The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 1, 2010). Vita. Thesis advisor: Habib Zaghouani. "May 2008" Includes bibliographical references.

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: The aim of this study was to investigate the effect of MBV and Coley’s Toxin on dendritic cells in vitro. The dendritic cell system of antigen presenting cells is the initiator and modulator of the immune response. The principle function of the dendritic cells is to present antigens to resting naïve T lymphocytes: these cells are the only APCs that prime naïve T cells and only mature DCs can carry out this function.Previous studies done on dendritic cells showed that bacterial peptides can induce the maturation of dendritic cells. With the results of these studies in mind we hypothesized that these two vaccines will also induce the maturation of dendritic cells. Chapter 1 is a literature review on the immune system explaining the organs and cells of the immune system. Chapter 2 includes a full description of DCs, the MBV and Coley’s toxin. Also included in this chapter is a short explanation of the principle of the technique being used for the identification and maturation of both mDCs and pDCs, namely the technique of flow cytometry. Chapter 3 describes the method for the phenotypic identification of DCs: the subsets are distinguished by their absence of expression of several lineage markers for lymphocytes, monocytes and NK cells and the expression of CD11c (in the case of myeloid DCs) and CD123 (in the case of plasmacytoid DCs). The inclusion of HLA-DR in addition to the previous described markers allows the discrimination of CD123+ DCs from basophils. The assay requires three tubes per sample which enables quick analysis of these rare subsets with a small sample volume. This assay was applied to peripheral blood samples obtained from healthy individuals and individuals with cancer, HIV and HIV and TB co-infected patients. Our results showed that the maturation status of DCs in HIV and lymphoma were low but those measured in the case of HIV + TB patients were even higher than in the control group. Chapter 4 and 5 describe the in vitro activation and maturation status of DCs following their incubation with bacterial-derived products. Interactions between DCs and microbial pathogens are fundamental to the generation of innate and adaptive immune responses and upon contact with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of human DCs to MBV and Coley’s Toxin. Previous studies showed DCs can be activated with killed Streptococcus pyogenes. With this study in mind it was hypothesized that the MBV and Coley’s Toxin used in this study might modulate DC maturation. The results of this study showed that the MBV and Coley’s toxin did induce the maturation of both pDCs and mDCs as measured by increased surface expression of costimulatory molecules such as CD80 and CD83. Chapter 6 presents the measurement of cytokines released after the PMBCs had been were incubated with Coley’s Toxin and Mixed Killed bacteria. The BD™ Cytometric Bead Array (CBA) flex set was used for the simultaneous detection of multiple soluble analytes. The results indicated that both Coley’s Toxin and the MBV activated the DCs and subsequently induced TH1 as well as a TH2 responses in the T cells present in the cell cultures. Finally, a general conclusion discussing the significance and implications of our results as well as possible future research required is discussed in Chapter 7. DCs are potent antigen presenting cells (APCs) which play a critical role in the regulation of the immune response. There is great interest in exploiting DCs to develop immunotherapies for cancer, chronic infections, immunodeficiency diseases and autoimmune diseases. / AFRIKAANSE OPSOMMING: Die doel van die studie was om die effek van ‘n