<p>Helminth exposure, allergy and asthma each induce cellular responses in lymphoid and peripheral tissues that give rise to type-2 inflammation. Essential molecular mediators of this response are type-2 cytokines interleukin(IL)-4 and IL-13 derived from various subsets of immune cells. In lymphoid tissues, CD4+ Tfh cells make IL-4 to elicit IgE and high-affinity IgG1 production. In peripheral sites of infection, group 2 innate lymphoid (ILC2) cells make IL-13 and Th2 cells make both IL-13 and IL-4. Together, these cells mediate smooth muscle contraction, mucus production and recruitment of other innate effector cells, all of which are hallmarks of type-2 inflammation. As central mediators of type-2 inflammation, understanding the cell-specific expression and molecular regulation of type-2 cytokines in CD4+ T cells and ILC2 cells may lead to new therapies that ameliorate allergic disease and helminth infections. </p><p>The AP-1 factor basic leucine zipper transcription factor ATF-like (BATF) has been identified as a pioneer factor in in vitro-generated Th17 cells. BATF facilitates chromatin remodeling at the IL-17 locus as well as loci of key Th17-associated lineage specifying factors. It has also been deemed essential to the generation of functional humoral immunity through the development of follicular helper T (Tfh) cells and germinal center B cells. However, the role of BATF in the development and function of other CD4+ T helper subsets and innate immune cells in vivo has remained unclear. I show here that mice deficient in BATF do not develop type-2 inflammation after exposure to the parasitic helminth Nippostongylus brasiliensis. Since type-2 cytokine expression by Th2 and ILC2 cells is essential for expedient helminth expulsion, I hypothesized that BATF likely has a role in the development and/or induction of cytokine expression in CD4+ Th2 cells and ILC2 cells. Consistent with this hypothesis, I found that BATF utilizes a novel mechanism to control Th2 cytokine expression in Th2 cells. Specifically, BATF promotes permissive epigenetic modifications to alter the chromatin landscape early during Th2 cell differentiation. In addition, my data show that BATF deficiency inhibits the activation of ILC2 cells, preventing ILC2-mediated helminth clearance. </p><p>In addition to uncovering BATF-mediated regulations of type-2 inflammation, my work has revealed new insight into the role of a second bZIP transcription factor, cMaf, during type-2 immunity. As mentioned above, helminth exposure elicits IL-4 production by both CD4+ Tfh and Th2 cells. Although type-2 cytokine transcription has been well characterized in Th2 cells, Tfh cell-mediated IL-4 production has yet to be fully defined. Importantly, I show that IL-4 production by Tfh cells is sustained upon deletion of classical IL-4 regulatory factors signal transducer and activator of transcription 6 (STAT6) and STAT5 and is not dependent on high GATA-3 expression. In sum, Tfh-driven IL-4 production is induced independent of classical pathways in Th2 cells. </p><p>Presently, the non-canonical transcription factors involved in IL-4 production by Tfh cells remain unclear. C-Maf works with BCL6, the master regulator of Tfh cells, to elicit Tfh formation. However, the precise role of c-Maf in Tfh cell fate and function remains unclear. So far, it has been shown that in Th2 cells, c-Maf binds to the IL-4 promoter and in Tfh cells, c-Maf binds to the CNS2 enhancer of the IL-4 locus to regulate IL-4 expression. Therefore, I hypothesized that c-Maf is important in non-canonical, GATA-3-independent IL-4 production by Tfh cells. </p><p>Here, I show that Tfh cells lacking canonical Th2 pathways for IL-4 expression express high levels of c-Maf and IL-4 transcript. Deletion of c-Maf in CD4+ T cells resulted in normal induction of BCL6 expression. Thus the initial stages of Tfh cell generation were induced. However, cMaf-deficient CD4+ T cells did not express important molecules associated with Tfh cell migration. Immunohistochemistry also confirmed that c-Maf deficiency inhibited CD4+ T cell migration from the paracortex into the B cell follicle. </p><p>These defects did not inhibit cMaf-deficient CD4+ T cells from making IL-4 transcript; however, IL-4 protein production was significantly impaired. Together, these results demonstrate that c-Maf is essential for Tfh cell-mediated immunity by promoting CD4+ T cell migration to the B cell follicles and the production of IL-4 protein in the germinal centers. </p><p>Collectively, the objective of my thesis research is to define the roles of the bZIP transcription factors BATF and c-Maf in type-2 inflammation. My data demonstrate that BATF is essential for the differentiation and function of Tfh, Th2, and ILC2 cells during helminth infection. Additionally, I have shown that c-Maf is required for Tfh function and CD4+ T cell migration to the B cell follicle. Thus, BATF and c-Maf are central to the development of humoral and peripheral type-2 inflammatory responses against helminth infection. Given the wide spectrum of disorders associated with type-2 inflammation, the identification of factors relevant to the development and function of Th2-, ILC2- and Tfh-driven allergic pathologies is broadly relevant. A comprehensive characterization of core factors like BATF and c-Maf provide new avenues in which to explore novel therapies to modulate type-2 inflammatory responses.</p> / Dissertation

