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TCR V8⁺ T Cells Prevent Development of Toxoplasmic Encephalitis in BALB/c Mice Genetically Resistant to the Disease¹

Hoil Kang^2,*,, Oliver Liesenfeld^3,*,, Jack S. Remington^*,, Jennifer Claflin, Xisheng Wang and Yasuhiro Suzuki^4,*,,

^* Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305; Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto, CA 94301; and Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
BALB/c are genetically resistant to development of toxoplasmic encephalitis (TE) when infected with Toxoplasma gondii, whereas CBA/Ca mice are susceptible. We compared TCR V chain usage in lymphocytes infiltrated into brains between these animals following infection. TCR V8⁺ cells were the most frequent T cell population in brains of infected, resistant BALB/c mice, whereas TCR V6⁺ T cells were more prevalent than V8⁺ T cells in brains of infected, susceptible CBA/Ca mice. Adoptive transfer of V8⁺ immune T cells, obtained from infected BALB/c mice, prevented development of TE and mortality in infected athymic nude mice that lack T cells. In contrast, adoptive transfer of V6⁺ immune T cells did not prevent development of TE or mortality in the nude mice. The protective activity of V8⁺ immune T cells was greater than that of the total V8^- population. In addition, V8⁺ immune T cells produced markedly greater amounts of IFN- than did the V8^- population after stimulation with tachyzoite lysate Ags in vitro. Thus, V8⁺ T cells appear to play a crucial role in the genetic resistance of BALB/c mice against development of TE.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The healthy brain is an immunologically privileged site because the blood-brain barrier excludes components of the peripheral immune system from the brain. The brain has its own specialized cells that produce cytokines and execute immunological effector functions (1, 2). However, when infection occurs, lymphocytes infiltrate into the brain. It has recently been shown that T cells in the inflamed brain are phenotypically distinct from those in other inflamed tissues (3). Thus, host defense in the brain appears to operate through a unique mechanism involving collaboration between specialized cells in the brain and lymphocytes that preferentially infiltrate into the brain. However, the mechanism(s) of defense of the brain against infection still remain to be defined.

Toxoplasma gondii, an intracellular protozoan parasite, forms cysts and establishes a latent, chronic infection preferentially in the brain after replication of the parasite in various organs during the acute stage of infection. The requirement for the immune system to maintain the latency of persistent infection is clearly evident from the reactivation of the infection in immunocompromised individuals, which results in the development of toxoplasmic encephalitis (TE)⁵ (4, 5). Thus, T. gondii provides an excellent model for analyzing the mechanism of host resistance of the brain against infection (6, 7, 8, 9, 10, 11). Resistance to development of TE during persistent infection is under genetic control in both humans (12, 13) and mice (14, 15, 16). In mice, strains with the H-2^b or H-2^k haplotypes develop progressive, necrotizing encephalitis, whereas strains with the H-2^a or H-2^d haplotypes do not. Because the H-2 complex encodes major recognition and immunoregulatory molecules, it is likely that the genes responsible for control of resistance against TE, or at least some of them, do so by regulating the immune response to the parasite. However, the mechanism of the genetic control of the resistance is still unclear. Because resistance of different strains of mice to development of TE does not correlate with their resistance to the acute acquired infection (17, 18), it is most likely that immunological mechanism(s) unique to the brain are operative in maintaining the latency of chronic infection and thereby serve to prevent development of TE.

One possibility that could occur in TE-resistant BALB/c and susceptible CBA/Ca mice is that T cells which infiltrate and expand in the brain of infected BALB/c mice recognize T. gondii Ags that are different from those recognized by T cells which infiltrate and expand in the brain of infected CBA/Ca mice. As a first step to assess this possibility, we compared TCR V chain usage in T cells infiltrated into brains of these animals following infection. We found marked differences in TCR V chain usage in the lymphocytes infiltrated into the brain, as well as in splenic T cells, between these mice during the course of infection. In the resistant strain, T cells bearing the V8 chain were the most frequent in the brain, and adoptive transfer of this T cell population alone was sufficient to prevent development of TE and mortality in infected, athymic nude mice.

