No / The expression and potential role of platelet membrane CD154 and sCD154 in atherosclerosis was investigated in patients with peripheral arterial disease. This prospective observational study measured the expression of platelet-bound CD154 and soluble CD154 (sCD154) in 39 patients with critical limb ischaemia (CLI, n = 15), stable intermittent claudication (SIC, n = 12) and age-matched controls (AMC, n = 12). Basal and agonist-stimulated CD154, P-selectin expression and fibrinogen binding was measured by whole blood flow cytometry, while sCD154 was measured in paired plasma samples by ELISA. Basal expression of CD154 on the platelet surface was enhanced in both groups of patients with peripheral arterial disease. However, the critical limb ischaemics showed the highest level of basal expression 0.7 ± 0.3 [median ± IQR] and was significantly increased compared to both stable intermittent claudicants and age-matched controls (P < 0.001). On agonist stimulation with either ADP or thrombin critical limb ischaemics demonstrated greater platelet reactivity and propensity to express CD154 compared to age-matched controls (P < 0.05). Confirmation of the cellular expression of CD154 results was obtained by measuring sCD154 concentrations in autologous plasma samples. Here plasma levels of sCD154 in critical limb ischaemics were significantly greater than both stable intermittent claudicants and age-matched controls (P < 0.005).

No description available.

L’insuffisance rénale aiguë (IRA) est une pathologie fréquente en réanimation. Elle est associée à une mortalité et une morbidité importante. Le sepsis en est la cause la plus fréquente. La compréhension de la physiopathologie du sepsis et de ses complications a beaucoup progressé ces dernières années mais ne s’est pas encore traduite par des avancées thérapeutiques significatives en pratique clinique. Le paradigme d’une altération de la perfusion sanguine comme paramètre clé de la constitution des lésions rénales a ainsi été remis en question, plusieurs travaux révélant que le débit sanguin rénal n’est pas toujours altéré en cas de sepsis, et qu’une IRA peut se développer en cas de débit sanguin rénal préservé, voire augmenté. Le sepsis est caractérisé par de profondes perturbations de la réponse immunitaire et une réaction inflammatoire disproportionnée. A l’origine de l’atteinte rénale, l’inflammation et les altérations de la microcirculation sont maintenant considérés comme des mécanismes physiopathologiques fondamentaux. Au-delà de leur rôle dans l’hémostase, la contribution des plaquettes sanguines à la réponse inflammatoire, au maintien de l’intégrité tissulaire et à la défense contre les infections a considérablement élargi le spectre de leurs compétences et en a fait des acteurs physiopathologiques potentiels dans le sepsis. Les plaquettes sanguines exercent la plupart de ces fonctions grâce à l’expression de nombreux médiateurs membranaires ou solubles. Parmi eux, le CD154 tient une place particulière : les plaquettes sont une source essentielle de CD154 dans l’organisme et il joue un rôle central dans la réponse inflammatoire. Nous proposons dans ce travail un aperçu de ces avancées physiopathologiques récentes et nous discutons de la contribution des plaquettes et du CD154 dans les atteintes microcirculatoires et les défaillances multi-viscérales dans le sepsis. Nous nous sommes intéressés au rôle pro-inflammatoire du CD154 en conditions d’hypoxie au niveau de l’épithélium tubulaire rénal. Des données récentes soulignent en effet l’importance de l’hypoxie dans la réaction inflammatoire. Le contrôle de la production d’interleukine (IL)-6, une cytokine centrale de la réponse inflammatoire, par le CD154 a été étudié dans un modèle de culture de cellules épithéliales tubulaires (CET) rénales. Un modèle murin d’IRA par ischémie/reperfusion rénale a également été mis au point et appliqué à des souris déficientes en CD154 et CD40. Nos travaux révèlent que le CD154 induit fortement la sécrétion d’IL-6 par les CET en conditions d’hypoxie et que les souris déficientes en CD154 régénèrent plus rapidement leur épithélium tubulaire après ischémie/reperfusion rénale. Ces résultats pourraient ouvrir la voie à de potentielles pistes thérapeutiques pour la prise en charge des IRA d’origine septique. / Acute kidney injury (AKI) is a common complication in critically ill patients and is associated with increased morbidity and mortality. Sepsis is the most common cause of AKI. The understanding of sepsis pathophysiology and its complications has progressed significantly in recent years but has not yet been translated into significant therapeutic advances in clinical practice. The traditional paradigm that sepsis-induced AKI is linked to renal hypoperfusion has been challenged by recent evidences showing that renal blood flow is not universally impaired during sepsis,and that AKI can develop in the presence of normal or even increased renal bloodflow. Sepsis is characterized by profound alterations of the immune response and adisproportionate inflammatory response. Inflammation and microcirculatorydysfunction are now considered as fundamental pathophysiological mechanisms atthe origin of renal injuries. Beyond haemostasis, the contribution of platelets ininflammation, tissue integrity and defence against infections has considerablywidened the spectrum of their role and made them potential physiopathologicalactors in sepsis. Platelets fulfil most of these functions through the expression ofmembrane-bound or soluble mediators. Among them, CD154 holds a peculiarposition, as platelets represent a major source of CD154 and as CD154 is a centralregulator of inflammation. Here, we provide an overview of these recentpathophysiological advances and discuss the platelets and CD154 contribution tomicrocirculatory alterations in multi-organ dysfunction in sepsis. We investigated thepro-inflammatory role of CD154 under hypoxic conditions in the renal tubularepithelium as recent data highlight the importance of hypoxia in the inflammatoryreaction. We studied the control of interleukin (IL)-6 production, a key cytokineinvolved in inflammation, by CD154 in oxygen deprivation conditions using a kidneytubular epithelial (TEC) cell line model. We also studied a murine model of kidneyinjury after ischemia/reperfusion, a model that was applied in CD154 and CD40deficient mice. We found that CD154 is a potent inducer of IL-6 secretion by TEC inhypoxia and that CD154-deficient mice regenerate earlier the tubular epithelium afterischemia/reperfusion injury. These findings may provide potential avenues for septicAKI management and therapy.

