Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 86-105). Also available online.

Allergy, as one of the worldwide most frequent pathologies, belongs to illnesses with constantly growing incidence among young children. In genetically predisposed individuals, dendritic cells are able to polarize the immune response of Th2 in contact with the allergen. Postnatal probiotic supplementation could be one the preventive measure to prevent the development of allergic diseases. It has been shown that introduction of selected probiotic strains or mixtures can prevent development of allergy. In this diploma thesis, the capacity of probiotic strain Escherichia coli O83:K24:H31 (E. coli O83) to support maturation of dendritic cells (DC) and polarization of immune responses was tested. Introduction of this probiotic vaccine called Colinfant Newborn appears to be suitable preventive measure, lowering allergy incidence in children with predisposition to development of allergy. The aim of this diploma thesis was to observe capacity of E. coli O83 to support maturation of the two main subpopulations of dendritic cells (myeloid dendritic cells - mDC and plasmacytoid dendritic cells - pDC) in cord blood of newborns of healthy mothers (children with relatively low risk for allergy development) and allergic mothers (children with relatively high risk for allergy development). To achieve this goal,...

Allergy as one of the most frequent pathologies worldwide belongs to illnesses with constantly increasing incidence even amongst young children. It develops in genetically predisposed individuals whose dendritic cells (DC) are, after contact with allergen, able to polarize the immune response predominantly to Th2, while Th1 response is supressed. One of the possible preventive measures to avoid an allergic disease developement could be an early postnatal supplementation of chosen probiotic bacterial strains or their mixtures. One of them is a well characterized strain Escherichia coli O83:K24:H31 (E. coli O83). Administering of this probiotic vaccine called Colifant Newborn is showing to be an effective prophylaxis to decrese the incidence of allergies in children with predisposition to their developement. The aim of my diploma thesis was to experimentally confirm the capacity of E. coli O83 to support maturation of two main subpopulations of newborn DC available from cord blood: myeloid dentritic cells - mDC and plasmacytoid dentritic cells - pDC. This DC subpopulations were isolated from cord blood of children born to healthy (non-allergic) mothers who had a low risk of allergy development or from children of allergic mothers who had an increased risk of allergy development. Subsequently, after...

Allergy, as one of the worldwide most frequent pathologies, belongs to illnesses with constantly growing incidence among young children. Identification of prognostic markers pointing to increased risk of allergy development, allows introduction of early preventive measures. Probiotic supplementation could be one the preventive measure. It has been shown that introduction of selected probiotic strains or mixtures can prevent development of allergy. In this diploma thesis, the capacity of probiotic strain Escherichia coli O83:K24:H31 (E. coli O83) to support maturation of dendritic cells and polarization of immune responses was tested. Introduction of this probiotic vaccine called Colinfant Newborn appears to be suitable preventive measure, lowering allergy incidence in children with predisposition to development of allergy. The capacity of E. coli O83 to support maturation of the two main subpopulations of dendritic cells (myeloid dendritic cells - mDC and plasmacytoid dendritic cells - pDC) in cord blood of newborns of healthy mothers (children with relatively low risk for allergy development) and allergic mothers (children with relatively high risk for allergy development) was measured by flow cytometry. The presence of cytokines and transcription factors characteristic for particular...

DCs play a pivotal role in orchestrating innate and adaptive antitumor immunity. Activated DCs can produce large amounts of various proinflammatory cytokines, initiate T-cell responses, and exhibit direct cytotoxicity against tumor cells. They also efficiently enhance the antitumoral properties of NK cells and T lymphocytes. Based on these capabilities, immunogenic DCs promote tumor elimination and are associated with improved survival of patients. Furthermore, they can essentially contribute to the clinical efficacy of immunotherapeutic strategies for cancer patients. However, depending on their intrinsic properties and the tumor microenvironment, DCs can be rendered dysfunctional and mediate tolerance by producing immunosuppressive cytokines and activating Treg cells. Such tolerogenic DCs can foster tumor progression and are linked to poor prognosis of patients. Here, we focus on recent studies exploring the phenotype, functional orientation, and clinical relevance of tumor-infiltrating conventional DC1, conventional DC2, plasmacytoid DCs, and monocyte-derived DCs in translational and clinical settings. In addition, recent findings demonstrating the influence of DCs on the efficacy of immunotherapeutic strategies are summarized.