	Materials and Methods

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Mice

Female BALB/c-background athymic nude (The Jackson Laboratory, Bar Harbor, ME), control BALB/c (The Jackson Laboratory), CBA/Ca (Bantin & Kingman, Fremont, CA), and Swiss-Webster mice (Taconic Farms, Germantown, NY) were 6–8 wk old when used. There were three to six mice for each experimental group.

Infection with T. gondii

Cysts of the ME49 strain were obtained from brains of Swiss-Webster mice that had been infected i.p. with 10 cysts for 2–3 mo. Mice were sacrificed by asphyxiation with CO₂, and their brains were removed and triturated in PBS (pH 7.2) (19). An aliquot of the brain suspension was examined for numbers of cysts, and after appropriate dilution in PBS, animals were infected with 10 cysts perorally by gavage. Athymic nude mice were treated with sulfadiazine in drinking water (400 mg/L) beginning 7 days after infection for 3 wk.

Flow cytometry

At 2, 4, or 8 wk after infection, mononuclear cells infiltrated into brains or spleen cells were obtained individually from three mice of each strain (BALB/c and CBA/Ca) as described previously (20). The cells were pretreated on ice for 10 min with 10 µl of predetermined optimal concentration of anti-FcII/IIIR mAb (2.4G2) to block non-Ag-specific binding of Abs to the FcII/IIIRs. Thereafter, the cells were incubated on ice for 30 min with 10 µl of optimal concentrations of FITC-conjugated mAbs to TCR V chains in combination with PE-conjugated mAbs to either CD4 or CD8. In some experiments, the cells were stained with FITC-conjugated mAb to TCR V8 in combination with PE-conjugated mAb to pan-NK cells (DX5). The mAbs were obtained from BD PharMingen (San Diego, CA). Analysis of stained cells was performed with a FACScan (BD Biosciences, Mountain View, CA). Dead cells were gated out on the basis of propidium iodide staining.

Purification and transfer of V8⁺, V6⁺, and the total V8^- population of immune T cells

Control BALB/c mice infected perorally with 10 cysts for 2–3 mo were challenged i.p. with 100 cysts. One week after the challenge infection, spleen cells were obtained from two or three mice, suspended in HBSS, and pooled. The total population of T cells was purified by treating the spleen cells with magnetic bead-conjugated anti-mouse CD4 (GK1.5) plus anti-mouse CD8 (53-6.7) mAbs (Miltenyi Biotec, Sunnyvale, CA) for MACS. Thereafter, V8⁺ and V6⁺ T cell subsets were purified by treating the T cells with either FITC-labeled, anti-V8 or -V6 mAbs, then sorted with FACSVantage (BD Biosciences). The total V8^- population of T cells was also purified in the same manner. The purity of the T cell subset in each of the purified preparations were >98%. A total of 1 x 10⁶ V8⁺ or V6⁺ immune T cells were injected i.v. from a tail vein to recipient nude mice at 9 and 2 days before discontinuation of treatment with sulfadiazine. In some experiments, 1 x 10⁵ V8⁺ or the total V8^- T cell population, or a total of 2 x 10⁷ immune spleen cells were injected into recipient nude mice in the same manner as V8⁺ immune T cells. For culture of V8⁺ T cells, V8⁺ and V8^- T cell subsets were purified as described above in this section or by treating the T cells with FITC-labeled anti-V8 mAb, then with magnetic bead-conjugated anti-FITC mAb (Miltenyi Biotec) for MACS. In this case, the V8^- T cells population was obtained by treating V8^- whole cells with magnetic bead-conjugated anti-CD4 and anti-CD8 for MACS.

