Des études récentes ont montré une implication de la neuropiline 1 (Nrp1)dans le contrôle de l'activation des lymphocytes T. Son invalidation s'accompagned'une aggravation de l'encéphalite auto-immune expérimentale (EAE). Lasémaphorine 3A (Sema-3A), ligand principal de la Nrp1, semble participer à uneboucle autocrine de rétro contrôle négatif de la prolifération des lymphocytes T.Cependant, peu d'études ont été réalisées chez l'homme pour déterminer dansquelle(s) situation(s) la Nrp1 est exprimée par les lymphocytes T. Notre travail aconsisté à étudier l'expression de la Nrp1 par les populations lymphocytaires Thumaines afin de comprendre à quel niveau peut avoir lieu ce rétro contrôle. Nousmontrons que les lymphocytes T régulateurs (Treg) chez l'homme n'expriment pas laNrp1, contrairement aux Treg murins. En revanche, la Nrp1 est exprimée par leslymphocytes T effecteurs après engagement avec l'antigène, soit au niveau desorganes lymphoïdes secondaires pour les lymphocytes T folliculaires helper (Tfh) eninteraction avec les lymphocytes B, soit au niveau des sites d'inflammationspériphériques pour les lymphocytes T effecteurs mémoires (TEM). Dans les deuxcas, cette expression survient en fin d'activation et pourrait servir de frein à uneactivation incontrôlée des lymphocytes T.D'autre part, nous avons abordé le rôle du récepteur alpha à l'IL-2 (CD25)dans l'homéostasie des lymphocytes T. L'étude chez la souris il2ra-/- a révélé un rôleimportant du CD25 pour la survie des Treg in vivo, mais aussi pour l'acquisition delymphocytes T mémoires. Seulement deux cas de déficience en CD25, associés àdes maladies auto-immunes, ont été décrits chez l'homme. Cependant, ces étudesn'ont pas abordé à quel niveau le CD25 intervient sur l'homéostasie des lymphocytesT. Nous complétons ces études par la présentation de trois nouveaux cas dedéficience en CD25 développant des maladies auto-immunes de type IPEX. Nousmontrons que le CD25 intervient activement dans le maintien des populations Tregnaïves et effectrices, mais aussi dans celui des populations lymphocytaireseffectrices mémoires.

Introdução: O adenocarcinoma pancreático é responsável pela maioria das neoplasias pancreáticas e está associado a um prognóstico extremamente pobre tanto devido à alta taxa de diagnósticos em estágio avançado quanto ao elevado índice de recidiva mesmo nos pacientes submetidos à ressecção com intenção curativa. Uma ferramenta que possa predizer adequadamente o prognóstico da doença é fundamental para uma melhor estratificação de risco. Evidências tem mostrado que a resposta inflamatória sistêmica está associada ao prognóstico de diversos tipos de câncer, sendo que a razão neutrófilos/linfócitos (NLR) e suas adaptações e a razão plaquetas/linfócitos (PLR) tem se mostrado promissores para este fim. Objetivo: O objetivo do presente estudo é avaliar o valor prognóstico das razões NLR, NLR derivada (dNLR) e PLR determinados por exames coletados no momento da internação e após tratamento quimioterápico paliativo numa população de pacientes com diagnóstico de adenocarcinoma pancreático, analisando ainda qual o valor de ponto de corte mais adequado para cada parâmetro. Além disso, pretendemos investigar se essas razões podem ter algum valor como fator preditivo de ressecabilidade no adenocarcinoma pancreático. Métodos: Foram coletados dados de pacientes com diagnóstico de adenocarcinoma pancreático confirmado por exame histopatológico atendidos no Hospital de Clínicas de Porto Alegre entre 2003 e 2013. As razões estudadas