The Early Growth Response genes 2 and 3 (Egr2/3) are zinc finger transcription factors that play an important role in the immune system. These transcription factors have reported functions in T cell receptor signaling, differentiation of effector T cell subsets and the development of lupus-like autoimmune diseases. Using CD2-Egr2-/- Egr3-/- mouse model, I investigate the development of inflammation pathology, differentiation of T follicular helper (Tfh) cells and the formation of germinal centers (GC) following viral challenge within these mice. The onset of inflammation pathology in CD2-Egr2-/- Egr3-/- mice was discovered to correlate with high levels of pro-inflammatory cytokines in the serum and the development of autoimmune diseases as previously reported by Li et al, 2012. Most importantly, a novel role for the Egr2/3 genes in the differentiation of T follicular helper (Tfh) cells was identified. Tfh cells are responsible for T cell dependent antibody immune response in the GC. They support the differentiation of GC B cells into plasma cells producing long lived high-affinity isotype-switched antibodies and memory B cells. Tfh cell differentiation is regulated by Bcl6 however; the regulators of Bcl6 during Tfh differentiation remain largely unknown. We have now discovered that Egr2/3 genes are required for Bcl6 expression during Tfh cell differentiation. In the absence of the Egr2 and 3 genes, Tfh cell differentiation is severely impaired and GC formation and functions were defective in response to Vaccinia Virus Western Reserve strain (VVWR) infection. Further investigation revealed that Egr2 regulated Bcl6 expression in a Tfh-specific manner as adoptive transfer of WT CD4+ T cells into Egr2-/- Egr3-/- mice was able to rescue Bcl6 expression, Tfh differentiation and GC formation. When the molecular mechanism of how Egr2 regulated Bcl6 was investigated, it was uncovered that Egr2 directly bound to the promoter region of Bcl6 gene in CD4 T cells to regulated Bcl6 expression. Indeed constitutive expression of either Egr2 or Bcl6 in CD2-Egr2-/- Egr3-/- CD4+ T cells rescued Tfh cell differentiation and GC formation. Our results inferred that the Egr2/3 genes are essential for Tfh differentiation and GC formation by regulating Bcl6 expression in CD4 T cells under Tfh condition. Our studies thus suggest that the Egr2/3 genes are paramount for minimising immunopathology and are also critical for efficient antibody production by regulating Tfh cell differentiation.

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.

Germinal center (GC) reactions are central features of T cell-driven B cell responses, and the site where antibody (Ab) producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of high affinity Abs. These processes require exquisite regulation not only to ensure the production of desired Abs, but to minimize unwanted autoreactive or low affinity Abs. To assess whether T regulatory cells (Treg) participate in the control of GC responses, immunized mice were treated with either an anti-glucocorticoid-induced TNFR-related protein (GITR) mAb or an anti-CD25 mAb to disrupt Treg activity. In both groups of treated mice, the GC B cell pool was significantly larger compared with control treated animals, with switched GC B cells composing an abnormally high proportion of the response. With these results indicating Tregs influence on GC dynamics, experiments were conducted to determine if Tregs were located in the GC, which subset of Treg was involved and by which mechanisms were their functions being effected. Within the spleens of immunized mice, CXCR5+ and CCR7- Tregs were documented by flow cytometry and Foxp3+ cells were found within GCs using immunohistology. Studies demonstrated administration of either anti-TGF-β or anti-IL-10R blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Tregs is important in controlling the GC response. Blockade of two Treg methods of suppression, PD-1/PD-L1 pathway and CTLA-4, also resulted in disrupted GCs, indicating the possible use of them for suppression by Treg. Collectively, these findings indicate that Tregs contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs.

