Most currently defined hematopoietic progenitor pools are heterogeneous, contributing to uncertainty regarding the development of certain blood cells. The origins of plasmacytoid dendritic cells, for instance, have long been controversial and progenitors exclusively committed to this lineage have never been described. We show here that the fate of hematopoietic progenitors is determined in part by their surface levels of 9-O-acetyl sialic acid. Pro-plasmacytoid dendritic cells were identified as lineage negative 9-O-acetyl sialic acid low progenitors that lack myeloid and lymphoid potential but differentiate into pre-plasmacytoid dendritic cells. The latter cells are also lineage negative, 9-O-acetyl sialic acid low cells but are exclusively committed to the plasmacytoid dendritic cell lineage. Levels of 9-O-acetyl sialic acid provide a distinct way to define progenitors and thus facilitate the study of hematopoietic differentiation.

Durant ce travail de thèse j’ai étudié des mécanismes de contrôle de la réponse inflammatoire qui, lorsqu’ils sont dérégulés, peuvent mener à une pathologie autoimmune/autoinflammatoire sévère, la Polyarthrite Rhumatoïde (PR). J’ai essentiellement analysé deux aspects de ces mécanismes : en premier lieu, j’ai pu participer à la démonstration de l’importance de l’endonucléase DICER (impliquée dans la biogénèse des microARNs - miRs), dans le contexte de la PR, particulièrement au niveau des synoviocytes type fibroblastique (FLS), des cellules résidentes de la cavité synoviale. J’ai aussi initié une étude visant à identifier, in vivo, l’intégralité des transcrits dont l’expression est régulée par des miRs dans les FLS de patients atteints de PR. En plus de fournir une vision globale de la régulation miR-dépendante dans ces cellules, ce travail permettra aussi d’identifier des cibles de miRs d’intérêt dans la PR en s’affranchissant des prédictions bio-informatiques qui peuvent s’avérer incorrectes. Un second axe de mon projet de thèse avait pour objectif de fournir une meilleure description du dialogue intercellulaire dans la cavité articulaire. Pour cela, je me suis plus particulièrement intéressé au rôle des cellules dendritiques plasmacytoïdes (pDCs) dans la physiopathologie de la PR. Ainsi j’ai pu démontrer un rôle protecteur de ces cellules (initialement décrites pour leurs fonctions dans la défense antivirale), dans le contexte de l’arthrite inflammatoire dans plusieurs modèles murins. Ces travaux m’ont permis de proposer une stratégie thérapeutique innovante, basée sur le recrutement articulaire des pDCs. Cette approche, non invasive (par application topique de crème contenant 5% d’imiquimod), s’est montrée efficace aussi bien pour la réduction des symptômes cliniques de l’inflammation que pour l’amélioration des marqueurs biologiques comme l’érosion osseuse. / During my PhD, I studied the mechanisms that control inflammation which, when disturbed, can lead to a severe autoimmune/ auto inflammatory disease, rheumatoid arthritis (RA). My work was focused on the analysis of two aspects in these mechanisms: first, I participated to an analysis of the roles of the endonuclease DICER (involved in the biogenesis of microRNAs – miRs) in the pathogenesis of RA, specifically in fibroblast-like synoviocytes (FLS), which are resident cells of the synovial cavity. I also initiated a study aiming at the identification of the FLS transcriptome which is regulated by miRs in RA patients. This approach will provide an overview of the miR-dependent regulation in these cells and enable the identification of in vivo validated miR-targeted mRNAs in RA. A second axis of my thesis project aimed at providing a better description of the intercellular dialogue in the joint cavity. For this, I was particularly interested in the role of plasmacytoid dendritic cells (pDCs) in the pathophysiology of RA. I demonstrated a protective role of these cells (initially described for their functions in antiviral defense) in the context of inflammatory arthritis in several mouse models. During this work, I had the opportunity to try an innovative therapeutic strategy based on the recruitment and activation of pDCs in the joints. This noninvasive and painless approach (topical application of cream containing 5% imiquimod) was effective in reducing inflammatory clinical symptoms and also improved biological markers such as bone erosion.