Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The purpose of this study is to evaluate the amount of transcripts from the Major Histocompatibility Complex (MHC) class I classical (BoLA-N *) and non-classical (BoLA-NC*), as well as of TNF-α (Tumor Necrosis Factor alpha) and TGF-β1 (Transforming Growth Factor beta 1) cytokines in bovine non-pregnant uterus, caruncles, cotyledons and blastocysts throughout the pregnancy period, in order to understand the immune and physiological mechanisms that occur in pregnancy in cows. The samples were analyzed by qPCR, the qualification (presence or absence) by conventional RT-PCR and the immune fluorescence with sheep monoclonal antibody against MHC class I. As positive control we used a sample of tissue from the bovine lymph node and/or spleen in all experimental tests. The amount of transcript BoLA-N* and BoLA-NC* was small (fold change 0 to 3) in blastocysts and cotyledons. Cotyledons transcription BoLA-NC* is greater than the transcription of BoLA-N*. Blastocysts transcription of BoLA-N* was higher than BoLA-NC*. Blastocysts transcribed more BoLA-NC2 than in cotyledons. Blastocysts not transcribed BoLA-NC4. Cotyledons transcribed BoLA-NC4 more than BoLA-NC2. The caruncles showed large amounts of transcripts (fold change 5 to 400), which were higher for pregnancy period of \"8-9\" month to BoLA-N* and one month for BoLA-NC*. Caruncles BoLA-NC2 transcription was higher than the BoLA-NC4. The uterus non pregnant had fewer transcripts BoLA-N* and BoLA-NC* than in caruncles. The uterus non pregnant no have transcripts of BoLA-NC2, but they had a small amount of transcripts of BoLA-NC4. Caruncles had large amounts of transcripts (fold change 50 to 500) of BoLA-NC2 with pregnancy age greater than one month; those are seemed to protect the fetus from rejection. TNF-α appears in greater amount in late gestation (8 and 9 months), showing the beginning of an inflammatory reaction, necessary to the adequate removal of the fetus and the placenta during delivery. Since TGF-β1 has a peak of expression in the sixth and seventh months of gestation, just when the fetus has its greatest growth, demonstrating a probable involvement of this cytokine in this phenomenon. / Com o intuito de entender os mecanismos imunofisiológicos da gestação em vacas esta tese teve como objetivo avaliar a quantidade de transcritos do Complexo de Histocompatibilidade Principal (MHC) de classe I clássico (BoLA-N*) e não clássico (BoLA-NC*), bem como das citocinas TNF-α (Fator de Necrose Tumoral alfa) e TGF-β1 (Fator de Crescimento Transformador beta 1) em úteros não gestantes, carúnculas, blastocistos e cotilédones bovino durante todo o período gestacional. Utilizou-se a quantificação por qPCR, a qualificação (presença ou ausência) via RT-PCR convencional e a imunofluorescência com anticorpo primário monoclonal MHC de classe I ovino. Como controle positivo foi utilizado uma amostra de tecido de linfonodo e/ou baço bovino em todos os ensaios experimentais. A quantidade de transcrito de BoLA de classe I clássico e não clássico foi pequena (fold change variando de 0 a 3) nos blastocistos e cotilédones. Nos cotilédones a transcrição de BoLA-NC* foi maior do que a transcrição de BoLA-N*. Nos blastocistos houve maior transcrição de BoLA-N* do que de BoLA-NC*. BoLA-NC2 transcreveu mais nos blastocisto do que nos cotilédones. BoLA-NC4 não transcreveu nos blastocistos. Os cotilédones transcreveram mais BoLA-NC4 do que BoLA-NC2. As carúnculas apresentaram grandes quantidades de transcritos (fold change variando de 5 a 400), que foram maiores nos períodos gestacionais de 8 a 9 para BoLA_N* e até 1 mês para BoLA-NC*. A transcrição de BoLA-NC2 foi maior do que a de BoLA-NC4 nas carúncula. O útero não gestante teve menor quantidade de transcritos de BoLA-N* e BoLA-NC* do que nas carúnculas. Não há transcrição de BoLA-NC2 no útero não gestante, mas há uma pequena quantidade de transcritos de BoLA-NC4 neste tecido. A presença de grandes quantidades de transcritos (fold change variando de 50 a 500) de BoLA-NC2 nas carúnculas, com idade gestacional maior do que um mês, parece proteger o feto da rejeição. Nas carúnculas parece existir um pico de transcrição de TGF-β1 no sexto e sétimo meses de gestação, justamente quando o feto tem seu maior crescimento, sinalizando um provável envolvimento desta citocina neste fenômeno. O TNF-α surge em maior quantidade no final da gestação, tanto nas carúnculas quanto nos cotilédones, demonstrando o início de uma reação inflamatória, necessária para a adequada expulsão do concepto e da placenta durante o parto. / Doutor em Imunologia e Parasitologia Aplicadas

