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

血友病A是一种先天性出血性疾病，由凝血因子VIII缺乏引起。大约30%的血友病A患者在替代治疗后产生针对因子VIII的抑制物。我们先前报道了新生儿暴露于因子VIII抗原可能通过血友病小鼠中的IFN-γ依赖性T细胞无反应性诱导抗原特异性免疫耐受（Madoiwa S，et al. JThromb Haemost 2：754- 762，2004）。免疫应答通过对自身反应性效应T细胞的负选择和对自身反应性调节T细胞的正选择在自身耐受中起关键作用。在这项研究中，我们专注于通过直接胸腺注射因子VIII诱导免疫耐受。在真实的实时高分辨率图像引导（Vevo 770，Visual Sonic Inc.）下将重组人因子VIII注射到血友病小鼠的胸腺中。我们测量了重复静脉注射因子VIII后的因子VIII抑制剂。分离CD 4+细胞、抗原呈递细胞（APC）和CD 4 + CD 25+细胞用于在因子VIII的体外刺激下的增殖和细胞因子测定。胸腺注射因子VIII的小鼠（n=17）中的抗因子VIII抑制性抗体滴度显著低于未注射的小鼠（n=18）（分别为14.6 ± 3.8 vs 184.5 ± 48.6 Bethesda单位/mL，p=0.0019）。当来自胸腺注射小鼠的CD 4+细胞与来自免疫原性小鼠的APC共培养时，它们不能增殖或产生响应于因子VIII的IL-2、IL-12和IFN-γ。来自胸腺处理的小鼠而不是来自幼稚小鼠的CD 4 + CD 25+细胞有效地抑制免疫原性小鼠来源的CD 4+细胞在APC存在下的增殖反应。胸腺内给予因子VIII可通过诱导因子VIII特异性调节性T细胞而导致免疫耐受。这种免疫耐受模型可能为预防因子VIII抑制剂提供新的操作基础。

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.