(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. Sorting was performed on the CD4⁺CD25^highCD127^low and CD4⁺CD25^highCD127^high populations. Sorting was also performed on the CD4⁺CD25^high population with 2 different gates. In the example provided for 1 MS patient, CD4⁺CD25⁺ T cells appear in orange, CD4⁺CD25⁺CD127^low T cells appear in green, and CD127^high cells appear in violet. The presence of CD127^high activated cells can be observed in the CD4⁺CD25^high sorted T cells when the gate is not stringent enough (4% gating stringency, area above the red line), while in the case of a 2% gating stringency (area above the blue line), only a few CD127^high cells remain within the CD4⁺CD25^high T cell subset. FSC, forward scatter. (B) CD4⁺CD25^– responder cells were stimulated with anti-CD3 antibody (0.1 μg/ml) in coculture with the top 2% of CD4⁺CD25^high sorted cells. Data are the mean of duplicate wells. Regulatory properties of CD4⁺CD25^high cells are comparable in both HIs (n = 10) and patients (n = 12). (C) Comparison of the proliferation of the top 2% of sorted CD4⁺CD25^high T cells in MS patients and HIs relative to the proliferation of the CD4⁺CD25^– T cell subset. The proliferation of CD4⁺CD25^high T cells was minimal and almost null in both patients and controls. Data are mean ± SD. (D) Intracellular FOXP3 staining was performed on the top 2% of sorted CD4⁺CD25^high T cells in 10 patients and 9 HIs. In B and D, the mean values for each group are indicated by horizontal lines.