L’hépatite B chronique touche environ 257 millions de personnes dans le monde. La perte de l’antigène HBs (AgHBs), marqueur de guérison fonctionnelle, n’est que très rarement observée, même sous traitement antiviral (3-16 %). Les protéines d’enveloppe du virus de l’hépatite B (VHB), formant l’AgHBs, sont très variables et cruciales pour le pouvoir infectieux du virus de l’hépatite B (VHB) et la physiopathologie. Nous avons émis l’hypothèse que cette variabilité pourrait expliquer, au moins partiellement, l’évolution de l’infection par le VHB, évaluée par la clairance de l’AgHBs, chez des patients traités ou non par analogues nucléos(t)idiques anti-VHB. Chez 29 patients infectés par différents génotypes du VHB (A, C et D), présentant différents profils cliniques (infection aigüe ou chronique, co-infection VHB/VIH) et thérapeutiques, une très grande variabilité des protéines d’enveloppe du VHB a été mise en évidence. Chez ces patients, la persistance de l’AgHBs était corrélée avec la présence de mutations et délétions localisées dans des régions des protéines d’enveloppe virale jouant un rôle important dans la reconnaissance du virus par le système immunitaire. Ces résultats renforcent l’hypothèse que l’étude des protéines d’enveloppe du VHB pourrait mettre en évidence des signatures moléculaires influençant le fitness du VHB et par conséquent l’évolution clinique de la maladie liée à l’infection par le VHB / Chronic hepatitis B affects about 257 million people worldwide. The loss of HBS antigen (HBsAg), a marker of the functional cure, is very rarely observed, even on anti-HBV treatment (3-16%). The hepatitis B virus (HBV) envelope proteins (HBsAg) are highly variable and crucial for the viral infectivity and pathogeny. We hypothesized that the HBV variability in the envelope proteins could explain, at least partially, the evolution of HBV infection, evaluated by HBsAg clearance, in patients treated or not by anti-HBV nucleos(t)idic analogues. For 29 patients infected with different HBV genotypes (A, C and D), presenting different clinical profiles (acute or chronic infection, HBV/HIV co-infection) and therapies, a very high variability of HBV envelope proteins was observed. In these patients, the persistence of HBsAg was correlated with the presence of mutations and deletions located in areas that play a key role in the viral recognition by the immune system. These results reinforce the hypothesis that the study of HBV envelope proteins could highlight molecular signatures influencing HBV fitness which would subsequently modify the clinical evolution of HBV-related disease

Made available in DSpace on 2016-08-10T10:38:33Z (GMT). No. of bitstreams: 1 CAROLINE STEGLICH RAMOS.pdf: 1233030 bytes, checksum: 0c294f9932ce70c185a878b013b4d356 (MD5) Previous issue date: 2012-02-07 / The breast cancer constitutes most common malignancy pathology among women where the knowledge of prognostic aspects suggest the clinical course of disease and patient survival. Detection of HLA-G has been described as prognostic factor for many cancers, this molecule, expresses in ectopic way in certain types of tumors, where induce the immunotolerance and, favors the tumoral immune escape. Thereby, this study aimed to evaluate the expression of the molecule HLA-G in infiltrating ductal breast carcinomas (CDI), as well as evaluate the polyphormism of the gene encoding HLA-G to determine possible correlation between these and the molecule expression. Thus, to meet these objectives, we evaluated the expression of HLA-G protein in forty-five patients with IDC by immunohistochemistry and also the insertion and deletion polymorphism of 14pb gene HLA-G in 80 samples of paraffin tumors, obtained from the Service of Anatomic