MECHANISM FOR SELECTIVE EXPANSION OF A PIG-A MUTANT IN PAROXYSMAL NOCTURNAL HEMOGLOBINEMIA

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Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disease characterized by hemolysis, venous thrombosis and marrow failure. A mechanism of hemolysis has been only elucidated. PNH cells lack glycosylphosphatidylinositol-anchored proteins (GPI-AP) including compliment regulatory proteins such as DAF and CD59, leading to increased sensitivity of erythrocytes to the lytic action of complement. The deficiency is caused by a somatic mutation of the PIG-A gene. Our current major concern is the mechanism by which a PNH clone expands. Increasing evidence has been shown that the presence of the mutation alone does not induce the expansion. To explain this issue, two hypotheses are proposed: a growth or a survival advantage theory. The latter indicates that PNH cells escape immune attack in the setting of immune-mediated bone marrow injury often observed in PNH patients. The present study was aimed to prove this hypothesis by verifying the escape mechanism in vitro.We first p … More repared GPI-AP-deficient cell lines and control counterparts fully recovered with the expression of GPI-AP by transfection of PIG-A cDNA, and examined the sensitivity of these cells to killing by natural killer (NK) cells using ^<51>Cr-release assay. To both primary and cultured NK cells, GPI-AP-deficient cells were less susceptible than their control counterparts. NK activity was completely abolished with concanamycin A and by calcium chelation, indicating that killing was preforin-dependent. There were no differences in major histocompatibility classes I expression or sensitivity to either purified perforin or to interleukin-2-activated NK cells between GPI-AP-deficient cells and control cells. From these results we infer that GPI-AP-deficient cells lack molecules needed for NK activation or to trigger perforin-medicated killing. Our experiments suggest that PIG-A mutations confer a relative survival advantage to a PNH clone, contributing to selective expansion of these cells in the setting of marrow injury by cytotoxic lymphocytes. (296 words) Less