L’idée qu’une cellule puisse effectuer la cytolyse de cellules transformées, comme une cellule Natural Killer (NK), tout en ayant la capacité de présenter des antigènes, comme une cellule dendritique (DC), peut sembler fantaisiste. Cependant, de telles cellules furent bel et bien identifiées chez la souris en 2006. Ces cellules, nommées Interferon-producing Killer Dendritic Cells (IKDC), furent l’objet d’une caractérisation extensive qui révéla leur énorme potentiel immunologique. La combinaison de fonctions associées à des cellules NK et à des DC a doté les IKDC d’un pouvoir antitumoral remarquable. D’ailleurs, il a été démontré que les IKDC sont plus efficaces que les cellules NK pour limiter la croissance tumorale. Ainsi, suite à leur découverte, les IKDC ont suscité beaucoup d’intérêt. Cependant, une controverse émergea sur la nature des IKDC. Plusieurs groupes indépendants tentèrent de reproduire les expériences attestant les fonctions de DC des IKDC, sans y parvenir. De plus, des études additionnelles révélèrent que les IKDC possèdent des similitudes très importantes avec les cellules NK. Ces observations ont mené la communauté scientifique à suggérer que les IKDC sont des cellules NK en état d’activation (aNK). Malgré cette controverse, les caractéristiques antitumorales des IKDC sont si uniques et considérables qu’il est primordial de poursuivre l’étude de ces cellules. Pour y arriver, il est essentiel de déterminer la nature des IKDC et de mettre fin à ce débat. Par la suite, il sera important d’identifier des façons de cibler spécifiquement les IKDC pour permettre leur usage dans le cadre de thérapies antitumorales. Ainsi, l’objectif de cette thèse est de définir l’identité des IKDC, puis de déterminer les facteurs génétiques responsables de la régulation de ces cellules. Nous avons démontré que les IKDC ne sont pas des cellules aNK, contrairement à ce qui avait été suggéré. Nous avons constaté que les IKDC prolifèrent activement et possèdent un phénotype unique, des caractéristiques associées à des cellules NK très immatures. Afin de déterminer si les IKDC peuvent acquérir un phénotype mature, nous avons effectué des expériences de transfert adoptif. Suite à leur injection in vivo, les IKDC acquièrent un phénotype de cellules matures, mais étonnamment, elles se différencient aussi en cellules NK. Ainsi, nous avons révélé que les IKDC sont un intermédiaire dans la différenciation des cellules NK. En parallèle, nous avons démontré que la proportion d’IKDC varie grandement entre des souris de fond génétique différent, indiquant que des facteurs génétiques sont impliqués dans la régulation de ces cellules. Nous avons alors effectué une analyse génétique qui a révélé que les IKDC sont régulées par des facteurs génétiques compris dans une région distale du chromosome 7. Les résultats présentés dans cette thèse constituent une avancée importante pour la recherche sur les IKDC. Ils ont permis de définir la nature des IKDC et d’identifier un intervalle génétique impliqué dans la régulation de ces cellules. Ces découvertes sont des connaissances précieuses pour l’identification des IKDC chez l’Homme et la création de nouvelles thérapies dans la lutte contre le cancer. / The idea that a cell could kill transformed cells, like a Natural Killer (NK) cell, all the while exhibiting also the capacity to present antigens to T cells, like a Dendritic Cell (DC), may seem farfetched. However, in mice, a cell presenting these specific properties was identified in 2006. These cells were named Interferon-producing Killer Dendritic Cells (IKDC) and extensive studies revealed that they were endowed with an important immunological potential. Indeed, the fact that IKDCs exhibit properties of both DC and NK cells conferred them with an exceptional anti-tumor potential. Notably, on a per cell basis, the in vivo anti-tumor activity of IKDCs is more efficient than NK cells. Therefore, following their identification, IKDCs showed great therapeutic promise. However, a debate on the cell lineage origin of IKDCs emerged. Several independent groups could not replicate the finding that IKDCs showed functional antigen-presentation properties similar to DCs. Also, additional studies revealed that IKDCs are very similar to NK cells. These and other observations led the scientific community to believe that IKDCs