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Infection with Toxoplasma gondii confers on the host lifelong protective immunity against reinfection. This suggests that prevention of toxoplasmosis is a realistic goal (22).

Studies showing the importance of systemic cell-mediated immunity in protection against toxoplasmosis are unequivocal (9, 11). Infection with T. gondii generally occurs via the oral route and triggers a cellular response in the gut (5, 6). Anti-T. gondii cellular mucosal immunity is strongly mediated by lymphocytes, mainly intraepithelial lymphocytes (IEL) (2, 19).

Some attempts to generate protective immunity against toxoplasmosis have used the peroral route (1). Since all mucosal surfaces have the same mucosal immune system, the intranasal (i.n.) route is a possible alternative for immunization (18). In a previous study, the use of SAG1, the major surface antigen of T. gondii, plus cholera toxin (CT) for i.n. immunization provided potent protection, reducing the cyst burden by 80% (8). Thus, protective immunity can be stimulated by i.n. immunization of the intestine, where the pathogen enters the host. This study was carried out to investigate the mucosal immunity induced by i.n. immunization with SAG1 plus CT.

Parasites. Cysts of the 76K T. gondii strain were obtained from the brains of orally infected CBA/J mice (Janvier, St Genest/Lisle, France). Tachyzoites of the virulent strain RH of T. gondii were harvested from peritoneal fluid of Swiss OF1 mice infected intraperitoneally and were used to prepare the sonicated antigen (toxoplasmic extract) (6).

Mouse immunizations. Eight CBA/J mice were immunized i.n. with 20 µg of T. gondii SAG1 plus 0.5 µg of CT (Sigma) at 10 µl per nostril under light ether anesthesia and boosted 4 weeks later in the same way. SAG1 was purified using an anti-SAG1 monoclonal antibody (MAb) affinity column (16), and the purity of the protein was demonstrated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis analysis in a PHAST system (Pharmacia), followed by silver staining of the gel (16).

Adoptive transfer of cells. Immunized CBA/J mice were killed on day 15 after the boost, and cell suspensions were prepared from cervical and mesenteric lymph nodes (CLN and MLN, respectively) and the small intestine. IEL were prepared as previously described (4). Cells (5 × 10⁵) were analyzed with an anti-CD8 MAb (53-5.8 clone; PharMingen) and an anti-Thy 1.2 MAb (53-2.1 clone; PharMingen) on a FACScan (Becton Dickinson, Mountain View, Calif.). Around 3 × 10⁷ and 2 × 10⁶ lymph node cells and IEL, respectively, were intravenously injected into CBA/J recipient mice (six per group).

Proliferative T-cell response. Single-cell suspensions were prepared 15 days after the boost by purifying the cells from CLN, MLN, and nasal tissue (NALT) from four immunized mice. To isolate NALT cells (27), mice were killed by ether asphyxia, the lower jaws were removed, and the palates were cut parallel to the septum and removed. The NALT from each side of the septum was gently removed with fine dissection forceps. Cell suspensions were prepared by passing the tissue suspended in RPMI 1640 medium through nylon net (Blutex; Tripett et Renaud) using a syringe. All cells (5 × 10⁵) were incubated for 5 days with different antigen (toxoplasmic extract) concentrations in RPMI 1640 medium (Gibco) containing 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µg of gentamicin per ml, 5 × 10⁵ M 2-mercaptoethanol, and 5% fetal calf serum. Proliferative responses were assessed over 18 h by measuring [³H]thymidine (18.5 kBq per well [Dositek]) incorporation. Incorporated radioactivity was measured in a liquid scintillation counter (LKB) following filtration on fiberglass filters. Results were expressed as the mean of triplicates ± the standard deviation.

CLN and MLN cells and IEL of i.n. immunized mice transfer protective immunity. CBA/J mice are resistant to acute T. gondii infection but develop a large number of brain cysts. The protection conferred by adoptive transfer of lymphocytes collected on day 42 from organs of i.n. immunized mice was evaluated. One month after infection, CBA/J mice given MLN or CLN cells from mice immunized with SAG1 plus CT exhibited significantly fewer cysts (50 and 60% reduction; ANOVA test, P < 0.0003 and P < 0.01, respectively) than naive mice or mice given lymph node cells stimulated with CT alone in vivo (Fig. 1). Thus, lymphocytes from CLN and MLN were stimulated by i.n. immunization with SAG1 plus CT and participated in the protection of recipient mice against T. gondii challenge. IEL from mice immunized with SAG1 plus CT were also transferred to recipient mice (Fig. 2). One month after the challenge, the protection conferred by IEL was similar to that obtained with MLN and CLN cells (about 50%) compared to controls.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Adoptive transfer of CLN and MLN primed with SAG1 plus CT to CBA/J mice. For MLN and CLN adoptive transfer, six naive mice were given around 3 × 107 cells from donor mice. They were challenged by the oral route with 80 cysts of strain 76K T. gondii 1 day after the transfer. Protection was assessed by counting the cysts in the brain 1 month later. Statistical significance was calculated by the ANOVA test as indicated by asterisks. For the SAG1-plus-CT group compared to the phosphate-buffered saline (PBS) or CT group: **, P < 0.0003; *, P < 0.01. The results are representative of two independent experiments.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Adoptive transfer of SAG1-plus-CT-primed IEL to CBA/J mice. Six naive CBA/J mice were given 2 × 106 primed or unprimed IEL from donors and challenged by the oral route with 80 cysts of strain 76K T. gondii 4 days after the transfer. These results are representative of two independent experiments. **, P < 0.01. PBS, phosphate-buffered saline.

Proliferative response of NALT, CLN, and MLN cells. In order to study activation of the mucosal compartments further, proliferative T-cell responses of NALT, CLN, and MLN were tested. Table 1 shows that a stronger specific proliferative response of NALT cells from the group immunized with SAG1 plus CT was observed when they were incubated with toxoplasmic extract compared to those from the group immunized with CT. Also, cells from the MLN and CLN of these mice also produced a cellular response to toxoplasmic extract incubation (Table 1). Thus, i.n. immunization seemed to induce a specific T-cell response in NALT, CLN, and MLN.

SAG1-primed IEL have mainly the CD8⁺ phenotype. IEL are mainly CD8⁺ cells, which comprise CD8⁺ and CD8⁺ cells (12, 15). A fluorescence-activated cell sorter analysis was performed to explore the change within the IEL population after i.n. immunization (Fig. 3). There was an increase in the Thy-1.2⁺ and CD8⁺ IEL subsets on day 11 following the boost in the groups immunized with SAG1 plus CT and CT. The phenotypic change that occurs within the IEL population after SAG1 immunization might indicate that intestinal IEL are sensitized by immunization by the i.n. route.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Phenotypic analysis of gut IEL subsets from CBA/J mice after immunization with SAG1 plus CT. Lymphocyte suspensions were prepared from four mice, and the relative percentages of Thy1.2+ or CD8+ fluorescent IEL were determined by cytofluorimetric analysis of 5 × 105 cells.