Protection from influenza A virus (IAV) challenge requires switched, high affinity Abs derived from long-lived memory B cells and plasma cells. These subsets are generated in germinal centers (GCs), hallmark structures of T helper cell-driven B cell immunity. A full understanding of the acute and persistent GC B cell reaction following respiratory IAV infection is lacking, as is the characterization of IAV-induced T follicular helper (TFH) cells that support GCs. Additionally, it remains unclear as to whether IAV-induced GC B cells are subject to control by regulatory T cells (Tregs). To address this, GC B cell and TFH cell responses were analyzed in mice following pulmonary challenge with IAV. Studies demonstrated that marked GC reactions were induced in lung-draining lymph nodes (dLNs), lung, spleen and nasal-associated lymphoid tissue (NALT), although the magnitude, kinetics, and isotype switching patterns of the response was site-specific, and largely depended on the magnitude of IAV-induced TFH cell populations. TFH cell magnitude peaked prior to that of GC B cells in all tissues, and TFH cells purified from dLNs generated IL-21 and IFN-gamma upon activation, although CD4+CXCR5- T effector cells produced higher levels of all cytokines. IgA+ GC B cells were infrequent in most sites, but composed a significant subset of the switched GC population in NALT. Further, splenectomized mice withstood a lethal recall challenge, suggesting the spleen to be unnecessary for long-term protection. Additionally, GC B cell populations were analyzed at distal time points to assess the understudied, persistent GC B cell response after IAV infection. Our analysis demonstrated that persistent GC B cell populations in mouse lungs directly correlated with infectious dose, pathogenicity of the virus, as well as the presence of long-term CD4+ T cell help. Finally, experiments showed that Tregs contribute to the control of GCs induced in the spleen by IAV challenge. This was demonstrated by a marked increase in the number of total and switched GC B cell numbers when Tregs were either depleted or disrupted in vivo proximal to IAV exposure.

La vaccination du tissu cutané présente un fort potentiel, car elle permet le ciblage de l’antigène aux populations de cellules dendritiques uniques et spécialisées de la peau, et le recrutement de cellules inflammatoires du sang.Les cellules T helper folliculaires (TFH) jouent un rôle crucial dans l’établissement de la réponse humorale. Cependant, les interactions cellulaires et moléculaires qui gouvernent leur induction dans un contexte de vaccination restent à élucider.Mon projet de thèse a eu pour but de mieux comprendre les mécanismes d’induction des réponses TFH et humorales, par l’étude des événements précoces ayant lieu aux sites d’immunisation et d’induction de l’immunité adaptative après vaccination cutanée. L’utilisation de modèles murins nous a permis d’évaluer la contribution de différentes populations de la peau, du ganglion, et du sang dans la mise en place de ces réponses après immunisation intradermique avec un antigène particulaire présentant l’antigène modèle p24 du VIH. Cette étude a révélé un rôle crucial des cellules de Langerhans et des cellules dendritiques migratoires de la peau dans l’induction de réponses TFH et humorales.Nous avons ensuite évalué la capacité de différentes formulations d’adjuvants à polariser la réponse TFH et humorale contre un antigène de l’enveloppe du VIH à fort potentiel vaccinal. L’utilisation de l’émulsion IFA favorise l’induction des cellules TFH et induit la production d’anticorps neutralisants des souches du VIH.Ces résultats soulignent l’importance de cibler les DCs de la peau par l’utilisation de voies de vaccination pertinentes et l’utilisation d’adjuvants capables de favoriser la réponse cellulaire TFH. / Skin vaccination is of great interest, as it enables targeting of the antigen to unique and specialized dendritic cell populations of the skin, as well as recruitment of inflammatory blood cells.T follicular helper (TFH) cells play a critical role in the setting of the humoral response. However, the cellular and molecular interactions that underlie their induction after vaccination remain unknown.My thesis project aimed at understanding the immune mechanisms by which skin vaccination could favor the induction of TFH and humoral immune responses by studying the early events that take place in tissue and lymph node.Using mice models, we evaluated the relative contributions of various populations from the skin, lymph node and blood in the setting of TFH cell responses after intradermal immunization with nanoparticles coated with p24 antigen from HIV. This revealed a crucial role of Langerhans cells and skin migratory dendritic cells in the induction of TFH and germinal center responses.We then evaluated the ability of different adjuvant formulations to polarize the TFH and humoral response against a promising vaccine antigen from HIV envelope protein. Emulsifying the antigen in IFA favors the induction of TFH cells and induces the production of neutralizing antibodies able to block viral infection.This work highlights the relevance of targeting skin dendritic cells by using relevant vaccination routes and adjuvant formulation able to induce TFH cell responses.