Pathology at Araújo Jorge Hospital, and a group of 191 healthy people was used as control. All the statistical analyses were made using the programs GraphPad Instat version 5.01 and Genepop version 2.0. Our results showed that the group of patients were inbalance in Hardy-Weinberg (p=0,01), due to the excess of heterozygotes (p=0,0007), there is not statistically significant difference in the allelic frequencies in locus HLA-G, when compared the group of patients with the control group, however, there were difference in the genotypics frequencies when comparing the same groups (p=0,012), being the genotypic frequency deletion/insertion significantly higher in the group of patients. Also, the expression of HLA-G was observed in 62% of the samples analyzed by immunohistochemistry and this expression was observed an mostly in tumor cells and also by intra-tumoral inflammatory infiltrate (p=0,03). The analysis showed that the expression of HLA-G was significantly higher in patients that had shorter survival (log rank = 0,03), after 5 years of follow up and still there was also a correlation between the expression of HLA-G and increased incidence of lymph node metastases (p=0,01). Thus, the results suggest that the HLA-G expression in patients with CDI may be associate to poor clinical evolution, with the reduced survival. / O câncer de mama constitui-se na patologia maligna mais comum entre as mulheres onde o conhecimento dos aspectos prognósticos sugerem o curso clínico da doença e a sobrevida dos pacientes. A detecção da molécula HLA-G tem sido descrita como fator prognóstico para inúmeras neoplasias, esta molécula, expressa de maneira ectópica em certos tipos de tumores, onde induz a imunotolerância e favorece o escape imunológico tumoral. Desse modo, esse estudo objetivou avaliar a expressão da molécula HLA-G em carcinomas ductais infiltrantes da mama (CDI), bem como, avaliar polimorfismo do gene codificador de HLA-G para determinar possível correlação entre estes e a expressão da molécula. Assim, para cumprir tais objetivos, avaliou-se a expressão da proteína HLA-G em quarenta e cinco pacientes com CDI por imunoistoquímica, e ainda, o polimorfismo de deleção e inserção de 14pb do gene HLAG, em 80 amostras de tumores parafinados, obtidas junto ao Serviço de Anatomia Patológica do Hospital Araújo Jorge e um grupo composto por 191 indivíduos saudáveis foi utilizado como controle. Todas as análises estatísticas foram feitas utilizando os programas GraphPad Instat versão 5.01 e Genepop versão 2.0. Os resultados mostraram que o grupo de pacientes encontrava-se em desequilíbrio de Hardy-Weinberg (p= 0,01), devido ao excesso de heterozigotos (p= 0,0007), não havendo diferença estatisticamente significante nas freqüências alélicas no loco HLA-G quando comparado o grupo de pacientes com o grupo controle, no entanto, houve diferença nas freqüências genotípicas ao comparar os mesmos grupos (p= 0,012), sendo a freqüência do genótipo deleção/inserção significantemente maior no grupo de pacientes. Também foi observada a expressão de HLA-G em 62% das amostras totais analisadas por imunoistoquímica e essa expressão foi apresentada principalmente, em células tumorais havendo, no entanto, também expressão pelas células do infiltrado inflamatório intratumoral (p= 0,03). As analises mostram que a expressão de HLA-G foi significantemente maior em pacientes que tiveram menor sobrevida (log rank= 0,03), após 5 anos de seguimento clínico e ainda houve correlação entre a expressão de HLA-G e aumento naincidência de metástases em linfonodos (p= 0,01). Com isso, os resultados sugerem que a expressão de HLA-G em pacientes com CDI pode estar associada à pior evolução clínica dos mesmos havendo redução na taxa de sobrevida.

