Abstract
The main complication of hemophilia A treatment is the development of neutralizing antibodies (inhibitors) against factor VIII (FVIII). Immune tolerance induction (ITI) is the prescribed treatment for inhibitor eradication, although its working mechanism remains unresolved. To clarify this mechanism, we compared blood samples of hemophilia A patients with and without inhibitors for presence of immunoregulatory cells and markers, including regulatory B-cells (Bregs), regulatory T-cells (Tregs), myeloid-derived suppressor cells (MDSCs), and expression of regulatory markers on T-cells (programmed cell death protein 1 [PD1], inducable T-cell costimulator, cytotoxic T-lymphocyte-associated protein 4 [CTLA4]), by use of flow cytometry. By cross-sectional analysis inhibitor patients (N = 20) were compared with inhibitor-negative (N = 28) and ex-inhibitor (N = 17) patients. In another longitudinal study, changes in immunoregulatory parameters were evaluated during ITI (N = 12) and compared with inhibitor-negative hemophilia A patients (N = 36). The frequency of Bregs, but not of Tregs nor MDSCs, was significantly reduced in inhibitor patients (3.2%) compared with inhibitor-negative (5.9%) and ex-inhibitor patients (8.9%; P < 0.01). CTLA4 expression on T-cells was also reduced (mean fluorescence intensity 133 in inhibitor versus 537 in inhibitor-negative patients; P < 0.01). Fittingly, in patients followed during ITI, inhibitor eradication associated with increased Bregs, increased Tregs, and increased expression of CTLA4 and PD1 on CD4+ T-cells. In conclusion, inhibitor patients express significantly lower frequency of Bregs and Tregs marker expression, which are restored by successful ITI. Our findings suggest that an existing anti-FVIII immune response is associated with deficits in peripheral tolerance mechanisms and that Bregs and changes in immunoregulatory properties of CD4+ T-cells likely contribute to ITI in hemophilia A patients with inhibitors.