Bone marrow continuously produces many B lymphocytes but the homeostatic mechanisms regulating this process are unknown. Two possible factors, the size of the B lymphocyte pool and antigenic stimulation, were examined, using radioautographic DNA-labeling with ('3)H-thymidine to quantitate lymphocyte turnover. In mice injected with anti-IgM antibodies from birth to delete all surface IgM-bearing B lymphocytes the rate of production of small lymphocytes per femur was indistinguishable from controls. In contrast, an injection of sheep erythrocytes (SRBC) in normal mice produced a transient increase in small lymphocyte turnover and increased numbers of both of immature B cells and of null small lymphocytes, as revealed by radioautographic labeling of surface IgM and Thy.1 antigen and of cytoplasmic (mu) chains. This response also occurred with other stimulants as well as in athymic and anti-IgM treated mice, but not in mice treated with silica to depress macrophage function. Chronic elevation or depression of extrinsic stimuli by repeated SRBC injection or feeding an elemental diet, respectively, produced corresponding changes in the level of the steady state of marrow lymphocyte production. The results indicate that the genesis of virgin B cells and other lymphocytes in bone marow is independent of feedback control from the peripheral B lymphocyte pool but may be amplified by environmental antigens, apparently an antigen non-specific effect mediated by macrophages.

Pristane, a pure alkane, is known to induce plasma cell dyscrasias in BALB/c mice. The present study aimed to investigate the effect of long-term dietary administration of Echinacea purpurea on immune and hemopoietic cell populations of the spleen and bone marrow, of normal and plasmacytoma-bearing mice. The results revealed quantitative cell lineage-specific changes in both the spleen and bone marrow of pristane-exposed animals. Moreover, the consumption of Echinacea resulted in cancellation of the pristane-mediated negative effects on NK cells and nucleated erythroid cells. Echinacea administration over the long-term resulted in a prolongation of the onset of old age mediated deaths by 120 days, and indeed resulted in a 30% increase in survival at age of 400 days when 55% of normal mice, consuming an untreated diet, were dead. In contrast, however, Echinacea did not improve the lifespan of plasmacytoma-bearing animals.

Delivery of transcription factor to cancer cells to reprogram gene expression may represent a novel strategy to augment the production of immune stimulatory cytokines and trigger a more potent antitumor response. In this study, we used a syngeneic mouse tumor model system involving the poorly immunogenic murine B16 tumor to evaluate whether delivery of the interferon regulatory factor 3 (IRF-3) can be used as an immunomodulator. The low immunogenicity of B16 melanoma cells may be due to their deficient cytokine expression, as well as their inefficient MHC-restricted epitope presentation. Gene-modified B16 melanoma cells were selected for their ability to express and to activate the IRF-3 protein. When injected into C57BL/6 mice, tumor growth was inhibited and tumors that developed from these mice had significant infiltration of inflammatory cells. Our observations demonstrated that gene transfer of IRF-3 into B16 melanoma could mediate important antitumor response by restoring both the deficient cytokine profile and the MHC class I protein expression.

