(A) CD4⁺ lymphocytes obtained from the peripheral blood of MS patients and healthy controls were stained with Pe-Cy7–conjugated anti-CD3, FITC-conjugated anti-CD8, Alexa Fluor 647–conjugated anti-CD25, and PE-conjugated anti-CD127. No statistically significant difference was observed in the frequency of CD4⁺CD25^highCD127^high T cells between MS patients (n = 25) and healthy controls (n = 19). Mean value is indicated for each group. (B) CD4⁺CD25^highCD127^low T cells or CD4⁺CD25^highCD127^high T cells were cocultured with irradiated autologous PBMCs and stimulated with anti-CD3 antibody. CD25^highCD127^low cells were isolated from 25 patients and 23 HIs. CD25^highCD127^high cells were isolated from 20 patients and 20 HIs. A significant difference was observed in the proliferation of CD25^highCD127^high T cells between MS patients and HIs (P = 0.017, Mann-Whitney U test). Bar graphs indicate the mean ± SD. (C) Suppression of proliferation of CD4⁺CD25^– cells by CD4⁺CD25^high cells was calculated in 13 patients and 15 HIs. The percentage of CD127^high cells present in the sorted CD4⁺CD25^high T cells was estimated in the same manner. A correlation was found between this percentage and the regulatory properties of CD4⁺CD25^high cells with a Pearson coefficient of r = –0.50 (P = 0.006, linear regression test). Black triangles represent data obtained from MS patients, and white squares represent data from HIs. (D) Intracellular FOXP3 staining was performed on CD4⁺CD25^highCD127^high T cells in 10 patients and 9 HIs. In B and D, horizontal lines indicate the mean.