Induction of Immune Tolerance by Neonatal Intravenous Injection of Human Factor VIII in Murine Hemophilia A

抑制性抗体形成是血友病A患者凝血因子VIII替代治疗最严重的并发症。因子VIII缺陷小鼠作为严重血友病A的模型，以表征对人因子VIII的免疫应答，并研究诱导免疫耐受的新方法。在出生后24小时内接受0.05单位/g体重人因子VIII静脉注射以及成年后重复注射的14只成年小鼠中，有13只未观察到抗因子VIII抑制性抗体和抗因子VIII IgG。相比之下，在产后第3天注射的13只小鼠中的7只中以及在所有未接受生殖治疗的成年小鼠中发现了高因子VIII抗体滴度（>50 Bethesda单位/mL）。在第0天分别用2倍（0.1单位/g体重）和10倍高剂量（0.5单位/g体重）的因子VIII初始处理时，9只小鼠中的1只和17只小鼠中的3只产生高滴度抗因子VIII抑制性抗体。人因子VIII特异性T细胞增殖反应是不存在的脾细胞获得的pharmacatally治疗的小鼠。然而，用破伤风类毒素免疫的耐受小鼠产生了高的抗破伤风类毒素抗体，表明耐受是因子VIII特异性的。脾细胞不能响应于因子VIII刺激而增殖或产生干扰素-γ，但仍分泌显著量的白细胞介素-2，并且通过添加外源性干扰素-γ或白细胞介素-12而恢复增殖，这表明缺乏因子VIII的抑制剂是由于干扰素-γ依赖性无能。这些研究表明，在第0天暴露于生理水平的因子VIII抗原可能对诱导免疫耐受是重要的。这种免疫耐受模型可能为在替代治疗期间预防因子VIII抑制剂的新方法提供基础。

Inhibitory antibody formation is the most serious complication of factor VIII replacement therapy in patients with hemophilia A. Factor VIII-deficient mice served as a model of severe hemophilia A to characterize the immune response to human factor VIII and to study new approaches for induction of immune tolerance. Neither anti-factor VIII inhibitory antibodies nor anti-factor VIII IgGs were observed in 13 of 14 adult mice that received 0.05 unit/g body weight of human factor VIII intravenously within 24 hours after birth, as well as repeated injections as adults. In contrast, high factor VIII antibody titers (>50 Bethesda units/mL) were found in 7 of 13 mice injected on day 3 post-partum and in all adult mice that had not been treated neonatally. One out of 9 mice and 3 of 17 mice developed high-titer anti-factor VIII inhibitory antibody, when they were initially treated with 2-fold (0.1 unit/g body weight) and 10-fold higher doses (0.5 unit/g body weight) factor VIII on day 0 respectively. A human factor VIII specific T cell proliferative response was absent in splenocytes obtained from neonatally treated mice. However, the tolerant mice immunized with tetanus toxoid developed high anti-tetanus toxoid antibody, demonstrating that tolerance is factor VIII specific. The splenocytes failed to proliferate or produce interferon-γ in response to factor VIII stimulation, yet still secreted significant amounts of interleukin-2 and were restored proliferation by the addition of exogenous interferon-γ or the interleukin-12, suggesting that the lack of inhibitor to factor VIII is due to interferon-γ dependent anergy. These studies indicate that exposure on day 0 to physiological levels of factor VIII antigen might be important for induction of immune tolerance. This immune tolerance model may provide a basis for new approaches regarding prevention of factor VIII inhibitors during replacement therapy.