L'évasion immunitaire tumorale joue un rôle central dans la progression tumorale et représente un obstacle majeur au succès des immunothérapies. Dans cette Thèse nous avons étudié le rôle du cytosquelette d’actine dans la résistance des cellules de cancer du sein à la lyse induite par les cellules "natural killers" (NKs). Nous avons trouvé que les cellules de cancer du sein résistantes échappent à l’attaques des cellules NKs par une accumulation importante et rapide d’actine près de la synapse immunologique, un processus que nous avons nommé "réponse actine". Nos analyses mécanistiques suggèrent que la réponse actine induit la polarisation d’autophagosomes vers la synapse immunologique et facilite ainsi la dégradation des molécules cytotoxiques sécrétées par les cellules NKs, tel que le ganzyme B, par autophagie. De plus, la réponse actine est associée au regroupement de ligands inhibiteurs à la synapse, suggérant qu’elle est au centre de plusieurs mécanismes de résistance. Dans leur ensemble, nos résultats constituent une base pour le développement d’approches thérapeutiques visant à interférer avec la réponse actine et à restaurer une réponse immunitaire anti tumorale efficace. / Tumor immune evasion plays a central role in cancer progression and is a major hurdle to effective immunotherapy. In this Thesis, we examine the role of the actin cytoskeleton in breast cancer cell resistance to natural killer (NK) cell-mediated cell lysis. We found that resistant breast cancer cells escape from NK-cell attack through a rapid and prominent accumulation of actin near the immunological synapse, a process we termed the “actin response”. Our mechanistic investigations suggest that the actin response drives autophagosome polarization toward the immunological synapse and thereby facilitates the autophagy-mediated degradation of NK cell-derived cytotoxic molecules such as granzyme B. In addition, the actin response was associated with inhibitory ligand clustering at the immunological synapse, suggesting that it is a common driver of different immune evasion mechanisms. Taken together, our data lays the groundwork for therapeutic approaches aimed at interfering with the actin response and restoring an effective anti-tumor immune response.

Nous sommes toujours aux prises avec la pandémie de SARS-CoV-2 plus de deux ans après son début. Le virus a depuis accumulé de nombreuses mutations qui ont mené à différentes souches virales au long de la pandémie. Plusieurs de ces mutations sont récurrentes: il y a un excès de mutations C > U dans les génomes viraux, certains codons sont fréquemment mutés vers différents acides aminés et certaines mutations sont convergentes, c’est-à-dire que la même substitution est apparue de manière indépendante sur différentes lignées. Dans ce mémoire, nous avons identifiés différentes manières par lesquelles le SARS-CoV-2 évolue à travers l’étude de ces mutations récurrentes et évaluons leur impact sur l’évasion immunitaire. Premièrement, nous avons déterminés que les mutations C > U sont responsable de l’introduction et du retrait préférentiel d’acides aminés spécifiques dans les épitopes viraux. Nous avons déterminé la significativité statistique de ces patrons de mutation à l’aide de simulations génomiques virales. Deuxièmement, nous avons participé à la surveillance des variants au Québec durant la deuxième vague de la pandémie, qui s’est déroulée d’août 2020 à mars 2021. C’était une période intéressante pour la diversité virale, puisque les restrictions de déplacement ont créé de multiples poches de variants locaux en compétition les uns avec les autres qui partagent des mutations convergentes. Notamment, nous reportons que les lignées B.1.160 et B.1.1.176 comptaient pour 50% des échantillons séquencés au sommet de la deuxième vague dans la province. Finalement, nous avons analysé les patrons mutationnels intra-hôte qui sont apparus de novo dans le contexte d’infections au SARS-CoV-2 de longue durée chez des patients atteints de cancers hématologiques. Une de ces patientes est une patiente québécoise infectée par B.1.160 et dans laquelle nous avons identifié la présence d’un réservoir viral. Nous avons également trouvé des éléments probants montrant différentes quasiespèces virales avec des propriétés d’évasion immunitaire. Nos résultats permettent de mieux comprendre les différentes manières dont les pressions sélectives façonnent l’évolution virale. / We are still living in the SARS-CoV-2 pandemic over two years after its start. The virus has since accumulated many mutations that have led to different viral strains throughout the pandemic. Several of these mutations are recurrent: there is an excess of C > U substitutions in viral genomes, some codons are frequently mutated to different amino acids, and some mutations are convergent, meaning that the same substitution has occurred independently on different lineages. In this thesis, we identified different ways in which SARS-CoV-2 evolves through these recurrent mutations and assess their impact on immune escape. First, we determined that C > U mutations drive the preferential introduction and removal of specific amino acids in viral epitopes. Using genetic simulations, we determined the statistical significance of these patterns. Second, we participated in the surveillance of variants in Quebec during the second wave of the pandemic that went from the end of August 2020 to the end of March 2021. This was an interesting period of viral diversity owing to imposed travel restrictions that created competition between multiple pockets of local strains that share convergent mutations. Notably, we found that lineages B.1.160 and B.1.1.176 account for 50% of samples sequenced at the height of the second wave in the province. Finally, we analyzed intra-host mutational patterns that arose de novo in the context of long-term infections of patients with hematological cancers, one of which was from Québec and infected by B.1.160. We have identified a pattern consistent with the presence of a viral reservoir in this patient. We have also found evidence of different viral quasispecies with immune escape properties. These results shed light on different ways in which selective pressures shape the evolution of SARS-CoV-2.

