Assessment of In Vitro Cytokine Response in Hemophilia A Patients With or Without Factor VIII Inhibitory Antibody

Factor VIII (FVIII) inhibitor antibodies are produced in a proportion of hemophilia A patients. Development of anti-FVIII inhibitor antibodies is a T cell-dependent response, mediated by FVIII specific CD4⁺ T cells. This study was performed to investigate the contribution of T helper (Th) cell-mediated cytokine response in inhibitor production. Peripheral blood mononuclear cells (PBMCs) were obtained from hemophilia A patients with (n = 14) or without inhibitor (n = 14) and from normal individuals (n = 14). Following stimulation of PBMCs with rFVIII and phytohemagglutinin (PHA) mitogen, the secreted cytokines, interferon-γ (IFN-γ), interleukin-10 (IL-10), and transforming growth factor-β1 (TGF-β1), in culture supernatant and the proliferative response were assessed using sandwich ELISA and ³H-thymidine incorporation, respectively. No significant proliferative response to FVIII was observed, whereas PHA induced a strong response in all groups. No cytokine secretion was observed in response to FVIII stimulation. Although PHA induced IL-10, TGF-β1 and IFN-γ secretion in all groups, the level of IFN-γ was significantly lower in hemophilia A patients than in normal individuals (p < 0.0001). The levels of TGF-β1 and IL-10 were similarly higher in patients compared with normal subjects, but the difference was not statistically significant. Lack of FVIII-induced proliferative response and cytokine production together with reduced secretion of PHA-induced IFN-γ in both groups of patients suggest involvement of nonspecific immunosuppression possibly due to hepatitis C virus (HCV) infection observed in the majority of patients.