L’immunothérapie est une avancée majeure dans le traitement des cancers, même s’il reste fondamental d’identifier de nouvelles cibles pour optimiser la réponse anti-tumorale. Des enzymes impliquées dans le métabolisme d'acides aminés ont été décrites comme pouvant inhiber ces réponses immunitaires et ainsi favoriser la progression tumorale. Elles sont donc devenues des cibles thérapeutiques de premier choix. L’IL-4 induced gene 1 (IL4I1) est une phénylalanine oxydase qui, au cours de cette dernière décennie, s’est dévoilée dans la littérature comme un véritable outil de la tolérance périphérique. Ses fonctions restent cependant largement inexplorées. Au cours de ma thèse, j’ai étudié les propriétés immunorégulatrices de l’IL4I1 en contexte tumoral dans un modèle de mélanome spontané (souris Ret), mais également dans la physiologie des lymphocytes B (LB). Dans un premier temps, mes résultats mettent en avant que l’activité enzymatique de l’IL4I1 dans les rates des souris Ret est corrélée avec la sévérité de la maladie. De façon intéressante, l’inactivation génétique de l’IL4I1 dans ce modèle (RetIL4I1KO) retarde le développement de la tumeur primaire, mais aussi la dissémination métastatique. Nous démontrons que l’IL4I1, principalement exprimée par les cellules myéloïdes, a la capacité de façonner la composante immunitaire du microenvironnement tumoral, et ce, par le recrutement préférentiel de cellules myéloïdes au détriment d’effecteurs lymphocytaires T. Par ailleurs, mes données chez les souris déficientes pour l’IL4I1 (IL4I1KO) ont mis en évidence un rôle crucial de l’IL4I1, régulant de nombreuses étapes de la biologie des LB. Les souris IL4I1KO présentent une sortie accélérée des LB de la moelle osseuse, entraînant l'accumulation de LB folliculaires en périphérie. Ces souris ont un taux sérique élevé d'immunoglobulines naturelles et des anticorps auto-réactifs. Nous démontrons aussi que l’IL4I1 contrôle la réaction du centre germinatif, la différenciation des plasmocytes et la production d'anticorps spécifiques en réponse à une immunisation par un antigène T-dépendant. In vitro, l’absence de l’IL4I1 dans les LB augmente l’activation des voies de signalisation en aval du récepteur à l’antigène des LB (BCR), ainsi que leur prolifération. Par conséquent, nos résultats révèlent une propriété clé de l’IL4I1, contrôlant négativement l'activation dépendante du BCR. Compte tenu de l’effet majeur de l’IL4I1 sur la biologie des LB, il reste important d’investiguer si l’hyperréactivité du BCR des LB déficients pour l’IL4I1 participe au meilleur contrôle de la progression tumorale. / Immunotherapy is one of the most promising advance in cancer treatments. It remains essential to identify new targets to maximize the anti-tumor immune response. Enzymes involved in amino acid metabolism have been described to impede these responses and thus promote tumor progression. They became good therapeutic targets. IL-4-induced gene 1 (IL4I1) is a phenylalanine oxidase which, over the last decade, was unveiled as a real tool for peripheral tolerance. However its functions remain largely unexplored. During my PhD thesis, I studied the immunoregulatory properties of IL4I1 in a tumoral context involving a spontaneous melanoma model (Ret mice), but also in the B cell physiology. First, my results highlight that the IL4I1 enzymatic activity is positively correlated with disease aggressiveness in the spleen of Ret mice. Interestingly, genetic inactivation of IL4I1 in this model (RetIL4I1KO) delayed the development of both primary tumor and metastatic dissemination. We demonstrate that IL4I1, mainly expressed by myeloid cells, has the ability to shape the immune compartment within the tumor microenvironment, through recruitment of myeloid cells instead of activated T cells. Furthermore, my data in mice deficient for IL4I1 (IL4I1KO) have emphasized a crucial role of IL4I1 in regulating many steps of B cell biology. Indeed, IL4I1KO mice exhibit an accelerated B cell egress from the bone marrow, resulting in the accumulation of peripheral follicular B cells. These mice have a high serum level of natural immunoglobulins and auto-reactive antibodies. We also demonstrate that IL4I1 controls the germinal center reaction, plasma cell differentiation and specific antibody production in response to a T-dependent antigen immunization. In vitro, the absence of IL4I1 in B cells increases their proliferation and the activation of signaling pathways upon B cell receptor (BCR) engagement. Thus, our results reveal a key role of IL4I1, which negatively controls the BCR-dependent activation. Regarding these effects of IL4I1 on B cell biology, it remains important to evaluate whether the BCR-dependent hyperreactivity in IL4I1KO mice contributes in the tumor control.

