INTRODUCTION

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The protozoan parasite Toxoplasma gondii is a significant cause of morbidity and mortality in AIDS patients and congenitally infected individuals (19, 21). Because T. gondii infections can be devastating to those who are immunodeficient or immunologically immature, there is an obvious motive to understand the mechanisms of resistance to this parasite that are normally employed by immunocompetent individuals. To this end, a number of studies have employed infection of laboratory mice as a model system with which to dissect immunological mechanisms of resistance to Toxoplasma. Thus, it has been found that acquired immunity to Toxoplasma, i.e., the ability to keep chronic infection in check and the capacity to resist challenge with virulent tachyzoites after vaccination, depends on T lymphocytes, particularly CD8⁺ T cells (8, 9, 24, 26, 34), and gamma interferon (IFN-) (9, 32, 33). Whether B cells and antibodies are important components of acquired resistance to T. gondii is much less clear, however.

There is little doubt that T. gondii infection elicits a specific antibody response. The Sabin-Feldman dye test (29), used clinically to diagnose infection with Toxoplasma, is simply an assay for serum antibodies which lyse tachyzoites in a complement-dependent manner (30). Both human and murine infections elicit not only immunoglobulin G (IgG) and IgM antibodies in serum but also IgA in secretions (3, 18, 20, 23). Still, it is not known whether any of these antibodies normally play a protective role against Toxoplasma. To address this question, Frenkel and Taylor (7) examined mice that were treated with anti-immunoglobulin from birth (µ-suppressed mice) and therefore that had no B cells. If such mice were infected with T. gondii and protected with chemotherapy to allow immunity to develop, the mice lived longer than T-cell-deficient mice after chemotherapy was stopped but still eventually died. However, if given immune serum, many of the mice survived. The authors concluded that antibodies may be able to provide some protection against chronic T. gondii infection. A number of passive immunization studies have been performed to determine the role of antibodies in immunity to Toxoplasma. For example, immune lymphoid cells or immune sera partially protected guinea pigs against a challenge with RH tachyzoites (27). Other studies in which mice were challenged with parasites after being given immune sera have produced mixed results regarding the protective potential of Toxoplasma-specific antibodies (6, 15, 17, 25). Additional studies have shown that monoclonal antibodies against various Toxoplasma antigens have the potential to protect unimmunized mice against a challenge with moderately virulent parasites and, to a lesser extent, with highly virulent parasites (12, 31). Such passive immunization experiments may reveal whether an anti-Toxoplasma antibody is capable of providing protection in the absence of an already developed cell-mediated immunity but do not address whether antibodies generated in the normal course of infection are required for protection of chronically infected mice or vaccinated and subsequently challenged mice.

To get at the question of whether antibodies or B cells are necessary for resistance to Toxoplasma infection, we have studied mice which have no B cells (µMT mice) as a result of a targeted mutation (14). Our results clearly demonstrate that B cells are required for resistance to T. gondii in a model in which mice are vaccinated with avirulent tachyzoites and later challenged with highly virulent tachyzoites. Our findings suggest that the role of B cells is to produce antibodies that block the infection of host cells by tachyzoites.

	MATERIALS AND METHODS

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Mice. Adult (>8-week-old) male and female B-cell-deficient mice with a targeted mutation in a transmembrane exon of the immunoglobulin µ chain gene (µMT mice [14]) were used. Mice were verified to be B cell deficient by flow cytometric analysis of peripheral blood lymphocytes. In addition, sera from B-cell-deficient mice were found to be devoid of T. gondii-specific antibodies when tested by enzyme-linked immunosorbent assay (ELISA). Also, fecal extracts from infected B-cell-deficient mice were found to be negative when tested for antibodies able to bind to tachyzoites in a flow cytometric assay (5). Thus, the B-cell deficiency of µMT mice was confirmed by their inability to produce antibodies specific for T. gondii in sera or intestinal secretions. In initial experiments, B-cell-deficient mice with a 129×B6 mixed genetic background were used. In later experiments, mice that were fully backcrossed to the C57BL/6J strain (B6) were used. Essentially the same results were obtained with both strains of mice. B6 mice were used as controls. B-cell-deficient J_HD/J_HD mice (4) having the BALB/c genetic background were also used, along with BALB/cByJ controls.

