Allergic asthma is associated with the presence of increased numbers of activated inflammatory cells, including mast cells, eosinophils and T-cells within the lung. The recently documented cytokine, stem cell factor (SCF) is critical for mast cell growth, and for chemoattraction of progenitor cells and eosinophils. We hypothesized that allergic asthma is associated with the increased expression of SCF and its receptor, c-kit. The expression of SCF and c-kit was studied within the airways of atopic asthmatics and non-asthmatic controls using in situ hybridization and immunocytochemistry. The results demonstrated significantly increased expression of SCF and c-kit in bronchial biopsies and BAL from asthmatics compared to controls. SCF was preferentially localized to epithelium, and was also found in macrophages. The results suggest that SCF may play a role in the pathogenesis of allergic asthma, and support the importance of epithelium in regulation of immune responses in the lung.

B cells are becoming increasingly recognized as important immune regulatory cells. Depending on their context of activation, the balance of B cell production of proinflammatory and anti-inflammatory cytokines is affected. Recent work has shown that Toll-like receptor (TLR) ligation is an important signal affecting B cell activation. We sought to determine the effect that the combination of antigen recognition, T cell help and TLR ligation would have on B cell response profiles of both control donors and Multiple Sclerosis (MS) patients. We found that TLR8 ligation resulted in enhanced B cell proliferation and survival combined with a marked suppression of B cell effector cytokines. TLR9 ligation resulted in similar enhancement in B cell proliferation that was combined with significantly increased effector cytokine production. These findings highlight the importance of TLR ligation on B cell activation and underscore the importance of understanding the effects of TLR activation on B cell biology. / Les cellules B deviennent de plus en plus reconnues comme étant des cellules immunorégulatrices importantes. Selon leur contexte d'activation, l'équilibre entre les cytokines pro-inflammatoires et anti-inflammatoires produites par les cellules B est affecté. Les travaux récents ont prouvé que l'activation des récepteurs "Toll-like" (communément appelés Toll-like receptors ou TLR) est un signal important affectant l'activation des cellules B. Nous avons cherché à determiner si l'activation des TLR combinée à l'identification d'antigène et l'aide des cellules T pouvait influencer les profils « effecteurs » des cellules B provenant de donneurs contrôles et de patients atteints de la sclérose en plaques (SP). Nous avons constaté que l'activation de TLR8 induit une augmentation de la prolifération et de la survie des cellules B, associée à une suppression marquée des cytokines effectrices sécrétées par les cellules B. L'activation de TLR9 a également induit une augmentation de la prolifération des cellules B, associée à une production sensiblement accrue de cytokines effectrices. Ces résultats accentuent l'important de l'activation des TLR sur l'activation des cellules B et soulignet l'importance de comprendre les effets de l'activation de TLR sur la biologies des cellules B.

Src family protein tyrosine kinases are involved in the regulation of a large number of cellular processes in response to extracellular stimuli. Their crucial role in the initiation of cellular activation of T-lymphocytes following stimulation of the T-cell receptor by an antigen has sparked great interest in better understanding the regulation of their catalytic activity and function. The protein tyrosine kinase Csk is the major negative regulator of Src-related kinases through its ability to phosphorylate a negative regulatory tyrosine, in their carboxy-terminal region. By virtue of its negative impact on Src-like enzymes, Csk is capable of down-regulating T-cell activation. Nevertheless, the mechanisms regulating the inhibition of T-cell receptor signalling by Csk are still poorly understood. The experiments presented in this thesis were aimed at improving our understanding of the regulation of Csk in T-lymphocytes. I have demonstrated that the SH3 and SH2 domains of Csk, which can mediate protein-protein interactions, are essential for its capacity to down-regulate T-cell activation. This observation led me to the identification of the first known ligand for the SH3 domain of Csk, the protein tyrosine phosphatase, PEP. Functional analysis of the Csk-PEP complex revealed that these two enzymes cooperate to inhibit signals generated by Src-related enzymes, thereby acting as potent negative regulators of T-cell activation. Taken together, the results obtained suggest that the inhibitory function of Csk is not only dependent on its ability to phosphorylate the carboxy-terminal tyrosine of Src-related enzymes, but also relates to its ability to associate with the protein tyrosine phosphatase PEP.* / *Originally published in DAI Vol. 61, No. 6. Reprinted here with corrected author name.

