, respectively. The T. gondii mutant, PruS1SPLACZ, was obtained by cotransfecting the parental PruHX- with the plasmid pSAG1-Gal, which contains the lacZ gene fused to the signal peptide of sag1, and pminiHXGPRT. For each transfection, 50–100 µg of BamHI-linearized plasmid DNA were used. For cotransfection, parasites were transfected with the lacZ-containing plasmid together with the selection plasmid pminiHXGPRT (14) at a 10:1 ratio. Both plasmids were linearized with BamHI before the transfection. Cotransfection was performed using restriction enzyme-mediated insertion in the presence of BamHI (22). From 24 to 48 h after electroporation, parasites were cultivated in the presence of 25 µg/ml mycophenolic acid and 40 µg/ml xanthine and cloned 7–10 days later by limiting dilution in 96-well microtiter plates containing HFF cells.
Stable transformants were analyzed for the presence of the recombinant protein at the respective cellular compartment of the parasites by indirect immunofluorescence assay. In addition, parasites were subjected to 5-bromo-4-chloro-3-indolyl b-D-galactoside (X-Gal) staining for the detection of -gal activity (16).
Indirect immunofluorescence assay
All manipulations were conducted at room temperature as described previously (23). In brief, after fixation with 4% paraformaldehye/0.05% glutaraldehyde in PBS, neutralization with 0.1 M glycine in PBS, permeabilization with 0.2% Triton X-100 in PBS, and blocking with 2% BSA/0.2% Triton X-100 in PBS, glass coverslips attached with the tachyzoite-infected HFF cells were incubated with rabbit polyclonal Abs directed against E. coli -gal (BioTrent, Cologne, Germany) and/or a mouse mAb against T. gondii BAG1, a bradyzoite-specific Ag, (24) (kindly provided by Dr. W. Bohne, Universität Göttingen, Goettingen, Germany), followed by FITC- or Alexa Fluor 594 reactive dye-conjugated goat anti-mouse or goat anti-rabbit Ab, respectively (Molecular Probes, Leiden, The Netherlands).
X-Gal staining
Tachyzoite-infected HFF cells with/without in vitro alkaline medium treatment and cyst-containing brain homogenates from chronically infected NMRI mice were fixed and subjected to X-Gal staining (16). In brief, brain tissue was isolated, minced through a cell strainer, and centrifuged. The pellet was resuspended in the fixative solution (2% formaldehyde/0.02% glutaraldehyde) and washed once with PBS before being resuspended in a substrate solution containing X-Gal (Sigma-Aldrich). After 2 h, the cells fixed in vitro were checked for blue color substrate formation. Brain homogenates were examined microscopically for blue-stained cysts after 12 h.
Screening for PruS4SPLACZ T. gondii expressing -gal after alkaline treatment
HFF cells attached on glass coverslips were infected with clones stably transfected with the expression vector pS4spLACZ-HX. After 6 h of incubation, T. gondii-infected cells were subjected to a pH shift by alkaline treatment as described before (25) for 3–5 days before testing for enzyme activity by X-Gal staining and the expression of both -gal protein and bradyzoite-specific marker BAG1 by indirect immunofluorescence assay. Stable clones expressing -gal selectively after alkaline treatment were selected for further in vivo analysis. The expression of -gal in brain tissue cysts was confirmed by -gal staining of cysts isolated from NMRI mice chronically infected with the selected clones.
Generation of MHC-tetramer reagents
MHC/peptide tetrameric complexes, Ld/-gal876–884, Kb/-gal96–103, and Kb/-gal497–504 were generated as described before (26). Briefly, recombinant Kb and Ld H chain and 2-microglobulin were expressed as insoluble inclusion bodies in E. coli and were further purified. The purified Ld H chain was refolded in vitro in the presence of a high concentration of the synthetic peptide TPHPARIGL (-gal876–884), whereas the purified Kb H chain was refolded in vitro in the presence of one of the peptides DAPIYTNV (-gal96–103) or ICPMYARV (-gal497–504) to form stable and soluble MHC/peptide complexes that are specifically biotinylated in vitro by adding the enzyme BirA (Avidity, Denver, CO), d-biotin, and ATP. Complexes were purified by gel filtration over a Superdex 200 HR column (Amersham Pharmacia Biotech, Freiburg, Germany). Purified biotinylated MHC/peptide complexes were multimerized with streptavidin-PE (Molecular Probes). Tetrameric complexes were buffer exchanged with PBS containing 0.02% sodium azide, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 0.5 mM EDTA, and adjusted to a concentration of 2 mg/ml by ultrafiltration. Peptides were supplied by Jerini (Berlin, Germany).
