Thèse (Ph. D.)--Université Laval, 2007. / Titre de l'écran-titre (visionné le 9 mai 2008). Bibliogr.

Würzburg, Univ., Diss., 2009. / Zsfassung in engl. Sprache.

No description available.

Le CD40 est une glycoprotéine transmembranaire de type I appartenant à la superfamille des récepteurs des facteurs de nécrose tumorale (TNFRs). Il est exprimé à la surface des cellules hématopoïétiques, principalement les cellules B, et les cellules nonhématopoïétiques telles que les cellules épithéliales et les fibroblastes. Son principal ligand, le CD154, est exprimé de façon transitoire à la surface de différents types cellulaires tels que les lymphocytes T activés. Le CD40 est capable d'interagir avec les deux formes du CD154: la forme membranaire et la forme soluble. Le CD40 joue un rôle important dans la réponse immunitaire humorale. Son engagement avec le CD154 induit la prolifération et la différenciation des cellules B, la commutation isotypique des anticorps, la formation du centre germinal, l’augmentation de la génération de cellules B mémoire et la survie des cellules B. D'autres études ont démontré son implication dans la mort cellulaire. En effet, nos études ont démontré que la signalisation via CD40 conduit à une mort rapide des cellules B, principalement observée dans les lignées de cellules B transformées par le virus d'Epstein-Barr (EBV) alors qu'il est bien documenté que son engagement sur des cellules B immatures les protège de la mort induite via le récepteur des cellules B (BCR). Il n’est cependant pas connu si l’effet apoptotique du CD40 se produit dans des lignées de lymphocytes B qui ne sont pas transformées par EBV. Le travail illustré dans ce mémoire porte sur l'étude du rôle du CD40 dans la mort cellulaire des Ramos qui est un modèle de cellules B immatures, EBV négatives, et à comprendre son influence sur la signalisation apoptotique induite via le BCR. Nos résultats montrent que le CD40 n’induit pas la mort des cellules Ramos mais leur activation par l’ester de phorbol (PMA) les sensibilise à la mort via le CD40, qui est plus significative suite à son interaction avec le ligand (CD154) résistant au clivage. Par contre, cette interaction est aussi capable d'inhiber la mort cellulaire induite via le BCR aussi bien sur les cellules au repos ou activées par le PMA. Cette inhibition de la mort cellulaire est comparable avec les deux formes du CD154. L’ensemble des études suggèrent que le CD40 peut réguler la réponse immune en induisant des signaux de survie nécessaires à la production d'anticorps et peut participer à la résolution de celle-ci en induisant la mort cellulaire des cellules B activées. Par ailleurs, le CD40 peut aussi constituer une cible thérapeutique pour les traitements des lymphomes B. / CD40 is a type I glycoprotein belonging to the tumor necrosis factor receptor (TNFRs) superfamily. The CD40 is expressed on hematopoietic cells, mainly B cells, and on nonhematopoietic cells such as epithelial cells and fibroblasts. Its classical ligand, CD154, is transiently expressed on different cell types such as activated T cells. CD40 is able to interact with both the membrane-bound and the soluble forms of CD154. CD40 plays an important role in the humoral immune response. Its ligation with CD154 induces B cell proliferation and differentiation, antibody class switching, germinal center formation, memory B cell generation and B cell survival. Other studies have demonstrated its implication in cell death. Indeed, our studies have demonstrated that signalling via CD40 leads to a rapid B cell death mainly observed in Epstein-Barr virus (EBV)- transformed B cell lines, although it is well documented that the engagement of CD40 on immature B cells rescues them from IgM-mediated death. However, it is unknown if CD40-mediated cell death occurs in non-EBV B cell lines. The work presented in this thesis focuses on the study of the role of CD40 in Ramos cell death, which is an immature B-cell model, EBV negative, and on its influence on the apoptotic signaling induced via the B-cell receptor (BCR). Our results show that CD40 is unable to induce Ramos cell death but Ramos activation with the phorbol ester (PMA) sensitises them to death via CD40, which is more significant following its interaction with the ligand (CD154) resistant to cleavage. However, CD40 interaction with CD154 is also able to protect both resting and activated Ramos from BCR-mediated death and this occurs equally well with both forms of CD154. Together these studies suggest that CD40 can regulate the immune response by delivering survival signals necessary for antibody production and can contribute to the resolution of the immune response by inducing death in activated B cells. Furthermore, CD40 can also represent a therapeutic target in B cell lymphomas.

