Adenoviral CD40L gene therapy (AdCD40L) is a strong inducer of anti-tumor immune responses via its activation of dendritic cells (DCs). Activated DCs can in turn activate T cells, which are key players in an efficient anti-tumor response. This thesis includes three papers that focus on different aspects of AdCD40L gene therapy. In the first paper, the infiltration of suppressive CD11b+Gr-1+ cells in orthotopic MB49 bladder tumors was investigated and found to be significantly reduced while activated T cells were increased when the tumors had been treated with local AdCD40L gene therapy. Further, AdCD40L could tilt the cells in the tumor microenvironment in favor of an efficient anti-tumor immunity (M1 macrophages and activated T cells) instead of an immunosuppressive environment (CD11b+Gr-1int/low myeloid cells and M2 macrophages). Immunotherapy combined with chemotherapy has shown promising results, and the second paper investigates the combination of AdCD40L gene therapy together with the chemotherapeutic drug 5-Fluorouracil (5-FU). A synergistic effect of the combination treatment on orthotopic MB49 bladder tumors could be demonstrated. The combination therapy resulted in decreased tumor growth, increased survival and systemic MB49-specific immunity, whereas AdCD40L or 5-FU therapy alone had a poor effect on tumor growth. Efficient AdCD40L therapy is dependent on high transduction efficiency in both cancer cells and cells present in the tumor microenvironment. In an attempt to enhance the transduction efficiency, and thereby the therapeutic efficacy, a modified adenovirus was developed for paper three. This modified Ad5PTDf35(mCD40L) could, in comparison with the unmodified Ad5(mCD40L), demonstrate increased transduction capacity of a variety of murine cells. Further, the ability of antigen presenting cells (APCs) to present antigens to T cells was improved after transduction with Ad5PTDf35(mCD40L).

Le CD40 est un membre de la famille des récepteurs du facteur de nécrose tumorale ("Tumour necrosis factor", TNF), initialement identifié sur des cellules de carcinome de la vessie. L'interaction du CD40 avec son ligand (CD40L) est d'une importance cruciale pour le développement des cellules B et de la commutation d'isotype au cours de la réponse immunitaire acquise. L'expression du complexe CD40/CD40L était initialement cru d'être limiter aux cellules du système immunitaire, mais aujourd'hui il est bien connu que ce complexe est également exprimé sur les cellules du système circulatoire et vasculaire, et est impliqué dans diverses réactions inflammatoires; de sorte que le CD40L est maintenant considéré comme une molécule thrombo-inflammatoire prédictive des événements cardiovasculaires. Les plaquettes expriment constitutivement le CD40, alors que le CD40L n'est exprimé que suite à leur l'activation. Il est ensuite clivé en sa forme soluble (sCD40L) qui représente la majorité du sCD40L en circulation. Il fut démontré que le sCD40L influence l'activation plaquettaire mais son effet exact sur la fonction plaquettaire, ainsi que les mécanismes cellulaires et moléculaires sous-jacents à son action demeurent inconnus. Ainsi, ce projet a été entrepris dans le but d’adresser les objectifs spécifiques suivants: 1) évaluer les effets in vitro du sCD40L sur l'activation et l'agrégation plaquettaire; 2) identifier les récepteurs plaquettaires impliqués dans l’action du sCD40L; 3) élucider les voies signalétiques intracellulaires induits par le sCD40L; 4) évaluer les effets du sCD40L sur la formation de thrombus in vivo. Nous avons trouvé que le sCD40L augmente fortement l'activation et l'agrégation des plaquettes en réponse à de faibles concentrations d'agonistes. Les plaquettes humaines traitées avec une forme mutante du sCD40L qui n'interagit pas avec le CD40, et les plaquettes de souris déficientes en CD40 ne furent pas en mesure d'induire de telles réponses, indiquant que le récepteur principal du sCD40L au niveau des plaquettes est le CD40. En plus, nous avons identifié la présence de plusieurs membres de la famille du facteur associé du récepteur du TNF ("TNF receptor-associated factor", TRAF) dans les plaquettes et nous avons montré que seulement le TRAF2 s'associe avec le CD40 suite à la stimulation par le sCD40L. Nos résultats indiquent aussi que le sCD40L agisse sur les plaquettes au repos par l'entremise de deux voies signalétiques distinctes. La première voie implique l'activation de la petite GTPase Rac1 et de sa cible en aval, soit