During the course of an immune response, CD4+ T helper cells differentiate into a number of subsets including: T helper 1 (TH1), TH2, TH17, and T follicular helper (TFH) populations. The functional diversity of CD4+ T effector cells results in a coordinated, pathogen-specific immune response. For example, the production of IFNγ by TH1 cells is vital for the clearance of intracellular pathogens, while TFH cell engagement with cognate B cells is required for germinal center (GC) formation and the generation of pathogen- and vaccine- induced antibody production. The development of CD4+ subsets is contingent on extracellular signals, in the form of cytokines, and downstream transcriptional networks responsible for promoting the unique gene expression profile for each subset while simultaneously suppressing alternative cell fates. However, the exact composition of, and stage-specific requirements for, these environmental cytokines and transcription factor networks in the governance of TFH cell differentiation remain incompletely understood. The work in this dissertation seeks to understand how cell-extrinsic cytokine signals and cell-intrinsic transcription factor activities are integrated to properly regulate TFH cell development. Here, we demonstrate that in response to decreased IL-2 and constant IL-12 signaling, T helper 1 (TH1) cells upregulate a TFH-like phenotype, including expression of the TFH lineage defining transcription factor Bcl-6. Intriguingly, our work established that signals from IL-12 were required for both the differentiation and function of this TFH-like population. Mechanistically, IL-12 signals are propagated through both STAT3 and STAT4, leading to the upregulation of the TFH associated genes Bcl6, Il21, and Icos, correlating with increased B cell helper activity. Conversely, exposure of these TFH-like cells to IL-7 results in the STAT5-dependent repression of Bcl-6 and subsequent inhibition of the TFH phenotype. Finally, we describe a novel regulatory mechanism wherein STAT3 and the Ikaros zinc finger transcription factors Ikaros and Aiolos cooperate to regulate Bcl-6 expression in these TFH-like cells. Collectively, the work in this dissertation significantly advances our understanding of the regulatory mechanisms that govern TFH cell differentiation, setting the basis for the rational design of novel immunotherapeutic strategies and increasingly effective vaccines. / Ph. D. / Specialized cells called T helper cells serve as a critical interface between the innate (first line of defense) and adaptive (specialized and long-term) immune systems. During the course of an infection, T helper cells are responsible for orchestrating the immune-mediated elimination of invading viruses, bacteria, and parasites. This wide breadth of functionality is achieved through the formation of distinct T helper subsets including T helper 1 (TH1), TH2, TH17, and T follicular helper (TFH) populations. Individual subsets have distinct developmental requirements and have unique functions within the immune system. For example, TFH cells are required for the production of effective antibodies that recognize invading pathogens, leading to their subsequent elimination. This naturally occurring process is the basis for a number of modern medical therapies including vaccination. Conversely, aberrant generation of antibodies that recognize host tissues can result in the onset of various autoimmune diseases including lupus, multiple sclerosis, and crohn’s disease. Due to the importance of TFH cells to human health, there is intense interest in understanding how these cells are formed. It is recognized that the generation of these therapeutically important immune cells is mediated by numerous cell-extrinsic andintrinsic influences, including proteins in their cellular environment called cytokines, and important proteins inside of the cell called transcription factors. However, as this is a complicated and multi-step process, many questions remain regarding the identity of these cytokines and transcription factors. The work in this dissertation seeks to understand how cellextrinsic cytokine signals and cell-intrinsic transcription factor activities are integrated to properly regulate TFH cell development. Collectively, this body of work significantly advances our understanding of the regulatory mechanisms that govern TFH cell differentiation, setting the basis for the rational design of novel immunotherapeutic strategies and increasingly effective vaccines.

