La différenciation terminale des lymphocytes B qui se déroule dans les centres germinatifs des organes lymphoïdes secondaires est l’étape ultime de la réponse T dépendante et aboutit à la production de plasmocytes (PC) à longue durée de vie qui sécrètent des anticorps hautement affins spécifiques de l’antigène et caractéristiques de la réponse immune adaptative. La transition d’une cellule B naïve vers un PC est gouvernée par un réseau de régulation génique bien décrit et est largement influencée par l’intégration de stimuli externes qui contrôlent le devenir des cellules B tels que l’interaction BCR-antigène et les cytokines produites par les cellules T. La stimulation précoce des lymphocytes B humains activés par IL-2, induit la différenciation en PC via une signalisation ERK prolongée entraînant la baisse d’expression de BACH2, un facteur de transcription clef des cellules B. La répression transitoire de BACH2 est suffisante pour déclencher la différenciation en plasmablastes en l’absence d’IL-2, suggérant ainsi qu’il joue un rôle de « verrou moléculaire » de la différenciation en PC. Il est à noter que cette répression forcée de BACH2 aboutit à la production de plasmablastes caractérisés par un phénotype lymphoplasmocytaire. Ce travail de recherche s’est focalisé sur la caractérisation des mécanismes moléculaires régulant l’expression de BACH2 via la voie de signalisation ERK induite par IL-2. Nous avons identifié ELK-1 comme un médiateur de la répression de BACH2 par la voie IL-2/ERK, comme l’atteste sa capacité à se lier avec un élément de régulation d’un enhancer localisé dans l’intron 1 de BACH2, induisant ainsi la répression de l’enhancer et déverrouillant la différenciation en PC. La caractérisation de cet enhancer de BACH2 a confirmé qu’il est régulé de manière dynamique au cours de la différenciation terminale B et qu’il est localisé dans une région sujette aux mutations suggérant qu’il pourrait être impliqué dans la lymphomagenèse. / The terminal differentiation of B cells, which takes places within germinal centres of secondary lymphoid organs, is the ultimate step of a T cell dependent response and results in the generation of long-lived plasma cells (PCs) that secrete protective, antigen-specific, high-affinity antibodies as part of adaptive immunity. The transition of a naive B cell into a PC is governed by a well-characterised gene regulatory network and is heavily influenced by the integration of externally received signals, including BCR-antigen binding and T cell help, such as cytokines which guide B cell fate. The early IL-2 priming of human primary activated B cells triggers PC differentiation through sustained ERK signalling resulting in the down regulation of B cell transcription factor BACH2. Transient BACH2 repression is sufficient to trigger plasmablast differentiation in the absence of IL-2 suggesting that it acts as a key lock of PC differentiation. Importantly, this enforced BACH2 repression results in the generation of plasmablasts with a lymphoplasmacytic phenotype. The focus of this thesis was to characterise the molecular mechanisms regulating BACH2 expression via the IL-2 ERK transduction pathway. We identify ELK-1 as the mediator of IL-2 ERK induced BACH2 downregulation as it binds to a regulatory enhancer element located within intron 1 of BACH2 instigating its repression and unlocking the PC programme triggering differentiation. The characterisation of this BACH2 enhancer confirms that it is dynamically regulated during PC differentiation and is located within a region targeted for mutation suggesting that it may have a potential role in lymphomagenesis.

