FOXP3 and NFAT1 in primary CD4⁺CD25⁺ T cells. (A) Purified CD4⁺CD25⁺ T cells from patients and healthy donors were analyzed by confocal microscopy for NFAT1 and FOXP3 expression. NFAT1 resided in the cytoplasm in unstimulated cells from control subject and FOXP3 was localized in the nucleus. All patients with aplastic anemia who were examined showed decreased or absent NFAT1 and FOXP3. Representative results are shown from at least 4 different experiments. In all experiments, patients' and control subjects' cells were stained in parallel. DIC indicates differential interference contrast; DAPI, 4′-6-diamidino-2-phenylindole. (B) Purified CD4⁺CD25⁺ T cells from patients and healthy donors were analyzed by confocal microscopy for FOXP3 expression after transient transfection with a wild-type NFAT1 construct. CD4⁺CD25⁺ T cells from healthy donors did not show any difference in FOXP3 expression after transfection with the NFAT1 construct. CD4⁺CD25⁺ T cells from patients showed significantly increased FOXP3 expression after the transfection. Representative results are shown from at least 4 different experiments. Patients' and control subjects' cells were transfected and stained in parallel. (C) NFAT1 levels in CD4⁺CD25⁻ T cells were comparable between patients and control subjects. (D) Cytoplasmic and nuclear extracts from CD4⁺CD25⁺ T cells (from healthy donors, n = 2) transfected with NFAT1-siRNA were analyzed by immunoblot for NFAT1 and FOXP3 expression, respectively. The NFAT1-knockdown CD4⁺CD25⁺ T cells showed decreased FOXP3 expression compared with the cells that were transfected with a control small inhibitory RNA. See “Patients, materials and methods, Confocal microscopy and T-cell transfections” for detailed image acquisition information.