were activated NK cells. Despite this controversy, IKDCs clearly exhibit a unique and outstanding anti-tumor potential, highlighting the relevance to further explore these cells. We must first close the debate regarding the lineage origin of IKDCs. We subsequently need to identify a means to specifically target IKDCs to facilitate their use in novel anti-tumor therapies. Thus, the objective of my thesis is first, to define the identity of IKDCs and second, to determine the genetic factors implicated in the regulation of these cells. For the first objective, we demonstrated that IKDCs do not represent activated NK cells, as previously suggested. We show that IKDCs are highly proliferative and exhibit a unique phenotype associated with very immature NK cells. In an attempt to verify if IKDCs could acquire a mature phenotype, we conducted an adoptive transfer experiment. We found that, after adoptive transfer, IKDCs adopt a mature phenotype, but also surprisingly differentiate into NK cells. These findings indicate that IKDCs represent an intermediate in NK-cell differentiation. For the second objective, we demonstrated that the IKDC proportion was highly variable between strains of different background origins, indicating that these cells are regulated by genetic factors. A genetic study revealed that genetic factors in distal arm of chromosome 7 associate with the proportion of IKDCs. The results presented in this thesis represent an important breakthrough for the research on IKDCs. They allowed to define the cell lineage origin of IKDCs and to identify a genetic region involved in the regulation of this cell type. These discoveries are valuable knowledge for the identification of human IKDCs and the development of novel anti-tumor therapies.

Introdução: A infecção pelo HIV causa um profundo comprometimento da resposta imune do hospedeiro, podendo levar à aids. Várias estratégias terapêuticas têm sido testadas ao longo dos anos, entre elas a imunoterapia com células dendríticas diferenciadas a partir de monócitos (MoDCs), pulsadas com HIV-1 inativado. Neste caso, a produção de vírus para o pulso das MoDCs consiste inicialmente no isolamento do vírus a partir de amostras de sangue do paciente e, em seguida, sua expansão em culturas de células CD4. Também deve ser considerado que quantidade excessiva de vírus pode ser tóxica para as MoDCs a serem pulsadas e do mesmo modo, quantidade insuficiente de vírus pode não ser efetiva para ativar uma resposta imune especifica. Neste contexto, a investigação do efeito de diferentes concentrações de vírus sobre o perfil fenotípico e funcional de MoDCs poderia auxiliar na determinação de uma quantidade ótima de vírus para o pulso das MoDCs e contribuir para o aperfeiçoamento da vacina terapêutica. Objetivo: Avaliar o efeito de diferentes quantidades de partículas virais, sobre o perfil fenotípico e funcional das MoDCs. Metodologia: Monócitos obtidos de indivíduos HIV+ foram diferenciados em MoDCs e pulsadas com HIV quimicamente inativado (3 partículas/MoDC, 30 partículas/MoDC, 300 partículas/MoDC). As células foram analisadas com relação ao perfil fenotípico, capacidade de internalizar p24, expressão de CD38, HLA-DR e CD69 e a produção de IFN-y por linfócitos T CD4+ e CD8+ autólogos. Resultados: O pulso com concentrações crescentes de vírus parece não interferir no perfil fenotípico e funcional das MoDCs. Conclusão: As diferentes quantidades de partículas virais utilizadas para o pulso parecem não ser tóxicas para as MoDCs estudadas, não tendo sido observadas diferenças com relação ao perfil fenotípico ou funcional das MoDCs / Introduction: The infection from HIV causes a profound impairment of the host immune response, which can lead to aids. Several therapeutic strategies have been tested over the years, including immunotherapy with monocyte - derived dendritic cells (MoDCs), pulsed with inactivated HIV-1. In this case, the production of virus for the pulse of the MoDCs initially consists of isolating the vírus from the patient\'s blood samples and then it into CD4+ cell cultures. It should also be considered that excessive amount of virus can be toxic to the MoDCs to be pulsed and likewise, insufficiently amount may not be effective for properly activate a specific immune response. In this context, the investigation of the effect of different virus