Changes in the ratio of regulatory (CD25highFoxP3+) and activated (CD25high) CD4+ T cells in (A) the PLF and (B) the spleen of 1-, 2–3-, or 4–11-month-old mice through the immunization period. Values in the plots show the mean ± SEM calculated from the data of 3–12 mice in each group. Significantly (p < 0.05) lower values in the 4–11-month-old group are indicated [(A)*2–3-month-old and 4–11-month-old versus 1-month-old, (B)#4–11-month-old versus 1-month-old and 2–3-month-old group]. (C)Representative flow cytometric contour plots show the distribution of CD4+ T cells based on CD25 and FoxP3 staining 12 days after the second PG immunization in the PLF and spleen. Percent (mean ± SEM) of regulatory (CD25highFoxP3+) CD4+ T cells (upper right quadrant), and activated (CD25high) CD4+ T cells (upper left quadrant) are shown. Values in the plots were calculated from the data of 3–12 mice (as indicated above the panels). Significant (p < 0.05) differences are indicated (†2–3-month-old and 4–11-month-old groups versus 1-month-old group; ‡4–11-month-old group versus 1-month-old and 2–3-month-old groups).