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive, rare malignancy. Exact incidence is unknown due to lack of diagnostic criteria. Typically, it involves skin and bone marrow and less likely, lymph nodes and visceral organs. We present a 76 year old male who started having a lesion on the left side of his back that was progressively enlarging. He initially started on antibiotic and topical medications for more than a month which did not help. Punch biopsy of the lesion was consistent with blastic plasmacytoid dendritic cell neoplasm, positive for CD2, CD5, CD7, CD43, weak CD58,Tdt, bcl-6. Patient denies fever, chills, night sweats, weight loss, change in appetite. Physical exam revealed a purplish lesion raised in the left upper back with multiple satellite-like purple lesions throughout the back. Laboratory showed white cell count 3.2 K/uL, hemoglobin 13 g/dL, platelet 135 K/uL. Bone marrow biopsy shows immature blastic neoplasm involving 15% of the bone marrow. Cytogenetics showed normal karyotype. Flow cytometry shows an immature lymphoid population with expression of CD4, CD56, and CD 123, negative for FLT3, IDH1, IDH2, NPM1 mutations. Positron emission tomography (PET) scan showed skin thickening with minimal FDG uptake in left posterior skin soft tissue of the chest near the shoulder with no other abnormal focal uptake and splenomegaly. BPDCN is a rare aggressive malignancy that is more common in older populations. The origin is from type 2 dendritic cells. Typical presentations are skin lesions, cytopenia, lymphadenopathy, and splenomegaly. Some of the cytological features of BPDCN include cloudy sky (blue cytoplasm with clearer areas), pseudopods, and microvacuoles. Confirmation of diagnosis is with immunophenotyping. Workup after diagnosis include complete blood count, liver and renal function, hepatitis panel, peripheral blood smear, bone marrow evaluation, systemic imaging, cerebrospinal fluid cytology. Treatment of BPDCN is challenging in this era. Most patients respond to chemotherapy, although they relapse. Tegraxofusp is suggested for remission induction therapy following allogeneic hematopoietic cell transplantation. Median overall survival is about one year. Only patients who underwent hematopoietic stem cell transplant had prolonged survival. Myelemia, old age and altered general state have worse prognosis.

<p>Vaccinia virus (VV) is the most thoroughly studied member of the Poxviridae family and the vaccine used to achieve the only successful eradication of a human disease. Over the years, it has proven itself as a useful tool for the study of antiviral immunity, vaccine development, and potentially cancer immunotherapy. VV is capable of eliciting a robust immune response; however the mechanisms by which VV accomplishes this task remain unknown. The overall goal of this thesis project is to determine how VV activates the innate immune system, and how this activation contributes to viral clearance in vivo. We determined that VV or VV-DNA activated the TLR8-MyD88 pathway in plasmacytoid dendritic cells (pDC), resulting in the production of type I interferons (IFN). We also demonstrated that TLR8-mediated production of type I IFN by pDC was crucial to efficient VV control and clearance in vivo. Moreover, we identified the polyA- and polyT-rich sequences in VV-DNA was the possible motif recognize by TLR8. Type I IFN, known for ability to establish the "antiviral state", are also critical mediators of NK cell activation. In the setting of VV infection, we demonstrated that direct action of type I IFN on NK cells, but not accessory cells such as DC, was necessary for NK cell activation in vivo. We further demonstrated that type I IFN-dependent activation of NK cells was required for optimal VV clearance in vivo. Given the importance of NK cells in anti-VV innate immunity, we next examined what role the TLR2-MyD88 pathway, critical for activation of cDC, played in the activation of NK cells. NK cells from TLR2-/- or MyD88-/- mice displayed a reduction in activation and cytolytic function, and this defect was independent of pro-inflammatory cytokine signaling. We were able to demonstrate that direct TLR2 signaling on NK cells was required for their optimal activation and function in response to VV infection. Moreover, we were able to demonstrate that TLR2-MyD88 signaling resulted in the activation of the PI3K-ERK pathway, which was necessary for NK cell cytotoxicity. In addition, we identified the NKG2D pathway as critical for efficient NK cell activation and function in response to VV infection, independent of the TLR2 pathway. Both the NKG2D and TLR2 pathways were crucial for optimal VV clearance and control in vivo. Collectively, this project illuminates the roles and mechanisms of the innate immune system in the control of VV in vivo.</p> / Dissertation