concentrations on the phenotypic and functional profile of MoDCs could assist in the determination of an optimal amount of virus for the pulse of the MoDCs and contribute to the improvement of the therapeutic vaccine. Objectives: To evaluate the effect of different amounts of viral particles on the phenotypic and functional profile of MoDCs. Methods: MoDCs generated from HIV+ individuals were differentiated into MoDCs and pulsed with chemically inactivated HIV (3 particles /MoDC, 30 particles /MoDC, 300 particles /MoDC). Cells were analyzed for phenotypic profile, ability to internalize p24, expression of CD38, HLA-DR and CD69, and the production of IFN-y by autologous CD4 + and CD8 + T lymphocytes. Results: The pulse with increasing concentrations of virus does not seem to interfere in the phenotypic and functional profile of the MoDCs. Conclusion: The different amounts of viral particles used for the pulse appear to be non-toxic to the MoDCs studied, and no differences were observed regarding the phenotypic or functional profile of the MoDCs

Cytomegalovirus (CMV) is a ubiquitous pathogen affecting over 95% of the world’s population. While infection is typically asymptomatic in healthy individuals, the virus persists life-long in its host and can be reactivated following withdrawal of immune control. As such, it remains a serious clinical concern in individuals who are immunocompromised, such as newborns and neonates, transplant and/or chemotherapy recipients, and HIV/AIDS patients. CMV also has the ability to cause immunosuppression, the mechanisms of which include defective antigen presentation to T cells and interference with haematopoiesis in the bone marrow (BM). Due to strict species specificity, murine CMV (MCMV) provides a relevant model for the study of CMV modulation of the immune system in vivo in its natural host. The type I interferons (IFNs) represent a major family of cytokines involved in the early response to MCMV infection. Their anti-viral activity and regulation of NK cell activation and cytotoxicity are of significant interest in the context of MCMV infection, as genetic resistance to MCMV is mediated by the ability of Ly49H+ NK cells to directly recognise and lyse infected cells. Chapter 2 comprises an analysis of acute MCMV infection in the absence of type I IFN activity. These studies were conducted in IFNAR1 and IFNAR2 deficient mice, which lack components of the type I IFN receptor. Data obtained from these studies confirmed the essential requirement for type I IFN in controlling viral titres, promoting expansion of splenic Ly49H+ NK cells, and inducing early activation of NK cell cytotoxicity. In addition, our data depicted an accumulation of infected myeloid cells in the absence of effective NK cell-mediated control. This was paralleled by a significant increase in the level of serum TNF-a and IFN-¿, an effect which in some cases has been linked to serious pathological disease. Thus, the data described in this chapter provide an insight into the consequences arising from delayed NK cell responses to MCMV infection in the absence of type I IFN. vii Type I IFN can also potentially affect BM haematopoiesis. BM atrophy and impairment of myelopoiesis are serious consequences of CMV infection. During acute MCMV infection we consistently observed a profound loss of splenic dendritic cells (DCs) in BALB/c mice. Since all DC subsets are derived from BM haematopoietic progenitor cells, the possibility that MCMV might interfere with BM haematopoiesis and DC differentiation was explored. Chapters 3 and 4 describe the impact of acute MCMV infection on BM progenitors, with particular emphasis on the differentiation capabilities of these cells in ex vivo culture systems. Chapter 3 focuses on the effect of MCMV infection on BM cellularity and frequency of specific BM progenitor populations. A thorough analysis of contributing factors, such as viral infection of BM cells, involvement of type I and II IFNs, progenitor cell trafficking and NK cell activity in the BM compartment, was conducted. Our results showed that a severe loss of BM cellularity occurs in MCMV-infected mice. Furthermore, when BM cells from MCMV-infected mice were cultured ex vivo in granulocyte macrophage-colony stimulating factor (GM-CSF), there was an impairment in their ability to differentiate into DCs.

