Expression of CD39 on human Treg cells. (A) Confocal image of CD39⁺Foxp3⁺cells. CD4⁺ cells of human PBMCs were stained for CD39 and Foxp3 and analyzed by laser confocal microscopy (see “Patients, materials and methods; Confocal microscopy” for image acquisition details). Nuclear staining was carried out with DAPI. (B) Suppressive capacity of CD4⁺CD39⁺ cells. The inhibitory activity of FACS-sorted populations of CD4⁺CD39⁺ (●) and CD4⁺CD39⁻ (○) was determined with CD4⁺CD25⁻ responder cells in a ³H-thymidine incorporation assay. Inhibition of proliferation is expressed as a percentage; the ratio between suppressor and responder cells is indicated. Error bars indicate standard deviation of data points. (C) CD39⁺ and CD39⁻ subpopulations within the Foxp3⁺ subset. PBMCs were gated as indicated into CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ subsets (left panel). The CD39 expression of the 2 subsets is shown in a double-staining with α-CD25 (right panel). (D) Correlation between CD39 and Foxp3 expression. The mean fluorescence intensities of CD39 and Foxp3 are shown for the subset of CD4⁺CD25^high cells. MFI values of data points were determined from the FACS data of a single donor by scanning the CD39 expression with a narrow gate for Foxp3 using the FlowJo analysis software. Line represents a hyperbola regression calculated by the Sigmaplot software package.