foram determinadas com base nos hemogramas coletados na internação dos pacientes e após dois ciclos de quimioterapia naqueles que foram submetidos a tratamento paliativo. Resultados: Na análise combinada de todos os pacientes incluídos no estudo, NLR basal, dNLR basal e PLR basal não mostraram evidência de ter impacto prognóstico na sobrevida (P= 0,394, P= 0,152, P= 0,177 respectivamente). No subgrupo de pacientes submetidos a quimioterapia paliativa, NLR, dNLR e PLR calculados pelos exames realizados após 2 ciclos de tratamento mostraram-se fatores prognósticos para sobrevida global (P=0,003, P=0,009 e P=0,001 respectivamente). Os pontos de corte mais adequados encontrados foram 4,11 para NLR (sensibilidade 83% e especificidade 75%), 362 para PLR (sensibilidade 91% e especificidade 62,5%) e 2,8 para dNLR (sensibilidade 87% e especificidade 62,5%). Nenhuma das razões se mostrou estatisticamente significativa como preditor para ressecabilidade (NLR, P=0,88; dNLR, P=0,99; PLR, P=0,64). Conclusões: As razões NLR, dNLR e PLR são úteis como marcadores prognósticos de sobrevida global em pacientes com adenocarcinoma pancreático submetidos a quimioterapia paliativa. Seu uso como preditor de ressecabilidade das lesões pancreáticas não foi demonstrado. / Background: Pancreatic adenocarcinoma is responsible for most of the pancreatic neoplasias and it is associated to an extremely poor prognosis due to diagnosis in advanced stage and the recurrence even among patients treated with curative intention. A prognostic tool is essential for a better risk stratification. Evidence has shown that systemic inflammatory response is associated to the prognosis of a variety of cancers and the neutrophil/lymphocyte ratio (NLR) and adaptations and the platelet/lymphocyte (PLR) ratio seem promising for this purpose. Objetive: The objective of this study is to evaluate the prognostic value of NLR, derived NLR (dNLR) and PLR determined by blood counts collected at hospital admission and after palliative chemotherapy in patients with pancreatic adenocarcinoma, analyzing the ideal cutoff value for each parameter. Also, we intend to investigate if those ratios have some role in predicting the resectability of pancreatic adenocarcinoma. Methods: Data were collected of patients who had diagnosis of pancreatic adenocarcinoma confirmed by histopathologic exam in Hospital de Clínicas de Porto Alegre between 2003 and 2013. The studied ratios were determined by blood counts collected at hospital admission and after two cycles of chemotherapy in patients submitted to palliative treatment. Results: In the combined analysis including all patients, basal NLR, dNLR and PLR did not have prognostic impact in overall survival (P=0,394, P=0,152, P=0,177 respectively). In subgroup analysis of patients submitted to palliative chemotherapy, NLR, dNLR and PLR determined by blood count collected after two cycles of chemotherapy were prognostic for overall survival (P=0,003, P=0,009, P=0,001 respectively). The ideal cutoff values found were 4,11 for NLR (sensibility 83%, specificity 75%), 2,8 for dNLR (sensibility 87%, specificity 62,5%) and 362 for PLR (sensibility 91%, specificity 62,5%). None of these ratios has shown to be able to predict resectability (NLR, P=0,88; dNLR, P=0,99; PLR, P=0,64). Conclusions: NLR, dNLR and PLR are useful as prognostic markers of overall survival in patients with pancreatic adenocarcinoma submitted to palliative chemotherapy. Its use as resectability predictor could not be demonstrated.