La présentation antigénique par le complexe majeur d’histocompatibilité (MHC) I est un processus ubiquitaire permettant la présentation de protéines endogènes qui reflètent l'état de la cellule à la surface cellulaire aux lymphocytes T CD8+ dans le contexte de la surveillance et la réponse immunitaires. Ainsi, l'expression des molécules du MHC I classiques est induite en réponse aux stimuli inflammatoires afin de favoriser la reconnaissance immunitaire et l'élimination des pathogènes. HFE est une molécule du MHC Ib non-classique qui sert de régulateur négatif de l'absorption du fer. HFE est associé au développement de l'hémochromatose héréditaire (HH), maladie associée au métabolisme du fer mais souvent accompagnée de défauts immunitaires. Ainsi, nous avons en premier lieu étudié l'impact de HFE sur la présentation antigénique par MHC I, afin d'expliquer en partie les défauts immunitaires liés à l'HH associée à HFEC282Y. Puis, compte tenu de l'impact de l'inflammation sur l'expression des molécules du MHC I classiques, nous avons étudié la régulation de l'expression de HFE en réponse aux stimuli inflammatoires induits par les cellules du sang périphérique mononucléées (PBMC). Nous avons mis au point un système d’expression antigénique dans lequel nous contrôlons l’expression de MHC I, de HFE et d’un antigène pour lequel nous avons généré des lymphocytes T CD8+ spécifiques. Nos résultats démontrent que la forme sauvage de HFE (HFEWT), contrairement à sa forme mutée (HFEC282Y), inhibe la reconnaissance de complexes MHC I/peptide (pMHC). Nous avons également démontré que l'inhibition de la reconnaissance est maintenue, indépendamment des niveaux d'expression de MHC I à la surface, d'une compétition pour la β2-microglobuline, de la capacité de HFE d'interagir avec le récepteur de la transferrine, de l'origine de l'antigène ou de l'affinité de celui-ci. Par ailleurs, nous avons identifié les domaines α1-2 de HFEWT comme étant responsables de l'inhibition de la reconnaissance antigénique. Par contre, la reconnaissance de peptides chargés de manière externe sur les molécules du MHC I présentes à la surface n'a démontré aucune inhibition en présence de HFEWT, suggérant que HFEWT pourrait affecter la reconnaissance en interférant avec le processus d'apprêtement antigénique intracellulaire. À l’inverse, nous avons souhaité déterminer si les lymphocytes T activés pouvaient influencer les niveaux d'expression de HFE. En termes de régulation de l'expression de HFE, nous avons établi que HFE est exprimé dans les tissus sains chez l'humain et induit chez les lignées de cancers du colon, du sein, du poumon, du rein et du mélanome. Par ailleurs, en co-cultivant des lymphocytes T activés avec ces lignées tumorales, nous avons démontré que l'expression de HFE est fortement inhibée dans toutes ces lignées tumorales lorsqu'exposées à des lymphocytes T activés. Finalement, la modulation de l'expression de HFE est indépendante du contact cellulaire et semble médiée en partie par le GM-CSF, l'IFN-γ et le TNF. En somme, ces résultats suggèrent que les lymphocytes T de l'hôte modulent l'expression de HFE dans le microenvironnement inflammatoire, ce qui pourrait promouvoir la reconnaissance des antigènes présentés sur les molécules du MHC I présentées aux lymphocytes T CD8+ antigène-spécifiques. De plus, ces études soulèvent la possibilité d'un nouveau rôle physiologique de HFEWT dans la voie de présentation antigénique par MHC I, qui pourrait moduler l'immunogénicité des antigènes et la réponse immunitaire cellulaire chez l'hôte. / MHC class I antigen presentation is an ubiquitous process by which cells present endogenous proteins to CD8+ T lymphocytes during immune surveillance and response. Accordingly, classical MHC I molecules are up-regulated in response to inflammatory stimuli to favor immune recognition and pathogen clearance. HFE is a non-classical, MHC Ib molecule which acts as a negative regulator of iron absorption. HFE has been linked to the development of hereditary hemochromatosis (HH), an iron overload disease often associated to immune defects. Firstly, we studied the impact of HFE expression on MHC I antigen presentation, as a hypothesis for HH-associated immunological defects observed in HFEC282Y-mutated HH patients. Secondly, we evaluated whether, like its classical MHC I counterparts, HFE expression could be modulated in response to peripheral blood mononuclear cell (PBMC) inflammation. We developed an antigen presentation system in which we control MHC I expression, HFE expression, and expression of a model antigen for which we have generated antigen-specific CD8+ T lymphocytes. Our results demonstrate that wild-type HFE (HFEWT), but not C282Y-mutated HFE (HFEC282Y), inhibits recognition of MHC I antigens. We further demonstrate that inhibition of antigen recognition is maintained regardless of MHC I surface levels, β2-microglobulin competition, HFE ability to interact with transferrin receptor, antigen origin, or epitope affinity. We identified the α1-2 domains of HFEWT as being responsible for inhibiting antigen recognition. However, recognition of externally peptide-pulsed 293-A2 remained uninhibited in presence of HFEWT, indicating that HFE may affect T cell recognition by interfering with intracellular antigen processing. We also questioned whether activated T lymphocytes may influence HFE expression. We established that HFE is widely expressed in healthy human tissues and induced in colon cancer, breast cancer, lung cancer, kidney cancer and melanoma cell lines. Furthermore, HFE mRNA expression was drastically inhibited in all tumor cell lines when exposed to activated T lymphocytes. Down-regulation of HFE mRNA expression was independent of cell contact and appears to be partially mediated by GM-CSF, IFN-γ, and TNF. Overall, these data suggest that host T lymphocytes may alter HFE expression levels in the inflammatory microenvironment, which could, in turn, promote recognition of MHC I antigens presented to antigen-specific CD8+ T lymphocytes. Accordingly, this could suggest a new physiological role for HFEWT in the MHC I antigen presentation pathway, which could modulate antigen immunogenicity and the cellular immune response.