la protéine kinase p38 activée par le mitogène ("p38 mitogen-activated protein kinase", p38 MAPK ), menant au changement de forme plaquettaire et à la polymérisation de l'actine; alors que la deuxième voie implique l'activation de la cascade signalétique du NF-kB. Par ailleurs, à la suite d'une lésion artérielle induite par le chlorure de fer, le sCD40L exacerbe la formation de thrombus et l'infiltration leucocytaire au sein du thrombus dans les souris du type sauvage, mais pas chez les souris déficientes en CD40. En conclusion, ce projet a permis d'identifier pour la première fois deux voies signalétiques distinctes en aval du CD40 plaquettaire et a permis d'établir leur implication dans l'activation et l'agrégation plaquettaire en réponse au sCD40L. De manière plus importante, ce projet nous a permis d'établir un lien direct entre les niveaux élevés du sCD40L circulant et la formation de thrombus in vivo, tout en soulignant l'importance du CD40 dans ce processus. Par conséquent, l'axe CD40/CD40L joue un rôle important dans l'activation des plaquettes, les prédisposant à une thrombose accrue en réponse à une lésion vasculaire. Ces résultats peuvent expliquer en partie la corrélation entre les taux circulants élevés du sCD40L et l'incidence des maladies cardiovasculaires. / CD40 is a member of the tumour necrosis factor (TNF) receptor family, originally identified on human bladder carcinoma cells. Interaction of CD40 with its ligand (CD40L) is of crucial importance for B cell development and immunoglobulin isotype switching during the adaptive immune response. Expression of the CD40/CD40L dyad was initially thought to be restricted to cells of the immune system, but today it is known to be also expressed on cells of the circulatory and vascular systems, and have important implications in various inflammatory reactions, such that CD40L is now regarded as a thrombo-inflammatory molecule and a reliable predictor of cardiovascular events. Platelets constitutively express CD40, whereas CD40L is expressed upon activation and subsequently cleaved into its soluble form (sCD40L), accounting for the majority of circulating sCD40L. Soluble CD40L has been shown to influence platelet activation but its precise effect on platelet function, and the underlying cellular and molecular mechanisms remain undefined; hence the purpose of this project. The specific aims of this study are: 1) to evaluate the in vitro effects of sCD40L on platelet activation and aggregation; 2) to determine the receptor(s) on platelets involved in the action of sCD40L; 3) to elucidate the intracellular signalling pathways induced by sCD40L; and 4) to evaluate the in vivo effects of sCD40L on thrombus formation. We have showed that sCD40L strongly enhances activation and aggregation of washed human platelets in response to sub-threshold concentrations of agonists. Human platelets treated with a mutated form of sCD40L that lacks CD40 binding, and platelets from CD40 deficient mice failed to elicit such responses, indicating that CD40 is the major platelet receptor for sCD40L. Moreover, we identified the presence of multiple members of the TNF receptor-associated factor (TRAF) in platelets and showed that only TRAF2 associates with CD40 after sCD40L stimulation. Interestingly, sCD40L primes resting platelets through two distinct signalling pathways. The first pathway involves activation of the small GTPase Rac1 and its downstream target p38 mitogen-activated protein kinase, leading to platelet shape change and actin polymerization; whereas the second pathway involves activation of the NF-κB signalling cascade. Furthermore, sCD40L exacerbates thrombus formation and leukocyte infiltration within the thrombus mass in wild-type mice but not in CD40 deficient mice following ferric chloride-induced arterial injury. In conclusion, we have identified for the first time two distinct signalling pathways downstream of platelet CD40, and established their implication in platelet activation and aggregation in response to sCD40L. Noticeably, we established a direct link between elevated levels of sCD40L and in vivo thrombus formation, while emphasizing the requirement of CD40 in this process. Therefore, the CD40/CD40L dyad plays an important role in platelet priming that predisposes platelets to enhanced thrombus formation in response to vascular injury. These results may partly explain the correlation between elevated circulating levels of sCD40L and the incidence of cardiovascular diseases.

