Relatively Low Sensitivity of CD109(-) Hematopoietic Stem/Progenitor Cells (HSPCs) to TGF-β: A Possible Mechanism Responsible for the Preferential Commitment of Piga Mutant HSPCs in Immune-Mediated Bone Marrow Failure

Abstract

[Background] An increase in the numbers of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] blood cells is often detected in patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS), and is associated with good response of their bone marrow (BM) failure to immunosuppressive therapy. Although some immune mechanisms are thought to play a role in the preferential commitment of hematopoietic stem/progenitor cells (HSPCs) with PIGA mutations in such BM failure patients, the exact mechanisms are unknown. Our previous studies suggested that GPI(-)T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) were less susceptible to TGF-ƒÀ-mediated inhibition of proliferation triggered by anti-CD3 and anti-CD28 antibodies than GPI(+)T cells of the same patient. The lower sensitivity of PIGA mutant HSPCs to TGF-ƒÀ, a cytokine capable of inhibiting the cell cycling of dormant HSPCs, than GPI(+) HSPCs may also explain the preferential commitment of GPI(-) HSPCs in immune-mediated BM failure. However, little is known about the GPI-APs that affect the sensitivity of HSPCs to TGF-ƒÀ.[Objectives/Methods] We assessed the roles of GPI-APs in the signal transduction of CD34(+) cells of a PNH patient and the myeloid leukemia cell line TF-1 in response to TGF-ƒÀ. We also assessed the TGF-ƒÀ-mediated inhibition of cell proliferation in TF-1 cells with or without a PIGA mutation. CD109, a GPI-AP that serves as a TGF-ƒÀ co-receptor in human keratinocytes, of TF-1 cells, was knocked out from TF-1 cells using a CRISPR-Cas 9 system, and the sensitivity to TGF-ƒÀ was compared between CD109(+) and CD109(-) TF-1 cells.[Results] The treatment of BM mononuclear cells from a florid PNH patient with TGF-ƒÀ induced SMAD2 phosphorylation in GPI(-) CD34(+) cells to a lesser degree than in GPI(+) CD34(+) cells (fold increase in pSMAD2 MFI, 1.0 vs. 2.6, Figure 1). TGF inhibited PIGA-mutant TF-1 (PNH-TF-1) proliferation to a lesser degree (percentage of inhibition, 19%}13%) than wild-type TF-1 cells (67%}3%) in an MTT-based proliferation assay. Transfection of PIGA into PNH-TF-1 cells restored GPI-AP expression as well as sensitivity to TGF-ƒÀ (53%}10% vs. 19%}13% in PNH-TF-1 cells). CD109 coimmunoprecipitated with TGF-ƒÀ in TF-1 cells, and its expression was confirmed on BM CD34+ cells of healthy individuals, particularly CD34+CD38+CD123-CD45RA- megakaryocyte-erythroid progenitor cells, as well as on TF-1 cells. The pSMAD2 induction in CD109(-) TF-1 cells by TGF-ƒÀ was less pronounced (relative increase in pSMAD2 MFI, 7.65}2.15 vs. 10.74}2.28) than that in CD109(+) TF-1 cells (Figure 2).[Conclusions] CD109 deficiency is involved in the lower sensitivity of GPI(-) HSPCs to TGF-ƒÀ than GPI(+) HSPCs. This deficiency may account for the preferential activation of PIGA mutant HSPCs in immune-mediated BM failure, in which TGF-ƒÀ suppresses activation of wild-type HSPCs. [Display omitted] [Display omitted] DisclosuresHosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.