The ability of cells within the adaptive immune system to develop into specialized subsets allow for a robust and tailored immune response in the advent of an infection or injury. Here, CD4+ T-cells are a crucial component within this system, with subsets such as TH1, TH2, TH17, TFH and TREG cells playing vital roles in propagating cell-mediated immunity. For example, TH1 cells are essential in combating intracellular pathogens such as viruses, while TFH cells communicate with B-cells to optimize antibody responses against an invading pathogen. The development (and functionality) of these subsets is ultimately dictated by the appropriate integration of extracellular cues such as cytokines with cell intrinsic transcription factors, thereby promoting the necessary gene profile. Moreover, the observation that T-helper cells could exhibit a flexible nature (i.e having shared gene profiles and effector functions) not only demonstrate the efficiency of our immune system but also how such flexibility could have unintended consequences during adverse events such as autoimmunity. An important mediator of such flexibility is cytokines. However, the complete network of factors that come together to co-ordinate cytokine mediated plasticity remain unknown. Thus, the work in this dissertation hope to delineate the factors that collaborate to regulate cytokine induced T-helper cell flexibility. As such, we see that in the presence of IL-2, the Ikaros Zinc Finger (IkZF) transcription factor Eos is upregulated in TH1 cells, with this factor playing a significant role in promoting regulatory and effector functions of TH1 cells. Moreover, we show that Eos forms a novel protein complex with STAT5 and promotes STAT5 activity in TH1 cells. However, depleting IL-2 from the micro-environment leads to the upregulation of two other members within the IkZF family, Ikaros and Aiolos. Aiolos in turn collaborate with STAT3, induces Bcl-6 expression within these cells, thus promoting these cells to exhibit characteristic features of TFH cells. The work in this dissertation hopes to advance our understanding of the regulatory mechanisms involved in cytokine mediated T-cell flexibility thereby hoping to open new avenues for the development of novel therapeutic strategies in the event of autoimmunity. / Ph. D. / T-helper (TH) cells are an important component of the immune system, as these cells aid in the fight against pathogens by secreting factors that either accentuate the inflammatory response during infection or attenuate immune responses post infection. Such effects are made possible because T-helper cells can differentiate into a variety of subsets, with each subset being an important mediator in maintaining immune homeostasis. For example, the T-helper cell subset called TH1 plays a vital role in the fight against intracellular pathogens such as viruses and certain parasites, while T-follicular helper (TFH) cells aid in the production of antibodies specific to the invading pathogen. The development of such subsets occur when cell extrinsic signals, called cytokines, lead to the activation or induction of cell intrinsic proteins called transcription factors. Interestingly, research over the years have shown that T-helper cells are highly adaptable in nature, with one subset having the ability to attain certain characteristic features of other subsets. This malleable nature of T-helper cells relies on several factors, with cytokines within the micro-environment being an important one. Although this form of flexibility is efficient and beneficial at times, it can also be detrimental, as such flexibility is known to promote certain autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and type 1 diabetes. Such detrimental effects are thought to be due to cytokines within the environment. Therefore understanding how cytokines influence the flexible nature of T-helper cells is important; as controlling such flexibility (either by regulating cytokines or the transcription factors activated as a consequence) could prevent the propagation of undesired T-helper cell functions. As such, the work in this dissertation hopes to uncover how one such cytokine, termed Interleukin-2 (IL-2) mediates the flexibility between TH1 and TFH cells. The work highlighted in this dissertation broadens our understanding of how cytokines influence T-helper cell development and flexibility, and consequently allows the design of novel therapeutic strategies to combat autoimmune diseases.