Le traitement antiplaquettaire à l’aspirine (ASA) est moins efficace chez certains patients coronariens, ce qui augmente leur risque de développer une thrombose. Des taux sanguins élevés de médiateurs thrombo-inflammatoires, tels que le sCD40L, peuvent expliquer de telles variabilités. Nous avons émis l’hypothèse que, en présence de taux élevés de sCD40L, l’efficacité de l’ASA peut être réduite. Ainsi, nous avons viser à déterminer les effets de l’ASA sur la signalisation et l’agrégation des plaquettes en présence de sCD40L. Les effets de l'ASA sur les plaquettes humaines traitées par le sCD40L, en réponse à des concentrations sub-optimales de collagène ou de thrombine, ont été évalués sur l'agrégation, la sécrétion de thromboxane A2 (TxA2) et la phosphorylation de la p38- « Mitogen-activated protein kinase » (MAPK), le facteur nucléaire-κB (NF-κB), la kinase activée par le TGF-β 1 (TAK-1) et la chaîne légère de la myosine (MLC). Le sCD40L a significativement augmenté la sécrétion de TxA2 dans les plaquettes, en réponse à des doses sub-optimales de collagène et de thrombine, ce qui a été inversé par l'ASA. L'ASA n'a pas inhibé la phosphorylation de p38-MAPK, NF-KB, TAK-1, que ce soit avec une stimulation par le sCD40L seul ou en présence des agonistes plaquettaires. Cependant, Le sCD40L a potentialisé l'agrégation plaquettaire, un effet complètement inversé et partiellement réduit par l'ASA en réponse au collagène et à la thrombine, respectivement. Les effets de l'ASA sur les plaquettes traitées par le sCD40L et stimulées par le collagène étaient liés à l'inhibition du changement de forme des plaquettes et la phosphorylation de la MLC. L'ASA n'affecte pas la signalisation du sCD40L dans les plaquettes, mais empêche son effet sur la sécrétion de TXA2 et l'agrégation plaquettaire en réponse au collagène, via un mécanisme impliquant l'inhibition de la MLC. Le ciblage de l'axe sCD40L dans les plaquettes peut avoir un potentiel thérapeutique chez les patients, présentant des taux élevés de sCD40L, qui ne répondent pas ou moins à l'ASA. / Antiplatelet therapy with Aspirin (ASA) is less efficient in some coronary patients, which increases their risk of developing thrombosis. Elevated blood levels of thrombo-inflammatory mediators, like sCD40L, may explain such variabilities. We hypothesized that in the presence of elevated levels of sCD40L, the efficacy of ASA may be reduced. Accordingly, this study was designed to determine the effects of ASA on sCD40L signalling and aggregation of platelets. The effects of ASA on sCD40L-treated human platelets, in response to suboptimal concentrations of collagen or thrombin, were assessed on aggregation, thromboxane A2 (TxA2) secretion, and phosphorylation of p38-MAPK, NF-κB, TGF-β-activated kinase 1 (TAK-1), and myosin light chain (MLC). sCD40L significantly elevated TxA2 secretion in platelets, in response to suboptimal doses of collagen and thrombin, which was reversed by ASA. ASA did not inhibit phosphorylation of p38-MAPK, NF-κB, TAK-1, either with sCD40L stimulation alone or with platelet agonists. However, sCD40L potentiated platelet aggregation, an effect completely reversed and partially reduced by ASA in response to collagen and thrombin, respectively. The effects of ASA in sCD40L-treated platelets with collagen were related to inhibition of platelet shape change and MLC phosphorylation. ASA does not affect platelet sCD40L signalling, but prevents its effect on TXA2 secretion and platelet aggregation in response to collagen, via a mechanism implying inhibition of MLC. Targeting sCD40L axis in platelets may have therapeutic potential in patients with elevated levels of sCD40L that are none or less responding to ASA.

