Demonstration of T-Cell dysfunction during acute toxoplasma infection

CELLULAR IMMUNOLOGY 98,422-433 (1986)

Demonstration of T-Cell Dysfunction during Acute Toxoplasma Infection’

JOHN CHAN,* JAY P. SIEGEL,? AND BENJAMIN J. LIJFI-*‘~

*Department of Medicine, Division of Infectious Diseases, State University of New York, Stony Brook, New York I1 794-8153; fFood and Drug Administration, Bethesda, Maryland 20817

Received September 11, 1985; accepted November 19, 1985

Mice were infected with the virulent RH and the relatively avirulent C56 strains of Toxoplasma gondii (TG). The concanavalin A (Con A)-stimulated lymphoproliferative response of these animals and interleukin-2 (IL2) production by their lymphocytes were assessed 3 and 6 days postinfection. The proliferative response of splenocytes (SC) and T-enriched cells from all infected groups was significantly (P < 0.05-0.005) depressed. Partial removal of macrophages (m$) or addition of indomethacin had no effect on the depressed proliferative response of SC from mice infected with the RH strain of TG for 6 days (RH6), and only partially improved that from the other infected groups. IL2 production of T-enriched cells, obtained by scrupulously removing rn4 using sequential adherence of SC to plastic and nylon wool, was markedly decreased in all infected mice. These data indicate that both rn& and T cells are involved in the immunodepression in toxoplasmosis. Except for the RH6 group, the depressed lymphoproliferative responses of all infected groups were entirely reconstituted by exogenous IL-2, but their peak response never reached that of the control group. Therefore, the decreased lymphoproliferation could not be explained solely by a defect in IL2 production. The proliferative response of the RH6 lymphocytes, in the presence of Con A, was significantly lower than that without Con A at each IL-2 concentration added. This suggests the presence of an active suppressor factor inducible by Con A. The RH strain of TG caused a greater degree of immunodepression than the C56 strain, suggesting an association between the virulence of different strains of TG with their ability to immunosuppress. Q 1986 Academic Press, Inc.

INTRODUCTION

Toxoplasma gondii (TG) is an obligate intracellular parasite which causes chronic infection. Cell-mediated immunity plays a prominent role in host defense against TG ( 1). Previous studies have demonstrated that cell-mediated immunity is depressed in toxoplasma infection in animals (2-6) and in humans (7,8). Furthermore, macrophages (m@) have been implicated as the suppressor cell responsible for the immunodepression seen in toxoplasma infection (9). While these studies have provided ample evidence to suggest that the cell-mediated immunity is depressed in toxoplasmosis, the pathophysiology of this immunodepression remains obscure. The purpose of our studies is

’ Presented in part at the National Meeting of the American Federation for Clinical Research, Washington, DC, May 1985.

’ To whom all correspondence should be addressed: Department of Medicine, Division of Infectious Diseases, Health Sciences Center, T-l 5 080, State University of New York, Stony Brook, N.Y. 11794-g 153.

422

000%8749/86 $3.00 Copyright 0 1986 by Academic Pns, Inc. All rights of reprcduction in any form reserved.

T-CELL DYSFUNCTION IN ACUTE MURINE TOXOPLASMA 423

to further characterize the mechanism of immunodepression in toxoplasmosis. In a series of experiments, we systemically studied the concanavalin A (Con A)-stimulated proliferative response of lymphocytes from mice infected with the virulent RH and the relatively avirulent C56 strains of TG, the production of interleukin-2 (IG2) by these lymphocytes, and the effect of exogenous recombinant IL-2 on the lymphocyte proliferative response of infected animals. In addition, we studied the effect of indomethacin on the proliferative response of lymphocytes from infected mice. Our data demonstrate that at least two cell populations are involved in the immunodepression of toxoplasmosis, macrophages and T cells. This immunodepression is associated with a decrease in Con A-induced lymphoproliferation and IL-2 production, and possibly, the development of an active suppressor mechanism. Finally, our results indicate that the virulent RH strain of TG is capable of causing a greater degree of immunodepression than the relatively avirulent C56 strain.

MATERIALS AND METHODS

Animals. Six- to eight-week-old C57B1/6 female mice were obtained from Charles River Breeding Laboratories (Wilmington, Mass.). Animals were age matched in each experiment.

