Genomic and functional impact of Trp53 inactivation in JAK2V617F myeloproliferative neoplasms.

Classical myeloproliferative neoplasms (MPNs) are characterized by the proliferation of myeloid cells and the risk of transformation into myelofibrosis or acute myeloid leukemia (AML) and TP53 mutations in MPN patients are linked to AML. However, JAK2V617F has been reported to impact the TP53 response to DNA damage, suggesting potential overlapping role of TP53 inactivation in MPN. We established a mouse model showing that JAK2V617F/Vav-Cre/Trp53-/- mice displayed a similar phenotype to JAK2V617F/Vav-Cre mice, but their proliferation was outcompeted in competitive grafts. RNA-Seq revealed that half of the genes affected by JAK2V617F were affected by p53-inactivation, including the interferon pathway. To validate this finding, mice were repopulated with a mixture of wild-type and JAK2V617F (or JAK2V617F/Vav-Cre/Trp53-/-) cells and treated with pegylated interferonα. JAK2V617F-reconstituted mice entered complete hematological remission, while JAK2V617F/Vav-Cre /Trp53-/--reconstituted mice did not, confirming that p53 loss induced interferon-α resistance. KEGG and Gene Ontology analyses of common deregulated genes showed that these genes were mainly implicated in cytokine response, proliferation, and leukemia evolution, illustrating that in this mouse model, the development of MPN is not affected by TP53 inactivation. Taken together, our results show that many genetic modifications induced by JAK2V617F are influenced by TP53, the MPN phenotype may not be. Trp53 loss alone is insufficient to induce rapid leukemic transformation in steady-state hematopoiesis in JAK2V617F MPN, and Trp53 loss may contribute to interferon resistance in MPN.

FOXP1 regulates oxidative stress, SIRT1 expression, and resistance to chemotherapies in acute myeloid leukemia cells.

Transcription factor Forkhead box P1 (FOXP1) belongs to the same protein family as the FOXOs that are well-known regulators of murine hematopoietic stem progenitor cell (HSPC) maintenance via dampening oxidative stress. FOXP1 and FOXOs can play opposite, or similar, roles depending on cell context; they can crossregulate each other's expression. In a previous study, we have shown that FOXP1 contributes to healthy human HSPC and acute myeloid leukemia (AML) cell growth. Here, we investigated the role of FOXP1 in HSPCs and AML cell oxidative stress defense in a human context. FOXP1 expression level was associated with an inferior survival outcome in patients with cytogenetically normal AML. FOXP1 knockdown enhanced superoxide anion levels of human-committed CD34+CD38+ cells but not stem cell-enriched CD34+CD38- HSPCs or AML cells in vitro. FOXP1 knockdown triggered enhanced NRF2 activity and increased cell oxidative stress. FOXP1 had no impact on FOXO1/3/4 expression in these cells; genetic and pharmacological inhibition of FOXOs did not change superoxide anion levels of human HSPCs or AML cells. Moreover, FOXP1 antioxidant activity was independent of changes in expression of superoxide dismutase 1 and 2 or catalase. Instead, FOXP1 upregulated expression of the stress sensor SIRT1 by stabilizing SIRT1 protein. FOXP1 loss sensitized AML cells to chemotherapy. Together, this study identified FOXP1 as a new safeguard against myeloid progenitor oxidative stress, which works independently of FOXOs but through SIRT1 and contributes to AML chemoresistance. It proposes FOXP1 expression/activity as a promising target to overcome drug resistance of AML HSPCs.

The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cells expansion.

The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFß-dependent and mediated by SMAD7, a TGFß- signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5'-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFß superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.

Multistage hematopoietic stem cell regulation in the mouse: A combined biological and mathematical approach.