The evaluation of human de novo T cell production is critical for a better understanding of T cell homeostasis and the immune reconstitution processes. The presence of a functional thymus post-puberty has been unsuspected due to the paucity of tools. This thesis provides direct evidence for a functional thymus, which can contribute to the diversity of the immune reconstitution in pathological situations where the immune system is severely destroyed. Through peripheral blood PCR-based quantification of TCR alpha and beta rearrangement excision/deletion circles (TREC), by-products of gene rearrangement events, we demonstrated that a diversified de novo T cell production occurs throughout life, even though thymic function decreases with age. De novo T cell production remains intact following allogenic hematopoietic stem cell transplantation (AHSCT) in the absence of graft-versus-host disease (GVHD) and therefore a reduced thymus function cannot be responsible for the long-lasting reduction in peripheral blood naive T cells observed in transplanted patients. As naive T cells from AHSCT patients have reduced levels of IL-7Ralpha chain (CD 127) expression, we propose that their low frequencies reflect an impaired naive T cell survival rather than thymic dysfunction as signaling through CD127 was previously reported to upregulate Bcl-2 expression. Evidence gathered in this thesis supports the concept that such naive T cells try to replenish themselves through enhanced levels of proliferation but fail to do so and likely die in the process. / Monitoring of the peripheral alpha and beta TREC ratio, a marker of intrathymic proliferation, demonstrated that HIV infection either induces the cellular depletion or inhibits the cell cycling of differentiating thymocytes. As intrathymic proliferation is important for both the magnitude and diversity of thymic function, the results of this thesis indicate that the replenishment of the naive T cell peripheral compartment through de novo T cell production is both quantitatively and qualitatively limited in HIV-infected individuals leading to the contraction of the peripheral T cell repertoire. / Although peripheral blood quantification of alpha and beta TREC can estimate peripheral blood RTE frequencies, reflective of thymopoiesis levels, it does not constitute a method that can lead to the characterization of this important T cell subset. To better understand the biology of RTEs, we engineered a transgene with restricted GFP expression in T cell that recently rearranged their TCR. This model would be very useful for the identification of molecules capable of modulating thymic function as well as serving as a source for obtaining a highly purified population of RTEs, then allowing the characterization of their gene expression profile. / Taken together, this thesis demonstrates the contribution of the adult thymus to immune reconstitution following AHSCT and during HIV infection.

The initiation of adaptive immune responses requires the interactions of T cells with antigen presenting cells (APC) in the context of an immunological synapse (IS). Naive T cell responses are dependent on the engagement of CD28 and CTLA-4 by CD86 and CD80, respectively amplifying and dampening the antigen specific signal. CD80 and CD86 cosignaling molecules display three major domains: a membrane distal IgV-like domain, a membrane proximal IgC-like domain and an intracellular domain. Crystallographic data has shown that only the IgV domain of CD80 and CD86 physically interacts with CTLA-4. However, extensive mutational analyses have also implicated the IgC domain in receptor binding and in the overall function of these molecules. The role of CD80 and CD86 within the IS and their exact molecular structure remains to be elucidated. The work presented in this thesis employs wild type, mutant, deleted and chimeric forms of CD80 and CD86 to characterize the role of their domains in molecular structure, receptor binding and overall cosignaling function in an antigen specific cellular interaction system. CD80 and CD86 are shown to be associated to the AFC cytoskeleton. A highly conserved K4 motif within CD86 is shown to be a cytoskeletal association motif. Moreover, CD86 is shown to physically interact with ERM proteins. Only cytoskeleton-linked CD86 localizes at the IS and induce IL-2 production. CD80 and CD86 molecular organization is clearly established using cytometry-based fluorescence resonance energy transfer (FCET) and biochemical approaches. CD80 exists as a mixed monomeric and dimeric population and CD86 as a monomer in live cells. The crucial role of CD80 and CD86 IgC domain in multimerization is revealed. Importantly, the molecular structure of these molecules correlates with their binding properties and cosignaling function. A functional picture of CD80 and CD86 domains emerges where the IgV is responsible for receptor binding, the IgC domain impacts dimerization, and the intracellular domain functionally links these proteins to the cytoskeleton. The findings presented in this thesis certainly contribute to the general understanding of cosignaling protein interactions and functions and may facilitate the design of structure-based immunotherapeutics.

