Identification and removal of a promiscuous CD4+T cell epitope from the C1 domain of factor VIII
Identification and removal of a promiscuous CD4+T cell epitope from the C1 domain of factor VIII
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DOI：
10.1111/j.1538-7836.2005.01309.x
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发表时间：
2005-05-01
影响因子：
10.4
通讯作者：
Baker, MP
中科院分区：
文献类型：
--
作者：
Jones, TD;Phillips, WJ;Baker, MP
背景资料：血友病患者中抑制物的形成是因子VIII（FVIII）替代疗法的严重并发症，并且是由FVIII特异性T辅助细胞驱动的过程。目的：精细定位整个FVIII蛋白中的T细胞表位，以研究工程改造抑制剂形成倾向降低的FVIII变体的可能性。患者和方法：T细胞系由5名已产生抑制剂的重度血友病患者产生，并针对跨越整个B结构域缺失（BDD）FVIII序列的重叠肽池筛选T细胞增殖。通过针对T细胞系筛选单个肽来解码阳性肽库。在一组健康供体中测试阳性肽及其突变体结合II类主要组织相容性复合体（MHC）和刺激T细胞增殖的能力。通过显色测定评估相应突变蛋白的活性。结果如下：一种肽，跨越FVIII氨基酸2098-2112，引起一个血友病供体的强烈反应，在一组健康供体中诱导强烈的T细胞反应，并与许多HLA-DR等位基因结合。在该肽中进行突变，去除其刺激健康供体的T细胞和结合MHC II类的能力，同时在掺入突变BDD-FVIII蛋白时保留全部活性。结论：整个FVIII蛋白的精细T细胞表位作图是可行的，尽管具有挑战性，并且该知识可用于产生潜在地具有降低的免疫原性的FVIII变体。
Background: The development of inhibitors in hemophiliacs is a severe complication of factor VIII (FVIII) replacement therapy and is a process driven by FVIII specific T helper cells. Objectives: To finely map T cell epitopes within the whole FVIII protein in order to investigate the possibility of engineering FVIII variants with reduced propensity for inhibitor development. Patients and methods: T cell lines were generated from five patients with severe hemophilia who had developed inhibitors, and were screened for T cell proliferation against pools of overlapping peptides spanning the entire B domain deleted (BDD) FVIII sequence. Positive peptide pools were decoded by screening individual peptides against the T cell lines. Positive peptides, and mutants thereof, were tested for their ability to bind major histocompatibility complex (MHC) Class II and stimulate T cell proliferation in a panel of healthy donors. The activities of the corresponding mutant proteins were assessed via chromogenic assay. Results: One peptide, spanning FVIII amino acids 2098-2112, elicited a vigorous response from one hemophiliac donor, induced strong T cell responses in the panel of healthy donors and bound to a number of HLA-DR alleles. Mutations were made in this peptide that removed its ability to stimulate T cells of healthy donors and to bind to MHC Class II while retaining full activity when incorporated into a mutant BDD-FVIII protein. Conclusions: Fine T cell epitope mapping of the entire FVIII protein is feasible, although challenging, and this knowledge may be used to create FVIII variants which potentially have reduced immunogenicity.