Gondii ESA-injection at G10 exhibited decreased number of Foxp3+ cells, but that of mice with T. gondii ESA-injection at G15 presented increased number of Foxp3+ cells, as compared with the control groups. These data provided evidence that the injection with T. gondii ESA at G10 could lead to diminished number of Tregs, but the injection at G15 resulted in the increased number of Tregs at the maternal-fetal interface.Figure 2. Effects of T. gondii ESA on the proportion and function of CD4+CD25+Foxp3+ T cells at different stages of pregnancy. All animals were killed at G18, 10457188 and their spleens, inguinal LN, and PBL were obtained. Lymphocytes from these tissues were 24195657 prepared and pooled as described in Materials and Methods. The cells were stained with CD4-FITC, CD25-APC and PE-Foxp3 Abs, respectively, and analyzed by flow cytometry. (A)Representative dot plots illustrating the regions and gating for the Madrasin site capture of cell phenotype data and intracellular Foxp3 expression. (B) Percentages and absolute number of CD4+CD25+Foxp3+-cells from spleens. (C) Percentages of CD4+CD25+Foxp3+-cells from inguinal LN, and PBL. (D) Responder CD4+CD25?T cells (16105/well) from naive mice were cultured with naive, Tetracosactide web irradiated APC (16105 cells/well) and CD4+CD25+T cells (56104 cells/well) harvested from mice with PBS or T. gondii ESA injection at G5, G10, G15, respectively. (E and F) The serum levels of IFN-c and IL-4 in pregnant mice injected with T. gondii ESA by ELISA. Data represent means 6 SD from groups of seven mice assayed individually. Statistical differences between groups are shown as follows: * p,0.05; ** p,0.01; *** p,0.001; # p.0.05. doi:10.1371/journal.pone.0069012.gT. gondii ESA Induced Tregs DysfunctionFigure 3. Foxp3 mRNA and protein levels at the maternal-fetal interface of mice with T. gondii ESA injection at G10 and G15. (A) Foxp3 expression levels in placentas from T. gondii ESA-injected and PBS-injected mice measured by real-time quantitative PCR. The data were normalized to individual b-actin mRNA expression and expressed as fold change relative to control mice. Data represent means 6 SD from groups of seven mice assayed individually. (B)Top panel, Foxp3 protein was analyzed by Western blot after the injection at G10 or G15 as indicated. Bottom panel, densitometric analysis of Foxp3 expression was conducted by Western blot. One representative result of three independent experiments performed is shown. (C) The distribution of Foxp3+-cells in the placentas of T. gondii ESA-injected and PBS-injected mice as determined by immunohistochemical staining. (D) The average number of Foxp3+-cells per field. Data represent means 6 SD from groups of four mice assayed individually. Statistical differences between groups are shown as follows: * p,0.05; **p,0.01; *** p,0.001. doi:10.1371/journal.pone.0069012.gThe Capacity of CD4+CD25+ Tregs Favors the Maintenance of PregnancyTo verify whether the diminished capacity of Tregs at G5 was causally associated with the fetal loss, we adoptively transferred CD4+CD25+T cells isolated from the spleens of normal pregnant mice, pregnant mice injected with T. gondii ESA at G5 or those at G15 into T. gondii ESA-injected pregnant mice at G5, respectively. First, we tested when the CD4+CD25+ Tregs decreased after the injection with T. gondii ESA. We found that the percentage of CD4+CD25+ Tregs significantly reduced to 1 at the first daypost injection (1 dpi) (Figure 4B). Hence, we transferred Tregs to the abortion-pro.Gondii ESA-injection at G10 exhibited decreased number of Foxp3+ cells, but that of mice with T. gondii ESA-injection at G15 presented increased number of Foxp3+ cells, as compared with the control groups. These data provided evidence that the injection with T. gondii ESA at G10 could lead to diminished number of Tregs, but the injection at G15 resulted in the increased number of Tregs at the maternal-fetal interface.Figure 2. Effects of T. gondii ESA on the proportion and function of CD4+CD25+Foxp3+ T cells at different stages of pregnancy. All animals were killed at G18, 10457188 and their spleens, inguinal LN, and PBL were obtained. Lymphocytes from these tissues were 24195657 prepared and pooled as described in Materials and Methods. The cells were stained with CD4-FITC, CD25-APC and PE-Foxp3 Abs, respectively, and analyzed by flow cytometry. (A)Representative dot plots illustrating the regions and gating for the capture of cell phenotype data and intracellular Foxp3 expression. (B) Percentages and absolute number of CD4+CD25+Foxp3+-cells from spleens. (C) Percentages of CD4+CD25+Foxp3+-cells from inguinal LN, and PBL. (D) Responder CD4+CD25?T cells (16105/well) from naive mice were cultured with naive, irradiated APC (16105 cells/well) and CD4+CD25+T cells (56104 cells/well) harvested from mice with PBS or T. gondii ESA injection at G5, G10, G15, respectively. (E and F) The serum levels of IFN-c and IL-4 in pregnant mice injected with T. gondii ESA by ELISA. Data represent means 6 SD from groups of seven mice assayed individually. Statistical differences between groups are shown as follows: * p,0.05; ** p,0.01; *** p,0.001; # p.0.05. doi:10.1371/journal.pone.0069012.gT. gondii ESA Induced Tregs DysfunctionFigure 3. Foxp3 mRNA and protein levels at the maternal-fetal interface of mice with T. gondii ESA injection at G10 and G15. (A) Foxp3 expression levels in placentas from T. gondii ESA-injected and PBS-injected mice measured by real-time quantitative PCR. The data were normalized to individual b-actin mRNA expression and expressed as fold change relative to control mice. Data represent means 6 SD from groups of seven mice assayed individually. (B)Top panel, Foxp3 protein was analyzed by Western blot after the injection at G10 or G15 as indicated. Bottom panel, densitometric analysis of Foxp3 expression was conducted by Western blot. One representative result of three independent experiments performed is shown. (C) The distribution of Foxp3+-cells in the placentas of T. gondii ESA-injected and PBS-injected mice as determined by immunohistochemical staining. (D) The average number of Foxp3+-cells per field. Data represent means 6 SD from groups of four mice assayed individually. Statistical differences between groups are shown as follows: * p,0.05; **p,0.01; *** p,0.001. doi:10.1371/journal.pone.0069012.gThe Capacity of CD4+CD25+ Tregs Favors the Maintenance of PregnancyTo verify whether the diminished capacity of Tregs at G5 was causally associated with the fetal loss, we adoptively transferred CD4+CD25+T cells isolated from the spleens of normal pregnant mice, pregnant mice injected with T. gondii ESA at G5 or those at G15 into T. gondii ESA-injected pregnant mice at G5, respectively. First, we tested when the CD4+CD25+ Tregs decreased after the injection with T. gondii ESA. We found that the percentage of CD4+CD25+ Tregs significantly reduced to 1 at the first daypost injection (1 dpi) (Figure 4B). Hence, we transferred Tregs to the abortion-pro.