Mice and infection
B6C (C57BL/6 x BALB/c F1; H-2bxd) and NMRI (outbred) mice (Janvier, Le Genest St Isle, France) were kept under specific pathogen-free conditions throughout the experiments. A total of 104 tachyzoites were injected i.p. into NMRI mice. Cysts were isolated from the brain of these animals 3–6 mo thereafter. Brain tissue was homogenized in HBSS (Life Technologies, Rockville, MD) before cyst counting. For all experiments, mice were infected orally with gavage.
Assessment of virulence of various T. gondii strains
The virulence of the various T. gondii transformants was analyzed by infecting B6C mice orally with cysts of the various clones (five cysts/mouse) and assessing both the survival of mice and the number of intracerebral cysts at day 30 postinfection (p.i.). For cyst counting, brain tissue was isolated from five mice per group and homogenized in a final volume of 2 ml of HBSS/brain. A total of 300 µl of each brain homogenate was examined microscopically for the presence of cysts.
Isolation of splenocytes and cerebral leukocytes
At the indicated days p.i., animals were anesthetized and intracardially perfused with 0.9% NaCl to remove contaminating intravascular leukocytes from the brain. Splenic leukocytes were isolated by passing spleens through a cell strainer (BD Biosciences, Heidelberg, Germany), and erythrocytes were lysed with ammonium chloride. Cerebral leukocytes were isolated from the brains as described previously (27). In brief, brain tissue was minced through a cell strainer, and leukocytes were separated by Percoll gradient centrifugation (Amersham Pharmacia Biotech).
Flow cytometry
The kinetics of -gal-specific CD8 T cells were determined by costaining isolated splenic and cerebral leukocytes with CD8-FITC and one of the generated tetramer reagents, Ld/-gal876–884-, Kb/-gal96–103-, and Kb/-gal497–504-PE, respectively. To analyze the activation state of -gal-specific CD8 T cells, splenic and cerebral T cells were costained with a rat anti-mouse CD8-CyChrome (clone 53-6.7), Ld/-gal876–884-PE, and either rat anti-mouse CD62L-FITC (clone MEL-14) or rat anti-mouse CD44-FITC (clone IM7). All Abs were obtained from BD Biosciences. Flow cytometric analysis was performed using a FACScan (BD Biosciences). Data were analyzed with the CellQuest 3.3 software (BD Biosciences).
ELISPOT assay
The frequency of IFN- producing splenic and cerebral -gal-specific CD8 T cells was determined by ELISPOT assay. Splenic and cerebral leukocytes at concentrations of 2 x 103, 2 x 104, and 2 x 105 cells/well, respectively, were placed in an ELISPOT plate coated with rat anti-mouse IFN- mAb (Biosource International, Camarilla, CA). Cerebral leukocytes were cocultured overnight with syngeneic B6C spleen cells from noninfected mice (4 x 105 cells/well) preloaded with 10-7 M final concentration of the Ld-restricted -gal876–884 peptide. ELISPOT plates were developed with biotin-labeled rat anti-mouse IFN- (BD Biosciences), peroxidase-conjugated streptavidin (Dianova, Hamburg, Germany), and amino-ethylcarbazole dye solution (Sigma-Aldrich). Spots were counted microscopically. The number of -gal876–884-specific T cells was expressed as the number of spots formed in each well per 104 leukocytes.