gemengde bakteriële vaksiene en Coley se toksiene op dendritiese selle te toets in vitro. Die dendritiese sel sisteem speel ‘n belangrike rol in die modulering en reaksie van die immuun sisteem.Die hoof funksie van dendritiese selle is om antigene bloot te stel aan naïewe ongeaktiveerde T selle. Slegs volwasse dendritiese selle kan die T selle aktiveer. Vorige studies het bewys dat bakteriële peptiedes die veroudering van die dendritiese selle kan induseer. Met die resultate in gedagte het ons gehipotiseer dat die twee vaksienes ook die maturasie van dendritiese selle kan induseer. Hoofstuk 1 is ‘n literatuur studie wat handel oor die organe en selle van die immuun sisteem. Hoofstuk 2 gee n volle beskrywing van dendritiese selle, die gemengde bakteriële vaksiene en Coley se toksiene. Ingesluit in die hoofstuk is die beskrywing van die prinsiep van die tegniek, vloei sitometrie, wat gebruik word vir die identifikasie en veroudering status van die dendritiese selle. Hoofstuk 3 beskryf ‘n vloei sitometrie metode vir die fenotipiese identifikasie van dendritiese selle. Dendritiese sel tipes kan onderskei word deur die afwesigheid van sekere merkers vir limfosiete, monosiete en NK selle. Plasmasitoïede dendritiese selle druk CD123 uit en miloïede dendritiese selle druk CD11c uit. HLA DR is ook ingesluit saam met die bogenoemde merkers om die dendritiese selle te onderskei van basofiele. Vir elke toets word slegs drie buise geprosesseer en dus kan die subklasse vinning geanaliseer word. ʼn Klein volume bloed word benodig vir die toests. Perifêre bloed is gebruik vir die toets op bloed monsters van 10 gesonde individue en individue met kanker, HIV en HIV en TB. Die resultate van die studie het getoon dat die maturasie status van die dendritiese selle in HIV en limfoom was, maar in die geval van HIV en TB pasïente was die maturasie status selfs hoër as die van die kontrole groep. Hoofstuk 4+5 beskryf die aktivering en maturasie status van die dendritiese selle na inkubasie met die bakteriële produkte. Interaksie tussen dendritiese selle en patogene speel ‘n belangrike rol in die aktivering van die immuunstelsel. Wanneer dendritiese selle in aanraking kom met bakterieë of bakteriële komponente, matureer die dendritiese sel wat lei tot the uitdrukking van stimulerings molekules, HLA molekules end die uitskeiding van sitokiene. Die uitdrukking van die molekules lei tot limfosiet ontwikkeling en differensiasie. In die studie het ons gekyk na die reaksie van menslike dendritiese selle in die teenwoordigheid van die gemende bakteriële vaksiene en Coley se toksiene. Vorige studies het bewys dendritiese selle word geaktiveer deur Streptococcus pyogenes. Met die resultate in gedagte het ons gehipotetiseer dat die gemengde bakteriële vaksiene en Coley se toksiene ook die maturasie van dendritiese selle kan induseer. Die resultate van die studie het bewys dat die gemengde bakteriële vaksiene en Coley se toksiene die veroudering van beide pDCs en mDCs induseer. Die uitdrukking van verouderings merkers CD80 en CD83 is gemeet. Hoofstuk 6 beskryf ‘n vloei sitometrie metode om die sitokiene te meet wat afgeskei word nadat selle geinkubeer het in die teenwoordigheid van Coley se toksiene en die gemengde bakteriële vaksiene.Die BDTM CBA Flex set metode het dit moontlik gemaak om meer as een sitokiene te meet in net een buis Die resultate het getoon dat albei die vaksienes ‘n TH1 en TH2 reaksie veroorsaak. Laastens volg‘n algemene afleiding waar ons kyk na die toepassing en implikasies van die resultate asook toekomstige navorsings moontlikhede,word bespreek in Hoofstuk 7 Dendritiese selle speel ‘n kritiese rol in die regulering van die immuun reaksie. Verdere studies kan nou gedoen word om dendritiese selle terapeuties toe te pas vir die behandeling van kanker, autoimmuniteit, immuun onderdrukkende siektes en kroniese siektes.

