Granule-dependent killing of Toxoplasma gondii by CD8+ T cells

Abstract

Immunization of mice with live bradyzoites of a low-virulent Beverley strain of Toxoplasma gondii has been shown to increase CD8+ T-cell mediated immunity against a highly virulent RH strain. We found that preimmunization with an RH homogenate further enhanced this immunity. Using this model, we investigated the mechanism of CD8+ T-cell mediated protection against T. gondii infection. Splenic cells from mice immunized with RH homogenate and live bradyzoites stimulated apoptosis of RH-infected J774A.1 macrophages in vitro, and at the same time, the immunization significantly suppressed the proliferation of parasites within macrophages, as assessed by measuring 3H-uracil uptake by the parasites. Splenic cells from the immunized mice produced larger amounts of interferon-gamma (IFN-gamma) than did naive splenic cells; however, the production of nitric oxide (NO) by RH-infected macrophages was not enhanced. The elimination of CD8+ T cells from splenic cells significantly reduced their inhibitory action on parasite proliferation as well as their cytotoxic activity against RH-infected macrophages, but it did not affect the production of IFN-gamma. Treatment of CD8+ T-enriched splenic cells from the immunized mice with concanamycin A, but not an anti-Fas ligand monoclonal antibody, significantly reduced their anti-proliferative and killing capabilities, suggesting that the CD8+ T cells induced by immunization with RH antigen and live bradyzoites of the Beverley strain may exert protection against T. gondii infection at least in part through granule-dependent cytotoxic activities.

Figure 1

Cytotoxic activity of splenic cells from mice immunized with RH homogenate and live bradyzoites of the Beverley strain against RH-infected macrophages. Splenic cells from naive mice (a, b) or mice immunized with RH homogenate and live bradyzoites (a, b) or live parasites alone (b) were restimulated with irradiated RH for 6 days in vitro. These cells were collected and mixed with target cells, ³H-labelled J774A.1 macrophages infected with the RH strain (non-immunized cells, open circles; immunized splenic cells, closed circles) at various effector : target cell ratios. The immunized splenic cells were depleted of CD4⁺ T cells or CD8⁺ T cells using immunomagnetic beads after restimulation and mixed with target cells (depletion of CD4⁺ T cells, closed triangles; depletion of CD8⁺ T cells, closed squares). After 4·5 hr incubation, % specific killing of target cells was calculated. Each value is the mean± SD of triplicate cultures. *P < 0·01, **P < 0·001 by Student's t-test, compared with non-immunized splenic cells. #P < 0·05 by Student's t-test, compared with immunized splenic control cells. Similar results were obtained from five separate experiments. (b) As the effector cells, CD8⁺ T-cell enriched splenic cells obtained by depletion of CD4⁺ T cells from mice immunized with live parasites alone or immunized with parasite homogenate plus live parasites after in vitro stimulation were used for the experiment (non-immunized cells, open circles; cells immunized with live bradyzoites, closed triangle; cells immunized with parasite homogenate and live parasites, closed circles). *P < 0·01, **P < 0·001 by Student's t-test, compared with non-immunized splenic cells. #P < 0·05 by Student's t-test, compared with cells from mice immunized with live parasites alone. Similar results were obtained from three separate experiments.

