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Analysis of mechanisms responsible for clonal expansion of PNH-type hematopoietic stem cells mediated by autoreactive T cells in patients with bone marrow failure

骨髓衰竭患者自身反应性T细胞介导的PNH型造血干细胞克隆扩增机制分析

基本信息

批准号：
17390275
负责人：
NAKAO Shinji
金额：
$ 9.79万
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：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390275/
关键词：
aplastic anemia DRS moesin paroxysmal nocturnal hemoglobinuria autoantibody cytotoxic T cell 細胞障害性T細胞

项目摘要

In an attempt to determine whether CD4^+ T cells specific to diazepam-binding inhibitor protein-1 (DRS-1) contribute to the survival advantage of paroxysmal nocturnal hemoglobinuria (PNH)-type stem cells, we established a lymphobastoid cell line with PIG-A mutation from an aplastic anemia (AA) patients showing a high titer antibodies speicific to DRS-1. We tried to transfect the PNH-type LCL cells with DRS-1 cDNA using various methods such as retroviral vectors and electroporation to use them as a target of DRS-1 specific CD4^+ T cells. However, all the methods failed to induce expression of DRS-1 gene in LCL cells, probably due to inhibitory mechanisms for the DRS-1 expression inherent to B lymphocytes. Thus, we suspended this experiments and focused on another candidate autoantigen, moesin.We screened the sera of AA patients possessing small populations of PNH-type cells for the presence of antibodies (Abs) which recognize proteins derived from a leukemia cell line, UT-7. Immunoblott … More ing using proteins derived from lysates or culture supernatants of UT-7 cells revealed the presence of IgG Abs specific to an 80 kD protein. Peptide mass fingerprinting identified this 80 kD protein as moesin. Enzyme-linked immunosorbent assay (ELISA) using recombinant moesin showed high titers of anti-moesin Abs in 25 (37%) of 67 AA patients. Moesin was secreted from several myeloid leukemia cell lines other than UT-7, such as OUN-1 and K562, as an exosomal protein. The presence of anti-moesin Abs was significantly correlated with the presence of PNH-type cells and anti-diazepam-binding inhibitor-related protein-1 (DRS-1) Abs. AA patients that did not show any of these three markers tended to respond poorly to immunosuppressive therapy. These findings suggest that a B-cell response to moesin, possibly derived from hematopoietic cells, frequently occurs in patients with AA and that detection of anti-moesin Abs in combination with other markers may be useful in diagnosing immune pathophysiology in AA patients. Less

为了确定地西泮结合抑制蛋白 1 (DRS-1) 特异性的 CD4^+ T 细胞是否有助于阵发性睡眠性血红蛋白尿 (PNH) 型干细胞的生存优势，我们从再生障碍性贫血 (AA) 患者中建立了具有 PIG-A 突变的淋巴母细胞系，显示出针对 DRS-1 的高效价抗体。我们尝试使用逆转录病毒载体和电穿孔等多种方法用DRS-1 cDNA转染PNH型LCL细胞，将其用作DRS-1特异性CD4^+ T细胞的靶标。然而，所有方法均未能诱导LCL细胞中DRS-1基因的表达，这可能是由于B淋巴细胞固有的DRS-1表达抑制机制所致。因此，我们暂停了这项实验，并专注于另一种候选自身抗原，moesin。我们筛选了拥有少量 PNH 型细胞的 AA 患者血清中是否存在识别源自白血病细胞系 UT-7 的蛋白质的抗体 (Abs)。使用源自 UT-7 细胞裂解物或培养上清液的蛋白质进行免疫印迹分析，结果显示存在 80 kD 蛋白质特异性的 IgG Ab。肽质量指纹识别将此 80 kD 蛋白质鉴定为 moesin。使用重组 moesin 的酶联免疫吸附测定 (ELISA) 显示 67 名 AA 患者中的 25 名 (37%) 具有高滴度的抗 moesin 抗体。 Moesin 作为外泌体蛋白从 UT-7 以外的几种髓系白血病细胞系（例如 OUN-1 和 K562）中分泌。抗 moesin 抗体的存在与 PNH 型细胞和抗地西泮结合抑制剂相关蛋白 1 (DRS-1) 抗体的存在显着相关。未表现出这三种标志物中任何一种的 AA 患者往往对免疫抑制治疗反应不佳。这些发现表明，B 细胞对可能源自造血细胞的 moesin 的反应经常发生在 AA 患者中，并且检测抗 moesin 抗体与其他标记物的结合可能有助于诊断 AA 患者的免疫病理生理学。较少的