Kawasaki Disease (KD) is a childhood disease leading to coronary arteritis. Elevated numbers of CD40L+ platelets in circulation is correlated with risk of heart damage. CD40L is a tumor necrosis family member that binds to CD40 and αIIbβ3, receptors which are also expressed on platelets. A single injection of Lactobacillus casei Cell Wall Extract (LCWE) induces a disease similar to KD in mice, where LCWE superantigen (SAg) reactive T-cells persist in the coronary artery. This phenotype is inconsistent with the fate of SAg-stimulated cells and is likely mediated by co-stimulation. This work shows that stimulation with a SAg induces platelet activation and CD40L expression in vitro. Furthermore, enhanced survival of SAg-reactive T-cells is demonstrated following antibody-mediated CD40L cross-linking. This effect is mediated via inhibition of the extrinsic apoptosis pathway. In addition, CD40 cross-linking is also reported to enhance SAg-reactive T-cell survival by enhancing CD86 expression on APCs and CD28 co-stimulation.

Kawasaki Disease (KD) is a childhood disease leading to coronary arteritis. Elevated numbers of CD40L+ platelets in circulation is correlated with risk of heart damage. CD40L is a tumor necrosis family member that binds to CD40 and αIIbβ3, receptors which are also expressed on platelets. A single injection of Lactobacillus casei Cell Wall Extract (LCWE) induces a disease similar to KD in mice, where LCWE superantigen (SAg) reactive T-cells persist in the coronary artery. This phenotype is inconsistent with the fate of SAg-stimulated cells and is likely mediated by co-stimulation. This work shows that stimulation with a SAg induces platelet activation and CD40L expression in vitro. Furthermore, enhanced survival of SAg-reactive T-cells is demonstrated following antibody-mediated CD40L cross-linking. This effect is mediated via inhibition of the extrinsic apoptosis pathway. In addition, CD40 cross-linking is also reported to enhance SAg-reactive T-cell survival by enhancing CD86 expression on APCs and CD28 co-stimulation.

Necrotic enteritis (NE) is an economically important enteric disease of broiler chicken primarily caused by a-toxin (Cpa) secreted by C. perfringens type A. Mice immunized with recombinant C-terminal domain of Cpa (CpaCD) had transient and fewer localized lesions upon challenge with C. perfringens type A. These results demonstrate the usefulness of CpaCD as an immunogen for vaccine development against NE for chickens. Chicken CD40 (chCD40) is mainly expressed on the surface of chicken antigen-presenting cells (APCs), and the interaction of chCD40 and chCD40L (natural ligand for chCD40) provides crucial activation signals for chicken B-cells. A hypothesis was proposed that in ovo vaccination with an adenovirus-vectored CpaCD vaccine capable of targeting immunogen to APCs through the CD40 pathway will improve protection against NE in chickens. One agonistic monoclonal anti-chCD40 antibody (designated 2C5) was produced and characterized. 2C5 not only detected expression of chCD40 on chicken APCs, but also induced NO synthesis in chicken HD11 macrophages and enhanced proliferation of serum-starved chicken DT40 B-cells. This demonstrated substantial functional equivalence of 2C5 with chCD40L. The potential of 2C5 as an immunological adjuvant was further assessed by targeting a hapten to chicken APCs in hopes of enhancing an effective IgG response. Seven-week old chickens were immunized subcutaneously once with a complex consisting of 2C5 and peptide, and relative quantification of the peptide-specific IgG response showed that this complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. An agonistic anti-chCD40 single-chain variable fragment (designated DAG1) was combined with an adenoviral delivery system to create a vaccine, Ad-(DAG1-Cp aCD-FLAG), for in ovo administration. The efficacy of in ovo vaccination of broilers with Ad-(DAG1-Cp aCD-FLAG) in controlling NE was evaluated by C. perfringens type A challenge at 18 days post-hatch. Neither statistically significant IgA / IgG response nor protection against C. perfringens type A challenge was found in the vaccinated birds. These preliminary data suggest that a super-optimal dose of Ad-(DAG1-Cp aCD-FLAG) may be the main issue, because Cpa-specific B-cells may undergo apoptosis through the CD40 pathway.