A cavidade peritoneal de camundongos abriga uma variedade de células do sistema imune. Inicialmente, devido as limitações metodológicas, acreditava-se que, aproximadamente, 90% das células peritoneais era representada por macrófagos. Em seguida, graças aos extensos estudos com células peritoneais, observou-se que, além dos macrófagos, o peritônio abrigava muitos linfócitos B, principalmente do subtipo B-1. Utilizando metodologias contemporâneas de FACS - citometria de fluxo, este trabalho mostra que, aproximadamente, 30% das células peritoneais são macrófagos, 55% são linfócitos B-1, dos quais 40% pertencem ao subtipo B-1a e 15% ao subtipo B-1b. Os 15%-20% restantes representam outros subtipos celulares, como linfócitos T, linfócitos B-2, linfócitos NK, eosinófilos, além da presença de outras populações de células que não foram possíveis de ser identificadas com os marcadores de superfície utilizados neste trabalho. Em contraste com a literatura, nossos estudos mostraram que os macrófagos da cavidade peritoneal de camundongos representam uma população heterogênea. Por meio da co-expressão de CD11b e F4/80, foi possível descrever dois subtipos funcionais de macrófagos, denominados aqui de PM-1 (CD11bhigh, F4/80high) e PM-2 (F4/8010W, CD11bint). As células PM-2 encontram-se em menor abundância (-10% do total de macrófagos) no peritônio não estimulado e, diferentemente de células PM-1, espraiam-se de forma alongada (bipolar) quando colocadas em cultura. Curiosamente, PM-2 são os únicos macrófagos peritoneais que expressam MHC-II e, ainda, 30% dessa população expressa CD11c, o marcador típico de células dendríticas. Já as células PM-1 possuem morfologia arredondada, estão em abundância no peritônio e produzem altas doses de NO após estímulo com LPS. Ambas as células são capazes de fagocitar bactérias in vivo, no entanto, as células PM-2 parecem mais eficientes, sendo capazes de internalizar quantidades maiores de bactérias. Após infecção in vivo, o número absoluto e a porcentagem de PM-2 aumenta muito no peritônio, chegando a representar metade dos fagócitos da cavidade peritoneal. Os macrófagos PM-1 e PM-2 parecem não representar monócitos já que não compartilham do fenótipo de monócitos presentes no sangue periférico. Deve-se ressaltar que, além das células PM-1 e PM-2, outras subpopulações do peritônio também expressam CD11b e F4/80. Com base na literatura atual, acredita-se que todas as células B-1 do peritônio expressam CD11b, uma molécula que é co-expressa com CD18 para formar o receptor de complemento e a molécula de adesão Mac-1. Entretanto, os estudos, apresentados neste trabalho, mostram que metade de cada subtipo de B-1 (B-1a e B-1b) não expressa CD11b. As células B-1 CD11b+ são maiores, mais granulosas e expressam quantidades maiores de IgM de superfície. As células CD11b+ possuem uma curiosa tendência de se juntar e formar duplas (doublets) fortemente ligadas entre si que estão presentes em abundância no peritônio de camundongos adultos. Além de ligarem entre si, as células B-1 podem formar duplas com macrófagos PM-1 e PM-2. Durante a ontogenia, as células CD11b- surgem primeiro no peritônio e são as progenitoras das células B-1 CD11b+. Após estímulo inflamatório (LPS í. v.) os linfócitos B-1 CD11b+ migram do peritônio para o baço, onde proliferam e transformam-se em células secretoras de anticorpos (plasmócitos). No peritônio, as células B-1 não são capazes de se diferenciar em plasmócitos (células CD138high). Os resultados apresentados aqui mostraram que, diferentemente dos macrófagos PM-2, os PM-1 são os responsáveis por inibir a formação de células secretoras de anticorpos derivadas de B-1. Em suma, nossos dados sugerem que, mediante estímulo inflamatório, as células B-1 maduras (CD11b+) migram do peritônio para o baço, afastandose de macrófagos PM-1. Já no baço, as células B-1 encontram um micro-ambiente favorável para se proliferarem e diferenciarem-se em plasmócitos, secretando a maioria dos anticorpos naturais vistos logo nas primeiras horas pós-estímulo. / Mouse peritoneal cavity (PerC) represents the source for a variety of cellular subsets of the immune system. In early studies, marred by Iimited methodological tools, it was thought that macrophages comprise roughly 90% of total PerC cells. Shortly thereafter, it was recognized that, beside macrophages, the mouse peritoneal cavity shelters large amount of B-lymphocytes, specially the B-1 subset. In essence, by applying contemporary technology, studies presented here show that, roughly 30% of PerC cells comprise macrophages, 55% comprise B-1 cells, which 40% represents the B-1 a subset and roughly 15% are B-1b cells. The remaining 15%-20% of PerC cells comprise a variety of known immune cells, including