Analysis of autoantigens in aplastic anemia : identification of an epitope recognized by CD4^+ T cells specific to hematopoietic progenitor cells

再生障碍性贫血中自身抗原的分析：鉴定造血祖细胞特异的 CD4^ T 细胞识别的表位

基本信息

批准号：
15390298
负责人：
NAKAO Shinji
金额：
$ 6.85万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390298/
关键词：
aplastic anemia autoantigen moesin DRS-1 HLA-DR15 PNH

项目摘要

To identify an 80-kD protein derived from a leukemia cell line, UT-7, that was recognized by scrum IgG of a plastic anemia (AA) patients, we purified this protein using immunoprecipitation and determined amino acid sequence with mass fingerprinting. The protein proved to be moesin. When patients' sera were screened using ELISA and recombinant moesin, 20 of 43 (46.5%) AA patients were positive for anti-moesin antibodies. The antibody was detected in 14 of 22 (64%) patients possessing increased paroxysmal nocturnal hemoglobinuria (PNH)-type cells which are known to be a marker for lmmunopathophysiology of AA while it was detected in only 3 of 18 (17%) patients without increased PNH-type cells.We also identified diazepam-binding inhibitor-related sequence-1 (DRS-1), as a candidate autoantigen of AA using immunoscreening of cDNA library derived from UT-7. Antibodies to this protein were detectable in 32 of 84 (38.1%) of AA patients possessing increased PNH-type cells. Epitope mapping using GST-fusion protein fragments derived from DRS-1 cDNA showed that a 26 mer peptide (amino acid 173-198) contained a hot spot of antibody epitopes which was recognized by nearly half of AA patients showing anti-DRS-1 antibodies. When peripheral blood lymphocytes were examined using ELISPOT assay for the presence of T-cell precursors specific to a DRS-1 peptide which has high affinity to HLA-DR15, two AA patients carrying HLA-DR15 and anti-DRS-1 antibodies showed high frequency of DRS-1-specific CD4^+ T cells. DRS-1-specific T cells generated from one of the two AA patients by stimulating T cells with the DRS-1 peptide showed cytotoxicity against an HLA-DR15^+ and DRS-1-expressing leukemia cell line KH88 in a dose-dependent manner. These findings indicate that in AA patients possessing HLA-DR15 and anti-DRS-1 antibodies, DRS-1-specific T cells may contribute to development of bone marrow failure.

为了鉴定源自白血病细胞系 UT-7 的 80 kD 蛋白质（可被再生障碍性贫血 (AA) 患者的血清 IgG 识别），我们使用免疫沉淀纯化该蛋白质，并通过质量指纹分析确定氨基酸序列。该蛋白质被证明是莫斯蛋白。当使用 ELISA 和重组 moesin 对患者血清进行筛选时，43 名 AA 患者中有 20 名 (46.5%) 抗 moesin 抗体呈阳性。在 22 名患有阵发性睡眠性血红蛋白尿 (PNH) 型细胞增多的患者中，有 14 名 (64%) 检测到了该抗体，而这种细胞已知是 AA 免疫病理生理学的标志物，而在 18 名没有阵发性睡眠性血红蛋白尿症 (PNH) 型细胞的患者中，只有 3 名 (17%) 检测到了该抗体。 PNH 型细胞增加。我们还鉴定了地西泮结合抑制剂相关序列 1 (DRS-1)，作为 PNH 型细胞的候选自身抗原AA 使用源自 UT-7 的 cDNA 文库进行免疫筛选。在 84 名 PNH 型细胞增多的 AA 患者中，有 32 名 (38.1%) 检测到了针对该蛋白的抗体。使用源自 DRS-1 cDNA 的 GST 融合蛋白片段进行表位作图显示，26 聚体肽（氨基酸 173-198）包含抗体表位的热点，该热点被近一半显示抗 DRS-1 抗体的 AA 患者识别。当使用 ELISPOT 检测检测外周血淋巴细胞中是否存在对与 HLA-DR15 具有高亲和力的 DRS-1 肽特异的 T 细胞前体时，两名携带 HLA-DR15 和抗 DRS-1 抗体的 AA 患者表现出高频率DRS-1 特异性 CD4^+ T 细胞。通过用 DRS-1 肽刺激 T 细胞，两名 AA 患者之一产生的 DRS-1 特异性 T 细胞对表达 HLA-DR15^+ 和 DRS-1 的白血病细胞系 KH88 具有剂量依赖性细胞毒性。这些发现表明，在拥有 HLA-DR15 和抗 DRS-1 抗体的 AA 患者中，DRS-1 特异性 T 细胞可能导致骨髓衰竭的发生。