Les basophiles sont les granulocytes les plus rares. Ils sont impliqués dans la polarisation des réponses immunitaires de type Th2, dans la différenciation des lymphocytes B et dans la protection contre les infections helminthiques. Les basophiles sont impliqués dans la modulation des réponses immunitaires, en particulier dans les maladies auto-immunes et inflammatoires. Des études récentes ont montré que les basophiles murins sont cellules présentatrices d’antigène (CPA) et induisent des réponses Th2 et IgE contre les allergènes et les infections helminthiques.Par conséquent, Nous avons exploré les fonctions des basophiles humains, en particulier comme CPA professionnelles. Les résultats montrent que les basophiles, contrairement aux cellules dendritiques et monocytes, n’expriment pas HLA-DR et les marqueurs de co-stimulations CD80 et CD86. De plus, la stimulation des basophiles par divers allergènes, comme des ligands de TLR et IgE, n’induit pas des changements dans l’expression de ces marqueurs. Enfin, nos résultats montrent que les basophiles ne favorise pas les réponses immunitaire de type Th2 ou Th17. Ainsi,notre étude montre que les basophiles humains circulant ne possèdent pas des fonctions de CPA professionnelles. Des plus, les basophiles sont impliqués dans la pathogenèse de maladies auto-immunes et inflammatoires dépendantes des réponses Th2 et médiées par les lymphocytes B. Puisque la dérégulation des basophiles joue un rôle important dans le développement des réponses immunitaires dans différentes conditions pathologiques, nous avons exploré les mécanismes de régulations qui modulent les fonctions les basophiles. En particulier, nous avons étudié le rôle suppresseur des lymphocytes T régulateurs (Tregs) CD4+CD25+FoxP3, des cellules clés dans la maintenance de l’homéostasie immune, sur les fonctions des basophiles. Nos résultats montrent que les fonctions des basophiles, contrairement à la majorité des cellules immunes, ne sont pas régulées par les Tregs. Bien au contraire, nos résultats montrent que les lymphocytes T favorisent l’activation des basophiles. En résumé, nous avons exploré de nouveaux mécanismes cellulaires et moléculaires impliqués dans la régulation des fonctions des basophiles humains. Ces résultats nous permettent de mieux comprendre le rôle des basophiles dans les conditions inflammatoires et dans le développement de nouvelles stratégies thérapeutiques. / Basophils are the rare granulocytes and play an important role in the polarization of Th2 responses, differentiation of B cells and protection against helminths. Basophils have a major influence on immune responses and various roles of these cells in autoimmune and inflammatory diseases are emerging. Recent reports showed that murine basophils function as antigen presenting cells (APCs) to induce Th2 and IgE responses to allergens and helminths. Therefore, I explored whether human basophils possess the features of APCs. I found that unlike dendritic cells (DCs) and monocytes, steady-state circulating human basophils did not express HLA-DR and co-stimulatory molecules CD80 and CD86. Basophils remained negative for these molecules following stimulation with various allergens, toll-like receptor ligands and IgE cross-linking.Unlike DCs, basophils did not promote Th2 and Th17 responses. Together, these results demonstrate the inability of circulating human basophils to function as professional APC. Further, basophils were also reported to be implicated in the pathogenesis of Th2 –associated and B cell-mediated autoimmune and inflammatory diseases. Considering the impact of dysregulated function of basophils on the outcome immune responses in various pathological conditions, it was essential to investigate the regulatory mechanisms by which basophil functions are kept in check. As CD4+CD25+FoxP3+ regulatory T cells (Tregs) are critical for the maintenance of immune homeostasis, I sought to investigate the interaction of Tregs with human basophils and its repercussion on basophil functions. My results indicated that unlike other immune cells that aresusceptible to Treg-mediated suppression, basophils are refractory to regulatory mechanism of Tregs. On the contrary I found that T cells could promote activation of basophils. My results thus provided an insight on cellular and molecular basis of regulation of human basophil functions. These data will have a repercussion in understanding role of basophils ininflammatory conditions and in designing therapeutic strategies.