Το Μυελοδυσπλαστικό Σύνδρομο (ΜΔΣ) αποτελεί νόσημα με διαταραχή σε επίπεδο αρχέγονου αιμοποιητικού κυττάρου (stem cell) που χαρακτηρίζεται από μη αποδοτική αιμοποίηση και κυτταροπενίες του περιφερικού αίματος που περιλαμβάνουν μία ή περισσότερες αιμοποιητικές σειρές. Διάφορες ανοσολογικές διαταραχές των ασθενών με ΜΔΣ, όπως, αυξημένη ευαισθησία σε βακτηριακές λοιμώξεις, αυτοάνοσα φαινόμενα και υψηλή συχνότητα κακοηθειών του λεμφικού ιστού, υποδεικνύουν αδυναμία των ασθενών με ΜΔΣ για ανοσολογική απάντηση, οι αιτίες των οποίων παραμένουν άγνωστες μέχρι σήμερα. Τα Δενδριτικά Κύτταρα (ΔΚ) είναι κύτταρα του ανοσολογικού μηχανισμού που προέρχονται από το μυελό των οστών. Ως αντιγονοπαρουσιαστικά κύτταρα (APC), είναι εξειδικευμένα για τη πρόσληψη, επεξεργασία, μεταφορά και παρουσίαση του αντιγόνου στα Τ λεμφοκύτταρα. Στη παρούσα μελέτη πραγματοποιήθηκε ανάλυση διαφορετικών ποσοτικών και λειτουργικών παραμέτρων των ΔΚ από ασθενείς με Μυελοδυσπλαστικό Σύνδρομο, in vivo ή in vitro. Αρχικά διερευνήθηκε ο αριθμός, ο φαινότυπος, η ικανότητα ενδοκύττωσης και η αλλογενής διεγερτική δυνατότητα των ΔΚ, προερχόμενων από μονοκύτταρα του περιφερικού αίματος (ΜοΔΚ) ασθενών με ΜΔΣ και υγιών μαρτύρων, σε διαφορετικά στάδια διαφοροποίησης. Τα μονοκύτταρα των ασθενών με ΜΔΣ χαρακτηρίστηκαν από μειωμένη ικανότητα διαφοροποίησης σε ΔΚ, λόγω του μειωμένου αριθμού των διαφοροποιημένων κυττάρων και τη χαμηλή έκφραση του CD1a αντιγόνου επιφανείας. Τα ΜοΔΚ των ΜΔΣ ασθενών παρουσίασαν χαμηλή έκφραση του υποδοχέα της μανόζης και μειωμένη ικανότητα ενδοκύττωσης. ΜοΔΚ των ΜΔΣ ασθενών επέδειξαν μειωμένη απάντηση ύστερα από διέγερση με TNF-α, καθώς η έκφραση των CD83, CD80 και CD54 αντιγόνων και η αλλοδιεγερτική ικανότητα ήταν μειωμένη, ενώ η επίδραση με LPS είχε ως αποτέλεσμα να εμφανίσουν φαινοτυπικά χαρακτηριστικά και ικανότητα διέγερσης των Τ-κυττάρων, όμοια με τα ΜοΔΚ των φυσιολογικών μαρτύρων. Σε δύο από τους ασθενείς με σύνδρομο 5q-, σχεδόν όλα τα μονοκύτταρα και τα ΜοΔΚ περιείχαν τη χρωμοσωμική διαταραχή, υποδηλώνοντας την προέλευσή τους από τον παθολογικό κλώνο. Στη συνέχεια διερευνήθηκε το δυναμικό πολλαπλασιασμού και διαφοροποίησης των CD34+ προγονικών κυττάρων του μυελού ασθενών με ΜΔΣ σε δενδριτικά κύτταρα (CD34-ΔΚ) σε υγρή καλλιέργεια παρουσία κυτοκινών. Παράλληλα, έγινε ανάλυση των κυκλοφορούντων ΔΚ περιφερικού αίματος στους ίδιους ασθενείς. Τα CD34+ προγονικά κύτταρα παρουσίασαν χαμηλή δυνατότητα ανάπτυξης ΔΚ in vitro, καθώς ο αριθμός των παραγόμενων ΔΚ ανά CD34+ κύτταρο ήταν χαμηλότερος συγκριτικά με τα δείγματα των υγιών μαρτύρων. Παρά την αυξημένη απόπτωση των προγονικών κυττάρων του μυελού των ΜΔΣ ασθενών, η επιβίωση και ο πολλαπλασιασμός των CD34+ κυττάρων στην καλλιέργεια, δεν συσχετίστηκε με την απόπτωση και αποτελεί αξιοσημείωτη παρατήρηση. Φαινοτυπικά, τα CD34-ΔΚ των ΜΔΣ ασθενών δεν διέφεραν από τα ΔΚ που παρήχθησαν από τα CD34+ κύτταρα του μυελού των φυσιολογικών μαρτύρων καθώς επέδειξαν όμοια έκφραση των CD83, CD80, CD40, HLA-DR και CD54 αντιγόνων. Κυτταροεπιλεγμένα CD1a+ κύτταρα ασθενών είχαν όμοια διεγερτική ικανότητα αλλογενών Τ κυττάρων με τα CD34-ΔΚ των φυσιολογικών ατόμων. Το ποσοστό των κυκλοφορούντων μυελοειδών- και πλασματοκυτταροειδών- ΔΚ στους ασθενείς με ΜΔΣ ήταν σημαντικά μειωμένο συγκριτικά με τους υγιείς μάρτυρες. Στους ασθενείς με 5q έλλειψη, τόσο τα CD34-ΔΚ, όσο και τα ΔΚ του αίματος, είχαν τη χρωμοσωμική