No description available.

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

Le CD40 ligand (CD40L) est une molécule thrombo-inflammatoire qui prédit des événements cardiovasculaires. Les plaquettes constituent la principale source du CD40L soluble (sCD40L) dans la circulation, avec la capacité d’influencer la fonction des plaquettes par l'intermédiaire de sa liaison à ses récepteurs plaquettaires : CD40, αIIbβ3 et α5β1. Nous avons précédemment démontré que la stimulation des plaquettes humaines avec le sCD40L induit une activation du nuclear factor kappa B (NF-κB), qui pourra jouer un rôle non génomique en amorçant les plaquettes. En effet, l’amorçage des plaquettes par le sCD40L augmente fortement l’activation et l’agrégation plaquettaire en réponse à des doses sous-optimales d’agonistes. Cependant, l’implication des différents récepteurs dans l’amorçage des plaquettes suite à l’activation du NF-κB par le sCD40L demeure inconnue. De plus, le transforming growth factor-B (TGF-B)-activated Kinase (TAK1), un régulateur majeur de l’inflammation, est activé par le sCD40L dans les cellules nucléées, et pourra ainsi être impliqué dans la signalisation sCD40L/NF-κB plaquettaire. D’autre part, le sCD40L se trouve en quantité élevée dans la circulation sanguine des patients coronariens. De plus, le traitement antiplaquettaire à l’Aspirine (ASA) est inefficace chez certains patients et son efficacité est réduite chez les patients à hauts risques d’évènements coronariens, ce qui augmente leur risque de sur-activation plaquettaire et de développer une thrombose. Des taux sanguins élevés de médiateurs thrombo-inflammatoires, tels que le sCD40L, peuvent expliquer de telles variabilités. Ainsi, ce projet a été entrepris dans le but d’élucider l’impact de l’axe sCD40L/NF-κB sur la fonction plaquettaire et sa modulation par l’ASA. Dans le premier volet de mon projet, nous avons identifié les récepteurs de sCD40L impliqués dans l'activation du NF-κB plaquettaire, leur signalisation en aval et leur implication dans l'agrégation plaquettaire. Nous avons montré que les plaquettes exprimaient les récepteurs du CD40L : CD40, αIIbβ3 et α5β1 et libéraient du sCD40L. Le sCD40L seul a induit une activation de NF-κB plaquettaire. Cet effet était absent des plaquettes de souris déficientes en CD40 (CD40-/-) et inhibé par le blocage de CD40, mais n'était pas affecté par le blocage de αIIbβ3 ou α5β1. L’axe sCD40L/CD40 a aussi activé TAK1 en amont de NF-κB. Dans les études fonctionnelles, le sCD40L seul n’a pas eu d’effet sur l’agrégation plaquettaire, mais a potentialisé l’agrégation en présence de doses sous-optimales de thrombine; cet effet a été aboli par des inhibiteurs de CD40, TAK1 et NF-κB. Cette première étude nous a permis de conclure que le sCD40L amorce les plaquettes via des voies de signalisation impliquant CD40/TAK1/NF-κB, ce qui prédispose les plaquettes à une activation et agrégation accrues en réponse à des stimuli thrombotiques. Dans le deuxième volet de mon projet, nous avons émis l’hypothèse qu’en présence de taux élevés de sCD40L, l’efficacité de l’ASA peut varier et avons visé à déterminer les effets de l’ASA sur la signalisation et l’agrégation des plaquettes en présence de sCD40L. Les effets de l'ASA sur les plaquettes humaines traitées au sCD40L, en réponse à des concentrations sous-optimales de collagène ou de thrombine, ont été évalués sur l'agrégation, la sécrétion de thromboxane A2 (TxA2) et la phosphorylation de p38 MAPK, NF-κB, TAK1 et la chaîne légère de la myosine (MLC). Le sCD40L a significativement augmenté la sécrétion de TxA2 par les plaquettes, en réponse à des doses sous-optimales de collagène et de thrombine; cet effet a été inversé par l'ASA. L'ASA n'a pas inhibé la phosphorylation de p38 MAPK, NF-κB, TAK1, que ce soit avec une