Culture of V8⁺ and V8^- T cells and detection of IFN- in the culture supernatants

Culture of V8⁺ and V8^- immune T cells and stimulation with T. gondii Ags was performed as previously described (20) with modification. Briefly, the T cells were purified from spleens of infected BALB/c mice as described in the paragraph above and suspended in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) with 10% FCS (Sigma-Aldrich) and penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (Fungizone; 0.025 µg/ml) (BioWhittaker, Walkersville, MD). Cells were placed into flat-bottom wells of 96-well plates (Costar, Cambridge, MA) at a cell density of 4 x 10⁵ cells/well with APC (plastic adherent cells) obtained from 4 x 10⁵ normal spleen cells. Cells were incubated with and without soluble T. gondii lysate Ags (20 µg/ml) for 72 h, and thereafter, the culture plates were centrifuged and the culture supernatants were collected. The concentration of IFN- in the culture supernatants was measured by ELISA using mAbs against IFN- (R4-6A2 as capture, XMG1.2 as secondary; BD PharMingen) (21).

Histopathology

At 7 days after discontinuation of treatment with sulfadiazine, mice were euthanized by asphyxiation with CO₂. Their brains were removed and immediately fixed in a solution containing 10% formalin, 70% ethanol, and 5% acetic acid. Two to four 5-µm thick sagittal sections (50- or 100-µm distance between sections) of the brain from each mouse were stained with immunoperoxidase stain by using rabbit IgG Ab against tachyzoite-specific SAG2 and evaluated for the numbers of areas of inflammation associated with tachyzoites. (19, 22). H&E-stained sections were evaluated for inflammatory changes.

Statistical analysis

Levels of significance for numbers of areas associated with tachyzoites in the brain, and amounts of IFN- in the culture supernatants were determined by Student’s t test or Alternate Welch t test. Alternate Welch t test was applied when SDs were significantly different between groups tested. Levels of significance for mortality in mice were determined using Fisher’s exact test. Differences which provided values of p < 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differences in TCR V chain usage in T cells infiltrated into the brains of BALB/c and CBA/Ca mice following infection with T. gondii

CBA/Ca (H-2^k haplotype; susceptible to TE) and resistant BALB/c (H-2^d haplotype; resistant to TE) mice were infected perorally with 10 cysts of the ME49 strain of T. gondii. At 2, 4, and 8 wk after infection, we compared TCR V chain usage in the lymphocytes infiltrated into the brains of these animals. Because TCR is responsible for recognition of Ags by T cells and because V region of TCR is responsible for determining the specificity of the receptor, we hypothesized that TCR V chain usage might differ in such T cells between these animals. In this model, only CBA/Ca mice begin developing progressive, necrotizing encephalitis by 4 wk after infection (14, 15, 23). In correlation with the difference in development of TE between these mice, the total number of T cells obtained from the brain after infection was greater in CBA/Ca than in BALB/c mice at each time point tested (2.50 ± 0.53 vs 0.90 ± 0.24 (x10⁶) at 2 wk, p < 0.0001; 2.98 ± 0.63 vs 1.39 ± 0.38 (x10⁶) at 4 wk, p < 0.001; 2.21 ± 0.97 vs 1.22 ± 0.32 (x10⁶) at 8 wk, p = 0.057).

Marked differences in TCR V chain usage were observed between these strains at each time point after infection. Fig. 1 shows results at 4 wk after infection. Similar results were seen at 2 and 8 wk after infection. T cells bearing V8 were most frequent in both CD4⁺ and CD8⁺ subsets infiltrated into brains of resistant BALB/c mice at all time points examined. In contrast, T cells bearing V4, V6, V10, or V14 were more frequent than those bearing V8 in brains of susceptible CBA/Ca mice. V6⁺ T cells were the most frequent population in the CBA/Ca mice at 4 wk after infection (Fig. 1). The frequencies of V8-bearing cells were significantly higher in BALB/c than in CBA/Ca mice in both CD4⁺ and CD8⁺ subsets (p < 0.0001 for both). The pattern of V chain expression in cerebral T cells was similar to that of splenic T cells (Fig. 1). However, some differences in the V chain usage between these T cells were noted; for example, the differences in the frequencies of V8-bearing cells in CD4⁺ T cells between BALB/c and CBA/Ca mice were significantly greater in brains (4.2 times) than in spleens (2.3 times) (p < 0.005), suggesting the presence of regulatory mechanisms in T cell recruitment into the brain following infection.