Acute T. gondii infection. Acute TG infection was established with the virulent RH and relatively avirulent C56 strains of TG (a gift from Jack S. Remington, Palo Alto, Calif.). The RH and C56 tachyzoites were harvested and prepared as previously described (10). Viability of the tachyzoites was determined by trypan blue exclusion, and was greater than 99%. Viable tachyzoites ( 103) were injected intravenously into C57B1/6 mice in 0.2 ml of phosphate-buffered saline (PBS; pH 7.2) via the lateral tail vein, 3 or 6 days prior to the experiments. Control mice received 0.2 ml PBS alone.

Splenocytes (SC) preparation. SC from three to eight animals were used as a source of cells for lymphocyte proliferation assay. SC were prepared as previously described ( 11) and suspended at a concentration of 5 X 1 O6 cells/ml in RPM1 medium (GIBCO, Grand Island, N.Y.) containing 10% heat-inactivated fetal calf serum (FCS; GIBCO), 100 units/ml of penicillin G, and 50 &ml of gentamicin (RPMI-FCS).

Nonadherent cells (NC) preparation. Plastic NC were prepared by incubating 10 ml of SC (5 X lo6 cells/ml) in a plastic petri dish (Lab Tek, Miles Scientific, Naperville, Ill.) at 37°C in a humidified atmosphere containing 5% COZ. After 1 hr the plastic NC were collected by gently washing the petri dish with warm PBS. These cells were centrifuged, washed with warm PBS, and resuspended at a concentration of 5 X lo6 cells/ml in RPMI-FCS. Nonspecific esterase stain of this nonadherent cell population was approximately 4% positive.

T-enriched cell preparation. T cells were enriched from SC by sequential adherence to plastic and nylon wool ( 12). Briefly, 2 ml of NC (5 X lo7 cells/ml) were transferred into a 1 O-ml plastic syringe packed with 0.6 g of sterile nylon wool (Fenwal Laboratory, Deerlield, Ill.). They were incubated for 1 hr at 37°C in a humidified atmosphere containing 5% COZ. The column was then eluted with 30 ml of warm RPMI-FCS over 30 min. The cells were resuspended at a concentration of 5 X lo6 cells/ml in RPMI-FCS. Nonspecific esterase stain of these cells was less than 1% positive. Cy- totoxicity assay using anti-Thy 1.2 antiserum (Accurate Chemicals, Hicksville, N.Y.) and rabbit complement (Accurate Chemicals) demonatrated approximately 80% Thy 1 .Zbearing cells in this population. Optimal dilutions of the anti-Thy 1.2 antiserum and complement were determined in preliminary experiments.

424 CHAN, SIEGEL, AND LUFI

Mucrophage preparation. Macrophages from peritoneal exudate cells (PEC) were used as accessory cells in proliferation assays of T-enriched cells. PEC from healthy C57B1/6 mice were harvested as described previously (1 l), washed in PBS, and resuspended in RPMI-FCS at a concentration of 2.5 X lo5 cells/ml. One-hundred microliters of this suspension was added to each well of a 96-well flat-bottomed tissue culture plate (Falcon, Oxnard, Calif.). These cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 . After 4 hr, NC were washed off with warm PBS before the addition of T-enriched cells in the lymphocyte proliferation assay. The number of PEC used per well, which gave a PEC:T-enriched cell ratio of 1:20, was found to be optimal for proliferation of T-enriched cells to Con A in preliminary experiments.

Reagents. Concanavalin A (Sigma, St. Louis, MO.) was dissolved in RPMI-FCS. Indomethacin (a gift from Merck, Sharp and Dohme, West Point, Pa.), was dissolved in distilled water at a concentration of 25 mg/ml. Sodium carbonate (Fisher Scientific Co., Springfield, N.J., 5.7 mg/ml solution) was then added at a ratio of 4:3 (sodium carbonate:indomethacin) by volume. This solution was then diluted 2.86-fold by distilled water. All solutions were filtered sterile before use. In the lymphocyte proliferation assays, Con A was used at final concentrations of 25, 12.5, and 3.125 &ml, and indomethacin at final concentrations of 5, 10, and 15 &ml.