The recruitment of peripheral blood lymphocytes (PBL) to sites of inflammation and their subsequent traffic into the lymphatic circulation is important in host defense. However, surprisingly little is known about their recruitment from the blood vasculature into inflamed tissue, and almost nothing about their egress from inflamed tissue via the lymphatic circulation. We showed that both human macrovascular and microvascular endothelial cells stimulated by TNF\(\alpha\) and IFN\(\gamma\), preferentially recruited memory T-lymphocytes (CD45RO positive cells) from a mixed pool of PBL. T-cells that had migrated across vascular endothelial cells subsequently utilised a combination of \(\beta\)1 and \(\beta\)2 integrins to traverse cytokine activated lymphatic endothelium. In addition we provide evidence that PGD2 was critical for the transmigration of lymphocytes through vascular endothelium. The process of trans-lymphatic migration was also significantly retarded in the presence of a function neutralising antibody against CCR7. Most importantly, we observed that memory T-cells showed a markedly enhanced capacity to migrate across lymphatic endothelium if they had first traversed a vascular endothelial cell barrier. We have shown that addition of exogenous PGD2 to isolated lymphocytes is able to restore the enhanced migration capacity of lymphocytes that have previously migrated through a vascular monolayer. The nature of the priming signal delivered by the process of migration across blood vessel endothelium remains to be fully identified, but is likely to be important in regulating the dynamics of an inflammatory response.

The phenomenon of cell type-specific DNA methylation has received much attention in recent years and a number of DNA methylation differences have been described between cells of the immune system. Of particular interest when studying DNA methylation are CpG islands (CGIs) which are distinct from the rest of the genome due to their elevated CpG content, generally unmethylated state and promoter association. In the instances when they become methylated this is associated with gene repression although it is unclear the extent to which differential methylation corresponds to differential gene expression. I have used an immune system model to assess the role of CGI methylation and the role of the methylation reader MBD2 in regulation of gene expression. A relatively small number of DNA methylation differences were seen between immune cell types with the most developmentally related cells showing the fewest methylation differences. Interestingly, the vast majority of CGI-associated cellspecific methylation occurred at intragenic CGIs located, not at transcription start sites, but in the gene body. Increased intragenic CGI methylation tended to associate with gene repression, although the precise reason for this remains unclear. Most differentially methylated CGIs were depleted for the active chromatin mark H3K4me3 regardless of their methylation state but some of these were associated with the silencing mark H3K27me3 when unmethylated. These findings suggest that intragenic CGIs are a distinct class of genomic element particularly susceptible to cell type-specific methylation. I also looked at the effect of removing the methyl- CpG binding domain protein MBD2 from immune system cells. Immune cells from Mbd2-/- mice showed a number of previously uncharacterised phenotypes as well as a number of differences in gene expression compared to wild-type animals. Most of these genes increased their expression in the absence of MBD2 consistent with MBD2’s role as a transcriptional repressor and Mbd2-/- Th1 cells showed increases in histone H3 acetylation compared to wild-type Th1 cells. This work provides an insight into the role played by cell-specific CGI methylation and MBD2 in regulating gene expression.

