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

Background: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen-specific immune tolerance by interferon-gamma (IFN-gamma)-dependent T-cell anergy in hemophilia A mice. Objective: The thymus plays crucial roles in self-tolerance, with negative selection of self-reactive effector T cells and positive selection of self-reactive regulatory T cells. We investigated the possibility of the induction of antigen-specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. Methods: Hemophilia A mice were injected with recombinant FVIII into the thymus under real-time high-resolution image guidance. Results: Anti-FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 +/- 2.3 vs. 122.5 +/- 27.6 BU mL(-1), respectively, P = 0.00078). The CD4(+) T cells from thymic-injected mice could not proliferate or produce interleukin (IL)-2, IL-12 and IFN-gamma in response to FVIII. The CD4(+)CD25(+) T cells generated from thymic-treated mice but not from naive mice efficiently suppressed the in vitro proliferative response of CD4(+) T cells and blocked the in vivo development of anti-FVIII antibodies in the adoptive transfer. Conclusion: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII-specific regulatory T cells.