We have reconciled steady-state and stress hematopoiesis in a single mathematical model based on murine in vivo experiments and with a focus on hematopoietic stem and progenitor cells. A phenylhydrazine stress was first applied to mice. A reduced cell number in each progenitor compartment was evidenced during the next 7 days through a drastic level of differentiation without proliferation, followed by a huge proliferative response in all compartments including long-term hematopoietic stem cells, before a return to normal levels. Data analysis led to the addition to the 6-compartment model, of time-dependent regulation that depended indirectly on the compartment sizes. The resulting model was finely calibrated using a stochastic optimization algorithm and could reproduce biological data in silico when applied to different stress conditions (bleeding, chemotherapy, HSC depletion). In conclusion, our multi-step and time-dependent model of immature hematopoiesis provides new avenues to a better understanding of both normal and pathological hematopoiesis.

Mitochondrial dynamics and reactive oxygen species initiate thrombopoiesis from mature megakaryocytes.

Blood platelets are essential for controlling hemostasis. They are released by megakaryocytes (MKs) located in the bone marrow, upon extension of cytoplasmic protrusions into the lumen of bone marrow sinusoids. Their number increases in postpulmonary capillaries, suggesting a role for oxygen gradient in thrombopoiesis (ie, platelet biogenesis). In this study, we show that initiation of thrombopoiesis from human mature MKs was enhanced under hyperoxia or during pro-oxidant treatments, whereas antioxidants dampened it. Quenching mitochondrial reactive oxygen species (mtROS) with MitoTEMPO decreased thrombopoiesis, whereas genetically enhancing mtROS by deacetylation-null sirtuin-3 expression increased it. Blocking cytosolic ROS production by NOX inhibitors had no impact. Classification according to the cell roundness index delineated 3 stages of thrombopoiesis in mature MKs. Early-stage round MKs exhibited the highest index, which correlated with low mtROS levels, a mitochondrial tubular network, and the mitochondrial recruitment of the fission activator Drp1. Intermediate MKs at the onset of thrombopoiesis showed high mtROS levels and small, well-delineated mitochondria. Terminal MKs showed the lowest roundness index and long proplatelet extensions. Inhibiting Drp1-dependent mitochondrial fission of mature MKs by Mdivi-1 favored a tubular mitochondrial network and lowered both mtROS levels and intermediate MKs proportion, whereas enhancing Drp1 activity genetically had opposite effects. Reciprocally, quenching mtROS limited mitochondrial fission in round MKs. These data demonstrate a functional coupling between ROS and mitochondrial fission in MKs, which is crucial for the onset of thrombopoiesis. They provide new molecular cues that control initiation of platelet biogenesis and may help elucidate some unexplained thrombocytopenia.

Transgenic Mice Expressing Human Proteinase 3 Exhibit Sustained Neutrophil-Associated Peritonitis.

Proteinase 3 (PR3) is a myeloid serine protease expressed in neutrophils, monocytes, and macrophages. PR3 has a number of well-characterized proinflammatory functions, including cleaving and activating chemokines and controlling cell survival and proliferation. When presented on the surface of apoptotic neutrophils, PR3 can disrupt the normal anti-inflammatory reprogramming of macrophages following the phagocytosis of apoptotic cells. To better understand the function of PR3 in vivo, we generated a human PR3 transgenic mouse (hPR3Tg). During zymosan-induced peritonitis, hPR3Tg displayed an increased accumulation of neutrophils within the peritoneal cavity compared with wild-type control mice, with no difference in the recruitment of macrophages or B or T lymphocytes. Mice were also subjected to cecum ligation and puncture, a model used to induce peritoneal inflammation through infection. hPR3Tg displayed decreased survival rates in acute sepsis, associated with increased neutrophil extravasation. The decreased survival and increased neutrophil accumulation were associated with the cleavage of annexin A1, a powerful anti-inflammatory protein known to facilitate the resolution of inflammation. Additionally, neutrophils from hPR3Tg displayed enhanced survival during apoptosis compared with controls, and this may also contribute to the increased accumulation observed during the later stages of inflammation. Taken together, our data suggest that human PR3 plays a proinflammatory role during acute inflammatory responses by affecting neutrophil accumulation, survival, and the resolution of inflammation.

PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells.