Ce travail examine les mécanismes de l’immunité innée impliqués dans l’hépatite aiguë virale du modèle murin d’infection par le virus de l’hépatite virale murine de type 3 (MHV-3). Afin de déterminer le rôle du TLR2 dans l’aggravation de l’hépatite, des infections avec le virus MHV-3 ont été réalisées in vivo chez des souris C57BL/6 et des souris déficientes pour le gène tlr2 et in vitro dans des macrophages et des hépatocytes infectés avec le virus MHV-3 et le virus moins virulent MHV-A59. Les niveaux de transcription et de traduction des senseurs microbiens, des interférons (IFN) de type I, des cytokines et/ou des chimiokines ont été évalués par qRT-PCR et ELISA. Les cellules ont été traitées avec des petits ARNs interférants (siRNAs) pour le TLR2 et le CEACAM1a ou mises en présence d’inhibiteurs des voies d’endocytose. Les résultats révèlent le rôle stimulateur du TLR2 pour la réplication virale, la production de cytokines pro-inflammatoires IL-6 et TNF-α et des chimiokines CXCL1, CXCL10 et CCL2. Un nouveau mécanisme d’échappement aux senseurs viraux dépendant du TLR2 a également été mis en évidence dans les macrophages et les hépatocytes lors de l’infection de ces cellules avec le virus MHV-3, et non pas avec le virus moins virulent MHV-A59. Ces différents travaux révèlent un nouveau rôle du TLR2 lors d’infections virales dans l'aggravation de la réponse inflammatoire tout en protégeant le virus des autres senseurs de la réponse immune innée. / This work investigates the innate immunity mechanisms involved in the Murine Hepatitis Virus type 3 (MHV-3) acute hepatitis disease model. In the goal of determining the role of the TLR2 receptor in aggravating the hepatitis disease, in vivo infections with the MHV-3 virus were performed on C57BL/6 mice or tlr2-/- gene knockout mice as well as in vitro infections of murine macrophages and hepatocytes with the MHV-3 and less virulent MHV-A59 serotypes. The levels of transcription and translation of different microbial sensors, type I interferons (IFN), cytokines and/or chemokines were measured by qRT-PCR and ELISA tests. Cells were treated with small interfering ARN (siRNA) for the TLR2 and/or CEACAM1a genes or treated with the different endocytosis inhibitors before infection. Our results reveal the stimulatory role of the TLR2 receptor on viral replication, production levels of the IL-6 and TNF-α cytokines and chemokines CXCL1, CXCL10 and CCL2. This study also reveals a new immune evasion mechanism via TLR2 in macrophages infected with MHV-3, but not with the lesser virulent MHV-A59 serotype. These different experiments have revealed a new role for the TLR2 receptor in viral infections wherein inflammatory responses are aggravated whilst shielding viral detection by other pathogens sensors of the innate immunity.