CD40 signals are required for productive immune responses but also play a role in autoimmune disease pathogenesis. The major goal of this research was to investigate the contribution of two receptors to the development of autoimmune disease: (1) LMP1, an oncogenic EBV-encoded mimic of CD40 and (2) a naturally-occurring polymorphism in CD40, P227A, which appears to confer LMP1-like properties to the CD40 receptor. Interestingly, hCD40-P227A is overrepresented in individuals of Mexican and South American descent. Although this allele is not directly associated with SLE incidence in Hispanic populations, patients of Hispanic ethnicity have a tendency toward increased severity of SLE symptoms, particularly lupus nephritis. This work reports the initial genetic description of CD40-P227A and characterizes its gain-of-function signaling properties in mouse and human B cells. In comparison to Wt-CD40 signaling, CD40-P227A signaling results in increased binding of TRAF3, TRAF5, and Act1, as well as enhanced secretion of IL-6, TNF-α, and Ig due to a selective hyperactivation of the JNK pathway. Whereas TRAF3 is normally a negative regulator of Wt-CD40 signaling, TRAF3 is a required positive regulator of CD40-P227A signaling as demonstrated in TRAF3-deficient B cells. LMP1 is an EBV-encoded CD40 mimic which signals in an amplified and sustained manner. Although EBV is latent in >90% of humans, EBV infection is associated with autoimmunity, particularly SLE. Upon flares of autoimmunity, EBV reactivates and LMP1 is expressed, yet the contribution of LMP1 to exacerbation of disease is unknown. LMP1 transgenic mice generated in our lab have an autoreactive phenotype but do not die early due to autoimmune disease. To test the hypothesis that LMP1 cooperates with other genes in the host to exacerbate autoimmunity, mCD40-LMP1 transgenic mice were bred to two lupus-prone strains of mice. LMP1 signaling was able to enhance the autoimmune phenotype of the B6.Sle1, but not the B6.Sle3 strain. These data suggest that LMP1 is redundant with genes within the Sle3 interval, but acts cooperatively with genes within the Sle1 interval to exacerbate autoimmunity. Together, the research foci of this dissertation examine how the CD40 pathways of B cell activation can be amplified and dysregulated, by either a viral mimic of CD40, a polymorphism in its signaling domain, or its cooperation with additional gene products. Differential usage of TRAF3 as a positive, rather than a negative, regulator of signaling appears to be one common mechanism by which this occurs. In conditions where enhanced CD40 signaling may be desirable such as during chronic infections, manipulation of TRAF3-CD40 signaling may serve to enhance immune responses. It is hoped that these studies can additionally reveal important information about the normal regulation of this powerful activation pathway.

Es sollten Checkpoint inhibierende anti-TNFRSF Rezeptor Antikörper-Fusionsproteine hergestellt und charakterisiert werden. Die agonistische Aktivität TNFR-spezifischer Antikörper wird maßgeblich durch eine Immobilisation über Fcγ-Rezeptoren beeinflusst. In dieser Arbeit erfolgte die Immobilisation der Antikörper-Fusionsproteine über den PD-L1. In funktionellen Assays konnte eine Aktivitätssteigerung der TNFR-spezifischen Domänen mittels PD-L1 vermittelter Immobilisation gezeigt werden. / Checkpoint inhibiting anti-TNFRSF receptor antibody fusion proteins should be prepared and characterized. The agonistic activity of TNFR-specific antibodies is significantly influenced by immobilization via Fcγ receptors. In this work, immobilization of the antibody fusion proteins was performed via the PD-L1. Functional assays revealed an increase in activity of TNFR-specific domains using PD-L1-mediated immobilization.

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanisms involved in the induction of allograft survival, we determined the fate of tracer populations of alloreactive T cell receptor (TcR) transgenic CD8+ T cells circulating in a normal microenvironment. In the first portion of this thesis, we observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive TcR transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4 and B7-l/2 by CTLA4-Ig, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive TcR transgenic CD8+ T cells. Also in support of our hypothesis, depletion of CD4+ T cells, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive TcR transgenic CD8+ T cells. We continued by examining the effects of IFN-γ, IL-10 and IL-4. None of these cytokines had any significant effect on the deletion of alloreactive TcR transgenic CD8+ T cells induced by co-stimulation blockade. The last part of this thesis studied the behavior of alloreactive TcR transgenic CD8+ T cells during the maintenance phase of allograft survival induced by our two-element protocol. Using a hematopoietic TcR transgenic chimera system, our results demonstrated that levels of alloreactive CD8+ T cells remained low throughout the maintenance phase. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by co-stimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade by co-stimulation blockade.