Introdução: O adenocarcinoma pancreático é responsável pela maioria das neoplasias pancreáticas e está associado a um prognóstico extremamente pobre tanto devido à alta taxa de diagnósticos em estágio avançado quanto ao elevado índice de recidiva mesmo nos pacientes submetidos à ressecção com intenção curativa. Uma ferramenta que possa predizer adequadamente o prognóstico da doença é fundamental para uma melhor estratificação de risco. Evidências tem mostrado que a resposta inflamatória sistêmica está associada ao prognóstico de diversos tipos de câncer, sendo que a razão neutrófilos/linfócitos (NLR) e suas adaptações e a razão plaquetas/linfócitos (PLR) tem se mostrado promissores para este fim. Objetivo: O objetivo do presente estudo é avaliar o valor prognóstico das razões NLR, NLR derivada (dNLR) e PLR determinados por exames coletados no momento da internação e após tratamento quimioterápico paliativo numa população de pacientes com diagnóstico de adenocarcinoma pancreático, analisando ainda qual o valor de ponto de corte mais adequado para cada parâmetro. Além disso, pretendemos investigar se essas razões podem ter algum valor como fator preditivo de ressecabilidade no adenocarcinoma pancreático. Métodos: Foram coletados dados de pacientes com diagnóstico de adenocarcinoma pancreático confirmado por exame histopatológico atendidos no Hospital de Clínicas de Porto Alegre entre 2003 e 2013. As razões estudadas foram determinadas com base nos hemogramas coletados na internação dos pacientes e após dois ciclos de quimioterapia naqueles que foram submetidos a tratamento paliativo. Resultados: Na análise combinada de todos os pacientes incluídos no estudo, NLR basal, dNLR basal e PLR basal não mostraram evidência de ter impacto prognóstico na sobrevida (P= 0,394, P= 0,152, P= 0,177 respectivamente). No subgrupo de pacientes submetidos a quimioterapia paliativa, NLR, dNLR e PLR calculados pelos exames realizados após 2 ciclos de tratamento mostraram-se fatores prognósticos para sobrevida global (P=0,003, P=0,009 e P=0,001 respectivamente). Os pontos de corte mais adequados encontrados foram 4,11 para NLR (sensibilidade 83% e especificidade 75%), 362 para PLR (sensibilidade 91% e especificidade 62,5%) e 2,8 para dNLR (sensibilidade 87% e especificidade 62,5%). Nenhuma das razões se mostrou estatisticamente significativa como preditor para ressecabilidade (NLR, P=0,88; dNLR, P=0,99; PLR, P=0,64). Conclusões: As razões NLR, dNLR e PLR são úteis como marcadores prognósticos de sobrevida global em pacientes com adenocarcinoma pancreático submetidos a quimioterapia paliativa. Seu uso como preditor de ressecabilidade das lesões pancreáticas não foi demonstrado. / Background: Pancreatic adenocarcinoma is responsible for most of the pancreatic neoplasias and it is associated to an extremely poor prognosis due to diagnosis in advanced stage and the recurrence even among patients treated with curative intention. A prognostic tool is essential for a better risk stratification. Evidence has shown that systemic inflammatory response is associated to the prognosis of a variety of cancers and the neutrophil/lymphocyte ratio (NLR) and adaptations and the platelet/lymphocyte (PLR) ratio seem promising for this purpose. Objetive: The objective of this study is to evaluate the prognostic value of NLR, derived NLR (dNLR) and PLR determined by blood counts collected at hospital admission and after palliative chemotherapy in patients with pancreatic adenocarcinoma, analyzing the ideal cutoff value for each parameter. Also, we intend to investigate if those ratios have some role in predicting the resectability of pancreatic adenocarcinoma. Methods: Data were collected of patients who had diagnosis of pancreatic adenocarcinoma confirmed by histopathologic exam in Hospital de Clínicas de Porto Alegre between 2003 and 2013. The studied ratios were determined by blood counts collected at hospital admission and after two cycles of chemotherapy in patients submitted to palliative treatment. Results: In the combined analysis including all patients, basal NLR, dNLR and PLR did not have prognostic impact in overall survival (P=0,394, P=0,152, P=0,177 respectively). In subgroup analysis of patients submitted to palliative chemotherapy, NLR, dNLR and PLR determined by blood count collected after two cycles of chemotherapy were prognostic for overall survival (P=0,003, P=0,009, P=0,001 respectively). The ideal cutoff values found were 4,11 for NLR (sensibility 83%, specificity 75%), 2,8 for dNLR (sensibility 87%, specificity 62,5%) and 362 for PLR (sensibility 91%, specificity 62,5%). None of these ratios has shown to be able to predict resectability (NLR, P=0,88; dNLR, P=0,99; PLR, P=0,64). Conclusions: NLR, dNLR and PLR are useful as prognostic markers of overall survival in patients with pancreatic adenocarcinoma submitted to palliative chemotherapy. Its use as resectability predictor could not be demonstrated.

