Glycosylphosphatidylinositol-anchored protein deficiency confers resistance to apoptosis in PNH

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.