Gamma interferon production, but not perforin-mediated cytolytic activity, of T cells is required for prevention of toxoplasmic encephalitis in BALB/c mice genetically resistant to the disease

Abstract

We previously showed the requirement of both T cells and gamma interferon (IFN-gamma)-producing non-T cells for the genetic resistance of BALB/c mice to the development of toxoplasmic encephalitis (TE). In order to define the role of IFN-gamma production and the perforin-mediated cytotoxicity of T cells in this resistance, we obtained immune T cells from spleens of infected IFN-gamma knockout (IFN-gamma(-/-)), perforin knockout (PO), and wild-type BALB/c mice and transferred them into infected and sulfadiazine-treated athymic nude mice, which lack T cells but have IFN-gamma-producing non-T cells. Control nude mice that had not received any T cells developed severe TE and died after discontinuation of sulfadiazine treatment due to the reactivation of infection. Animals that had received immune T cells from either wild-type or PO mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-gamma(-/-) mice developed severe TE and died as early as control nude mice. T cells obtained from the spleens of animals that had received either PO or wild-type T cells produced large amounts of IFN-gamma after stimulation with Toxoplasma gondii antigens in vitro. In addition, the amounts of IFN-gamma mRNA expressed in the brains of PO T-cell recipients did not differ from those in wild-type T-cell recipients. Furthermore, PO mice did not develop TE after infection, and their IFN-gamma production was equivalent to or higher than that of wild-type animals. These results indicate that IFN-gamma production, but not perforin-mediated cytotoxic activity, by T cells is required for the prevention of TE in genetically resistant BALB/c mice.

FIG. 1.

Mortality (A) and development of TE (B) in T. gondii-infected, sulfadiazine-treated athymic nude mice with adoptive transfer of immune T cells from infected IFN-γ^−/− or wild-type BALB/c mice. Athymic nude mice were infected with 10 cysts of the ME49 strain perorally and were treated with sulfadiazine for 3 weeks, beginning 7 days after infection. Nine and 2 days before the discontinuation of sulfadiazine treatment, mice received an intravenous injection of 10⁷ immune T cells from infected wild-type or IFN-γ^−/− mice (see Materials and Methods). Histological studies were performed 5 or 6 days after the discontinuation of treatment with sulfadiazine. Two to four sagittal sections (distance between sections, 50 μm) were stained with an immunoperoxidase stain by using rabbit anti-T. gondii IgG and were 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 are shown in the figure and were used for statistical analysis to compare differences between groups. The data shown are representative of two separate experiments. There were four to five mice in each experimental group in each experiment.

FIG. 2.

Histological changes in brains of T. gondii-infected, sulfadiazine-treated athymic nude mice with adoptive transfer of immune T cells from infected IFN-γ^−/− (A) or PO (B) mice. Athymic nude mice were infected with 10 cysts of the ME49 strain perorally and were treated with sulfadiazine for 3 weeks, beginning 7 days after infection. Nine and 2 days before the discontinuation of sulfadiazine treatment, mice received an intravenous injection of 10⁷ immune T cells from the donor animals (see Materials and Methods). Histological studies were performed 6 days (the recipients of IFN-γ^−/− T cells) or 90 days (the recipients of PO T cells) after the discontinuation of treatment with sulfadiazine. Sections were stained with hematoxylin and eosin. The experiments were performed twice, and there were four to six mice in each group in each experiment.

FIG. 3.

Mortality (A) and development of TE (B) in T. gondii-infected, sulfadiazine-treated athymic nude mice with adoptive transfer of immune T cells from infected PO or wild-type BALB/c mice. Athymic nude mice were infected with 10 cysts of the ME49 strain perorally and were treated with sulfadiazine for 3 weeks, beginning 7 days after infection. Nine and 2 days before the discontinuation of sulfadiazine treatment, mice received an intravenous injection of 10⁷ immune T cells from infected wild-type or PO mice (see Materials and Methods). Histological studies were performed 90 days after the discontinuation of treatment with sulfadiazine. For control animals without cell transfer, the histological studies were done 6 or 7 days after discontinuation of the treatment. Two to four sagittal sections (distance between sections, 50 μm) were stained with an immunoperoxidase stain by using rabbit anti-Toxoplasma IgG and were 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 are shown in the figure and were used for statistical analysis to compare differences between groups. The data shown are representative of two separate experiments. There were four to six mice in each experimental group in each experiment.

FIG. 4.

Production of IFN-γ by T cells purified from spleens of nude mice that had received immune T cells from PO or wild-type BALB/c mice. Athymic nude mice were infected with 10 cysts of the ME49 strain perorally and were treated with sulfadiazine for 3 weeks, beginning 7 days after infection. Nine and 2 days before the discontinuation of sulfadiazine treatment, mice received an intravenous injection of 10⁷ immune T cells from infected wild-type or PO mice (see Materials and Methods). Ninety days after the discontinuation of sulfadiazine, T cells were purified from the spleens of the recipient animals and then stimulated with tachyzoite lysate Ags (20 μl/ml) in the presence of antigen-presenting cells (plastic-adherent cells from spleens of normal BALB/c mice) (see Materials and Methods). The experiment shows means ± standard deviations of triplicate cultures. The data shown are representative of two separate experiments.

FIG. 5.

Expression of IFN-γ mRNA in brains of T. gondii-infected, sulfadiazine-treated nude mice after adoptive transfer of immune T cells from infected PO or wild-type BALB/c mice. Athymic nude mice were infected with 10 cysts of the ME49 strain perorally and were treated with sulfadiazine for 3 weeks, beginning 7 days after infection. Nine and 2 days before the discontinuation of sulfadiazine treatment, mice received an intravenous injection of 10⁷ immune T cells from donor animals (see Materials and Methods). Ninety days after the discontinuation of sulfadiazine treatment, their brains were analyzed for the amounts of mRNAs for β-actin and IFN-γ (see Materials and Methods). NC, negative control; PC, positive control. There were three or four mice in each experimental group.

FIG. 6.

Mortality (A) and production of IFN-γ by T and spleen cells (B) in infected PO and wild-type BALB/c mice. Mice were infected with 10 cysts of the ME49 strain perorally. T cells were purified from the spleens of the animals 6 months after infection and then stimulated with tachyzoite lysate Ags (20 μl/ml) in the presence of antigen-presenting cells (plastic-adherent cells from spleens of normal BALB/c mice) (see Materials and Methods). Total spleen cells were also stimulated with the lysate Ags. The experiment shows means ± standard deviations of triplicate cultures. The data shown are representative of two separate experiments.