Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH
Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH
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DOI：
10.1016/j.exphem.2008.09.002
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发表时间：
2009-01-01
影响因子：
2.6
通讯作者：
Brodsky, Robert A.
中科院分区：
文献类型：
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作者：
Savage, William J.;Barber, James P.;Brodsky, Robert A.
目标。探讨猪- a突变对阵发性夜间血红蛋白尿（PNH）克隆扩增的贡献。材料与方法。使用来自PNH患者的自体效应物或来自健康对照的同种异体效应物，通过细胞介导的杀伤实验，采用How细胞术检测PNH患者的原代CD34（+）造血祖细胞的膜联蛋白v阳性。为了明确评估猪- a突变在克隆优势发展中的作用，并解决继发性突变和差异免疫攻击的混淆因素，这些因素可能会混淆原代细胞的实验，我们建立了一个可诱导的猪- a CD34（+）骨髓细胞系TF-1。在暴露于异体效应物、NK92细胞（一种依赖于白细胞介素2的细胞系，具有活化的自然杀伤[NK]细胞的表型和功能）、肿瘤坏死因子（tnf - α）和γ辐照后，评估细胞凋亡抵抗。annexin-V染色及caspase 3/7活性检测细胞凋亡。在PNH患者中，缺乏糖基磷脂酰肌醇（GPI）锚定蛋白（GPI- ap(-)）的CD34（+）造血祖细胞比GPI- ap (+) CD34（+）前体更容易受到来自同一患者的自体（8% vs 49%, p < 0.05）和异体（28% vs 58%, p < 0.05）细胞介导的杀伤。在诱导型猪-a模型中，GPI-AP(-) TF-1细胞在异体细胞介导的杀伤、nk92介导的杀伤、tnf - α和γ辐射的作用下，比诱导型GPI-AP(+) TF-1细胞表现出更少的凋亡。当GPI-AP（-）细胞产生效应物时，GPI-AP(-) TF-1细胞保持了对凋亡的抵抗，证明GPI-AP不是PNH免疫攻击的目标。应激诱导的GPI-AP ULBP1和ULBP2激活免疫效应物的特异性抗体阻断nk92介导的杀伤，可以部分抑制nk92介导的杀伤。克隆竞争实验表明，在tnf - α促凋亡的条件下，突变体克隆随着时间的推移而扩大。PIG-A突变通过赋予造血祖细胞在促凋亡应激下的生存优势，促进PNH的克隆扩增。(C) 2009 ISEH -血液学和干细胞学会。Elsevier Inc.出版。
Objective. Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).Materials and Methods. Primary CD34(+) hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by How cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34(+) myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interieukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity.Results. In PNH patients, CD34(+) hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP(+) CD34(+) precursors to autologous (8% vs 49%; p < 0.05) and allogeneic (28% vs 58%; p < 0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP(+) TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha.Conclusion. PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses. (C) 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.