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Plus de 20 million de personnes à travers le monde sont infectées par le virus T-lymphotrope humain de type 1 (HTLV-1), causant des leucémies à cellules T dans ~5 % des individus infectés. Le virus de la leucémie bovine (BLV), structurellement et fonctionnellement proche de HTLV-1, induit des leucémies à cellules B dans des modèles animaux suite à une infection naturelle (bovin) ou expérimentale (mouton). Les mécanismes moléculaires responsables du potentiel oncogène de ces deux virus restent largement incompris. Dans les deux maladies, leucémies T chez l’homme, leucémies B chez l’animal, le site intégration du virus dans les cellules leucémiques est très variable. Il est donc généralement admis que le potentiel oncogène du provirus est principalement lié à l’activité de l’oncoprotéine virale Tax. De manière paradoxale cependant, ni HTLV-1 ni BLV n’expriment de protéines virales au stade tumoral. Dans ce travail, nous avons étudié le transcriptome non codant des leucémies induites par BLV et HTLV-1 par séquençage à haut débit. Dans la première partie, nous démontrons que le provirus BLV n’est en fait pas silencieux dans les cellules tumorales. BLV produit un ensemble de dix microARNs (miRNAs) très abondants et extrêmement conservés dans toutes les tumeurs. Cette observation constitue la première description de miRNAs encodés par un rétrovirus. Les microARNs encodés par BLV sont transcrits par la RNA Polymérase III, stratégie qui permet leur production de façon indépendante de celle des messagers viraux ainsi que leur expression abondante dans le contexte tumoral caractérisé par l’absence d’activité RNA Polymérase II. Nous avons ensuite montré que, comme HTLV-1, BLV produit des transcrits encodés par le brin négatif du provirus. L’analyse par séquençage ARN à haut débit (RNA-Seq) de tumeurs induites par BLV montre l’absence d’expression virale à partir du promoteur viral situé dans le LTR 5’. Cependant, elle révèle la présence de deux transcrits viraux anti-sens non, Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/nonPublished

Mon travail de thèse a consisté à caractériser une sous-population de lymphocytes T régulateurs naturels de phénotype CD8+CD25+.Nous avons purifié les CD8+CD25+ nTregs et vérifié par cytométrie de flux leur expression en FOXP3 et CTLA-4. Puis nous avons pu montrer que ces cellules possèdent des propriétés suppressives dans un test d’inhibition de la prolifération de lymphocytes T activés allogéniquement. Les lymphocytes CD8+CD25+ nTregs expriment les gènes FOXP3, CTLA-4, GARP et CCL-4 et les cytokines IL-10 et TGF-β. Par contre, les gènes CD28, ICOS, FOXO1 et Helios sont sous-exprimés dans les nTregs CD8+CD25+ par rapport aux lymphocytes T CD8+CD25-. Nous avons établi une signature micro-ARN qui comprend 10 micro-ARNs différentiellement exprimés :7 micro-ARNs sous-exprimés "miR-9, -24, -31, -155, -210, -335 et -449 " et 3 micro-ARNs surexprimés " miR-214, -205 et -509". De plus, nous avons pu explorer la relevance biologique de cette signature micro-ARN en montrant dans un premier temps que les miRs "-31, -24, -210, -335" ciblent spécifiquement la région 3'UTR de FOXP3, de même les miR-9 et miR-155 ciblent la région 3'UTR de CTLA-4, et les miR-24, et -335 ciblent la région 3'UTR de GARP. Ceci a été fait par des expériences de co-transfections suivies d'une mesure de l'activité rapportrice luciférase. De plus, nous avons pu démontrer par des expériences de transduction lentivirale ex vivo, de cellules T primaires, que des micro-ARNs de la signature régulent l’expression de FOXP3, CTLA-4 et GARP dans les Tregs naturels CD8+CD25+ humains. Cette étude montre l'importance des micro-ARNs dans la régulation post-transcriptionnelle des gènes impliqués dans la fonction régulatrice des lymphocytes T régulateurs., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