Acquired immunodeficiency syndrome AIDS-related illnesses are a leading cause of infectious disease mortality worldwide. The development of a safe and effective prophylactic HIV vaccine is an imperative global public health priority fraught with significant obstacles because the answers to fundamental immunological questions remain unknown. One such gap in knowledge includes the identification of the elusive correlates of immune protection against HIV.Untreated HIV infection is characterized by severe dysregulation of the antiviral immune response that begins during the earliest stages of infection. A rare subset of HIV infected individuals demonstrate sustained ability to control HIV replication and/or maintain stable CD4+ cell counts without therapy. Determining the genetic and immunological bases underlying their benign disease course will aid in the development of novel anti-viral strategies and suggest ways the immune system can be manipulated in a vaccination setting to support the development of protective immunity. Epidemiological studies suggest that licensed NK cells may play a significant role in disease progression by associating the co-carriage of certain KIR/HLA combined genotypes with favorable disease outcomes. The projects described in this thesis provide a functional basis in support of these epidemiological data and contribute to our understanding of how interactions between protective HLA alleles and NK cell receptors may enhance the control of viremia by NK cells. In chapter II, I investigate whether T-cell immune activation levels account for the heterogeneity in longitudinal changes in the rates of CD4 counts among HIV-infected elite controllers (EC) with undetectable viral load (VL) and demonstrate that EC with protective HLA or KIR/HLA combined genotypes exhibit elevated immune activation levels which may be indicative of beneficial antiviral immune responses. Chapters III and IV explore novel mechanisms through which licensed NK cells can influence HIV disease progression by demonstrating that KIR/HLA receptor-ligand combinations affect the NK cell functional potential of HIV infected slow progressors (SP). As mediators of the innate and adaptive immune response, understanding the mechanisms that may underlie the development of protective immunity by NK cells is key. The work presented in this thesis contributes to our understanding of how protective HLA alleles interact with NK cells to influence HIV pathogenesis and provide insights as to the type of immunity an HIV vaccine should recapitulate. / Les maladies liées au syndrome de l'immunodéficience acquise (SIDA) sont principalement responsables de la mortalité par maladies infectieuses dans le monde. La mise au point d'un vaccin préventif sécuritaire et efficace contre le VIH reste un problème urgent et prioritaire détenant des obstacles majeurs pour la santé publique mondiale, car on ignore les réponses aux questions fondamentales sur l'immunologie. Un tel fossé dans la science inclut la détermination des corrélats indéfinissables quant à la protection immunitaire contre le VIH.Une rare cohorte d'individus infectés par le VIH démontre une capacité continue de limiter la réplication du VIH et/ou de maintenir un taux stable de cellules CD4+ sans traitement. Déterminer les bases génétiques et immunologiques qui sous-tendent la progression de leur maladie bénigne favorisera la création de stratégies antivirales inédites et évoquera des façons dont le système immunitaire peut être manipulé dans un contexte de vaccination dans le but d'appuyer le renforcement d'une immunité protectrice.Une infection par le VIH non traitée se caractérise par une dérégulation sévère de la réponse immunitaire antivirale qui débute pendant les premières phases de l'infection. Les études épidémiologiques avancent que les cellules NK sont susceptibles de jouer un rôle important dans la progression de la maladie en associant l'expression de certaines combinaisons des génotypes KIR/HLA avec des résultats favorables de la maladie. Les projets décrits dans cette thèse fournissent une base utile en faveur des données épidémiologiques et contribuent à notre compréhension de la manière dont les interactions entre les allèles HLA et les récepteurs des cellules NK sont susceptibles d'améliorer le contrôle de virémie par les cellules NK.Dans le chapitre II, j'examine si les niveaux d'activation immunitaire des cellules T expliquent l'hétérogénéité dans les changements longitudinaux des taux des lymphocytes CD4 parmi les « contrôleurs élite » (EC) ayant une charge virale indétectable et je démontre que ces derniers, qui possèdent des gènes HLA protecteurs ou une association des génotypes KIR/HLA, montrent des niveaux d'activation immunitaire élevés, ce qui indiqueraient que des réponses immunes antivirales sont bénéfiques. Les chapitres III et IV fournissent à l'appui de cellules NK la progression lente de la maladie et démontrent que les combinaisons des récepteurs-ligands KIR/HLA influencent le potentiel fonctionnel des patients infectés du VIH à progression lente. En tant que médiateurs des réponses immunitaires innées et adaptives, comprendre les mécanismes qui sous-tendent la progression de l'immunité protectrice des cellules NK est la clé. Le travail présenté dans cette thèse contribue à notre compréhension de la manière dont les allèles HLA interagissent avec les cellules NK pour influencer la pathogénèse du VIH et donnent un aperçu quant au type d'immunité qu'un vaccin contre le VIH devrait récapituler.

