FoxP3 expression of CD4+CD25+/highCD127low/-T cells in different compartments and gating strategy for CD25highcells. Samples were stained with anti-CD4–fluorescein isothiocyanate (FITC) (clone 2A3), anti-CD25–allophycocyanin (APC) (clone RDR5) and anti-CD127–peridinin chlorophyll protein complex cychrome 5.5 (anti-CD127-PerCP) (clone RDR5) to identify CD4+CD25+/highCD127low/-cells. After undergoing fixation and permeabilisation, cells were further stained with phycoerythrin-labelled FoxP3 (clone 259D/C7). The cells in these analyses were gated for CD4+CD25+/highCD127low/-regulatory T cells, as shown in Figure 1. Cells stained with isotype controls are shadowed in the histograms. The boldface line represents the FoxP3+ T cells. Four samples were analysed for each compartment. Representative histograms for (a) peripheral blood (PB), (b) synovial fluid (SF) and (c) synovial membrane (SM) are shown. The numbers in the histograms indicate the percentage of gated cells expressing FoxP3+. (d) to (f) Staining pattern for CD4-FITC and CD25-APC is shown. Representative dot plots of one patient are shown. Gated areas indicate the CD25high cell populations in (d) PB, (e) SF and (f) SM.