Die effiziente Präsentation von Tumorepitopen stellt einen kritischen Faktor zur Eliminierung von Tumorzellen durch die CD8+ cytotoxische T-Lymphozyten (CTL) vermittelte Immunantwort dar. Eine wichtige Rolle spielt in diesem Zusammenhang die effiziente Generierung von Tumorepitopen durch das Ubiquitin-Proteasom-System (UPS). Veränderungen von Komponenten des UPS, die an der Degradation und Prozessierung von Antigenen beteiligt sind, können daher zur Immunevasion von Tumorzellen gegenüber CTL führen. In der vorliegenden Arbeit wurden zwei unterschiedliche UPS-assoziierte Immunevasionsmechanismen des malignen Melanoms identifiziert, die auf einer ineffizienten Präsentation des immundominanten Tumorepitops Melan-A26-35 basieren. Ein Mechanismus beruht auf den unterschiedlichen katalytischen Eigenschaften von Proteasomsubtypen, deren Expression durch INFgamma induziert wird. Proteasomen, die einerseits die Immunountereinheiten beta1i und/oder beta2i beinhalten, oder anderseits mit dem Proteasomaktivator 28 (PA28) assoziiert sind, führen zu einer drastisch reduzierten Generierung des Tumorepitops Melan-A26-35. In beiden Fällen ist dies auf eine ineffiziente Prozessierung des N-Terminus des Epitops zurückzuführen. Der andere Immunevasionsmechanismus steht im Zusammenhang mit der ER-assoziierten Degradation (ERAD), die den retrograden Transport von Proteinen aus dem Endoplasmatischen Retikulum (ER) ins Cytosol zur proteasomalen Degradation beinhaltet. Durch Immunselektion mittels Melan-A26-35-spezifischen CTL wurden cytolyseresistente Melanomzellen identifiziert, deren Resistenz auf eine defiziente ER-assoziierte Degradation zurückzuführen ist. Dieser Defekt beruht auf einer verminderten Expression von ERAD-Komponenten, deren Reduktion die Verfügbarkeit des Antigens Melan-A zur proteasomalen Degradation und Generierung des immundominanten Epitops Melan-A26-35 wesentlich limitiert. / Efficient presentation of tumor epitopes by MHC class I molecules on the cell surface is a prerequisite for the elimination of tumor cells by cytotoxic CD8+ T lymphocytes. The generation of these epitopes requires the degradation and processing of proteins by the ubiquitin proteasome system (UPS). Therefore alterations of UPS components can lead to tumor escape from immune recognition as a result of decreased epitope generation. In the present thesis two different UPS connected immune escape mechanisms of melanoma cells were identified. Both are based on an impaired generation of the immunodominant epitope Melan-A26-35 derived from Melan-A/MART-1 tumor antigen. One mechanism is mediated by the expression of different INFgamma-inducible proteasome immunosubunits leading to the formation of intermediate proteasome subtypes, which differ in their cleavage site preferences. Purified proteasomes harboring the immunosubunits beta1i and/or beta2i show a dramatic decrease in the generation of the Melan-A26-35 epitope. In addition, the INFgamma induced association of proteasomes with the proteaosome activator 28 (PA28) results in a reduced epitope generation. Both mechanisms are induced by an inefficient processing of the epitope’s N-terminus. The second immune escape mechanism is caused by defects of the ER-associated degradation pathway (ERAD). ERAD mediates the transport of ER-proteins back to the cytosolic compartment for proteasomal degradation. Via immunselection of tumor cells with Melan-A26-35 specific CTL, cytolysis resistant cells were identified. Resistance to CTL mediated lysis was shown to be connected to a decreased expression of ERAD components. This defect of the ERAD pathway limits the availability of the Melan-A protein and as consequence the generation of the immunodominant Melan-A26-35 epitope by proteasomes.