Lymphocyte proliferation assay. One-hundred microliters of lymphocytes in RPMI- FCS (5 X 1 O6 cells/ml) was distributed in 96-well round-bottomed tissue culture plates (Coming Glass, Coming, N.Y.) in triplicate in the presence of 100 ~1 of RPMI-FCS containing Con A. They were incubated at 37°C in a humidified atmosphere containing 5% COz. For T-enriched cells, flat-bottomed tissue culture plates (Falcon) were used. After 48 hr, the cells were pulsed with 1 &i of [3H]thymidine (New England Nuclear, Boston, Mass. 6.7 Ci/mmol). In preliminary experiments, this time point was found optimal for the proliferative responses of lymphocytes from both infected and uninfected groups of mice. Eighteen hours after pulsing, cells were harvested by a PHD cell harvester (Cambridge Technology, Cambridge, Mass.). Incorporation of [3H]thymidine in cultures was determined in a Beckman LS 8000 liquid scintillation counter (Beckman Instruments, Palo Alto, Calif.), and the results were expressed as the difference in counts per minute (Acpm) in response to Con A minus the back- ground from unstimulated cultures. The results are expressed in counts per minute f the standard deviation from three replicate cultures. In each experiment the peak Acpm between groups was compared. The percentage depression was calculated by the equation:

Acpm of infected mice Acpm of control mice

x loo *

Significance of differences in [3H]thymidine incorporation as well as percentage depression between groups was determined by the Student t test.

Interleukin-2 production. The Con A-stimulated IL-2 production by lymphocytes from infected and healthy mice was compared. In these experiments, 5 X 10’ cells in 100 ~1 of RPMI-FCS were cultured with 100 ~1 of RPMI-FCS containing Con A (final concentrations: 25, 12.5, and 0 &ml) in 96-well flat-bottomed tissue culture plates. The supematant was harvested after 36-40 hr of incubation and measured for IL2 activity (units/ml) by an assay using the IL-2-dependent T-cell line, CTLL-2 (13). The percentage of IL-2 production was calculated by the equation:


% of IL-2 production = IL-2 activity of lymphocytes from infected mice IL-2 activity of lymphocytes from control mice

x loo *

Significance of differences in the percentage of IL-2 production between groups was determined by the Student t test.

Interleukin-2 assay. IL-2 activity was determined in a microassay measuring the capacity of each sample to induce thymidine incorporation by an IL-2dependent murine T-cell line, CTLL-2 ( 13). Samples were tested in parallel with our laboratory reference standard and IL-2 levels were determined by comparison of the activity of the samples with that of the standard in the manner previously described (14). The potency of our IL-2 standard was established by testing in comparison with a primary IL-2 reference standard provided by the Biological Resources Branch of the Biological Response Modifiers Program of the National Cancer Institute.

RESULTS

Eflect of Acute TG Infection on the Proliferative Response of Lymphocytes to Con A

To evaluate the cell-mediated immune status in mice infected with TG in our model, we studied the Con A-stimulated proliferative response of SC from mice infected with RH tachyzoites for 6 days (RH6), 3 days (RH3), and from mice infected with C56 tachyzoites for 6 days (C56-6) and 3 days (C56-3). As shown in Fig. 1, all infected groups demonstrated a marked and significant depression compared to the control group (P < 0.005). The degree of depression of RH6 SC (96.7 f 4.1%) was significantly greater than that of the RH3 SC (64.3 + 8.9%, P It O.OOS), C56-6 SC (75.2 ? 17.6%, P < O.OOS), and C56-3 SC (58.2 + 15.7%, P < 0.005). Figure 2 shows the results of a set of representative experiments of [3H]thymidine incorporation by the SC from the various infected groups of mice.

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426 CHAN, SIEGEL, AND LLJFI.

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FIG. 2. Effect of acute TG infection on the Con A-stimulakxi proliferative response of splenocytes. Each bar represents the mean [‘Hlthymidine incorporation from a triplicate culture. The standard deviation of the mean is shown.

Plastic Nonadherent Cells

In order to evaluate whether rn4 played a role in the depression of lymphocyte proliferation during acute toxoplasma infection, we studied. the effect of the removal of plastic adherent cells on the proliferative response of SC from infected mice. As shown in Fig. 3, partial removal of plastic adherent cells had no effect on the proliferative response of RH6 SC, and mildly, but not significantly, decreased the degree of depression of the RH3 SC (from 68.7 to 56.4%, P > 0.05), C56-3 SC (66 to 44%, P > 0.05), and C56-6 SC (85 to 78.5%, P > 0.25).