La plupart des chimiothérapies sont connues pour exercer une immunosuppression en plus de leur effet cytotoxique, car elles ciblent sans distinction les cellules à prolifération rapide telles que les cellules tumorales ainsi que les cellules du système immunitaire. Cependant, la radiothérapie ainsi que certaines chimiothérapies comme les anthracyclines ou l’oxaliplatine ont montré une propriété immunostimulante, grâce à l’induction d’une mort cellulaire dite immunogène. Cette mort cellulaire se caractérise par la libération de signaux de danger par la cellule mourante qui vont activer le système immunitaire. Tout d’abord, l’exposition de la calréticuline à la surface de la cellule va être un signal de phagocytose pour les cellules dendritiques. Les cellules succombant à la mort cellulaire libèrent aussi HMGB1 qui en se liant à TLR4 permet un apprêtement et une présentation des antigènes tumoraux efficace. Ensuite, la libération d’ATP qui agit sur les récepteurs P2RX7 permet l’activation de l’inflammasomme NLRP3 et conduit à la sécrétion d’IL-1β indispensable pour l’activation des lymphocytes T CD8+ sécrétant de l’IFN-γ. L’autophagie est un processus de dégradation permettant de limiter l’instabilité génomique et l’initiation de cancers. L’autophagie, peut être induite après un stress du réticulum endoplasmique, qui est nécessaire à l’exposition de la calréticuline lors de la mort cellulaire immunogène. Nous avons donc cherché à évaluer l’importance de l’autophagie après traitement aux chimiothérapies immunogènes. Nous avons montré que l’autophagie est requise pour induire la libération d’ATP lors de la mort cellulaire immunogène. De plus, nous avons montré que l’ATP libéré par les cellules mourantes après traitement aux chimiothérapies immunogènes permet le recrutement de cellules de type monocyte inflammatoire (CD11b+Ly6ChighLy6G-) ainsi que de leurs précurseurs. En outre, l’ATP est un facteur important dans la différenciation de ces cellules en cellules dendritiques inflammatoires. Les cellules CD11b+Ly6ChighLy6G- ont montré une grande capacité à présenter les antigènes tumoraux aux lymphocytes T CD8+ permettant leur activation. Les cellules déficientes pour l’autophagie n’ont quant à elles pas permit le recrutement de cellules dendritiques dans les tumeurs ni l’activation de lymphocytes T CD8+. Ces travaux ont permit de montrer l’importance de l’autophagie pour mettre en place une réponse immunitaire anti-tumorale spécifique lors du traitement avec des chimiothérapies immunogènes. De plus, nous avons montré que l’ATP est impliqué dans le recrutement et la différenciation de cellules avec un phénotype de monocytes inflammatoires. L’ensemble de ces résultats apporte de nouveaux éléments dans la caractérisation du processus de mort cellulaire immunogène. / Most of the cytotoxic agents used in cancer therapy are known to be immunosuppressive, because of their unspecific targeting of rapidly dividing cells, like tumor cells, but also cells of the immune system. However, radiotherapy, as well as some chemotherapeutic agents such as anthracyclines or oxaliplatine were reported to have immunostimulatory effects, thanks to their capacity to induce immunogenic tumor cell death. This type of cell death is characterized by the release of danger signals from the dying tumor cell, which will activate the immune system. As a first event, exposure of calreticulin from the dying tumor cell will act as an « eat-me » signal for dendritic cells. Once released, the nuclear protein HMGB1 will bind to TLR4, facilitating antigen processing and presentation. The dying tumor cells will also release ATP, which acts on P2X7 receptors and activates NLRP3 inflammasomme, leading to IL-1 release, necessary for IFN-- producing CD8+ T cells activation. Autophagy is a degradation process limiting genomic instability and cancer initiation. It can be induced after a stress of the endoplasmic reticulum (ER). ER-stress is also involved in calreticulin exposure during immunogenic cell death, so we aimed at understanding the role of autophagy in immunogenic cell death. We found that autophagy is required for the release of ATP after treatment with immunogenic chemotherapies. Moreover, we showed that ATP released from the dying cells is necessary for the recruitment of immune cells with inflammatory monocyte phenotype (CD11b+Ly6ChighLy6G-), as well as their precursors. ATP was also important for the differentiation of these inflammatory monocytes into dendritic cells. These CD11b+Ly6ChighLy6G- cells were efficient in presenting the tumor antigens to CD8+ T cells, and to induce a tumor-specific immune response. However, autophagy-deficient cells were not able to recuit dendritic cells or to induce CD8+ T cells activation. These studies showed the importance of autophagy in tumor-specific immune response, after treatment with immunogenic chemotherapies. We also reported that ATP is involved in the recruitment and differentiation of cells with inflammatory monocytes phenotype. Altogether, these results give new insights in the concept of immunogenic cell death.

