(A) Comparison of the percentage of CD4⁺CD25^high T cells from MS patients and controls. The cut-off for high-staining CD25 was placed at 6 × 10³ of mean fluorescence intensity. No statistical difference can be shown between the groups (patients, n = 21; HI, n = 17). (B) Regulatory properties of CD4⁺CD25^high cells were examined in 11 untreated patients with RR-MS and 11 healthy controls. Cocultures of CD4⁺CD25^– and CD4⁺CD25^high cells were performed at a 1:1 ratio and under anti-CD3 stimulation. Proliferation was measured by incorporation of ³H-thymidine after 5 days of incubation. The percentage of suppression of responding cell proliferation (CD4⁺CD25^–) by CD4⁺CD25^high cells was determined as 1 – (proliferation of coculture / proliferation of responder population alone) × 100, where proliferation is expressed as cpm. CD4⁺CD25^high T cells from MS patients exhibited less suppressive activity when the gate for sorting was positioned as shown in Supplemental Figure 1 (P < 0.05, Mann-Whitney U test). Mean values in A and B are indicated by horizontal lines. (C) Comparison of the proliferation of CD4⁺CD25^high T cells in MS patients and HIs relative to the proliferation of the CD4⁺CD25^– T cell subset. Proliferation of CD4⁺CD25^high cells was not significantly different between patients (n = 11) and HIs (n = 11). When the top 4% of CD4⁺CD25^high T cells were sorted, the cells were not fully anergic, suggesting the presence of activated T cells. Data represent mean ± SD.