Splenocytes in the Presence of Indomethacin

Since prostaglandins have been implicated to participate in the macrophage-mediated suppression of murine lymphocyte proliferation (15), we evaluated the role of rn9 in the immunodepression in acute toxoplasmosis by studying the effect of indomethacin, a prostaglandin inhibitor, on the proliferative response of SC from infected and healthy mice.

In these experiments, SC from various infected groups and uninfected controls were cocultured in the presence of Con A with or without indomethacin. These experiments were performed twice and yielded similar results. Figure 4 shows the data of one of two sets of experiments performed. As can be seen, addition of indomethacin resulted in a marked and significant increase in the proliferative response of SC from RH3, C56-6, and C56-3 mice (P < 0.005), but had no effect on that from RH6 mice. Con- currently, addition of indomethacin caused a marked decrease in the degree of depression of the proliferative response of SC from the RH3 mice (from 66.4 to 20.80%), C56-6 mice (67.5 to 31.1%), and C56-3 mice (73.7 to 24.7%) and had no effect on that from the RH6 mice (99 to 97%).

T-Enriched Cells

These data.sug%est that the rn4 is only partiahy responsible for the immunodepression in acute toxoplasma infection since partial removal of rn4 or addition of indomethacin


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did not fully restore the proliferative response of SC to Con A in RH3, CM-6, and C56-3 mice and had no effect on the response of SC from RH6 mice. To confirm this hypothesis, we scrupulously removed rn$ by sequential adherence of SC to plastic and nylon wool, and then evaluated the Con A-stimulated proliferative response of the resulting T-enriched cells from healthy and infected mice. In these experiments, T-enriched cells were cultured with normal rn& which acted as accessory cells. As

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PIG. 4. Effect of indomethacin on the Con A-stimulated proliferative response of splenocytes (SC) from TG-infected mice. Each bar represents the mean [3H]thymidine incorporation from a triplicate culture. The standard deviation of the mean is shown.

428 CHAN, SIEGEL, AND LUFT

shown in Fig. 5, the T-enriched cells from all infected mice demonstrated a marked and significant depression (P < 0.05 to 0.005). The degree of depression of RH6 T- enriched cells (89.2 f 12.5%) was significantly greater than that of the other infected mice (RH-3 39.0 + 19.7%, C56-6 43.2 + 18.4%, C56-3 29.3 f 5.8%; P < 0.01 to 0.005). Because these findings strongly suggest that there is a T-cell defect in acute toxoplasma infection, and because proliferation of T lymphocytes in response to T- cell mitogens requires the presence of IL-2 (16, 17) a product of T lymphocytes (18, 19), we evaluated the production of IL-2 by lymphocytes from infected mice.

Effect of Acute TG Infection on IL-2 Production by Murine Lymphocytes

In these experiments, the IL-2 production by SC, plastic NC, and T-enriched cells from various infected groups of mice was compared to that from healthy mice. As shown in Fig. 6, there was a marked decrease in IL-2 production by both SC and NC of all infected groups compared to controls. Splenocytes from RH6 mice demonstrated the greatest decrease in IL-2 production. Partial removal of plastic adherent cells had little effect on the percentage of IL-2 production by SC from any infected group. Furthermore, IL-2 production by T-enriched cells from various infected groups and uninfected controls after rigorous removal of rn4 by sequential adherence of SC to plastic and nylon wool was measured in the presence of rn4 from uninfected mice. Figure 7 shows the results of a set of representative experiments. As can be seen, there was a marked decrease in the production of IL-2 from all infected groups compared to controls. T-enriched cells from RH6 mice demonstrated the greatest decrease in IL-2 production. Since IL-2 is a lymphokine produced by T lymphocytes (18, 19), and our experiments were performed in the presence of healthy peritoneal rnb which acted as accessory cells, these data give further evidence that there is a T-cell defect in acute toxoplasmosis.

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FIG. 5. Effect of acute TG infection on the Con A-stimulated proliferative response of T-enriched cells. Each point represents the results of an individual experiment. The percentage of depression was calculated by Eq. [I] outlined under Materials and Methods.


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FIG. 6. Con A-induced IL2 production by splenocytes (SC) and nonadherent cells from TG-infected mice. Each point represents the result of an individual experiment.

