GPI-Anchored Protein Deficiency Confers Resistance to Apoptosis in PNH.
Authors: Savage William J , Barber James , Cheng Linzhao , Chen Guiben , Thoburn Christopher , Hu Rong , Mukhina Galina , Matsui William , Hess Allan , Jones Richard , Brodsky Robert A. 
Published: April 2020

Abstract
OBJECTIVE: Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).
INTRODUCTION: Whether PNH cells are inherently less susceptible to apoptosis remains controversial. Studies using PNH mouse models and lymphoid cell lines have failed to find a difference in apoptosis, but reports using K562 cells have found a relative resistance. Studies using primary CD34+ cells from PNH patients have also found a relative resistance of PNH cells, but not in comparison to CD34+ cells from normal controls. We designed experiments 1) to control for genetic heterogeneity among PNH and control cell lines, 2) to address the potential methodological issues associated with studying lymphocytes, which are rarely affected in PNH, and 3) address inconsistency comparing normal and PNH progenitors that have experienced different in vivo environments.
METHODS: GPI-anchored protein (GPI-AP) positive and negative 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 selection in vivo, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Using a doxycycline-inducible wild type PIG-A tet-SUPER transgene expression system, in which GPI-AP− and GPI-AP+ cells are isogenic, we assessed apoptosis resistance and clonal expansion in response to various pro-apoptotic stimuli. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 effectors, TNF-α, and γ-irradiation. Apoptosis was measured by annexin V/PI staining (effector experiments) or caspase 3/7 activity (TNF-α and γ-irradiation experiments). Blocking experiments of NK92-mediated killing utilized mAb to ULBP1 and ULBP2, as per Hanaoka et al. Clonal competition experiments tracked wild type and PIG-A mutant TF-1 cells using flow cytometry after exposure to TNF-α as a surrogate for immune attack.
RESULTS: In PNH patients, GPI-AP− CD34+ hematopoietic progenitors were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% versus 49%, p&lt;0.05) and allogeneic (28% versus 58%, p&lt;0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP− TF-1 cells exhibited less apoptosis than GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing (35% less), NK92-mediated killing (24% less), TNF-α (14% less), and γ-irradiation (24% less). All differences were statistically significant by the paired t test with p&lt;0.05. For allogeneic effector killing experiments, 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. In the NK92-mediated killing model, 24% of the apoptosis resistance was lost with ULBP1 and ULBP2 blockade (p&lt;0.05). No change in ULBP1 and ULBP2 expression was observed in TNF-α and γ-irradiation experiments. Clonal competition experiments demonstrate that the mutant clone expands by 72% over two days under pro-apoptotic conditions with TNF-α (p&lt;0.05).
CONCLUSIONS: PIG-A mutations contribute to the clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under pro-apoptotic stresses. Lack of GPI-anchored ULBP1 and ULBP2 expression contributes to the apoptosis resistance in a cell-mediated killing model but is not solely responsible for the apoptosis resistance. The global apoptosis resistance translated into expansion of the PNH clone in an in vitro model of immune attack.