Determination of a Key Antigen for Immunological Intervention To Target the Latent Stage of Toxoplasma gondii

Abstract

Toxoplasma gondii, an obligate intracellular protozoan parasite, establishes a chronic infection by forming cysts preferentially in the brain. Up to one third of the human population worldwide is estimated to be chronically infected with this parasite. However, there is currently no drug effective against the cyst form of the parasite. In addition, the protective immunity against the cysts remains largely unknown. We analyzed the molecular mechanisms by which the immune system detects host cells harboring the cysts to eliminate the latent stage of the parasite using mice with the H-2^d haplotype, which are genetically resistant to the infection. Our study revealed that CD8⁺ immune T cells bearing TCR Vβ8.1, 8.2 chain have a potent activity to remove T. gondii cysts from the brain. Our studies also uncovered that H-2L^d is the major Ag-presenting molecule to CD8⁺ T cells for initiating cyst elimination, and that CD8⁺Vβ8.1, 8.2⁺ immune T cells recognize the N-terminal region (aa 41-152) of dense granule protein 6 (GRA6Nt) of the parasite presented by the H-2L^d molecule. Furthermore, CD8⁺ immune T cells induced by immunization with recombinant GRA6Nt were eventually capable of removing the cysts from the brain when transferred to infected immunodeficient mice lacking T cells. Thus, GRA6Nt is a novel and potent Ag to activate CD8⁺ T cells capable of removing T. gondii cysts. These observations offer a basis for immunological intervention to combat chronic infection with T. gondii by targeting the persistent cysts of the parasite.

FIGURE 1

CD8⁺ T cells bearing TCR Vβ8.1, 8.2 chain are most abundant in the brains of T. gondii-infected SCID mice following a systemic transfer of CD8⁺ T cells, and this T cell population is capable of removing T. gondii cysts from the brains of the recipient SCID mice. (A) SCID mice were infected and treated with sulfadiazine beginning at 10 days after infection to establish a chronic infection by forming cysts in their brains. The animals received CD8⁺ immune T cells (6.7 × 10⁶ cells) purified from the spleens of chronically infected BALB/c mice intravenously at 3 weeks after infection. Seven days later, TCR Vβ usage of the T cells that infiltrated into their brains were examined by flow cytometry. (B) Frequencies of perforin-expressing cells in each of CD8⁺Vβ8.1, 8.2⁺ T cell population and a total population of CD8⁺Vβ8.1, 8.2⁻ T cells. **P<0.01 by Student’s t test. (C–F) Infected and sulfadiazine-treated SCID mice received CD8⁺Vβ8.1, 8.2⁺ immune T cells (0.9 × 10⁶ cells) at 3 weeks after infection. (C) Numbers of T. gondii cysts, (D) amounts of mRNA for bradyzoite-specific BAG1, (E) amounts of mRNA for perforin, and (F) amounts of mRNA for granzyme B in their brains were examined on the day (Day 0) and 12 days after the cell transfer (Day 12) (n=4 or 6). Data represent the mean ± SEM. (C) *Pc<0.05 between the controls without the T cell transfer (Day 0) vs. the experimental group with the T cell transfer (Day 12), and between the controls without the T cell transfer (Day 12) vs. the experimental group with the T cell transfer (Day 12) by Mann-Whitney test. (D–F), *Pc<0.05 and **Pc<0.01 by Mann-Whitney test.

FIGURE 2

CD8⁺Vβ8.1, 8.2⁺ immune T cells secrete perforin and granzyme B by recognizing rGRA6Nt of T. gondii. (A and C) Perforin and (B and D) granzyme B levels of the culture supernatants of CD8⁺Vβ8.1, 8.2⁺ T hybridoma (5 × 10⁴ cells/well) following stimulation with (A and B) previously identified epitopes of T. gondii antigens (epitope HPGSVNEFDF [HF10] of GRA6, epitope SPMNGGYYM [SM9] of GRA4, epitope IPAAAGRFF [IF9] of ROP7, epitope TPTENHFHL [SP0534] of SAG1 and epitope YPESGPVNL [SP0536] of SAG3 at 1 μg/ml) (–24) (n=3), or (C and D) rGRA6Nt, rGRA6Ct, rGRA2 at 1 μg/ml, or a total lysate antigens of tachyzoites (TLA) at 20 μg/ml in the presence of antigen-presenting cells (APC) from uninfected wild-type BALB/c mice (n=3). (E) Perforin and (F) granzyme B levels of the culture supernatants of the primary CD8⁺Vβ8.1, 8.2⁺ immune T cells (1 × 10⁵ cells/well) purified from the spleens of infected BALB/c mice following stimulation with rGRA6Nt, rGRA6Ct (1 μg/ml), or TLA (20 μg/ml) in the presence of antigen-presenting cells (APC) from uninfected wild-type BALB/c mice (n=3 except for rGRA6Ct [n=2]). (G) Perforin and (H) granzyme B levels of the culture supernatants of the primary CD8⁺Vβ8.1, 8.2⁺ immune T cells and a total population of CD8⁺Vβ8.1, 8.2⁻ T cells (1 × 10⁵ cells/well) purified from the spleens of infected BALB/c mice following stimulation with rGRA6Nt (n=3). Data represent the mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 by one-way ANOVA with Newman-Keuls Post test. In Fig. 2F, granzyme B levels in the culture with TLA were low when compered to those in the culture with rGRA6Nt but they were 14.5 times greater than those in the control culture with PBS (P<0.001 by Student’s t test).

