The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) poses a major challenge in hemophilia A (HA) treatment. The formation of FVIII inhibitors is a CD4+ T-cell dependent mechanism which includes antigen presenting cells (APCs), B- and T-helper lymphocytes. APCs present FVIII derived peptides on major histocompatibility complex class II (MHC-II) to CD4+ Tcells. We previously established a mass spectrometry based approach to delineate the FVIII repertoire presented on HLA-DR and HLA-DQ. In this study, specific attention was directed towards the identification of FVIII peptides presented on HLA-DP. A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocytes-derived dendritic cells (moDCs) from HLA-typed healthy donors. Using this method, we identified 176 to 1352 different HLA-DP presented peptides per donor, including 26 different FVIII derived peptides. The most frequently presented peptides derived from the A3, and C2 domains of FVIII. Comparison of the FVIII repertoire presented on HLA-DP with that presented on HLA-DR revealed considerable overlap but also suggested preferential presentation of specific peptides on either HLA-DR or HLA-DP. Fourteen FVIII peptides presented on HLA-DP were synthesized and evaluated for their binding ability to the commonly expressed HLA-DP4 molecule which is highly prevalent in the Caucasian population. Peptide binding studies showed that 7 of 14 peptides competed with a reference peptide to HLADP4. Interestingly, an A3 domain derived peptide bound with high affinity to HLA-DP4 positioning this peptide as a prime candidate for the development of novel peptide-based tolerogenic strategies for FVIII inhibitors.

中文翻译：

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在 HLA-DP 上呈递的 FVIII 肽，并鉴定与常见 HLA-DP4 具有高亲和力的 A3 结构域肽结合。


针对凝血因子 VIII （FVIII） 的中和抗体（抑制剂）的开发对血友病 A （HA） 治疗构成了重大挑战。FVIII 抑制剂的形成是一种 CD4+ T 细胞依赖性机制，包括抗原呈递细胞 （APC）、B 和 T 辅助性淋巴细胞。APC 在 CD4+ T 细胞的主要组织相容性复合物 II 类 （MHC-II） 上呈递 FVIII 衍生肽。我们之前建立了一种基于质谱的方法来描述 HLA-DR 和 HLA-DQ 上呈现的 FVIII 库。在这项研究中，特别关注 HLA-DP 上呈递的 FVIII 肽的鉴定。通过将人 FVIII 与来自 HLA 型健康供体的未成熟单核细胞衍生的树突状细胞 （moDC） 孵育来生成天然加工的 FVIII 肽数据集。使用这种方法，我们为每个供体鉴定了 176 至 1352 种不同的 HLA-DP 呈递肽，包括 26 种不同的 FVIII 衍生肽。最常出现的肽来源于 FVIII 的 A3 和 C2 结构域。HLA-DP 上呈现的 FVIII 库与 HLA-DR 上呈现的 FVIII 库的比较揭示了相当大的重叠，但也表明 HLA-DR 或 HLA-DP 上特异性肽的优先呈现。合成了 HLA-DP 上呈递的 14 种 FVIII 肽，并评估了它们与在白种人人群中高度普遍的常用 HLA-DP4 分子的结合能力。肽结合研究表明，14 种肽中有 7 种与 HLADP4 的参比肽竞争。有趣的是，与 HLA-DP4 高亲和力结合的 A3 结构域衍生肽将该肽定位为开发基于 FVIII 抑制剂的新型基于肽的耐受性策略的主要候选肽。