Evelyne Lauret

Notch/Delta4 Interaction in Human Embryonic Liver CD34+ CD38− Cells: Positive Influence on BFU‐E Production and LTC‐IC Potential Maintenance

We investigated whether Notch signaling pathways have a role in human developmental hematopoiesis. In situ histochemistry analysis revealed that Notch1, 2, and 4 and Notch ligand (Delta1–4, and Jagged1) proteins were not expressed in the yolk sac blood islands, the para‐aortic splanchnopleure, the hematopoietic aortic clusters, and at the early stages of embryonic liver hematopoiesis. Notch1–2, and Delta4 were eventually detected in the embryonic liver, from 34 until 38 days postconception. Fluorescence‐activated cell sorter analysis showed that first‐trimester embryonic liver CD34+CD38low cells expressed both Notch1 and Notch2. When these cells were cultured on S17 stroma stably expressing Delta4, a 2.6‐fold increase in BFU‐E number was observed at day 7, as compared with cultures with control stroma, and this effect was maintained for 2 weeks. Importantly, exposure of these cells to Delta4 under these conditions maintained the original frequency and quality of long‐term culture‐initiating cells (LTC‐ICs), while control cultures quickly resulted in the extinction of this LTC‐IC potential. Furthermore, short‐term exposure of embryonic liver adherent cells to erythropoietin resulted in a dose‐dependent increase in Delta4 expression, almost doubling the expression observed with untreated stroma. This suggests that Delta4 has a role in the regulation of hematopoiesis after a hypoxic stress in the fetus.

Telomere dysfunction and telomerase reactivation in human leukemia cell lines after telomerase inhibition by the expression of a dominant-negative hTERT mutant.

As activation of telomerase represents a key step in the malignant transformation process, experimental models to develop anti-telomerase drugs provide a rational basis for anticancer strategies. We analysed the short and long-term efficacy of a stably expressed dominant-negative mutant (DN) of the telomerase catalytic unit (hTERT) in UT-7 and U937 human leukemia cell lines by using an IRES-e-GFP retrovirus. As expected, telomerase inactivation resulted in drastic telomere shortening, cytogenetic instability and cell growth inhibition in all e-GFP positive DN clones after 15–35 days of culture. However, despite this initial response, 50% of e-GFP positive DN clones with short telomeres escaped from crisis after 35 days of culture and recovered a proliferation rate similar to the control cells. This rescue was associated with a telomerase reactivation inducing telomere lengthening. We identified two pathways, one involving the loss of the DN transgene expression and the other the transcriptional up-regulation of endogenous hTERT with persistence of the DN transgene expression. Although this second mechanism appears to be a very rare event (one clone), these findings suggest that genomic instability induced by short telomeres after telomerase inhibition might enhance the probability of activation or selection of telomere maintenance mechanisms dependent on hTERT transcription.

Distinct Effects of the Soluble Versus Membrane‐Bound Forms of the Notch Ligand Delta‐4 on Human CD34+CD38low Cell Expansion and Differentiation

Although Notch ligands are considered to activate signaling through direct cell‐cell contact, the existence of soluble forms has been demonstrated. However, their roles remain controversial: soluble forms have been reported to mimic the biological activity of membrane‐bound form, whereas other studies rather suggested an antagonistic activity toward their full‐length counterparts. We previously observed that membrane‐bound Delta4‐expressing S17 stroma (mbD4/S17) reduced human CD34+CD38low cell proliferation and favored self‐renewal. Here, we assessed the effects of a soluble form of Delta4 (solD4) by exposing CD34+CD38low cells to S17 feeders engineered to express solD4 (solD4/S17). In contrast to mbD4/S17, (a) solD4/S17 increased 10‐fold cell production after 2 weeks, through enhanced cell proliferation, and (b) it did not preserve colony‐forming cell and long‐term culture‐initiating cell potential of output CD34+ cells. mbD4 and solD4 appeared to also differ in their signaling. Indeed, mbD4, but not solD4, strongly activated both CSL (the nuclear mediator of Notch signaling) in Hela cells overexpressing Notch1 and transcription of some classic Notch target genes in CD34+CD38low cells. Furthermore, both biological effects and CSL activation elicited by mbD4 were strictly dependent upon the γ‐secretase complex, whereas solD4 enhanced cell expansion in a partially γ‐secretase‐independent manner. Altogether, these results suggest that part of solD4 activity did not rely upon canonical Notch pathway.