Dendritic cells (DCs) comprise a distinct lineage of potent antigen-presenting mononuclear phagocytes that serve as both mediators of innate immune responses and key facilitators of the adaptive immune response. DCs play both immunogenic and tolerogenic roles through their dual ability to elicit pathogen-specific T cell immunity as well as induce regulatory T cell (Treg) responses to promote tolerance in the steady state. The aim of the work presented here is to examine the normal regulatory mechanisms of DC development and function, starting with the dissection of mechanisms behind an aberrantly activated developmental pathway, followed by the exploration of new mechanisms governed by two candidate transcription factors. The first chapter of the thesis focuses on the growth factor receptor Flt3, an essential regulator of normal DC development in both mice and humans, and concurrently one of the most commonly mutated proteins found in acute myeloid leukemia (AML). We investigated the effect of its most common activating mutation in AML, the Flt3 internal tandem duplication (Flt3-ITD), and found that this mutation caused a significant cell-intrinsic expansion of all DC populations. This effect was associated with an expansion of Tregs and the ability to dampen self-reactivity, with an inability to control autoimmunity in the absence of Tregs. Thus, we describe a potential mechanism by which leukemia can modulate T cell responses and support Treg expansion indirectly through DCs, which may compromise immunosurveillance and promote leukemogenesis. The subsequent chapters explore the basic molecular mechanisms of DC development by using Flt3 expression as a guide to uncover new candidates involved in the DC transcriptional program. We show that Myc family transcription factor, Mycl1, is largely dispensable for DC development and function, contrary to recent published findings that propose a role in proliferation and T cell priming. On the other hand, we find that conditional deletion of our second candidate gene, an Ets family transcription factor, has diverse effects on DC development, monocyte homeostasis, and cytokine production. Overall, our studies highlight an unexpected molecular link between DC development and leukemogenesis, and elucidate novel mechanisms controlling DC differentiation and function.

La macroautophagie est un processus catabolique, situé au carrefour entre l’homéostasie et le métabolisme cellulaire. Dans le système immunitaire, elle joue des rôles spécialisés, en contribuant notamment à la régulation de l’inflammation et la présentation antigènique. En utilisant deux modèles murins, nous avons pu démontrer que la macroautophagy est nécessaire à la lignée lymphocytaire B et contribue à l’élaboration d’une réponse humorale optimale. En effet, la macroautophagie contribue à la survie des cellules sécrétrices d’antigènes, notamment la population plasmocytaire, ainsi que des cellules à longue durée de vie, telles que les lymphocytes B mémoire. Nous avons également démontré qu’une forme d’autophagie non-canonique était nécessaire pour la présentation efficace d’antigènes particulaires reconnus par le récepteur des lymphocytes B. Dans ce contexte, la machinerie macroautophagique contribue à la polarisation du cytosquelette des lymphocytes B, afin de former une synapse immunologique, nécessaire au chargement efficace du complexe majeur d’histocompatibilité de classe II, et ainsi, à la présentation antigènique. A l’aide d’un troisième modèle de souris transgénique, nous avons caractérisé un rôle jusqu’alors inconnu de la macroautophagie dans le maintient de l’homéostasie des cellules de Langerhans. L’inhibition de la macroautophagie altère la survie de ces cellules, en les exposant à potentiel stress du réticulum endoplasmique, potentiellement non compensé. En somme, nous avons démontré que la macroautophagie était un acteur majeur au sein des cellules présentatrices d’antigènes. / Macroautophagy is a catabolic process at the crossroad between homeostasis and metabolism. In the immune system it also possesses specialized roles such as inflammation regulation and antigen presentation. Here we demonstrated in two mice models that macroautophagy is integral to B cell lineage for proper humoral responses. Indeed it insures the survival of secreting cells such as plasma cells and long living cells such as memory B cells. We also report that non-canonical autophagy is also needed for an efficient presentation of particulate antigen recognized by the B cell receptor. In this context it drives B cell cytoskeleton polarization to form an immune synapse necessary for the efficient loading of class two major histocompatibility complexes and the subsequent antigen presentation. Using a third mice model we unveiled a yet uncharacterized function of macroautophagy in Langerhans cells, a subset of epidermal dendritic cells, homeostasis. Macroautophagy inhibition impairs their survival by exposing them to a potentially uncompensated endoplasmic reticulum stress response. Altogether we demonstrated that macroautophagy is a major actor in several types of antigen presenting cells.