IFN-γ is a cytokine with important roles in the innate and adaptive immune responses. This cytokine is secreted by activated T cells, NK cells and macrophages. Studies on the regulation of human IFN-γ expression had been previously focused on the promoter region. Consequently, the role of microRNAs (miRs) in this regulation has not been investigated yet. As miR-24 and miR-181 were found to have potential target sites in IFN-γ mRNA 3'UTR, we assessed their impact on IFN-γ expression by co-stimulating PB CD4+ T cells with anti-CD3, anti-CD28, IL-12, and IL-18. This co-stimulation cocktail induced an abundant secretion of IFN-γ together with a down-regulation of miR-24, and miR-181. Existence of a link between these two phenomena was further substantiated by transfection and transduction assays that showed that these two miRs negatively regulate IFN-γ expression by directly binding to their target sites in the mRNA. Thus, identifying target sites for miR-24 and miR-181 in IFN-γ-3'UTR points to a novel regulatory mechanism of this crucial gene. © 2014 American Society for Histocompatibility and Immunogenetics., SCOPUS: ar.j, info:eu-repo/semantics/published

Background: Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8+CD25+ Treg cells and the impact of microRNAs on molecules associated with immune regulation.Methods: We purified human natural CD8+ Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8+CD25+FOXP3+CTLA-4+ natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3′-UTR of important Treg cell-associated genes.Results: The human CD8+CD25+ natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo.Conclusions: We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer., SCOPUS: ar.j, info:eu-repo/semantics/published

Cardiovascular disease linked to atherosclerosis is the leading cause of death worldwide. Atherosclerosis is mainly linked to dysfunction in vascular endothelial cells and subendothelial accumulation of oxidized forms of LDL. In the present study, we investigated the role of myeloperoxidase oxidized LDL (Mox-LDL) in endothelial cell dysfunction. We studied the effect of proinflammatory Mox-LDL treatment on endothelial cell motility, a parameter essential for normal vascular processes such as angiogenesis and blood vessel repair. This is particularly important in the context of an atheroma plaque, where vascular wall integrity is affected and interference with its repair could contribute to progression of the disease. We investigated in vitro the effect of Mox-LDL on endothelial cells angiogenic properties and we also studied the signalling pathways that could be affected by analysing Mox-LDL effect on the expression of angiogenesis-related genes. We report that Mox-LDL inhibits endothelial cell motility and tubulogenesis through an increase in miR-22 and heme oxygenase 1 expression. Our in vitro data indicate that Mox-LDL interferes with parameters associated with angiogenesis. They suggest that high LDL levels in patients would impair their endothelial cell capacity to cope with a damaged endothelium contributing negatively to the progression of the atheroma plaque., SCOPUS: ar.j, info:eu-repo/semantics/published

MicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding RNA molecules whose deregulation of expression can contribute to human disease including the multistep processes of carcinogenesis in human. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as type 2 diabetes, pulmonary disease, colorectal cancer, and gastric cancer among others; however, defining a plasma miRNA signature in acute myeloblastic leukemia (AML) that could serve as a biomarker for diagnosis or in the follow-up has not been done yet., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Confidentiel, Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/nonPublished