PARV4 is a novel human parvovirus initially identified in an intravenous drug user at risk of HIV infection. PARV4 is a small single stranded DNA virus principally absent from the general population, but common in HCV- and HIV-infected individuals. Until 2009, most published PARV4 studies related to the prevalence of PARV4 in various risk groups. PARV4 has been detected in the liver of HCV-patients and the bone marrow of HIV-patients. Parvovirus B19, the closest related virus, elicits a strong immune response and can cause serious disease. Thus, this project was initiated to characterise the immune response to PARV4, and investigate the clinical significance of this virus. Cohorts of HCV-infected, HIV-infected, HCV-HIV co-infected, healthy and acute parvovirus B19-infected individuals were screened for humoral and cellular responses in both acute and chronic PARV4 infection. HCV- and HIV-related disease progression was also assessed relative to PARV4 infection. This study demonstrates that the highest prevalence of PARV4 infection is found in HCV-HIV co-infected intravenous drug users, and provides additional evidence for parenteral transmission. I present here the first data on the cellular immune response to PARV4 in acute and chronic infection and define PARV4 as a persistent virus. Although no clear correlation could be found between PARV4 and HCV or HIV disease progression, the high prevalence rates emphasize the importance of investigations into emerging infections such as PARV4.

HCV infects 170 million persons worldwide and is a serious global health problem. Genotype-3a is the dominant genotype in newly diagnosed infections within the UK and has a high response rate to interferon therapy, with up to 70% patients achieving a sustained virological response (SVR). The reason(s) for this are unknown; therefore the aim was to assess host and viral factors that determine treatment outcome of subtype-3a infection. Full-length subtype-3a viral sequence analysis identified 2 novel regions of hypervariability within E2 - HVR495 and HVR575, that are subject to positive selection pressure. A 5 amino-acid insertion found only in subtype-3a and a putative glycosylation site were contained within HVR575. These data suggest that HVR495 and HVR575 may serve as major antigenic sites in subtype-3a HCV infection. Successful treatment of chronic subtype-3a infection was not associated with pre-treatment quasispecies diversity and complexity, PePHD, HVR495 or HVR575 sequence. Different patterns of quasispecies variation were observed in patients that failed treatment. Subtype-3a specific CD8+ T-cell responses in chronic infection target non-structural proteins, in contrast to pre-dominant genotype-1 core-specific CD4+ T-cell responses. SVR was associated with a decline in subtype-3a specific and non-specific T-cell responses, and also total lymphocyte counts, which all recovered after treatment. These data do not support the theory that clearance of subtype-3a is associated with an enhancement of antiviral T-cell responses. Overlapping peptides detected a greater number of subtype-3a T-cell responses compared with peptides representing putative predicted CD8 epitopes. Therefore subtype-3a HCV is distinct from genotype-1 in terms of genome sequence, effect of treatment on quasispecies and subtype-3a specific T-cell responses, further emphasising the importance in understanding this distinct subtype.

The current worldwide epidemic of Mycobacterium tuberculosis infection is a huge global health problem. Widespread BCG vaccination remains a useful tool in combating this epidemic; however, its variable efficacy requires urgent development of novel vaccines against Mycobacterium tuberculosis. Such a candidate vaccine is a serotype 5 adenovirus expressing antigen 85A from M. tuberculosis (Ad85A). In animal models Ad85A confers significant protection when administered intra-nasally. The work in this thesis demonstrates that intra-nasal immunisation with Ad85A results in inhibition of M. tuberculosis growth in the lung early after infection, in contrast to the late inhibition induced by parenterally administered vaccines. Early inhibition correlates with the presence in the lung of a highly activated population of antigen-specific CD8 T cells, maintained for at least 6 months post-immunisation by persistent antigen. For intra-nasal Ad85A to be effective, the vaccine must be delivered into the lower respiratory tract, as immunisation targeting only the nasal-associated lymphoid tissue (NALT) does not result in protection. Following a change of animal facility, the lung immune response to intra-dermal immunisation with Ad85A increased and this route of immunisation now induced protection, though growth of M. tuberculosis was inhibited only late after infection. However, this response and protection can be altered by exposure to environmental mycobacteria. Further experiments showed that simultaneous respiratory and parenteral immunisations (SIM) act additively, where local lung immunity inhibits the growth of M. tuberculosis early after infection and systemic immunity protects later. SIM regimes generate greatly improved protection over either immunisation alone and do not depend on priming and boosting.