Despite the actions of central tolerance during thymic selection, it is clear that the peripheral T cell repertoire contains significant numbers of self-reactive T cells. The immune system needs to curtail the risk of autoimmune disease by controlling the activity of these self-reactive T cells. Various mechanisms are in place to achieve this control (peripheral tolerance). Activation of CD4+ T cells requires two signals; engagement of the T cell receptor (TCR) with an appropriate peptide:MHC complex (signal 1), and the aggregate effect of multiple signals generated following ligation of costimulatory and coinhibitory molecules (signal 2). Both signals are required for the generation of a productive T cell response and both are provided by the professional antigen presenting cell, the dendritic cell (DC). T cells are fully activated upon receiving both signal 1 and 2, but are rendered tolerant when they receive only signal 1. This can be exploited therapeutically through the administration of peptides to induce tolerance in peptidereactive T cells. Administration of peptide with an adjuvant provides both signal 1 and 2, and leads to a sustained T cell response against the administered peptide (immunity). However, if the same peptide is administered in soluble form, only signal 1 is provided, leading to the establishment of T cell tolerance. The studies in this thesis explore the role of both signal 1 and signal 2 in peptide-induced T cell tolerance. Previous data from our laboratory have highlighted PD-1 and RANKL as costimulatory molecules which could play a role in peptide-induced T cell tolerance. Here we show that PD-1, an important coinhibitory molecule, plays a vital role in restraining peripheral T cell expansion under conditions leading to T cell immunity. However, in contrast to data from other studies, we demonstrate that PD-1 plays no role in the induction, establishment or maintenance of peptide-induced T cell tolerance. We show that the costimulatory receptor ligand pair RANK:RANKL plays a role in the balance between T cell tolerance and immunity; as administration of anti-RANKL was seen to potentiate both tolerance and immunity. We also explored the effect of altering the affinity of a peptide for MHC on the induction of peptide tolerance. We demonstrate that use of a peptide with a high-affinity for MHC induces tolerance via a novel, non-deletional mechanism of peptide-tolerance induction. Importantly, we show that the high-affinity peptide can form peptide- MHC complexes which persist in a biologically relevant form for fourteen days following peptide administration. We suggest that this leads to chronic stimulation of peptide-reactive T cells which promotes acquisition of a novel tolerant phenotype. Collectively the work described in this thesis demonstrates the important roles both signal 1 and 2 play in therapeutic-tolerance induction and how the qualitative and quantitative alteration of these signals can alter T cell fate and/or responsiveness.

Les hypothèses retraçant les mécanismes physiopathologiques de la maladie de Behçet (MB), une vascularite inflammatoire non liée aux anticorps anti-cytoplasme des polynucléaires neutrophiles (ANCA), sont multiples. Cette étude propose une compilation exhaustive des mécanismes immunopathologiques décrits dans la littérature contemporaine, et fournit un résumé détaillé des aspects cliniques de la maladie et de ses différents traitements. Cette étude inclut également une analyse statistique de 20 signatures de protéines proposées comme biomarqueurs potentiels de la MB. Vingt-deux patients avec une MB active (MBA) et 46 patients avec une MB inactive (MBI), répondant aux critères de l’International Criteria for Behçet Disease (2013), ainsi que 47 donneurs sains (DR) et 98 patients subissant une angiographie coronaire (AC) ont fourni des échantillons de sérums pour une étude de dosage multiplex. Les résultats indiquent que les protéines sériques ICAM-1, SAA, THBD, et VCAM-1 jouent un rôle essentiel dans la différenciation entre les patients MB et les DR. De même, Caldesmon, Clusterin, CRP, IL-8, SELP et SICMA3 permettent un tri entre les patients MB et AC. Les modèles de signatures des biomarqueurs proposés dans cette étude et qui séparent entre les patients atteints par la MB, les DR et / ou les AC, représentent une nouvelle piste pour le développement de tests sériques pour la MB, avec une sensibilité et une spécificité élevées. Ceci peut éventuellement compléter les outils de diagnostic clinique établis. Ces résultats apportent une contribution significative à l’interprétation actuelle de la pathogénie de la MB en tant que vascularite auto-immune non-ANCA. Cette enquête fournit un bilan à la fois qualitatif et quantitatif aux cliniciens et aux chercheurs dans ce domaine. / Hypotheses concerning the specific pathophysiological mechanisms of Behçet’s Disease (BD), a non-antineutrophil cytoplasmic antibodies (ANCA) inflammatory vasculitis, are numerous. This study offers an exhaustive review of the disease in an attempt to recap the immunopathological pathways described by extant literature, and provides a detailed summary of the clinical aspects of, and treatment options for the disease. In addition, this investigation completed a statistical analysis of 20 protein signatures that were proposed as potential biomarkers for BD. Twenty-two patients with active BD (BDA) and 46 patients with inactive BD (BDI) fulfilling the International Criteria for Behçet's Disease, 47 healthy donors (HD), and 98 coronary angiography patients (CA) provided serum samples for a multiplex assay study. Findings indicate that serum proteins ICAM-1, SAA, THBD, and VCAM-1 play a significant role in differentiating BD patients from HD. Likewise, Caldesmon, Clusterin, CRP, IL-8, SELP, and sICAM-3 segregate between BD and CA. The biomarker predictive models proposed in this study that segregate between BD, HD, and / or CA represent a significant avenue for the development of sera testing specific to BD with a high level of sensitivity and specificity. This may serve as a supplement to established clinical diagnostic tools. These results represent a noteworthy complement to the current interpretation of the pathogenesis of BD as an autoimmune non-ANCA vasculitis. This investigation provides expert clinicians and researchers with both qualitative and quantitative outcomes.

