Les traitements antirétroviraux combinés limitent la morbidité et la mortalité liées au SIDA après l'infection par le VIH, mais une hyperactivation du système immunitaire persiste, notamment au niveau des cellules myéloïdes, en corrélation avec une morbidité et une mortalité métabolique et cardio-vasculaire plus précoces que celles de la population générale. Il existe deux types de VIH ; l'infection par le VIH-2, prévalente en Afrique de l'Ouest et dans des communautés émigrées de cette région, conduit moins rapidement et moins fréquemment au SIDA que l'infection par le VIH-1, pour des raisons de relation hôte-virus encore à élucider. Lors de l'infection aigüe par le VIH-1, un pic sérique d'interféron (IFN) de type I et d'autres cytokines est observé, puis régulé négativement. Il est accompagné d'une forte réponse des gènes stimulés par l'IFN, qui persiste lors du passage à l'infection chronique, avec hyperactivation du système immunitaire. Les IFN de type I sont produits par toutes les cellules, mais en particulier par les cellules dendritiques plasmacytoïdes (pDC). Les IFN de type III (lambda) sont liés à la résolution de l'infection par le VHC, mais leur production en réponse au VIH reste à explorer. Durant mon doctorat, j'ai montré pour la première fois qu'in vitro, le VIH-1 et le VIH-2 stimulent la production d'IFN-lambda par les PBMC de donneurs sains de façon comparable. Chez des donneurs sains, les pDC produisent ces IFN de façon intrinsèque, mais pas les autres DC myéloïdes. Les pDC ont également un rôle dans la réponse immunitaire adaptative contre le VIH. L’équipe a en effet montré qu’elles peuvent effectuer la présentation croisée d’antigènes de cellules mortes infectées par le VIH, comme le font les autres DC. Elles peuvent ainsi activer des réponses T cytotoxiques spécifiques qui vont éliminer les cellules infectées. J’ai étudié ce mécanisme et montré que l’activation spécifique des lymphocytes T par les pDC est potentialisée par une pré-activation non-spécifique. En effet, les pDC s’activent en présence du virus et sécrètent des cytokines qui pré-activent une production intracellulaire d'IFN-gamma par les lymphocytes T CD8. Ces lymphocytes ne relarguent l'IFN-gamma qu'après reconnaissance spécifique par leur récepteur T d'un complexe peptide-CMH. Les résultats de cette thèse pourraient donner une place aux IFN-lambda ou à leur inhibition dans l'arsenal thérapeutique contre le VIH, ainsi qu'à l'activation des pDC pour conduire à une meilleure détection et élimination des réservoirs viraux par présentation croisée. / Combined antiretroviral treatments limit AIDS-related morbidity and mortality after HIV infection. But hyperactivation of the immune system persists, notably within the myeloid cell compartment, in correlation with metabolic and cardiovascular morbidity, which occurs earlier than in the general population. Two types of HIV have been described: HIV-2 infection, prevalent in West Africa and in emigrated communities originating from this area, leads less frequently and less rapidly to AIDS compared to HIV-1 infection, because of host-virus, which still need to be characterized. During acute HIV-1 infection, in the plasma, peak levels of type I Interferon (IFN) and other cytokines are observed, then they are down-modulated. A strong IFN stimulated gene (ISG) response is also observed. During chronic infection, the ISG response persists, with hyperactivation of the immune system. Type I IFN are produced by all cell types, but more specifically by plasmacytoid Dendritic Cells (pDC). Certain type III IFN (lambda) gene variants correlate with clearance of HCV infection, but IFN-lambda production in response to HIV remained to be studied. During my PhD, I showed for the first time that, in vitro, HIV-1 and HIV-2 induce IFN-lambda production by healthy donors PBMC at comparable levels. Plasmacytoid DC from healthy donors produce these IFN intrinsically, but not conventional DC. Plasmacytoid DC also have a role in the induction of adaptive immune responses against HIV. Our team demonstrated that they can crosspresent antigens from HIV infected apoptotic cells, like other DC. They can therefore activate specific cytotoxic T cell responses which eliminate infected cells. I studied this mechanism and showed that the activation of specific T cell by pDC is potentiated by non-specific pre-activation. Indeed, pDC become activated in the presence of virus and secrete cytokines which pre-activate intracellular IFN-gamma production by CD8 T cells. IFN-gamma is then secreted only after cognate MHC-peptide-T cell receptor interaction. The results of this thesis potentially give a role to IFN-lambda or their blockade in HIV treatment, and to the activation of pDC to induce better detection and elimination of HIV reservoirs through crosspresentation.