Le lymphome angioimmunoblastique à cellules T (AITL) est un lymphome périphérique à cellules T agressif dont les symptômes sont la lymphadénopathie et l'hypergammaglobulinémie. Actuellement, les patients atteints du AITL ont des options de thérapeutiques limitées et des résultats cliniques défavorables, avec un taux de survie sur 5 ans d'environ 30%. Les cellules tumorales du AITL proviennent de cellules T CD4+ appelées cellules T auxiliaires folliculaires (Tfh). Les cellules Tfh sont essentielles dans le centre germinatif (GC), où elles facilitent l'expansion et la différentiation des cellules B en plasmocytes. Cette fonction d'aide est soutenue par de nombreuses protéines dérivées des cellules Tfh et des programmes de transcription qui pourraient aussi fonctionner dans les cellules tumorales du AITL. Par conséquent, la perturbation des principaux mécanismes de signalisation soutenant l'identité des cellules Tfh et leurs interactions avec les cellules B pourrait inhiber la croissance du AITL. Des études ont démontré que les cellules hyperactives de type Tfh provoquent une accumulation de cellules immunitaires telles que les cellules B, les plasmocytes et les macrophages dans les tumeurs. Cependant, le microenvironnement du AITL n'a pas été bien étudié et il n'a pas été vérifié si certaines cellules immunitaires pourraient être utilisées pour arrêter la croissance de la tumeur. Bien que l’on trouve des cellules Tfh circulantes dans l’AITL humain, le taux de propagation peut varier d’un patient à l’autre. Ainsi, une possibilité est la présence de mécanismes de surveillance immunitaire s'opposant à la progression de la tumeur. En accord avec cette hypothèse, un signal positif pour la phagocytose nommé SLAMF7 (contrebalancé par la voie inhibitrice CD47-SIRPα) est exprimé dans un sous-ensemble de patients atteints du AITL. Toutefois, la corrélation entre les différents niveaux d'expression du SLAMF7 et l'amélioration des résultats pour les patients n'a pas été étudiée. En utilisant des souris Roquinsan/+, qui développent spontanément l’AITL, nous avons étudié le rôle des mécanismes de signalisation immunitaire dans les cellules tumorales de type Tfh et du microenvironnement tumoral. Nous avons cherché à inhiber les protéines et les voies de signalisation typiques des cellules Tfh dans les tumeurs afin d'évaluer la valeur thérapeutique potentielle. Nous avons aussi étudié le rôle de la phagocytose dépendante des macrophages dans le contexte SLAMF7 et comment la modulation de la signalisation de CD47-SIRPα peut améliorer l'efficacité de la phagocytose des cellules tumorales. Notre hypothèse centrale est qu'en supprimant les programmes fondamentaux des cellules Tfh ou en favorisant l'élimination phagocytaire des cellules tumorales de type Tfh, nous pouvons favoriser la régression de la tumeur. Nous avons démontré que les tumeurs AITL nécessitent des protéines d’identité des cellules Tfh essentielles telles que le facteur de transcription Bcl6 et la protéine adaptatrice SAP, ainsi que la communication entre les cellules T et B (T-B). Même en l'absence de GC classiques, les cellules tumorales de type Tfh ont apporté un soutien aux cellules B. Cela est démontré par des titres élevés d'IgG et l'accumulation de cellules précurseurs des plasmocytes dans les tumeurs. Nous avons trouvé des preuves de l'opposition entre la surveillance immunitaire et l'évasion au sein des tumeurs de type AITL, car les cellules Tfh augmentent l’expression de la molécule inhibitrice CD47 tandis que les macrophages stimulent le niveau de SLAMF7. Les cellules de type AITL ont été phagocytées plus efficacement in vitro quand la signalisation du CD47 était bloquée. En résumé, nous démontrons que les voies de signalisation importantes pour l'identité des cellules Tfh et la communication entre les cellules T et B sont essentielles pour la progression de l’AITL et suggèrent qu’une surveillance immunitaire continue par les macrophages peut influencer