Le myélome multiple (MM) est une néoplasie B caractérisé par l'accumulation d'un clone plasmocytaire dans la moelle osseuse. Cette pathologie demeure incurable d'où la nécessité d'identifier de nouvelles cibles thérapeutiques. C'est notamment dans cette optique que nous avons initié, au sein du laboratoire, un travail de comparaison des profils d'expression génique des plasmocytes tumoraux purifiés de malades avec ceux de plasmocytes normaux et de lymphocytes B, ce qui permettra l'identification de nouvelles voies importantes pour la biologie du MM et donc de nouvelles cibles thérapeutiques potentielles.<br />Par cette approche, nous avons mis en évidence un rôle essentiel des membres de la famille BAFF/APRIL et de leurs récepteurs (BCMA, BAFF-R et TACI) dans la biologie du MM. Les cellules de MM expriment les récepteurs alors que les ligands sont principalement produits par les cellules de l'environnement médullaire. L'utilisation d'un inhibiteur spécifique de BAFF/APRIL a permis de montrer que ces facteurs de croissance sont importants pour la survie et la prolifération des cellules tumorales. TACI apparaît être le récepteur principal pour médier l'effet de BAFF et APRIL dans le MM. Une forte expression de TACI par les cellules de MM est associée à une signature génique de plasmocytes matures alors que les plasmocytes tumoraux présentant une faibles expression de TACI ont une signature génique de plasmablastes proliférants. Nous avons montré que syndecan-1, un protéoglycane à chaînes héparane sulfate joue un rôle essentiel dans la biologie du MM en permettant l'accumulation de fortes concentrations de facteurs de croissance à la surface des cellules. Nous avons identifié que syndecan-1 joue un rôle de corécepteur pour APRIL et TACI supportant ainsi la croissance des cellules de MM. <br />Ces travaux offrent de nouvelles perspectives thérapeutiques pour le MM et ont débouché sur un essai clinique de phase I/II, au CHU de Montpellier, utilisant un inhibiteur de la voie BAFF/APRIL dans le MM.

The idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of chronic muscle diseases with frequent extramuscular organ involvement that contributes to serious prognosis. The presence of autoantibodies and composition of muscle infiltrates both support autoimmune nature of the disease and pathogenic role of B lymphocytes. Besides the traditional diagnostic subgroups, autoantibody characterised phenotype subsets have been identified with presumed similar pathogenic mechanisms. The best known is the antisynthetase syndrome which is characterised by presence of myositis, antisynthetase autoantibodies (with anti-Jo-1 being the most frequent), interstitial lung disease and other extramuscular manifestations. BAFF (B cell-Activating Factor of the TNF Family) is a key factor in B cell homeostasis modulation. In high levels, it allows survival of autoreactive B cell clones and thus participates in the pathogenesis of autoimmune diseases. Its expression is induced by type I interferons (IFN-1). The aim of the PhD thesis was to explore the role of BAFF in pathogenesis of IIMs by analysis of its serum levels, the receptors for BAFF in muscle tissue, their associations to IFN-1 and expression of BAFF gene mRNA transcription variants in peripheral blood cells. Further aspect was to study a possible...

La sclérodermie systémique (ScS) est une maladie autoimmune rare qui se caractérise par une fibrose cutanée et parfois pulmonaire. Nous avons tout d’abord évalué le rôle de BAFF, une cytokine impliquée dans le développement des lymphocytes B (LB), dans la fibrose pulmonaire induite par la bléomycine chez la souris. Nous avons démontré que BAFF était augmenté en réponse à la bléomycine et que les souris BAFF-/- ou traitées par le BAFF-R-Ig sont protégées de la fibrose pulmonaire. Ensuite, nous avons évalué si les LB et BAFF pouvaient moduler la production de collagène par des fibroblastes de peau isolés de patients atteints de ScS. Nous avons démontré que les LB augmentent la production de collagène et de cytokines impliquées dans la fibrose cutanée et l’ajout de BAFF augmente cet effet des LB sur les fibroblastes. Enfin, nous avons étudié la régulation de l’expression de BAFF par les microARNs. Nos résultats montrent que les miR-30a*, d* et e* ciblent directement l’ARNm de BAFF. / Systemic sclerosis (SSc) is a rare autoimmune disease characterized by skin fibrosis and occasionally pulmonary fibrosis. We first assessed the role of BAFF, a cytokine involved in B cell maturation, in bleomycin-induced pulmonary fibrosis in mice. We showed that BAFF was increased in response to bleomycin and that BAFF-/- mice or BAFF-R-Igtreated mice are protected from pulmonary fibrosis. Then, we assessed whether B cells and BAFF could regulate collagen production by skin fibroblasts isolated from SSc patients. We demonstrated that B cells increase collagen production and cytokines involved in skin fibrosis. The addition of BAFF increases the effect of B cells on fibroblasts. Lastly, we studied the regulation of BAFF expression by microRNAs. Our results show that miR-30a*, d* and e* directly target the BAFF mRNA.