A enzima indolamina 2,3-dioxigenase (IDO) é responsável pela degradação de triptofano (TRP) pela via das quinureninas. O aumento da expressão de IDO é a grande responsável pela resposta de tolerância imunológica e pode ser induzida por IFN-γ e lipopolissacarídeos (LPS). É conhecido que a IDO participa do mecanismo de imuno escape de células tumorais, sendo relatada como marcadora de progressão tumoral. Desta forma, a inibição da atividade da IDO para a restauração da imunidade anti-tumoral do hospedeiro tem sido considerada uma estratégia para a terapêutica antineoplásica. N,N-dimetiltriptamina (DMT) e triptamina (TRY) são compostos indólicos de origem endógena, provenientes de uma rota paralela de metabolismo do triptofano - a rota das triptaminas, que ocorre principalmente no sistema nervoso central. O presente estudo teve como objetivo avaliar se DMT e TRY modulariam a atividade de IDO. Os resultados obtidos demonstraram ações inibitórias para ambos os compostos e as cinéticas enzimáticas revelaram Ki de 506µM para o DMT e de 156µM para a TRY, com perfis inibitórios característicos de inibidores não-competitivos clássicos. A atividade inibitória foi também observada sobre a enzima IDO expressa constitutivamente, ou induzida por IFN-γ, em células da linhagem de glioblastoma humano A172. Nesta mesma linhagem, em estudo paralelo do grupo, foi avaliada a influência de DMT e TRY sobre a expressão gênica da enzima IDO. Concluímos que nas células a ação inibitória dos compostos avaliados é exercida diretamente sobre sua atividade enzimática, sem redução de sua transcrição. Os resultados deste estudo também serviram como base para estudos da atividade inibitória da DMT e TRY em sistema de co-cultura das células A172 com células mononucleares humanas, onde foi observada redução significativa da atividade enzimática e da proliferação das células tumorais. Em conclusão mostramos a possibilidade de que a via das triptaminas, através de seus metabólitos, possa contribuir com a regulação endógena da via das quinureninas e que o DMT e a TRY deveriam ser avaliados como candidatos a inibidores farmacológicos da enzima IDO e/ou como protótipos para a síntese de novos inibidores. / Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme via the kynurenine pathway. Increased IDO expression is largely responsible for the immune tolerance response and can be induced by IFN-γ and lipopolysaccharide (LPS). It is known that IDO participates in the mechanism of tumor immune tolerance and have been reported as a tumor progression marker. Thus, to restore the anti-tumor immunity of the host, IDO inhibition has been considered as a strategy for anticancer therapy. N, N-dimethyltryptamine (DMT) and tryptamine (TRY) are endogenous indolic compounds originated from a parallel route of tryptophan metabolism - the tryptamines route, which occurs mainly in the central nervous system. This study aimed to assess whether DMT and TRY modulate the IDO activity. The results showed inhibitory actions for both compounds and enzyme kinetics revealed Ki = 506µM to DMT and Ki = 156µM to TRY, with inhibitory profiles characteristic of classical non-competitive inhibitors. The inhibitory activity was also observed on the IDO constitutively expressed or induced by IFN-γ in A172 human glioblastom cell line. Using the same line, in parallel group study, we evaluated the influence of DMT and TRY on the IDO gene expression. We conclude that in cells the evaluated compounds inhibitory action is exerted directly on its enzymatic activity without reduction of its transcription. The results of this study also served as the basis for studies of DMT and TRY inhibitory activity in A172 cells with human mononuclear cells co-culture system, in which was observed significant reduction in enzyme activity and tumor cells proliferation. In conclusion we show the possibility that the tryptamines route, through its metabolites, may contribute to quinurenines pathway endogenous regulation and that DMT and TRY should be evaluated as IDO pharmacological inhibitors candidates and / or as prototypes to new inhibitors synthesis.

