MECHANISM FOR SELECTIVE EXPANSION OF A PIG-A MUTANT IN PAROXYSMAL NOCTURNAL HEMOGLOBINEMIA

阵发性睡眠性血红蛋白尿症（PNH）是一种以溶血、静脉血栓形成和骨髓衰竭为特征的造血干细胞疾病。仅阐明了溶血机制。PNH细胞缺乏糖基磷脂酰肌醇锚定蛋白（GPI-AP），包括补体调节蛋白，如CD 59和CD 59，导致红细胞对补体溶解作用的敏感性增加。这种缺陷是由PIG-A基因的体细胞突变引起的。我们目前主要关注的是PNH克隆扩展的机制。越来越多的证据表明，突变的存在本身并不能诱导扩增。为了解释这个问题，提出了两个假设：增长或生存优势理论。后者表明PNH细胞在PNH患者中经常观察到的免疫介导的骨髓损伤的情况下逃避免疫攻击。本研究的目的是通过体外实验验证这种逃避机制来证明这一假说。 ...更多信息 通过转染PIG-A cDNA，使GPI-AP缺陷细胞系和对照细胞完全恢复GPI-AP表达，并使用13 Cr-释放测定法检测这些细胞对自然杀伤（NK）细胞杀伤的敏感性<51>。对于原代和培养的NK细胞，GPI-AP缺陷的细胞比它们的对照对应物更不敏感。NK活性完全废除与康卡那霉素A和钙螯合，表明杀伤preforin依赖。GPI-AP缺陷细胞和对照细胞之间的主要组织相容性I类表达或对纯化穿孔素或白细胞介素-2激活的NK细胞的敏感性没有差异。从这些结果中，我们推断GPI-AP缺陷细胞缺乏NK激活或触发穿孔素介导的杀伤所需的分子。我们的实验表明，PIG-A突变赋予相对生存优势的PNH克隆，有助于选择性扩增这些细胞的骨髓损伤的细胞毒性淋巴细胞的设置。(296减

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disease characterized by hemolysis, venous thrombosis and marrow failure. A mechanism of hemolysis has been only elucidated. PNH cells lack glycosylphosphatidylinositol-anchored proteins (GPI-AP) including compliment regulatory proteins such as DAF and CD59, leading to increased sensitivity of erythrocytes to the lytic action of complement. The deficiency is caused by a somatic mutation of the PIG-A gene. Our current major concern is the mechanism by which a PNH clone expands. Increasing evidence has been shown that the presence of the mutation alone does not induce the expansion. To explain this issue, two hypotheses are proposed: a growth or a survival advantage theory. The latter indicates that PNH cells escape immune attack in the setting of immune-mediated bone marrow injury often observed in PNH patients. The present study was aimed to prove this hypothesis by verifying the escape mechanism in vitro.We first p … More repared GPI-AP-deficient cell lines and control counterparts fully recovered with the expression of GPI-AP by transfection of PIG-A cDNA, and examined the sensitivity of these cells to killing by natural killer (NK) cells using ^<51>Cr-release assay. To both primary and cultured NK cells, GPI-AP-deficient cells were less susceptible than their control counterparts. NK activity was completely abolished with concanamycin A and by calcium chelation, indicating that killing was preforin-dependent. There were no differences in major histocompatibility classes I expression or sensitivity to either purified perforin or to interleukin-2-activated NK cells between GPI-AP-deficient cells and control cells. From these results we infer that GPI-AP-deficient cells lack molecules needed for NK activation or to trigger perforin-medicated killing. Our experiments suggest that PIG-A mutations confer a relative survival advantage to a PNH clone, contributing to selective expansion of these cells in the setting of marrow injury by cytotoxic lymphocytes. (296 words) Less