Induction of factor VIII specific unresponsiveness by intrathymic factor VIII injection in murine hemophilia A

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Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against factor VIII following replacement therapy. We have previously reported that neonatal exposure of factor VIII antigen might induce antigen specific immune tolerance by IFN-γ dependent T cell anergy in hemophilic mice (Madoiwa S, et al. J Thromb Haemost 2:754-762,2004). Thymus plays crucial roles of self-tolerance with negative selection of self-reactive effector T cells and positive selection of self-reactive regulatory T cells. In this study, we focused on the induction of immune tolerance by direct thymic injection of factor VIII. Hemophilia mice were injected recombinant human factor VIII into thymus under the real time high resolution image guidance (Vevo 770, Visual Sonic Inc.). We measured the factor VIII inhibitors after repeated intravenous injection of factor VIII. The CD4+ cells, antigen presenting cells (APCs) and CD4+CD25+ cells were isolated for the proliferation and cytokine assays under in vitro stimulation of factor VIII. Anti-factor VIII inhibitory antibody titers were significantly lower in mice (n=17) with thymic injection of factor VIII than in mice (n=18) without injection (14.6 ± 3.8 vs 184.5 ± 48.6 Bethesda units/mL, respectively, p=0.0019). The CD4+ cells from thymic injected mice could not proliferate or produce IL-2, IL-12, and IFN-γ in response to factor VIII, when they were co-cultured with APCs from immunogenic mice. The CD4+CD25+ cells from thymic treated mice but not from naive mice efficiently suppressed the proliferative response of immunogenic mice derived CD4+ cells in the presence of APCs. Intrathymic administration of factor VIII could result in immune tolerance by factor VIII specific regulatory T cells induction. This immune tolerance model may provide a basis of new manipulation for the prevention of factor VIII inhibitors.