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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CD4^+ T细胞对地西潘结合抑制剂蛋白-1 （DRS-1）的特异性是否有助于突发性夜间血红蛋白尿（PNH）型干细胞的生存优势，我们从再生障碍性贫血（AA）患者中建立了具有猪- a突变的淋巴样细胞系，该细胞系显示出对DRS-1的高滴度抗体。我们尝试用逆转录病毒载体和电穿孔等多种方法转染pnh型LCL细胞，将其作为DRS-1特异性CD4^+ T细胞的靶细胞。然而，所有方法均未能诱导LCL细胞中DRS-1基因的表达，这可能与B淋巴细胞固有的抑制DRS-1表达的机制有关。因此，我们暂停实验，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患者血清中存在的抗体（Abs），这些抗体可识别来自白血病细胞系UT-7的蛋白质。免疫印迹：使用UT-7细胞裂解液或培养上清液中提取的蛋白质进行更多的检测，发现存在针对80 kD蛋白的IgG抗体。肽质量指纹图谱鉴定该80 kD蛋白为moesin。重组moesin酶联免疫吸附试验（ELISA）显示，67例AA患者中25例（37%）抗moesin抗体滴度高。Moesin作为外泌体蛋白从除UT-7以外的几种髓系白血病细胞系（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）抗体的存在显著相关。未显示这三种标志物的AA患者对免疫抑制治疗的反应往往较差。这些发现表明，b细胞对moesin的反应，可能来源于造血细胞，经常发生在AA患者中，检测抗moesin抗体结合其他标志物可能有助于诊断AA患者的免疫病理生理。少