Células híbridas dendríticas-tumorais vem sendo usadas em vacinas terapêuticas contra o câncer e tem mostrado resultados promissores, porém seu comportamento e funções são insuficientemente conhecidos. O presente trabalho pretende estudar a interação dessas células híbridas com diferentes subpopulações de linfócitos T em um ambiente 3D com colágeno tipo III. Nossos resultados indicaram que as células híbridas do grupo Fusão Tumoral interagiram tanto com linfócitos CD4 quanto com CD8. Linfócitos CD4 interagiram 2 vezes mais no grupo Fusão Linhagem do que nos outros grupos e sua interação foi de apenas com um único linfócito. Linfócitos CD8 do grupo Fusão Tumoral interagiram mais tempo quando comparado aos outros grupos. Não foi observada IL-2 na culturas onde as células híbridas estavam presentes e a proliferação de linfócitos no grupo Fusão Tumoral também não foi observada. Assim, a cocultura 3D foi capaz de demonstrar a importância de um protocolo personalizado para cada tipo de tumor, levando em consideração as características únicas de cada tumor. / Dendritic cell-tumor cell hybrids have been used in therapeutic vaccines against cancer and has shown promising results, but their behavior and functions are insufficiently known. In this context, this study aims the interaction of these hybrid cells with different subpopulations of T cells in a 3D environment with collagen type III. Our results indicated that cells in Tumor Fusion group interacted with both CD4 and CD8 T lymphocytes. CD4 lymphocytes interacted 2 times more in the SKBR-3 Fusion group than another groups and their interaction was only with a single lymphocyte. CD8 lymphocytes interaction with the Tumor Fusion group resulted in longer interactions when in comparison with the another groups. No IL-2 was observed in cultures where the hybrid cells were present and the proliferation of lymphocytes in Tumor Fusion group was not observed. Thus, the 3D co-culture was able to demonstrate the importance of a custom protocol for each type of tumor, taking into consideration the unique characteristics of each one.

Endogenous retroviruses (of the HERV-W family) represent about 8% (1%) of our genome. Their endogenous and exogenous forms (MSRV, Multiple Sclerosis-associated RetroVirus) can alter the regulation of the immune system and be involved in inam- matory and autoimmune pathologies (Multiple Sclerosis). The MSRV envelope protein (ENV) stimulates T lymphocytes by acting as a superantigen. It also interacts with mono- cytes and dendritic cells via membrane receptors, thereby provoking inammatory cytokine production. Our studies are based on the characterisation of the immunological cascade leading from the interaction of the viral envelope with its receptor to the pathological inammatory reaction. The work presented in this thesis combines an in vitro cellular and molecular approach with an in vivo validation using an animal model (mouse). The chosen animal model is Experimental Autoimmune Encephalomyelitis (EAE) in which the complete Freund's adjuvant can be replaced by ENV. Its effects on the murine organism are evaluated on several levels: analysis of behaviour (clinical score) and brain (IRM), cellular and molecular analysis of the immune system. Furthermore, we want to generate a transgenic mouse model expressing different ENVs (MSRV/HERV) under the control of different promoters since MSRV/HERV are found only in great apes. This model, of which the rst steps of elaboration are part of this work, will allow us to study the behaviour of the ENV over-expressing animals as well as their brain and the effects on the immune system.

<p>The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (T_H) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. </p><p>To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65_495-503 peptide, a recombinant adenovirus coding for pp65, <i>in vitro</i> transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8⁺ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4⁺ T_H cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and T_H cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells <i>ex vivo</i> from virtually all stem cell donors for adoptive T cell transfer. </p><p>I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8⁺ T cells even when starting from undetectable frequencies (<0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8⁺ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer. </p>

<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>

The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (TH) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65495-503 peptide, a recombinant adenovirus coding for pp65, in vitro transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8+ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4+ TH cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and TH cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells ex vivo from virtually all stem cell donors for adoptive T cell transfer. I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8+ T cells even when starting from undetectable frequencies (&lt;0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8+ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer.