Eflect of IL-2 on the Proliferative Response of Lymphocytes from Infected and Healthy Mice

Having established the fact that the immunodepression in acute toxoplasmosis is associated with a decrease in Con A-stimulated lymphoproliferation and IL-2 production, we studied the effect of exogenous IL-2 on the proliferative response of lymphocytes from infected mice and evaluated the relationship between these two phe-

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430 CHAN, SIEGEL, AND LUFT

nomena. In these experiments, increasing concentrations of recombinant IL-2 (kindly supplied by Cetus Corp., Emeryville, Calif.) were added to SC and T-enriched cells from infected and healthy mice, and their proliferative responses to Con A compared. All experiments were repeated once or twice, and yielded similar results. Data shown here are those from a representative experiment. As can be seen in Fig. 8, addition of IL-2 to a final concentration of 80 units/ml resulted in a 196 f 75% increase in the Con A-stimulated proliferative response of SC from healthy mice compared to that without addition of IL-2. Exogenous IL-2 fully reconstituted the depressed proliferative response of SC from RH3, C56-3, and C56-6 mice; however, the peak proliferative responses of SC from infected mice treated in this manner were all lower than those of similarly treated SC from control mice. In contrast, RH6 SC were unresponsive to exogenous IL-2. Addition of IL-2 to T-enriched cells from both healthy and infected groups of mice resulted in a pattern of response similar to that of SC (Fig. 9).

In these experiments, exogenous IL-2 had a proliferative effect on both SC and T- enriched cells independent on Con A. Of interest is the effect of IL-2 on the Con A- stimulated proliferative response of RH6 SC and T-enriched cells. Table 1 shows that the proliferative response of both SC and T-enriched cells from RH6 mice, in the presence of Con A, was significantly less than that without Con A at each concentration of IL-2 added. Without IL-2, the proliferative response of these lymphocytes was still lower when Con A was present, but not significantly so. These data suggest that Con A might stimulate the production of a suppressor factor by the RH6 T-enriched cells, which is capable of causing depression of lymphoproliferative response.

DISCUSSION

It has been previously demonstrated that cell-mediated immunity is depressed in toxoplasma infection in animals (2-6) and in humans (7,8). Recently, the macrophage has been implicated as the suppressor cell responsible for immunodepression in toxo-

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FIG. 8. Effect of exogenous IL-2 on the Con A-stimulated proliferative response of splenocytes (SC) from TG-infected mice. Each point is the mean [3H]thymidine incorporation from a triplicate culture; the standard deviation of the mean is shown. RH6 SC was unresponsive to exogenous IL2.


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plasmosis (9). However, the precise mechanism responsible for the depressed cell- mediated immunity during this infection remains unclear. The results of these studies in mice demonstrate that there are at least two cell populations involved in the immunodepression of toxoplasmosis, macrophages and T cell. All infected groups of mice demonstrated a significant depression of the proliferative response of their splenocytes to the T-cell mitogen, Con A. This confirms the known immunodepressed state in toxoplasmosis and is in agreement with the data of Strickland et al. (2), which showed that the proliferative response of murine splenocytes to T-cell mitogens was markedly depressed in toxoplasmosis.

Our data also support the findings of Suzuki and Kobayashi (9), which demonstrated the suppressive effect of macrophages on lymphocyte proliferation to nonspecific and toxoplasma-specific antigens in murine toxoplasmosis. However, our findings indicate that the macrophage is not the sole suppressor factor in the immunosuppression due

TABLE 1

Effect of IL-2 on the Con A-Stimulated Proliferative Response of RH6 Splenocytes and T-Enriched Cells

[‘H]Thymidine incorporation (cpm + SD)

IL-2 added (unit/ml)

Splenocytes T-Enriched cells

+Con A -Con A P +Con A -Con A P

0 15186 + 1391 18975 + 5037 >0.05 6550 k 785 7139 + 763 >0.05 10 33875 f 2086 52219 + 2755 <0.005 17474 + 1893 32131 + 1692 <0.005 20 42588 + 4375 55740 + 2432 <O.Ol 18096 + 1346 53643 + 8341 <0.005 40 38296 + 1442 60855 rk 2293 <0.005 20747 + 877 105599 f 16814 <0.005 80 35108 + 4918 50651 + 2319 <0.005 17312 + 953 78412k 11311 <0.005

432 CHAN, SIEGEL, AND LUIT

to acute infection with T. gondii. Our data also demonstrate that indomethacin has no effect on the proliferative response of RH6 splenocytes, and only partially, but significantly, increases the proliferation of cells from the other infected groups of mice.