T cells are an essential part of the adaptive immune system and protect the body from intracellular infections. The specificity with which αßTCR-bearing T cells recognize cognate antigen presented on MHC molecules is paramount to maintaining the balance between mounting effector functions against pathogens and establishing peripheral tolerance to self. The mechanism by which T cells translate qualitative differences in TCR:pMHC binding to sensitive proximal signalling events which ultimately result in specific Tcell effector responses to infected cells but not to self is mostly unknown. To address how T cell signalling responds to qualitative differences in TCR triggering by pMHC, I established a system of stimulating T cells bearing the 1G4 TCR specifically in vitro with a panel of four NY-ESO-1_156-165 peptide variant MHC tetramers. Single amino acid substitutions to the NY-ESO-1_156-165 peptide conferred a maximum 35-fold difference in the monomeric affinity for the 1G4 TCR. The system allows the highly controlled investigation of very rapid TCR proximal signalling events simultaneously and quantitatively using flow cytometry. Stimulations with pMHC tetramers showed rapid sensitive sequential signalling responses which were able to confer ligand discrimination. Very early signalling events such as CD3ζ phosphorylation showed analogue responses to the different affinity pMHC tetramers. Later signalling events including phospho-ERK showed a distinct on/off switch-like response. The amplitude of the very early analogue signalling responses determined the extent of later digital ERK signals. This indicates that a certain analogue signalling threshold must be passed to result in T cell activation. The thymocyte protein Themis has been shown proximal TCR signalling to modulate thymocyte selection thresholds. Its deletion results in profound defects in positive thymocyte selection. Themis locates to the LAT signalosome of the TCR signalling cascade via Grb2, yet its molecular function is unknown. Employing the system I established, I demonstrate that Themis-k/d cells show increased levels of CD3z-chain phosphorylation, phospho-ERK signalling and signal-induced apoptosis which was independent of the ERK signal. This shows that Themis is a global attenuator of proximal TCR signalling. We are currently investigating possible associations of Themis to proteins phosphastases such as SHP-1 which could attenuate TCR proximal protein tyrosine signalling events.

Protein-protein recognition plays a central role in the surveillance of self and non-self in the mammalian immune system and ultimately in cellular survival within the organism. Two systems of fundamental importance to the immune system are the Tumour Necrosis Factor (TNF) and the T cell receptor (TCR) families. High-throughput methods developed within the Oxford Protein Production Facility have been successfully applied to the production of members of the TNF receptor and ligand superfamilies for structural characterisation. The TNF receptor DR6 was successfully refolded from E.coli inclusion bodies using a rapid-dilution technique and yielded diffraction quality crystals. Data collected from these crystals will be used to obtain an x-ray crystallographic model of DR6. Vascular Endothelial Growth Inhibitor (VEGI) was produced as a soluble recombinant protein in E.coli, and formed a number of poorly diffracting crystals, it is hoped that further trials and optimization of conditions will lead to improved data quality. Lymphotoxin β receptor was produced in a Eukaryotic system. This has shed light on the complications posed by signal peptide cleavage and glycosylation on the production of protein for crystallization trials. TNF superfamily proteins are ideal targets for the design of novel therapeutic agents due to their involvement in a number of disease pathologies. Various methods of molecular docking and small molecule design were applied to the search for potential inhibitors of receptor binding for the TNF ligand proteins TRAIL and BAFF. A number of potential drug leads were identified from the National Cancer Institute drug database. The Natural Killer (NK) T cell restricted TCRs recognise CD1d-presented glycolipid. Determination of the crystal structures of the invariant NK TCR and the NK restricted TCRs 5E and 5B shows that these proteins adopt the canonical structures of class I MHC restricted TCRs. This suggests that the binding of CD1d-glycolipid by these receptors will conform to the same model of binding seen for the class I MHC restricted TCRs.