ανωμαλία. Τα παραπάνω αποτελέσματα υποδηλώνουν ότι η διαδικασία παραγωγής δενδριτικών κυττάρων από το μυελό (‘δενδριτοποίηση’) των ασθενών με ΜΔΣ, είναι μέρος της κλωνικής διαταραχής με αποτέλεσμα την μη αποδοτική παραγωγή ΔΚ από τα προγονικά κύτταρα του μυελού και το χαμηλό ποσοστό των κυκλοφορούντων πρόδρομων ΔΚ. Όλες οι ΔΚ υποομάδες προέρχονται από τον παθολογικό κλώνο και χαρακτηρίζονται από ποσοτικές και ποιοτικές ανωμαλίες. Το σύνολο αυτών των διαταραχών που παρατηρήθηκαν στα ΔΚ πολύ πιθανόν να συμβάλει στη διαταραγμένη ανοσολογική απάντηση έναντι παθογόνων οργανισμών, στην επιβίωση και στην επικράτηση του παθολογικού κλώνου, όπως επίσης και στην εμφάνιση αυτοάνοσων φαινομένων, που παρατηρούνται στους ασθενείς με ΜΔΣ. / Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis and blood cytopenias involving one or several myeloid lineages. Various immune disturbances in MDS such as increased susceptibility to bacterial infections, autoimmune phenomena and high incidence of lymphoid malignancies reveal an underlying defect of the immune response in MDS patients, the reasons for which still remain unclear. Dendritic cells (DCs) are bone marrow derived cells. As the most potent antigen presenting cells (APC), they are specialized for the uptake, processing, transport and presentation of Ag to T cells. In the present study different quantitative and functional parameters of DCs in patients with MDS were analyzed either in vivo or in vitro. The number, phenotype, endocytic ability, and allostimulatory capacity of DCs derived from peripheral blood monocytes (MoDCs) were investigated in patients with MDS and healthy controls at different stages of differentiation using the maturation stimuli-TNF-á and LPS. Monocytes in MDS showed low potential to differentiate into DCs, as determined by low cell yield and CD1a expression. MDS-MoDCs exhibited low expression of Mannose receptor and reduced endocytic capacity. When stimulated with TNF-á, MoDCs obtained from MDS patients showed a diminished response with low CD83, CD80 and CD54 expression and allostimulatory capacity, whereas in the presence of LPS MDS-MoDCs acquired phenotypic characteristics and ability to stimulate T-cells similar to MoDCs derived from controls. In two patients with 5q- syndrome the vast majority of both monocytes and MoDCs were positive for the 5q deletion, suggesting that they originate from the malignant clone. Second, we investigated the potential of bone marrow CD34+ progenitors in patients with MDS to proliferate and differentiate into DCs in a liquid cytokine supplemented culture system and also analyzed the status of blood DC subsets in those patients. CD34+ progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34+ cell was significantly lower compared to controls. Interestingly, although the increased apoptotic level of bone marrow progenitors in MDS, the survival and proliferation of CD34+ cells in culture was not correlated to the degree of apoptosis. Phenotypically the MDS CD34-DCs did not differ from DCs obtained from normal BM CD34+ cells, exhibiting similar expression of CD83, CD80, CD40, HLA-DR, and CD54. FACsorted CD1a+ cells from MDS patients were as efficient stimulators of allogeneic T cells as normal CD34-DCs. The percentage of both circulating DC subsets, MDCs and PDCs in MDS patients was extremely diminished compared to controls. In