B-2, T, NK and eosinophils. In addition, there were some other cellular subsets that could not be identified in these studies, probably due to limited cell surface molecules analyzed. Surprisingly, the data presented here show that peritoneal macrophages represent a highly heterogeneous population. Based on the co-expression of both CD11b and F4/80, we have identified two functionally distinct subsets of peritoneal macrophages, named here as PM-1 (CD11bhigh, F480high) and PM-2 (CD11bint, F4/80low). PM-2 are less abundant (-10% within total macrophages) in the PerC of unstimulated mouse, however, unlike PM-1, these cells are able to spread in spindle (bipolar) morphology upon sorting and in vitro culture. Curiously, PM-2 is the only PerC macrophage that expresses surface MHC-II. In addition, one third of PM-2 population expresses CD11c, a universal marker for dendritic cells. In turn, PM-1 cells are round shaped cells, represent the majority of PerC macrophages (-90%) and secrete high amounts of NO upon LPS stimulation. In vivo phagocytosis assay revealed that both cells could internalize bacteria, however, PM-2 cells showed to be more efficient, in that it was able to phagocyte higher amounts of bacteria when compared to PM-1. After i.p. in vivo stimulation, the absolute number and percentage of PM-2 cells increase drastically in the peritoneum, reaching almost half of the total PerC macrophages. Importantly, PM-1 and PM-2 macrophages seem not to represent PerC monocytes since it does not share any of the phenotype expressed by blood monocytes. Interestingly, in addition to PM-1 and PM-2, some other cellular subsets in the PerC, such as eosinophils, are able to express CD11b and F4/80. PerC B-1 cells have long been known to express CD11b, which is co-expressed with CD18 to form the Mac-1/CR3 complement receptor and adhesion molecule. However, although all PerC B-1 cells are commonly believed to express CD11b, we show here that nearly half of the cells in each of the PerC B-1 subsets (B-1a and B-1b) do not express this surface receptor. The CD11b+ cells in each B-1 subset are larger, more granular and express higher levels of surface IgM than the CD11b- B-1 cells. Surprisingly, the CD11b+ B-1 subset has the curious tendency to initiate the formation of tightly associated doublets that are present at high frequency in adult PerC. In addition to binding to each other, B-1 cells can also form doublets with PM-1 and PM-2. During ontogeny, CD11b- B-1 cells arise earlier in the mouse PerC and are the progenitors of CD11b+ B-1 cells. Upon LPS stimulation, PerC CD11b+ B-1 cells migrate to the spleen where they proliferate and differentiate into antibody-secreting cells (plasma cells). Within the PerC, B-1 cells are not able to differentiate into plasma cells (CD138high cells). The data shown in here reveal that, unlike PM-2, PM-1 macrophages are the cellular subset responsible for inhibiting the formation of B-1-derived antibody-secreting cells. Altogether, our data suggest that, upon inflammatory stimuli, mature CD11b+ B-1 cells migrate from the PerC to the spleen, avoiding the inhibitory effect of PM-1 cells. In the spleen, B-1 cells find an appropriate environment to proliferate and terminally differentiate into antibody-secreting cells, thus, secreting the majority of immunoglobulin seen in few hours after in vivo stimulation.

A cavidade peritoneal de camundongos abriga uma variedade de células do sistema imune. Inicialmente, devido as limitações metodológicas, acreditava-se que, aproximadamente, 90% das células peritoneais era representada por macrófagos. Em seguida, graças aos extensos estudos com células peritoneais, observou-se que, além dos macrófagos, o peritônio abrigava muitos linfócitos B, principalmente do subtipo B-1. Utilizando metodologias contemporâneas de FACS - citometria de fluxo, este trabalho mostra que, aproximadamente, 30% das células peritoneais são macrófagos, 55% são linfócitos B-1, dos quais 40% pertencem ao subtipo B-1a e 15% ao subtipo B-1b. Os 15%-20% restantes representam outros subtipos celulares, como linfócitos T, linfócitos B-2, linfócitos NK, eosinófilos, além da presença de outras populações de células que não foram possíveis de ser identificadas com os marcadores de superfície utilizados neste trabalho. Em contraste com a literatura, nossos estudos mostraram que os macrófagos da cavidade peritoneal de camundongos representam uma população heterogênea. Por meio da co-expressão de CD11b e F4/80, foi possível descrever dois subtipos funcionais de macrófagos, denominados aqui de PM-1 (CD11bhigh, F4/80high) e PM-2 (F4/8010W, CD11bint). As células PM-2 encontram-se em menor abundância (-10% do total de macrófagos) no peritônio não estimulado e, diferentemente