B virus (Macacine herpesvirus 1, Cercopithecine herpesvirus 1, herpes B virus) is an Old World monkey simplex virus endemic in macaques. B virus infection in its natural host, macaque, is very similar to HSV-­‐1 infection in humans causing mild or asymptomatic infection. On the other hand, zoonotic infection in humans results in death in the absence of early initiation of antiviral drugs. Viruses evade host immune responses in order to survive and propagate. Most herpes viruses including HSV-­‐1 down-­‐regulate major histocompatibility complex class I (MHC class I) surface expression on infected cells in order to prevent CD8+ T-­‐cell recognition and subsequent cell lysis. MHC class I molecules bind to the inhibitory receptors of NK cells and prevent NK cell activity. Thus, this mechanism protects HSV-­‐1 infected cells from CD8+ T-­‐cell lysis, making them sensitive to natural killer (NK) cell cytotoxicity. To investigate if B virus pathogenicity is a result of novel immune evasion mechanisms employed by B virus, we determined NK cell regulation during B virus infection. To this end, our experiments demonstrate that B virus does not down-­‐ regulate MHC I expression as effectively as HSV-­‐1, leading us to hypothesize that B virus in-­‐ fected cells are resistant to NK cell activity. We examined the expression of MHC I chain related genes (MICA/ MICB), which are activation ligands to NKG2D receptors on NK cells. Our results show that there is no significant difference in MICA and MICB expression between HSV-­‐1 and B virus infected cells. Furthermore, we tested for the up-­‐regulation of cytokines and chemokines responsible for NK cell activation and migration. Our results indicate a significant up-­‐regulation of IFN-­‐α from PBMCs co-­‐cultured with HSV-­‐1 infected cells, which plays an important role in activating NK cells. NK cells within these PBMCs up-­‐regulate perforin release indicative of NK cell activity. PBMCs co-­‐cultured with B virus infected cells do not up-­‐regulate any cytokines or chemokines responsible for NK cell activity. As a result the NK cells within these PBMCs do not significantly up-­‐regulate perforin release. These results demonstrate that B virus employs a novel immune evasion mechanism to subvert NK cell activity.

No description available.

Les éléments transposables (TE) sont des séquences répétitives représentant environ 45% des génomes humain et murin. Il est généralement assumé que leur expression est réprimée dans les cellules somatiques pour protéger l’intégrité du génome, et cette régulation épigénétique est fréquemment perdue dans les cancers, menant à la surexpression des TEs dans les tumeurs. Puisque l’expression aberrante des TEs est associée à l’infiltration de la tumeur par les cellules immunitaires, les TEs sont considérés comme des cibles prometteuses d’immunothérapies du cancer. Une meilleure description de l’expression des TEs dans les tissus somatiques ainsi que dans le thymus, l’organe responsable du développement de la tolérance au soi des lymphocytes T, est toutefois nécessaire pour évaluer la capacité des TEs d’induire des réponses immunitaires et déterminer si l’expression des TEs est belle et bien spécifique aux tumeurs. L’objectif de cette thèse est donc de brosser un portrait exhaustif de l’expression des TEs dans les tissus somatiques humains ainsi que dans le thymus. Pour ce faire, des données transcriptomiques et immunopeptidomiques ont été analysées pour mieux comprendre les interactions entre les TEs et les lymphocytes T à l’état basal. Nos résultats ont montré que l’expression des TEs est répandue dans les tissus somatiques humains, bien que leur niveau d’expression varie d’un tissu à l’autre et que plusieurs TEs sont exprimés de façon tissu-spécifique. De plus, les TEs peuvent être traduits et présentés par le CMH-I à la surface de cellules non-cancéreuses. Nous avons aussi déterminé que les TEs ont trois fonctions potentielles dans le thymus : ils pourraient fournir des sites de liaison à un grand nombre de facteurs de transcription dans toutes les populations cellulaires du thymus, ils stimuleraient la sécrétion d’IFN ɑ/β par les pDCs thymiques, et ils contribuent aux sélections positive et négative des thymocytes. Nos travaux illustrent la complexité des interactions entre les TEs et le système immunitaire adaptatif. Finalement, étant donnée l’expression répandue des TEs dans les tissus somatiques, nos travaux soulignent l’importance d’établir la tolérance des lymphocytes T à l’égard des TEs pour éviter des réactions auto-immunes. / Transposable elements are repetitive sequences representing around 45% of the human and murine genomes. It is generally assumed that their expression is repressed in somatic cells to preserve genomic integrity, but this epigenetic regulation is frequently lost in cancer cells, leading to the aberrant expression of TEs in tumors. As aberrant TE expression is associated with tumor infiltration by immune cells, TEs are considered as promising cancer