Indiana University-Purdue University Indianapolis (IUPUI) / Human immunodeficiency virus (HIV) primarily infects T helper (Th) cells. Decrease in the number of Th cells is the hallmark of HIV infection. Latent reservoirs of human immunodeficiency virus (HIV) are the leading barrier towards eradication of HIV infection. T Follicular helper (Tfh) cells are a subset of Th cells that function to provide aid to B cells for their maturation, affinity selection and antibody class switch. Several studies have shown that Tfh cells are a major reservoir of latent as well as productive hiv infection. But in contrast to the fate of other Th cell subsets, the frequency of Tfh cells was shown to have increased during HIV infection which could not be attributed to their reduced susceptibility to HIV infection. The hypothesis was that Tfh cells possess a unique metabolic phenotype that protects them from HIV induced cell death. Transcriptome analysis of Th subsets from human donors and showed that Tfh cells rely less on glycolysis for their energetic requirements and instead have increased transcription of fatty acid synthesis genes. This finding was corroborated by seahorse extracellular flux assay. The results shoId that glycolysis was not essential for Tfh cell differentiation in-vitro. The observed increase in Tfh cell frequency could not be attributed to increased Tfh differentiation upon HIV infection since HIV infection inhibited the differentiation of both non-Tfh and Tfh cells. The results found that bypassing the glycolytic pathway by providing Tfh cells with Galactose in the medium protected ex-vivo infected primary tonsillar cells from HIV induced cell death. This protection could be partly explained by the induction of Baculovirus IAP repeat containing 5 (BIRC5) when the cells utilized Galactose instead of Glucose. The studies together show that Tfh cells have an oxidative metabolic phenotype which protects them from HIV induced cell death in part by induction of BIRC5 expression.

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Les cellules T folliculaires helper (Tfh) jouent un rôle central dans la mise en place de réponses humorales efficaces. En effet, les Tfh participent à la sélection des lymphocytes B permettant le développement de lymphocytes B mémoires et d’anticorps de haute affinité. Les Tfh représentent ainsi une cible prometteuse pour la mise en place de nouvelles stratégies thérapeutiques, notamment pour augmenter l’efficacité de la vaccination. Ainsi, il apparaît crucial de mieux comprendre les étapes menant à leur développement, en particulier chez l’Homme. L’initiation de la polarisation Tfh se déroule dans les organes lymphoïdes secondaires et met en jeu les cellules myéloïdes mononucléaires (MMC). Les MMC présentes dans les organes lymphoïdes comprennent les macrophages résidents et trois sous populations de DC résidentes : les cDC1 (CD141+), les cDC2 (CD1c+) et les pDC. Nous nous sommes plus particulièrement intéressés au rôle respectif des sous populations de MMC humaines dans l’induction de la polarisation Tfh. Ainsi, les travaux effectués au cours de ma thèse avaient pour objectifs dans un premier temps d’analyser la capacité des différentes populations de MMC à induire la polarisation Tfh, afin de mettre en évidence de potentielles spécialisations fonctionnelles. Dans un second temps, nous nous sommes concentrés sur les mécanismes moléculaires impliqués dans l’induction par les MMC de la polarisation Tfh. Nous avons montré une spécialisation fonctionnelle des cDC2 et des macrophages des amygdales pour la polarisation Tfh. Toutefois, des différences ont été observées entre les cDC2 et macrophages, puisque les macrophages induisent la sécrétion par les lymphocytes T d’une grande quantité de CXCL13 par rapport au cDC2, qui sont plus efficaces pour induire la production d’IL21. Nous avons pu également montrer que les cDC2 et macrophages sécrétaient des cytokines précédemment identifiées comme ayant un rôle dans l’induction des Tfh telles que IL12p70, ActivinA et TGFβ. Afin de confirmer le rôle de ces cytokines dans la polarisation induite par les MMC d’amygdales, nous avons utilisé des anticorps bloquants dans nos expériences de polarisation T helper. Ainsi, nous avons confirmé le rôle de l’IL12p70, de l’Activin A et du TGFβ dans l’induction des Tfh humains. Nos résultats suggèrent également un rôle de l’Activin A et de TGFβ dans l’induction de la sécrétion de CXCL13, alors que l’IL12p70 serait impliqué dans l’induction de la sécrétion d’IL21. Nos résultats suggèrent aussi l’existence de deux sous populations de Tfh caractérisées soit par l’expression d’IL21 soit par l’expression de CXCL13. Les travaux réalisés au cours de ma thèse enrichissent ainsi les connaissances sur la spécialisation fonctionnelle des sous populations de DC et des macrophages humains, et apportent de nouveaux éléments pour la compréhension de la différenciation des Tfh humains. / T follicular helper cells (Tfh) play a key role in the establishment of efficient humoral responses. Indeed, Tfh are involved in B lymphocyte selection allowing the development of high affinity memory B cells and antibodies. Tfh are promising targets for new therapeutic strategies, especially to increase the effectiveness of vaccination. Thus, it is crucial to better understand the stages leading to their development, especially in human. Initiation of Tfh polarisation occurs in secondary lymphoid organs and involves mononuclear myeloid cells (MMC). MMC from secondary lymphoid organs include resident macrophages and three subsets of resident Dendritic Cells (DC): cDC1 (CD141+), cDC2 (CD1c+) and pDC. We were particularly interested in human MMC subsets respective roles in the induction of Tfh polarisation. Thus, the work carried out during my thesis aimed first at analysing the ability of different populations of MMC to induce Tfh polarisation, in order to highlight potential functional specialisations. In a second step, we focused on the molecular mechanisms involved in Tfh polarisation by MMC. We have shown a functional specialisation of cDC2 and tonsillar macrophages for Tfh polarisation. However, differences have been observed between cDC2 and macrophages, since macrophages induce secretion by T cells of a large amount of CXCL13 compared to cDC2, which are more effective in inducing IL21 production. We have also been able to show that cDC2 and macrophages secreted cytokines previously shown to play a role in Tfh induction such as IL12p70, ActivinA and TGFβ. In order to confirm the role of these cytokines in Tfh polarisation induced by tonsil MMCs, we used blocking antibodies in our T helper polarisation experiments. Thereby, we confirmed the role of IL12p70, Activin A and TGFβ in the induction of human Tfh. Our results also suggest a role for Activin A and TGFβ in inducing secretion of CXCL13, whereas IL12p70 would be involved in the induction of IL21 secretion. Besides, our results suggest the existence of two Tfh subsets characterised by expression of either IL21 or CXCL13. The work performed during my thesis broadens the knowledge on the functional specialisation of human DC subsets and macrophages, and provides new insight into the differentiation of human Tfh.