<p>Dysregulated inflammation underlies the pathogenesis of a myriad of human diseases ranging from cancer to autoimmunity. As coordinators, executers and sentinels of host immunity, T cells represent a compelling target population for immune-modulation. In fact, the antigen-specificity, cytotoxicity and promise of long-lived of immune-protection make T cells ideal vehicles for cancer immunotherapy. Interventions for autoimmune disorders, on the other hand, aim to dampen T cell-mediated inflammation and promote their regulatory functions. Although significant strides have been made in targeting T cells for immune-modulation, current approaches remain less than ideal and leave room for improvement. In this dissertation, I seek to improve on current T cell-targeted immunotherapies, by identifying and preclinically characterizing their mechanisms of action and in vivo therapeutic efficacy.</p><p>CD8+ cytotoxic T cells have potent antitumor activity and therefore are leading candidates for use in cancer immunotherapy. The application of CD8+ T cells for clinical use has been limited by the susceptibility of ex vivo-expanded CD8+ T cells to become dysfunctional in response to immunosuppressive microenvironments. To enhance the efficacy of adoptive cell transfer therapy (ACT), we established a novel microRNA-targeting approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor Blimp-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR-23a was upregulated in tumor-infiltrating CD8+ T cells, and its expression correlated with impaired antitumor potential of patient CD8+ T cells. We determined that tumor-derived TGF-&#946; directly suppresses CD8+ T cell immune function by elevating miR-23a and downregulating Blimp-1. Functional blockade of miR-23a in human CD8+ T cells enhanced granzyme B expression; and in mice with established tumors, immunotherapy with just a small number of tumor-specific CD8+ T cells in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CD8+ T cells that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF&#946;-mediated tumor immune-evasion pathway.</p><p>Having established that miR-23a-inhibition can enhance the quality and functional-resilience of anti-tumor CD8+ T cells, especially within the immune-suppressive tumor microenvironment, we went on to interrogate the translational applicability of this strategy in the context of chimeric antigen receptor (CAR)-modified CD8+ T cells. Although CAR T cells hold immense promise for ACT, CAR T cells are not completely curative due to their in vivo functional suppression by immune barriers &#8210; such as TGF&#946; &#8210; within the tumor microenvironment. Since TGF&#946; poses a substantial immune barrier in the tumor microenvironment, we sought to investigate whether inhibiting miR-23a in CAR T cells can confer immune-competence to afford enhanced tumor clearance. To this end, we retrovirally transduced wildtype and miR-23a-deficient CD8+ T cells with the EGFRvIII-CAR, which targets the PepvIII tumor-specific epitope expressed by glioblastomas (GBM). Our in vitro studies demonstrated that while wildtype EGFRvIII-CAR T cells were vulnerable to functional suppression by TGF&#946;, miR-23a abrogation rendered EGFRvIII-CAR T cells immune-resistant to TGF&#946;. Rigorous preclinical studies are currently underway to evaluate the efficacy of miR-23a-deficient EGFRvIII-CAR T cells for GBM immunotherapy. </p><p>Lastly, we explored novel immune-suppressive therapies by the biological characterization of pharmacological agents that could target T cells. Although immune-suppressive drugs are classical therapies for a wide range of autoimmune diseases, they are accompanied by severe adverse effects. This motivated our search for novel immune-suppressive agents that are efficacious and lack undesirable side effects. To this end, we explored the potential utility of subglutinol A, a natural product isolated from the endophytic fungus Fusarium subglutinans. We showed that subglutinol A exerts multimodal immune-suppressive effects on activated T cells in vitro: subglutinol A effectively blocked T cell proliferation and survival, while profoundly inhibiting pro-inflammatory IFN&#947; and IL-17 production by fully-differentiated effector Th1 and Th17 cells. Our data further revealed that subglutinol A might exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells, but not in innate immune cells or fibroblasts. Additionally, we demonstrated that subglutinol A significantly reduced lymphocytic infiltration into the footpad and ameliorated footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of subglutinol A as a novel therapeutic for inflammatory diseases.</p> / Dissertation