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.

Crohn's disease, a chronic inflammation of the bowel, is a multi-factorial condition where uncontrolled immune responses to luminal bacteria occur in genetically predisposed individuals. The first observable clinical signs are small ulcers that form at a specialised form of epithelium, follicle-associated epithelium (FAB). The FAB covers immune inductive sites, Peyer's patches, which function primarily as sensory areas that sample the externaI gut environment. Dendritic cells are one of the key cells that are involved in sensing luminal contents and orchestrating the gut immune system. The main aim of this thesis was to determine whether the barrier of the FAB is breached in Crohn's disease and if dysfunctional immune regulators, namely dendritic cells, playaroIe in initiating and/or maintaining the chronic intestinal inflammation. Using biopsies and surgical specimens, we were able to show that in Crohn's disease, there was an increased transmucosaI transport of Escherichia coli compared to specimens from ulcerative colitis and non-inflammatory bowel disease (IBD) controIs. Dendritic cells internalised a higher percentage of bacteria that had translocated across the FAB in the Crohn's samples. Furthermore, significantly higher concentrations of TNF-u was released upon bacterial stimulation by tissues from patients with Crohn's disease than in controIs. We went on to characterise the dendritic cells present in the Peyer's patches of patients with Crohn's disease. We found an accumulation of both immature and mature dendritic cells beneath the FAB, in the sub-epithelial dome (SED). Normally, mature dendritic cells migrate towards T cell-rich areas. However, we observed mature dendritic cells accumulating in the SED because they lacked the CCR7 migratory receptor. Furthermore, they were more prone to take-up bacteria, and produced TNF-α. To study the function of mucosal dendritic cells, we performed isolation experiments and mixed Iymphocyte reactions. Dendritic cells from both the ileum and blood of patients with active Crohn's had reduced capacity for inducing T cell proliferation than non-IBD controIs. Blood dendritic cells of patients in remission had normalised function that was similar to dendritic cells from healthy controls. The SAMPl/YitFc mice, considered an appropriate murine model for Crohn's disease, had an inherent permeability defect that increased with the chronicity of intestinaI inflammation. However unlike in human Crohn's disease, dendritic cells did not seem to playaroIe in murine ileitis. This thesis highlights the accumulation of the actively surveying dendritic cells that are prone to bacterial internalisation, and points to their possible different functional roles in active versus in-active disease; thereby confirming dendritic cells as one ofthe key components in the pathogenesis ofCrohn's disease.