RNA-binding proteins (RBPs) have emerged as important regulators of invertebrate adult stem cells, but their activities remain poorly appreciated in mammals. Using a short hairpin RNA strategy, we demonstrate here that the 2 mammalian RBPs, PUMILIO (PUM)1 and PUM2, members of the PUF family of posttranscriptional regulators, are essential for hematopoietic stem/progenitor cell (HSPC) proliferation and survival in vitro and in vivo upon reconstitution assays. Moreover, we found that PUM1/2 sustain myeloid leukemic cell growth. Through a proteomic approach, we identified the FOXP1 transcription factor as a new target of PUM1/2. Contrary to its canonical repressive activity, PUM1/2 rather promote FOXP1 expression by a direct binding to 2 canonical PUM responsive elements present in the FOXP1-3' untranslated region (UTR). Expression of FOXP1 strongly correlates with PUM1 and PUM2 levels in primary HSPCs and myeloid leukemia cells. We demonstrate that FOXP1 by itself supports HSPC and leukemic cell growth, thus mimicking PUM activities. Mechanistically, FOXP1 represses the expression of the p21-CIP1 and p27-KIP1 cell cycle inhibitors. Enforced FOXP1 expression reverses shPUM antiproliferative and proapoptotic activities. Altogether, our results reveal a novel regulatory pathway, underscoring a previously unknown and interconnected key role of PUM1/2 and FOXP1 in regulating normal HSPC and leukemic cell growth.

Architectural and functional heterogeneity of hematopoietic stem/progenitor cells in non-del(5q) myelodysplastic syndromes.

Myelodysplastic syndromes (MDSs) are hematopoietic stem cell disorders in which recurrent mutations define clonal hematopoiesis. The origin of the phenotypic diversity of non-del(5q) MDS remains unclear. Here, we investigated the clonal architecture of the CD34+CD38- hematopoietic stem/progenitor cell (HSPC) compartment and interrogated dominant clones for MDS-initiating cells. We found that clones mainly accumulate mutations in a linear succession with retention of a dominant subclone. The clone detected in the long-term culture-initiating cell compartment that reconstitutes short-term human hematopoiesis in xenotransplantation models is usually the dominant clone, which gives rise to the myeloid and to a lesser extent to the lymphoid lineage. The pattern of mutations may differ between common myeloid progenitors (CMPs), granulomonocytic progenitors (GMPs), and megakaryocytic-erythroid progenitors (MEPs). Rare STAG2 mutations can amplify at the level of GMPs, from which it may drive the transformation to acute myeloid leukemia. We report that major truncating BCOR gene mutation affecting HSPC and CMP was beneath the threshold of detection in GMP or MEP. Consistently, BCOR knock-down (KD) in normal CD34+ progenitors modifies their granulocytic and erythroid differentiation. Clonal architecture of the HSPC compartment and mutations selected during differentiation contribute to the phenotypic heterogeneity of MDS. Defining the hierarchy of driver mutations provides insights into the process of transformation and may guide the search for novel therapeutic strategies.

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.

The Notch Delta-4 ligand helps to maintain the quiescence and the short-term reconstitutive potential of haematopoietic progenitor cells through activation of a key gene network.

Understanding the role of Notch and its ligands within the different bone marrow niches could shed light on the mechanisms regulating haematopoietic progenitor cells (HPCs) maintenance and self-renewal. Here, we report that murine bone marrow HPCs activation by the vascular Notch Delta-4 ligand maintains a significant proportion of cells specifically in the G0 state. Furthermore, Delta-4/Notch pathway limits significantly the loss of the in vivo short-term reconstitutive potential upon transplantation of Delta-4 activated HPCs into lethally irradiated recipient mice. Both effects are directly correlated with the decrease of cell cycle genes transcription such as CYCLIN-D1, -D2, and -D3, and the upregulation of stemness related genes transcription such as BMI1, GATA2, HOXB4 and C-MYC. In addition, the transcriptional screening also highlights new downstream post-transcriptional factors, named PUMILIO1 and -2, as part of the stem signature associated with the Delta-4/Notch signalling pathway.

