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，该蛋白是由塑性贫血（AA）患者的Scrum IgG识别出的，我们使用免疫沉淀和确定具有质量指纹的氨基酸序列纯化了该蛋白质。蛋白质被证明是moesin。当使用ELISA和重组Moesin筛选患者血清时，43例AA患者中有20名（46.5％）患者对抗Moesin抗体呈阳性。在22名（64％）患者中有14例（64％）患者中检测到了抗体（DRS-1），作为AA的候选自动抗原，使用源自UT-7的cDNA库的免疫镜头。在拥有PNH型细胞增加的AA患者中，有32名（38.1％）中有32例（38.1％）中有32例可检测到该蛋白的抗体。使用从DRS-1 cDNA得出的GST融合蛋白片段的表位图表表明，26个MER肽（氨基酸173-198）包含抗体表位的热点，近一半的AA患者识别出抗-DRS-1抗体。当使用ELISPOT测定法检查外周血淋巴细胞以对具有HLA-DR15具有高亲和力的T特异性的T细胞前体进行T细胞前体，两名携带HLA-DR15的AA患者和抗-DRS-1抗体显示出抗-DRS-1抗体的DRS-1抗体高频率显示出DRS-1-1-特异性CD4^+ T细胞的高频。通过用DRS-1肽刺激T细胞的DRS-1特异性T细胞，以剂量依赖性的方式刺激TS-1肽的T细胞对HLA-DR15^+和表达DRS-1表达白血病细胞系KH88的细胞毒性。这些发现表明，在具有HLA-DR15和抗DRS-1抗体的AA患者中，DRS-1特异性T细胞可能有助于骨髓衰竭的发展。