MACAQUE LYMPHOCYTES TRANSDUCED BY A CONSTITUTIVELY EXPRESSED INTERFERON BETA GENE DISPLAY AN ENHANCED RESISTANCE TO SIVMAC251 INFECTION

We are developing a method of gene therapy of HIV infection based on the low constitutive expression of an interferon beta (IFN-beta) gene in HIV target cells. Herein we report the first step in the development of a relevant animal model, provided by the macaque (Macaca fascicularis) infected with a pathogenic SIVmac251 isolate. To avoid the possibility of in vivo rejection of macaque lymphocytes expressing Hu IFN-beta, we have PCR-amplified and sequenced the Ma IFN-beta-coding sequence, and placed it under the control of a PstI-NruI 0.6-kb fragment of the murine H-2Kb gene promoter in the MFG-K(b)MaIFNbeta retroviral vector. Lymphocytic CEMX174 cells, transduced by coculture on packaging cells with this construct, harbored a mean of 0.07 to 1.2 copies of the IFN-beta transgene per cell, and were characterized by an IFN production ranging from 75 to 750 units per 5 x 10(5) cells per 3 days. The IFN-beta-transduced populations displayed an enhanced resistance against the pathogenic SIVmac251 isolate. Control experiments showed that the enhanced resistance could not be ascribed to the Ma IFN-beta released during the 3 days of coculture by the packaging cells, or to the mere transduction with a retroviral vector. Macaque lymphocytes transduced by the MFG-K(b)MaIFNbeta retroviral vector by coculture on packaging cells, acquired a mean number of IFN-beta transgene copies per cell ranging from 0.03 to 0.1. Such transduction led to the release of IFN-beta into the culture medium, ranging from 10 to 20 units per 5 x 10(5) cells per 3 days. This increased the anti-SIV resistance of the lymphocytes, as demonstrated by a decreased p27 antigen release into the culture medium, without affecting lymphocyte proliferation.

CXCR4/CXCL12 axis counteracts hematopoietic stem cell exhaustion through selective protection against oxidative stress

Hematopoietic stem cells (HSCs) undergo self-renewal to maintain hematopoietic homeostasis for lifetime, which is regulated by the bone marrow (BM) microenvironment. The chemokine receptor CXCR4 and its ligand CXCL12 are critical factors supporting quiescence and BM retention of HSCs. Here, we report an unknown function of CXCR4/CXCL12 axis in the protection of HSCs against oxidative stress. Disruption of CXCR4 receptor in mice leads to increased endogenous production of reactive oxygen species (ROS), resulting in p38 MAPK activation, increased DNA double-strand breaks and apoptosis leading to marked reduction in HSC repopulating potential. Increased ROS levels are directly responsible for exhaustion of the HSC pool and are not linked to loss of quiescence of CXCR4-deficient HSCs. Furthermore, we report that CXCL12 has a direct rescue effect on oxidative stress-induced HSC damage at the mitochondrial level. These data highlight the importance of CXCR4/CXCL12 axis in the regulation of lifespan of HSCs by limiting ROS generation and genotoxic stress.

Low autocrine interferon beta production as a gene therapy approach for AIDS: Infusion of interferon beta-engineered lymphocytes in macaques chronically infected with SIVmac251

BackgroundThe aim of this study was to evaluate gene therapy for AIDS based on the transduction of circulating lymphocytes with a retroviral vector giving low levels of constitutive macaque interferon β production in macaques chronically infected with a pathogenic isolate of SIVmac251.ResultsTwo groups of three animals infected for more than one year with a pathogenic primary isolate of SIVmac251 were included in this study. The macaques received three infusions of their own lymphocytes transduced ex vivo with the construct encoding macaque IFN-β (MaIFN-β or with a vector carrying a version of the MaIFN-β gene with a deletion preventing translation of the mRNA. Cellular or plasma viremia increased transiently following injection in most cases, regardless of the retroviral construct used. Transduced cells were detected only transiently after each infusion, among the peripheral blood mononuclear cells of all the animals, with copy numbers of 10 to 1000 per 106 peripheral mononuclear cells.ConclusionLong-term follow-up indicated that the transitory presence of such a small number of cells producing such small amounts of MaIFN-β did not prevent animals from the progressive decrease in CD4+ cell count typical of infection with simian immunodeficiency virus. These results reveal potential pitfalls for future developments of gene therapy strategies of HIV infection.