Autoimmune demyelinating diseases of the nervous system are a major cause for neurological impairment in humans. Recent evidence indicates that CD8+ T cells may contribute significantly to pathogenesis in these conditions but the mechanisms by which CD8+ T cells induce damage remain to be established. To understand the mechanisms by which CD8+ T cells may induce nervous tissue injury in vivo, we study an animal model (referred to as L31) that spontaneously develops CD8+ T cell-mediated demyelination and axonal damage in the central nervous system (CNS). In this project, we revealed that CD8+ T cells located in clusters at specific sites within the CNS of L31 mice and their location was associated with areas of demyelination. We showed that oligodendrocytes, but not neurons, from L31 mice up-regulated their expression of major histocompatibility complex class I (MHC I) molecules and had thus the potential to interact with CNS-infiltrating CD8+ T cells in a MHC I-dependent manner before clinical manifestations of disease. We reported that active caspase 3 was present in oligodendrocytes, suggesting that oligodendrocytes undergo apoptosis in L31 mice. We found that CNS-infiltrating CD8+ T cells from L31 mice were activated effector cells and we provided evidence showing that these cells degranulated in situ; nonetheless, we found that demyelination did not occur through either perforin- or Fas/FasL-dependent mechanisms. We also determined the upregulation of the chemokines CCL5 and CXCL9 and the chemokine receptor CXCR3 in the CNS of L31 mice and established that absence of CXCR3 expression was sufficient to inhibit CD8+ T cell accumulation in the CNS, microglial activation, demyelination and disease development in the L31 model, although CXCR3 deficiency did not alter the functional phenotype of transgenic CD8+ T cells. In addition, no compensatory effect for CCR5 could be detected in L31 mice. Finally, we established the presence of CD8+ T cells and tissue damage / Les maladies autoimmunitaires de démyélinisation du système nerveux sont une cause majeure pour les déficiences neurologiques chez les humains. De récentes preuves indiquent que les lymphocytes T CD8+ peuvent contribuer significativement dans la pathogénèse de ces conditions mais les mécanismes par lesquelles ces cellules induisent des dommages reste à déterminer. Pour comprendre les mécanismes par lesquelles les cellules CD8+ T peuvent induire des lésions dans le tissue nerveux in vivo, on étudie un modèle animal (nommé L31) dont la démyélinisation et l'endommagement des neurones se développent de façon spontanée, dans le système nerveux central (SNC). Dans ce projet, on a remarqué que les lymphocytes T CD8+ sont localisés en grappes aux emplacements spécifiques dans le SNC des souris L31 et ils sont associés avec des lésions de démyélinisation. On a observé que les oligodendrocytes, mais non les neurones, des souris L31 augmentent leur expression des molécules de complexe majeur d'histocompatibilité de classe I (CMH I), et ils ont le potentiel d'interagir avec les cellules T CD8+ infiltrant le SNC de manière dépendante du CMH I avant la manifestation clinique de la maladie. On a constaté que les lymphocytes T CD8+ infiltrant le SNC sont des cellules effectrices activées et on a montré que ces cellules dégranulent in situ; cependant, on a démontré que la démyélinisation ne se produise pas par des mécanismes dépendants de perforine ni de Fas/FasL. On a déterminé l'augmentation des chimiokines CCL5 et CXCL9 et le récepteur de chimiokines CXCR3 dans le SNC des souris L31 et on a établit que l'expression de CXCR3 est nécessaire pour l'activation des microglies, l'accumulation des cellules T CD8+ dans le SNC, la démyélinisation et le développement de la maladie dans les souris L31, bien que le déficit en CXCR3 n'altère pas le phénotype fonctionnelle des lymphocytes T CD8+ transgéniques. Finalement, on a confirm

