CD4⁺CD25⁺ T cells in patients with aplastic anemia and in healthy control subjects. (A) Peripheral blood mononuclear cells from patients with aplastic anemia and healthy control subjects were stained with anti-CD4 and anti-CD25 antibodies followed by intracellular anti-FOXP3 antibodies. The upper gate on the dot plots (gate a) represents CD4⁺CD25hi⁺ T cells and the lower gate (gate b) the CD4⁺CD25⁺ T cells. FOXP3 expression was gated on CD4⁺CD25hi⁺ T cell population (gate a). Representative dot plots are shown from a healthy control subject, a patient with aplastic anemia, and another patient before and after immunosuppressive treatment. Tx, treatment. (B,C) Relative numbers of CD4⁺CD25hi⁺ T and CD4⁺CD25hi⁺ FOXP3⁺ T cells from all healthy control subjects and patients with aplastic anemia examined are shown. All patients were examined before receiving any immunosuppressive treatment. Differences between patients and control subjects were statistically significant (P < .001). (D) CD4⁺CD25⁺ T cells' nuclear extracts from healthy volunteers and patients with aplastic anemia were analyzed for FOXP3 expression. All patients examined had significantly decreased FOXP3 protein levels compared with healthy donors (P < .001). All patients' samples were analyzed side-by-side with healthy donors' samples. (E) Cytoplasmic extracts from CD4⁺CD25⁺ T cells from patients and healthy donors were analyzed by immunoblot for NFAT1 expression. All patients examined showed diminished or undetectable NFAT1 protein levels compared with healthy control subjects (P < .001). All patients' samples were run side-by-side with control subjects' samples. There were no differences in TLR2 expression between patients and control subjects. (F) RNA from CD4⁺CD25⁺ T cells from patients with aplastic anemia and control subjects was analyzed for FOXP3 gene expression in quantitative polymerase chain reaction experiments. Patients with aplastic anemia showed decreased FOXP3 mRNA/actin copies compared with healthy control subjects (P = .03). Horizontal lines represent mean values. (G-I) The densitometric intensities of immunoblot results from all the subjects studied for FOXP3, NFAT1, and TLR2 expression are collectively presented. Horizontal lines represent mean values. Results are plus or minus standard error of the mean (± SEM).