stimulation plaquettaire par le sCD40L seul ou en présence des agonistes. Le sCD40L a potentialisé l'agrégation plaquettaire, un effet complètement inversé et partiellement réduit par l'ASA en réponse à une dose sous-optimale de collagène et de thrombine, respectivement. Les effets de l'ASA sur les plaquettes traitées au sCD40L avec du collagène étaient liés à l'inhibition du changement de forme des plaquettes et à la phosphorylation de la MLC. En résumé, l'ASA n'affecte pas la signalisation plaquettaire du sCD40L, mais empêche son effet sur la sécrétion de TxA2 et l'agrégation plaquettaire en réponse au collagène, via un mécanisme impliquant l'inhibition de la MLC. En conclusion, ce projet nous a permis de déterminer que l’amorçage des plaquettes par le CD40L via l’activation de NF-κB dépend du récepteur CD40 et la signalisation via TAK1. Ainsi, l’axe sCD40L/CD40/TAK1/NF-κB potentialise l’activation et l’agrégation en réponse à des stimuli thrombotiques, ce qui peut favoriser l’occurrence d’événements athéro-thrombotiques chez les patients coronariens. De plus, étant donné que l’ASA n’a pas d’effet sur la signalisation via l’axe sCD40L, le ciblage de cet axe dans les plaquettes peut avoir un potentiel thérapeutique chez les patients coronariens présentant des taux élevés de sCD40L et ne réagissant pas ou moins à l'ASA. / CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets are the main source of soluble CD40L (sCD40L) in the circulation, with the ability to influence platelet function through its binding to platelet receptors: CD40, αIIbβ3, and α5β1. We have previously demonstrated that stimulation of human platelets with sCD40L induces activation of nuclear factor kappa B (NF-κB), which may play a non-genomic role in priming platelets. Indeed, platelet priming by sCD40L strongly enhances platelet activation and aggregation in response to suboptimal doses of agonists. However, the involvement of the different receptors in platelet priming following activation of NF-κB by sCD40L remains unknown. In addition, the transforming growth factor-B (TGF-B) -activated Kinase (TAK1), a major regulator of inflammation, is activated by sCD40L in nucleated cells, and may be involved in sCD40L/NF-κB signaling. On the other hand, sCD40L is high in the bloodstream of coronary patients, who receive Aspirin (ASA) for secondary prevention. However, antiplatelet therapy with ASA is ineffective in some patients and its efficacy is reduced in patients at high risk for coronary events, increasing their risk of platelet over-activation and developing thrombosis. High blood levels of thrombo-inflammatory mediators, such as sCD40L, may explain such variability. Thus, we undertake this project to elucidate the impact of the sCD40L/NF-κB axis on platelet function and its modulation by ASA. In the first part of our project, we aimed to identify the sCD40L receptors involved in the activation of platelet NF-κB, their downstream signaling and their involvement in platelet aggregation. We have shown that platelets express CD40L receptors: CD40, αIIbβ3 and α5β1 and release sCD40L. sCD40L alone induced activation of platelet NF-κB. This effect was absent in CD40-/- mouse platelets and inhibited by CD40 blockade, but was not affected by αIIbβ3 or α5β1 blockade. The sCD40L/CD40 axis has also activated TAK1 upstream of NF-kB. In functional studies, sCD40L alone had no effect on platelet aggregation but potentiated aggregation in the presence of suboptimal thrombin doses. The inhibitors of CD40, TAK1 and NF-κB abolished this effect. This first study concluded that sCD40L primes platelets via CD40/TAK1/NF-κB signaling pathways, which predisposes platelets to increased activation and aggregation in response to thrombotic stimuli. In the second part of our project, we hypothesized that in