Measurement of CTL activity
At day 23 p.i. with the strains PruHX-, PruS1SPLACZ, and PruS4SPLACZ, leukocytes were isolated from the brains and the spleens. P815 (H-2d) cells were used as target cells and were coated with 10-7 M -gal876–884 peptide in MEM-
supplemented with 10% FCS at 37°C, 5% CO2. During the last hour of peptide incubation, P815 cells were labeled with 51Cr (100 µCi/1 x 106 cells) (Amersham Pharmacia Biotech). Thereafter, target cells were washed three times with MEM- supplemented with 10% FCS to remove unbound peptide and extracellular 51Cr. Isolated leukocytes and target cells were incubated at E:T ratios of 200:1, 100:1, 30:1, 10:1, 3:1, 1:1, and 0.3:1 in triplicate. After incubation at 37°C with 5% CO2 for 5 h, 100 µl of the supernatant from each well were collected, and the released 51Cr was counted in a gamma counter (Beckman Coulter, Munich, Germany). The specific release was calculated according to the following formula: 100 x [(test release - spontaneous release)/(maximal release - spontaneous release)] where test release was in the presence of effector cells, spontaneous release was in the presence of medium alone, and maximal release was in the presence of detergent.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression vectors pS1LACZ-HX and pS4spLACZ-HX were generated from the plasmid pSAG1-Gal as illustrated in Fig. 1A. Three transgenic clones of T. gondii, namely PruS1LACZ, PruS1SPLACZ, and PruS4SPLACZ, were generated by transfecting the parental PruHX - T. gondii with the respective expression vectors. PruS1LACZ expressed -gal in the tachyzoite cytoplasm under the control of the tachyzoite-specific sag1 promoter (Fig. 1B); tachyzoites of PruS1SPLACZ clone secreted -gal under the control of the tachyzoite-specific sag1 promoter (Fig. 1B); and bradyzoites of the PruS4SPLACZ secreted -gal under the control of the bradyzoite-specific sag4 promoter both in vivo and in vitro (Fig. 1, B and C). The predicted expression of -gal in the various parasitic clones was controlled by 1) location of -gal expression by an indirect immunofluorescence assay in vitro (Fig. 1, B and C) and 2) the stage-specific expression of -gal in vitro in tachyzoites and bradyzoites as well as in tissue cysts by -gal staining (Fig. 1, B and C).
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Impact of the cellular compartment of the expressed Ag for the induction of a CD8 T cell response
To analyze the impact of the compartment in which the respective T. gondii Ags are expressed, i.e., the parasite cytoplasm or the parasitophorous vacuole, on the CD8 T cell response, mice were infected orally with the PruHX- control PruS1SPLACZ (vacuolar/secretory -gal expression) and PruS1LACZ (cytoplasmic -gal expression) clones, respectively. Infection with the PruS1SPLACZ clone induced a strong -gal876–884-specific CD8 T cell response (Fig. 2). At day 9 p.i., 0.5% of all CD8 T cells were specific for -gal876–884 peptide. At day 21 p.i., the highest frequency of -gal876–884-specific CD8 T cells (2.6%) was observed with a decline to 0.9% of the total number of CD8 T cells until day 49 p.i. Infection with PruS1LACZ or PruHX - T. gondii did not induce a -gal876–884-specific CD8 T cell response. These results suggest that the secretion of a T. gondii protein into the parasitophorous vacuole is a prerequisite for the induction of an Ag-specific CD8 T cell response.
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In additional experiments, the kinetics and hierarchy of -gal-specific CD8 T cells specific for all three identified MHC class I restricted peptides, namely the Ld-restricted -gal876–884, the Kb-restricted -gal96–103, and the Kb-restricted -gal497–504, were monitored by MHC class I tetramer staining.
At day 7 p.i. with PruS1SPLACZ T. gondii, CD8 T cells specific for the Ld-restricted -gal876–884 peptide were detectable in the spleen (Fig. 3). Thereafter, their frequency increased sharply with a peak of 4% of the total CD8 T cell population up to day 23 p.i. The frequency of -gal876–884-specific CD8 T cells declined rapidly from day 25 to 28 p.i. and thereafter returned gradually to baseline levels. However, low numbers of specific CD8 T cells corresponding to 0.3% of all CD8 T cells persisted until day 180 p.i. On the contrary, CD8 T cells specific for the Kb-restricted -gal96–103 peptide remained undetectable throughout the entire course of infection, and only a small peak of 0.5% CD8 T cells specific for the Kb-restricted peptide -gal497–504 was detected at day 14 p.i. (data not shown). Thereafter, the frequency of CD8 T cells specific for the -gal497–504 epitope declined to background levels. To confirm the frequencies of CD8 T cells specific for -gal, an IFN- ELISPOT assay was performed at days 14 and 25 p.i. These experiments confirmed the data obtained by tetramer staining. Splenic leukocytes produced IFN- upon restimulation with -gal876–884 peptide at days 14 and 25 p.i. Some splenic leukocytes produced IFN- after restimulation with -gal497–504 peptide at day 14 p.i., and no IFN- production was observed upon restimulation with -gal96–103 peptide (data not shown).