Differential contributions of STAT5A and STAT5B to stress protection and tyrosine kinase inhibitor resistance of chronic myeloid leukemia stem/progenitor cells.

STAT5 fulfills essential roles in hematopoietic stem cell (HSC) self-renewal and chronic myeloid leukemia (CML), a prototypical stem cell malignancy. However, the specific contributions of the two related genes STAT5A and STAT5B have not been determined. In this study, we used a RNAi-based strategy to establish participation of these genes to CML disease and persistence following targeted therapy. We showed that STAT5A/STAT5B double-knockdown triggers CML cell apoptosis and suppresses both normal and CML HSC long-term clonogenic potential. STAT5A and STAT5B exhibited similar prosurvival activity, but STAT5A attenuation alone was ineffective at impairing growth of normal and CML CD34(+) cells isolated at diagnosis. In contrast, STAT5A attenuation was sufficient to enhance basal oxidative stress and DNA damage of normal CD34(+) and CML cells. Furthermore, it weakened the ability to manage exogenous oxidative stress, increased p53 (TRP53)/CHK-2 (CHEK2) stress pathway activation, and enhanced prolyl hydroxylase domain (PHD)-3 (EGLN3) mRNA expression. Only STAT5A and its transactivation domain-deficient mutant STAT5AΔ749 specifically rescued these activities. STAT5A attenuation was also active at inhibiting growth of CML CD34(+) cells from patients with acquired resistance to imatinib. Our findings show that STAT5A has a selective role in contributing to stress resistance through unconventional mechanisms, offering new opportunities to eradicate the most primitive and tyrosine kinase inhibitor-resistant CML cells with an additional potential to eradicate persistent stem cell populations.

Cotargeting signaling pathways driving survival and cell cycle circumvents resistance to Kit inhibitors in leukemia.

Oncogenic mutations leading to persistent kinase activities are associated with malignancies. Therefore, deciphering the signaling networks downstream of these oncogenic stimuli remains a challenge to gather insights into targeted therapy. To elucidate the biochemical networks connecting the Kit mutant to leukemogenesis, in the present study, we performed a global profiling of tyrosine-phosphorylated proteins from mutant Kit-driven murine leukemia proerythroblasts and identified Shp2 and Stat5 as proximal effectors of Kit. Shp2 or Stat5 gene depletion by sh-RNA, combined with pharmacologic inhibition of PI3kinase or Mek/Erk activities, revealed 2 distinct and independent signaling pathways contributing to malignancy. We demonstrate that cell survival is driven by the Kit/Shp2/Ras/Mek/Erk1/2 pathway, whereas the G(1)/S transition during the cell cycle is accelerated by both the Kit/Stat5 and Kit/PI3K/Akt pathways. The combined use of the clinically relevant drugs NVP-BEZ235, which targets the cell cycle, and Obatoclax, which targets survival, demonstrated synergistic effects to inhibit leukemia cell growth. This synergy was confirmed with a human mast leukemia cell line (HMC-1.2) that expresses mutant Kit. The results of the present study using liquid chromatography/tandem mass spectrometry analysis have elucidated signaling networks downstream of an oncogenic kinase, providing a molecular rationale for pathway-targeted therapy to treat cancer cells refractory to tyrosine kinase inhibitors.

Notch/Delta4 signaling inhibits human megakaryocytic terminal differentiation.