View larger version (41K): [in this window] [in a new window]   FIGURE 1. TCR V chain usage in lymphocytes infiltrated into brains and splenic lymphocytes in T. gondii-infected BALB/c and CBA/Ca mice that are genetically resistant and susceptible to development of TE, respectively. At 2, 4, and 8 wk after infection with 10 cysts of the ME49 strain, lymphocytes infiltrated into the brain of mice were isolated individually from three animals from each group, and their TCR V chain usage was analyzed by flow cytometry as described (17 ). The mean value from three mice was calculated for each TCR V chain. The panel shows these values at 4 wk after infection. Similar results were seen at 2 and 8 wk after infection.

Protective activity of V8⁺ immune T cells of resistant BALB/c mice against development of TE

We examined whether V8-bearing T cells, which are the most frequent population in brains of infected BALB/c mice, play an important role in their genetic resistance to development of TE. For this purpose, we used an experimental model of reactivation of T. gondii infection in the brain of infected, sulfadiazine-treated immunodeficient mice (19). V8⁺ immune T cells were purified from spleens of infected BALB/c mice and transferred into infected, sulfadiazine-treated athymic nude (BALB/c-background) mice that lack T cells. Control nude mice that had not received the immune T cells developed severe TE and died after discontinuation of sulfadiazine treatment (Fig. 2). In contrast, nude mice that had received V8⁺ immune T cells did not develop TE (Figs. 2B and 4A) and survived (Fig. 2A). These results indicate that V8⁺ immune T cells of BALB/c mice have a potent protective activity to prevent reactivation of chronic T. gondii infection in the brain of the athymic nude mice.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Mortality (A) and development of TE (B) in T. gondii-infected, sulfadiazine-treated athymic nude mice with or without adoptive transfer of V8+ immune T cells. Athymic nude mice were infected with 10 cysts of the ME49 strain perorally and treated with sulfadiazine for 3 wk beginning 7 days after infection. Nine and 2 days before discontinuation of sulfadiazine treatment, mice received an i.v. injection of 1 x 106 V8+ immune T cells from infected, control BALB/c mice (see Materials and Methods). Histological studies were performed 7 days after discontinuation of treatment with sulfadiazine. Two to four sagittal sections (distance between sections of 50 µm) were stained with immunoperoxidase stain by using tachyzoite-specific SAG2 Ab and evaluated for the numbers of areas of inflammation associated with tachyzoites. The mean value from these sections for each mouse was calculated as the number per section. These values were shown in the figure and were used for statistical analysis to compare differences between groups. The data shown are the representative of two separate experiments. There were three or four mice in each experimental group in each experiment.

View larger version (125K): [in this window] [in a new window]   FIGURE 4. Histological changes in brains of T. gondii-infected, sulfadiazine-treated athymic nude mice with adoptive transfer of either V8+ (A) or V6+ (B) immune T cells. Athymic nude mice were infected with 10 cysts of the ME49 strain perorally and treated with sulfadiazine for 3 wk beginning 7 days after infection. Nine and 2 days before discontinuation of sulfadiazine treatment, mice received an i.v. injection of 1 x 106 V8+ or V6+ immune T cells from infected BALB/c mice (see Materials and Methods). Histological studies were performed 7 days after discontinuation of treatment with sulfadiazine. H&E stain. A large area of necrosis of brain tissue is shown in B. The data shown are the representative of two separate experiments. There were three or four mice in each experimental group in each experiment.