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-gal under the control of the tachyzoite-specific SAG1 promoter induced a dominant Ld-restricted -gal876–884-specific CD8 T cell response reaching a maximum at day 23 p.i. and a subdominant Kb-restricted -gal497–504-specific CD8 T cell response, which peaked as early as day 14 p.i. Impact of stage-specific Ag expression for the induction of a CD8 T response
To analyze whether a parasite-specific CD8 T cell response is exclusively directed against a tachyzoite-specific Ag or whether a bradyzoite-specific Ag can also be a target for recognition by CD8 T cells, mice were infected orally with T. gondii clones secreting -gal either as tachyzoites (PruS1SPLACZ) or bradyzoites (PruS4SPLACZ) as well as the parental PruHX- control strain.
In noninfected mice, -gal876–884-specific CD8 T cells were undetectable in the spleen and in the brain (data not shown). At day 15 p.i. with PruS1SPLACZ, when a significant number of leukocytes including CD8 T cells had already been recruited to the brain (Ref.7 and present study, data not shown), 2.0 and 4.2% of total CD8 T cells in the spleen and the brain stained positively with Ld/-gal876–884 tetramer, respectively (Fig. 4, A and B). In both the spleen and the brain, the percentage of tetramer -gal876–884-positive CD8 T cells reached a peak at day 23 p.i. when 2.1 and 5.2% of the total CD8 T cells were tetramer positive, respectively (Fig. 4). The peak of intracerebral tetramer-positive CD8 T cells coincided with the peak of CD8 T cell infiltration into the brain (D. Schlüter, unpublished results). Thereafter, the percentage of -gal876–884-specific CD8 T cells declined in the spleen from 1.3 (day 38 p.i.) to 0.7% of all CD8 T cells at day 60 p.i. (Fig. 4A). In the brain, the frequency of -gal876–884-specific CD8 T cells declined slightly to 3.5% of all CD8 T cells at day 38 p.i. and remained at this frequency (3.7%) until day 60 p.i. (Fig. 4B). The majority of the splenic and cerebral Ld/-gal867–884 tetramer-positive CD8 T cells expressed high levels of CD44 and no CD62L, indicating that they were activated (data not shown).
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-gal876–884-specific CD8 T cells were not detectable throughout the course of infection (Fig. 4, A and B). Thus, only tachyzoites, but not bradyzoites secreting -gal, induced a primary CD8 T cell response, which declined in the spleen during the infection but remained at a high and stable level in the brain of chronically infected mice.
-gal-secreting bradyzoites increase the number of -gal876–884-specific CD8 T cells in mice primed with tachyzoites secreting -gal
To analyze whether bradyzoites induce an expansion of Ag-specific CD8 T cells, which had already been primed by a primary infection with tachyzoites, mice were orally infected with cysts of the clone PruS1SPLACZ to develop a primary -gal876–884-specific CD8 T cell response. Thereafter, mice were orally reinfected with either the PruS4SPLACZ, the PruS1SPLACZ, or the PruHX-.
At day 41 after primary infection, i.e., the day 0 of reinfection, 3.2% of all cerebral CD8 T cells were specific for -gal876–884 (Fig. 5A). At day 7 after secondary infection, the frequency of -gal876–884-specific CD8 T cells had increased to 5.1% of all CD8 T cells in mice infected with the PruS1SPLACZ T. gondii and to 6.8% of all CD8 T cells in mice infected with the PruS4SPLACZ T. gondii, respectively (Fig. 5B). In contrast, without reinfection and upon reinfection with the PruHX- strain, the frequency of cerebral -gal876–884-specific CD8 T cells decreased to 2.8 and 2.3%, respectively, at day 7 after secondary infection (Fig. 5B). The increase of -gal-specific cerebral CD8 T cells at day 7 after secondary infection was a transient phenomenon, because at day 35 after secondary infection with PruS1SPLACZ or PruS4SPLACZ T. gondii, the frequency of -gal876–884-specific CD8 T cells declined to 2.7 and 3.1%, respectively. These frequencies corresponded to control animals without secondary infection (2.9% of all CD8 T cells) or to mice that were reinfected with the PruHX- strain (1.9% of all CD8 T cells) (Fig. 5B). In contrast to the brain, a reinfection with PruS4SPLACZ T. gondii did not result in an increase of the frequency of -gal876–884-specific CD8 T cells in the spleen (data not shown).
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-gal stimulate the expansion of the -gal-specific CD8 T cell population in the brain upon secondary infection only after priming with tachyzoites secreting -gal.