Förfrågan efter nya och mer avancerade applikationer är en pågående process vilket leder till en konstant utveckling av nya material. För att förstå relationen mellan en applikations egenskaper och dess sammansättning krävs full förståelse och kontroll över materialets uppbyggnad. En sådan kontroll över uppbyggnaden hos material hittas i en undergrupp till dendritiska polymerer som kallas dendrimerer. I den här doktorsavhandlingen belyses nya metoder för att framställa dendrimer med hjälp av selektiva kemiska reaktioner. Sådana selektiva reaktioner kan hittas inom konceptet klickkemi och har i detta arbete kombinerats med traditionell anhydrid- och karbodiimidmedierad kemi. Denna avhandling diskuterar en accelererad tillväxtmetod, dendrimerer med inre och yttre reaktiva grupper, simultana reaktioner och applikationer baserade på dessa dendritiska material. En accelererad tillväxtmetod har utvecklats baserad på AB2- och CD2-monomerer. Dessa monomerer tillåter tillväxt av dendrimerer utan att använda sig av skyddsgruppkemi eller aktivering av ändgrupper. Detta gjordes genom att kombinera kemoselektiviteten hos klickkemi tillsammans med traditionell syraklorid kopplingar. Dendrimerer med inre alkyn- eller azidfunktionalitet syntetiserades genom att använda AB2C-monomerer. Den dendritiska tillväxten skedde med hjälp av karbodiimidmedierad kemi. Monomererna som användes bär på en C-funktionalitet, alkyn eller azid, och på så sätt byggs får interiören i de syntetiserade dendrimeren en inneburen aktiv funktionell grupp. Ortogonaliteten hos klickkemi användes för att sammanfoga monomerer till en dendritisk struktur. Traditionell anhydridkemi- och klickemireaktioner utfördes samtidigt och på så sätt kunde dendritiska strukturer erhållas med färre antal uppreningssteg. En ljusemitterande dendrimer syntetiserades genom att koppla azidfunktionella dendroner till en alkynfunktionell cyclenkärna. Europiumjoner inkorporerades i kärnan varpå dendrimerens fotofysiska egenskaper analyserades. Mätningarna visade att den bildade triazolen hade en sensibiliserande effekt på europiumjonen. Termiska studier på några av de syntetiserade dendrimerer utfördes för att se om några av dem kunde fungera som templat vid framställning av isoporösa filmer. / The need for new improved materials in cutting edge applications is constantly inspiring researchers to developing novel advanced macromolecular structures. A research area within advanced and complex macromolecular structures is dendrimers and their synthesis. Dendrimers consist of highly dense and branched structures that have promising properties suitable for biomedical and electrical applications and as templating materials. Dendrimers provide full control over the structure and property relationship since they are synthesized with unprecedented control over each reaction step. In this doctoral thesis, new methodologies for dendrimer synthesis are based on the concept of click chemistry in combination with traditional chemical reactions for dendrimer synthesis. This thesis discusses an accelerated growth approach, dendrimers with internal functionality, concurrent reactions and their applications. An accelerated growth approach for dendrimers was developed based on AB2- and CD2-monomers. These allow dendritic growth without the use of activation or deprotection of the peripheral end-groups. This was achieved by combining the chemoselective nature of click chemistry and traditional acid chloride reactions. Dendrimers with internal azide/alkyne functionality were prepared by adding AB2C monomers to a multifunctional core. Dendritic growth was obtained by employing carbodiimide mediated chemistry. The monomers carry a pendant C-functionality (alkyne or azide) that remains available in the dendritic interior resulting in dendrimers with internal and peripheral functionalities. The orthogonal nature of click chemistry was utilized for the simultaneous assembly of monomers into dendritic structures. Traditional anhydride chemistry and click chemistry were carried out concurrently to obtain dendritic structures. This procedure allows synthesis of dendritic structures using fewer purification steps. Thermal analyses on selected dendrimers were carried out to verify their use as templates for the formation of honeycomb membranes. Additionally, a light emitting dendrimer was prepared by coupling of azide functional dendrons to an alkyne functional cyclen core. A Europium ion was incorporated into the dendrimer core, and photophysical measurements on the metal containing dendrimer revealed that the formed triazole linkage possesses a sensitizing effect. / QC 20100629

The focus of this work was to functionally characterize the CD300e receptor expressed in human monocytes and myeloid dendritic cells and investigate the implications that receptor engagement has on their biology. We provide evidence formally supporting that CD300e functions as an activating receptor capable of regulating the innate immune response by triggering various pro- inflammatory functions including intracellular calcium mobilization, superoxide anion production, pro-inflammatory cytokine release and up-regulation of co-stimulatory molecules in myeloid cells. We also report that ligation of CD300e on the surface of monocytes results in their differentiation to functional M&#934;2-like macrophages by an autocrine mechanism that involves M-CSF and its receptor (CD115). / L'objectiu d'aquest treball ha estat caracteritzar funcionalment el receptor CD300e expressat en monòcits i cèl·lules dendrítiques mieloides humanes, així com investigar les implicacions que l'activació d'aquest receptor pot tenir en la seva biologia. Demostrem formalment que el receptor CD300e funciona com un receptor activador capaç de regular la resposta immune innata activant diverses funcions proinflamatòries, incloent la mobilització de calci intracel·lular, la producció d'anió superòxid, la secreció de citocines proinflamatòries i la inducció de molècules coestimuladores en cèl·lules mieloides. També descrivim que l'activació del receptor CD300e a la superfície dels monòcits provoca la seva diferenciació cap a macròfags funcionals del tipus M&#934;2 gràcies a un mecanisme autocrí que funciona a través del M-CSF i el seu receptor (CD115).