Bacille Calmette-Guérin (BCG) is the only vaccine currently in use against tuberculosis. Despite its use for nearly a century, the scope of the global tuberculosis pandemic demonstrates that the vaccine is not providing the necessary protection. Improving BCG requires the identification of bacterial determinants of immunogenicity and protection, as well as analysis of how the vaccine stimulates the relevant host pathways.Work done previously by our laboratory has established that BCG daughter strains have undergone multiple deletions during its ongoing in vitro evolution. Following the initial attenuating mutation, the vaccine lost Region of Difference 2 (RD2) during on-going propagation. This is hypothesized to have resulted in the over-attenuation of the vaccine. To test this, we disrupted RD2 in Mycobacterium tuberculosis H37Rv and evaluated its phenotype in cellular and animal models. These experiments suggested candidate genes for complementation into BCG strains naturally lacking RD2. Vaccine-challenge studies and immunological assays were used to determine what role RD2 played in vaccine protection. Our findings revealed that RD2 is required for full virulence in M. tuberculosis and modulates host innate immunity. However, the presence of RD2 does not increase pulmonary protection in BCG, even though it increases the immunogenicity of the vaccine.Additionally, previous studies revealed that the host intracellular receptor NOD2 (Nuclear Oligomerization Domain 2) is critical for mediating innate and adaptive immune response to M. tuberculosis. Therefore we hypothesized that NOD2 also was important for vaccination. To test this, we vaccinated Nod2+/+ and Nod2-/- animals and then challenged them with an aerosol dose of M. tuberculosis. We characterized the effect of Nod2 disruption on bacterial burden, pathology, and adaptive immunity. Our experiments revealed that the loss of NOD2 resulted in increased pathology in vaccinated animals following infection with M. tuberculosis. Furthermore, they suggested that NOD2-mediated control of pathology is accomplished through the action of T-cells.Overall these studies further our knowledge of the in vitro evolution of the BCG vaccine, and how this affects protection. Additionally, our investigation of the role of a host receptor in vaccination highlights the importance of understanding the interactions between host and pathogen for vaccine-induced protection. / Le vaccin Bacille de Calmette-Guérin (BCG) est le seul vaccin disponible destiné à protéger contre la tuberculose. Malgré l'usage du vaccin face a la pandemie globale de la tuberculose indique qu'il ne fournit pas la protection requise. Cependant, l'ampleur de l'épidémie mondiale suggère que l'efficacité du vaccin est faible. L'améelioration du vaccin BCG néecessite l'identification de facteurs bactériens de virulence et l'élucidation de la façon dont le vaccin induit une réponse immunitaire dans l'hôte. Des travaux effectués antérieurement dans notre laboratoire ont établi que les souches filles de BCG ont subi de nombreuses délétions au cours de l'évolution continue in vitro. Suite à la première mutation, la Région de Différence 2 (RD2) est perdue pendant la propagation de la bactérie. Nous avons émis l'hypothèse que cela explique l'atténuation élevée du souche. Pour vérifier notre hypothèse, nous avons perturbé RD2 de M. tuberculosis H37Rv et évalué son phénotype dans des modèles cellulaires et animaux. Ces expériences identifient des gènes candidats pour la complémentation des souches de BCG naturellement déficientes en RD2. Des études utilisant des provocations par vaccin et des tests immunologiques ont été utilisées pour déterminer le rôle joué par RD2 dans la protection vaccinale. Nos résultats ont révélé que RD2 est nécessaire pour la virulence complète de M. tuberculosis et qu'elle module l'immunité innée. Cependant, la présence de RD2 n'augmente pas la protection pulmonaire avec BCG, même si RD2 augmente l'immunogénicité du vaccin.De plus, des études antérieures ont révélé que NOD2 (Nuclear Oligomerization Domaine 2), un récepteur intracellulaire de l'hôte, est essentiel pour la médiation des réponses immunitaires innées et adaptatives contre M. tuberculosis. Donc, nous avons supposé que NOD2 était aussi important pour la vaccination. Pour confirmer cette hypothèse, nous avons vacciné les souris Nod2 + / + et Nod2-/- , et par la suite provoqué ces souris avec une dose aérosol de M. tuberculosis. Nous avons caractérisé l'effet d'une perturbation de NOD2 sur la charge bactérienne, la pathologie, et l'immunité adaptative. Nos expériences démontrent que la perte de NOD2 mène à une pathologie aggravée chez les animaux vaccinés suite à une infection par M. tuberculosis. De plus, nos résultats suggèrent que le contrôle de la pathologie attribué à NOD2 est réalisé grâce à l'action des lymphocytes T.Les études réalisées ici améliorent notre compréhension de l'évolution in vitro du vaccin BCG et comment ce processus peut affecter la protection antibactérienne. De plus, notre enquête sur le rôle d'un récepteur de l'hôte dans la vaccination souligne l'importance de la compréhension des interactions entre l'hôte et les agents pathogènes pour la protection vaccinale.