immunotherapy targets. However, a better description of TE expression in somatic tissues and in the thymus, the organ responsible of T cell self-tolerance induction, is required to evaluate the potential of TEs to induce immune responses as well as the tumor specificity of TE expression. Thus, this thesis’ objective is to draw an exhaustive profile of TE expression in human somatic tissues and in the thymus. To do so, we analyzed transcriptomic and immunopeptidomic data to better understand interactions between TEs and T cells at steady state. Our work shows that TE expression is widespread in human somatic tissues, even though their expression level varies between tissues and many TEs are expressed in a tissue-specific manner. Additionally, TEs are translated and presented by the MHC-I on the surface of non-malignant cells. We also determined that TEs have three potential functions in the thymus: they could provide transcription factor binding sites in all cell populations of the thymus, they might induce the constitutive IFN ɑ/β secretion of thymic pDCs, and they contribute to both positive and negative selections of thymocytes. Altogether, our work illustrates the complexity of the interactions between TEs and the vertebrate adaptive immune system. Given the widespread expression of TEs in somatic tissues, this thesis highlights the importance of establishing T cell tolerance towards TE sequences to avoid autoimmune reactions in peripheral tissues.

Le complexe majeur d’histocompatibilité de type I (CMH I) est une glycoprotéine dont le rôle est de présenter des peptides endogènes au récepteur de cellules T (TCR) des cellules T CD8. La régulation de l’expression du CMH I a été exclusivement étudiée chez les cellules hématolymphoïdes. Cependant, ce processus reste peu élucidé chez les cellules épithéliales (ECs) malgré leur rôle important dans la défense de l’hôte contre divers pathogènes. Dans ce présent travail, nous avons effectué une analyse approfondie de l’expression du CMH I dans les ECs primaires fraîchement prélevées du thymus, de la peau, du colon et des poumons. Nos analyses de cytométrie en flux révèlent une grande variabilité de l’expression du CMH I à la surface des ECs primaires. Nous avons démontré que l’expression du CMH I est 10 à 100 fois plus élevée à la surface des ECs thymiques (TECs) que les ECs extrathymiques. Nous avons également observé aussi que l’expression élevée du CMH I à la surface des TECs est principalement due à l’interféron lambda (IFN-λ), produit en conditions physiologiques dans le thymus. Nous avons révélé aussi que l’absence de la voie d’IFN-λ induit de l’auto-immunité chez la souris. En effet notre étude ouvre la voie vers une exploration plus approfondie de l’impact de l’IFN-λ dans les fonctions thymiques. D’autre part, les ECs subissent continuellement de l’apoptose afin d’éliminer les cellules endommagées et restaurer l’intégrité de l’épithélium. Cependant, les ECs apoptotiques représentent une source majeure d’auto-antigènes (AAg) ce qui en fait une cible indéniable des maladies auto-immunes. Dans ce présent travail, nous avons évalué dans quelle mesure les cellules présentatrices d’antigènes (APCs) thymiques (TEC médullaires (mTECs) et les cellules dendritiques thymiques (tDCs)) contribuent dans la tolérance centrale à l’égard des antigènes exprimés par les ECs extrathymiques. Nous avons trouvé que les APCs thymiques expriment environ 93% des gènes exprimés dans les ECs extrathymiques. Cependant, nous avons révélé une fraction des gènes (environ 7%) dans le transcriptome des ECs extrathymiques qui n’est pas exprimé par les APCs thymiques. Ces gènes sont capables de générer des peptides associés au CMH I (MAPs) quoique dans une moindre mesure que les gènes partagés avec les APCs thymiques. Dans l’ensemble, cette étude fournit la première tentative de caractérisation du transcriptome des ECs extrathymiques et les APCs thymiques, capables de générer des MAPs. / The major histocompatibility complex class I (MHC I) is a cell surface glycoprotein involved in the presentation of endogenously derived peptides, to the T-cell receptor TCR of CD8 T cells. However, the regulation of the MHC I expression has been studied almost exclusively in hematolymphoid cells and very little is known about this process in epithelial cells (ECs). In the present work, we performed a deep analysis of MHC I expression in primary ECs freshly harvested from the thymus, skin, gut, and lung. In fact, we found that the superior MHC I expression in TECs is driven mainly by interferon lambda (IFN-λ) produced under steady-state conditions in the thymus. Moreover, Ifnlr1−/− mice present autoimmune manifestations. Our study paves the way for a more detailed exploration of the impact of IFN-λ signaling in thymic functions. On the other hand, ECs continually undergo apoptosis to delete damaged cells while restoring the epithelium integrity. However, apoptotic ECs are a major source of autoantigens (AAgs) which makes them an undeniable target