Airway hyperresponsiveness (AHR) is one of the primary features of allergic airways disease. Despite continuous allergen exposure atopic asthmatics do not develop progressively worsening AHR. The mechanism(s) that limit AHR are unknown. Two valid candidates are regulatory T cells (Treg) and antigen presenting cells (APC). Dendritic cells (DC) are the main APC within the airways. Presentation of allergens to T cells can result in the differentiation and expansion of different subsets of T cells including effector Treg cells. The precise role of Treg and DC in the attenuation of allergen-induced AHR remains unknown. The general aim of this thesis is to investigate mechanisms to limit AHR in a murine model of atopic asthma. Specific aims are to: 1. develop a murine model of allergen-induced attenuation of AHR, 2. determine the potential role of regulatory T cells (Treg) in allergen-induced AHR attenuation, and 3. determine the potential role of airway dendritic cells (DC) in allergen-induced AHR attenuation. Balb/c mice were sensitised with intraperitoneal Ovalbumin (OVA) in aluminium hydroxide and challenged with a single, 3-weeks or 6-weeks of OVA aerosols. Aerosols were 1% OVA in sterile saline delivered for 30 minutes for three days per week. Animals were sacrificed 24 hours after the final aerosol for measurements of lung function and Methacholine (MCh) responsiveness (low-frequency forced oscillation technique), collection of bronchoalveolar lavage fluid (BALF) and serum. '...' In contrast, 6-weeks of OVA challenges decreased Treg numbers back to control levels. Adoptive transfer of 1x106 Treg taken from DLN of 3-week challenged mice attenuated AHR in single-OVA recipients (p<0.05). Furthermore, in vivo depletion of Treg in 3-week OVA challenged mice restored AHR (p<0.05 compared with control). Similar proportions of CD4+ T cells became activated following both aerosol regimes, however total numbers of airway CD4+ T cells were decreased (p<0.05), and OVA-specific CD4+ T cell proliferation in DLN was reduced (p<0.05) after 3-weeks versus one OVA aerosol. Analysis of antigen handling by airway APC populations showed antigen uptake (OVA-647) and processing (DQ-OVA) by macrophages and airway DC subsets to be down-regulated (p<0.05) after 3-weeks of OVA aerosols. In addition, adoptive transfer of Treg into single-OVA recipients did not affect antigen handling by airway APC populations. These data suggest that Treg are responsible for allergen-induced attenuation of AHR in vivo in established airways disease. AHR attenuation was associated with an altered function of airway DC, resulting in reduced antigen capture and processing, leading to limited clonal expansion of antigen-specific CD4+ T cells with limited production of Th2 cytokines. Furthermore, Treg were not directly responsible for the down-regulation of allergen capture in the airways. In conclusion, knowledge of the role of Treg and DC in attenuation of AHR could potentially result in improved and more directed therapies for the attenuation of AHR in atopic asthmatics.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 12, 2007) Vita. Includes bibliographical references.

Graduation date: 1994

Diss. Stockholm : Karolinska institutet, 2007.