Metalloproteinases (MPs) include the families of matrix metalloproteinases (MMPs) and metalloproteinase-disintegrins (ADAMS). MPs are implicated in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Both MS and EAE involve central nervous system (CNS) infiltration, microglial activation, and expression of chemokines and cytokines including CC chemokine ligand 2 (CCL2) and interferon-gamma (IFNgamma). MPs mediate cellular infiltration of the CNS parenchyma and regulate activity of chemokines and cytokines. This thesis describes studies of MPs in EAE and other animal models of neuroinflammation. / Gene expression of the majority of MPs was upregulated in the CNS of mice with EAE. In contrast, four of the six membrane-bound MMPs (MT-MMPs) were downregulated. MMP-8, MMP-10, MMP-12, ADAM-12, TIMP-1, and all MT-MMPs were selected for further analysis. Macrophages were identified as a major source of MMP-12 and the tissue inhibitor of MPs-1 (TIMP-1), and granulocytes as a major source of MMP-8. ADAM-12 was expressed primarily by T cells. All but one of the MT-MMPs were expressed by microglia, and three MT-MMPs were downregulated by microglia in EAE. Five of the MT-MMPs were downregulated in transgenic mice overexpressing IFNgamma specifically in the CNS. / MPs were also regulated in non-immune models of CNS infiltration that did not involve production of IFNgamma. After entorhinal cortex stab lesion, which results in prominent influx of leukocytes to the injured area, three MT-MMPs were significantly downregulated. In transgenic (Tg) mice that overexpress CCL2 specifically in the CNS, leukocytes spontaneously cross the endothelial basement membrane of the blood-brain barrier (BBB) and accumulate in the perivascular space surrounding CNS vessels, but the mice do not show clinical symptoms. Pertussis toxin (PTx) given intraperitoneally induced encephalopathy and weight loss in CCL2 Tg mice. This involved leukocyte migration across the glia limitans into the brain parenchyma. PTx induced expression of TIMP-1, ADAM-12 and MMPs 8 and 10 in brains of CCL2 Tg mice, whereas there was no significant change in expression of MT-MMPs. Weight loss and parenchymal infiltration induced by PTx were significantly inhibited by the broad-spectrum MP inhibitor BB-94/Batimastat. / These studies identify cellular sources of MPs in neuroinflammation and links stages of cellular CNS infiltration to distinct MP profiles.