for autoimmune attacks. In the present study, we evaluated the extent to which thymic antigen presenting cells APCs (medullary TECs (mTEC) and thymic dendritic cells (tDCs)) contribute to central tolerance against extrathymic ECs-antigens. We report that thymic APCs express about 93.5% of genes expressed in extrathymic ECs whereas 7 % of genes expressed in extrathymic ECs are silent in thymic APCs. We found that these extrathymic ECs- unique genes are efficiently able to produce MHC I-associated peptides (MAPs), albeit to a lower extent than the genes expressed by thymic APCs. In summary, this study provides the first characterization of the self-transcriptome expressed by extrathymic ECs and thymic APCs (mTECs and tDCs), and able to generate MAPs.

Il existe plusieurs défis au développement d’une thérapie visant à stimuler l’immunité cellulaire. Dans la prévention contre certains virus et en immunothérapie du cancer, l’induction de lymphocytes T spécifiques est cependant primordiale. Dans la première partie de l’étude, nous avons porté notre attention sur la compréhension de la présentation croisée par le complexe majeur d’histocompatibilité de classe I (CMH I) médiée par des particules pseudo-virales (VLP) composées de la protéine de surface de potexvirus à laquelle nous avons ajouté un épitope de la protéine M1 du virus de l’influenza ou un épitope de la protéine gp100 du mélanome. Cette VLP se caractérise par sa capacité à stimuler, sans l’aide d’adjuvant, le système immunitaire et de présenter de façon croisée l’épitope inséré dans sa protéine de surface et ce, indépendamment de l’activité du protéasome. Nous avons, tout d’abord, comparé les propriétés de présentation antigénique croisée des VLP formées du virus de la mosaïque de la malva (MaMV) à celles des VLP du virus de la mosaïque de la papaye (PapMV). Les résultats confirment que ces propriétés sont partagées par plusieurs membres de la famille des potexvirus malgré des divergences de séquences (Hanafi et al. Vaccine 2010). De plus, nous avons procédé à des expériences pour préciser le mécanisme menant à la présentation de l’épitope inséré dans les VLP de PapMV. Les résultats nous confirment une voie vacuolaire dépendante de l’activité de la cathepsine S et de l’acidification des lysosomes pour l’apprêtement antigénique. L’induction de l’autophagie par les VLP semble également nécessaire à la présentation croisée par les VLP de PapMV. Nous avons donc établi un nouveau mécanisme de présentation croisée vacuolaire dépendant de l’autophagie (Hanafi et al. soumis Autophagy). En second lieu, en immunothérapie du cancer, il est aussi important de contrôler les mécanismes d’évasion immunitaire mis en branle par la tumeur. Nous avons spécifiquement étudié l’enzyme immunosuppressive indoleamine 2,3-dioxygénase (IDO) (revue de la littérature dans les tumeurs humaines; Hanafi et al. Clin. Can. Res 2011) et son inhibition dans les cellules tumorales. Pour ce faire, nous avons tenté d’inhiber son expression par la fludarabine, agent chimiothérapeutique précédemment étudié pour son activité inhibitrice de l’activation de STAT1 (signal transducers and activators of transcription 1). Étonnamment, nos résultats ont montré l’inhibition d’IDO dans les cellules tumorales par la fludarabine, indépendamment de l’inhibition de la phosphorylation de STAT1. Nous avons démontré que le mécanisme d’action dépendait plutôt de l’induction de la dégradation d’IDO par le protéasome (Hanafi et al. PlosOne 2014). Les travaux présentés dans cette thèse ont donc portés autant sur la compréhension d’une nouvelle plateforme de vaccination pouvant médier l’activation de lymphocytes T CD8+ cytotoxiques et sur le contrôle d’une immunosuppression établie par les cellules tumorales pour évader au système immunitaire. Ces deux grandes stratégies sont à considérer en immunothérapie du cancer et la combinaison avec d’autres thérapies déjà existantes pourra permettre une meilleure réponse clinique. / There are several challenges to the development of therapies aimed at stimulating cellular immunity. In viral infection prevention and in cancer immunotherapy, the induction of specific T lymphocytes is, however, of paramount importance. In the first part of this study, we focused our attention on understanding the major histocompatibility complex class I (MHC I) cross-presentation mediated by virus-like particles (VLP) composed of potexvirus coat protein, in which we had inserted an epitope from the M1 protein of the Influenza virus or an epitope from gp100, a tumour antigen of melanoma. This particular VLP is characterized by its ability to stimulate the immune system with no adjuvant and its cross-presentation of the inserted epitope independently of proteasome activity. First, we compared the antigenic cross-presentation properties of Malva mosaic virus (MaMV) VLPs to that of Papaya mosaic virus (PapMV) VLPs. The results confirm that cross-presentation mechanisms are shared among different members of the potexvirus family despite