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

PACAP et VIP sont deux peptides structuralement reliés qui appartiennent à la classe de neuropeptides comprenant la sécrétine, le glucagon et le growth hormone releasing factor. VIP et PACAP ont de nombreuses fonctions biologiques au niveau du système nerveux et sont considérés comme des agents neurotransmetteurs, sécrétagogues, neuroprotecteurs, neurotrophiques, mitogéniques et comme agents différenciant. VIP et PACAP utilisés à des concentrations de l’ordre du nano-molaire sont décrits comme agents anti-inflammatoires. Dans cette thèse, nous montrons que les neuropeptides PACAP et VIP utilisés à des doses élevées de l’ordre du μ-molaire agissent également comme des molécules pro-inflammatoires au niveau des monocytes humains.Nous montrons aussi qu’au sein des monocytes humains, le PACAP agit par l’intermédiaire du récepteur VPAC-1. Les voies de signalisation activées incluent de façon proximale la PLC et la PI3-kinase et de façon plus distale les voies ERK et p38 des MAP-Kinases et les voies dépendantes de focal adhésion kinase associées à la protéine du cytosquelette paxilline. PACAP induit également un pic calcique résultant d’une mobilisation intra- et extracellulaire de calcium.Au niveau fonctionnel, nous montrons quelle PACAP augmente la production de radicaux libres et l’expression membranaire de l’intégrine CD11b impliquée dans l’adhésion et dans la régulation de nombreuses voies métaboliques.Nous montrons également que de faibles doses de PACAP de l’ordre du nM sont suffisantes pour désensibiliser les monocytes à des concentrations plus importantes de PACAP, concentrations connues pour avoir un effet pro-inflammatoire. Le PACAP a ainsi un double rôle, anti-inflammatoire à faible dose et pro-inflammatoire à dose plus élevé.Nous avons de plus investigué la présence d’une transactivation au niveau des cellules monocytaires. Dans ces cellules, PACAP comme rapporté dans les cellules neuronales, utilise la signalisation NGF/TrkA. PA, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Currently, in most situations, the autologous immune system is unable to eradicate the residual leukemic burden persisting after chemo-radiotherapy, but a balance can be established between leukemic and immune cells leading to a clinical remission for several months or years. If this balance is broken, a clinical relapse can occur. The high incidence of relapses in human cancers demonstrates the frequent inefficacy of the immune system to control these residual cells. In this context, allogeneic hematopoietic stem cell transplantation (HSCT) has been proven to be the most effective way to reinforce the immune reaction against leukemia, graft-versus-leukemia (GVL) effect and, so, achieve a definitive eradication of the residual disease in a significant proportion of patients. Indeed, the whole concept of HSCT evolved from an organ transplant concept (to replace a defective ill organ with a new healthy one) to the concept of creating an extraordinary immunotherapeutic platform in which the donor immune system contributes to the eradication of the residual leukemic cells. Thus, the past and present issues remain those of finding the best immunomodulatory modalities to achieve a full engraftment, a powerful GVL effect and no or moderate graft-versus-host disease (GVHD). Different ways to reach this goal, such as post transplant cytokine modulation, specific or global cellular depletion of the graft and post transplant global or specific donor immune cell add-backs, are still extensively studied. Nevertheless, the persistent high relapse rate (RR) observed in leukemia patients after HSCT remains the most important cause of death before transplant-related toxicities. Moreover, since only about 40 to 70% (depending on the ethnic context) of patients with high-risk hematological malignancies, eligible for allogeneic HSCT, have a fully HLA-matched sibling or matched unrelated donor (MUD), a great deal of effort has been invested to make the use of an alternative haploidentic, Doctorat en Sciences médicales, info:eu-repo/semantics/nonPublished

The differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

info:eu-repo/semantics/published

Mature dendritic cells (DCs) represent, by far, the most potent antigen-presenting cells. The development of clinical grade techniques to produce them in large numbers has rendered possible their use in clinical trials. It is therefore crucial to assess the DCs characteristics according to the methodology used to generate them, to improve the comparison and standardization of these trials. We thus compared DCs generated and matured in culture plates (pla-DCs) or in clinical grade bags (bag-DCs) by analyzing, their secretion of bioactive interleukin (IL)-12 and their capacity to induce in-vitro primary responses. We also used several molecular techniques to better characterize the functional differences between the 2 type of DCs. Mature bag-DCs displayed a mature phenotype, but did not secrete significant amounts of IL-12 and failed to initiate primary immune responses. Molecular analyses performed on immature bag-DCs showed them already engaged in a particular maturation process (early activation of nuclear factor κ B and β-catenin). Using microarrays, we found underexpression of receptors for the maturation cocktail in bag-DCs. In mature bag-DCs, we found crucial genes (IL-12, chemokines, and costimulatory and adhesion molecules) down-regulated. Electrophoertic mobility shift assay and Western blots showed a normal activation profile in mature pla-DCs, but not in bag-DCs where the Mek/Erk pathway was still activated. Our results strongly suggest that differentiation of monocytes into DCs in bags generates immature DCs already engaged in an inefficient type of activation, with down-regulation of genes involved in response to the maturation cocktail. This results in mature DCs unable to induce TH1-type responses. Copyright © 2010 by Lippincott Williams & Wilkins., SCOPUS: ar.j, info:eu-repo/semantics/published