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Le virus d’Epstein-Barr (EBV) est un herpèsvirus humain qui infecte plus de 90% de la population mondiale. Il est décrit comme associé à plusieurs pathologies cancéreuses humaines comme les carcinomes nasopharyngés et gastriques et divers lymphomes, comme le lymphome de Burkitt, les lymphomes NK/T et certains lymphomes de Hodgkin. Le lymphome T angio-immunoblastique (LTAI), un cancer des cellules T folliculaires helper TFH, contient souvent des cellules B porteuses de l’EBV. Mais jusqu’à présent le rôle de l’EBV dans la pathogenèse de cette maladie reste inconnu. Dans ce contexte, notre travail avait pour objectif de déterminer si l’EBV associé au LTAI présentait une particularité laissant envisager son rôle dans cette pathologie. Pour ce faire, nous avons étudié la séquence complète de l’EBV au sein d’échantillons de LTAI et comparé les résultats à ceux obtenus pour d’autres lymphomes (B, NK/T) ainsi qu’aux séquences publiées. Le séquençage a tout d’abord été réalisé sur 7 lignées cellulaires positives pour l’EBV, afin de valider la technique, et a ensuite été appliqué aux échantillons d’adénopathies de 40 patients atteints de syndrome lymphoprolifératif, parmi lesquels 20 souffraient de LTAI. L’enrichissement en génome viral a été réalisé par capture à l’aide de sondes spécifiques du génome de l’EBV. Ensuite les librairies ont été synthétisées et séquencées sur les plateformes Illumina MiSeq et NextSeq. Dans un deuxième temps, nous avons réalisé l’assemblage de novo des reads et déterminé la séquence complète du virus majoritaire dans chaque échantillon. Les données obtenues ont été analysées bioinformatiquement. D’une manière intéressante, le virus a été trouvé clonal ou quasi-clonal dans les LTAI alors que les lymphocytes B étaient dans la plupart des cas polyclonaux. En outre, le profil de mutations trouvé présentait des similitudes avec ce qui était trouvé pour les autres lymphomes associés à l’EBV, notamment au niveau des épitopes cibles des cellules de l’immunité suggérant un processus de sélection de la souche virale identique à celui d’une tumeur clonale associée à l’EBV. Ceci pourrait jouer un rôle important dans l’échappement au système immunitaire du virus dans ce contexte multicellulaire complexe. La présence de cellules B polyclonales avec un EBV clonal dans un compartiment T tumoral clonal pourrait relever d’une double sélection tumorale, endogène T et exogène EBV clonal, et pourrait suggérer l’existence de cross-talk entre les cellules B-T. / More than 90% of the world's population is infected by Epstein-Barr virus (EBV), a human herpesvirus. EBV is thought to be implicated in the pathogenesis of several human malignancies including epithelial tumors such as nasopharyngeal and gastric carcinomas as well as lymphoproliferative diseases such as Burkitt's lymphoma, NK/T lymphomas and some Hodgkin lymphomas. In angioimmunoblastic T-cell lymphoma (AITL), a peripheral neoplasm of follicular helper T (TFH) cells, a recurrent finding is the presence of EBV-positive B lymphocytes at the beginning of the disease. However, whether this EBV infection of B cells plays a role in AITL pathogenesis remains unclear. In this context, our work aimed to determine if the EBV associated with the AITL presented an oncogenic profile allowing us to consider its role in this pathology. To do this, we sequenced the whole EBV genomes in AIL samples and compared the results to those obtained for other lymphomas (B, NK / T) as well as to previously published sequences. Sequencing was first performed on 7 EBV-positive cell lines to validate the technique, and then was applied to lymphadenopathy specimens from 40 patients with lymphoproliferative disease, of whom 20 had AITL. Enrichment of the viral genome was performed by capture using specific EBV genome probes. The libraries were synthesized and sequenced on Illumina MiSeq and NextSeq platforms. In a second step, we performed de novo assembly and determined the sequence of the virus in each sample. The data obtained were analyzed bioinformatically. Interestingly, the virus was found to be clonal or quasi-clonal in AITL, while B cells were in some cases polyclonal. In addition, the mutational pattern was similar to other EBV-associated lymphomas, especially at the level of the target epitopes of immune cells suggesting a process of selection of the viral strain identical to that of a clone tumor associated with EBV. This could play an important role in the virus escape from the immune system in this context. The presence of polyclonal B cells with clonal EBV in a clonal tumor T cell compartment could be a dual tumor selection; or that is endogenous T and exogenous clonal EBV, and could therefore suggest the existence of a cross-talk between B-T cells.