Parasites and immunizations. Mice were immunized by intraperitoneal (i.p.) injections of 2 × 10⁴ ts-4 strain tachyzoites (29). Mice were challenged by i.p. injection of 2 × 10³ RH strain tachyzoites. Tachyzoites were maintained in cultures of Hs68 human fibroblasts (ATCC CRL 1635) in HEPES-buffered RPMI 1640 medium supplemented with heat-inactivated fetal bovine serum (FBS; 10%), L-glutamine, and penicillin-streptomycin at 33 (ts-4) or 37°C (RH) in a humidified 5% CO₂ atmosphere. To test whether immune serum inhibits the infection of cultured cells, ts-4 tachyzoites were cultured in immune or normal (nonimmune) serum (10% [vol/vol]) for 10 min, washed, and added to monolayers of Hs68 fibroblasts cultured on coverslips in supplemented RPMI 1640 medium (10% FBS and antibiotics) containing 10% immune or normal serum. Cells and tachyzoites were cultured at 37°C and 5% CO₂ for progressive lengths of time, washed, and stained for microscopy.

Spleen cell cultures. Spleen cell suspensions were prepared by crushing spleens through stainless steel mesh and suspending cells in HEPES-buffered Hanks' balanced salt solution. Erythrocytes were removed by hypo-osmotic lysis from cells pelleted by centrifugation. Washed leukocytes were counted and suspended in HEPES-buffered RPMI 1640 containing 10% FBS, 50 µM 2-mercaptoethanol, and antibiotics. Spleen cells were placed in 96-well flat-bottom plates (6 × 10⁵/well) in triplicate wells with either concanavalin A (5-µg/ml final concentration), ts-4 tachyzoites (1 × 10⁵ per well), or medium alone in a total volume of 200 µl per well. Supernatants were harvested after 72 h and stored frozen at 20°C until assayed for cytokine content.

IFN- assay. Levels of IFN- in sera, peritoneal lavage fluids, and spleen cell culture supernatants were measured by ELISA (13).

Flow cytometry. Spleen cells were prepared as described above. To obtain peripheral blood leukocytes, a few drops of blood were collected and erythrocytes were lysed hypo-osmotically. Leukocytes were pelleted in a microcentrifuge, washed, and incubated with antibodies to detect both CD45RB (16A; phycoerythrin [PE] conjugated; Pharmingen) and CD4 [GK1.5; fluorescein isothiocyanate (FITC)-conjugated F(ab')₂; prepared at this institute] or CD8 [ATCC TIB210; FITC-conjugated F(ab')₂; prepared at this institute]. Additional samples were stained with anti-CD19 (1D3, PE conjugated; Pharmingen) and anti-mouse IgG plus IgM plus IgA [FITC-conjugated F(ab'); Southern Biotechnology Associates]. Lymphocytes were identified by forward scatter-side scatter characteristics.

Statistics. Unless otherwise stated, differences in means between groups were analyzed by Student's t test. Survival data were analyzed by the Mann-Whitney U test.

	RESULTS

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Vaccinated B-cell-deficient mice are susceptible to a challenge infection with virulent tachyzoites. Tachyzoites of the temperature-sensitive ts-4 strain of Toxoplasma have been widely used to immunize mice for analysis of mechanisms of immunity to a later challenge with highly virulent Toxoplasma. Virtually all unimmunized mice will die from a challenge with as few as 10 highly virulent RH tachyzoites, the parent strain of ts-4 (28), but ts-4-immune immunocompetent mice can resist a challenge with several orders of magnitude more RH tachyzoites. Therefore, to investigate whether B cells are required for vaccination-induced immunity to T. gondii, we immunized B-cell-deficient mice with ts-4 tachyzoites and challenged them at later times with an i.p. injection of RH tachyzoites. Results shown in Fig. 1a are representative of five such experiments using B-cell-deficient µMT mice and B6 controls and show clearly that ts-4-immunized B-cell-deficient µMT mice are highly susceptible to RH challenge, whereas B-cell-sufficient B6 mice are not.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Susceptibility of ts-4-vaccinated B-cell-deficient mice to challenge with virulent tachyzoites. (a) Groups of five µMT and B6 mice were vaccinated with ts-4 tachyzoites and challenged with RH tachyzoites 6 weeks later. The median survival time of vaccinated µMT mice (8 days; range, 7 to 18 days) was significantly (P < 0.05) shorter than that of vaccinated B6 mice (>100 days). Data are representative of five such experiments. In all experiments, the survival of vaccinated B-cell-sufficient mice was significantly longer than that of vaccinated µMT mice. In some experiments, the survival of vaccinated µMT mice was statistically longer than that of naive controls, although the difference was quantitatively small. (b) Groups of male and female BALB/c background JHD/JHD mice (n = 22) and B-cell-sufficient BALB/cByJ (n = 9) mice were vaccinated with ts-4 tachyzoites and challenged 52 days later, together with nonimmune JHD/JHD controls (n = 6), with virulent RH tachyzoites. The median survival time of vaccinated JHD/JHD mice (11 days; range, 7 to 24 days) was significantly (P < 0.01) shorter than that of vaccinated BALB/c controls (>98 days) but significantly (P < 0.01) longer than that of naive JHD/JHD controls (7.5 days; range, 7 to 11 days).