Functional capacity of -gal867–884-specific CD8 T cells induced by infection with tachyzoites secreting -gal
To analyze the functional capacity of both splenic and cerebral -gal867–884-specific CD8 T cells, their IFN- production and cytotoxic activity were determined. At day 25 after infection with the clone PruS1SPLACZ, 1.1% of the splenic and 3.0% of the cerebral leukocytes were Ld/-gal867–884 tetramer-positive CD8 T cells (data not shown), and 64/104 (0.64%) of all splenic and 267/104 (2.67%) of all cerebral leukocytes produced IFN- upon restimulation with the -gal867–884 peptide (Fig. 6A). Both splenic and cerebral leukocytes isolated from mice infected with PruS1SPLACZ T. gondii were able to lyse target cells loaded with -gal867–884 peptide (Fig. 6, B and C), and the cytotoxic activity paralleled the percentage of tetramer Ld/-gal867–884-positive cells in both the spleen and the brain (Fig. 6, B and C). In control mice infected with PruHX- or PruS4SPLACZ T. gondii, -gal867–884-specific CD8 T cells were not detectable by tetramer staining. In addition, splenic and cerebral CD8 T cells isolated from these mice did not produce IFN- upon restimulation with the -gal867–884 peptide and did not lyse -gal867–884-loaded target cells (Fig. 6, B and C).
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-gal induce IFN--secreting, cytotoxic -gal876–884-specific CD8 T cells in both the spleen and the brain of infected animals. The frequency of -gal867–884 tetramer-positive cells was directly correlated to the number of -gal867–884-specific IFN--producing cells as well as the level of -gal867–884-specific cytotoxicity. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-gal, a model Ag that contains one Ld and two Kb epitopes (18, 19, 20), under specific conditions in a low-virulent cyst-forming type II strain of T. gondii. The combined use of -gal as a model Ag and the infection of B6C (H-2bxd) mice with different -gal-expressing clones of T. gondii allowed us for the first time to define the frequency, kinetics, and hierarchy of an Ag-specific CD8 T cell response with epitope-specific precision in toxoplasmosis.
Secreted, but not cytoplasmic, -gal expressed by tachyzoites induced a primary -gal-specific CD8 T cell response. The preferential priming of an Ag-specific CD8 T cell response by a secretory, but not cytoplasmic, Ag has also been demonstrated in the kinetoplastid T. cruzi and in Salmonella (12, 13). However, both secreted and cytoplasmic Ags of Listeria monocytogenes were able to prime an Ag-specific CD8 T cell response (11). Therefore, the impact of the compartmentalization of an Ag on the induction of an Ag-specific CD8 T cell response is pathogen defined and may be dependent on the intracellular life cycle and survival strategy of the pathogen. The observation that only a secreted Ag induced a CD8 T cell response in murine toxoplasmosis implies that an effort to predict and identify intrinsic T. gondii epitopes should focus on the secretory Ag of the parasite.
Upon oral infection with cysts, which corresponds to the natural route of infection, only tachyzoites, but not bradyzoites secreting -gal, induced a CD8 T cell response. These data clearly illustrate that the stage-specific expression of an Ag is a major factor determining the induction of a CD8 T cell response in toxoplasmosis. Thus, intestinal and intracerebral bradyzoite-containing cysts were insufficient to induce an Ag-specific CD8 T cell response. Several mutually nonexclusive factors, including the kinetics of bradyzoite/tachyzoite conversion, the organ distribution of these parasitic stages, the amount of -gal produced by either bradyzoites or tachyzoites, as well as the transport or efflux of secreted Ags across the cyst wall or parasitophorous vacuolar membrane may account for this result. After oral infection, bradyzoites switch to tachyzoites within 12–18 h (1), and this rapid stage conversion may result in the production of too low amounts of Ag, which is insufficient for the induction of a bradyzoite-specific CD8 T cell response. Thereafter, predominantly tachyzoites, but not bradyzoites, multiply in the gastrointestinal tract, disseminate in the host, and infect multiple parenchymatous and lymphatic organs (1, 2). The conversion of some tachyzoites into bradyzoites, which eventually form intracellular tissue cysts in parenchymatous but not in lymphatic organs, is not initiated before day 6 p.i. Moreover, the cyst wall may prevent the efflux or transport of secreted Ag into the host cell cytoplasm where the MHC class I processing pathway is located, whereas the parasitophorous membrane allows the efflux of small molecules between 1.3 and 1.9 kDa from the vacuolar space into the host cell cytoplasm (28, 29).