de células PM-1, espraiam-se de forma alongada (bipolar) quando colocadas em cultura. Curiosamente, PM-2 são os únicos macrófagos peritoneais que expressam MHC-II e, ainda, 30% dessa população expressa CD11c, o marcador típico de células dendríticas. Já as células PM-1 possuem morfologia arredondada, estão em abundância no peritônio e produzem altas doses de NO após estímulo com LPS. Ambas as células são capazes de fagocitar bactérias in vivo, no entanto, as células PM-2 parecem mais eficientes, sendo capazes de internalizar quantidades maiores de bactérias. Após infecção in vivo, o número absoluto e a porcentagem de PM-2 aumenta muito no peritônio, chegando a representar metade dos fagócitos da cavidade peritoneal. Os macrófagos PM-1 e PM-2 parecem não representar monócitos já que não compartilham do fenótipo de monócitos presentes no sangue periférico. Deve-se ressaltar que, além das células PM-1 e PM-2, outras subpopulações do peritônio também expressam CD11b e F4/80. Com base na literatura atual, acredita-se que todas as células B-1 do peritônio expressam CD11b, uma molécula que é co-expressa com CD18 para formar o receptor de complemento e a molécula de adesão Mac-1. Entretanto, os estudos, apresentados neste trabalho, mostram que metade de cada subtipo de B-1 (B-1a e B-1b) não expressa CD11b. As células B-1 CD11b+ são maiores, mais granulosas e expressam quantidades maiores de IgM de superfície. As células CD11b+ possuem uma curiosa tendência de se juntar e formar duplas (doublets) fortemente ligadas entre si que estão presentes em abundância no peritônio de camundongos adultos. Além de ligarem entre si, as células B-1 podem formar duplas com macrófagos PM-1 e PM-2. Durante a ontogenia, as células CD11b- surgem primeiro no peritônio e são as progenitoras das células B-1 CD11b+. Após estímulo inflamatório (LPS í. v.) os linfócitos B-1 CD11b+ migram do peritônio para o baço, onde proliferam e transformam-se em células secretoras de anticorpos (plasmócitos). No peritônio, as células B-1 não são capazes de se diferenciar em plasmócitos (células CD138high). Os resultados apresentados aqui mostraram que, diferentemente dos macrófagos PM-2, os PM-1 são os responsáveis por inibir a formação de células secretoras de anticorpos derivadas de B-1. Em suma, nossos dados sugerem que, mediante estímulo inflamatório, as células B-1 maduras (CD11b+) migram do peritônio para o baço, afastandose de macrófagos PM-1. Já no baço, as células B-1 encontram um micro-ambiente favorável para se proliferarem e diferenciarem-se em plasmócitos, secretando a maioria dos anticorpos naturais vistos logo nas primeiras horas pós-estímulo. / Mouse peritoneal cavity (PerC) represents the source for a variety of cellular subsets of the immune system. In early studies, marred by Iimited methodological tools, it was thought that macrophages comprise roughly 90% of total PerC cells. Shortly thereafter, it was recognized that, beside macrophages, the mouse peritoneal cavity shelters large amount of B-lymphocytes, specially the B-1 subset. In essence, by applying contemporary technology, studies presented here show that, roughly 30% of PerC cells comprise macrophages, 55% comprise B-1 cells, which 40% represents the B-1 a subset and roughly 15% are B-1b cells. The remaining 15%-20% of PerC cells comprise a variety of known immune cells, including B-2, T, NK and eosinophils. In addition, there were some other cellular subsets that could not be identified in these studies, probably due to limited cell surface molecules analyzed. Surprisingly, the data presented here show that peritoneal macrophages represent a highly heterogeneous population. Based on the co-expression of both CD11b and F4/80, we have identified two functionally distinct subsets of peritoneal macrophages, named here as PM-1 (CD11bhigh, F480high) and PM-2 (CD11bint, F4/80low). PM-2 are less abundant (-10% within total macrophages) in the PerC of unstimulated mouse, however, unlike PM-1, these cells are able to spread in spindle (bipolar) morphology upon sorting and in vitro culture. Curiously, PM-2 is the only PerC macrophage that expresses surface MHC-II. In addition, one third of PM-2 population expresses CD11c, a universal marker for dendritic cells. In turn, PM-1 cells are round shaped cells, represent the majority of PerC macrophages (-90%) and secrete high amounts of NO upon LPS stimulation. In vivo phagocytosis assay revealed that both cells could internalize bacteria, however, PM-2 cells showed to be more efficient, in that it was able to phagocyte higher amounts of bacteria when compared to PM-1. After i.p. in vivo stimulation, the absolute number and percentage of PM-2 cells increase drastically in the peritoneum, reaching almost half of