Increased age-associated B cells in acquired AA. (A) PBMC from patients and healthy individuals were stained with anti-CD38, anti-CD11c, anti-CD19, and anti-CD21 monoclonal antibodies followed by intracellular anti–T-bet monoclonal antibody for the ABCs, with anti-CD4, anti-CXCR5, anti-ICOS, and anti-PD1 monoclonal antibodies for the cTfh and with anti-CD4, anti-CD25, anti-CD127 monoclonal antibodies for the Tregs. Patients with AA (n = 21) displayed an increased frequency of ABCs and increased cTfh compared with HDs (n = 20). Patients with AA showed a lower percentage of CD4⁺CD25^hiCD127^lo T cells than healthy donors. The relative numbers from all study subjects are shown. The 2 patients in remission showed a comparable percentage of ABC to that observed in healthy individuals. All samples from patients and healthy donors were run in parallel during the same experiments. (B) Patients with AA showed increased T-bet expression in the ABC population compared with the T-bet expression in healthy individuals. The mean fluorescence intensity of T-bet expression is shown. The 2 patients in remission showed comparable levels of T-bet expression in ABC to those observed in healthy individuals. (y-axis; MFI T-bet in the ABC population). (C) Ets-1 protein levels were examined in protein extracts from the PBMC of patients and healthy controls. Western blot analysis revealed that patients with AA (n = 12) had increased Ets-1 protein levels compared with controls (n = 4). Two patients in remission (Y1 and Y2) showed comparable Ets-1 protein levels to those of healthy individuals. Increased Ets-1 protein levels are associated with increased T-bet expression in ABC in patients with AA. (D) Densitometry analysis of the western blots (Ets1/actin) showed statistically significant increased levels of Ets-1 protein in patients with AA compared with those of controls. The 2 patients in remission had comparable Ets-1 protein levels to those of healthy individuals. (E) The increased percentage of ABC (x-axis: % of ABC) in patients with AA correlates with increased Ets-1 protein levels (y-axis: Ets-1/actin protein levels) (R² = 0.345). (F-G) PBMCs from patients with AA at diagnosis before any treatment (n = 3) and healthy individuals (n = 3) were left overnight in media or stimulated with phorbol myristate acetate and ionomycin. The cells were collected and analyzed using flow cytometry for the intracellular expression of IFN-γ. A representative experiment is shown from 1 patient and 1 healthy subject. The cells were stained with anti-CD38, anti-CD11c, anti-CD19, and anti-CD21 monoclonal antibodies, followed by intracellular staining for IFN-γ. The gating of IFN-γ expression in the ABC population is shown. Patients with AA showed increased intracellular levels of IFN-γ in the ABC compared with those in healthy controls. Stimulated cells showed a statistically significant increased IFN-γ levels compared with healthy subjects. Data from all patients and controls analyzed are shown in Panel F. AA, aplastic anemia; AA Rem, patients with AA in remission; ABC, age-associated B cells; ABCs+IFN-γ, percentage of intracellular IFN-γ levels in age-associated B cells; cTfh, circulating T follicular helper cells; HD, healthy donor; OD Ets1/Actin, densitometry analysis of Ets1 protein levels to actin levels; Tregs, regulatory T cells.