The effects of Notch signaling on human megakaryocytic and erythroid differentiation were investigated by exposing human CD34(+) progenitor cells to an immobilized chimeric form of the Notch ligand, Delta-like4 (Dll4Fc). Exposure of human cord blood CD34(+) cells to Dll4Fc induced a modest enhancement of erythroid cell production. Conversely, under megakaryocytic culture conditions, Dll4Fc strongly impaired platelet production by reducing the generation of mature CD41a(+)CD42b(+) megakaryocytes (MKs) and platelet-forming cells. The inhibitory activity of Dll4 on terminal MK differentiation was confirmed by culturing CD34(+) cells onto Dll-4-expressing stroma cells (engineered to express the membrane-anchored form of Dll4). The reduced production of mature CD41a(+)CD42(+) cells was rescued by inhibiting Notch signaling either with the N-N-(3,5-difluorophenacetyl-L-alanyl)-S-phenylglycine t-butyl ester γ-secretase inhibitor or the dominant-negative version of Mastermind. Dll4 impaired the generation of mature CD41a(+)CD42b(+) cells and proplatelet formation without affecting earlier steps of MK differentiation, such as production of megakaryocytic/erythroid progenitors and colony-forming units-MKs. This blockade was accompanied by a modulation of the transcriptional program of megakaryocytic differentiation. All these results indicate that Dll4/Notch signaling inhibits human terminal MK differentiation.

Human mesenchymal stem cells license adult CD34+ hemopoietic progenitor cells to differentiate into regulatory dendritic cells through activation of the Notch pathway.

The mechanisms underlying the immunomodulatory functions of mesenchymal stem cells (MSC) on dendritic cells (DC) have been shown to involve soluble factors, such as IL-6 or TGF-beta, or cell-cell contact, or both depending on the report referenced. In this study, we intend to clarify these mechanisms by examining the immunosuppressive effect of human adult MSC on adult DC differentiated from CD34(+) hemopoietic progenitor cells (HPC). MSC have been shown to inhibit interstitial DC differentiation from monocytes and umbilical CD34(+) HPC. In this study, we confirm that MSC not only halt interstitial DC but also Langerhans cell differentiation from adult CD34(+) HPC, as assessed by the decreased expression of CD1a, CD14, CD86, CD80, and CD83 Ags on their cell surface. Accordingly, the functional capacity of CD34(+) HPC-derived DC (CD34-DC) to stimulate alloreactive T cells was impaired. Furthermore, we showed that 1) MSC inhibited commitment of CD34(+) HPC into immature DC, but not maturation of CD34-DC, 2) this inhibitory effect was reversible, and 3) DC generated in coculture with MSC (MSC-DC) induced the generation of alloantigen-specific regulatory T cells following secondary allostimulation. Conditioned medium from MSC cultures showed some inhibitory effect independent of IL-6, M-CSF, and TGF-beta. In comparison, direct coculture of MSC with CD34(+) HPC resulted in much stronger immunosuppressive effect and led to an activation of the Notch pathway as assessed by the overexpression of Hes1 in MSC-DC. Finally, DAPT, a gamma-secretase inhibitor that inhibits Notch signaling, was able to overcome MSC-DC defects. In conclusion, our data suggest that MSC license adult CD34(+) HPC to differentiate into regulatory DC through activation of the Notch pathway.

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+CD38(low) cell proliferation and favored self-renewal. Here, we assessed the effects of a soluble form of Delta4 (solD4) by exposing CD34+CD38(low) 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+CD38(low) cells. Furthermore, both biological effects and CSL activation elicited by mbD4 were strictly dependent upon the gamma-secretase complex, whereas solD4 enhanced cell expansion in a partially gamma-secretase-independent manner. Altogether, these results suggest that part of solD4 activity did not rely upon canonical Notch pathway.

Association between a cell-seeded collagen matrix and cellular cardiomyoplasty for myocardial support and regeneration.

