B cell depletion therapy with rituximab significantly decreases new disease activity in multiple sclerosis (MS) patients; however, these benefits do not correlate with a reduction in circulating or cerebrospinal fluid antibody levels. These findings implicate antibody-independent, pro-inflammatory roles of B cells in MS. Furthermore, in other autoimmune diseases, such as systemic lupus erythematosus (SLE), B cell depletion reportedly resulted in increased function of circulating regulatory T (Treg) cells. Since MS patients have been found to exhibit deficient Treg function, we hypothesized that activated B cells in MS patients can abnormally suppress the function of Treg cells. Therefore, B cell depletion with rituximab allows for restored Treg function, and the prevention of new autoimmune disease activity. In particular, we postulated that the abnormal pro-inflammatory cytokine profile secreted by MS B cells was responsible for defective Treg function. To begin studying the potential relationship between B and Treg cells, I optimized and validated an in vitro human B cell activation assay, as well as a human Treg suppression assay in healthy controls, to subsequently determine the effects of supernatants from differentially activated B cells on Treg suppressive function.Human B cells were isolated using magnetic-activated cell sorting (MACS), then stimulated with B cell crosslinking antibody (X), CD40 ligand (40), a combination of the two (X40), or CpG-nucleotides. Their supernatants were collected and responses found to support previously published findings. Treg and T responder (Tresp) cells were isolated using both MACS and fluorescence-activated cell sorting (FACS) techniques; then stimulated in coculture with and without B cell supernatants; and proliferation was determined by either standard beta scintillation counting (3H-TdR) or carboxyl-fluorescein succinimidyl ester (CFSE) dilution of Tresp cells. We found that Treg cells isolated using the MACS technique contain high numbers of contaminating CD4+CD25neg Tresp cells; are non-proliferative when stimulated alone; and do not robustly suppress Tresp cell proliferation. In contrast, FACS-isolated Treg cells have higher purity and are suppressive of Tresp cell proliferation and cytokine secretion. Suppression could be modulated by treating cells with either IL-10 to promote suppression, or Pam3Cys to decrease suppression, establishing the dynamic range of the suppression assay. When supernatants from B cells activated with CpG-nucleotides or CD40 ligation were added to the suppression assay, we did not find any significant changes. As such, this may reflect a more subtle biology than can be captured within this assay and still requires further investigation. / La thérapie au rituximab visant la déplétion des lymphocytes B diminue la sévérité de la sclérose en plaques (SP) de façon significative. Cependant, ces avantages ne sont pas accompagnés d'une réduction du niveau d'anticorps présents dans le sérum ou dans le liquide céphalo-rachidien. Ces résultats suggèrent certains rôles des lymphocytes B dans la SP indépendants de la production d'anticorps et qui seraient pro-inflammatoires. Dans le lupus érythémateux disséminé (LED), la déplétion des lymphocytes B entraine une hausse des fonctions des lymphocytes T régulateurs (Treg) circulants. Puisque les patients atteints de la SP démontrent un fonctionnement déficient de leurs Treg, nous émettons l'hypothèse que l'activation des lymphocytes B chez ces patients pourrait avoir un impact négatif sur le fonctionnement des lymphocytes T régulateurs. Ainsi, la déplétion des lymphocytes B grâce au rituximab permettrait le rétablissement du fonctionnement des Treg et préviendrait de nouvelles maladies auto-immunes. Notamment, nous avons postulé que les lymphocytes B des patients atteints de la SP produisent un profil anormal de cytokines pro-inflammatoires, et que ceci serait responsable de la dysfonction des Treg. Pour commencer l'étude de la relation entre les lymphocytes B et Treg, j'ai optimisé et validé un test d'activation de lymphocytes B humains in vitro, ainsi qu'un test de suppression des Treg humain chez des contrôles sains afin de déterminer les effets des surnageants provenant de lymphocytes B activés différemment sur la suppression de fonctions chez les Treg.Des lymphocytes B humans ont été isolés à l'aide d'un tri cellulaire magnétique (MACS), et ont été stimulés avec un anticorps lié aux récepteurs des lymphocytes B (X), CD40 ligand (40), une combinaison des deux (X40), ou avec des nucléotides CpG. Les surnageants recueillis démontrent des réponses cellulaires similaires aux résultats publiés auparavant. Les lymphocytes T régulateurs et lymphocytes T répondeurs ont été isolés en combinant MACS et un tri cellulaire fluorescent (FACS), puis ont été stimulés en culture ensemble avec ou sans surnageants de lymphocytes B. La prolifération a été déterminée par soit l'incorporation de la thymidine tritiée, ou par d'ester de 5,6-carboxyfluorescéine diacétate succinimidyl (CFSE). Nous avons trouvé que les lymphocytes T régulateurs isolés en utilisant la technique MACS contiennent un grand nombre de lymphocytes T répondeurs CD4+CD25neg, ne prolifèrent pas lorsqu'ils sont stimulés seuls, et n'étouffent pas la prolifération des lymphocytes T répondeurs. En revanche, les cellules Treg isolées par FACS sont d'une plus grande pureté et inhibent de la prolifération des lymphocytes T répondeurs ainsi que leur sécrétion de cytokines. Cette suppression peut être modulée en traitant les lymphocytes avec IL-10 afin de promouvoir la suppression, ou avec Pam3Cys afin d'atténuer la suppression, établissant une gamme dynamique au test de suppression. Lorsque les surnageants des lymphocytes B activés à l'aide de nucléotides CpG ou de CD40 ligand ont été ajoutés au test de suppression, nous n'avons trouvé aucun changement significatif. À ce titre, ceci pourrait refléter une biologie plus subtile qui ne peut être captée au sein de ce test, exigeant une enquête plus approfondie.

HIV infection causes the progressive decline in total CD4 T cells, leading to immunodeficiency, failure of the immune system to control pathogens and ultimately death. This loss has been attributed to a number of factors such as direct viral CD4 T cell cytopathogenicity, increased immune activation resulting in T cell exhaustion, as well as a possibly impaired function of the thymus which would halt the input of new diverse T cells. This last element has, however, been disputed as the existing methods to measure thymic function have important technical and interpretational limitations. In order to circumvent these caveats, we have developed a novel non-invasive assay based on the ratio of T cell receptor excision circles (TREC) which are episomal DNA molecules excised during TCR rearrangement: the sj/betaTREC ratio. More specifically, the sj/betaTREC ratio accurately monitors thymic activity by measuring the extent of intrathymic proliferation that occurs between the TCRB and TCRA rearrangements. Using this tool, we have uncovered an important thymic defect that occurs early in HIV infection and persists into the chronic phase. We reveal that this defect is characterized by a severe blockade within a key proliferative phase that occurs during the earlier phases of T cell development which impacts on the quantitative parameters of the sj/betaTREC ratio. We have also shown that, during the first months of infection, the failing thymus is counterbalanced by a peripheral compensatory mechanism maintaining recent thymic emigrants (RTEs) numbers, most likely by increasing their survival rate. As the disease progresses with a persistently weak thymus, this compensatory mechanism is lost, resulting in the loss of RTEs. This contributing to the total CD4 T cell decline observed in therapy-naive HIV-infected patients. Moreover, a functional thymus observed within a subset of patients is associated with the maintenance of CD4 T cells, further illustrating the role of the thymus in the outcome of HIV disease. Under successful antiretroviral therapy, the re-establishment of high levels of intrathymic proliferation is intimately linked to the rate of CD4 T cell immune reconstitution illustrating the important contribution of the thymus to an effective CD4 T cell repopulation. / The elucidation of these HIV-induced perturbations within RTE homeostasis prompted us to seek out a specific phenotype that would permit subsequent functional analysis and comparison of these cells to other naive T cells. We found that high expression of CD31 on a subset of naive CD4 T cells identifies RTEs and discriminates these cells as a seemingly functionally distinct naive T cell population, with different cytokine (i.e. IL-2R) and chemokine (i.e. CXCR4) receptors expression patterns. In addition, chronic HIV infected patients have low counts of RTEs that are partially restored under highly active retroviral therapy, supporting our previous findings. / Overall, this thesis exposes for the first time, using novel tools, the existence and the nature of a persistent thymic defect in HIV infection and the important repercussions on RTE homeostasis, both playing key roles in the HIV-induced CD4 T cell loss. The work also demonstrates that the administration of antiretroviral therapy partly reverses this impaired thymic activity which, once reestablished, contributes significantly to an effective immune restoration. Importantly, the findings presented in this thesis stress the need as well as provide the tools for the development of novel strategies aimed at enhancing thymic activity that will ultimately provide more efficient therapeutic means for treating immuno-compromised individuals. / Keywords. HIV, thymus, thymocytes, recent thymic emigrants, TREC, immune reconstitution, T cell homeostasis, naive T cells, beta-selection, intrathymic proliferation.

Antigen specific CD4+ T cells adoptively transfer airway inflammation comprised mainly of lymphocytes and eosinophils. The ability of these transferred T cells to induce inflammation is dependent on the cytokines they express particularly Th2 cytokines. In order to better understand the mechanism by which adoptively transferred T cells induce airway inflammation, we chose to modulate the expression (GATA-3) and activity (STAT-6) of two key regulators of Th2 cytokine production. To modify expression of GATA-3, we used a bicistronic retroviral vector encoding GATA-3 and enhanced green fluorescent protein (EGFP). As a control, we used a retrovector encoding EGFP alone. By coupling in vitro antigen stimulation with retroviral transduction we generated antigen specific CD4+ T cells expressing EGFP alone or GATA-3 and EGFP. When transferred into naïve recipients that were subsequently challenged, these transduced CD4+ T cells induced lung inflammatory responses with an increase in both CD4+ lymphocytes and eosinophils. This antigen specific inflammatory response was enhanced in animals receiving T cells overexpressing GATA-3. Analysis of the infiltrating cells also revealed that the EGFP+ T cells were present in the lung following antigen challenge, comprising only a small fraction of the CD4+ T cells recruited to the lung during the antigen response. Thus, GATA-3 amplifies antigen-specific inflammatory responses in the airways by augmenting the ability of antigen specific T cells to recruit inflammatory cells to the lung following antigen challenge. To modify the activity of STAT-6 we used chimeric cell penetrating peptides containing a poly-arginine protein transduction domain (PTD) coupled to a sequence predicted to bind and inhibit STAT-6 activity (SIP-1). Using fluorescein-tagged SIP-1, we demonstrate that the poly-arginine PTD efficiently translocates to the cytoplasm within an hour. In vitro, antigen-induced IL-4 production was inhibited in SIP-1-treated spleno / Les cellules CD4+ T à antigènes spécifiques transfèrent par adoption l'inflammation pulmonaire constituées principalement de lymphocytes et d'éosinophiles. L'habileté de celles-ci à transférer des cellules T pour induire l'inflammation est dépendante de leur expression de cytokines Th2. De manière à mieux comprendre le mécanisme par lequel les cellules T transmises par adoption induisent l'inflammation pulmonaire, nous avons choisi de moduler l'expression de GATA-3) ou l'activité de (STAT-6) des deux régulateurs-clés de production de cytokine Th2. Afin de modifier l'expression de GATA-3 dans les cellules T destinées au transfert par adoption, nous avons utilisé un rétrovirus recombinant concentré avec une filtration par centrifugeuse. Ce procédé a dramatiquement augmenté leurs titres et ainsi leur habileté à transduire les cellules CD4+ T en culture primaire. Nous avons utilisé un rétrovirus recombinant qui encode la GATA-3 et / ou la protéine fluorescente verte (EGFP). En couplant in vitro la stimulation d'antigènes avec la transduction par vecteur viral, nous avons généré des cellules CD4+ T à antigènes spécifiques exprimant de l'EGFP seul ou bien de la GATA-3 et de l'EGFP. Lorsque transféré dans un rat qui avait subséquemment été provoqué avec des antigènes, ces cellules CD4+ T induisent une réaction aux inflammations pulmonaires avec une augmentation des lymphocytes et éosinophiles. Cette réaction inflammatoire fut accrue chez les animaux recevant les cellules T surexprimant la GATA-3. L'analyse des cellules infiltrantes a aussi révélé que bien que les cellules EGFP+ étaient présentes dans les poumons suivant la provocation par antigènes, elles étaient constituées seulement d'une petite fraction de cellules CD4+ T recrutées dans les poumons. Ainsi, la GATA-3 amplifie la réaction inflammatoire des poumons induite par antigènes en augmentant l'habileté des cellules T à antigènes spécifiques à recruter

Leishmaniasis is an important parasitic disease for which no test with 100% sensitivity and/or specificity exists. We studied pre- and post-treatment sera (n=49) from patients with Old and New World cutaneous leishmaniasis (CL) as well as pre-treatment sera (n=45) from visceral leishmaniasis (VL) cases from Nepal. In all cases, samples were fractionated, bound on CM10 and IMAC30 arrays and read by surface-enhanced, laser desorption and ionization, time-of-flight mass spectrometry (SELDI-TOF-MS). Data were analyzed using CiphergenExpress (creating M/z scatter plots, p-values and ROC) and Biomarker Pattern Software (building decision trees). We attempted to visualize on gel all biomarkers detected in both software programs. Three of the 14 most promising candidate biomarkers for CL and 4 of 15 candidate biomarkers for VL were visible. These bands were cut and sequenced by LC-MS-MS. This allowed us to identify various proteins as possible biomarkers in CL and VL patients. / La leishmaniose est une maladie parasitaire pour laquelle aucun test de sensibilité et/ou spécificité égale à 100% n'existe. Nous avons étudié des sérums (n = 49) pré- et post-traitement de patients atteints de leishmaniose cutanée (LC) de l'ancien et du nouveau monde de même que des sérums (n = 45) prétraitement de cas de leishmaniose viscérale (LV) du Népal. Tous les échantillons ont été fractionnés et sont retenues sur une surface chromatographique phase solide appelée ‘Chip' (CM10 et IMAC30) et lus par SELDI-TOF-MS (Surface Enhanced Laser Desortion and Ionization, Time-of-Flight Mass Spectrophotometry). Les résultats ont été analysés par les programmes Ciphergen Express (en utilisant des graphique M/z, p-values et ROC) et Biomarker pattern Software (en créant des arbres décisionels). Nous avons tenté de visualiser sur gel tous les marqueurs biologiques détectés par les deux programmes d'analyse. Trois des 14 marqueurs biologiques pour LC et 4 des 15 marqueurs biologiques pour VL ont pu être visualisés. Les bandes ont été sectionnées et séquencées par LC-MS-MS. Ceux ci nous permis aidé à identifier des marqueurs biologiques dans les LC et LV patients.

The generation of an Ab response is modulated by contact and cytokine mediated T help for B cells. Here we show that murine splenic small resting B cells do not express mRNA for, or bear IL-2R. Accordingly, these cells do not respond to IL-2. T- contact events induce IL-2R expression on B cells and this is inhibited by blocking of CD40, MHC II or CD54. Although CD40 ligation on its own induces B cell proliferation, it does not confer IL-2 responsiveness. In contrast, signalling through MHC II and CD54 synergizes with IL-5 to induce functional IL-2R on B cells. Moreover, physiological, gp39$ rm sp{low},$ T help for B cell IL-2 responsiveness is equivalently dependent on ligation of CD40, CD54 and MHC II, and requires prior sIg signalling. IL-5 synergizes with either LPS or Th to render B cell responses to IL-2 autonomous of further stimulus. Thus, expression of a functional IL-2R is a marker of B cell activation which appears to be tightly regulated through sIg signals and T-contact events and can be modulated by cytokines.

The studies presented in this thesis examine the mechanisms of T cell immunosuppression during the graft-versus-host reaction (GVHR). GVHR was induced by the injection of parental lymphoid cells into F1 hybrid recipient mice. The ensuing acute reaction consisted of an initial immunoproliferative phase, followed by the development of profound immunosuppression and histopathological lesions of epithelial and lymphoid tissues. Survivors of the acute reaction developed the persistent immune abnormalities characteristic of chronic GVHR. / We have found that the T cell protein tyrosine kinases p56$ rm sp{lck}$ and p59$ rm sp{fyn}$, involved in signal transduction through the T cell receptor (TCR), are downregulated in the T cells of mice during GVHR. The reduction of lck and fyn was prevented by adrenalectomy of the recipients, and a similar reduction could be induced in normal (non-GVH-reactive) mice by an injection of exogenous cortisone. These findings suggested that the GVHR-induced elevation in endogenous glucocorticoid levels could trigger the decrease of T cell lck and fyn, resulting in a T cell signalling defect during GVHR. In fact, we have demonstrated that glucocorticoids induced a decrease in lck and fyn in T cell clones in vitro. Thus, it appeared that the early GVHR-induced T cell unresponsiveness was due to the glucocorticoid-dependent downregulation of T cell lck and fyn. / We have investigated changes in T cell maturation and selection in the GVHR-dysplastic thymus, which may account for the persistent immune abnormalities of chronic GVHR. Thymocyte TCR expression and usage were aberrant during GVHR; changes included decreased expression of CD3 on CD4$ sp+$8 thymocytes, inconsistent TCR V$ beta$ usage, and appearance of phenotypically autoreactive mature thymocytes. These abnormalities, suggestive of defective positive and negative selection, are likely to result from the GVHR-induced decrease in thymic class II MHC expression. Altered T cell education may lead to the long-term peripheral T cell defects observed in chronic GVHR. Lastly, we report that GVHR-induced cutaneous injury was exacerbated by irradiation of the target tissue, suggesting that in clinical GVHR, irradiation may intensify tissue damage, including thymic epithelial lesions; this could potentially lead to more serious alterations in thymic function, and thus to longer lasting, more severe peripheral T cell immune deficiency.

Cadherins are a family of cell adhesion molecules (CAMs) that mediate calcium dependent cell-cell interactions. Amongst the many functions of cadherins are several well-established roles in the immune system, such as thymocyte development, and the heterotypic interaction between T cells and dendritic cells. To date, it is not known whether cadherins are expressed on B lymphocytes. CAMs play important roles in B cell maturation, by mediating cell adhesion with stromal cells of the microenvironment and initiating signalling cascades that promote cell survival. In the present study, N-cadherin expression was examined during B lymphopoiesis. / We have demonstrated that a subset of CD19+ B cells in human marrow, but not in peripheral blood lymphocytes (PBLs) express N-cadherin, suggesting loss or down-regulation of N-cadherin as B lymphocytes mature and enter the circulation. In contrast, PBLs derived from B cell chronic lymphocytic leukemic (B-CLL) patients continue to express N-cadherin. To test whether N-cadherin may be re-expressed on B cells with a more mature phenotype, we characterized its expression on Daudi, Raji, and Ramos B cells. N-cadherin mRNA and protein was found in Daudi cells but not in Raji or Ramos cells. Finally, the inhibition of N-cadherin mediated adhesion results in an increase in programmed cell death. Maturation of B lymphocytes to antibody-secreting cells involves stringent selection as 95% of developing B lymphocytes are deleted by apoptosis. We surmise that N-cadherin may contribute to protection from cell death during the course of B cell maturation.

Anti-phospholipid antibodies (aPL) occur in patients with the anti-phospholipid syndrome, and are directed against various combinations of phospholipids and phospholipid-binding proteins (e.g., beta2-glycoprotein I and prothrombin). Lupus anticoagulants (LA), a subset of aPL, exhibit anticoagulant properties in vitro, but are strikingly procoagulant in vivo. We have previously demonstrated that some aPL bind specifically to apoptotic, but not viable, thymocytes in the presence of beta2-glycoprotein 1. Here, we demonstrate that prothrombin binds selectively to the surface of apoptotic Jurkat cells, and supports the binding of LA-positive murine monoclonal antibodies (mAb) and patient-derived IgG to apoptotic cells. Despite similar LA activity and reactivity with apoptotic cells, the mAb differed in affinity and specificity. One mAb was highly reactive with prothrombin alone, while the other required anionic phospholipid for elevated binding. These results demonstrate that aPL recognize multiple epitopes on apoptotic cells, suggesting that apoptotic antigens contribute to the induction and/or perpetuation of aPL.

The i&barbelow;nterf&barbelow;eron&barbelow; (IFN) inducible d&barbelow;ouble s&barbelow;tranded (ds) RNA activated protein kinase PKR inhibits protein synthesis by phosphorylating the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha). The introduction of dominant negative mutants of PKR leads to cell transformation and tumor formation in nude mice, and is thought to be due to the deregulation of general protein synthesis. In addition to translational control, PKR has been implicated in several signaling pathways leading to gene transcription. For example, a link between PKR and NF-kappaB activation was first detected when PKR knockout mouse embryonic fibroblasts were deficient in NF-kappaB DNA binding upon dsRNA mediated activation. Recently, PKR has been shown to induce IkappaB kinase (IKK) activity resulting in the induction of NF-kappaB mediated gene transcription. The mechanism of activation of IKK by PKR has been suggested to be independent of PKR's catalytic capacity and may be due to a protein-protein interaction between PKR and IKK. The effects of dominant negative mutants of PKR (PKRDelta6/PKRLS4/PKRLS9) on the activation of NF-kappaB was examined in NIH3T3 cells. (Abstract shortened by UMI.)

The purpose of this investigation was to elucidate the role of the oligosaccharides of Class II MHC glycoproteins in allostimulation. Plasma membranes (PM) from the Daudi cell line were chemically deglycosylated using anhydrous hydrogen fluoride (HF). Subsequently, native and deglycosylated (dgl) Class II MHC molecules were affinity purified from their respective PM and inserted into the PM of peripheral blood leukocytes (PBL) which were used as stimulators in the mixed leukocyte reaction (MLR). Stimulator and responder cells were from the same donor. Both forms of the antigen were found to elicit a proliferative and cytolytic (CML) response but the dgl antigen did so to a lesser extent. Thus, it seemed as though the oligosaccharide side chains of Class II MHC molecules may not be required for allostimulation. A similar reduction in the cytolytic response was obtained when effector cells generated by the native antigen were used against targets that were stripped of N-linked oligosaccharides by tunicamycin (TM) pretreatment. Twenty-four clones were raised against the native antigen. Three clones gave proliferative responses only to the native antigen while two clones responded equally to both native and dgl antigen. In CML studies the three clones lysed normal targets but failed to lyse TM-treated target cells while the two clones did not discriminate between the two targets. Accordingly, the three clones were termed anti-MHC oligosaccharide clones while the two clones were termed anti-MHC polypeptide clones. In inhibition of CML experiments using the dgl supernatant which contained Daudi PM oligosaccharide or using a anti-MHC class II monoclonal antibody; CML reactivity of the anti-MHC oligosaccharide clones was blocked by the dgl supernatant but not by the anti-MHC MoAb. On the other hand the anti-MHC polypeptide clones were only inhibited by the anti-MHC MoAb whether against intact or TM-treated targets. These studies strongly indicate that glycoconjugates of