Systemic lupus erythematosus (SLE) is an autoimmune, inflammatory disease characterized by autoantibody production and an activated type I interferon system. Cardiovascular disease (CVD) is as a major cause of morbidity and mortality. The aim of this thesis was to identify genetic risk factors for CVD in SLE. The role of T cells in regulation of the interferon-α (IFNα) production by plasmacytoid dendritic cells (pDCs) was also investigated.    In paper I, a thicker intima, thinner media and increased intima/media ratio was found in young premenopausal women with SLE compared to healthy controls indicating increased cardiovascular risk. As traditional ultrasound assessment of the common carotid intima-media thickness (CCA-IMT) in SLE has given conflicting results separate measurement of the intima and media can be a useful tool to identify SLE patients at increased risk of CVD.    In paper II, an association was demonstrated in SLE between a STAT4 risk allele and ischemic cerebrovascular disease and presence of anti-phospholipid antibodies (aPL). The association remained after adjustment for traditional CVD risk factors. A possible mechanism for this association is that the risk allele leads to increased production of aPL, which promotes thromboembolism.    In paper III, a genetic locus in IRF8 was identified to be associated to coronary heart disease (CHD) in SLE. The association remained after adjustment of other CHD risk factors.  Patients with the IRF8 risk variant had increased CCA-IMT, more carotid plaques and reduced frequency of circulating B cells. Weaker binding of nuclear protein to the risk allele was demonstrated, suggesting a regulatory function of the IRF8 risk variant.    In paper IV, activated T cells were found to strongly enhance the IFNα production by pDC stimulated with RNA-containing immune complexes via GM-CSF and IL-3. Activated SLE T cells enhanced the IFNα production to the same extent as T cells from healthy controls. This finding together with previous observations in SLE of increased levels of GM-CSF and IL-3 suggests that T cells contribute to the activated type I interferon system in SLE.    In conclusion, this thesis demonstrates that genetic predisposition is important for CVD in SLE and describes a new role for T cells in the pathogenesis of SLE.

Le cancer de l’ovaire est immunogène et constitue un bon modèle pour étudier l’immunité antitumorale. Nous avons effectué une étude comparative et systématique de la fréquence, du phénotype, de la fonction et de l’impact sur la survie des cellules dendritiques plasmacytoïdes (pDC) et des lymphocytes T régulateurs (Treg) dans le sang, l’ascite et la tumeur. Nous avons observé que les pDC s’accumulent dans les ascites et sont présentes dans certaines tumeurs alors qu’elles sont profondément déplétées dans le sang des patientes. La présence de pDC associées aux tumeurs (TApDC) est un facteur pronostique indépendant associé à une survie sans progression (SSP) plus courte. De plus, les TApDC, mais pas les pDC d’ascite, sont altérées dans leur fonction innée principale de production d’IFN-α en réponse aux TLR ligands in vitro et induisent le développement de lymphocytes T CD4+ producteurs d’IL-10 responsables d’une tolérance immune favorisant la progression tumorale. Les Treg s’accumulent dans les ascites et les tumeurs de l’ovaire mais leur taux dans le sang est comparable aux donneurs sains. Leur accumulation dans les tumeurs et non dans les ascites est un facteur pronostique indépendant associé à une SSP plus longue. Les TATreg ont un phénotype activé et inhibent la production d’IL-10 par les lymphocytes T CD4+ conventionnels associés aux tumeurs. De façon intéressante, les patientes dont les tumeurs augmentent l’infiltration par les Treg Foxp3+ après chimiothérapie néoadjuvante ont une rechute retardée suggérant qu’en plus d’un effet antitumoral direct, la chimiothérapie induit une réponse immune / Ovarian cancer (OC) is