marked differences in their sequences (Hanafi et al. Vaccine 2010). Furthermore, we have conducted experiments to clarify the mechanism leading to the cross-presentation of the inserted epitope in PapMV VLPs. The results confirm a vacuolar pathway dependent on cathepsin S activity and on lysosomal acidification for antigen presentation. Autophagy induction by VLPs is also important to PapMV VLP antigen cross-presentation. We have herein described a new vacuolar MHC I cross-presentation pathway dependent on autophagy (Hanafi et al. in preparation). Secondly, in cancer immunotherapy, it is crucial to control immune evasion mechanisms that are initiated by the tumour. We have specifically studied the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) (human cancer literature review in Hanafi et al. Clin. Can. Res. 2011), and its inhibition in tumour cells. To this end, we inhibited its expression using fludarabine, a chemotherapeutic agent previously studied for its inhibitory effect on STAT1 (signal transducers and activators of transcription 1) phosphorylation. Surprisingly, our results demonstrate that IDO inhibition in cancer cells by fludarabine was independent of STAT1 phosphorylation. We showed that the mechanism of action was rather dependent on the induction of IDO degradation by the proteasome (Hanafi et al. PlosOne 2014). The work presented in this thesis provides a better understanding of how a new vaccine platform can mediate cytotoxic CD8+ T lymphocytes activation and the control of the problem of immunosuppression by tumour cells for the evading of the immune system. These two main strategies are to key for the consideration of cancer immunotherapy in combination with other existing therapies, as these should allow a better clinical response to cancer treatment.

Japanese encephalitis virus (JEV) is one of the major causes of encephalitis in Asia. JEV causes serious inflammation of the brain, which may lead to permanent brain damage and has a high mortality rate. Almost 3 billion people live in JE endemic areas and JEV causes an estimated 20,000 cases of disease and 6000 deaths per year. JEV is a positive stranded RNA virus belonging to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. JEV transmission occurs through a zoonotic cycle involving mosquitoes and vertebrate amplifying hosts, chiefly pigs and ardeid birds. Humans are infected when bitten by an infected mosquito and are dead end hosts. The role of humoral and cell mediated immune responses during JEV infection have been studied by several groups. While the humoral responses play a central role in protection against JEV, the cell mediated immune responses contributing to this end are not fully understood. The MHC molecules have been known to play predominant roles in host responses to viral infections and the consequences of virus infection on the expression of MHC molecules are varied. The expression of MHC-I molecules is known to decrease upon infection with many viruses such as HIV, MCMV, HCMV, Adv, and EBV. In contrast, infection with flavivirus such as West Nile Virus (WNV) has been shown to increase the cell surface expression of both MHC-I and MHC-II molecules. It has been reported previously that WNV infection increases the cell surface expression of adhesion molecules such as ICAM-1, VCAM-1 as well as E-Selectin and these changes were mediated directly by WNV and not by soluble cytokines. In contrast to classical MHC-I molecules, the nonclassical MHC-I molecules do not belong to a single group of structurally and functionally homologous proteins and normally have lower cell surface expression. Earlier studies have shown that the expression of nonclassical MHC-I molecules were induced during infection with JHM strain of mouse hepatitis virus (MHV). However, the functional significance of this induction is unclear. Expression of nonclassical MHC-I molecules upon flaviviral infection is not very well understood. In this thesis, evidence is presented that JEV infection induces the expression of both classical and nonclassical MHC-I molecules on primary mouse brain astrocytes, mouse embryonic fibroblasts (MEFs) and H6 (hepatoma cell). The levels of adhesion molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results clearly demonstrate that JEV infection induces their expression on astrocytes, MEFs and H6. The role of NF-κB and type-I IFNs in the induction of classical and nonclassical MHC-I molecules as well as molecules involved in antigen processing and presentation were also analyzed and our results demonstrated that type-I IFN mediated signaling is responsible for the induction of these molecules during JEV infection. Chapter 1 discusses the innate and adaptive immune system, the role of classical and nonclassical MHC molecules in the initiation of immune response and diverse strategies adapted by different viruses to evade the immune response. It also includes a detailed discussion about the IFN and NF-κB signaling pathways and their modulation by viral