The T-enriched cells from all infected groups of mice also demonstrated a marked and significant decrease in the proliferative response to Con A, suggesting that there is an intrinsic defect in the T cells of mice with acute toxoplasma infection independent of the macrophage. We feel that the decreased responsiveness to Con A of these T- enriched cells is not likely to be caused by contaminating macrophages because the nonspecific esterase stain of these cells was less than 1% positive. Furthermore, we repeated experiments using L-leucine methyl ester (a lysosomotropic compound that depletes macrophages effectively (20-22)) as a macrophage depletor before adherence to a nylon wool column and obtained similar results in the T-enriched cell proliferation assay (data not shown). The involvement of IL- 1 can also be excluded because the T- enriched cell proliferation assay was performed in the presence of healthy peritoneal macrophages, which acted as the source of IL- 1.

Data from these studies show that exogenous recombinant IL-2, to a final concentration of 80 units/ml, has no effect on the depressed proliferative response of the RH6 T-enriched cells. In addition, although the proliferative response of T-enriched cells from the other infected groups of mice could be entirely reconstituted by exogenous IL-2, the peak lymphoproliferative response achieved in the infected groups was always less than that in the control group (Fig. 9). Therefore, the decreased Con A-induced proliferative response cannot be solely explained by a defect in IL-2 production. Other possible explanations are (1) decreased affinity between the IL-2 receptor and IL-2 in infected animals, (2) decreased expression of the IL-2 receptor (indeed, a suppressor factor that inhibits T-cell proliferation has been described and was the thought to exert its suppressor effect by inhibition of the expression of IL-2 receptor during activation (23)), and (3) the presence of an inhibitor(s), a suppressor cell, or a soluble suppressor factor that actively suppresses the lymphoproliferative response of these T-enriched cells. Con A-induced suppressor cells that limit IL-2 production have been described in vitro in mice (24). Alloantigen-induced suppressor factor(s) that inhibit T-cell proliferation as well has also been described (23). This factor(s) seems to exert its suppressor effect by inhibition of IL2 production as well as the expression of the IL-2 receptor during activation. Finally, suppressor T cells have also been implicated in mice infected with mycobacteria (25) and in patients with leprosy (26, 27). This last hypothesis is especially attractive in the case of the RH6 mice. When exogenous IL-2 was added to cultures, the proliferative response of RH6 splenocytes and T- enriched cells in the presence of Con A was significantly depressed compared to that without Con A at each concentration of IL-2 added (Table 1). Therefore, there appears to be present in the splenocytes of RH6 mice a suppressor factor that can be promoted by Con A. This phenomenon, however, was not observed in the other groups of infected mice. It is conceivable that the development of such a suppressor mechanism requires a certain virulence factor that is present in the more virulent RH strain but not the less virulent C56 strain of T. gondii, or that there is a quantitative difference in a virulence factor. Our data also showed that RH6 lymphocytes demonstrated the greatest degree of unresponsiveness to Con A and exogenous IL-2, and the greatest decrease in IL-2 production. Therefore, there may be an association between the virulence of different strains of T. gondii with their ability to immunosuppress.

Decreased lymphocyte proliferation and IL-2 production have been reported in animals infected with various intracellular parasites. Murine malaria infection (28),


Mycobacterium lepraemurium infection (29,30), trypanosomiasis (3 1, 32), and leish- maniasis (33) have all been associated with decreased production of IL-2. In these studies, we have demonstrated that decreased lymphocyte proliferation and IL-2 production occur in mice acutely infected with T. gondii. At a cellular level, we have demonstrated that both macrophages and T cells are involved in the pathogenesis of immunodepression in this infection, causing a decrease in Con A-stimulated lymphocyte proliferation which can, in part, be attributed to a decrease in IL-2 production. The decreased lymphocyte proliferation may also be due to the development of an active suppressor cell, and/or a soluble suppressor factor, which is preferentially induced during acute infection with T. gondii.

Our model of murine toxoplasmosis may be important in understanding the alter- ations of the immunoregulatory circuits in parasitic infections and the pathophysiology of chronic parasitic diseases.

ACKNOWLEDGMENT We thank Ms. Ruth Latour for her expert secretarial and editorial assistance.

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