FIGURE 3

The H-2L^d molecule presents T. gondii antigens including rGRA6Nt to CD8⁺Vβ8.1, 8.2⁺ immune T cells. (A) Perforin and (B) granzyme B levels in the culture supernatants of the CD8⁺Vβ8.1, 8.2⁺ T hybridoma (5 × 10⁴ cells/well) following stimulation with rGRA6Nt (1 μg/ml) in the presence of antigen-presenting cells (APC) from uninfected wild-type BALB/c or dm2 mice (n=3). (C) Perforin and (D) granzyme B levels in the culture supernatants of the primary CD8⁺Vβ8.1, 8.2⁺ T cells (1 × 10⁵ cells/well) purified from the spleens of infected BALB/c mice following stimulation with tachyzoite-infected J774 macrophage cell line in the presence of anti-H-2L^d mAb (25 μg/ml) or isotype control Ab (n=3). Data represent the mean ± SEM. (A) ***P<0.0001 by Student’s t test. (B) ***P=0.0004 by Student’s t test. (C and D) *P<0.05, **P<0.01 and ***P<0.001 by one-way ANOVA with Newman-Keuls Post test.

FIGURE 4

The H-2L^d molecule presents T. gondii antigens to CD8⁺ immune T cells for removing cysts of the parasite from the brain. SCID and dm2 mice were infected and treated with sulfadiazine beginning at 7 or 10 days after infection to establish a chronic infection by forming cysts in their brains. The animals received CD8⁺ immune T cells purified from the spleens of chronically infected BALB/c mice intravenously at 3 weeks after infection. (A) Numbers of T. gondii cysts, (B) amounts of mRNA for bradyzoite-specific BAG1, (C) amounts of mRNA for perforin, and (D) amounts of mRNA for granzyme B in their brains were examined on the day (Day 0) and 7 days after the cell transfer (Day 7). Data represent the mean ± SEM. (A) The results from two independent experiments with a transfer of 4.2 × 10⁶ or 5.7 × 10⁶ CD8⁺ immune T cells are combined (n=7–10). **Pc<0.01 and ***Pc<0.001 by Mann-Whitney test. (B–D) The results from one of the two experiments are shown (n=4). *P<0.05 by Mann-Whitney test.

FIGURE 5

CD8⁺ immune T cells induced by immunization with rGRA6Nt-primed BM-DC remove T. gondii cysts from the brains of infected SCID mice. SCID mice were infected and treated with sulfadiazine beginning at 10 days after infection to establish a chronic infection by forming cysts in their brains. The animals received CD8⁺ immune T cells from uninfected BALB/c mice, which had received an immunization with BM-DC pre-cultured with and without rGRA6Nt, at 3 weeks after infection. (A) Numbers of T. gondii cysts and (B) amounts of mRNA for bradyzoite-specific BAG1 in their brains were examined at one day before (Day −1) and 7 days after (Day 7) the cell transfer. The data from two independent experiments with a transfer of CD8⁺ immune T cells (2.7–3.3 × 10⁶) are combined (n=8–10). (C) Perforin and (D) granzyme B levels of the culture supernatants of the spleen cells of the recipient SCID mice at 7 days after receiving the CD8⁺ immune T cells. There were 4–5 mice in each experimental group, and the culture was performed using pooled cells for each group (5 wells for each group). Data represent the mean ± SEM. (A) **Pc<0.01 by Mann-Whitney test. (B) *Pc<0.05 between the control group without the T cell transfer (Day 7) and the experimental groups with the T cell transfer from donors immunized with GRA6Nt-primed BM-Dc (Day 7), and between the control groups without the T cell transfer on Day -1 and Day 7 by Mann-Whitney test. (C and D) **P<0.01 by one-way ANOVA with Newman-Keuls Post test.