infection. Finally, the genome organization, epidemiology, transmission cycle, pathogenesis and pathology, clinical features, humoral as well as cell mediated immune response to JEV infection and the current vaccine status to JEV infection are briefly discussed. Chapter 2 describes the general materials and methods used in this study. It includes the details of the reagents and cell lines used in the experiments. It also discusses the various techniques such as RT-PCR, FACS analysis, EMSA and ELISA. Chapter 3 focusses on the validation of different knockout MEFs used in the study as well as confirming the purity of primary astrocyte cultures established from pub brains. The susceptibility of various cells to JEV infection has also been investigated. Our results confirmed the authenticity of all the cells and the purity of primary astrocyte cultures used in the study. Our results also indicated that all the cells used in the study are susceptible to JEV infection. Chapter 4 discusses the expression of MHC and related genes involved in immune response upon JEV infection of primary mouse brain astrocytes, MEFs and H6. Chapter 4 demonstrates for the first time that JEV infection induces the expression of nonclassical MHC-I or class Ib molecules namely Qa-1, Qb1 and T10 in addition to the induction of classical MHC-I molecules. In contrast to WNV, there was no increase in the cell surface expression of MHC-II molecules upon JEV infection of primary mouse brain astrocytes. JEV infection also induces the expression of adhesion molecules as well as molecules involved in antigen processing and presentation namely Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10. Chapter 5 demonstrates that JEV infection induces NF-κB activation in astrocytes and MEFs. Studies using MEFs deficient in classical and alternate pathways of NF-κB activation indicate that JEV activates the classical pathway of NF-κB activation and is dependent on canonical lKKβ/IKK2 activity. JEV infection of astrocytes, MEFs and H6 induces the production of type-I IFNs. To determine the mechanism of type-I IFN induction during JEV infection, MEFs deficient in NF-κB signaling and IFN signaling were used. Results indicate that type-I IFN production in MEFs occurs by both NF-κB dependent and independent mechanisms. In contrast, the production of IFN-α was completely abrogated in IFNAR-\- MEFs whereas IFN-β production was greatly reduced. Production of type-I IFNs in IFNGR-\- MEFs is also reduced upon JEV infection but the reason for this is unclear. Chapter 6 demonstrates that JEV induced expression of classical MHC-I molecules occurs by type-I IFN mediated signaling. This result is in contrast to WNV infection, in which both NF-κB and type-I IFNs are involved in the induction of classical MHC-I molecules. Type-I IFNs were also shown to be involved in the induction of nonclassical MHC molecules namely, Qa-1 and Qb1 during JEV infection. In contrast, the expression of T10, another nonclassical MHC molecule occurs independent of type-I IFN signaling. The expression of molecules involved in antigen processing and presentation namely, Tap1, Tap2, Lmp2 and Lmp7 was type-I IFN-mediated, whereas the expression of Tapasin and Lmp10 was mediated by both type-I IFN dependent and independent mechanisms. The expression of VCAM-1 was dependent on NF-κB mediated signaling. Chapter 7 precisely describes the underlying mechanism of induction of MHC and various other related molecules and their significance during JEV infection. In addition, it also includes a working model for the induction of these molecules during JEV infection. In summary, this is the first study in which the mechanism of JEV mediated induction of classical as well as nonclassical MHC molecules has been studied in detail. This study clearly demonstrated that type-I IFNs are involved in the induction of classical and nonclassical MHC-I molecules during JEV infection. The functional significance of this JEV mediated induction of classical MHC-I molecules is unclear, but it has been proposed that this is to escape from the action of NK cells. The absence of MHC-II induction during JEV infection could be important because it may lead to the initiation of an immune response which is different from that induced during other viral infections which induce the expression of MHC-II molecules. In contrast to classical MHC-I molecules, the functional and biological significance of nonclassical MHC-I molecules are poorly studied. Nonclassical MHC-I molecules play an important role in bridging adaptive and innate immune response. So the nonclassical MHC molecules induced during JEV infection may play an important role in the initiation of immune response during JEV infection. The role these nonclassical MHC-I molecules in antigen presentation during JEV infection is not known. These nonclassical antigens are also recognized by NK and γδT cells, thus the expression of nonclassical MHC-I molecules during JEV infection might also confer a protective role.