the total PerC macrophages. Importantly, PM-1 and PM-2 macrophages seem not to represent PerC monocytes since it does not share any of the phenotype expressed by blood monocytes. Interestingly, in addition to PM-1 and PM-2, some other cellular subsets in the PerC, such as eosinophils, are able to express CD11b and F4/80. PerC B-1 cells have long been known to express CD11b, which is co-expressed with CD18 to form the Mac-1/CR3 complement receptor and adhesion molecule. However, although all PerC B-1 cells are commonly believed to express CD11b, we show here that nearly half of the cells in each of the PerC B-1 subsets (B-1a and B-1b) do not express this surface receptor. The CD11b+ cells in each B-1 subset are larger, more granular and express higher levels of surface IgM than the CD11b- B-1 cells. Surprisingly, the CD11b+ B-1 subset has the curious tendency to initiate the formation of tightly associated doublets that are present at high frequency in adult PerC. In addition to binding to each other, B-1 cells can also form doublets with PM-1 and PM-2. During ontogeny, CD11b- B-1 cells arise earlier in the mouse PerC and are the progenitors of CD11b+ B-1 cells. Upon LPS stimulation, PerC CD11b+ B-1 cells migrate to the spleen where they proliferate and differentiate into antibody-secreting cells (plasma cells). Within the PerC, B-1 cells are not able to differentiate into plasma cells (CD138high cells). The data shown in here reveal that, unlike PM-2, PM-1 macrophages are the cellular subset responsible for inhibiting the formation of B-1-derived antibody-secreting cells. Altogether, our data suggest that, upon inflammatory stimuli, mature CD11b+ B-1 cells migrate from the PerC to the spleen, avoiding the inhibitory effect of PM-1 cells. In the spleen, B-1 cells find an appropriate environment to proliferate and terminally differentiate into antibody-secreting cells, thus, secreting the majority of immunoglobulin seen in few hours after in vivo stimulation.

Le plasmocyte (PC), stade terminal de la différenciation du lymphocyte B induite par l'antigène, est la cellule effectrice de l'immunité humorale responsable de la production des anticorps. La population plasmocytaire se divise en deux grands sous-types différant par leur durée de vie et par leur localisation anatomique. On distingue ainsi les PC à durée de vie courte ou PC effecteurs (dans les tissus lymphoïdes secondaires) et les PC à longue durée de vi (LLPC ou PC à mémoire) localisés principalement dans la moelle osseuse. Ces derniers contribuent à la mémoire humorale en continuant à sécréter des anticorps protecteurs après résolution de l'infection. Nos résultats expérimentaux montrent que les Ags thymo-dépendants (TD) et les Ag thymo-indépendants (TI) induisent des PC médullaires exprimant des caractéristiques phénotypiques et fonctionnelles différentes. Ainsi, l'expression d'une forme membranaire fonctionnelle du récepteur antigénique (BCR) persiste sur les LLPC TI alors qu'elle est perdue sur les LLPC TD. Cette fonctionnalité nouvelle portée par les PC médullaires TI n'est pas dépendante de la sous population lymphocytaire B recrutée ni de la nature de l'Ag, mais de l'isotype IgM. Nos résultats montrent également que cette population de PC médullaires a les caractéristiques des LLPC : ces PC sont quiescents, ils demeurent dans la moelle osseuse jusqu'à 180 jours et sont phénotypiquement semblables aux LLPC. Nos travaux ont permis de montrer que les LLPC à IgM sont capables de reconnaître l'Ag et que l'engagement de leur BCR par l'Ag conduit à la production d'IL10 / Plasma cells (PC) represent the terminal differentiation stage of B lymphocytes. Their canonical function is to secrete antibodies (Abs). PC differentiation is driven by remodeling of the B cell transcriptional program, highlighted by the induction of the transcriptional repressor Blimp-1 and repression of Pax5, considered as the guardian of B cell identity. The dogma holds that PC, as opposed to B cells, have lost the Ag recognition capacity because they have switched from expression of a membrane-bound Ag receptor (mBCR) to production of the secreted form of the BCR (Abs). Here, we have compared the phenotypical and functional attributes of memory PC generated by the T cell-dependent (TD) and T-cell independent (TI) forms of the hapten NP. Our data show that TI NP-specific bone marrow (BM) PC generated by NP-dextran retain an Ag-binding capacity comparable to that of B cells long after immunization while TD NP-specific BM PC do not. We found that this difference is not imputable to the structure of the immunogen but is a specific feature of IgM-expressing PC, which are prominent in response to TI Ag. Upon Ag recognition in vitro, the mBCR of IgM+ BM PC promotes: i) Ca++ mobilization, ii) phosphorylation of Syk and Blnk, iii) Ag internalization and phosphorylation of the late endosomal kinase Erk. Finally, we demonstrate that Ag recall in vivo induces significant changes in the gene expression profile of NP-specific IgM+ BM PC with evidence for activation of a cytokine production program characterized in particular by up-regulation of the CCL5 and IL10 transcripts. In conclusion, our data show that IgM-expressing BM PC can sense Ag and may be driven to express a regulatory function upon Ag recall