During the course of infection with chronic pathogens such as Hepatitis C virus (HCV), Hepatitis B virus (HBV) and HIV, virus-specific CD8^&plus; T cells differentiate into heterogeneous dysfunctional subpopulations. Advances in multi-parameter flow cytometry have allowed these subpopulations to be further classified into classes of exhausted T cells, primarily by their expression of multiple inhibitory receptors. However, the exact phenotype of CD8^&plus; T cells during exhaustion is an area of great interest as many inhibitory receptors are also expressed on functional CD8^&plus; T cells. Discovering novel and specific markers of T cell exhaustion is fundamental in developing strategies to restore CD8^&plus; T cell function in chronic viral infections. Recently, genome wide expression profiles have identified broad molecular phenotypes in exhausted T cells that could not have been discovered by flow cytometry alone. I show how similar genomic approaches identify and further characterise the ectonucleotidase CD39 as a novel marker of CD8^&plus; T cell exhaustion in chronic viral infection. I show that CD39 is highly expressed in HCV and HIV-specific CD8^&plus; T cells and that CD39^&plus; CD8^&plus; T cells are enriched with gene signatures of exhaustion. CD39 is highly co-expressed with multiple inhibitory receptors including PD-1, enzymatically active on CD8^&plus; T cells in HCV infection and positively correlated with viral load in both HCV and HIV. I also demonstrate the discovery of a novel CD39^High population of cells in the mouse model of chronic Lymphocytic Choriomenigitis Virus (LCMV) infection, which express the highest degrees of PD-1, LAG3 and 2B4 in the CD39^&plus; fraction. Thus, CD39 is a novel and specific marker of severe CD8^&plus; T cell exhaustion in human and animal models of chronic viral infection.

Les lymphomes T cutanés constituent un ensemble hétérogène de lymphomes non-hodkinniens. Les lymphomes T cutanés sont définis par une prolifération clonale de lymphocytes T malins et de cellules NK (natural killers) de localisation cutanée. Dans l'Union Européenne, l'indication du bexarotène par voie orale est le traitement des manifestations cutanées des lymphomes cutanés T épidermotropes (LCT), au stade avancé (ou dès un stade précoce aux États Unis). L'objectif de ce travail a été d'étudier les effets indésirables du bexarotène chez les patients atteints de lymphome cutané. Nous nous sommes plus particulièrement intéressés aux interactions du bexarotène avec le système de la coagulation et avec le métabolisme lipidique.Dans la première partie de nos travaux, nous avons étudié l'origine de la coagulopathie induite par le bexarotène. Nous avons montré que le bexarotène inhibe les facteurs IX et X de la coagulation, ce qui provoque le prolongement du temps de coagulation ; ces effets pourraient être à l'origine de la coagulopathie observée chez les patients traités au bexarotène.Dans la deuxième partie, nous avons montré que le bexarotène interagit également avec le métabolisme lipidique par l'activation du CETP et par l'inhibition de la lipoprotéine lipase, ce qui provoque une chlolésterémie et une triglyéridémie.Enfin, nous avons présenté les résultats préliminaires de l'essai clinique organisé au niveau de la région ouest de la France sur les effets indésirables du bexarotène.Les résultats de ce travail mettent en évidence l'importance de la structure chimique du bexarotène (effets de charges) dans la neutralisation des facteurs de coagulation d'une part et dans le mécanisme d'interaction avec le métabolisme lipidique (CETP et lipoprotéine lipase) d'autre part. La compréhension des mécanismes moléculaires conduisant à une coagulopathie et à la dyslipidémie représente un enjeu majeur pour prévenir la toxicité du bexarotène et pour permettre une meilleure prise en charge du patient. / The cutaneous T cell lymphomas are a heterogeneous group of non-lymphoma hodkinniens. The cutaneous T cell lymphomas are defined by a clonal proliferation of malignant T cells and NK (natural killer) cells from skin location. In the European Union, the indication of oral bexarotene is the treatment of cutaneous manifestations of cutaneous T-cell lymphomas epidermotropic (LCT) to advanced (or at an early stage in the United States). The objective of this work was to study the adverse effects of bexarotene in patients with cutaneous lymphoma. We are particularly interested in the interactions of bexarotene with the coagulation system and lipid metabolism.In the first part of our work, we studied the origin of the coagulopathy induced by bexarotene. We have shown that bexarotene inhibited factors IX and X of the coagulation causing the extension of clotting time and coagulopathy that was observed in bexarotene-treated patients. In a second part, we showed that Bexarotene induces in vitro cholesteryl ester transfer protein activity, and suppress lipoprotein lipase activity in human plasma.Finally, we presented the preliminary results of the clinical trial conducted at the western region of France on the adverse effects of bexarotene.The results of this study highlight the importance of the chemical structure of bexarotene (charge effects) in the neutralization of coagulation factors mechanisms on the one hand and in its interaction with lipid metabolism (CETP and lipoprotein lipase) on the other hand. Understanding the molecular mechanisms leading to coagulopathy and dyslipidemia is a major issue to prevent the toxicity of bexarotene and to elicit better management of the bexarotene-treated patient.

<p>CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.</p><p>microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases. </p><p>To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis. </p><p>A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.</p><p>Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.</p> / Dissertation