an immunogenic disease and represents a good model for studying antitumoral immunity. We performed a systematic comparison between plasmacytoid dendritic cells (pDC) and regulatory T cells (Treg) in blood, ascites, and tumors in term of frequencies, phenotypes, functions, and impact on outcome of OC patients. We found that pDC accumulate in ascites and are present in some tumors whereas they are profoundly depleted in patients’ blood. Their presence within tumors (but not ascites) is deleterious because associated with early relapse of OC patients. Moreover, Tumor associated pDC (TApDC) but not ascite pDC were altered in their innate function, i.e. the production of IFN-α in response to TLR ligands in vitro, and they induce the development of IL-10+ CD4+T cells. All these results suggest that TApDC but not ascite pDC induce immune tolerance allowing cancer progression. Treg accumulate in ascites and tumors but their levels in patients’ blood were not increased. Their accumulation in tumors, but not ascites, was an independent prognostic factor associated with delayed relapse. TATreg showed an activated phenotype and inhibit IL-10 production by CD4+conventional TAT cells. Interestingly, patients whose tumor infiltration by Foxp3+ Treg is increased after neoadjuvant chemotherapy showed delayed relapse suggesting that chemotherapy, in addition to its direct antitumoral effect, induces an immune response

Les cellules dendritiques plasmacytoïdes (pDCs) sont caractérisées par leur capacité unique de sécrétion massive d’interféron de type I (IFN-I) suite à la stimulation des Tolllike récepteurs (TLR) 7 et 9. Un rôle fondamental des pDCs a été démontré dans le lupus érythémateux systémique via la production d’IFN-I. Les pDC expriment le récepteur de forte affinité aux immunoglobulines de type E (IgE), FcεRI, impliqué dans la régulation négative de la sécrétion d’IFN-I. L’objectif de notre étude est d’explorer, dans le contexte lupique, les effets du traitement par les IgE sur les fonctions des pDC, particulièrement sur la production d’IFN-I. In vitro, le traitement des pDC par des IgE monoclonales permet la surexpression du FcεRI à leur surface et diminue le taux de transcrits des TLR7/9 et de l’IRF7. De plus, les pDC traitées par des IgE diminuent leur production d’IFN-I et l’expression de marqueurs de maturation, induites par leur stimulation par des ligands des TLR7/9 et des complexes immuns lupiques. En outre, ces pDC pré-traitées par des IgE induisent la différenciation de LT4 naïfs allogéniques en LT4 produisant de l’IL-10. In vivo, les patients lupiques en phase quiescente de la maladie présentent des taux plus élevés d’IgE totales comparés aux patients en phase active (indépendamment d’allergies et d’infestations parasitaires). Chez les patientslupiques, le taux d’IgE totales est inversement corrélé au taux d’anti-ADN et à l’activité de la maladie (SLEDAI). L’ensemble de nos résultats suggère un rôle protecteur des IgE dans le lupus à travers la modulation de la réponse inflammatoire des pDC. / Plasmacytoid dendritic cells (pDCs) are characterized by their unique ability to produce large amounts of type I interferon (IFN-I) upon Toll-like receptors (TLR) 7 and 9 triggering. A fundamental role for pDCs has been shown in systemic lupus erythematosus (SLE) through IFN-I production. pDCs express the high affinity Fc receptor for immunoglobulin E (IgE), FcεRI, involved in the negative regulation of IFN-I secretion. The objective of our study is to investigate, in the context of SLE, the effects of IgE treatment on pDCs functions, especially on IFN-I production. In vitro, monoclonal IgE treatment of pDCs upregulate their surface expression of FcεRI and decrease transcripts levels of TLR7/9 and IRF7. IgE-treated pDCs decrease IFN-α secretion and downregulate maturation markers expression induced by TLR7/9 and immune complexes triggering. Moreover, the coculture of IgE pretreated pDCs with allogeneic naive LT4 promotes their differentiation into IL-10-secreting cells. In vivo, patients with quiescent SLE have higher IgE levels than patients with active disease (independently of allergy or parasitic infection). In SLE patients, IgE levels are inversely correlated to anti-DNA antibodies and disease activity (SLEDAI). All together, our data suggest a protective role for IgE in SLE through the modulation of the inflammatory response by pDC.