Introdução: O adenocarcinoma pancreático é responsável pela maioria das neoplasias pancreáticas e está associado a um prognóstico extremamente pobre tanto devido à alta taxa de diagnósticos em estágio avançado quanto ao elevado índice de recidiva mesmo nos pacientes submetidos à ressecção com intenção curativa. Uma ferramenta que possa predizer adequadamente o prognóstico da doença é fundamental para uma melhor estratificação de risco. Evidências tem mostrado que a resposta inflamatória sistêmica está associada ao prognóstico de diversos tipos de câncer, sendo que a razão neutrófilos/linfócitos (NLR) e suas adaptações e a razão plaquetas/linfócitos (PLR) tem se mostrado promissores para este fim. Objetivo: O objetivo do presente estudo é avaliar o valor prognóstico das razões NLR, NLR derivada (dNLR) e PLR determinados por exames coletados no momento da internação e após tratamento quimioterápico paliativo numa população de pacientes com diagnóstico de adenocarcinoma pancreático, analisando ainda qual o valor de ponto de corte mais adequado para cada parâmetro. Além disso, pretendemos investigar se essas razões podem ter algum valor como fator preditivo de ressecabilidade no adenocarcinoma pancreático. Métodos: Foram coletados dados de pacientes com diagnóstico de adenocarcinoma pancreático confirmado por exame histopatológico atendidos no Hospital de Clínicas de Porto Alegre entre 2003 e 2013. As razões estudadas foram determinadas com base nos hemogramas coletados na internação dos pacientes e após dois ciclos de quimioterapia naqueles que foram submetidos a tratamento paliativo. Resultados: Na análise combinada de todos os pacientes incluídos no estudo, NLR basal, dNLR basal e PLR basal não mostraram evidência de ter impacto prognóstico na sobrevida (P= 0,394, P= 0,152, P= 0,177 respectivamente). No subgrupo de pacientes submetidos a quimioterapia paliativa, NLR, dNLR e PLR calculados pelos exames realizados após 2 ciclos de tratamento mostraram-se fatores prognósticos para sobrevida global (P=0,003, P=0,009 e P=0,001 respectivamente). Os pontos de corte mais adequados encontrados foram 4,11 para NLR (sensibilidade 83% e especificidade 75%), 362 para PLR (sensibilidade 91% e especificidade 62,5%) e 2,8 para dNLR (sensibilidade 87% e especificidade 62,5%). Nenhuma das razões se mostrou estatisticamente significativa como preditor para ressecabilidade (NLR, P=0,88; dNLR, P=0,99; PLR, P=0,64). Conclusões: As razões NLR, dNLR e PLR são úteis como marcadores prognósticos de sobrevida global em pacientes com adenocarcinoma pancreático submetidos a quimioterapia paliativa. Seu uso como preditor de ressecabilidade das lesões pancreáticas não foi demonstrado. / Background: Pancreatic adenocarcinoma is responsible for most of the pancreatic neoplasias and it is associated to an extremely poor prognosis due to diagnosis in advanced stage and the recurrence even among patients treated with curative intention. A prognostic tool is essential for a better risk stratification. Evidence has shown that systemic inflammatory response is associated to the prognosis of a variety of cancers and the neutrophil/lymphocyte ratio (NLR) and adaptations and the platelet/lymphocyte (PLR) ratio seem promising for this purpose. Objetive: The objective of this study is to evaluate the prognostic value of NLR, derived NLR (dNLR) and PLR determined by blood counts collected at hospital admission and after palliative chemotherapy in patients with pancreatic adenocarcinoma, analyzing the ideal cutoff value for each parameter. Also, we intend to investigate if those ratios have some role in predicting the resectability of pancreatic adenocarcinoma. Methods: Data were collected of patients who had diagnosis of pancreatic adenocarcinoma confirmed by histopathologic exam in Hospital de Clínicas de Porto Alegre between 2003 and 2013. The studied ratios were determined by blood counts collected at hospital admission and after two cycles of chemotherapy in patients submitted to palliative treatment. Results: In the combined analysis including all patients, basal NLR, dNLR and PLR did not have prognostic impact in overall survival (P=0,394, P=0,152, P=0,177 respectively). In subgroup analysis of patients submitted to palliative chemotherapy, NLR, dNLR and PLR determined by blood count collected after two cycles of chemotherapy were prognostic for overall survival (P=0,003, P=0,009, P=0,001 respectively). The ideal cutoff values found were 4,11 for NLR (sensibility 83%, specificity 75%), 2,8 for dNLR (sensibility 87%, specificity 62,5%) and 362 for PLR (sensibility 91%, specificity 62,5%). None of these ratios has shown to be able to predict resectability (NLR, P=0,88; dNLR, P=0,99; PLR, P=0,64). Conclusions: NLR, dNLR and PLR are useful as prognostic markers of overall survival in patients with pancreatic adenocarcinoma submitted to palliative chemotherapy. Its use as resectability predictor could not be demonstrated.