Les neutrophiles contribuent à l'inflammation protectrice et pathologique. Ce projet de thèse consiste à déterminer le rôle des neutrophiles dans des modèles d'inflammation systémique graves et potentiellement mortelles, induite par le lipopolysaccharide (LPS, endotoxémie) ou par des complexes immuns antigène-anticorps (anaphylaxie). L'anaphylaxie est une réaction allergique qui peut être IgE- et/ou IgG-dépendante. L’endotoxémie est un modèle pertinent de l'inflammation au cours de maladies graves. Pour étudier les neutrophiles in vivo, nous avons utilisé un nouveau modèle murin de neutropénie inductible. Nous montrons que les neutrophiles et la Myélopéroxidase qu’ils produisent ont un rôle protecteur dans le choc endotoxique, indépendamment de l'environnement microbiologique. A l'inverse, les neutrophiles peuvent contribuer à l'anaphylaxie induite par les IgG chez la souris. Comme les récepteurs pour les IgG (FcγR) murins sont très différents des humains, nous avons développé un modèle de souris knock-in dans lequel les FcγR murins a été remplacé par les FcγR humains, activateurs et inhibiteur. Chez ces souris, nous montrons que des IgG humaines peuvent induire une anaphylaxie: le FcγRIIA a un rôle dominant, via l'activation des neutrophiles, et les médiateurs PAF et histamine. En parallèle, nous développons un modèle murin d’anaphylaxie à un curare, le Rocuronium, utilisé en clinique. Au même temps, dans une étude clinique, les résultats d’analyses des échantillons sanguins des patients suspectés d’avoir subi une anaphylaxie au curare soutien notre hypothèse de travail: que l’activation des neutrophiles par des IgG spécifiques est impliquée dans l'anaphylaxie humaine. / Neutrophils are agents of protective and pathological inflammation. This thesis work aimed to determine the role of neutrophils during severe, potentially fatal models of systemic inflammation induced by lipopolysaccharide (LPS, endotoxemia) or by IgG immune complexes (anaphylaxis). Anaphylaxis is a severe allergic reaction that may proceed via IgE- or IgG-dependant pathways. Endotoxemia is a model relevant to inflammation during critical illness. To study neutrophils in vivo, we employed a new mouse model of inducible neutropenia. We found, surprisingly, that neutrophils and neutrophil-derived MPO protect against the severity of endotoxic shock, independently of the microbiological environment, suggesting that neutrophils limit inflammation during endotoxemia. Conversely, neutrophils can contribute to IgG-induced anaphylaxis in mice. As mice and human IgG receptors (FcγR) are very different, we developed a novel mouse strain in which targeted insertion of human FcγR into the murine loci recapitulated hFcγR expression. Herein, using these mice, this work demonstrates that anaphylaxis induced by hIgG proceeds within a native context of activating and inhibitory hFcγRs, and that neutrophil activation via FcγRIIA is a dominant pathological pathway, involving the mediators PAF and histamine. Finally, we describe ongoing development of a mouse model of anaphylaxis in response to Rocuronium, a curare-based neuromuscular blocking agent (NMBA). In addition, as part of a collaborative clinical study we analysed blood samples from patients suspected of NMBA-induced anaphylaxis, finding evidence for the activation of a neutrophil- and IgG-dependent axis during human anaphylaxis.