Most T cells are produced in the thymus, where they proceed through several differentiation steps to shape the TCR repertoire. The first thymocyte subset to express a TCR complex is characterized by the expression of CD4 and CD8 co-receptors, CD4+CD8+ thymocytes. At this stage, the CD4+CD8+ thymocytes' fate relies on the affinity/avidity of their TCR towards self-peptide/MHC complexes. Indeed, elimination of thymocytes by apoptosis occurs in death by neglect, due to the absence of TCR-peptide/MHC interactions of sufficient affinity/avidity, and during negative selection, upon interactions of strong affinity/avidity. In contrast, weak affinity/avidity binding of TCR with its ligand would lead to positive selection followed by thymocyte maturation. This dissertation centers on understanding the factors involved in the elimination of thymocytes at the CD4+CD8+ stage of thymic differentiation, namely death by neglect and negative selection. / Particularly, we address the role of CD45 in thymocyte deletion. Ligation of CD45 in the absence of TCR signaling has been shown to induce mature T cell death. Thus, we have evaluated the consequences of CD45 ligation on thymocytes in the absence of TCR triggering, and shown that it induces the death of thymocytes with rapid kinetics. Moreover, this non-necrotic form of cell death is independent of caspases and does not lead to DNA fragmentation. Based on these results, we propose a role for CD45 in the induction of death by neglect. Next, we have evaluated the contribution of factors involved in negative selection. In particular, the individual impact of TCR-ligand affinity, TCR-ligand density, and costimulation in the process of negative selection was studied, We found that, with ligands of physiological affinity, costimulation is required for the induction of negative selection. In addition, our results suggest that increasing TCR ligand density or costimulation does not always promote thymocyte deletion. Finally, we studied some aspects of the intracellular activation signaling pathways leading to thymocyte apoptosis. Specifically, caspase-3 was shown to participate in negative selection, and regulation of Nur77 activity on a physiological mammalian DNA response element was identified, In summary, this work contributes to understanding the regulation of the apoptosis-induction in developing thymocytes.

The CD4 molecule is expressed on a subset of T lymphocytes which recognize their cognate antigen in the context of MHC class II molecules. It is widely accepted that the interaction between CD4 and MHC class II molecules enhances T cell response to specific antigen. CD4 is non-covalently associated with the src-related tyrosine kinase p56$ rm sp{lck}.$ Using the T cell clone 2.10, we have shown that CD4 can sequester lck and inhibit anti-TcR induced proliferation if not co-aggregated with the TcR. Our results suggest that MHC class II molecules through their simultaneous interaction with the TcR and CD4 potentiate T cell activation by bringing the CD4/lck complex to the proximity of the TcR. This cellular system was also used to demonstrate that the extracellular domain of CD4 can also regulate the initiation of T cell activation independently of its interaction with MHC class II molecules. Indeed, cells expressing chimeric molecules composed of the epidermal growth factor receptor (EGFR) extracellular domain and the CD4 cytoplasmic tail were still responding to anti-TcR simulation in the absence of co-aggregation. The role of the extracellular domain of CD4 was further demonstrated in experiments in which the HIV-1 envelope glycoprotein gp120 was used to inhibit antigenic stimulation of CD4-independent T cell responses. This inhibition was occurring whether CD4 is associated with lck or not, suggesting that gp120 is modulating a CD4 function other than association with lck and which is related to its extracellular portion. In addition to its effect on T cell activation, interaction between gp120 and CD4 modulates HIV replication at a post-transcriptional level. The CD4/lck association is required for this effect as the virus replicates much more efficiently in cells bearing a CD4 which is not associated with lck. Activation of lck through the CD4/gp120 interaction may thus be responsible for the induction of latency. Nef, another HIV protein playing a critical r

