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

为了确定地西潘结合抑制蛋白-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症(PNH)型干细胞的生存优势，我们建立了一株带有PIG-A突变的淋巴母细胞系，该细胞系来自一例表现出DRS-1特异性高滴度抗体的再生障碍性贫血(AA)患者。我们尝试用逆转录病毒载体和电穿孔等多种方法将DRS-1基因导入PNH型LCL细胞，作为DRS-1特异性CD4^+T细胞的靶细胞。然而，所有方法都未能诱导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，并将重点放在另一种候选自身抗原Moesin上。我们对拥有少量PNH类型细胞的AA患者的血清进行了筛选，以确定是否存在识别来自白血病细胞系UT-7的蛋白质的抗体(Abs)。免疫印迹…进一步使用来自UT-7细胞裂解产物或培养上清液的蛋白，发现存在80kD蛋白的特异性Ig G抗体。多肽质量指纹图谱鉴定该80kD蛋白为Moesin。应用重组moesin进行酶联免疫吸附试验(ELISA)，67例AA患者中有25例(37%)检测到高滴度的抗moesin抗体。Moesin是由UT-7以外的几个髓系白血病细胞株分泌的胞外蛋白，如OUN-1和K562。抗Moesin抗体的存在与PNH型细胞和抗安定结合抑制物相关蛋白-1(DRS-1)抗体的存在显著相关。没有显示这三个标志物中的任何一个的AA患者对免疫抑制治疗的反应往往很差。这些结果表明，再生障碍性贫血患者中经常发生B细胞对moesin的反应，可能来源于造血细胞，抗moesin抗体的检测与其他标志物的联合检测可能有助于AA患者的免疫病理生理诊断。较少

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