View larger version (82K): [in this window] [in a new window]   FIG. 2.   Cytospins of peritoneal lavage fluids from vaccinated µMT and B6 mice challenged with virulent RH tachyzoites. (a) A moribund µMT mouse vaccinated 60 days prior to challenge with RH tachyzoites. Lavage fluids were harvested on day 10 after challenge. (b) Apparently healthy identically treated B6 mouse. (c) A moribund unvaccinated control µMT mouse 7 days after RH challenge. Arrows, tachyzoites. Magnification, ×400.

Vaccinated B-cell-deficient mice are not deficient in IFN- production in response to T. gondii in vivo. Because IFN- is required for resistance to challenge with RH tachyzoites in ts-4-immunized mice (9), we examined whether there was deficient production of IFN- in vaccinated µMT mice. ts-4-immunized and unimmunized µMT and B6 mice were challenged with RH tachyzoites and their sera were collected and their peritoneal cavities were flushed 7 days after challenge (experiment 1) or 9 to 10 days later, as mice were becoming moribund (experiment 2). Fluids were analyzed for IFN- by ELISA (Table 1). In neither compartment was the level of IFN- less in immunized µMT mice than in immunized B6 mice, and was in fact significantly greater in µMT mice except in peritoneal lavages in experiment 1. It was also found that the percentage of CD8⁺ peripheral blood T cells from ts-4-vaccinated µMT mice expressing relatively lower levels of the CD45RB marker (indicative of prior antigen exposure) did not differ significantly 50 days after vaccination from the corresponding percentage of the population in vaccinated B-cell-sufficient mice (Fig. 3), or at any of several times during the first 5 weeks after vaccination (P. C. Sayles and L. L. Johnson, unpublished data). Similarly, the total numbers of CD45RB^loCD8⁺ cells in the two groups were not different. Thus activation of the critical CD8⁺ T-cell population in peripheral blood did not appear to be impaired in B-cell-deficient mice.

View this table: [in this window] [in a new window]   TABLE 1.   In vivo production of IFN- by vaccinated and naive µMT and B6 mice challenged with virulent RH tachyzoites

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Activated peripheral blood T cells (PBL) in ts-4-vaccinated µMT and B6 mice. Peripheral blood cells taken from unvaccinated mice and vaccinated mice 50 days after immunization with 2 × 104 ts-4 tachyzoites were examined by flow cytometry. Cells were stained with CD4- or CD8-specific FITC-conjugated F(ab')2 antibodies and PE-conjugated anti-CD45RB. Bars show the percentages of CD45RBlo cells within the CD4+ and CD8+ lymphocyte populations (determined by forward scatter-side scatter characteristics). Asterisks, significant (P < 0.05) differences from corresponding populations in naive mice.

View this table: [in this window] [in a new window]   TABLE 2.   IFN- production in vitro by spleen cells from vaccinated µMT and B6 micea

Toxoplasma-immune serum enhances resistance to RH challenge in vaccinated B-cell-deficient mice. The foregoing results establish that B cells are required by immunized mice to resist an RH challenge but not to produce IFN- or to activate CD8⁺ T cells. Moreover, several T-cell-dependent functions appear to be normal in µMT mice in our experience, including resistance to primary and secondary intravenous Listeria monocytogenes infection and rejection of allogeneic skin grafts (Sayles and Johnson, unpublished data). These findings suggest that the defect in µMT mice, with respect to resistance to Toxoplasma, may lie in their inability to produce antibodies, rather than resulting from an indirect effect of B-cell deficiency on T-cell-dependent immunity. Therefore, we hypothesized that providing immunized µMT mice with Toxoplasma-immune serum would improve their resistance to RH challenge. Results presented in Fig. 4 show that Toxoplasma-immune serum significantly lengthened the survival time of vaccinated µMT mice beyond that of nonimmune µMT mice or vaccinated µMT mice given nonimmune serum.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Survival of ts-4-vaccinated µMT mice given immune or nonimmune serum. Groups of 5 µMT mice were vaccinated with ts-4 and challenged with RH tachyzoites 37 days later, along with unimmunized controls. Vaccinated mice were given 0.5 ml of Toxoplasma-immune serum (log10 IgG2a titer = 4.72 by ELISA) or nonimmune serum i.p. on days 1, 0, 2, 4, 7, and 10 relative to RH challenge. Survival of mice given immune serum (median survival time, 27 days; range, 18 to 41 days) was significantly (P < 0.05) prolonged relative to that of mice given nonimmune serum (median survival time, 13 days; range, 4 to 14 days).

