Resistance of Hematopoietic Progenitors to Fas-Mediated Apoptosis Is Actively Sustained by NFκB with a Characteristic Transcriptional Signature

Abstract

Umbilical cord blood (UCB) is a good source of hematopoietic progenitors with increasing implementation in the clinical transplant setting. This study evaluates the molecular mechanisms of progenitor resistance to apoptosis triggered by Fas cross-linking. CD34(+) and lineage-negative progenitors survive short-term ex vivo incubation and are not induced into apoptosis by Fas cross-linking. Furthermore, brief exposure of UCB cells to Fas-ligand for 24-48 h does not impair quantitative severe combine immune deficiency (SCID) reconstitution activity and appears to foster myelomonocyte reconstitution. The transcriptome of Fas receptor-positive CD34(+) cells that survived an apoptotic challenge showed significant transcriptional upregulation of caspase-8, mucosa-associated lymphoid tissue lymphoma translocation gene-1 (MALT1), HtrA2, and GSK3β in addition to higher levels of c-FLICE inhibitory protein (FLIP), Bcl-2, and cytosolic inhibitor of apoptosis protein (cIAP) in all Fas-positive cells. Most prominent is the transcriptional upregulation of several key components the NFκB1 pathway including the membrane receptors TGF-β, interleukin-1 (IL-1), and TCR, the associated factor TNF receptor-associated factor-6 (TRAF6), and the converting enzymes TGF-β-activated kinase-1 (TAK1), double-stranded RNA-activated protein kinase (PKR), and α-catalytic subunit of IκB kinase (IKKα), that promote activation and nuclear translocation of this transcription factor. These data indicate that hematopoietic progenitors are not insensitive to apoptosis but are actively shielded from the extrinsic and intrinsic apoptotic pathways. This may occur through inherent transcriptional upregulation of the entire NFκB pathway in the presence of competent apoptotic signaling.

Negative selection by apoptosis enriches progenitors in naïve and expanded human umbilical cord blood grafts

“…In contrast to apoptotic signaling in differentiated cells that mediates homeostatic negative regulation, murine and human precursors are inherently insensitive to apoptosis. [17][18][19][20] Signaling mediated by the Fas, TNF and TNF-Related Apoptosis-Inducing Ligand (TRAIL) receptors triggers progenitor activity and synergizes with other inductive factors in promoting hematopoietic differentiation. 19,21,22 This mechanism links injury signals, including TNF-family ligands, with the activation of the hematopoietic system to ensure prompt recovery from hypoplasia.…”

“…It also explains the marked upregulation of these receptors under conditions of stress hematopoiesis 23 and ubiquitous expression in most primitive progenitors. 17,20,21 The identified physiological roles of receptor/ligand interactions in the transplant setting include cell interaction with bone-marrow stroma 24 and autocrine-and paracrine-trophic signaling. [18][19][20][21][22] In addition to these early activities, hematopoietic progenitors deficient in Fas and TNF receptors fail to mediate durablemultilineage reconstitution.…”

“…17,20,21 The identified physiological roles of receptor/ligand interactions in the transplant setting include cell interaction with bone-marrow stroma 24 and autocrine-and paracrine-trophic signaling. [18][19][20][21][22] In addition to these early activities, hematopoietic progenitors deficient in Fas and TNF receptors fail to mediate durablemultilineage reconstitution. 17,18 Insensitivity of hematopoietic progenitors to apoptosis can be used to improve the outcome of transplants, conferring immune privilege to the graft 24,25 and fostering progenitor activity.…”

“…17,18 Insensitivity of hematopoietic progenitors to apoptosis can be used to improve the outcome of transplants, conferring immune privilege to the graft 24,25 and fostering progenitor activity. [19][20][21][22] A pretransplant apoptotic challenge with Fas-ligand (FasL) and TNF-α reduces significantly the incidence and severity of GvHD in haploidentical-and xenogeneic-murine transplants, 26,27 and improves early myeloid reconstitution. 20,22 In this study we assessed possible implementation of apoptotic signaling in several aspects of UCB cell transplantation.…”

“…[19][20][21][22] A pretransplant apoptotic challenge with Fas-ligand (FasL) and TNF-α reduces significantly the incidence and severity of GvHD in haploidentical-and xenogeneic-murine transplants, 26,27 and improves early myeloid reconstitution. 20,22 In this study we assessed possible implementation of apoptotic signaling in several aspects of UCB cell transplantation. Following documentation of the insensitivity of UCB progenitors to apoptotic signaling, 20 we assessed the influence of FasL, TNF-α and their combination on the various lineages as a possible approach to graft preparation for transplantation.…”