Stimuler le système immunitaire est un enjeu majeur dans le traitement des leucémies aigües. Nous avons centré notre étude sur la leucémie aigüe myéloïde (LAM) et la leucémie à cellules dendritiques plasmacytoïdes (LAPDC). Dans une première partie, nous avons étudié les médicaments immunomodulateurs (IMiDs) utilisés dans le traitement du myélome multiple et des syndromes myélodysplasiques à délétion 5q. Les IMiDs présentent des propriétés anti-angiogéniques, anti-prolifératives, pro-apoptotiques, et immunomodulatrices en particulier sur les cellules NK (Natural Killer). Nous avons évalué les effets anti-leucémiques des IMiDs (lenalidomide et pomalidomide) dans le but d’améliorer l’activité cytotoxique des NK dans la LAM. Nous avons mis en évidence une altération de la survie des blastes de LAM par les IMiDs in vitro, et dans un modèle in vivo de greffe dans les souris immunodéficientes NOD/SCID/IL2rg-/- (NSG). Nous avons également montré une sensibilisation par les IMiDs des blastes de LAM à la lyse par les NK allogéniques, indépendamment de la cible moléculaire connue, le cereblon. Le traitement des blastes de LAM par IMiDs stimule les fonctions NK. Enfin, nous avons décrit des modifications phénotypiques induites par les IMiDs sur les récepteurs NK, et une diminution d’expression de HLA-classe I sur les cellules de LAM. Ces résultats encouragent la poursuite du développement des IMiDs dans la LAM, en particulier les associations stimulant les fonctions NK. Dans une seconde partie, nous avons développé un modèle murin de LAPDC dans la souris NSG. Cet outil préclinique est indispensable dans l’élaboration de stratégies d’immunothérapie dans les leucémies aigües. / Boosting the Immune System is a major challenge in the treatment of acute leukemias. We focused our study on acute myeloid leukemia (AML) and plasmacytoid dendritic cell leukemia (BPDCN). In the first part, we studied immunomodulatory drugs (IMiDs) that are currently used in the treatment of patients with myeloma and myelodysplastic syndrome with 5q deletion. IMiDs exhibit anti-angiogenesis, anti-proliferative, pro-apoptotic, and immunomodulatory properties especially on NK cells and T lymphocytes. We investigated the anti-leukemic effects of two IMiDs (lenalidomide and pomalidomide) in order to improve NK cell cytotoxic activity in AML. We have shown that IMiDs impaired survival of AML blasts in vitro, and in vivo in NOD/SCID/IL2rg-/- (NSG) murine model. In addition, IMiDs treatment sensitized AML blasts to allogeneic NK cell mediated lysis, independently of Cereblon, the known molecular target of IMiDs. IMiDs treatment of AML blasts enhanced NK cell functions such as degranulation and cytokine production. Finally, we have described phenotypic changes induced by IMiDs on NK receptors, and a down-regulation of HLA-class I on AML blasts. These results encourage continuing investigation for the use of IMiDs in AML, especially in combination with immunotherapies based on NK cells. In a second part, we have developed a murine model of plasmacytoid dendritic cell leukemia (BPDCN) in NSG mice. Murine model of leukemia are essential preclinical tools in the development of new immunotherapies in acute leukemias.

