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Fig. 1. CVS induces anxiety-like behaviors. Anxiety levels were quantified following 24 days of CVS or nonstress conditions. The elevated plus maze (A–B) and open field tests (C) were performed as described in Materials and methods. Bar graphs represent means ± SEM of 20–21 mice in each group, pooled from three independent experiments. p-values were calculated by Student's t-test. **p < 0.01; ***p < 0.001.

Fig. 2. Splenocyte and CD4 T-cell loss in CVS-exposed mice. Spleen atrophy (A–B: number of splenocytes; C–D: spleen weight; E–F: spleen weight/body weight ratio) was assessed in female (left panels) and male (right panels) mice following 24 days of CVS or nonstressed conditions. Bar graphs represent means ± SEM of 11 mice in each group, pooled from two independent experiments. p-values were calculated by Student's t-test. * *p < 0.05; *p < 0.01; ***p < 0.001.

Fig. 3. Defining CD4 Treg cells based on CD25 and CD127 expression. CD4+ T cells were first gated out of splenocytes and blood (A) and are shown as percentage of levels measured in the nonstressed group (B). Percentage of CD127CD4+ T cells was then analyzed for CD4+CD25high, CD4+CD25low and CD4+CD25 T cells (C–D). Quantification analysis (E) shows that CD4+CD127 T cells are enriched within the CD25low and to a further extent within the CD25high T-cell subsets, as compared with CD4+CD25 T cells. Data represent 10 mice from two independent experiments. p-values were calculated by Student's t-test. ***p < 0.001.

Fig. 4. CD127 T cells are enriched within the CD25+/FoxP3+ Treg cells. Transgenic mice expressing enhanced green florescent protein (E-GFP) under the control of the mouse Foxp3 promoter were used to analyze the frequency of CD127 T cells within the CD25+/FoxP3+ T cells. Splenocytes were first gated for CD4 (A) and then CD4+ T cells were gated for CD25 and CD127 (B). Analysis of T cells expressing Foxp3 within each of the subpopulations (a–f) is shown in (C). Notice that the frequency of CD4+/FoxP3+ T cells is higher within the CD127 T cells than within the CD127+ T cells (a versus d (CD25high T cells); b versus e (CD25low T cells) and c versus f (CD25 T cells)). (D) Quantification analysis of the data shown in (C). Data represent 10 mice pooled from two independent experiments. p-values were calculated by Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5. CVS reduces the frequency of blood-derived Treg cells. Blood samples were drawn from both nonstressed and stressed mice before and following EAE. PBLs were then harvested, stained for CD4, CD25, and CD127 and subsequently analyzed by flow cytometry. (A–B) Analysis of CD4+CD25+ T cells. (A–B) The frequency of CD127 cells among CD4+CD25+ T cells. (C) The CD127/CD127+ ratio within the CD4+ T-cell subsets. (D) The frequency of CD25+CD127+ and CD25+CD127 cells among CD4+ T cells. (E) CD127/CD127+ ratio among CD4+CD25+ T cells before and following EAE. (A–D) Data are shown as means ± SEM of 17 mice per group, pooled from three independent experiments. (E) Data represent means ± SEM of 6–8 mice per group. p-values were calculated by Student's t-test *p < 0.05; **p < 0.01; ***p < 0.001.

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