La cavité buccale constitue une porte d’entrée et un site réservoir majeur pour une diversité de virus. Mis à part l’herpèsvirus simplex, peu d’études virologiques détaillent le rôle des virus pathogènes de la sphère orale dont le virus d’Epstein-Barr. Le consensus est que les inflammations orales surviennent principalement en réaction à des infections bactériennes et fongiques provoquant une dysbiose du biofilm oral. Toutefois, ce modèle n’est pas parfait et il explique mal les récidives observées après décontamination, le découplage entre dysbiose et poussées inflammatoires, la proximité de dents atteintes et saines dans un environnement bactérien similaire. L’hypothèse d'un mécanisme synergique combinant virus et bactéries a été proposée pour la parodontite. Selon ce modèle, l’action complémentaire des 2 types d’agents infectieux sur le microenvironnement immunitaire favoriserait la chronicité inflammatoire et l’évolution de la maladie. Nos travaux se concentrent sur l’étude de l’infection de la cavité orale par EBV. EBV est un virus ubiquitaire infectant l’homme de manière persistante. La cavité orale est le site privilégié pour sa réplication avec une production salivaire quasi-constante. Outre un rôle transformant majeur, l’implication de EBV est aussi suspectée pour plusieurs maladies inflammatoires. Tout d’abord, nous nous sommes intéressés au lichen plan oral (LPO), une maladie auto-immune dont l'étiopathogénie n’est pas clairement établie. Sur des biopsies de patients atteints de LPO (n=99), nous avons démontré par hybridation in situ que le LPO est fréquemment infecté par EBV (74%), notamment les formes cliniques érosives. Nous montrons que le degré d’infiltration des lésions par des cellules infectées par EBV (EBV+) est corrélé avec les paramètres inflammatoires et que les cellules EBV+ infiltrées sont des plasmocytes. Cela apporte un élément nouveau dans la mesure où les plasmocytes sont reconnus comme des cellules immunitaires régulatrices majeures. Nous décrivons des profils cytokiniques différents entre LPO infectés ou non, sans qu’il soit possible à ce stade de pouvoir impliquer directement les plasmocytes. Nous confirmons par microscopie électronique que les plasmocytes hébergent les stades tardifs du cycle d’EBV et produisent des virions dans le LPO, suggérant un mécanisme d’amplification locale de l'infection. Ensuite, nous nous sommes intéressés à la parodontite, maladie inflammatoire chronique commune qui détruit la structure parodontale et provoque le déchaussement dentaire. Cette maladie est clairement identifiée comme facteur aggravant de nombreuses maladies systémiques. Nos études avaient déjà mis en évidence un lien direct entre infection EBV et la sévérité de la parodontite. Mes travaux ont permis de montrer que le parodonte inflammatoire est infiltré par des cellules EBV+ qui semblent être majoritairement des plasmocytes. La présence de plasmocytes producteurs de virus au sein de la lésion inflammatoire pourrait expliquer l’infection des épithéliums adjacents. De plus, une étude clinique menée sur une petite cohorte de patients traités pour parodontite met en évidence une corrélation entre la diminution de la charge EBV salivaire et l’amélioration clinique de patients après traitement. Si ce résultat se confirme, il constituerait un argument supplémentaire en faveur d’une contribution de l’infection parodontale par EBV à la globalité de la charge EBV salivaire. En conclusion, nos données mettent en évidence la présence quasi constante d’EBV dans deux types de lésions orales inflammatoires. Cette infection virale contribue à aggraver une situation inflammatoire locale associée ou pas à une dysbiose bactérienne. L’observation majeure concerne la présence, souvent massive, de plasmocytes infectés dont le rôle reste à identifier. Ces observations constituent des avancées significatives qui permettent d’étayer un nouveau modèle de pathogénie orale associant virus et bactéries. / The oral cavity is a major entry point and reservoir site for a variety of viruses. Apart from herpesvirus simplex, few virological studies detail the role of oral pathogenic viruses, including Epstein-Barr virus. The consensus is that oral inflammation occurs mainly in response to bacterial and fungal infections causing dysbiosis of the oral biofilm. However, this model is not perfect and does not explain well the recurrences observed after decontamination, the decoupling between dysbiosis and inflammatory outbreaks, the proximity of affected and healthy teeth in a similar bacterial environment. The hypothesis of a synergistic mechanism combining viruses and bacteria has been proposed for periodontitis. According to this model, the complementary action of the 2 types of infectious agents on the immune microenvironment would promote inflammatory chronicity and disease progression. Our work focuses on the study of oral cavity infection with EBV. EBV is a ubiquitous virus that persistently infects humans. The oral cavity is the preferred site for its replication with almost constant saliva production. In addition to a major transformative role, EBV's involvement is also suspected for several inflammatory diseases. First, we focused on oral plan lichen (OPL), an autoimmune disease whose etiopathogeny is not clearly established. On biopsies of patients with OPL (n=99), we demonstrated by in situ hybridization that OPL is frequently infected with EBV (74%), particularly in erosive clinical forms. We show that the degree of infiltration of lesions by EBV-infected cells (EBV+) is correlated with inflammatory parameters and that the infiltrated EBV+ cells are plasma cells. This brings a new element to the extent that plasma cells are recognized as major regulatory immune cells. We describe different cytokinic profiles between infected and uninfected OPL, although it is not possible at this stage to directly involve plasma cells. We confirm by electron microscopy that plasma cells host the late stages of the EBV cycle and produce virions in the OPL, suggesting a local amplification mechanism of infection. Then, we focused on periodontitis, a common chronic inflammatory disease that destroys the periodontal structure and causes tooth loosening. This disease is clearly identified as an aggravating factor in many systemic diseases. Our studies had already shown a direct link between EBV infection and the severity of periodontitis. My work has shown that the inflammatory periodontium is infiltrated by EBV+ cells, which appear to be predominantly plasma cells. The presence of virus-producing plasma cells within the inflammatory lesion may explain the infection of adjacent epithelia. In addition, a clinical study conducted on a small cohort of patients treated for periodontitis showed a correlation between the decrease in salivary EBV load and the clinical improvement of patients after treatment. If this result is confirmed, it would be an additional argument in favour of a contribution of periodontal EBV infection to the overall salivary EBV burden. In conclusion, our data show the almost constant presence of EBV in two types of inflammatory oral lesions. This viral infection contributes to worsening a local inflammatory situation associated or not with bacterial dysbiosis. The main observation concerns the presence, often massive, of infected plasma cells whose role remains to be identified. These observations represent significant advances that support a new model of oral pathogenesis combining viruses and bacteria.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18899号 / 医博第4010号 / 新制||医||1009(附属図書館) / 31850 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 三森 経世, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

La commutation de classe (CSR) est une étape clef de la réponse immunitaire. Ce phénomène va permettre de changer le type d’immunoglobuline (Ig) produite en réponse à un antigène donné. Ces Ig seront ensuite produites par les plasmocytes, qui constituent le stade ultime de la différenciation de la lignée cellulaire B. Lors de dérèglements de la prolifération de ces cellules, certaines Ig monoclonales anormales peuvent être produites et conduire à des situations pathologiques. La première partie de ce travail s’inscrit dans une logique de compréhension des éléments minima requis pour l’établissement de ce phénomène de CSR. Grace à un modèle animal d’insertion dirigée dans le locus kappa murin, naturellement ciblé par l’enzyme AID responsable de ce phénomène, nous avons mis en évidence que la présence de deux régions « switch » transcrites et fortement mutées par AID, n’était pas suffisante pour permettre ce phénomène. Un modèle murin reproduisant une maladie due à une Ig anormale a aussi été établi. Ce modèle de HCDD (Heavy Chain Deposition Disease) nous a permis de mettre en évidence la nécessité de la délétion du CH1 des chaînes lourdes d’Ig pour la génération des dépôts et nous a également permis