Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH

William J. Savage, James P. Barber, Galina L. Mukhina, Rong Hu, Guibin Chen, William Matsui, Chris Thoburn, Allan D. Hess, Linzhao Cheng, Richard J. Jones, Robert A. Brodsky

Research output: Contribution to journalArticlepeer-review


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 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)-α, and γ-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-α, and γ-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-α. Conclusion: PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.

Original languageEnglish (US)
Pages (from-to)42-51.e1
JournalExperimental Hematology
Issue number1
StatePublished - Jan 2009

ASJC Scopus subject areas

  • Molecular Biology
  • Hematology
  • Genetics
  • Cell Biology
  • Cancer Research

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