Acute myelogenous leukemia (AML) five year survival ranges between 20-30% despite the administration of the most intensive chemotherapy regimen, hence the need to develop new therapeutic strategies. Substantial inter-patient variation in sensitivity to AVE9633, a very promising anti-CD33 immunoconjugate, was observed. The goal of this project is to identify the mechanisms underlying resistance of AML cells to AVE9633. Using the AVE9633 sensitive AML cell line, HL60, and its resistant variant HL60/s, we found that both internalize the immunotoxin to a similar extent. The uncoupling of the antibody from the CD33 receptor is significantly lower in HL60/s. CD33 degradation was seen to be reduced in the resistant cell line while Suppressor Of Cytokine Signalling 3, SOCS3, playing a role in CD33 degradation, is only expressed in the sensitive cell line. In conclusion, antibody-receptor uncoupling and CD33 degradation defects were shown to be associated with resistance to AVE9633 in the HL60/s cell line. The identification of these mechanisms of resistance could provide means to screen-out patients most likely to benefit from treatment with AVE9633 and identify new approaches to treat patients refractory to current cancer treatments. / La Leucémie Myéloïde Aigue (LMA) est associée à une survie après cinq ans qui varie entre 20 et 30% en dépit de l’administration des régimes de chimiothérapie les plus intensifs, d’où la nécessité de développer de nouvelles stratégies thérapeutiques. Une importante variation de sensibilité cellulaire à l’AVE9633, un immunoconjugué anti-CD33 très prometteur, a été observée entre les patients. Le but de ce projet est d’identifier les mécanismes responsables de la résistance des cellules de LMA à l’AVE9633. En employant la lignée LMA sensible à AVE9633, HL60, et sa variante résistante, HL60/s, nous avons découvert que les deux lignées internalisent autant d’immunotoxine. Le découplage de l’anticorps du récepteur est significativement moindre dans la lignée résistante HL60/s. La dégradation de CD33 est réduite chez HL60/s alors que la protéine « Suppressor Of Cytokine Signalling 3 », SOCS3, jouant un rôle dans la dégradation de CD33, est uniquement exprimée dans la lignée cellulaire sensible. En conclusion, nous avons démontré que le découplage anticorps-récepteur et la dégradation de CD33 sont associés à la résistance de la lignée HL60/s à l’AVE9633. L’identification de ces mécanismes de résistance devrait pouvoir nous offrir des moyens d’identifier les patients les plus susceptibles de bénéficier d’un traitement par AVE9633 et de découvrir de nouvelles façons de traiter les patients ne répondant point aux traitements actuels du cancer.

Rheumatoid Arthritis (RA) is an autoimmune disease of unknown etiology that is characterized by chronic inflammation of the joints and by the presence, in high titers, of an autoantibody known as the rheumatoid factor (RF). The human cytomegalovirus, a member of the human Herpesviridae family, has been proposed as a potential environmental agent involved in the induction of RF production. The report of an anti-CMV antibody having structural homology to a major group of human paraproteins with RF activity, as detected by the expression of RF-associated idiotypes and by variable region sequences, provided a possible structural relationship between CMV infection and RF production. / In this thesis, the structural characterization of eight human hybridoma anti-CMV antibodies and their possible relationship to RFs is presented. All eight antibodies recognized the viral matrix phosphoprotein, known as pp65, which has previously been demonstrated to be highly immunogenic during the natural infection. Seven of these antibodies expressed a restricted number of RF-associated idiotypes. The HCV-2 anti-CMV antibody expressed the greatest number of RF-associated idiotypes and was most similar to RFs of the "PO" idiotypic family. All eight antibodies were composed of different $ rm V sb{H}/V sb{L}$ pairs, with evidence of antigen-selected somatically-induced mutations in the majority of cases. When the nucleotide sequences of these anti-CMV antibodies were compared to previously reported rearranged immunoglobulin sequences, the majority were found to share the highest degree of identity with antibodies possessing RF activity. This data provides evidence that there is extensive overlap between the autoimmune and anti-pathogen antibody repertoires. / Comparisons of the anti-CMV antibodies with homologous RF sequences revealed that most of the amino acid differences could be accounted for by putative somatic mutation events and different junctional diversity in the third complementarity determining region (CDR3) of the antibody heavy chains. This was supported by the cloning and sequencing of the variable regions of a human hybridoma RF, known as C304, which was derived from a patient with active RA. The C304 heavy and light chain sequences had high identity with one of the anti-pp65 antibodies (HCV-3) and with a previously reported antibody directed against the human herpes simplex virus (HSV). The sequences diverged at points of suspected somatic mutations and, extensively, in the CDR3 of the heavy chains. / Although no evidence was obtained that could definitively implicate human herpes viruses in the induction of RFs, this work has provided an analysis of the relationship between idiotype expression and antigenic specificity. This data has enabled the identification of the sequence differences between RFs and anti-viral antibodies that potentially play a role in determining antigenic specificity and provides the basis for an experimental model to address the issue of how primary amino acid sequence determines the specificity of human antibodies.

