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Objective: To investigate the effect of excreted/secreted antigens (ESAs) from Toxoplasma gondii RH strain and TgCtwh3 strain on apoptosis of CD4+CD25+ regulatory T cells and interferon-γ (IFN-γ) secretion., Methods: ESAs of Toxoplasma gondii RH strain and TgCtwh3 strain were prepared. Splenic mononuclear cells were isolated from C57BL/6 mice and randomly divided into RH ESA group（2×106 cells/well with addition of 10 μg/ml RH ESA）, TgCtwh3 ESA group （2×106 cells/well with addition of 10 μg/ml TgCtwh3 ESA） and control group（2×106 cells/well with addition of 10 μg/ml ovalbumin）. Flow cytometry was performed to examine the early apoptosis of CD4+CD25+ regulatory T cells after treatment for 48 h and 72 h. ELISA was conducted to determine the level of IFN-γ in the supernatant after treatment for 72 h. In another experiment, the 3 groups of splenic mononuclear cells were added with 10 μg/ml anti-IFN-γ antibody for 72 h and flow cytometry was performed to examine the early apoptosis of CD4+CD25+ regulatory T cells. Meanwhile, splenic mononuclear cells from IFN-γ knockout and wild-type C57BL/6 mice were also divided into the above-described 3 groups, and flow cytometry was performed to examine the early apoptosis of CD4+CD25+ regulatory T cells after treatment for 72 h., Results: The concentrations of RH ESA and TgCtwh3 ESA were 0.54 mg/ml and 2.14 mg/ml, respectively. Flow cytometry showed that the early apoptosis rate of CD4+CD25+ regulatory T cells in the RH ESA group and the TgCtwh3 ESA group after 48 h treatment was （12.90±1.26）% and （9.71±1.04）%, respectively （P＜0.05）, both significantly higher than that in control group （4.48±0.48）% （P<0.01） . The early apoptosis rate of CD4+CD25+ regulatory T cells after 72 h in the RH ESA group was（15.21±1.11）%, significantly higher than that in the TgCtwh3 ESA group[（11.02±0.92）%] （P<0.05） and the control group[（10.10±1.49）%]（P<0.01）. ELISA showed that the level of interferon-γ in the RH ESA group and the TgCtwh3 ESA group after 72 h was（4 764.0±118.7） pg/ml and （3 629.0±33.6） pg/ml, respectively （P＜0.01）, both significantly higher than that in the control［（679.4±30.6） pg/ml］（P<0.01）. Flow cytometry revealed lower early apoptosis rate of CD4+CD25+ regulatory T cells in the RH ESA group added with anti-IFN-γ antibody［（10.44±1.44）%］ compared with that without the addition of the antibody［（14.96±0.83）］（P<0.05）. But this difference was not observed for the TgCtwh3 ESA group. Moreover, the RH ESA-induced apoptosis rate of regulatory T cells from IFN-γ knockout mice［（10.64±0.55）%］ was significantly lower than that from the wild-type mice ［（15.21±1.11）%］（P<0.01）. But this difference was not found for the TgCtwh3 ESA treatment., Conclusion: T. gondii RH ESA induces apoptosis of CD4+CD25+ regulatory T cells and IFN-γ secretion, and these effects are stronger than those of TgCtwh3 ESA. The T. gondii ESA-induced IFN-γ stimulates generation of anti-Toxoplasma immunity during acute Toxoplasma infection through mediation of regulatory T cell apoptosis.

The proteomic techniques have been widely used in Toxoplasma gondii research since the past decade, providing proteomic data that facilitate understanding of T. gondii activities. Currently, the global proteomic studies of T. gondii are mainly confined to the tachyzoite and the oocyst stages. The subproteomic research involves some important antigens, such as the soluble tachyzoite antigen, glycoproteins, and immunoproteins, etc. The differential proteomics of T. gondii is mainly focused on identifying differentially-expressed proteins in different T. gondii strains. This review summarizes the current status of proteomic research on T. gondii, with specific focuses on global proteomic, subproteomic, and differential proteomic findings. In addition, this review gives an overview on web-based resources that provide proteomic data and support for studies on T. gondii, and finally discusses future prospects of T. gondii proteomics applications.