Vaccinated mice lacking Fc receptors or deficient in C5 are resistant to RH challenge. There are several possible mechanisms by which antibodies could function protectively in vaccinated mice, including facilitation of Fc receptor-dependent uptake of parasites by phagocytic cells or killing by cytotoxic cells (ADCC), lysis of extracellular tachyzoites in a complement-dependent manner, and direct blocking of tachyzoite entry into host cells. To examine the first two of these possibilities, mice lacking Fc receptors or mice lacking the fifth component of complement were immunized with ts-4 tachyzoites and challenged with RH tachyzoites. Both kinds of mice were adequately protected against the RH challenge (Fig. 5 and 6), which argues that neither Fc receptor-mediated phagocytosis or ADCC nor antibody-and-complement-dependent lysis of tachyzoites is a likely mechanism by which antibodies function protectively in ts-4-immunized, RH-challenged mice.

View larger version (19K): [in this window] [in a new window]   FIG. 5.   Survival of vaccinated FcR/ mice challenged with virulent tachyzoites. Groups of five male mice with a targeted mutation in the gene for the  chain common to FcR and FcRIII (FcR /) or with a targeted mutation in the gene for FcRII, along with nonmutant B6129F2 controls, were vaccinated with ts-4 tachyzoites and, together with unimmunized controls, were challenged with RH tachyzoites 49 days later. Vaccinated FcR/ mice survived (median survival time of each group, >56 days) significantly (P < 0.05) longer than unvaccinated controls (median survival time, 8 days; range, 8 to 9 days) and as long as vaccinated nonmutant controls (median survival time, >56 days).

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Survival of vaccinated C5-deficient mice challenged with virulent tachyzoites. Groups of five C5-deficient male mice (B10.D2o and DBA/2J) and C5-sufficient B6 controls were vaccinated with ts-4 and challenged with RH tachyzoites 48 days later, together with unvaccinated controls. Vaccinated C5-deficient mice survived significantly longer than unvaccinated controls (median survival time, 8 days; range, 8 to >50 days) and as long as vaccinated C5-sufficient controls (median survival time, >50 days).

Toxoplasma-immune serum inhibits infection of cultured cells by tachyzoites. To determine whether Toxoplasma-specific antibodies directly affect the ability of tachyzoites to enter host cells, tachyzoites were incubated in serum from mice immunized against Toxoplasma or from unimmunized mice and then added to monolayers of Hs68 human fibroblasts on coverslips. Mouse serum (either immune or nonimmune) was present in the culture medium at 10% final concentration. Coverslips were removed at progressive intervals and washed, and the cells were stained and examined microscopically for the presence of tachyzoites. Results from one of three such experiments are shown in Fig. 7, where it may be seen that immune serum almost completely inhibited tachyzoite entry into host cells but that nonimmune serum did not.

View larger version (136K): [in this window] [in a new window]   FIG. 7.   Immune serum-mediated inhibition of infection of cultured fibroblasts by ts-4 tachyzoites. (a) Immune serum at 45 h of incubation. (b) Nonimmune serum at 45 h of incubation. Arrows, intracellular and extracellular tachyzoites. Magnification, ×400.

View larger version (31K): [in this window] [in a new window]   FIG. 8.   Inhibition of infection of cultured fibroblasts by serum from immune B6 mice but not by serum from vaccinated µMT mice. Numbers in the upper right of each panel denote the percentages of infected (fluorescent) cells and the mean fluorescence intensity of the infected population.