the presence of high levels of sCD40L; the effectiveness of ASA may vary. Accordingly, we aimed at determining the effects of ASA on signaling and aggregation of platelets in the presence of sCD40L. The effects of ASA on human platelets treated with sCD40L, in response to suboptimal concentrations of collagen or thrombin, were evaluated on aggregation, thromboxane A2 (TxA2) secretion and phosphorylation p38 MAPK, NF-κB, TAK1 and myosin light chain (MLC). sCD40L significantly increased TxA2 secretion by platelets in response to suboptimal doses of collagen and thrombin; this effect has been reversed by the ASA. ASA did not inhibit the phosphorylation of p38 MAPK, NF-κB, TAK-1, with platelet stimulation by sCD40L either alone or in the presence of agonists. sCD40L potentiated platelet aggregation, an effect completely reversed and partially reduced by ASA in response to a suboptimal dose of collagen and thrombin, respectively. In addition, the effects of ASA on sCD40L-treated platelets with collagen related to inhibition of platelet shape change and phosphorylation of MLC. In summary, ASA does not affect platelet signaling via sCD40L, but prevents its effect on TXA2 secretion and platelet aggregation in response to collagen, via a mechanism involving inhibition of MLC. In conclusion, this project allowed us to determine that platelet priming by sCD40L via activation of NF-κB depends on the CD40 receptor and signaling via TAK1. Thus, the sCD40L/CD40/TAK1/NF-κB axis potentiates activation and aggregation in response to thrombotic stimuli, which may promote occurrence of atherothrombotic events in coronary patients. In addition, since ASA has no effect on platelet signaling via the sCD40L axis, targeting of this axis in platelets may have therapeutic potential in coronary patients with high levels of sCD40L that are none or less responding to ASA.

Indiana University-Purdue University Indianapolis (IUPUI) / The immune system protects the body from foreign organisms. T cells and B cells are integral components of the ability of the immune system to generate focused immune responses. The development of specialized subsets of T helper cells is governed by transcription factors. Previous work demonstrated a requirement for the transcription factor PU.1 in the development of IL-9-secreting Th9 cells. Work in this dissertation demonstrates that the Th9 subset is not stable in vitro, and that PU.1 expression decreases during long-term culture. To examine a role for PU.1 in Th9-independent immunity we examined a model of multiple sclerosis termed experimental autoimmune encephalomyelitis (EAE). Mice that lack PU.1 expression in T cells (Sfpi1lck-/- mice) demonstrated more severe disease with attenuated recovery compared to control mice, and this was accompanied by an increase of T cells in the central nervous system. We also observed that following multiple routes of immunization Sfpi1lck-/- mice had increased numbers of T follicular helper (Tfh) cells and increased germinal center responses. This correlated with increased expression of the cytokine IL-21 and the surface protein CD40L in T cells that lacked PU.1 expression and resulted in increased numbers of germinal center B cells and antigen-specific antibody titers compared to control mice. The increased germinal center B cells and antibody titers were attenuated with blocking CD40L antibody but not with neutralizing IL-21 antibody. These results suggest that PU.1 limits the expression of CD40L on Tfh cells to regulate the humoral immune response. Together, the data in this dissertation demonstrate Th9-independent functions of PU.1. Moreover, this work shows that transcription factors promoting the development of one subset of T helper cells can simultaneously have negative effects on distinct T cell lineages.

The tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were initially discovered as proteins that inducibly interact with the intracellular region of TNF receptors (TNFRs). Because the TNFRs lack intrinsic catalytic activity, the TRAFs are hypothesized to orchestrate signaling activation downstream of the TNFR superfamily, however their mechanism of activation remains unclear (Inoue et al., 2000; Bishop, 2004). Originally, the TRAFs were compared to the signal transducers and activators of transcription (STAT) protein family, due to their sequence homology, and the presence of multiple RING- and zinc-finger domains, suggesting that their function may be to regulate transcriptional activity (Rothe et al., 1994; Hu et al., 1994; Sato et al. 1995; Cheng et al., 1995). However, subsequent research focused predominantly on their cytoplasmic functions, and more recently on their function as E3 ubiquitin ligases (Pineda et al., 2007). In my research, I analyzed the subcellular localizations of the TRAFs following CD40 ligand (CD40L)-stimulation, and found that TRAF2 and 3 rapidly translocate into the nucleus of primary neurons and Neuro2a cells. Interestingly, similar analysis conducted in pre-B lymphocytes (Daudi cells) revealed a different response to CD40L-stimulation, with TRAF2 and 3 being rapidly degraded within 5-minutes of stimulation. These findings are significant because they demonstrate for the first time that the TRAFs translocate into the nucleus and suggest that they may function within the nucleus in a cell-specific manner. I next analyzed the ability of TRAF2 and 3 to bind to DNA, and found that they both bind to chromatin and the NF-kappaB consensus element in Neuro2a cells, following CD40L-stimulation. Similar analyses of the chromatin binding of TRAF2 and 3 in Daudi cells revealed that they were rapidly degraded, similar to the results from my analysis of their subcellular localization. These findings show for the first time that the TRAFs interact with DNA, and therefore support the hypothesis that the TRAFs may function within the nucleus as transcriptional regulators. Finally, I analyzed the ability of the TRAFs to regulate transcriptional activity by luciferase assay. Previous studies showed that overexpression of TRAF2 and 6 could induce NF-kappaB transcriptional activity; however researchers have not been able to determine the mechanism by which they do so. In my studies, I found that every TRAF can directly regulate transcriptional activity either as co-activators or co-repressors of transcription, in a cell- and target protein-specific manner. Additionally, I found that TRAF2 can act as a transcriptional activator, and that its ability to regulate transcription is largely dependent upon the presence of its RING-finger domain. In conclusion, these studies have revealed an entirely novel function for the TRAFs as immediate-early transcriptional regulators. Future research into the genes that are regulated by the specific TRAF complexes will further elucidate how the TRAFs regulate TNFR signaling, as well as whether dysfunctions in TRAF signaling may be associated with known disorders. If specific TRAF complexes are found to regulate specific genes, then pharmacological targeting of the individual TRAF complexes may allow for the highly specific inhibition of signaling events downstream of the TNFRs, without compromising overall receptor signaling, transcription factor pathways, or cellular systems.

