Increased resistance of PIG-A- bone marrow progenitors to tumor necrosis factor α and interferon γ: Possible implications for the in vivo dominance of paroxysmal nocturnal hemoglobinuria clones

Background and Objectives. Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder due to a PIG-A gene mutation, resulting in deficient expression of GPI-anchored-proteins. Both immune-mediated suppression of hematopoiesis and cytokine alterations have been reported in aplastic anemia, a disease closely related to PNH whereas no data are available on PNH itself. The aim of this study was to investigate the effect of exogenous cytokines on clonogenic activity in PNH. Design and Methods. We evaluated burst-forming units-erythroid (BFU-E) and colonyforming units-granulocyte-macrophage (CFU-GM) in bone marrow mononuclear cells (BMMC) from 5 PNH patients and 5 controls, alone or in the presence of transforming-growth-factor (TGF)-β, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and specific antibodies. Molecular analysis of the PIG-A gene was performed by polymerase chain reaction (PCR) and direct sequencing on every single colony. Results. Patients' cells showed less clonogenic activity than did control cells. In PNH, addition of TGF-β inhibited both BFU-E and CFU-GM; IFN-γ and TNF-α inhibited BFU-E alone. In patients cytokines modulated normal and mutated clones differently: TGF-β reduced the number of PIG-A^- and PIG-A⁺ colony-forming-cells (CFC), whereas TNF-α and IFN-γ reduced PIG-A⁺ CFC only. BMMC from patients showed higher TGF-β production than did BMMC from controls. Interpretation and Conclusions. TGF-β could contribute to the genesis of the unfavorable bone marrow microenvironment but does not seem to play a role in the in vivo dominance of PIG-A deficient cells. Mutated clones were more resistant to the inhibitory effects of IFN-γ and TNF-α, suggesting that PNH cells may have a growth advantage in an unfavorable microenvironment.