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The following supplementary material is available as part of the online article (full text) from: http://www.blackwell-synergy.com

Fig. S1. Gating strategy for phenotypic analysis of CD25 subsets. An initial gate to define CD25hi using an internal control was created on dot-plots of CD25 staining of whole lymphocytes, with the lower limit of the gate set at the upper limit of CD25 expression by CD4-lymphocytes. This gate was added to CD4 cells alone, and gates created for the CD25hi2% population to be used for functional analysis (i.e. the 2% CD4+ T cells expressing the highest levels of CD25), CD25 cells and CD25int. These gates were created using anti-CD25-fluorescein isothiocyanate (FITC) when the marker of interest was identified using phycoerythrin (PE)-labelled antibodies (anti-glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR), CTLA-4 and CCR7). The filled grey plot in the histograms represents the isotype control, the black line staining of the CD25 subset.

Fig. S2. Depletion of CD25hi cells from whole peripheral blood mononuclear cells (PBMC); 1·5 × 107 PBMC were incubated for 30 min at 4°C with 50 μl of anti-CD25 antibody-coated beads. To demonstrate the efficiency of CD25hi depletion, whole PBMC were stained with anti-CD4 and anti-CD25, and a CD25hi gate created using the upper limit of CD25 expression in CD4 lymphocytes as the lower limit of CD25hi expression. The first column shows the CD4/CD25 dot-plot of whole lymphocytes used to create the CD25hi gate in a control and a patient with active disease. The second column shows the percentage within this gate in whole CD4+ T cells and the gates used to define CD25int and CD25. The third column shows the percentage of cells remaining in each of these gates after depletion and the fourth column shows the CD4+ T cells removed from the depleting beads.

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