La polyarthrite rhumatoïde (PR) est une maladie inflammatoire chronique caractérisée par des atteintes articulaires. Les synoviocytes, cellules qui tapissent la membrane synoviale, deviennent réfractaires à l‘apoptose et, en produisant la cytokine inflammatoire Interleukin 6 (IL-6), participent à la chronicité de l‘inflammation qui est à l‘origine de la perte osseuse dans la PR.Le zinc (Zn) et le cadmium (Cd) partagent leurs propriétés physico-chimiques et leurs mécanismes de transport cellulaire. Le Zn régule des fonctions du système immunitaire, contrairement au Cd avec des propriétés sur le système immunitaire peu décrites dans la littérature.Notre travail vise à identifier les mécanismes impliqués dans la modification de l‘homéostasie des métaux dans les synoviocytes par l‘inflammation et les conséquences de cette altération.L‘inflammation induit l‘accumulation des métaux dans les synoviocytes en augmentant l‘expression de l‘importeur ZIP-8. En revanche, l‘expression de l‘exporteur ZnT1 et des metallothionéines (MTs, régulateurs de l‘homéostasie des métaux) est dépendante de la présence des métaux. L‘affinité Cd-MTs permet une accumulation irréversible du Cd dans les cellules qui réduit leur prolifération et la production d‘IL-6.Les effets antiprolifératif et anti-inflammatoire du Cd ont été testés dans le modèle d‘arthrite chez le rat où une seule injection intra-articulaire de Cd à faible dose prévient la perte osseuse et réduit les scores cliniques d‘arthrite. Cela pourrait représenter une nouvelle approche thérapeutique pour le traitement de la PR et d'autres pathologies caractérisées par une hyperplasie et une inflammation localisées / Rheumatoid arthritis (RA) is a chronic inflammatory disease. Synoviocytes, cells forming the inner layer the synovium, become refractory to apoptosis and participate in the chronicity of inflammation through the production of IL-6. The perpetuation of inflammation causes an important induction of bone loss in joints.Zinc (Zn) and Cadmium (Cd) share many physico-chemical properties and cell transport mechanism. Zn is known as a regulator of the normal function of the immune system, while Cd properties on the immune system are not well defined.Our aim was to provide information on the metal homeostasis mechanisms in synoviocytes during chronic inflammation and on the consequences of metal homeostasis changes. After studying the differential effect of Zn and Cd at the cellular level, we could provide an innovative tool to control synoviocyte contribution to rheumatoid arthritis, which was tested on an in vivo model of arthritis.Results show that IL-17/TNF-a combination drives the accumulation of metals inside synoviocytes through the enhancing of ZIP-8 importer expression and regardless of the concentration of metals in the culture medium. In contrast, the expression of the metal exporter ZnT1 and of the homeostasis regulators metallothioneins (MTs) was primarily dependent on metal levels.Addition of Zn stimulated the inflammatory response, while addition of Cd can reduce both viability and inflammation.The anti-proliferative and anti-inflammatory properties of Cd were used in the rat model of arthritis as intra-articular treatment to reduce local inflammation and joint destruction and it may represent a new local therapeutic approach for RA treatment

Le sepsis est la réaction inflammatoire généralisée secondaire à une infection. C’est une pathologie fréquente et grave qui fait intervenir le système immunitaire. L’action de l’immunité innée se fait par l’activation et le recrutement des monocytes, des cellules mononuclées circulantes qui modulent le phénomène inflammatoire. La mobilisation des monocytes fait intervenir les cytokines chimiotactiques (chimiokines) et leurs récepteurs. Nous nous sommes spécifiquement intéressé dans ce travail au rôle de l’expression monocytaire des récepteurs aux chimiokines CCR2 et CX3CR1 au cours du sepsis. Pour ce faire, nous avons utilisé des modèles murins de sepsis et analysé le rôle d’un polymorphisme génétique de CX3CR1 dans une cohorte de malades atteints de sepsis. Nous avons montré qu’au cours du sepsis, les monocytes présentaient une augmentation de l’adhérence aux parois vasculaire contrôlée par le récepteur CX3CR1. Nous avons également montré que les monocytes inflammatoires jouaient un rôle essentiel dans la régulation du phénomène inflammatoire au cours du sepsis en protégeant le rein des lésions septiques. Cette protection est médiée par l’expression de CX3CR1. L’allèle I249 du gène Cx3cr1, à l’origine d’une augmentation des propriétés adhésives du couple CX3CR1/CX3CL1, est un