No description available.

T Zellen können in zwei Hauptpopulationen mit unterschiedlichen Aufgaben unterschieden werden. CD4+ T Zellen exprimieren im Zuge ihrer Aktivierung CD40L, welches ein zentraler kostimulatorischer Rezeptor zur Induktion von B-Zell basierter humoraler Immunität, APC Aktivierung und einer effizienten Effektor CD8+ T Zell Entwicklung ist („Helfer-Funktion“). Im Gegensatz dazu sind die zytotoxischen CD8+ T Zellen dazu vorbestimmt, infizierte oder maligne Zellen direkt abzutöten. Jedoch wurde eine Fraktion von CD8+ T Zellen identifiziert, die nach Aktivierung CD40L exprimiert. Bisher ist nicht verstanden, wie in solchen CD8+ T Zellen a) die CD40L Expression reguliert ist, b) wann und wie die Fähigkeit CD40L zu exprimieren implementiert wird und c) was die Folgen für das Immunsystem sind. In dieser Arbeit konnten wir zeigen, dass sowohl in CD4+ als auch in CD8+ T Zellen die CD40L Expression durch DNA-Methylierung regulatorischer Regionen des CD40LG Lokus reguliert wird. Die Demethylierung zentraler Elemente wird im Thymus implementiert, manifestiert sich mit der T-Zell Reifung und geht mit einer zunehmenden Stabilität der CD40L Expression einher. Erhöhte Expression von CD5 und NUR77 in CD40L+ CD8+ SP Thymozyten weisen auf eine positive Selektion mit hoher Affinität gegen Selbst-peptide während der Reifung im Thymus hin, welche das weitere Schicksal der CD40L exprimierenden CD8+ T Zellen beeinflusst. Naive CD40L+ CD8+ T Zellen besitzen ein anderes TCR Repertoire als CD40L- CD8+ T Zellen und reifen im Zuge ihrer Aktivierung bevorzugt zu Gedächtniszellen mit Zytokin- und Chemokinrezeptorprofilen von Tc2, Tc17 und Tc22 Zellen heran. Mit ihrem nicht-zytotoxischen Phänotyp und ihrer Genexpressionsignatur ähneln diese Zellen stark Helfer-CD4+ T Zellen und können von den klassisch zytotoxischen Tc1 und Tc17+1 Zellen durch ihre IL-6R und fehlende SLAMF7 Expression sowie der Expression von Markern die auf eine Fähigkeit in die Haut zu wandern schließen lassen, unterschieden werden. Zusammenfassend zeigen wir hier, dass naive CD8+ T Zellen von den frühsten Entwicklungsstadien im Thymus an nicht homogen sind und die Fähigkeiten über CD40L Expression eine Helferfunktion auszuüben beziehungsweise über die Sekretion zytolytischer Moleküle Zielzellen abzutöten unabhängig vom CD4+ or CD8+ T-Zell Status sind. Zellen mit Zytokin- und Genexpressionsignaturen, die mit denen der CD8+ Helfer-T Zellen übereinstimmen, wurden von uns und anderen in Geweben (Haut, Lunge) identifiziert und tragen zu den verschiedensten autoinflammatorischen Erkrankungen bei. Diese Arbeit insinuiert daher die Notwendigkeit einer grundlegenen Neubewertung der CD8+ T Zell Fähigkeiten und Funktionen in Immunantworten. / The T cell compartment consists of two major subsets with diverse assignments. CD4+ T cells express CD40L upon activation, a central co-stimulatory receptor to induce B cell mediated humoral immunity, activate APCs and prime efficient effector CD8+ T cell development (“helper function”). In contrast, cytotoxic CD8+ T cells are predetermined to kill infected or malignant cells directly. However, a fraction of CD8+ T cells expressing CD40L upon activation was identified. So far, it is not understood in CD8+ T cells a) how CD40L expression is regulated, b) when and how the ability of CD40L expression is implemented and c) what are the implications for the immune system. In this thesis, we found that CD40L expression is regulated by DNA-methylation of regulatory regions of the CD40LG locus in CD4+ as well as CD8+ T cells. The de-methylation of central elements is implemented in the thymus and increases with T cell maturation reflected by enhanced stability of CD40L expression. Elevated CD5 and NUR77 expression of CD40L+ CD8+ SP thymocytes suggests that high affine detection of self-peptides during positive selection in the thymus implements CD40L expression ability and predetermines the fate of the CD40L imprinted CD8+ T cells. CD40L+ naïve CD8+ T cells differ in their TCR repertoire from their CD40L- counterparts and preferentially mature into memory cell subsets with cytokine and chemokine receptor profiles of Tc2, Tc17 and Tc22 cells. With their non-cytotoxic phenotype and gene expression signatures, the CD40L+ memory CD8+ T cell subsets Tc2, Tc17 and Tc22 widely resemble helper CD4+ T cells and can be distinguished from classical cytotoxic Tc1 and Tc17+1 cells by their IL-6R and absent SLAMF7 expression and their skin migratory phenotype. Altogether, we demonstrate that from the earliest developmental stages in thymus onwards naive CD8+ T cells are not homogenous and the abilites to provide “CD40L based help” or “cytotoxicity mediated killing” are independent of the CD4+ or CD8+ T cell status. Cells with helper-type CD8+ T cell cytokine and gene-expression signatures were found at barrier sites (skin, lung) by us and others where they contribute to multiple autoinflammatory diseases. Therefore, this work insinuates the need to revisite CD8+ T cell capablities and function in immune responses.