Le syndrome de Gougerot-Sjögren (SGS) est une maladie autoimmune (MAI) systémique inflammatoire chronique caractérisée principalement par la diminution des secrétions salivaires et lacrymales, aboutissant à une sécheresse de la bouche et des yeux. Elle affecte principalement les femmes autour de la ménopause. Au niveau physiologique, le tissu épithélial des glandes salivaires (GS) est infiltré par des lymphocytes, ce qui provoque l’apoptose des cellules épithéliales (CE). L’épigénétique pouvant jouer un rôle important dans le développement des MAI comme le SGS, nous avons analysé l’expression d’éléments rétroviraux endogènes humains (HERV) et des microARN. Par ailleurs, afin de mieux comprendre les mécanismes cellulaires et moléculaires impliqués dans les interactions lymphocytes-CE au niveau de la GS pathologique, nous avons mis en place un modèle de co-culture in vitro entre des CE et des lymphocytes B ou T (LB, LT). Nous avons également étudié le rôle de BAFF sur les CE qui expriment l’un de ses récepteurs, BR3.Dans un premier temps, nous avons montré que dans les GS des patients, les HERV et les miARN ont un profil d’expression distinct de celui des personnes saines, montrant ainsi une participation de l’épigénétique dans la pathologie. Dans un deuxième temps, nous avons montré que les CE entrent en apoptose suite à l’interaction directe avec les lymphocytes B etT, en faisant intervenir la voie Fas pour les LT et la voie de la PKCδ pour les LB. En lien avec l’apoptose induite par les LB, nous avons démontré que BAFF est impliqué dans la survie des CE, et que le blocage de sa signalisation ou la sous-expression de son récepteur conduit à la mort des CE. Nous pouvons supposer que les LB entrent en compétition avec les CE pour le signal de survie apporté par BAFF, et que les CE qui s’en trouvent privées meurent. Finalement, nous avons montré que plusieurs formes de BAFF sont produites parles CE et reconnues de manière différente par les anticorps anti-BAFF. Il pourrait s’agir d’isoformes plus ou moins glycosylés ou de variants de BAFF. Cependant d’autres études sont nécessaires afin de les identifier. / Sjögren’s syndrome (SS) is an inflammatory systemic autoimmune disease (AID), mainly characterized by the decrease of salivary and lachrymal secretions. This leads to dry mouthand dry eyes. This chronic disease principally affects women around menopause. At the physiological level, salivary gland (SG) epithelial tissue is infiltrated by lymphocytes, leading to epithelial cells (EC) apoptosis. As epigenetics can play an important role in AID- such asSS development, we investigated human endogenous retroviral elements (HERV) and microRNA expression. Furthermore, in order to better understand the cellular and molecular mechanisms implied in lymphocyte-EC interactions in pathological SG, we set up an in vitroco-culture model between EC and B and T cells. We also studied BAFF role on EC which express one of its receptors, BR3. First, we demonstrated that in SS SG, HERVs and miRNAs show a distinct profile from healthy controls, indicating that epigenetics is implied in the pathology. Secondly, we showed that EC undergo apoptosis following direct interaction with T and B cells, via two distincts ignaling pathways: Fas pathway for T cells and PKCδ pathway for B cells. Linked with Bcell-induced apoptosis, we demonstrated that BAFF is implied in EC survival, and that BAFFsignaling blocking or BR3 down-regulation leads to EC death. We may suppose that B cells compete with EC for survival signaling from BAFF, and that EC die from lack of it. Finally, we showed that several forms of BAFF are expressed by EC and that they are differentially recognized by anti-BAFF antibodies. These may be more or less glycozylated BAFF isoformsor BAFF variants. Further studies are needed to identify them.

Das Verständnis der Immunpathogenese des systemischen Lupus erythematodes (SLE) hilft, das komplexe Krankheitsgeschehen zu verstehen und neue Therapiestrategien zu entwickeln. Die Krankheitsmanifestationen des SLE sind im Wesentlichen Folge von Autoantikörpern, Immunkomplexen und Zytokinen. Insbesondere die Neigung zu unterschiedlichen Autoantikörpern macht das Wesen der Erkrankung aus; die genauen Spezifitäten der Autoantikörper führen zu ganz unterschiedlichen Organmanifestationen. Diese Übersichtsarbeit stellt den klinisch relevanten Stand des Wissens zur SLE-Pathogenese dar – mit dem Ziel, ein für den klinischen Einsatz nützlichesModell zu etablieren, das auch hilft, die neuen Therapieansätze einzuordnen.