Further studies on the mechanism of CFU-S determination—I. Pluripoietin(s) responsible for CFU-S determination toward granulopoiesis after fractionated doses of ARA-C

Fractionated doses of Ara-C orient CFU-S differentiation towards granulopoiesis and megakaryocytopoiesis in vivo in contrast to the preferential channelling towards erythropoiesis after a similar dose of Ara-C given as a single injection. In this paper, we show that pluripoietin found in the serum of mice treated with fractionated doses of Ara-C is responsible for the choice of CFU-S differentiation towards granulopoiesis. Therefore, we confirm our previous results involving single doses of Ara-C, that humoral factors can commit CFU-S preferentially towards one of the cell lineages. It is not as yet known whether there is a specific pluripoietin for each cell lineage or whether the same pluripoietin has a different effect according to its concentration in the organism. The difference in response of CFU-S commitment to single and fractionated doses of Ara-C is not due to the difference in the kinetic status of CFU-S:CFU-S are cycling after both treatments.

Modifications of CFU-S differentiation pathways by pluripoietins: Influence of treatment protocols

Similar total doses of Ara-C given as a single injection or given in a fractionated protocol have reverse effects on CFU-S differentiation pathways. Whereas a single dose of 20 mg channels CFU-S towards erythropoiesis, 5 X 5 mg given at 8 or 24 hour intervals channels CFU-S to granulopoiesis and megakaryocytopoiesis. It therefore seems possible to manipulate CFU-S differentiation not only by varying the inducing agents but also by varying the protocols using the same agent. Hypotheses to explain the mechanisms of CFU-S regulation are presented.

Further studies on the mechanism of CFU-S determination—II. Feedback regulation

In previous papers, we have demonstrated that the determination of CFU-S differentiation is under the control of humoral factors, which we have called pluripoietins. The activity of these regulators varies according to the experimental protocol. The aim of this work was to determine the mechanisms involved in the regulation of pluripoietin secretion after each treatment. The results suggest that the production of pluripoietin(s) is under the control of a feedback mechanism, originating, at least in part, in the progenitor compartments. After one dose of 20 mg of Ara-C, CFU-S determination is channelled towards erythropoiesis when the BFU-E compartment is more depleted than the GM-CFC compartment. During fractionated doses of Ara-C, at the time of the third injection, GM-CFC survival is lower than BFU-E survival and CFU-S orient their differentiation towards granulopoiesis. After bleeding, there is no difference of survival between the two compartments and CFU-S determination is not changed. These results seem to indicate that CFU-S determination is modified in order to restore normal homeostasis of the hemopoietic tissues by preferentially replenishing the more depleted compartment. This is most likely achieved by the secretion of pluripoietin(s) that vary in either their nature or their concentration according to the events occurring in the more mature compartments.

Further studies on the mechanism of CFU-S determination—III. CFU-S cell lineage determination is not influenced by GM-CSF, EPO, multi-CSF nor WEHI 3B conditioned medium

CFU-S proliferation is under the control of factors such as stimulators and inhibitors whereas commitment of CFU-S is controlled by pluripoietins. We have studied the effects in vitro of Epo, GM-CSF, multi-CSF and WEHI 3B conditioned medium in order to elucidate their eventual role on CFU-S proliferation and cell lineage determination. Neither Epo nor GM-CSF are able to induce the entry of CFU-S into cycle. Multi-CSF and WEHI 3B conditioned medium (similar to interleukin 3) stimulate CFU-S into DNA synthesis. However, we were able to show that the stimulator we have studied is different from IL 3. Epo, GM-CSF, multi-CSF and WEHI 3B CM have no effect on CFU-S commitment. Therefore, these four hemopoietic regulators are different from the pluripoietins we have previously described and have no influence on CFU-S determination.