Figure 2

Anti-parasitic activity of splenic cells from mice immunized with parasite homogenate and live bradyzoites of the Beverley strain. Splenic cells from mice immunized with RH homogenate plus live parasites (a, b) or live parasites alone (b) and splenic cells from naive mice (a, b) were restimulated with irradiated RH strain for 6 days in vitro. These cells were collected and mixed with RH-infected J774A.1 macrophages (non-immunized cells, open circles; immunized splenic cells, closed circles). The immunized splenic cells were depleted of CD4⁺ T or CD8⁺ T cells using immunomagnetic beads after restimulation and mixed with target cells (depletion of CD4⁺ T cells, closed triangles; depletion of CD8⁺ T cells, open squares) at various effector : target cell ratios. After 4·5 hr incubation, ³H-uracil (0·5 µCi/well) was added to each well, and the cells were further incubated for 14 hr. Then the uptake of ³H-uracil by parasites was measured. Each value is the mean ± SD of triplicate cultures. *P < 0·01 by Student's t-test, compared with non-immunized splenic cells. #P < 0·01 by Student's t-test, compared with immunized splenic control cells. Similar results were obtained from five separate experiments. (b) As the effector cells, CD8⁺ T-enriched splenic cells obtained by depletion of CD4⁺ T cells from mice immunized with live parasites alone or immunized with parasite homogenate plus live parasites after in vitro stimulation were used for the experiment (non-immunized cells, open circles; cells immunized with live bradyzoites alone, closed triangle; cells immunized with parasite homogenate and live parasites, closed circles). *P < 0·05, **P < 0·01 by Student's t-test, compared with non-immunized splenic cells. #P < 0·05, #P < 0·01 by Student's t-test, compared with cells from mice immunized with live parasites alone. Similar results were obtained from three separate experiments.

Figure 3

Production of IFN-γ. Splenic cells from naive mice or mice immunized with RH homogenate and live bradyzoites were restimulated with irradiated RH strain for 6 days in vitro. These cells were collected and mixed with RH-infected J774A.1 macrophages at the effector : target cell ratio of 25 : 1. The immunized splenic cells were depleted of CD4⁺ T cells or CD8⁺ T cells using immunomagnetic beads after restimulation and incubated with RH-infected J774A.1 macrophages. After 20 hr incubation, the concentration of IFN-γ protein in the culture supernatant was measured by ELISA. Each value is the mean ± SD of triplicate cultures. Similar results were obtained from three separate experiments. *P < 0·05 by Student's t-test, compared with non-immune splenic cells. #P < 0·05 by Student's t-test, compared with immune splenic control cells.

Figure 4

Effects of L-NMMA and anti-IFN-γ mAb on CD8⁺ T cell-enriched splenic cells. Splenic cells from mice immunized with RH homogenate and live bradyzoites were restimulated with irradiated RH for 6 days in vitro, and then the cells were collected and depleted of CD4⁺ T cells using immunomagnetic beads. L-NMMA or anti-IFN-γ mAb was added to the mixture of these CD8⁺ T cell-enriched splenic cells and target cells, J774A.1 macrophages infected with the RH strain, at the effector : target cell ratio of 25 : 1. After 4·5 hr incubation, ³H-uracil (0·5 µCi/well) was added to each well, and the cells were further incubated for 14 hr. Then the uptake of ³H-uracil by parasites was measured. Each value is the mean ± SD of triplicate cultures. *P < 0·05 by Student's t-test, compared with non-immunized splenic cells.

Figure 5

Effects of CMA and anti-FasL on CD8⁺ T cell-enriched splenic cells. Splenic cells from mice immunized with RH homogenate and live bradyzoites were restimulated with irradiated RH for 6 days in vitro, and then the cells were collected and depleted of CD4⁺ T cells using immunomagnetic beads. These CD8⁺ T -enriched splenic cells were treated with CMA or anti-FasL mAb before incubation with target cells, ³H-labelled J774A.1 macrophages infected with the RH strain, at the effector : target cell ratio of 25 : 1. After 4·5 hr incubation, % specific killing of target cells was calculated (a). CD8⁺ T cell-enriched splenic cells treated with CMA or anti-FasL mAb were incubated with RH-infected J774A.1 macrophages at the effector : target cell ratio of 25 : 1. After 4·5 hr incubation, ³H-uracil (0·5 µCi/well) was added to each well, and the cells were further incubated for 14 hr. Then the uptake of ³H-uracil by parasites was measured (b). Each value is the mean± SD of triplicate cultures. *P < 0·05 by Student's t-test, compared with untreated CD8⁺ T cells-enriched cells. #P < 0·05 by Student's t-test, compared with non-immunized splenic cells.