Inefficiency of V6⁺ immune T cells of resistant BALB/c mice for prevention of TE

To examine whether the protective activity observed in V8⁺ immune T cells was common in T cells bearing other TCR V chains, V6⁺ immune T cells were purified from spleens of infected BALB/c mice and transferred into infected, sulfadiazine-treated nude mice in the same manner as for V8⁺ T cells. V6-bearing T cells were chosen as a control because they were one of the populations observed most frequently in brains of infected, susceptible CBA/Ca mice as shown in Fig. 1. Nude mice that had received V6⁺ immune T cells developed severe TE (Figs. 3B and 4B) and died as early as control nude mice that had not received any T cells (Fig. 3A). The numbers of areas of inflammation associated with tachyzoites did not differ between brains of the nude mice with and without the cell transfer (Fig. 3B). These results indicate that V6⁺ population of immune T cells of BALB/c mice do not have a potent protective activity against development of TE as do V8⁺ immune T cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Mortality (A) and development of TE (B) in T. gondii-infected, sulfadiazine-treated athymic nude mice with or without adoptive transfer of V6+ immune T cells. Athymic nude mice were infected with 10 cysts of the ME49 strain perorally and treated with sulfadiazine for 3 wk beginning 7 days after infection. Nine and 2 days before discontinuation of sulfadiazine treatment, mice received an i.v. injection of 1 x 106 V6+ immune T cells from infected BALB/c mice (see Materials and Methods). Histological studies were performed 7 days after discontinuation of treatment with sulfadiazine (see Fig. 1). The data shown are the representative of two separate experiments. There were three or four mice in each experimental group in each experiment.

Comparison of the protective activity against development of TE between V8⁺ and V8^- populations of immune T cells

To further assess the importance of V8⁺ T cells in genetic resistance of BALB/c mice to development of TE, we compared the protective activities of V8⁺ immune T cells with that of the total V8^- T cell population. V8⁺ and V8^- populations of immune T cells were purified from spleens of infected BALB/c mice and transferred into infected, sulfadiazine-treated nude mice. In this study, the number of cells transferred was one-tenth of that used in the experiments shown in Figs. 2–4. Because of the lower number of cells transferred, nude mice that had received V8⁺ T cells eventually died after discontinuation of sulfadiazine treatment, but survived significantly longer than control animals that had not received any T cells (p < 0.05) (Fig. 5). Mice that had received the total V8^- population died as quickly as did the control animals (Fig. 5), and the time to death did not differ between these two groups.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Mortality in T. gondii-infected, sulfadiazine-treated athymic nude mice with adoptive transfer of either V8+ or the total V8- population of immune T cells. Athymic nude mice were infected with 10 cysts of the ME49 strain perorally and treated with sulfadiazine for 3 wk beginning 7 days after infection. Nine and 2 days before discontinuation of sulfadiazine treatment, mice received an i.v. injection of 1 x 105 V8+ or V8- immune T cells from infected BALB/c mice (see Materials and Methods). There were four mice in each experimental group.

Comparison of IFN- production between V8⁺ and V8^- populations of immune T cells

We previously reported the importance of IFN- in genetic resistance of BALB/c mice against development of TE (24). Because of the potent protective activity of V8⁺ T cells against the disease, we next performed an in vitro study to compare the ability of V8⁺ and V8^- T cells to produce IFN- in response to T. gondii Ags. V8⁺ immune T cells obtained from infected BALB/c mice produced large amounts of IFN- when stimulated with parasite Ags in vitro (Fig. 6). V8^- population also produced IFN-; however, the amounts of the cytokine produced by this population were significantly less than those produced by V8⁺ population (p < 0.005) (Fig. 6).

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Production of IFN- by V8+ and the total V8- population of immune T cells following stimulation with tachyzoite lysate Ags in vitro. V8+ and V8- populations of immune T cells were stimulated with tachyzoite lysate Ags (20 µl/ml) in the presence of APC (plastic-adherent cells from spleens of normal BALB/c mice) (see Materials and Methods). The data shown are the mean values from two separate experiments.