Proteins, which are T cell-dependent antigens, preferentially induce antibodies of the IgG1 class in mouse, whereas LPS, which is a T-independent antigen, preferentially induces IgG3 and IgG2b. Interaction between CD40 on B cells and CD40 ligand (CD40L) on T cells has been shown to mediate T cell contact help for B cell proliferation, differentiation and immunoglobulin isotype switching. In addition, it has been shown that membranes from activated T cells induce germline γ1 transcripts, and that CD40 signaling induces germline γ1 transcripts. These results indicate that T cell contact help mediated by CD40 ligand (CD40L)-CD40 interaction may contribute to this preferential IgG1 isotype selection in response to T-dependent antigens by inducing transcription of germline Ig γ1 transcripts. Here we show that signaling via CD40 increases expression of a transiently transfected luciferase reporter plasmid driven by the germline γ1 promoter in M12.4.1 B lymphoma cells. By linker scanning mutation analysis of the promoter, we have identified a CD40 responsive region (CD40RR) which is able to confer inducibility by CD40L to a minimal c-fos promoter. The CD40RR contains three NF-кB-binding sites, each of which is required for maximal induction of the γ1 promoter activity by CD40L. Binding of the NF-кB/Re1 proteins p50, Re1A, c-Re1 and Re1B to the CD40RR can be induced by CD40 signaling in M12.4.1 cells or in splenic B cells. Co-transfection of expression plasmids for p50 together with Re1A or Re1B, but not p50 alone or p50 and c-Re1, transactivates the CD40RR in transient transfection assays in M12.4.1 cells. These data demonstrate NFкB/Re1 proteins activated by CD40 engagement play an important role in regulation of the germline γ1 promoter. Further support for this conclusion is provided by the finding that treatment of splenic B cells with NF-кB inhibitors prevents induction of germline γ1 transcripts by CD40L. Although LPS also induces NF-кB activation, it poorly induces germline γ1 promoter activity in M12.4.1 cells and it also poorly induces germline γ1 transcripts in splenic B cells and in the mouse B cell line, 1B4.B6. Western blot analyses show that LPS predominantly activates p50 and c-Re1, whereas CD40L induces all NF-кB/Re1 proteins (Re1A, Re1B, c-Re1 and p50). Likewise, in nuclear extracts from LPS-treated cells, p50/cRe1 and p50/p50 dimers are the major NF-кB/Re1 proteins which bind to the promoter for germline γ1 transcripts in electrophoretic mobility shift assays, whereas in nuclear extracts from CD40L-treated cells, p50/Re1A and p50/Re1B dimers are the major complexes. Reporter gene assay by over expressing NF-кB/Re1 fusion proteins indicates that p50/Re1A and p50/Re1B dimers, but not p50/c-Re1 or p50/p50 dimer, can transactivate the germline γ1 promoter. Despite their inability to activate the promoter, p50/c-Re1 and p50/p50 can bind to the promoter and suppress the transactivation activity of p50/Re1A and p50/Re1B. Therefore, the effect of NF-кB activation on the germline γ1 promoter depends on the Relative amounts of transactivating and non-trans activating NF-кB/Re1 dimers. The inability of LPS to induce germline γ1 transcripts can be explained by induction of non-transactivating NF-кB/Re1 dimers and the ability of CD40L to activate the promoter by a greater induction of Re1A and Re1B Re1ative to c-Re1.

T and B-lymphocytes play an important role in an adaptive immune response. Communication between these two cells may result in either a humoral immune response or tolerance. Communication between T and B-lymphocytes involves a number of inducible cell surface molecules on both T and B-lymphocytes. It was the aim of this project to gain a greater understanding of the role of CD40 in the dynamic communication that occurs between naïve T-lymphocytes and resting B-lymphocytes during cognate communication. Because in vivo antigen specific T-lymphocytes are at low frequency, it is difficult to examine antigen-specific naïve T-lymphocytes. Thus, an in vitro system employing naïve antigen-specific T cell receptor (TCR) transgenic T cells and small resting B-lymphocytes that did not express CD40 was devised to examine the role of CD40 in cognate communication between naïve T-lymphocytes and resting B-lymphocytes. Upon recognition of antigen on resting B-lymphocytes that expressed CD40, T-lymphocytes proliferated, expressed the activation antigens CD69 and CD25, and remained responsive to subsequent antigen challenge. In the absence of CD40, resting B-lymphocytes did not induce sustained proliferation or sustained expression of the activation markers CD69 and CD25 on naïve T-lymphocytes, and their recovery was decreased compared to naïve T-lymphocytes that recognized antigen on resting B-lymphocytes that expressed CD40. Naïve T-lymphocytes, however, remained responsive to subsequent antigen challenge after recognition of antigen on resting CD40-/- B-lymphocytes. Recognition of antigen on resting CD40-/- B-lymphocytes also resulted in increased recovery and antigen responsiveness of T-lymphocytes when compared to controls without antigen, The role of CD40 in sustaining activation of naïve T-lymphocytes may be unique to resting B-lymphocytes, since proliferation of naïve T-lymphocytes in response to dendritic cells that did not express CD40 was similar to proliferation of naïve T-lymphocytes in response to dendritic cells that expressed CD40. The mechanism by which CD40 sustained activation of naïve T-lymphocytes was investigated by examining the induction of various costimulatory molecules on resting CD40+/- and CD40-/- B-lymphocytes during cognate interaction with naive T-lymphocytes. Induction of B7-1, upregulation of CD44 and ICAM-1, and sustained but not initial induction of B7-2 required that CD40 be expressed on resting B-lymphocytes. Expression of B7-1 and CD44H was not required for proliferation of naïve T-lymphocytes in response to antigen presented on resting B-lymphocytes. However, sustained expression of B7-2 was crucial for proliferation of naïve T-lymphocytes in response to antigen presented on resting B-lymphocytes.

No description available.