	DISCUSSION

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Our findings show that B cells are required for immunity to an intraperitoneal challenge with virulent Toxoplasma parasites in mice vaccinated with attenuated tachyzoites. A requirement for B cells, in addition to cell-mediated immunity, has also been reported for mice infected with Plasmodium chabaudi (37) and Schistosoma mansoni (11). In several of the experiments performed in this study (e.g., Fig. 1b), B-cell-deficient ts-4-vaccinated mice challenged with RH tachyzoites survived significantly longer than unimmunized mice. This finding suggests that, even in the complete absence of B cells, some amount of host resistance, presumably T cell mediated, is developed but that it is insufficient to completely protect the mice. We have also observed that unimmunized mice given repeated injections of Toxoplasma-immune serum have very little resistance to challenge with RH tachyzoites (Sayles and Johnson, unpublished data). Thus, mice apparently require both immune T cells and antibody-producing B cells for effective resistance to an i.p. challenge with virulent tachyzoites.

CD8⁺ T cells and IFN- are known to be important components of resistance to Toxoplasma. Although µMT mice have numerically fewer splenic T cells than similarly vaccinated B6 mice (Table 2), the absence of B cells during infection does not seem to compromise the ability of vaccinated, challenged mice to activate the T cells they possess or to make substantial quantities of IFN- in serum or at the site of the challenge infection in the peritoneal cavity (Table 1). It should be noted that despite the deficiency of activated CD4⁺ and CD8⁺ T cells in the spleens of vaccinated µMT mice, we have found that these mice have numbers of activated (CD45RB^lo) peripheral blood CD8⁺ T cells that are equivalent to those in vaccinated B6 mice (approximately 2.3 × 10⁶/ml). Moreover, the estimated total number of activated peripheral blood CD8⁺ T cells in vaccinated µMT mice was greater than that in spleens of vaccinated B6 mice. This may explain why the levels of IFN- in peritoneal lavage fluids and sera of vaccinated µMT mice were not less than those of vaccinated B6 mice (Table 1), despite the deficiency of activated splenic T cells.

Our findings are most compatible with the hypothesis that the critical protective role of B cells is to produce anti-Toxoplasma antibodies. We believe that antibodies directly block the ability of tachyzoites to infect host cells, rather than functioning as part of an Fc receptor-dependent or complement-dependent anti-Toxoplasma effector mechanism. Further evidence against a role for complement-dependent killing of tachyzoites was provided by the observation that heat inactivation of immune serum did not lessen its ability to prevent infection of fibroblasts in vitro (Fig. 8). It should be mentioned, however, that our in vivo experiments testing whether Fc receptors and C5 are essential components of protection in this model were performed using mice having genetic backgrounds different from those of µMT and B6 mice. We therefore view our conclusions regarding the lack of need for Fc receptors or C5 in vivo as provisional.

The hypothesis that Toxoplasma-specific antibodies are a source of protection in this vaccination-challenge model predicts that other mice with impaired capacity to produce Toxoplasma-specific antibodies might also be poorly resistant to a challenge infection. Interestingly, we have found that mice with targeted mutations in genes for CD40 or CD40 ligand, which have B cells but which do not make isotype-switched antibodies (IgG or IgA) when vaccinated with ts-4 tachyzoites, are highly susceptible to a challenge infection with RH tachyzoites (P. C. Sayles, G. W. Gibson, and L. L. Johnson, submitted for publication). Similarly, mice inoculated with ME49 cysts as neonates have far lower titers of Toxoplasma-specific serum antibodies after reaching adulthood than do controls vaccinated as adults and die much sooner after an RH challenge (L. L. Johnson, unpublished observations). While T-cell defects may underlie the susceptibility of mice in both of these models, low titers of Toxoplasma-specific antibodies or their absence might also contribute to the observed high susceptibility to RH challenge.

Although a lack of B cells does not lead to a deficiency of IFN- production in sera or peritoneal lavages of infected mice relative to controls, it may, in fact, cause higher levels of this cytokine to be made locally (Table 1). Thus, we do not rule out the possibility that overproduction of cytokines associated with a Th1 response may contribute to the morbidity and mortality of the B-cell-deficient mice. Indeed, IFN--dependent immunopathology of the intestine has been reported in B6 mice fed large numbers of Toxoplasma cysts (16) and interleukin 10 gene knockout mice acutely infected with an i.p. inoculation of parasites (10). Experiments are in progress to investigate whether an excessive Th1-type response contributes to the mortality of B-cell-deficient RH-challenged mice or whether tissue damage, in livers or lungs, for example, is a result of poorly controlled tachyzoite proliferation.