Stimulation induces T-bet expression in healthy controls but not in aplastic anemia patients. (A) Stimulation conditions with PMA and ionomycin did not have any effect on T-bet levels in patients with aberrant expression of T-bet (patient no. 7). In patients with lower T-bet levels, stimulation induced further T-bet expression (patient no. 6). No significant differences in STAT1 protein levels were detected between aplastic anemia patients and controls. (B) Healthy controls did not express T-bet in unstimulated T cells. T-bet expression was induced after stimulation with PMA and ionomycin. Stimulation conditions induced T-bet expression in patients with lower T-bet protein levels (aplastic anemia no. 8). (C-E) Patients with myelodysplastic syndrome (MDS), refractory anemia with excess of blasts (RAEBs), sickle cell anemia (SCA), and autoimmune hemolytic anemia (AIHA) showed undetectable T-bet levels in immunoblot experiments comparable with healthy controls and patients in remission (patient no. 21). (F) CD8⁺ T cells express increased T-bet levels compared with CD4⁺ T cells. (G) T-bet protein levels are not affected by in vitro maintenance after 24 hours without T-cell stimulation.