facteur protecteur dans la survenue d’insuffisance rénale aiguë chez le malade atteint de sepsis. Collectivement, ces travaux confirment un rôle régulateur des monocytes inflammatoires au cours du sepsis et identifient de potentielles nouvelles cibles thérapeutiques. / Sepsis is the generalized inflammatory response secondary to an infection. This is a common and serious condition that involves the immune system. The action of innate immunity in sepsis is mediated by the activation and recruitment of monocytes, which are mononuclear circulating cells, which modulate the inflammatory process. The mobilization of monocytes involves chemotactic cytokines (chemokines) and their receptors. This work was specifically focused on the role of monocyte expression of chemokine receptors CCR2 and CX3CR1 in sepsis. To this end, we used mouse models of sepsis and analyzed the role of a common genetic polymorphism of CX3CR1 in a cohort of patients with sepsis.We have shown that in sepsis, monocytes’ motility was modified with an increase of their adhesion to vascular walls that was controlled in part by CX3CR1. We have also shown that inflammatory monocytes play a key role in the regulation of the inflammatory phenomenon in sepsis and that they protected the kidney from septic lesions via a CX3CR1 mediated adhesion mechanism. The I249 allele of CX3CR1, confering increased adhesive properties to monocytes, is a protective factor regarding the occurrence of acute kidney injury in septic patients. Collectively, these data confirm a a regulatory role for inflammatory monocytes during sepsis and identify potential new therapeutic targets.

As the principal antigen-presenting cells to T cells, dendritic cells (DCs) have a key role in the balance of immunity and autoimmunity. They are essential in two major, converse roles - eliciting T cell immune responses to pathogenic material, and maintaining peripheral tolerance to self-tissue by inhibiting self-reactive T cells. These functions involve the processing of pathogenic or self antigens and subsequent presentation of antigenic peptides on MHC to antigen-specific T cells. DC recognition of conserved pathogenic markers induces a mature phenotype that governs immunogenic presentation to T cells and, consequently, the adaptive immune response. In contrast, DC recognition of self tissue suppresses maturation, instead inducing a tolerogenic phenotype that induces self antigen-specific T cell to die, become anergised, or converted to T regulatory cells. Apoptotic cells are the major source of self-antigen for the maintenance of peripheral tolerance, and their defective clearance by DCs is implicated in autoimmunity. Apoptotic cells are thought to actively suppress maturation of DCs and inhibit the possible immune responses promoted by proinflammatory mediators released from necrotic cells. However, the immune function of apoptotic cells and their relative influence over necrotic cells are highly contested, partially due to the complex nature of immunogenicity arising from the sourcing and generation of apoptotic cells. In this investigation, various methods of inducing apoptosis and necrosis are evaluated. Definitive methods of inducing well-characterised cell death are then employed to compare the effects of apoptotic and necrotic cells on dendritic cells and in vitro and in vivo immune responses. Reported here are in vitro findings that support previous reports of the anti-inflammatory response of DCs to apoptotic cells, and the inflammatory response of DCs to necrotic cells. The previously-reported inhibitory effect of apoptotic cells on LPS-induced secretion of Th1 cytokines is supported here, but the inhibitory effect of apoptotic cells on LPS-induced upregulation of co-stimulatory molecules is contested. Novel findings describe the upregulation of DC expression of co-inhibitory molecules induced by both apoptotic cells and necrotic cells. Apoptotic cells, but not necrotic cells, had a suppressive effect on CpG-induced upregulation of co-stimulatory molecules and pro-inflammatory cytokines. Apoptotic cells suppressed the capacity of untreated and CpG-treated, but not LPS-treated, DCs to elicit IFNγ production by T cells. Apoptotic cells, but not necrotic cells, induced regulatory T cells and partially restored their CpG-suppressed induction. Finally, apoptotic cell-modulation of DCs inhibited the induction of autoimmunity in a novel modification of an in vivo model of diabetes. Interestingly, novel evidence for the possibility of necrotic cell-induced tolerance by means of direct T cell killing is addressed.

Pas de résumé / No abstract