A enzima indolamina 2,3-dioxigenase (IDO) é responsável pela degradação de triptofano (TRP) pela via das quinureninas. O aumento da expressão de IDO é a grande responsável pela resposta de tolerância imunológica e pode ser induzida por IFN-γ e lipopolissacarídeos (LPS). É conhecido que a IDO participa do mecanismo de imuno escape de células tumorais, sendo relatada como marcadora de progressão tumoral. Desta forma, a inibição da atividade da IDO para a restauração da imunidade anti-tumoral do hospedeiro tem sido considerada uma estratégia para a terapêutica antineoplásica. N,N-dimetiltriptamina (DMT) e triptamina (TRY) são compostos indólicos de origem endógena, provenientes de uma rota paralela de metabolismo do triptofano - a rota das triptaminas, que ocorre principalmente no sistema nervoso central. O presente estudo teve como objetivo avaliar se DMT e TRY modulariam a atividade de IDO. Os resultados obtidos demonstraram ações inibitórias para ambos os compostos e as cinéticas enzimáticas revelaram Ki de 506µM para o DMT e de 156µM para a TRY, com perfis inibitórios característicos de inibidores não-competitivos clássicos. A atividade inibitória foi também observada sobre a enzima IDO expressa constitutivamente, ou induzida por IFN-γ, em células da linhagem de glioblastoma humano A172. Nesta mesma linhagem, em estudo paralelo do grupo, foi avaliada a influência de DMT e TRY sobre a expressão gênica da enzima IDO. Concluímos que nas células a ação inibitória dos compostos avaliados é exercida diretamente sobre sua atividade enzimática, sem redução de sua transcrição. Os resultados deste estudo também serviram como base para estudos da atividade inibitória da DMT e TRY em sistema de co-cultura das células A172 com células mononucleares humanas, onde foi observada redução significativa da atividade enzimática e da proliferação das células tumorais. Em conclusão mostramos a possibilidade de que a via das triptaminas, através de seus metabólitos, possa contribuir com a regulação endógena da via das quinureninas e que o DMT e a TRY deveriam ser avaliados como candidatos a inibidores farmacológicos da enzima IDO e/ou como protótipos para a síntese de novos inibidores. / Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme via the kynurenine pathway. Increased IDO expression is largely responsible for the immune tolerance response and can be induced by IFN-γ and lipopolysaccharide (LPS). It is known that IDO participates in the mechanism of tumor immune tolerance and have been reported as a tumor progression marker. Thus, to restore the anti-tumor immunity of the host, IDO inhibition has been considered as a strategy for anticancer therapy. N, N-dimethyltryptamine (DMT) and tryptamine (TRY) are endogenous indolic compounds originated from a parallel route of tryptophan metabolism - the tryptamines route, which occurs mainly in the central nervous system. This study aimed to assess whether DMT and TRY modulate the IDO activity. The results showed inhibitory actions for both compounds and enzyme kinetics revealed Ki = 506µM to DMT and Ki = 156µM to TRY, with inhibitory profiles characteristic of classical non-competitive inhibitors. The inhibitory activity was also observed on the IDO constitutively expressed or induced by IFN-γ in A172 human glioblastom cell line. Using the same line, in parallel group study, we evaluated the influence of DMT and TRY on the IDO gene expression. We conclude that in cells the evaluated compounds inhibitory action is exerted directly on its enzymatic activity without reduction of its transcription. The results of this study also served as the basis for studies of DMT and TRY inhibitory activity in A172 cells with human mononuclear cells co-culture system, in which was observed significant reduction in enzyme activity and tumor cells proliferation. In conclusion we show the possibility that the tryptamines route, through its metabolites, may contribute to quinurenines pathway endogenous regulation and that DMT and TRY should be evaluated as IDO pharmacological inhibitors candidates and / or as prototypes to new inhibitors synthesis.