Removal of Toxoplasma gondii cysts from the brain by perforin-mediated activity of CD8+ T cells

Abstract

Chronic infection with Toxoplasma gondii is one of the most common parasitic infections in humans. Formation of tissue cysts is the basis of persistence of the parasite in infected hosts, and this cyst stage has generally been regarded as untouchable. Here we provide the first evidence that the immune system can eliminate T. gondii cysts from the brains of infected hosts when immune T cells are transferred into infected immunodeficient animals that have already developed large numbers of cysts. This T cell-mediated immune process was associated with accumulation of microglia and macrophages around tissue cysts. CD8(+) immune T cells possess a potent activity to remove the cysts. The initiation of this process by CD8(+) T cells does not require their production of interferon-gamma, the major mediator to prevent proliferation of tachyzoites during acute infection, but does require perforin. These results suggest that CD8(+) T cells induce elimination of T. gondii cysts through their perforin-mediated cytotoxic activity. Our findings provide a new mechanism of the immune system to fight against chronic infection with T. gondii and suggest a possibility of developing a novel vaccine to eliminate cysts from patients with chronic infection and to prevent the establishment of chronic infection after a newly acquired infection.

Figure 1

Immune spleen cells and T cells have the ability to reduce the numbers of cysts in the brains of recipient nude mice and the anti-cyst activity of immune T cells is associated with an accumulation of mononuclear cells on T. gondii cysts. Athymic nude mice were infected with 20 cysts of the ME49 strain perorally and treated with sulfadiazine beginning at 7 days after infection for the entire period of the observation. The animals received 2 × 10⁷ of immune spleen cells (A) or 0.7 to 1 × 10⁷ of immune T cells (C−G) obtained from chronically infected BALB/c mice at 3 and 4 weeks after infection. A: Numbers of cysts in the brains of infected nude mice at 1 day before the first cell transfer (Day −1) and 1 month after the second cell transfer (Day 39). As an additional control, euthymic BALB/c mice were infected, and numbers of cysts in their brains were examined at Day 39 without cell transfer. A half brain of each mouse was suspended in 0.5 ml of PBS and numbers of cysts in 60 μl (3 samples of 20 μl each) of the brain suspensions were counted microscopically. The data represent means ± SD. *P < 0.05 compared with control nude mice without cell transfer, Day −1 and Day 39. B: Representative cyst detected at Day −1. Each cyst detected had a typical cyst wall (arrow). C: Numbers of cysts in the brains of infected nude mice at one day after the first cell transfer (Day 1) and 1 month after the second cell transfer (Day 36). The data represent means ± SD. The data shown are representative of four independent experiments. **P < 0.01 compared with control nude mice without cell transfer, Day 1 and Day 36. D: Reverse transcription-PCR detecting mRNA for bradyzoite-specific BAG1 at Day 39. E−G: Accumulation of mononuclear cells on and within T. gondii cysts in the brains of infected nude mice at 2 days after a transfer of immune T cells (0.7 × 10⁷ cells) (n = 3). E and F: H&E stain. Scale bar = 50 μm. G: Immunostaining for BAG1 to detect bradyzoites (some are highlighted with arrows). H: A representative of T. gondii cysts observed in the brains of nude mice that had received normal T cells (n = 2) or those that had not received any T cells (n = 2) (H&E stain). At least five sections (25 μm or more distance between sections) were examined on each brain sample.

Figure 2

CD8⁺ T cells remove T. gondii cysts from the brains of recipient nude mice and their protective activity does not require IFN-γ but requires perforin. A: Numbers of T. gondii cysts and (B) the amounts of BAG1 mRNA in the brains of infected, sulfadiazine-treated SCID mice at 1 day before and 7 days after a transfer of normal or immune CD8⁺ T cells (3.5 × 10⁶ cells) from uninfected or chronically infected BALB/c mice. The cell transfer was performed at 3 weeks after infection. C: Numbers of T. gondii cysts and (D) the amounts of BAG1 mRNA in the brains of infected, sulfadiazine-treated SCID mice at 1 and 7 days after a transfer of CD8⁺ immune T cells (2.1 × 10⁶ cells) from infected IFN-γ^−/− or wild-type BALB/c mice. E: Numbers of T. gondii cysts and (F) the amounts of BAG1 mRNA in the brains of infected, sulfadiazine-treated SCID mice at 1 day before and 7 days after a transfer of CD8⁺ immune T cells (2.1 × 10⁶ cells) from infected perforin-deficient (PO) or wild-type BALB/c mice. All data represent mean ± SD. The data shown in (E) are representative of two independent experiments. *P < 0.005, **P < 0.0005, ***P = 0.0001, ****P < 0.0001 compared with control nude mice without cell transfer, Day 7.