l’évolution de la maladie. Des études futures pourraient explorer la possibilité de combiner des inhibiteurs de l'activité des cellules Tfh ou T-B avec des médicaments qui stimulent l'activité phagocytaire antitumorale pour améliorer l'efficacité thérapeutique du traitement. / Angioimmunoblastic T cell lymphoma (AITL) is an aggressive peripheral T cell lymphoma manifesting with symptoms such as generalized lymphadenopathy and hypergammaglobulinemia. Currently, AITL patients have limited treatment options and poor clinical outcomes with a 5-year survival rate around 30%. AITL tumor cells derive from a subset of CD4+ T cell, the T follicular helper (Tfh) cell. Tfh cells are essential in germinal centers (GC), where they facilitate B cell expansion and differentiation into plasma cells. This helper function is supported by numerous Tfh cell-derived proteins and transcriptional programs which may still be operational in AITL tumor cells. Therefore, disrupting key signaling mechanisms sustaining Tfh cell identity and their ability to interact with B cells could inhibit AITL tumor growth. Studies have demonstrated that these hyperactive Tfh-like cells lead to the accumulation of immune cell subsets such as B cells, plasma cells, and macrophages within tumor lymph nodes. Nevertheless, the AITL tumor microenvironment itself has not been well-studied and whether some immune cells could be harnessed to impede tumor growth has not been tested. In human AITL, although circulating Tfh cells have been reported, the rate of tumor spreading can vary between patients. As such, one possibility is the presence of immune surveillance mechanisms opposing tumor progression. In line with this idea, SLAMF7, a positive signal for macrophage-mediated phagocytosis (counterbalanced by the inhibitory CD47-SIRPα pathway), is expressed in a subset of AITL patients. Despite this, whether differing levels of SLAMF7 expression correlates with improved patient outcomes has not been investigated. Using Roquinsan/+ mice, a spontaneous AITL-like mouse model, we addressed the role of immune signaling mechanisms within Tfh-like tumor cells and the surrounding tumor microenvironment that would promote tumor regression. First, we aimed to inhibit signature Tfh cell proteins and downstream signaling pathways in developed AITL-like tumors to evaluate potential therapeutic value. Second, we investigated the role of macrophage-mediated phagocytosis in the context of SLAMF7 and how modulating CD47-SIRPα signaling may enhance the efficiency of AITL tumor cell engulfment. Our central hypothesis is that by removing fundamental Tfh cell supporting programs from tumor cells or by promoting the phagocytic removal of Tfh-like tumor cells we can favour tumor regression and impair future growth. Through this work, we demonstrated that AITL-like tumors continuously require critical Tfh cell identity proteins such as transcription factor Bcl6 and adaptor protein SAP, as well as T cell-B cell (T-B) crosstalk. Importantly, despite the absence of conventional GCs, Tfh-like tumor cells provided functional support to B cells as evidenced by elevated IgG titers and accumulation of plasma cell precursors in tumors. We also found evidence of opposition between immune surveillance and evasion within AITL-like tumors as Tfh-like cells upregulated inhibitory CD47 levels while macrophages increased expression of prophagocytic SLAMF7. Moreover, AITL-like tumor cells were more efficiently phagocytosed in vitro when CD47 signaling was blocked. Taken together, we demonstrate that pathways important for Tfh cell identity and T-B communication are critical for AITL-like disease progression and suggest that ongoing macrophage-mediated immune surveillance may influence disease outcomes. Future studies may explore combining inhibitors of Tfh cell activity or T-B crosstalk along with drugs which boost antitumor phagocytic activity to further improve the therapeutic efficacy of treatment.