cases with the 5q deletion both CD34-DCs and blood DCs harbor the cytogenetic abnormality. The results indicate that “dendritopoiesis” in MDS is affected by the transformation process resulting in ineffective production of DCs from bone marrow progenitors with low circulating blood precursors. All DC subsets were derived from the malignant clone and exhibited quantitative and qualitative abnormalities. This constellation of DCs defects probably contribute to the defective immune response against pathogens, escape and expansion of the malignant clone, as well as autoimmune phenomena, observed in MDS patients.

L’idée qu’une cellule puisse effectuer la cytolyse de cellules transformées, comme une cellule Natural Killer (NK), tout en ayant la capacité de présenter des antigènes, comme une cellule dendritique (DC), peut sembler fantaisiste. Cependant, de telles cellules furent bel et bien identifiées chez la souris en 2006. Ces cellules, nommées Interferon-producing Killer Dendritic Cells (IKDC), furent l’objet d’une caractérisation extensive qui révéla leur énorme potentiel immunologique. La combinaison de fonctions associées à des cellules NK et à des DC a doté les IKDC d’un pouvoir antitumoral remarquable. D’ailleurs, il a été démontré que les IKDC sont plus efficaces que les cellules NK pour limiter la croissance tumorale. Ainsi, suite à leur découverte, les IKDC ont suscité beaucoup d’intérêt. Cependant, une controverse émergea sur la nature des IKDC. Plusieurs groupes indépendants tentèrent de reproduire les expériences attestant les fonctions de DC des IKDC, sans y parvenir. De plus, des études additionnelles révélèrent que les IKDC possèdent des similitudes très importantes avec les cellules NK. Ces observations ont mené la communauté scientifique à suggérer que les IKDC sont des cellules NK en état d’activation (aNK). Malgré cette controverse, les caractéristiques antitumorales des IKDC sont si uniques et considérables qu’il est primordial de poursuivre l’étude de ces cellules. Pour y arriver, il est essentiel de déterminer la nature des IKDC et de mettre fin à ce débat. Par la suite, il sera important d’identifier des façons de cibler spécifiquement les IKDC pour permettre leur usage dans le cadre de thérapies antitumorales. Ainsi, l’objectif de cette thèse est de définir l’identité des IKDC, puis de déterminer les facteurs génétiques responsables de la régulation de ces cellules. Nous avons démontré que les IKDC ne sont pas des cellules aNK, contrairement à ce qui avait été suggéré. Nous avons constaté que les IKDC prolifèrent activement et possèdent un phénotype unique, des caractéristiques associées à des cellules NK très immatures. Afin de déterminer si les IKDC peuvent acquérir un phénotype mature, nous avons effectué des expériences de transfert adoptif. Suite à leur injection in vivo, les IKDC acquièrent un phénotype de cellules matures, mais étonnamment, elles se différencient aussi en cellules NK. Ainsi, nous avons révélé que les IKDC sont un intermédiaire dans la différenciation des cellules NK. En parallèle, nous avons démontré que la proportion d’IKDC varie grandement entre des souris de fond génétique différent, indiquant que des facteurs génétiques sont impliqués dans la régulation de ces cellules. Nous avons alors effectué une analyse génétique qui a révélé que les IKDC sont régulées par des