However, upon oral infection bradyzoites secreting -gal increased the frequency of -gal-specific CD8 T cells in animals that were already infected with a T. gondii strain secreting -gal tachyzoite specifically. This finding illustrates that the conditions for the induction and restimulation of T. gondii-specific CD8 T cells differ with respect to the stage specificity of the Ag. Because the infection with -gal-secreting tachyzoites induced -gal-specific CD8 T cells persisting in a lymphatic organ, i.e., the spleen, as well as in a parenchymatous organ, i.e., the brain, -gal produced by bradyzoites after reinfection may be rapidly recognized by these CD8 T cells and foster an expansion of this cell population.
Both in brain and spleen, -gal-specific CD8 T cells peaked at day 23 p.i. Compared with many bacterial and viral infections, this peak in the spleen is rather late (26, 30) and occurs at a time point when the splenic parasitic load, which peaks around day 10 p.i., has already declined (1). This unusual kinetics may be explained by the fact that proliferation of splenic T cells is actively inhibited by macrophage-derived nitric oxide and IL-10 between days 7 and 14 p.i. (31, 32). Beyond day 23 p.i., the frequency of -gal-specific CD8 T cells dropped rapidly, which may be caused by a fast contraction of the Ag-specific CD8 T cell population in the spleen as well as a recruitment of -gal-specific CD8 T cells to the brain (7). In fact, the formation of intracerebral T cell infiltrates in TE strictly depends on the recruitment of peripheral T cells to the brain. In the brain, the frequency of -gal-specific CD8 T cells also peaked at day 23 p.i. and persisted at high levels thereafter. Thus, intracerebral CD8 T cells specific for a secreted Ag of tachyzoite were not eliminated from the brain in chronic TE, i.e., when the parasite persists as bradyzoites within cysts. The persistence of these CD8 T cells is consistent with the observation that intracerebral T cells form a stable cell pool in chronic TE, which is only slowly downsized by a low level of apoptosis (7). Because -gal-specific CD8 T cells were functionally active and rapidly produced IFN- (the major cytokine in resistance against T. gondii (33)) upon peptide restimulation and also killed peptide-pulsed APCs, they appear to play an important role in the control of intracerebral parasites in acute and chronic TE. Because the intracerebral conversion of bradyzoites to tachyzoites is a major risk for chronically infected hosts—in AIDS patients the loss of T cell-mediated immunity against T. gondii is considered to be the major factor leading to a lethal necrotizing TE by reactivated T. gondii (34)—the intracerebral persistence of tachyzoite-specific CD8 T cells may be important for the rapid elimination of reactivated T. gondii. This assumption is experimentally supported by the crucial protective role of CD8 T cells in the TE of retrovirus-infected immunocompromised mice (35, 36). The failure of CD8 T cells to completely eliminate T. gondii from the brain may be caused by an immune evasion of tachyzoites in which CD8 T cells switch into bradyzoites, which do not induce a primary -gal-specific CD8 T cell response, as well as the formation of cysts in neurons (37, 38), which in general lack expression of MHC class I and II Ag (39).
The observation that a heterologous Ag of T. gondii is successfully targeted to the parasitophorous vacuole and subsequently induces a strong Ag-specific CD8 T cell response indicates that T. gondii may serve as a vaccine vector against diseases in which CD8 T cells are protective. In fact, T. gondii has recently been probed for its potential to serve as a vaccine vector in murine malaria (40). However, the application of T. gondii as a vaccine vector might potentially be limited by the fact that resistance to T. gondii as well as strong and persisting Ag-specific CD8 T cell responses are genetically restricted to Ld, as observed in the present and previous studies (8, 41).
Collectively, the finding that a heterologous Ag secreted under control of the tachyzoite-specific SAG1 promoter induces Ag-specific functionally active CD8 T cells, which persist in the brain in chronic TE, has substantial implications for both the development of T. gondii-based vaccines and the interaction of T. gondii and the host CD8 T cell response. Although, it has to be kept in mind that endogenous tachyzoite- and bradyzoite-specific proteins differ from each other and also from -gal, which may also have an impact on the ensuing CD8 T cell response.
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2 Address correspondence and reprint requests to Dr. Dirk Schlüter, Institut für Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Mannheim, Universität Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany. E-mail address: dirk.schlueter{at}imh.ma.uni-heidelberg.de
3 Abbreviations used in this paper: gal, galactosidase; HFF, human foreskin fibroblasts; p.i., postinfection; TE, Toxoplasma encephalitis; HXGPRT, hypoxanthine-xanthine-guanine-phosporbosyl-transferase; X-Gal, 5-bromo-4-chloro-3-indolyl b-D-galactoside.
Received for publication September 13, 2002. Accepted for publication December 6, 2002.
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