Orientador: Alexandre Leite Rodrigues de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T17:19:57Z (GMT). No. of bitstreams: 1 Simoes_GustavoFerreira_D.pdf: 65115196 bytes, checksum: 18f51cababf3e248cceb806b8faef631 (MD5) Previous issue date: 2012 / Resumo: Atualmente, muito se sabe sobre o acometimento muscular na DMD, mas poucos estudos estão voltados para os efeitos no Sistema Nervoso Central (SNC), mais especificamente no microambiente do motoneurônio medular. Sabe-se que durante a evolução da doença, o terminal axonal, na junção neuromuscular, entra em um ciclo de denervação (retração) e reinervação (brotamento). A possibilidade de modulação do MHC I se apresenta como uma nova estratégia de influenciar positivamente o processo de plasticidade sináptica após lesões do Sistema Nervoso Periférico (SNP) e SNC. Tal modulação pode ser realizada através da utilização ou desenvolvimento de drogas específicas. O fator estimulador de colônias glanulocitárias (G-CSF) é uma glicoproteína que foi descrita há mais de vinte anos, possui aprovação do ANVISA (Agência Nacional de Vigilância Sanitária) e é comumente utilizada para tratar neutropenia, ou para transplantes de medula óssea. O GCSF possui um efeito neuroprotetor aparentemente multimodal, incluindo-se a atividade anti-apoptóptica em neurônios, regeneração da vascularização, efeito antiinflamatório e estimulação de neurogênese endógena, sendo capaz de atuar efetivamente no processo de regeneração do sistema nervoso. No presente trabalho, foram utilizados camundongos MDX. Os camundongos foram distribuídos em 4 grupos (axotomia + G-CSF; Axotomia; Controle + G-CSF e Controle), com n=10. Incluiu-se para imunoistoquímica o grupo placebo, onde os animais receberam uma dose diária de 200?m, via subcutânea, de glicose a 25%. Nossos resultados indicam que redução de sinapses nos motoneurônios alfamedulares e aumento da astrogliose circunjacente aos neurônios alfa-medulares, seja decorrente da desconexão parcial entre o orgão alvo e o corpo neuronal durante o período de ciclos de degeneração/regeneração muscular que ocorrem a partir das primeiras semanas de vida nos camundongos MDX. Estes ciclos podem repercutir retrogradamente nos corpos celulares dos motoneurônios alfa-medulares, provocando uma série de alterações denominadas cromatólise. A axotomia do nervo isquiático resulta num aumento significativo da expressão de MHC I nas duas linhagens estudadas. Contudo, nos animais MDX, este aumento é menor, comparativamente à linhagem C57BL/10. Quando tratados com G-CSF a expressão de MCH I ficou maior em relação aos grupos não tratados e, isso pode indicar um papel ativo da droga no potencial regenerativo após a lesão. Também podemos sugerir que, apesar dos animais MDX apresentarem uma menor função motora em relação aos animais controle, os resultados indicam que o tratamento com G-CSF é capaz de reduzir os efeitos inflamatórios e atuar positivamente no processo de regeneração nervosa periférica após esmagamento do nervo isquiático / Abstract: Currently, much is known about the muscular involvement in DMD, but few studies have focused on the effects on the central nervous system (CNS), specifically in the microenvironment of spinal motor neurons. It is known that during the course of the disease, the axon terminal at the neuromuscular junction, enters a cycle of denervation (retraction) and reinnervation (sprouting). The possibility of modulation of MHC I presents itself as a new strategy to positively influence the process of synaptic plasticity after injury Peripheral Nervous System (PNS) and CNS. Such modulation may be accomplished through the use or development of special drugs. The granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which was first described more than twenty years, has approval from ANVISA (Agência Nacional de Vigilância Sanitária) and is commonly used to treat neutropenia, or bone marrow transplants. The G-CSF has a multimodal neuroprotective effect l, including the anti-apoptotic activity in neurons, regeneration of vascularization, anti-inflammatory effect and stimulation of endogenous neurogenesis, being able to act effectively in the process of regeneration of the nervous system. In this study, we used MDX mice. The mice were divided into 4 groups (axotomy + G-CSF; axotomy, Control + G-CSF and Control), with n = 10. Included immunohistochemistry to the placebo group, where the animals received a daily dose of 200?m, subcutaneously, glucose 25%. Our results indicate that reduction of synapses in the alpha motoneurosn and increased astrogliosis , either due to partial disconnection between the target organ and the neuronal body during the cycles of degeneration /regeneration muscle that occur from first weeks of life in MDX mice. These cycles can pass retrogradely in alpha motoneurons cell bodies, causing a series of changes called chromatolysis. The sciatic nerve axotomy results in a significant increase of MHC I expression in both strains studied. However, in MDX strain, this increase is smaller, compared to C57BL/10. After treatment with G-CSF the expression of MCH I got bigger compared to untreated groups, and this may indicate an active role in the regenerative potential of the drug after injury. Also we suggest that while the animals present MDX a smaller motor function compared to control animals, the results indicate that treatment with G-CSF is capable of reducing the inflammatory effects and act positively on peripheral nerve regeneration process after nerve crush sciatic. Also our results indicate that treatment with G-CSF is able to reduce the inflammatory effects and act positively on peripheral nerve regeneration process after nerve crush sciatic / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural