Antiphospholipid syndrome (APS) is defined by the presence of antiphospholipid antibodies (aPL) and clinical manifestations. It is unclear why some patients have high levels of aPL, but no clinical symptoms, while others develop APS. Recent studies have demonstrated T cell reactivity to beta2-glycoprotein I (beta2GPI) in patients with APS. To understand why a T cell response develops selectively in some individuals the requirements for antigen presentation and T cell reactivity were evaluated in a murine model of aPL induction. aPL were readily induced in immunized mice, and required MHC class II, but not CD1, antigen presentation. In contrast, induction of a measurable T cell response to beta2GPI necessitated hyperimmunization in the presence of a polyclonal activator. T cells reacted more to reduced, than native, beta2GPI, suggesting that disulfide bond reduction facilitates antigen processing. These results suggest that a T cell response to beta2GPI occurs only in individuals repeatedly exposed to antigen in a proinflammatory environment.

Proinflammatory cytokines by Th1 cells and macrophages are involved in the pathogenesis of several organ-specific autoimmune diseases. Clinically, cytokine therapy has the potential of influencing disease outcome by altering the balance between proinflammatory versus immunosuppressive cytokine profiles. Somatic cytokine gene therapy is an attractive alternative to cytokine immunotherapy, because it eliminates the need for frequent protein injections, and generates more constant cytokine levels in vivo which may reduce toxicity and increase therapeutic efficacy. To study the immunoregulatory effects of TGF-beta1 and IL-4 in vivo, we used a novel method of i.m. cytokine gene therapy in 2 experimental models of Th1 cell-mediated autoimmune diseases: murine diabetes and EAE. In our first model, i.m. TGF-beta1 gene administration is effective at suppressing a DTH response, and at protecting NOD mice from autoimmune insulitis and diabetes. In this model, disease protection was associated with a decreased intrapancreatic IL-12 and IFNgamma mRNA expression. In our second model, TGF-beta1 and IL-4-IgG1 gene therapy resulted in anti-encephalitogenic effects in mice with MBP-induced EAE. TGF-beta1 gene delivery had pronounced downregulatory effects on MBP-stimulated T cell proliferation and production of IFN-gamma and TNF-alpha. IL-4-IgG1 gene delivery also suppressed these responses and enhanced endogenous secretion of IL-4. Cytokine gene therapy resulted in a decrease in the severity of CNS inflammatory lesions. With either treatment, CNS IL-12 and IFNgamma mRNA expression was significantly diminished, while IL-4 and TGF-beta1 mRNA levels were increased compared to control mice. In summary, i.m. delivery of cytokine plasmid vectors suppressed pathogenic Th1 responses, while enhancing the production of protective, regulatory cytokines in peripheral lymphoid and/or target tissues. Somatic cytokine gene therapy proved to be an effective therapeutic strategy of cytokine deliv

A molecular interplay between the signaling components utilized by cytokine receptors, the pre-T cell receptor (pre-TCR), and the T cell receptor (TCR) is presumed to be required for proper regulation of thymocyte development. The earliest intrathymic progenitors are known to depend on cytokine signaling. However, the mechanisms regulating cytokine signaling during thymopoiesis have not been characterized. Moreover, the contribution of cytokine signaling to the maturation of CD4-CD8- thymocytes into CD4+CD8+ thymocytes, mainly controlled by the pre-TCR, is uncertain. Overexpression of SOCS-1, a negative regulator of cytokine signaling, in developing thymocytes resulted in decreased proliferation following pre-TCR signaling, revealing a novel role for cytokine signaling at this developmental stage. Moreover, pre-TCR signaling negatively regulates SOCS-1 expression, establishing a link between the pre-TCR and mechanisms regulating cytokine signaling. During the later stages of thymocyte development, signals delivered by the pre-TCR and TCR assume a dominant regulatory role. Although structurally similar to the TCR, the pre-TCR possesses distinct biological properties. The major difference between these receptors is the presence of the pTalpha chain in the pre-TCR, which is structurally different from the TCRalpha chain present in the TCR. The connecting peptide domain of pTalpha regulates the interaction between the pre-TCR and the TCR&zgr; subunit. In addition, the structural differences between pTalpha and TCRalpha constitute a post-translational regulatory mechanism to prevent the coexpression of pre-TCR and TCR at the cell surface of CD4+CD8+ thymocytes, as pTalpha cannot pair with TCRbeta in the presence of TCRalpha. The competitive advantage of TCRalpha relates to the position of the bridging cysteine residue within its connecting peptide domain. Thus, the structural features of pTalpha regulate the assembly and surface expression of the pre-TCR, and endow it

The HIV-1 Nef protein is a virulence factor and major determinant of pathogenicity of the virus. Nef impairs T cell function by uncoupling TcR signaling from antigen stimulation and by down regulating CD4 on the cell surface. In Nef infected thymocytes, the activity of intracellular T signaling molecule Lck is increased in basal conditions and attenuated with stimulation. This phenomenon contributes to partial T cell activation allowing viral replication meanwhile preventing full activation of the cell in order to prevent cell death. In addition, Nef infection results in intracellular accumulation of Lck and delocalization from its regular compartments. In the first part of this study we use biochemical techniques and confocal microscopy to study other proteins that are involved in the tightly regulated Lck localization and activity in a normal thymocytes to attempt to understand the pathway in the context of Nef infection. We find that the delocalization of Lck disrupts the regulatory feedback loop of the protein and subsequently interferes with the activation of downstream mediators. TcR signaling and Lck are also important for thymic selection and progression from CD4+CD8+ double positive to CD4+ and CD8+single positive thymocytes. In the second part of this study, we show that the loss of CD4+ thymocytes results in the loss of an incredibly important mediator of central tolerance, the transcription factor AIRE. We correlate this loss of AIRE to an impairment of maturation of thymic epithelial cells and a profound loss of expression of tissue restricted antigens necessary for inducing thymic tolerance. Such loss in the thymus is directly correlated to autoimmunity in mouse models and human studies. This study proposes a novel mechanism for autoimmunity frequently observed in patients with HIV/AIDS. / La protéine Nef du VIH-1 joue un rôle important dans la pathogenèse du virus en modulant les voies de signalisation du récepteur de la lymphocyte T et en baissant l'expression du corécepteur CD4 à la membrane. Ainsi, dans une cellule infectée par Nef, l'activation de la molécule Lck est augmentée avant la stimulation de la cellule et diminuée après stimulation par rapport une cellule normale. Cette activation partiale faitque le virus peut répliquer en évitant l'apoptose. De plus, Nef mène à une accumulation intracellulaire de la protéine Lck ce qui la délocalise de ses compartiments habituels. Dans la première partie de cette étude, nous employons des essais biochimiques ainsi que la microscopie confocale afin d'étudier les protéines que sont impliquées dans la régulation précise de Lck pour que nous puissions comprendre sa signalisation sous le contrôle de Nef. Nous avons trouvé que la délocalisation de Lck perturbe la rétroaction régulatrice de la protéine ce qui interfére avec l'activation des messagers secondaires. La signalisation du lymphocyte T et de la protéine Lck sont importantes pour la séléction des thymocytes et la progression des cellules doubles positives CD4+ CD8+ aux cellules simples positives CD4+ ou CD8+. Dans la deuxième partie de ce mémoire, nous démontrons que la perte des thymocytes CD4+ mène à une perte d'une protéine essentielle à la tolérance centrale immunitaire, AIRE. Nous avons lié cette perte d'AIRE à une déficience des cellules épithéliales médullaires matures. Une telle perte est directement corrélée à l'autoimmunité dans les modèles de souris et les études chez l'homme. Notre étude suggère un nouveau mécanisme pour l'autoimmunité que l'on voit souvent chez les patients qui sont atteints du VIH-1.

Food allergy, an immune-mediated adverse hypersensitivity reaction to ingested food, is an emerging global health problem that not only causes the disease to susceptible individuals but also causes psychological impacts and financial damage to their families. Food allergy incidence is significantly increased in the past decade and currently estimated at 6-8% in 1 year old children, and 2-4% in older children and adults. Food allergy is the leading cause of anaphylaxis in children and severe cases may lead to fatal anaphylaxis reactions. Although continuous efforts have been made to better understand the nature of allergens, predisposing environmental factors, and the host's immune response mechanisms, there is no cure available for food allergy today. In this study, we have investigated three food allergy reducing strategies by supplementing probiotics, low doses of allergens and omega-3 poly unsaturated fatty acids (ω-3 PUFAs) in cow's milk protein sensitized Balb/c mice. Lactobacillus rhamnosus GG (LGG) supplementation had tendency to promote Th1 response while VSL#3 provided more potent allergy reducing effects via inducing intestinal secretory IgA (sIgA). Low doses of allergen administration offered suppression of allergen-specific immune responses via Treg-mediated active suppression, indicated by suppressing both allergen-specific Th1 response [reduced BLG-specific serum IgG2a and elevated IL-12(p40)], and Th2 response [lower BLG-specific serum IgE and IgG]. Interestingly, mice received both VSL#3 and low doses of allergen exhibited both allergen-specific active suppression effects and higher sIgA production. We then investigated the effects of different levels of ω-3 and ω-6 PUFAs in the energy and fat rich Western-style diet on food allergy development. Observation of elevated BLG-specific serum immunoglobulins in all experimental mice indicated that both ω-3 and ω-6 PUFAs failed to prevent the development of allergen-specific immune response. However, ω-3 PUFAs alleviated anaphylactic reactions and the severity of allergic reaction as indicated by the unchanged rectal temperature, lower hypersensitivity scores, and Th1-favoured immune responses in BLG-sensitized O3H mice. In general, this study revealed the promising strategies for treatments and prevention against food allergy in the near future. / Les allergies alimentaires, réactions dues à une hypersensibilité du système immunitaire après ingestion d'un certain type d'aliments, constituent un problème de santé publique croissant qui, en plus de dégrader la santé des individus susceptibles, engendre un impact psychologique et financier pour les familles touchées. L'incidence des allergies alimentaires a significativement augmenté ces dernières années et le taux est actuellement estimé être de 6-8% chez les jeunes enfants d'1 an et de 2-4% chez les autres enfants et les adultes. Les allergies alimentaires représentent la première cause d'anaphylaxie chez l'enfant et plusieurs cas d'allergies peuvent causer une réaction anaphylactique fatale. Bien que des efforts continus aient été faits pour tenter de mieux comprendre la nature des allergènes, les facteurs environnementaux prédisposant aux allergies et les mécanismes de la réponse immunitaire, aucun traitement contre les allergies alimentaires n'est disponible à l'heure actuelle. Dans cette étude, nous avons testé trois différentes stratégies visant à réduire l'allergie alimentaire, à savoir l'utilisation de probiotiques, de faibles doses d'allergènes et d'acides gras polyinsaturés oméga-3 chez des souris Balb/c sensibilisées aux protéines de lait de vache. L'administration de Lactobacillus rhamnosus GG (LGG) a montré une tendance à promouvoir une activation des cellules Th1 alors que VSL#3 a engendré une plus forte réduction de l'allergie via l'induction de la sécrétion intestinale d'IgA (sIgA). L'administration de faibles doses d'allergènes a provoqué une suppression de la réponse immunitaire dirigée contre les allergènes via l'activation des cellules Treg, ceci étant suggéré par la suppression des réponses immunitaires Th1 spécifique [diminution des IgG2a BLG-spécifiques et augmentation des IL-12(p40)] et Th2 spécifique [diminution des IgE et IgG BLG-spécifiques]. De plus, les souris ayant reçu à la fois VSL#3 et des faibles doses d'allergènes ont montré une suppression des effets de la réponse allergénique et une production plus élevée de sIgA. Nous avons ensuite étudié les effets de différents niveaux d'oméga-3 et d'oméga-6 dans un régime alimentaire de type occidental riche en énergie et en matières grasses sur le développement des allergies alimentaires. L'observation d'une élévation des immunoglobulines BLG-spécifiques chez toutes les souris expérimentales ont indiqué que les oméga-3 et -6 n'ont pas réussi à empêcher le développement de la réponse immunitaire dirigée contre les allergènes. Cependant, les oméga-3 ont diminué les réactions anaphylactiques et le degré de sévérité de la réaction allergique, ceci étant suggéré par un score d'hypersensibilité plus faible, une absence de changement de la température rectale et une réponse immunitaire favorisée par les cellules Th1. Pour conclure, cette étude a révélé des stratégies prometteuses pour le traitement et la prévention des allergies alimentaires dans les années à venir.

Several lines of evidence suggest that CD8+ T cells could contribute to the pathogenesis of many autoimmune diseases of the nervous system. Our group studies a mouse model (L31 mice) that spontaneously develops neurological symptoms associated with demyelinated lesions in the CNS. We have shown that the demyelinating disease in L31 mice is T cell-dependent with CD8+ T cells acting as effector T cells in the pathological process. Because L31 mice exhibit motor dysfunction indicated by hind limb clasping and difficulty walking, we wanted to determine whether the PNS was also affected. A flow cytometry approach determined that CD8+ T cells accumulate in the CNS of L31 mice from early on in life while they only appear in the PNS once mice display neurological symptoms. Furthermore, immunofluoresecent studies of the CNS and PNS of these mice recapitulate the flow cytometry data and demonstrate the large extent of demyelination in the PNS of symptomatic L31 mice, which is associated with CD8+ T cell accumulation. However, it is also evident that these CD8+ T cell clusters are associated with macrophage infiltration in the PNS of symptomatic L31 mice and that these macrophages have a mature phagocytic phenotype. We hypothesize that epitope spreading during the course of this CD8+ T cell-mediated demyelinating disease may be responsible for the PNS pathology. It will be of further interest to determine the T cell receptor specificity of CNS and PNS infiltrating lymphocytes and the role of PNS infiltrating macrophages. / Plusieurs études suggèrent que les cellules T CD8+ pourraient contribuer à la pathogenèse de plusieurs maladies auto-immunes du système nerveux. Notre groupe étudie un modèle murin (souris L31) qui développe spontanément des symptômes neurologiques associés à des lésions de démyélinisation dans le SNC. Cette maladie requiert la présence de lymphocytes T et ce sont la sous-population de lymphocytes T CD8+ qui agissent comme cellules effectrices dans le processus pathologique. Parce que les souris L31 présentent des troubles moteurs tels que le serrement des pattes arrières quand les souris sont soulevées et des difficultés à marcher, nous avons voulu déterminer si le SNP est également affecté. Une approche de cytométrie en flux a déterminé que les cellules T CD8+ s'accumulent dans le SNC des souris L31 très tôt alors qu'ils n'apparaissent dans le SNP que lorsque les souris présentent des symptômes neurologiques. De plus, des études d'immunofluorescence sur coupe en congélation du SNC et du SNP récapitulent les données de cytométrie en flux et démontrent la grande étendue de la démyélinisation dans le SNP des souris L31 symptomatiques, qui est associé à l'accumulation en amas de lymphocytes T CD8+. Il est également démontré que ces amas de cellules T CD8+ sont associés à l'infiltration de macrophages dans le SNP de souris L31 symptomatiques et que ces macrophages ont un phénotype phagocytaire et mature. Nos données suggèrent que la présentation de nouveaux épitopes au cours de la demyelinisation dans le SNC pourrait être responsable de la pathologie qui se développe plus tard dans le SNP. Il serait important dans le futur de déterminer la spécificité du récepteur pour l'antigène des lymphocytes T CD8+ qui s'accumulent dans le SNC et le SNP et ainsi que d'étudier le rôle des macrophages infiltrant le SNP.

Previous studies suggest that IL-12, the potent Th1-inducing cytokine, IFN-gamma, TNF-alpha and nitric oxide (NO) contribute to resistance against blood-stage malaria. Early Th1 responses correlate with resistance, whereas predominantly Th2 responses are associated with susceptibility. In the present studies, the requirements for endogenous IL-12 and its role in host defense against blood-stage P. chabaudi AS malaria were investigated. Our results reveal, for the first time, significant differences in the kinetics of endogenous IL-12 p70 synthesis and splenic IL-12R beta1 and beta2 mRNA expression between resistant B6 and susceptible A/J mice that correlate with the polarization of Th responses observed in these hosts during early blood-stage malaria. The spleen was found to be the major source of systemic IL-12 in infected B6 hosts. In addition, significant differences were observed between acutely infected B6 and A/J hosts, on a per cell basis, in IL-12 p70 release by splenic macrophages in vitro. Importantly, these differences correlated with greater malaria parasite-induced IFN-gamma synthesis in vitro by spleen cells from infected B6 mice. Furthermore, systemic IL-12 production and Th1 responses were found to be unimpaired in P. chabaudi AS infected mice deficient in TNFR compared to wild type controls. However, LPS, but not PRBC, -induced NO synthesis by splenic macrophages, was significantly reduced in infected TNFR deficient hosts. Finally, compared to controls receiving chloroquine (CQ) alone, the mechanism(s) of combined low dose IL-12 and CQ therapy for malaria-infected A/J mice was found to involve increased splenocyte expression of IL-12R beta1 and beta2 and IFN-gamma mRNA, together with enhanced parasite antigen-induced synthesis of IFN-gamma by spleen cells in vitro. This novel IL-12 and CQ therapeutic strategy resulted in significantly reduced parasitemia, enhanced survival, and was effective against established blood-stage malaria. Taken together

Plasmodium chabaudi AS infections in mice result in lethal and non-lethal infections in a strain-dependent fashion. Resistant C57BL/6 mice exhibit moderate levels of peak parasitemia and anemia and survive the infection. Susceptible A/J mice develop significantly higher parasitemias, severe anemia and succumb to infection. Resistance is controlled by a single or tightly linked, dominant, autosomal, non-H-2 linked locus(i), and is associated with the development of enhanced splenomegaly during infection and enhanced reticulocytic response to phenylhydrazine-induced hemolytic anemia. In this thesis, the relationship between the splenic erythropoietic response and resistance to infection is examined. Impairment of splenic erythropoiesis is demonstrated in susceptible A/J mice. This impairment could not be explained by a deficiency in erythropoietin production or in the mobilization of erythroid progenitor cells from the marrow to the spleen. The kinetics and mechanism of the inhibition of erythropoiesis was examined using an in vitro erythroid proliferation assay. Increased inhibition was associated with the rise in parasitemia and was found to be invariant in resistant and susceptible mice. Evidence is presented to suggest that the inhibitor is erythroid-specific and is distinct from IL-1, TNF and IFN-gamma. In transfusion experiments, evidence was obtained for the importance of anemia in the mortality of susceptible A/J mice and in the induction of parasitologic crisis. In splenectomy and immune cell transfer experiments, evidence is presented for the conclusion that an intact spleen is required for the induction but not the expression of B cell-dependent immunity to Plasmodium chabaudi AS.

No description available.

Studies of proteoglycan-induced arthritis in mice revealed two previously unidentified features: polymorphonuclear leucocytes in knee joint cavities and sacroiliac joint involvement. Immunological studies of cellular responses to the proteoglycan revealed a lack of response prior to arthritis which was produced by a further injection. Further studies revealed that depletion of suppressor/cytotoxic T cells in vitro led to an increased response to antigen. Cellular responses in immune and preimmune mice involve helper T lymphocytes. The preimmune but not immune responses wee inhibited by mouse serum and involved a helper T cell dependent response. It did not involve detectable cell death or suppressor/cytotoxic T lymphocytes. Bacteria isolated from mice did not produce arthritis and showed no immune cross-reactivity with proteoglycan.