The mammalian immune system has developed diverse strategies to protect the gastrointestinal tract, as this tissue locale represents a huge absorptive surface and is susceptible to microbial breach. Paradoxically, one key aspect of this protective strategy is the maintenance of selected commensal microorganisms. These commensals serve essential roles in digestion, interfere with pathogenic microbial invasion and stimulate development of the host immune system. Therefore, immune responses which deplete these commensal populations are detrimental to the host. One effective intestinal immune response which selectively promotes the survival of commensals is production of antibodies of the IgA isotype which bind to bacteria without triggering inflammatory cytokines. Proteins of the tumor necrosis factor (TNF) family such as Lymphotoxin and BAFF contribute to the induction of IgA responses. Lymphotoxin is required for generation and organization of most organized lymphoid tissues, where B cell differentiation occurs, while BAFF is necessary for B cell survival and induces B cells to produce IgA. In this thesis, I describe work I have done in examining the roles of the TNF family members Lymphotoxin, BAFF and two related TNF family member cytokines, LIGHT and APRIL, in the regulation of IgA production in mice and in humans. Specifically, LIGHT over-expression drives immense production of IgA, leading to renal deposition of immune complexes in mice. Similar to LIGHT, BAFF over-expression drives increases in IgA production in the intestine, however I have shown that the effects of the BAFF pathway on IgA hyper-production are independent of LIGHT activity. Secondly, examining the phenotype of BAFF-over-expressing mice, I have shown that this phenotype resembles human IgA nephropathy (IgAN) and is dependent on intestinal commensals. Finally, I have described a lymphotoxin-dependent chemokine system in the intestinal lamina propria that could be responsible for organizing cells for the development of IgA responses in this mucosal site.

Systemic lupus erythematosus (SLE) and Sj?gren?s syndrome (SS) are both heterogeneous autoimmune diseases with strong B cell aspects. A proportion of SLE and SS patients exhibit elevated serum BAFF (B cell activating factor of the TNF family); BAFF plays a key role in B cell homeostasis, survival and tolerance. BAFF transgenic (Tg) mice develop nephritis and salivary gland destruction that resemble aspects of SLE and SS respectively. Autoimmune disease development in BAFF Tg mice correlates with marginal zone (MZ) B cell expansion and the abnormal presence of MZ-like B cells outside of the spleen. The role of MZ B cells in BAFF induced autoimmune disease was analysed by crossing BAFF Tg mice with Lymphotoxin-β knockout mice (creating LTβ-BTg mice) which lack most peripheral lymph nodes, and also lack MZ B cells as a result of disrupted splenic architecture. LTβ-BTg mice were not protected against nephritis but exhibited reduced salivary gland infiltration and destruction. Indicating that the development of sialadenitis but not nephritis in BAFF Tg mice is MZ B cell dependent. Nephritis development in LTβ-BTg mice was associated with the detection of B-1 B cells in the inflamed kidneys. As B-1a B cell survival is dependent on the spleen, the contribution of B-1a B cells to nephritis development in BAFF Tg mice was assessed by crossing BAFF Tg mice to congenitally asplenic Hox11-/- mice (creating Hox11 -BTg mice). The absence of a spleen and B-1a B cells in Hox11-BTg mice delayed the nephritis development. In contrast, splenectomy of BAFF Tg mice at 12 weeks of age did not alter nephritis onset. In these mice B-1a B cells persisted in the peritoneal cavity and MZ-like B cells were detected in the periphery 8 months after surgery. In summary, nephritis development in BAFF Tg mice is unaltered by the absence of MZ B cells, but delayed in the absence of a spleen, MZ and B-1a B cells. Thus, B-1a and B-1b B cells may be potential targets for the treatment of nephritis in SLE patients with elevated BAFF.

