Immunization with the amino-terminus region of dense granule protein 6 (GRA6) of Toxoplasma gondii activates CD8+ cytotoxic T cells capable of removing tissue cysts of the parasite through antigen presentation by human HLA-A2.1

Abstract

CD8⁺ T cells from HLA-A2.1-transgenic mice, but not wild-type mice, immunized with the amino-terminus region (aa 41-152) of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii secreted large amounts of perforin and granzyme B in response to GRA6Nt through antigen presentation by HLA-A2.1 in vitro. When those CD8⁺ T cells were transferred into chronically infected HLA-A2.1-expressing NSG mice deficient in T cells, cerebral cyst burden of the recipients of HLA-A2.1-transgenic T cells, but not of WT T cells, became significantly less than that of control mice with no cell transfer. Furthermore, the significant reduction of the cyst burden by a transfer of the HLA-A2.1-transgenic CD8⁺ immune T cells required an expression of HLA-A2.1 in the recipient NSG mice. Thus, antigen presentation of GRA6Nt by human HLA-A2.1is able to activate anti-cyst CD8⁺ T cells that eliminate T. gondii cysts through antigen presentation by human HLA-A2.1.

Keywords: CD8(+) T cells; Dense granule protein 6; HLA-A2; Protective immunity; Tissue cysts; Toxoplasma.

Fig. 1.. Immunization with rGRA6Nt of T. gondii activates CD8⁺ cytotoxic T cells to this antigen through antigen presentation by human HLA-A2.1 molecule.

Transgenic mice expressing human HLA-A2.1 and WT mice were immunized intraperitoneally with 50 μg of rGRA6Nt three times with 4-week intervals. Two weeks after the third immunization, CD8⁺ T cells were purified from their spleens and cultured in 96 well-culture plates (3 × 10⁵ cells/well) with or without rGRA6Nt (5 μg/ml) for 72 h in the presence of splenic innate immune cells (1.5 × 10⁵ cells/well) from infected, sulfadiazine-treated immunodeficient NSG mice (deficient in T, B, and NK cells) expressing the HLA-A2.1 as APCs. CD8⁺ immune T cells purified from 3 mice were pooled for the cultures. There were 5 wells in each experimental group. The concentrations of perforin (A) and GzmB (B) in the culture supernatants were measured by ELISA. The results presented are from one experiment. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.. CD8⁺ immune T cells activated through antigen presentation of rGRA6Nt of T. gondii by human HLA-A2.1 molecule have the capability to remove tissue cysts of the parasite.

The HLA-A2.1-transgenic and WT mice were immunized intraperitoneally with 50 μg of rGRA6Nt twice (panel E) or three times (panels A-D) with 4-week intervals. Two weeks after the final immunization, CD8⁺ T cells were purified from their spleens and injected intravenously into infected HLA-2.1-NSG mice (2 × 10⁶ cells/mouse) from a tail vein at 4 weeks after infection. An additional group of the HLA-A2.1-NSG mice did not receive any T cells as a control. These NSG mice were under treatment with sulfadiazine beginning at 5 days after infection to establish a chronic infection and maintain the parasite as the cysts until the end of the study. Seven days after the T cell transfer, the brains of the HLA-A2.1-NSG mice were applied for quantification of mRNA levels for bradyzoite (cyst)-specific BAG1 (A and E), CD8β (B), perforin (C), and GzmB (D) by real time RT-PCR. The results from three (panels A-D) or two (panel E) independent experiments are combined. Number of mice in each experimental group was 10–12 mice (panels A-D) and 7 or 8 mice (panel E). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.. T. gondii cyst elimination by adoptive transfer of CD8⁺ T cells from rGRA6Nt-immunized HLA-A2.1-transgenic mice requires an expression of the HLA-A2.1 in the recipients.

HLA-A2.1-transgenic mice were immunized intraperitoneally with 50 μg of rGRA6Nt twice with a 4-week interval. Two weeks after the second immunization, CD8⁺ T cells (2 × 10⁶ cells) purified from their spleens were injected intravenously into infected HLA-2.1-NSG mice and control NSG mice not expressing the HLA-A2.1 from a tail vein at 4 weeks after infection in the recipients. An additional group of the HLA-A2.1-NSG and control NSG mice did not receive any T cells as a control. These NSG mice were under treatment with sulfadiazine beginning at 5 days after infection to establish a chronic infection and maintain the parasite as the cysts until the end of the study. Seven days after the CD8⁺ T cell transfer, the brains of the recipient HLA-A2.1-NSG and control NSG mice were applied for quantification of mRNA levels for bradyzoite (cyst)-specific molecules, BAG1 (A), CD8β (B), Prf1 (C), and GzmB (D) by real time RT-PCR. Number of mice in each experimental group was 4 mice. *P < 0.05, **P < 0.01, ***P < 0.001.