L’infection par le VIH-1 est un problème majeur de la santé publique qui touche plus de 35 millions de personnes à l’échelle mondiale. La réplication du VIH-1 est déclenchée par l’activation du promoteur LTR, qui contient deux sites de liaison pour le facteur de transcription NF-κB. Ces sites de liaison sont hautement conservés dans le génome du VIH-1, illustrant ainsi l’importance de NF-κB dans l’activité transcriptionelle des gènes du VIH-1 et la production de nouvelles particules virales. La withaferin A (WA) est une substance bioactive extraite de la plante Withania somnifera, qui possède des propriétés pharmacologiques non négligeables dans la régulation de la réponse immunitaire. Des études récentes ont démontré que le potentiel anti-inflammatoire de la WA est dû principalement à l’inhibition de la voie de NF-κB. Le but de ce projet est de déterminer l’effet de la WA sur la réplication du VIH-1 dans les cellules T, qui sont les cibles principales du virus. Des essais de transfections transitoires de cellules T Jurkat E6.1 avec des plasmides contenant le promoteur du VIH-1 ayant différentes constructions de NF-κB, ont démontré que la WA peut réduire l’activité du promoteur d’une manière dépendante de NF-κB. Quant à la production de particules virales, des essais d’infection avec des virus pseudotypés démontrent que la WA diminue la production virale jusqu’à 90% dans des cellules T stimulées avec PMA/PHA et TNF-α, tandis que les mutants ayant des sites de liaison défective pour NF-κB ne sont pas affectés. Des essais de retardement sur gel ainsi que des immunobuvardages de type Western ont montré que la WA altère l’habilité de NF-κB à transloquer dans le noyau, ce qui se traduit par l’inhibition de la synthèse de l’IκB-α, protéine inhibitrice de NF-κB, phénomène sous contrôle étroite de ce facteur de transcription. Ces résultats suggèrent que la WA pourrait permettre une diminution de la réplication virale d’une manière dépendante de NF-κB et ainsi empêcher la propagation du virus aux cellules T chez les individus infectés.

No description available.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Le développement de nouvelles stratégies d’immunothérapie représente de nos jours un enjeu majeur de santé publique. Dans ce contexte, les lymphocytes T Natural Killer invariant (iNKT) exercent de puissantes activités immuno-modulatrices. Ces cellules ont la particularité de reconnaitre par l’intermédiaire de leur récepteur T (TCR), des (glyco) lipides, présentés par la molécule CD1d exprimée par les cellules présentatrices d’antigènes (APC), notamment les cellules dendritiques (DC). Initialement découvert à partir d’une éponge marine pour ses activités anti-métastatiques, l’alpha-galactosylcéramide (a-GalCer ou KRN) induit rapidement une sécrétion massive, par les cellules iNKT, de cytokines immunomodulatrices telles l’IFN-g, conduisant à la transactivation de nombreuses cellules immunitaires, notamment les cellules Natural Killer (NK), les DC ou encore les lymphocytes Tgd. Cette propriété unique permet aux cellules iNKT de contrôler, chez la souris, le développement des réponses immunes, notamment la réponse anti-tumorale. Face à ces résultats encourageants, des essais cliniques chez l’homme ont été réalisés mais les résultats se sont avérés décevants. Actuellement, il existe deux stratégies pour cibler un antigène à une population cellulaire particulière : 1) le ciblage passif, basé sur la taille des particules ou leur composition et 2) le ciblage actif, basé sur les fortes interactions anticorps/antigène ou ligand/récepteur. Nous avons testé les deux types de ciblages et avons utilisé le même polymère pour constituer nos vecteurs : le PLGA ou poly(lactic coglycolic acid). Dans une première étude (ciblage passif), nous avons comparé l’efficacité de l’encapsulation de l’a-GalCer dans des particules de tailles différentes: des nano (NP) et microparticules (MP). L’a-GalCer vectorisé dans les NP et les MP est endocyté par les DC (voie des clathrines) et active les cellules iNKT in vitro et in vivo mais ne peuvent empêcher leur anergie. Ces résultats décevants nous ont conduits à opter pour le ciblage actif des DC. Dans un premier temps, étant donné les études controversées sur le rôle des DC dans l’anergie des cellules iNKT, nous avons revisité l’implication de ces dernières dans la primo-activation/anergie des cellules iNKT. Nous confirmons ainsi le rôle primordial de DC dans la primo-activation des cellules iNKT mais surtout, nous montrons qu’elles n’induisent pas leur anergie. Représentant une population hétérogène, nous montrons que parmi les DC, les DC CD8a+ sont de puissantes activatrices des cellules iNKT. Nos résultats nous ont ainsi menés à délivrer spécifiquement l’a-GalCer aux DC CD8a+. Pour cela, nous avons greffé sur les NP de