Clinical and Molecular Hemostasis Research Group, Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada.
Janeway Children's Health and Rehabilitation Centre, St. John's, NL, Canada.
Front Immunol. 2020 Feb 7;11:138. doi: 10.3389/fimmu.2020.00138. eCollection 2020.
The development of neutralizing antibodies (inhibitors) against factor VIII (FVIII) is a major complication of hemophilia A treatment. The sole clinical therapy to restore FVIII tolerance in patients with inhibitors remains immune tolerance induction (ITI) which is expensive, difficult to administer and not always successful. Although not fully understood, the mechanism of ITI is thought to rely on inhibition of FVIII-specific B cells (1). Its efficacy might therefore be improved through more aggressive B cell suppression. FcγRIIB is an inhibitory Fc receptor that down-regulates B cell signaling when cross-linked with the B cell receptor (BCR). We sought to investigate if recombinant FVIII Fc (rFVIIIFc), an Fc fusion molecule composed of FVIII and the Fc region of immunoglobulin G1 (IgG1) (2), is able to inhibit B cell activation more readily than FVIII. rFVIIIFc was able to bind FVIII-exposed and naïve B cells from hemophilia A mice as well as a FVIII-specific murine B cell hybridoma line (413 cells). An anti-FcγRIIB antibody and FVIII inhibited binding, suggesting that rFVIIIFc is able to interact with both FcγRIIB and the BCR. Furthermore, incubation of B cells from FVIII-exposed mice and 413 cells with rFVIIIFc resulted in increased phosphorylation of SH-2 containing inositol 5-phosphatase (SHIP) when compared to FVIII. B cells from FVIII-exposed hemophilia A mice also exhibited decreased extracellular signal-regulated kinase (ERK) phosphorylation when exposed to rFVIIIFc. These differences were absent in B cells from naïve, non-FVIII exposed hemophilic mice suggesting an antigen-dependent effect. Finally, rFVIIIFc was able to inhibit B cell calcium flux induced by anti-Ig F(ab). Our results therefore indicate that rFVIIIFc is able to crosslink FcγRIIB and the BCR of FVIII-specific B cells, causing inhibitory signaling in these cells.
针对因子 VIII (FVIII) 的中和抗体 (抑制剂) 的产生是血友病 A 治疗的主要并发症。恢复抑制剂患者 FVIII 耐受性的唯一临床治疗方法仍然是免疫耐受诱导 (ITI)，但该方法昂贵、难以实施且并非总是有效。尽管尚未完全了解，但 ITI 的机制被认为依赖于 FVIII 特异性 B 细胞 (1) 的抑制。因此，通过更积极的 B 细胞抑制，其疗效可能会得到改善。FcγRIIB 是一种抑制性 Fc 受体，当与 B 细胞受体 (BCR) 交联时，可下调 B 细胞信号传导。我们试图研究重组 FVIII Fc (rFVIIIFc) 是否能够比 FVIII 更易于抑制 B 细胞活化，rFVIIIFc 是由 FVIII 和免疫球蛋白 G1 (IgG1) 的 Fc 区组成的 Fc 融合分子 (2)。rFVIIIFc 能够结合来自血友病 A 小鼠的 FVIII 暴露和幼稚 B 细胞以及 FVIII 特异性鼠 B 细胞杂交瘤系 (413 细胞)。抗 FcγRIIB 抗体和 FVIII 抑制结合，表明 rFVIIIFc 能够与 FcγRIIB 和 BCR 相互作用。此外，与 rFVIIIFc 孵育来自 FVIII 暴露的小鼠和 413 细胞的 B 细胞，与 FVIII 相比，SH-2 包含的肌醇 5-磷酸酶 (SHIP) 的磷酸化增加。当暴露于 rFVIIIFc 时，来自 FVIII 暴露的血友病 A 小鼠的 B 细胞也表现出细胞外信号调节激酶 (ERK) 磷酸化减少。这些差异在来自非 FVIII 暴露的无血友病小鼠的 B 细胞中不存在，提示存在抗原依赖性效应。最后，rFVIIIFc 能够抑制抗 Ig F(ab) 诱导的 B 细胞钙流。因此，我们的结果表明 rFVIIIFc 能够交联 FVIII 特异性 B 细胞的 FcγRIIB 和 BCR，导致这些细胞中的抑制性信号传导。
