Detection of circulating CD4+CD25low/-GITR+cells in SLE patients . (A) Flow-cytometry analysis of anti-GITR and anti-CD25-stained CD4+ T lymphocytes, freshly isolated from blood of a representative SLE patient, reveals the presence of a CD25-GITR+ cell subpopulation. Left panel: gating strategy. Gated CD4+ T lymphocytes were stained with isotype control Abs (middle panel) or anti-GITR and anti-CD25 Abs (right panel). (B) the levels of CD25 expression were evaluated in the upper left and lower left quadrants and MFI is reported. (C) mRNA expression of CD25 (FAM6 fluorochrome) in the indicated subpopulations was evaluated in quadruplicate with real-time PCR. In the same tube, expression of the housekeeping gene HPRT1 (VIC fluorochrome) was evaluated for normalization. Values of CD25 expression (striped column, CD25highGITR-; black column, CD25low/-GITR+) is shown as fold increase of mRNA levels in the positively sorted subpopulations over mRNA levels in effector (CD4+CD25-GITR-) T cells, arbitrarily set equal to 1. Data shown are mean ± SD of four SLE patients. ***P <0.001.