Savage William J, Barber James P, Mukhina Galina L, Hu Rong, Chen Guibin, Matsui William, Thoburn Chris, Hess Allan D, Cheng Linzhao, Jones Richard J, Brodsky Robert A
Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2196, USA.
Exp Hematol. 2009 Jan;37(1):42-51. doi: 10.1016/j.exphem.2008.09.002. Epub 2008 Nov 14.
Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).
Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow 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 interleukin-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.
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.
PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.
研究磷脂酰肌醇聚糖A（PIG-A）突变在阵发性睡眠性血红蛋白尿（PNH）克隆性扩增中的作用。
采用流式细胞术检测PNH患者的原发性CD34⁺造血祖细胞膜联蛋白-V阳性情况，在细胞介导的杀伤试验中使用PNH患者的自体效应细胞或健康对照的异体效应细胞。为了具体评估PIG-A突变在克隆优势发展中的作用，并解决可能混淆原代细胞实验的二次突变和差异免疫攻击等混杂因素，我们建立了一种可诱导的PIG-A CD34⁺髓系细胞系TF-1。在暴露于异体效应细胞、NK92细胞（一种具有活化自然杀伤细胞表型和功能的白细胞介素-2依赖细胞系）、肿瘤坏死因子（TNF）-α和γ射线照射后，评估细胞的抗凋亡能力。通过膜联蛋白-V染色和半胱天冬酶3/7活性检测细胞凋亡情况。
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⁺前体细胞对来自同一患者的自体（8%对49%；p<0.05）和异体（28%对58%；p<0.05）细胞介导的杀伤更不敏感。在可诱导的PIG-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射线照射后，比诱导产生的GPI-AP⁺ TF-1细胞表现出更少的凋亡。当效应细胞针对GPI-AP(-)细胞产生时，GPI-AP(-) TF-1细胞仍保持抗凋亡能力，这表明GPI-AP并非PNH中免疫攻击的靶点。NK92介导的杀伤作用被激活免疫效应细胞的应激诱导型GPI-AP ULBP1和ULBP2的特异性抗体部分抑制。克隆竞争实验表明，在TNF-α诱导的促凋亡条件下，突变克隆会随着时间的推移而扩增。
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，促进了PNH中的克隆性扩增。
