DataSheet_2_Neutralizing Antibodies Against Factor VIII Can Occur Through a Non-Germinal Center Pathway.pdf

Humoral immunity to factor VIII (FVIII) represents a significant challenge for the treatment of patients with hemophilia A. Current paradigms indicate that neutralizing antibodies against FVIII (inhibitors) occur through a classical CD4 T cell, germinal center (GC) dependent process. However, clinical observations suggest that the nature of the immune response to FVIII may differ between patients. While some patients produce persistent low or high inhibitor titers, others generate a transient response. Moreover, FVIII reactive memory B cells are only detectable in some patients with sustained inhibitor titers. The determinants regulating the type of immune response a patient develops, let alone how the immune response differs in these patients remains incompletely understood. One hypothesis is that polymorphisms within immunoregulatory genes alter the underlying immune response to FVIII, and thereby the inhibitor response. Consistent with this, studies report that inhibitor titers to FVIII differ in animals with the same F8 pathogenic variant but completely distinct backgrounds; though, how these genetic disparities affect the immune response to FVIII remains to be investigated. Given this, we sought to mechanistically dissect how genetics impact the underlying immune response to FVIII. In particular, as the risk of producing inhibitors is weakly associated with differences in HLA, we hypothesized that genetic factors other than HLA influence the immune response to FVIII and downstream inhibitor formation. Our data demonstrate that FVIII deficient mice encoding the same MHC and F8 variant produce disparate inhibitor titers, and that the type of inhibitor response formed associates with the ability to generate GCs. Interestingly, the formation of antibodies through a GC or non-GC pathway does not appear to be due to differences in CD4 T cell immunity, as the CD4 T cell response to an immunodominant epitope in FVIII was similar in these mice. These results indicate that genetics can impact the process by which inhibitors develop and may in part explain the apparent propensity of patients to form distinct inhibitor responses. Moreover, these data highlight an underappreciated immunological pathway of humoral immunity to FVIII and lay the groundwork for identification of biomarkers for the development of approaches to tolerize against FVIII.

针对因子VIII（FVIII）的体液免疫对治疗血友病A患者构成重大挑战。现有范式表明，针对FVIII的中和抗体（抑制剂）的产生是通过经典的CD4 T细胞、生发中心（GC）依赖性过程。然而，临床观察表明，患者对FVIII的免疫反应性质可能存在差异。部分患者产生持续的低或高抑制剂滴度，而另一些患者则产生短暂的反应。此外，仅在某些具有持续抑制剂滴度的患者中可检测到FVIII反应性记忆B细胞。调控患者所发展免疫反应类型的决定因素，更不用说这些患者免疫反应差异的具体机制，尚不完全明了。一种假设是，免疫调节基因中的多态性改变了针对FVIII的潜在免疫反应，进而影响抑制剂反应。与此一致，研究表明，具有相同F8致病变异但背景完全不同的动物中，FVIII的抑制剂滴度存在差异；尽管如此，这些遗传差异如何影响针对FVIII的免疫反应仍有待研究。鉴于此，我们旨在从机制上剖析遗传如何影响针对FVIII的潜在免疫反应。特别是，由于产生抑制剂的风险与HLA差异弱相关，我们假设除了HLA之外的其他遗传因素会影响针对FVIII的免疫反应以及下游抑制剂的形成。我们的数据显示，编码相同MHC和F8变异的FVIII缺陷小鼠产生不同的抑制剂滴度，并且形成的抑制剂反应类型与生成GC的能力相关。有趣的是，通过GC或非GC途径形成抗体的过程似乎并非由于CD4 T细胞免疫的差异，因为这些小鼠对FVIII中免疫优势表位的CD4 T细胞反应相似。这些结果表明，遗传可以影响抑制剂发展的过程，并在一定程度上解释了患者形成不同抑制剂反应的潜在倾向。此外，这些数据突显了体液免疫针对FVIII的一个未被充分认识的免疫途径，并为识别生物标志物以开发针对FVIII的耐受化方法奠定了基础。