The proportion of CD4+CD25+Foxp3+ Tregs was significantly increased in the spleens of mice with diabetes for 4 months. Splenocytes were freshly prepared at different time points after the onset of diabetes and stained for CD4, CD8, CD25 and Foxp3. (a) Absolute cell numbers as well as the percentages of CD4+ T cells or CD8+ T cells in the splenocytes of control and diabetic mice at indicated time points. (b) Representative FACS stain for CD25 and Foxp3 on gated CD4+ T cells in the spleen. (c) The percentage of CD4+CD25+Foxp3+ Tregs out of total CD4+ T cells in splenocytes at indicated time points. Data are shown as mean±s.d. (n=10), for one representative out of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001, compared with the identical control mice. FACS, fluorescence-activated cell sorting; Treg, T regulatory cell.