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.

La réponse humorale est altérée lors de l’infection par le virus de l’immunodéficience humaine (VIH). Les lymphocytes T CD4+ folliculaires helper (Tfh) sont impliqués dans la maturation des lymphocytes B (LB) dans les organes lymphoïdes secondaires. Mon travail de thèse s’est articulé autour de deux axes complémentaires visant à étudier les Tfh et les réponses B mémoires lors de l’infection par le VIH. J’ai d’abord étudié les Tfh spléniques lors de la phase chronique de l’infection par le VIH. J’ai mis en évidence une augmentation des populations de Tfh dans les rates de patients VIH+. D’autre part l’infection par le VIH a un impact sur le profil transcriptionnel des Tfh de la rate et la production de cytokines impliquées dans la différenciation des LB, suggérant un défaut fonctionnel des Tfh. Parallèlement, la maturation des LB est altérée dans les rates VIH+. Dans le second axe de ma thèse, j’ai étudié les réponses B mémoires anti-VIH dans différentes cohortes de patients VIH+ : Elite controller (EC) contrôlant l’infection sans traitements et des patients VIH+ traités. J’ai mis en évidence que les EC préservent naturellement leurs compartiments B mémoires et que les réponses B mémoires spécifiques du VIH sont maintenues dans le sang de ces patients. Les réponses B mémoires IgG1+ anti-VIH sont majoritaires chez les EC, tandis que les réponses IgG2+ et IgG3+ sont plus rares. Ces travaux permettent une meilleure compréhension de la physiopathologie de l’infection par le VIH en apportant de nouveaux éléments sur la fonctionnalité des Tfh et les réponses B mémoires anti-VIH. / HIV infection is associated with a defect of humoral response. T follicular helper cells (Tfh) support multiple steps of B cell maturation and antibody production. My work was divided in two complementary axes aiming to characterize Tfh and memory B cell responses in HIV-infected patients.I identified several Tfh populations in HIV+ and HIV- spleens by FACS. These three populations were increased in HIV+ spleen. I also evidenced an impact of HIV infection on transcriptional profile and a compromised production of B cell differentiation-related cytokines by splenocytes from HIV+ donors. These results suggest Tfh functions impairment during HIV-infection. In parallel, we noticed an altered maturation of B cells in HIV+ spleens. In a cohort study, we compared memory B cell responses in the blood of Elite controllers (EC) who naturally control HIV and treated HIV+ patients. I evidenced that EC naturally preserve their memory B cell compartments. In contrast to anti-HIV IgG2 and IgG3 secreting B cells, most EC exhibit a high frequency of anti-HIV IgG1 secreting B cells. My work highlights a defective Tfh differentiation, which might explain why B cell maturation is severely affected in HIV-progressors. The status of HIV-controller seems associated with the presence of an IgG1 B cell memory response. Further work will highlight whether Tfh functions are preserved in EC.

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.