<p>Patients with systemic lupus erythematosus (SLE) have elevated levels of interferon (IFN)-α in blood and IFN-α-producing cells in tissues. In the present thesis, we investigate the mechanisms behind the upregulated IFN-α-production in SLE and also show that the IFN-α system is activated in primary Sjögren’s syndrome (pSS), with IFN-α-producing cells in the major affected organ, the salivary glands. The IFN-α is a type I IFN, a family of cytokines counteracting especially viral infections, by acting directly on infected cells, and via many immunomodulatory effects. The latter may also contribute to autoimmune processes.</p><p>The type I IFNs are usually produced upon recognition of microbial structures. In SLE, however, DNA-containing immune complexes (ICs) that induce IFN-α production are found. Many autoantibodies in SLE and pSS are directed to nucleic acids or to DNA/RNA-binding proteins. We show that also RNA in complex with autoantibodies from SLE or pSS patients (RNA-IC) induces IFN-α-production. The RNA could be either in the form of RNA-containing material released from apoptotic or necrotic cells or as a pure RNA-containing autoantigen, the U1 small nuclear ribonucleoprotein particle. </p><p>The IFN-α-production induced by RNA-IC occurred in plasmacytoid dendritic cells (PDCs), also termed natural IFN-producing cells (NIPCs), via binding to Fcγ-receptor IIa, endocytosis and triggering of Toll-like receptors (TLRs), probably TLR7 and TLR9. The RNA-IC may also have other effects, and we found that they induce prostaglandin E2 (PGE2) production in monocytes and tumor necrosis factor (TNF)-α in both monocytes and NIPC/PDC. The PGE2 downregulated the IFN-α induction in NIPC/PDC, and the IFN-α induction was increased in monocyte-depleted cell cultures. </p><p>The findings presented in this thesis aids in the understanding of the mechanisms behind the activated IFN-α system in SLE and other autoimmune diseases, and shows that also pSS is one of these diseases.</p>

Patients with systemic lupus erythematosus (SLE) have elevated levels of interferon (IFN)-α in blood and IFN-α-producing cells in tissues. In the present thesis, we investigate the mechanisms behind the upregulated IFN-α-production in SLE and also show that the IFN-α system is activated in primary Sjögren’s syndrome (pSS), with IFN-α-producing cells in the major affected organ, the salivary glands. The IFN-α is a type I IFN, a family of cytokines counteracting especially viral infections, by acting directly on infected cells, and via many immunomodulatory effects. The latter may also contribute to autoimmune processes. The type I IFNs are usually produced upon recognition of microbial structures. In SLE, however, DNA-containing immune complexes (ICs) that induce IFN-α production are found. Many autoantibodies in SLE and pSS are directed to nucleic acids or to DNA/RNA-binding proteins. We show that also RNA in complex with autoantibodies from SLE or pSS patients (RNA-IC) induces IFN-α-production. The RNA could be either in the form of RNA-containing material released from apoptotic or necrotic cells or as a pure RNA-containing autoantigen, the U1 small nuclear ribonucleoprotein particle. The IFN-α-production induced by RNA-IC occurred in plasmacytoid dendritic cells (PDCs), also termed natural IFN-producing cells (NIPCs), via binding to Fcγ-receptor IIa, endocytosis and triggering of Toll-like receptors (TLRs), probably TLR7 and TLR9. The RNA-IC may also have other effects, and we found that they induce prostaglandin E2 (PGE2) production in monocytes and tumor necrosis factor (TNF)-α in both monocytes and NIPC/PDC. The PGE2 downregulated the IFN-α induction in NIPC/PDC, and the IFN-α induction was increased in monocyte-depleted cell cultures. The findings presented in this thesis aids in the understanding of the mechanisms behind the activated IFN-α system in SLE and other autoimmune diseases, and shows that also pSS is one of these diseases.