Interleukin-(IL)-9 is a pleiotropic cytokine secreted by activated Th2 cells. Interleukin-9 and the α-chain of the IL-9 receptor (IL-9Rα) have been shown to affect the differentiation pathway of various cell types including human T cells, eosinophils, and mast cells, all key players in allergic inflammation. However, there is little information regarding the differentiation effect of IL-9 on B cells. Specifically, there are no reports describing the expression of the IL-9Rα on human B lymphocytes. To gain a better understanding of the effect of IL-9 on human B lymphocytes and its role in the B-Th immune synapse in the germinal center, we sought to determine the expression of the IL9Rα on human tonsillar B cells. This allows us to study the expression of the IL-9Rα on B cells at different stages of their antigen-driven maturation, in the context of B-T cell interactions. Human tonsillar B cells were fractionated, using a discontinuous Percoll density gradient, into three populations representing different stages of B lymphocyte maturation: low density (LD) (recovered in the 30-50% Percoll fraction), which represents follicular mantle (FM) B cells and germinal center (GC) B cells, medium density (MD) (50-60%), which are primarily centroblasts and centrocytes, and high density (HD) (>60%) or more mature B cells. We have determined the expression of the IL-9Rα chain by immunocytochemistry, FACS analysis and immunohistochemistry. Immunocytochemistry was performed on fresh1y isolated purified human tonsillar B cells using a monoclonal antibody to the α chain of the IL-9R. This clearly demonstrated the presence of the IL9Rα protein on tonsillar B cells. Using FACS analysis, unfractionated as well as fractionated B cells showed positive immunoreactivity. However, IL-9Rα expression was predominantly higher in the LD fraction of the B cells (39%), as compared to the MD (22%), and HD fractions (16%). These findings prompted us to further investigate the precise localization of the IL-9Rα positive B cells within the germinal centers. For this purpose, immunohistochemistry was performed on sections of human tonsils. Using double staining with anti-IL-9Rα and anti-CD20 (Alkaline Phosphatase anti-Alkaline Phosphatase APAAP) as well as with anti-IL-9Rα and anti-CD19 (immunofluorescence), we found that CD20+/CD19+ B cells within the secondary lymphoid follicles, and especially cells on the edge of these follicles, displayed IL-9Rα. Furthermore, analysis of CD38 and IgD expression on IL-9Rα positive cells by three color flow cytometry showed that IL-9Rα was expressed on FM B cells (CD38-IgD+) and GC cells (CD38+Igb+). Upregulation of phosphotyrosine levels in LD cells stimulated with IL-9 as well as signal transducers and activators of transcription (STATs) phosphorylation was detected by immunoblotting and flow cytometry respectively. The results show a specific upregulation of STAT-5 and STAT3 phosphorylation in LD cells and not in total B cells, MD or HD cells. In order to better define the effect of IL-9 on follicular B cells, we hypothesized that IL-9 played a role in the B-Th immune synapse in GC by promoting germinal center isotype switching, and affecting IgE production consequently. In order to directly assess the effect of IL-9 on IgE production, LD cells, which are germinal center cells expressing the highest level of IL9Rα in our analysis, were stimulated with an antibody to CD40 and IL-9 in the presence or absence of IL-4 and IgE production was measured by ELISA. Although IL-9 was unable to induce IgE secretion by itself, it potentiated IgE production mediated by IL-4. This suggests a synergistic role for IL-9 with IL-4 in modulating IgE production and B cell differentiation. This was accompanied by an upregulation of IL-9R and CD27 expression on LD cells throughout the culture period specifically following CD40 and IL-4 stimulation. No further increase in expression levels was observed in the presence of IL-9. Furthermore, CD27 upregulation occurs almost entirely on IL-9R positive cells, pointing to an important role for IL-9 downstream of CD40 and IL-4 signaling in B cell differentiation. The observed increase in IgE production was not due to an increase in cell number as IL-9 was not able to potentiate IgM-activated LD B cell proliferation, nor anti-CD40-activated LD B cell proliferation. Moreover, no synergistic effect for IL-9 on IL-4 induced LD B cell proliferation was observed. However, IL-9 protected LD B cells from Fas-induced apoptosis, suggesting an important role for IL-9 in B cell survival. In summary, IL-9 synergizes with IL-4 to augment IgE production in LD cells, it does so by enhancing cell survival rather than by increasing cell number. These data show for the first time that a functional IL-9Rα chain is expressed on human follicular germinal center B cells. Taken together, our studies establish a new understanding for the role of IL-4, a cytokine linked to increased susceptibility to allergy and asthma, in B cell maturation and IgE production.