No description available.

CD154 joue un rôle important dans la pathogenèse de plusieurs maladies auto-immunes, ainsi que des dysfonctionnements vasculaires. CD154 est un membre de la famille du facteur de nécrose tumorale (tumor necrosis factor, TNF) d'une importance cruciale dans l'immunité humorale. Cependant, CD154 partage également des fonctions critiques inflammatoires grâce à son interaction avec son récepteur CD40 ou des partenaires de liaison récemment identifiés, à savoir αIIbβ3, α5β1 et αMβ2. Ces réponses impliquent CD154 comme un facteur clé dans les maladies inflammatoires chroniques, notamment les maladies auto-immunes et la thrombose. L’interruption de l'interaction de CD154 avec ses récepteurs par anticorps anti-CD154 inhibe de manière significative le développement de ces maladies, bien que des effets secondaires graves ont été associés à ces traitements. Pour remédier à ces effets indésirables, d'autres approches comme des souris defficientes (Knockout), oligonucléotide antisens et ARNi ciblage ont été développés. Ces approches ont été axées sur l’interaction de CD154 avec CD40 et ne traitent pas l'interaction de CD154 avec ses autres récepteurs. Par conséquent, il existe un besoin de nouveaux traitements pour la prévention/abrogation des maladies inflammatoires ou auto-immunes qui cibles tous les récepteurs de CD154. Notre groupe a profondément étudié l'interaction structurelle/fonctionnelle de CD154 avec ses différents récepteurs. Étant donné l'importance de la structure trimérique de CD154 pour son activité biologique, nous avons généré une forme monomère de la molécule qui peut se lier spécifiquement à un récepteur sans induire l'activation intracellulaire. Cet agent est un outil thérapeutique potentiel pour le traitement de maladies reliées au CD154, tels que les événements thrombotiques. / CD154 has emerged as an important player in the pathogenesis of several autoimmune diseases, as well as vascular dysfunctions. CD154 is a member of the tumour necrosis factor family of pivotal importance in humoral immunity. However, CD154 also shares critical inflammatory functions through its interaction with its classical CD40 receptor or recently identified binding partners, namely αIIbβ3, α5β1 and αMβ2. These responses imply CD154 as a key factor in chronic inflammatory disorders including autoimmune diseases and thrombosis. Disrupting the interaction of CD154 with its receptors through anti-CD154 Abs significantly inhibits the development of these diseases, albeit serious side effects have been associated with these therapies. To overcome these adverse effects, other approaches such as knockout, antisense oligonucleotide and siRNA targeting were developed. These approaches were focused on the CD154/CD40 interaction and did not address the interaction of CD154 with its other receptors. Thus, there is a need for novel CD154 treatments for the prevention/abrogation of inflammatory or autoimmune diseases that address all CD154 receptors. Our group profoundly investigated the structural/functional interaction of CD154 with its various receptors. Given the importance of the trimeric structure of CD154 for its biological activity, we generated monomeric form of the molecule that can specifically bind to one receptor without inducing intracellular activation. This agent represents a potential therapeutic tool for the treatment of CD154-mediated diseases such as thrombotic events.