Autoimmune Polyendocrine Syndrome Type 1(APS I) is a monogenic autosomal recessive autoimmune disorder which is the result of mutations in the autoimmune regulator (AIRE) gene. Symptoms of the disease include circulation of multiple organ specific autoantibodies, which leads to the breakdown of several tissues, including the adrenal cortex and the parathyroid glands. The patients also develop a number of non-endocrine disorders. This study has investigated the peripheral tolerance mechanisms controlled by the AIRE gene in Aire deficient mice, an animal model of the disease. The B cell Activating Factor (BAFF), which is a cytokine involved in B cell survival and growth, is elevated in Aire-/- mice, resulting in an increased release of autoantibodies and B cell proliferation. Therefore the BAFF level differences between TCR-/- and B6 mice was studied, and the results showed significantly higher levels of BAFF in TCR-/- mice. This is not in accordance with earlier studies. ICOS and ICOSL are involved in the activation of follicular T helper cells. The expression of ICOSL on different subpopulations of DC from mice was studied to evaluate the possible influence of AIRE expression on the T cells in the spleen. The results showed that ICOSL is significantly higher expressed in peripheral 33D1+ DCs in Aire-/- mice, showing that AIRE has a role in the over-activation of the follicular T helper cells, which can lead to autoantibody production and inflammation. These results show that AIRE is involved in peripheral tolerance.

The mammalian immune system has developed diverse strategies to protect the gastrointestinal tract, as this tissue locale represents a huge absorptive surface and is susceptible to microbial breach. Paradoxically, one key aspect of this protective strategy is the maintenance of selected commensal microorganisms. These commensals serve essential roles in digestion, interfere with pathogenic microbial invasion and stimulate development of the host immune system. Therefore, immune responses which deplete these commensal populations are detrimental to the host. One effective intestinal immune response which selectively promotes the survival of commensals is production of antibodies of the IgA isotype which bind to bacteria without triggering inflammatory cytokines. Proteins of the tumor necrosis factor (TNF) family such as Lymphotoxin and BAFF contribute to the induction of IgA responses. Lymphotoxin is required for generation and organization of most organized lymphoid tissues, where B cell differentiation occurs, while BAFF is necessary for B cell survival and induces B cells to produce IgA. In this thesis, I describe work I have done in examining the roles of the TNF family members Lymphotoxin, BAFF and two related TNF family member cytokines, LIGHT and APRIL, in the regulation of IgA production in mice and in humans. Specifically, LIGHT over-expression drives immense production of IgA, leading to renal deposition of immune complexes in mice. Similar to LIGHT, BAFF over-expression drives increases in IgA production in the intestine, however I have shown that the effects of the BAFF pathway on IgA hyper-production are independent of LIGHT activity. Secondly, examining the phenotype of BAFF-over-expressing mice, I have shown that this phenotype resembles human IgA nephropathy (IgAN) and is dependent on intestinal commensals. Finally, I have described a lymphotoxin-dependent chemokine system in the intestinal lamina propria that could be responsible for organizing cells for the development of IgA responses in this mucosal site.

Systemic lupus erythematosus (SLE) and Sj?gren?s syndrome (SS) are both heterogeneous autoimmune diseases with strong B cell aspects. A proportion of SLE and SS patients exhibit elevated serum BAFF (B cell activating factor of the TNF family); BAFF plays a key role in B cell homeostasis, survival and tolerance. BAFF transgenic (Tg) mice develop nephritis and salivary gland destruction that resemble aspects of SLE and SS respectively. Autoimmune disease development in BAFF Tg mice correlates with marginal zone (MZ) B cell expansion and the abnormal presence of MZ-like B cells outside of the spleen. The role of MZ B cells in BAFF induced autoimmune disease was analysed by crossing BAFF Tg mice with Lymphotoxin-β knockout mice (creating LTβ-BTg mice) which lack most peripheral lymph nodes, and also lack MZ B cells as a result of disrupted splenic architecture. LTβ-BTg mice were not protected against nephritis but exhibited reduced salivary gland infiltration and destruction. Indicating that the development of sialadenitis but not nephritis in BAFF Tg mice is MZ B cell dependent. Nephritis development in LTβ-BTg mice was associated with the detection of B-1 B cells in the inflamed kidneys. As B-1a B cell survival is dependent on the spleen, the contribution of B-1a B cells to nephritis development in BAFF Tg mice was assessed by crossing BAFF Tg mice to congenitally asplenic Hox11-/- mice (creating Hox11 -BTg mice). The absence of a spleen and B-1a B cells in Hox11-BTg mice delayed the nephritis development. In contrast, splenectomy of BAFF Tg mice at 12 weeks of age did not alter nephritis onset. In these mice B-1a B cells persisted in the peritoneal cavity and MZ-like B cells were detected in the periphery 8 months after surgery. In summary, nephritis development in BAFF Tg mice is unaltered by the absence of MZ B cells, but delayed in the absence of a spleen, MZ and B-1a B cells. Thus, B-1a and B-1b B cells may be potential targets for the treatment of nephritis in SLE patients with elevated BAFF.