PLGA un anticorps anti-DEC205, récepteur lectinique fortement exprimé par les DC CD8a+. In vitro et in vivo, les NP/DEC205/a-GalCer induisent une plus forte activation des cellules iNKT comparativement à l’a-GalCer libre. De même, la co-délivrance d’a-GalCer et d’ovalbumine (OVA) au sein des DC CD8a+ améliore les propriétés adjuvantes de l’a-GalCer en induisant des réponses humorale et cellulaire (lymphocytes T CD8+) spécifiques de l’OVA plus importantes comparativement à la délivrance des deux composés sous leur forme libre. Finalement, de façon intéressante, nous montrons que suite à une primo-activation par les NP/DEC205/a-GalCer, les cellules iNKT sont capables de répondre de nouveau à une seconde stimulation, traduisant l’absence d’anergie des cellules iNKT. En conclusion, nos résultats indiquent que la délivrance spécifique de l’a-GalCer aux DC CD8a+ amplifie la primo-activation des cellules iNKT tout en évitant la mise en place du phénomène d’anergie et ouvrent de nombreuses perspectives dans le cadre de thérapies anti-tumorales et anti-infectieuses. / Nowdays, the development of new immunotherapy strategies represent a major issuein public health. In this context, the invariant Natural Killer T lymphocytes (iNKT) have strongimmunomodulatory properties. This cell population recognizes (glycol)lipid presented by theCD1d molecule expressed by antigen presenting cell (APC) as dendritic cells (DC). Initiallyfound in a marine sponge for its anti-metastatic activities, alpha-galactosylceramide (-GalCer or KRN) induces a massive cytokine production (IFN-, IL-4, IL-17) by iNKT cells.This cytokine burst lead to downstream activation of numerous immune cells like naturalkiller cell (NK), DC or CD8+ T cells. Through this property, iNKT cells regulate numerousimmune responses, including anti-tumoral response. Based on encouraging results in themouse model, -GalCer has been used in anti-tumour therapy in human. Although the drugwas well tolerated, no or moderate clinical responses were observed in patients repeatedlyinoculated with -GalCer. As observed in the mouse system, one potential explanation forthis disappointing observation may lie in the induction of a long-term anergy of human iNKTcells, thus preventing cytokine release upon a recall stimulation. Although controversial,various studies suggest that this phenomonen should be due to a lack of delivery of -GalCer into dendritic cells (DC) and so its presentation by non adequate antigen presentingcell (APC) as B cells. The objective of our work was to optimize -GalCer activity by avoidingiNKT anergy using vectorisation approach. Actually, there are 2 strategies using nanotechnologies to target an antigen to a specific cell population: 1) passive targeting based on the size of the particles, their composition and their surface charge and 2) active targeting based on the strong interactions between an antibody and its antigen or a ligand and its receptor. We have tested the two strategies and therefore we use the same composition of our particles: PLGA or poly(lactic co glycolic acid). This biodegradable and biocompatible molecule is already used in therapy.In a first study (passive targeting), we compared the efficiency of -GalCer encapsulation inparticles with different size: nano (NP) and microparticle (MP). Vectorised -GalCer in NPand MP rapidly activates iNKT cells in vitro. Both type of particles are uptake by DC via aclathrin dependent mechanism. In in vivo approaches, NP/-GalCer and MP/-GalCeractivate iNKT cells but unfortunately could not prevent iNKT cell anergy. These disappointingresults led us to use an active targeting. In first time, because of controversial role of DC iniNKT anergy, we have revisited the role of DC in iNKT primo-activation and anergy. Weconfirm the primordial role of DC in iNKT primo-activation but especially we show that DC donot induce iNKT anergy. DC are heterogeneous and we show that among DC, CD8a+ DCsubpopulation are potent iNKT cells activation. Our results led us to deliver specifically a-GalCer to CD8+ DC. For this, anti-DEC205 antibodies were covalently linked to the surfaceof PLGA NP, DEC205 being highly expressed by CD8+ DC. In vitro and in vivo,NP/DEC205/-GalCer induce a stronger iNKT cell activation relative to free -GalCer (or NPIgG/-GalCer). Moreover, -GalCer and ovalbumin co-delivery in CD8+ DC improve -GalCer adjuvanticity leading to more important humoral and cellular responses. Interestingly,after a primo-activation by NP/DEC205/-GalCer, iNKT cells are able to respond to secondstimulation thus avoiding iNKT cell anergy.In conclusion, our results indicate that specific -GalCer delivery to CD8+ DC improve iNKT cells primo-activation and avoid anergy phenomenon. These findings open several perspectives in anti-tumoral and anti-infectious therapies.