de montrer que l’efficacité des thérapies à base d’inhibiteur de protéasome observée chez les patients atteint de HCDD, était en partie due à l’Ig pathogène elle-même, qui induit une élévation du stress du réticulum endoplasmique (UPR) au sein des plasmocytes producteurs de ces Ig. / Class Switch Recombination (CSR) is a key step during the immune response. CSR results in a switch to a more specific Ig isotype in response to a specific antigen. Plasma cells, the ultimate stage of B cell lineage differentiation, will synthesize this Ig. During plasma cell disorders, the production of an abnormal monoclonal Ig can lead to pathogenic situations. The aim of the first part of this study is to determine the minimal requirements for CSR induction with a mouse model in which we inserted a “switch cassette” composed of two transcribed S regions into a kappa locus which is naturally targeted by AID. However, despite efficient transcription and AID targeting of S regions, the “switch cassette” was not sufficient to induce effective CSR. We also developed a mouse model of HCDD (Heavy Chain Deposition Disease) which reproduced typical Randall-type renal lesions due to production of a pathogenic truncated heavy chain. This model demonstrated that the effective response to proteasome inhibitors observed in patients, is the consequence of the presence of a truncated HC that sensitizes plasma cells to this type of therapy through an elevated unfolded protein response (UPR).

Le lupus érythémateux disséminé est une maladie auto-immune systémique très invalidante dont les atteintes sont multiples, les plus fréquentes étant cutanées, articulaires et rénales. Dans ce type de maladie, le système immunitaire, hyperactif, ne se limite pas à lutter contre des agents extérieurs mais s'attaque à ses propres cellules, entre autres par le biais d'auto-anticorps. Ces anticorps délétères sont produits par des plasmocytes, cellules issus de la différenciation des lymphocytes B. Ce processus se déroule principalement au sein des centres germinatifs (GC) dans les organes lymphoïdes secondaires, et fait intervenir de nombreux acteurs moléculaires et cellulaires. Mon projet de thèse a porté sur l'étude de la contribution du GC et de ses constituants, tels que les cellules auxiliaires folliculaires (Tfh) et l'IL-21, au cours du lupus. Au cours de ce travail, nous avons mis en évidence une altération à la fois quantitative et qualitative des cellules Tfh chez des patients lupiques et dans un modèle murin, altération entre autres responsable de taux anormalement élevés d'IL-21. Nous avons également observé une sensibilité accrue des cellules B de souris lupiques à cette cytokine, dont la cause est une surexpression de molécules clés telles que STAT3, et dont la conséquence est un surcroit de différenciation plasmocytaire. Tous les éléments sont donc présents pour favoriser l'interaction "Tfh-B" et la réaction du GC, et amplifier la réponse autoimmune. Enfin, la découverte de l'existence de GC ectopiques fonctionnels dans les reins de souris lupiques permet d'envisager l'existence de réponses locales au sein même des organes cibles. Les données obtenues, fondamentales, sont prometteuses et laissent entrevoir de nouvelles perspectives de biothérapies, plus ciblées, pour le traitement de la maladie lupique. / Systemic lupus erythematosus is a disabling chronic autoimmune disease characterized by B cell hyperactivity leading to the production of autoantibodies, some of which exerting pathogenic effects. Autoantibodies are produced by plasma cells, which originate from the differentiation of B cells through a process that mainly takes place in germinal centers (GC) in secondary lymphoïd organs and involves many molecular and cellular parameters. The aim of my PhD project was to analyze the individual contribution of GC components, such as follicular helper T cells (Tfh) and IL-21, to lupus development. During this work, we have shown both a quantitative and qualitative impairment of Tfh cells in lupus patients and in a mouse model, leading, among other things, to high IL-21 levels. We also observed that B cells from lupus mice display a specific intrinsic sensitivity to this cytokine, due to over-expression of key molecules such as STAT3, which results in increased plasma cell differentiation. Thus, all elements are gathered that favor "Tfh-B" cell interactions and the GC reaction, and therefore the autoimmune response. Finally, the discovery of functional ectopic GC in the kidneys of lupus mice allows envisaging that local responses occur within the target organs and likely participate to kidney injury. The fundamental data we obtained are promising and anticipate new and better targeted biotherapies for lupus treatment.