Frequencies of pan-NK marker-positive cells in V8⁺ population of T cells that infiltrated into the brain

Because V8 is one of the TCR V chains frequently used by NKT cells (25), we examined, by flow cytometry, whether V8⁺ T cells that infiltrated into the brain of infected BALB/c mice express the pan-NK cell marker. Few (<1%) of the V8⁺ T cells were positive for the NK cell marker.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study revealed distinct differences in TCR V chain usage in T cells infiltrated into brains of TE-resistant BALB/c and TE-susceptible CBA/Ca mice following infection with T. gondii. V8⁺ T cells were the most frequent population in brains of infected BALB/c mice, whereas this was not the case in the CBA/Ca mice; T cells bearing four other V chains were more abundant than V8⁺ T cells in these animals. The frequencies of V8⁺ T cells were significantly greater in BALB/c than in CBA/Ca mice. These differences in TCR V chain usage suggest that T. gondii Ags recognized by T cells infiltrated into the brain may differ between the TE-resistant and -susceptible strains of mice.

Significantly greater numbers of lymphocytes were collected from brains of CBA/Ca than those of BALB/c mice during the course of infection, including 2 wk after infection, which is the time that CBA/Ca mice had not developed severe TE. Despite having a greater number of lymphocytes recruited into the brain, CBA/Ca mice could not control T. gondii in their brains and developed TE. Therefore, it appears that recruitment of a selected, protective population(s) of T cells, rather than total number of T cells recruited, is important for prevention of TE.

Adoptive transfer of V8⁺ immune T cells from infected BALB/c mice into infected, sulfadiazine-treated athymic nude mice prevented their development of TE and mortality. Control nude mice that had not received any T cells suffered severe encephalitis due to reactivation of T. gondii infection and all died after discontinuation of sulfadiazine. These results clearly indicate that V8⁺ T cells in infected BALB/c mice have a potent protective activity against development of TE, and that this T cell population alone is sufficient to prevent the encephalitis. This is the first demonstration of prevention of an infectious disease in the brain by T cells expressing a specific TCR V chain.

In contrast to V8⁺ immune T cells, adoptive transfer of the comparable number of V6⁺ immune T cells did not prevent development of TE and mortality in the infected nude mice. Furthermore, the protective activity of V8⁺ immune T cells was greater than that of the total V8^- population of immune T cells. In the latter study, we observed a tendency for prolonged time to death in the infected nude mice that had received the total V8^- T cells at the dose we used. Therefore, if greater numbers of the total V8^- T cells are used for the cell transfer, those cells would reduce or prevent the pathology and mortality. Even V6⁺ T cells might provide a partial protection when much greater numbers of the cells are transferred. However, the data in the present study indicate that there are quantitative differences in the protective activity among T cell populations bearing different TCR V8 chains, and that the cells bearing TCR V8 chain are a potent protective population. Because V8⁺ T cells alone were sufficient to prevent TE as mentioned above, and because V8⁺ T cells are the most frequent population in the T cells infiltrated into the brains of these animals, the T cell population bearing TCR V8 chain most likely plays a crucial role in the genetic resistant of BALB/c mice against development of TE, although other T cell populations also appear to be involved in the resistance.

The importance of V8⁺ T cells in the resistance of BALB/c mice is further supported by the evidence of their potent activity of production of IFN-, which is required for prevention of TE. The present study revealed that V8⁺ immune T cells produced significantly greater amounts of this cytokine than did the total V8^- T cell population following stimulation with tachyzoite lysate Ags in vitro. It appears that V8⁺ T cells recognize major protective Ag(s) of T. gondii. As mentioned above, V8⁺ T cells are the most frequent population of T cells infiltrated into the brain in infected BALB/c mice. Thus, this T cell population is most likely an important producer of IFN- in the brain of these animals following infection. In this regard, by flow cytometric analysis, V8⁺ T cells were the largest population (19.7%) in the T cells that were positive for intracellular IFN- in the brains of infected BALB/c mice (J. Claflin and Y. Suzuki, unpublished data).

