The phenotype and functions of CD25^highCD4⁺ T cells. (A) Analysis of Tr for intracellular expression of FOXP3. The threshold of CD25 fluorescence intensity for the analysis gate of Tr was set above the maximum level of fluorescence emitted from CD25-labeled CD8⁺ T cells. For all the patient samples, more than 90% of the CD25^highCD4⁺ T cells were FOXP3⁺ (histogram). Gray-shaded histogram depicts the background fluorescence intensity of the isotype control antibody. (B) Suppression of anti-CD3–induced proliferation of CD25⁻CD4⁺CD3⁺ T cells by different relative numbers of Tr. The 100% value on the y-axis indicates the maximal anti-CD3–induced proliferation (cpm) of CD25⁻CD4⁺CD3⁺ T cells without Tr present (1:0). The mean percentages (± SD) of the maximal response assessed at different ratios of these 2 CD4⁺ T-cell subpopulations are depicted for patients (MM; n = 9) and control subjects (C; n = 5). The degree of Tr-dependent inhibition of proliferation was not significantly different between the groups. (C) The Tr-mediated suppression of proliferation was accompanied by decreased concentrations of Th cytokines in the culture supernatants and, in most cases, elevated levels of TGF-β. The concentrations of cytokines in cultures with Tr present (1:1) are expressed in percentage of the concentrations obtained in cultures without Tr (1:0), which is plotted for each patient (△) and control subjects () for the respective cytokine. (Occasional increases in the concentration of TGF-β of more than 100% are plotted as 100% for graphics reasons.)