facteurs génétiques compris dans une région distale du chromosome 7. Les résultats présentés dans cette thèse constituent une avancée importante pour la recherche sur les IKDC. Ils ont permis de définir la nature des IKDC et d’identifier un intervalle génétique impliqué dans la régulation de ces cellules. Ces découvertes sont des connaissances précieuses pour l’identification des IKDC chez l’Homme et la création de nouvelles thérapies dans la lutte contre le cancer. / The idea that a cell could kill transformed cells, like a Natural Killer (NK) cell, all the while exhibiting also the capacity to present antigens to T cells, like a Dendritic Cell (DC), may seem farfetched. However, in mice, a cell presenting these specific properties was identified in 2006. These cells were named Interferon-producing Killer Dendritic Cells (IKDC) and extensive studies revealed that they were endowed with an important immunological potential. Indeed, the fact that IKDCs exhibit properties of both DC and NK cells conferred them with an exceptional anti-tumor potential. Notably, on a per cell basis, the in vivo anti-tumor activity of IKDCs is more efficient than NK cells. Therefore, following their identification, IKDCs showed great therapeutic promise. However, a debate on the cell lineage origin of IKDCs emerged. Several independent groups could not replicate the finding that IKDCs showed functional antigen-presentation properties similar to DCs. Also, additional studies revealed that IKDCs are very similar to NK cells. These and other observations led the scientific community to believe that IKDCs were activated NK cells. Despite this controversy, IKDCs clearly exhibit a unique and outstanding anti-tumor potential, highlighting the relevance to further explore these cells. We must first close the debate regarding the lineage origin of IKDCs. We subsequently need to identify a means to specifically target IKDCs to facilitate their use in novel anti-tumor therapies. Thus, the objective of my thesis is first, to define the identity of IKDCs and second, to determine the genetic factors implicated in the regulation of these cells. For the first objective, we demonstrated that IKDCs do not represent activated NK cells, as previously suggested. We show that IKDCs are highly proliferative and exhibit a unique phenotype associated with very immature NK cells. In an attempt to verify if IKDCs could acquire a mature phenotype, we conducted an adoptive transfer experiment. We found that, after adoptive transfer, IKDCs adopt a mature phenotype, but also surprisingly differentiate into NK cells. These findings indicate that IKDCs represent an intermediate in NK-cell differentiation. For the second objective, we demonstrated that the IKDC proportion was highly variable between strains of different background origins, indicating that these cells are regulated by genetic factors. A genetic study revealed that genetic factors in distal arm of chromosome 7 associate with the proportion of IKDCs. The results presented in this thesis represent an important breakthrough for the research on IKDCs. They allowed to define the cell lineage origin of IKDCs and to identify a genetic region involved in the regulation of this cell type. These discoveries are valuable knowledge for the identification of human IKDCs and the development of novel anti-tumor therapies.