The objective of cellular cardiomyoplasty is to regenerate the myocardium using implantation of living cells. Because the extracellular myocardial matrix is deeply altered in ischemic cardiomyopathies, it could be important to create a procedure aiming at regenerating both myocardial cells and the extracellular matrix. We evaluated the potential of a collagen matrix seeded with cells and grafted onto infarcted ventricles. A myocardial infarction was created in 45 mice using coronary artery ligation. Animals were randomly assigned to 4 local myocardial treatment groups. Group I underwent sham treatment (injection of cell culture medium). Group II underwent injection of human umbilical cord blood mononuclear cells (HUCBCs). Group III underwent injection of HUCBCs and fixation onto the epicardium of a collagen matrix seeded with HUCBCs. Group IV underwent fixation of collagen matrix (without cells) onto the infarct. Echocardiography was performed on postoperative days 7 and 45, followed by histological studies. Echocardiography showed that the association between the cell-loaded matrix and the intrainfarct cell implants was the most efficient approach to limiting postischemic ventricular dilation and remodeling. Ejection fraction improved in both cell-treated groups. The collagen matrix alone did not improve left ventricular (LV) function and remodeling. Histology in Group III showed fragments of the collagen matrix thickening and protecting the infarct scars. Segments of the matrix were consistently aligned along the LV wall, and cells were assembled within the collagen fibers in large populations. Intramyocardial injection of HUCBCs preserves LV function following infarction. The use of a cell-seeded matrix combined with cell injections prevents ventricular wall thinning and limits postischemic remodeling. This tissue engineering approach seems to improve the efficiency of cellular cardiomyoplasty and could emerge as a new therapeutic tool for the prevention of adverse remodeling and progressive heart failure.

Inhibition of HIV replication: a powerful antiviral strategy by IFN-beta gene delivery in CD4+ cells.

In this study, we demonstrated the efficiency and feasibility of a gene therapy protocol against HIV infection using the antiviral effects of IFN-beta expression. Lentiviral vectors containing the human or the simian IFN-beta sequences under the influence of the murine moderate H2-kb promoter were constructed. To examine the capacity of IFN-beta to inhibit the replication of HIV in human CD4(+) cells, a transduction protocol permitting to efficiently transduce CD4(+) cells or PBMC (85+/-12% of CD4(+)-transduced cells) with a moderate expression of IFN-beta was developed. Results indicate that enforced expression of IFN-beta has no negative effects in terms of apoptosis and proliferation. In human CD4(+) cells, it drastically inhibits (up to 99.9%) replication after challenging with different strains of HIV-1. The expression of exogenous IFN-beta leads to an amplification of the CD4(+) cells (11-fold) and to a drastic decrease of the p24 protein. Micro-array analyses indicated that antiviral effect of IFN-beta could be due to a major regulation of the inflammatory response. These results are encouraging for the development of a clinical study of gene therapy against AIDS using IFN-beta.

The Notch ligand delta-1 is a hematopoietic development cofactor for plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) play an important role in innate and adaptive immunity, prompting interest in mechanisms controlling the production of this lineage of cells. Notch signaling via one of the Notch ligands, delta-like 1 (delta-1), influences the hematopoietic development of several lymphoid and myeloid lineages, but whether or not delta-1 affects the formation of pDCs is unknown and was tested here. Human CD34+ progenitor cells were cultured onto delta-1-expressing OP9 stroma in the presence of flt-3 ligand and IL-7, and this efficiently generated BDCA-2+ CD123+ CD4+ CD11c- cells with the characteristic morphology of pDCs, expressing toll-like receptor-9 (TLR9), pre-Talpha mRNAs, and secreting CpG-induced IFN-alpha. Delta-1 augmented the numbers of BDCA-2+ cells produced without affecting their proliferation, and the effect was blocked by gamma-secretase inhibition. The development of pDCs was stroma-, delta-1-, and cytokine-dependent and could be induced from committed lymphoid progenitor cells, which responded to delta-1 by opposite changes in pDC- and B-cell production. Our results identify delta-1 as a novel factor enhancing pDC hematopoiesis and delineate a new role for Notch signaling in lymphopoiesis by showing its opposite effect on pDC and B lineage determination.

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(+)CD38(low) 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.

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

BACKGROUND: The 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 beta production in macaques chronically infected with a pathogenic isolate of SIVmac251. RESULTS: Two 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-beta (MaIFN-beta or with a vector carrying a version of the MaIFN-beta 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. CONCLUSION: Long-term follow-up indicated that the transitory presence of such a small number of cells producing such small amounts of MaIFN-beta 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.