Thesis advisor: Thomas C. Chiles / B-lymphocytes respond to environmental cues for their survival, growth, and differentiation through receptor-mediated signaling pathways. Naïve Blymphocytes must acquire and metabolize external glucose in order to support the bioenergetics associated with maintaining cell volume, ion gradients, and basal macromolecular synthesis. The up-regulation of glycolytic enzyme expression and activity via engaged B-cell receptor mediated-events was glucose-dependent. This suggests an essential role for glucose energy metabolism in the promotion of B cell growth, survival, and proliferation in response to extracellular stimuli. In addition, the activity of ATP-citrate lyase (ACL) was determined to be crucial for ex vivo splenic B cell differentiation to antibody-producing cells wherein B cells undergo endomembrane synthesis and expansion. This investigation employed knockout murine models as well as chemical inhibitors to determine the signaling components and enzymes responsible for glucose utilization and incorporation into membrane lipids. These results point to a critical role for phosphatidylinositol 3- kinase (PI3K) in orchestrating cellular glucose energy metabolism and glucosedependent de novo lipogenesis for B lymphocyte responses. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Welkin Johnson / Thesis advisor: Thomas Chiles / B cell activation is an energetically demanding process during which B lymphocytes undergo reprogramming and shift from a resting state to a highly proliferative, metabolically active state. Little is known about the metabolic reprogramming process or the role extracellular nutrients play in the activation response. Here we demonstrate that there are distinct requirements for the nutrients L-glutamine and glucose during activation. We show that cells activated in glucose-depleted conditions are still able to undergo growth and signaling events. In contrast, we show that extracellular L-glutamine is essential for all but the earliest activation events, and cells cultured in L-glutamine-deprived conditions are unable to enter the cell cycle. Consistently, we show that extracellular supplementation of the cell-permeable derivative of α-ketoglutarate (α-KG), a glutaminolytic product, is able to rescue cell activation in the absence of glutamine. We also show the induction of the high affinity amino acid transporter ASCT2 is required for glutamine uptake following B cell receptor (BCR) crosslinking. Specifically, we found that halting glutamine uptake or processing by inhibiting ASCT2 or the glutaminolytic enzyme glutaminase causes activation defects that parallel those observed in glutamine deprived conditions, indicating a requirement for glutaminolysis during the very early stages of activation. We found that -KG does not contribute to epigenetic remodeling, but is necessary for mammalian target of rapamycin complex 1 (mTORC1) activation. In turn, mTORC1 activity is required for upregulation of the glucose transporter Glut1 during the initial 24 hours of activation, as well as increased glucose uptake. These findings indicate a distinct metabolic profile that begins with glutamine uptake, and acts through mTORC1 signaling to later promote glucose uptake. Finally, we show that nutrients contribute to functional differentiation events during B cell activation. Glucose is required to support biogenesis of the endoplasmic reticulum as well as differentiation into plasma-like cells, while glutamine is required to support differentiation into IL-10 secreting regulatory B cell subsets. The requirement for glutamine for in vitro B10 cell differentiation is the first reported link between nutrient signaling and regulatory B cell development, and is a novel finding in the field. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.