The evidence for the potent protective activity of V8⁺ T cells and the inefficiency of V6⁺ T cells in prevention of TE in genetically resistant BALB/c mice in the present study does not necessarily mean that the same situation occurs in susceptible CBA/Ca mice. The activities of these T cell populations in CBA/Ca mice could differ from those in BALB/c mice because of their differences in the MHC molecules expressed. Further studies are needed to address the role of these T cell populations in the genetic susceptibility of CBA/Ca mice to TE.

V8 is one of the TCR V chains preferentially used by NKT cells (25). In the present study, few (<1%) of V8⁺ T cells that infiltrated into the brain of infected BALB/c mice were found to be positive for pan-NK cell marker. Therefore, it appears that NKT cells would not play a major role, if any, in the protective activity of the V8⁺ T cell population in the brain of infected mice. In relation to this, Nakano et al. (26) recently reported the possibility that NKT cells play a suppressive role on IFN--mediated, protective immunity against acute acquired infection with T. gondii in BALB/c mice.

T cells bearing TCR V8 chain are also known to be one of the T cell populations that recognize bacterial and viral superantigens (27, 28). Denkers et al. (29) previously reported a superantigen activity in tachyzoite soluble Ags. However, in their studies, the T cell population which responded to the superantigen of T. gondii is that bearing the TCR V5 chain. Furthermore, they reported that the T cell population that responded to the superantigen becomes anergic in the chronic stage of infection (30). Because, in the present study, V8⁺ immune T cells were purified from chronically infected mice, it is likely that the T. gondii Ag(s) recognized by the protective V8⁺ T cells are not the superantigen-like molecule(s).

As mentioned above, genetic resistance of inbred strains of mice against development of TE does not correlate with their resistance against acute acquired infection, suggesting that different mechanisms in host resistance are operative during these two different stages of infection. In relation to our finding of the importance of V8⁺ T cells for genetic resistance of BALB/c mice against development of TE, an occurrence of clonal expansion of CD8⁺ T cells was recently demonstrated in the brain of multiple sclerosis patients, by analyzing TCR V region sequences of T cells infiltrated into active lesions (31). In Theiler’s murine encephalomyelitis virus-infected mice, a model of multiple sclerosis, clonal expansion of T cells was also suggested in demyelinating lesions in the brain (32). In a murine model of cerebral malaria, T cells bearing TCR V8.1 chain play a pathogenic role (33). Immune responses with limited population(s) of T cells that effectively infiltrate into the brain and recognize the limited epitope(s) may play an important role in the immunopathogenesis in the brain in various situations.

	Acknowledgments

 
We thank T. A. Nguyen, S. Lim, and P. Chu for their technical assistance.

	Footnotes

 
¹ This work was supported by Public Health Service Grants AI47730 and AI04717, and a grant from Universitywide AIDS Research Program, University of California (R00-PAM-015).

² Current address: Department of Internal Medicine, Ageo Kosei Hospital, 421-1 Jitoukata, Ageo-Shi, Saitama 362-0051, Japan.

³ Current address: Klinikum Benjamin Franklin der Freie Universitat Berlin, Abteilung für Medizinische Mikrobiologie und Infektionsimmunologie, Institut für Infectionsmedizin, Hindenburgdamm 27, 12203 Berlin, Germany.

⁴ Address correspondence and reprint requests to Dr. Yasuhiro Suzuki, Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Prices Fork Road, Blacksburg, VA 24061. E-mail address: ysuzuki{at}vt.edu

⁵ Abbreviation used in this paper: TE, toxoplasmic encephalitis.

Received for publication September 17, 2002. Accepted for publication February 5, 2003.

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