The goal of this body of research was to elucidate the mechanism by which CD4+ T cells provide help for CD8+ cytotoxic T lymphocyte (CTL) responses in different immunization types. The establishment of diseases, such as chronic infections and cancers, is attributed to severe loss of or dysfunctions of CD4+ T cells. Even in acute infections, CD4+ T cell deficiency leads to poor memory responses. While the role of CD4+ T cells is being increasingly appreciated in these diseases, the timing and nature of CD4+ T help and associated molecular mechanisms are not completely understood. Growing evidence suggests that, depending on the type of infections or immunizations, the requirements of CD4+ T cells can vary for optimal CD8+ CTL responses. In order to understand the modulatory effects of CD4+ T cells for optimal CD8+ CTL responses, two distinct immunization types were chosen. These include: 1) non-inflammatory dendritic cell (DC) immunization, which fails to provide inflammatory/danger signals; and 2) inflammatory adenovirus (AdV) immunization, which provides profound inflammatory/danger signals. This allowed us to study CD4+ T cell’s participation under different inflammatory conditions. The studies described in Chapters 2 and 3 of this thesis were performed to further understand the concept of how CD4+ T cells mediate optimal CD8+ CTL responses. This has been called the “new dynamic model of CD4+ T helper – antigen (Ag)-presenting cells (Th-APCs),” proposed in 2005 by our laboratory. The study described in Chapter 2 shows that Th-APCs participate not only in augmenting CTL-mediated immune responses, perhaps during early phase, but also in regulating cellular immunity, perhaps during a later phase. Through enhanced IL-2, CD80 and CD40L singnaling, and weaker peptideMHC I (pMHC) signaling, Th-APCs stimulated naïve CD8+ T cells to differentiate into effector CTLs, capable of developing into, central memory CTLs. Th-APC-stimulated CD4+ T cells behaved like Th cells in function, augmenting the overall magnitude of CTL responses. In contrast, Th-APCs were able to kill DCs and other Th-APCs, predominantly through perforin-mediated pathway. The experiments described in Chapter 3 revealed a novel co-operative role of cognate Th-CTL interactions, contrary to previously known immune-regulatory mechanisms among Th-Th or CTL-CTL interactions. In our experiments, Th cells, via CD40L, IL-2, and acquired pMHC-I signaling, enhanced CTL survival and transition into functional memory CTLs. Moreover, RT-PCR, flow cytometry and western blot analysis demonstrate that increased survival of Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of FasL and TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (NFATc1, Bcl-10, Casp-3, Casp-4, Casp-7) genes/ molecules. Finally, helped CTLs were also able to induce protection against highly metastasizing tumor challenge, explaining why memory CTLs generated under cognate Th1’s help show survival and recall advantages. The studies in Chapter 4 showed how the precursor frequency (PF) of CD8+ T cells impacts CD4+ T helper requirements for functional CTL responses. At endogenous PF, CD4+ T helper signals were necessary for both primary and memory CTL responses. At increased PF, CD4+ T help, and its CD40L but not IL-2 signal became dispensable for primary CTL responses. In contrast, memory CTL responses required CD4+ T cell signals, largely in the form of IL-2 and CD40L. Thus, these results could impact the development of novel immunotherapy against cancers, since their efficacy would be determined in part by CD4+ T help and CD8+ T cell PF. Finally, the study showed the importance of CD4+ T cells for multiple phases of AdV transgene product-specific CTL responses. These include: a) cognate CD4+ T cells enhanced CTL responses via IL-2 and CD40L signaling during primary, maintenance and memory phases; b) polyclonal CD4+ T environment enhanced the survival of AdV-specific CTL survival, partially explaining protracted CTL contraction phase; and c) during the recall phase, the CD4+ T environment, particularly memory CD4+ T cells, considerably enhanced not only helped, but also unhelped, memory CTL expansion. Thus, these results suggest the participation of both cognate and polyclonal CD4+ T cells for multiple phases of AdV-specific CTLs. Taken together, the current work delineated the critical roles of CD4+ T cells in different stages of CTL responses and in the development of anti-tumor immunity. The results presented here will significantly advance our current understanding of immunity to cancers, autoimmunity and chronic infections, since pathogenesis of these diseases is largely determined by CD4+ T helper functions. As most immunization procedures use the principle that is based on functions of memory cells, the knowledge gained from this work will also have a major impact on designing vaccines against intractable diseases, including cancers and chronic infections. Moreover, in advanced tumors, vaccines developed using this knowledge may act synergistically with other cancer treatments such as irradiation, chemotherapy and microsurgery, minimizing their side effects and prolonging the lives of patients.