Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH

作者：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^*

来源：Experimental Hematology, 2009, 37(1): 42-51.

DOI：10.1016/j.exphem.2008.09.002

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.

• 出版日期2009-1