title: Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH.
abstract: OBJECTIVE
  Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).
  
  
  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)-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.
  
  
  CONCLUSIONS
  PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.
authors.affiliation: Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2196, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Savage
authors.forename: James P
authors.initials: JP
authors.lastname: Barber
authors.forename: Galina L
authors.initials: GL
authors.lastname: Mukhina
authors.forename: Rong
authors.initials: R
authors.lastname: Hu
authors.forename: Guibin
authors.initials: G
authors.lastname: Chen
authors.forename: William
authors.initials: W
authors.lastname: Matsui
authors.forename: Chris
authors.initials: C
authors.lastname: Thoburn
authors.forename: Allan D
authors.initials: AD
authors.lastname: Hess
authors.forename: Linzhao
authors.initials: L
authors.lastname: Cheng
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Jones
authors.forename: Robert A
authors.initials: RA
authors.lastname: Brodsky
mesh_terms: D000328:Adult; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053179:Caspase 7 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005260:Female; D005720:Gamma Rays; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006457:Hemoglobinuria, Paroxysmal / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D009154:Mutation; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D017304:Annexin A5; D008565:Membrane Proteins; D014409:Tumor Necrosis Factor-alpha; C080086:phosphatidylinositol glycan-class A protein; C505407:CASP3 protein, human; D053148:Caspase 3; D053179:Caspase 7