La réponse immunitaire de type humorale se déclenche suivant certaines infections virales et bactériennes de même que suivant une immunisation. Au niveau cellulaire, ce type de réponse favorise la formation de petites structures, nommées centres germinatifs (CG), qui se développeront dans les organes lymphoïdes secondaires (OLS) tels que la rate et les ganglions. Ces CG sont orchestrés par la présentation des antigènes étrangers par les cellules dendritiques et les cellules dendritiques folliculaires (FDC), aux cellules T et B respectivement, ainsi que par des interactions complexes survenant entre ces lymphocytes T et B. Suivant ce processus, les lymphocytes B quittant les CG se différencieront soient en plasmocytes sécréteurs d’anticorps de fortes affinités ou en cellules B mémoires qui assureront une protection lors d’une seconde exposition face à un antigène étranger ayant précédemment été rencontré. Plusieurs évidences suggèrent que la qualité de la réponse humorale est influencée par des variants génétiques. Par exemple, des études quantifiant les titres d’anticorps suivant la vaccination ont observé que ces titres variaient en fonction de différents groupes ethniques. Toutefois, malgré ces évidences, la contribution de la génétique quant à la variation des aspects cellulaires de la réponse humorale demeure incomplète. En utilisant douze lignées de souris génétiquement éloignées, nous avons donc évalué l'impact de la variabilité génétique sur les aspects cellulaires de cette réponse humorale, et ce, à l'état d'équilibre et suivant l’immunisation avec un antigène étranger. Pour ces deux conditions, nous avons quantifié, par cytométrie en flux, le nombre ainsi que la composition cellulaire (cellules B, plasmocytes et cellules T auxiliaires folliculaires) des CG contenus dans plusieurs OLS ainsi que dans la moelle osseuse des différentes lignées de souris. Après immunisation, le positionnement cellulaire au sein des CG de la rate a également été évalué par immunofluorescence. Nos résultats indiquent que le nombre et la taille des CG après immunisation ainsi que la composition cellulaire de ces CG à l’état d’équilibre et suivant l’immunisation varient entre les différentes lignées de souris à l’étude. Comme les douze lignées de souris ont été soumises aux mêmes conditions, ces résultats suggèrent que les variants génétiques, étant différents d’une lignée de souris à une autre, sont responsables des variations que nous avons observées au niveau des aspects cellulaires de la réponse humorale. Ce projet permettant de mieux comprendre l’impact de la variabilité génétique sur certains aspects de la réponse humorale pourrait ultimement mener à une amélioration des approches vaccinales chez les individus répondant moins bien à un certain type de vaccination. / The humoral immune response is triggered following certain viral and bacterial infections as well as following immunization. At the cellular level, this type of response promotes the formation of small structures, called germinal centers (GC), which develop into secondary lymphoid organs such as the spleen and lymph nodes. These GC are orchestrated by the presentation of foreign antigens by dendritic cells and follicular dendritic cells (FDC), to T and B cells respectively, and by subsequent interactions between these T and B lymphocytes. Following this process, B cells leaving the GC will differentiate into high-affinity antibody-secreting plasma cells or memory B cells that will provide protection upon a second exposure to a previously encountered foreign antigen. There is some evidence to suggest that the quality of the humoral response is influenced by genetic variants. For example, studies quantifying antibody titers following vaccination have observed that these titers vary across different ethnic groups. However, despite this evidence, the contribution of genetics to the variation of the cellular aspects of the humoral responses remains incomplete. Using twelve genetically divergent mouse strains, we therefore evaluated the impact of genetic variability on the cellular aspects of this humoral response at steady state and following immunization with a foreign antigen. For these two conditions, we quantified, by flow cytometry, the number as well as the cellular composition (B cells, plasma cells and T follicular helper cells) of the GC contained in several SLO and in the bone marrow of the different mouse strains. After immunization, cell positioning within the GC of the spleen was also assessed by immunofluorescence. Our results indicate that the number and size of GC after immunization as well as the cellular composition of these GC at steady state and following immunization vary between the different mouse strains studied. As the twelve mouse strains were subjected to the same conditions, these results suggest that the genetic variants, being different from one mouse strain to another, are responsible for the variations that we observed in the cellular aspects of the humoral response. This project, which allows us to better understand the impact of genetic variability on some aspects of the humoral response, could ultimately lead to an improvement in vaccine approaches in individuals who respond less well to a certain type of vaccination.