T cells expressing CD4 recognize antigens presented by class II following the contact of CD4 with non-polymorphic regions of class II. CD4 enhances T cell activation by acting as an adhesion molecule (co-ligand function), or by bringing the CD4 associated p56$ sp{lck}$ to the vicinity of the TCR (co-receptor function). / To dissect the molecular interactions which lead to CD4 function(s), wild-type (WT) and mutant CD4 molecules were expressed in the CD4-dependent 3DT52.5.8 T cell hybridomas. Results showed that multiple sites on CD4 encompassing the CDR1, the CDR3 regions of D1 and the FG loop of D2 are involved in class II interaction. The opposite face containing the CDR2 region also plays a role, either as another class II binding site, or the TCR docking site, or in another function of CD4. Co-receptor function requires a much larger site on CD4, compared to co-ligand function. A stretch of 15 amino acids which links D2 and D3 of CD4 appears to be very important for maintaining CD4 conformation, or to provide CD4 the flexibility required for its interaction with other cell surface molecules, including class II, the TCR, etc. / Crystallographic and functional studies have suggested that CD4 may dimerize, although biochemical evidence is lacking. To investigate the CD4 dimerization issue both human and mouse CD4 WT were co-expressed in 3DT52.5.8 cells. Surprisingly this led to a severe disruption of CD4 functions, although it has been shown that both human and mouse CD4 molecules are capable of interacting with human class II efficiently. As expected, co-expression of h-CD4 WT with class II-interaction-deficient CD4 mutants within the CDR1, CDR3 and the FG loop did not rescue CD4 functions. However, co-expression of CD4 WT with mutants from the CDR2 region resulted in an enhanced response. This result suggests that CDR2 mutants do not dimerize with WT molecule, therefore cannot behave as a dominant negative mutant, which is not the case for class II-interaction-deficient mutants from the CDR1, CDR3 and FG loop. Based on these results we suggest a model whereby dimerization involves, at least in part the CDR2 region. Final confirmation of this model awaits further structural data.

Colonic mucins bind to the E. histolytica Gal/GalNAc adherence lectin and inhibit amoebic adherence to and lysis of epithelial cells. High $M sb{ rm r}$ rat colonic mucins isolated from Sepharose 4B and subfractionated by Cellex-E (ECTEOLA) ion-exchange chromatography demonstrated a minor neutral and a major acidic species with distinct amino acid composition and immunogenicity. Virulent E. histolytica trophozoites elicited a generalized secretory response of both neutral and acidic mucin species which was confirmed by thin-section histochemistry. Such activity may function to deplete the host's protective layer to facilitate invasion. In rats immunized against E. histolytica, enhanced mucin secretion was demonstrated by an increase in the secretion of $ sp3$H-glucosamine labelled glycoproteins (25%) and high $M sb{ rm r}$ Sepharose 4B mucins (27%). Ion-exchange chromatography and histochemistry confirmed a pronounced and generalized secretion of both mucin species. Increased mucin secretion in immunized animals may function as a host defense mechanism to prevent invasion or aid in parasite expulsion.

<p> The binding of antigen (Ag) to the B cell receptor (BCR) induces the activation of intracellular signaling and the reorganization of the actin cytoskeleton. However, the function of actin reorganization and the mechanisms by which BCR signaling and actin reorganization is coupled have not been well studied. This thesis has investigated how BCR signaling regulates actin reorganization and how actin remodeling in turn influences BCR signalig. My studies show that the key stimulatory signaling molecule of the BCR, Bruton's tyrosine kinase (Btk), is critical for actin polymerization at the activation surface and BCR clustering and B cell spreading, events that are essential for signaling initiation and amplification. The key inhibitory signaling molecule, SH2-containing phosphatidylinositol-5 phasphatase (SHIP-1), is important for removal of F-actin from the activation surface, and actin-mediated B cell contraction and the formation of BCR central clusters. SHIP-1 suppresses actin polymerization by inhibiting Btk-dependent activation of Wiskott-Aldrich syndrome protein (WASP). These results suggest that BCR signaling can regulate B cell morphology and surface BCR clustering via modulationg actin dynamics. To understand the roles of actin reorganization in BCR signaling, I investigated the effects of gene knockout of the two actin regulators, WASP and its homolog, neuronal (N)-WASP. My results show that both WASP and N-WASP are required for optimal BCR clustering, B cell spreading, and BCR signaling, but they play distinct roles. WASP promotes actin polymerization, B cell spreading, BCR clustering, and signaling amplification, and N-WASP inhibits actin polymerization at the activation surface and promotes B cell contraction, BCR central cluster formation, and signaling attenuation. Importantly, B cell-specific N-WASP knockout causes increases in the levels of autoantibody. In addition, WASP and N-WASP negatively regulate each other, compete for Arp2/3, and are inversely regulated by Btk and SHIP-1. Taken together, these results demonstrate that the balance of stimulatory and inhibitory BCR signaling controls actin dynamics and organization through regulating the activities of WASP and N-WASP. Actin remodeling in turn amplifies BCR signaling activation or down regulation by modulating B cell morphlogy and the organization of surface BCRs.This research reveals a bidrectional feedback loop between BCR signaling and the actin cytoskeleton. </p>