Penicillin Binding Proteins as Danger Signals: Meningococcal Penicillin Binding Protein 2 Activates Dendritic Cells through Toll-Like Receptor 4

Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml±0.1), CD80 (LOGEC50 = 4.88 µg/ml±0.15) and CD86 (LOGEC50 = 5.36 µg/ml±0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7±5.1% cells versus 12±2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed tha

Targeting TMEM176B Enhances Antitumor Immunity and Augments the Efficacy of Immune Checkpoint Blockers by Unleashing Inflammasome Activation.

Although immune checkpoint blockers have yielded significant clinical benefits in patients with different malignancies, the efficacy of these therapies is still limited. Here, we show that disruption of transmembrane protein 176B (TMEM176B) contributes to CD8+ T cell-mediated tumor growth inhibition by unleashing inflammasome activation. Lack of Tmem176b enhances the antitumor activity of anti-CTLA-4 antibodies through mechanisms involving caspase-1/IL-1β activation. Accordingly, patients responding to checkpoint blockade therapies display an activated inflammasome signature. Finally, we identify BayK8644 as a potent TMEM176B inhibitor that promotes CD8+ T cell-mediated tumor control and reinforces the antitumor activity of both anti-CTLA-4 and anti-PD-1 antibodies. Thus, pharmacologic de-repression of the inflammasome by targeting TMEM176B may enhance the therapeutic efficacy of immune checkpoint blockers.Uruguay INNOVA 2, Fondo Maria Viñas and Clemente Estable from ANII, as well as grants from CABBIO, PEDECIBA, ECOS-SUD and FOCEM (MERCOSUR Structural Convergence Fund), COF 03/11 to MH, The Harry J Lloyd Foundation to MRG and the Instituto Nacional del Cancer to YDM, Agencia de Promoción Científica y Tecnológica to GAR and MRG, Fundación Bunge & Born and Fundación Sales to GA

Strategy to achieve biomarker-driven immunosuppression after solid organ transplantation by an academic-industry partnership within the European BIO-DrIM consortium

Solid organ transplantation has emerged as the “gold standard” therapy for end-stage organ failure as it improves both quality of life and survival. Despite the progress in short-term graft survival, that is closely associated with the impressive reduction of acute rejections within the first year, long-term graft and patient survival remain almost un-changed and unsatisfactory. Incomplete control of chronic allograft injury but particularly the adverse effects of long-term immunosuppression, such as graft toxicity, diabetes, cardiovascular events, infections, and tumours continue to challenge the long-term success. In general, immunosuppression is applied as one-size-fits-all strategy. This can result in over- and under-immunosuppression of patients with low and high alloresponsiveness, respectively. Trial- and -error strategies to minimize or even completely wean of immunosuppression have a high failure rate. Consequently, there is an unmet medical need to develop biomarkers allowing objective risk stratification of transplant patients. To achieve this goal, we engaged in an academic-industrial partnership. The central focus of the European-wide BIO-DrIM consortium (BIOmarker-Driven IMmmunosuppression) is the implementation of biomarker-guided strategies for personalizing immunosuppression to improve the long-term outcome and to decrease the adverse effects and costs of chronic immunosuppression in solid organ transplant patients. The concept includes four innovative investigator-driven clinical trials designed by the consortium

Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications

Identification et étude de nouveaux médiateurs du rejet et de l'induction de tolérance dans un modèle d'allogreffe chez le rat

La connaissance approfondie des mécanismes immunologiques menant au rejet ou à la tolérance d'une allogreffe représente un enjeu capital pour améliorer le succès de la transplantation d'organes chez l'homme, avec pour objectif d'éviter les traitements immunosuppresseurs. La première partie de cette thèse a été consacrée à l'étude du rôle d'une nouvelle molécule de la famille du TNF, TRANCE, dans le rejet d'allogreffe vascularisée chez le rat. Nous montrons que la voie TRANCE-RANK participe aux mécanismes du rejet aigu ainsi qu'au développement des lésions de rejet chronique, indépendament de la voie CD40L-CD40. La seconde partie, constituant l'objectif principal de cette thèse, consistait à isoler des gènes spécifiquement exprimés dans des allogreffes cardiaques dans un modèle d'induction de tolérance par transfusion de sang du donneur (TSD) chez le rat. Une méthodologie de recherche de gènes différentiellement exprimés a été mise en place, en utilisant notamment les techniques d'Hybridation Soutractive Suppressive et de PCR quantitative en temps réel. Nous montrons que la chimiokine Fractalkine et son récepteur CX3CR1 son fortement exprimés dans les greffons tolérés, et que les cellules CD8+ du receveur, requises pour la tolérance, sont responsables de cette induction. Nous avons d'autre part identifié une molécule de fonction inconnue, que nous avons appelée TORID (TOlerance-Related and InduceD transcript), puis démontré son appartenance à la famille du CD20. TORID est localisée dans l'enveloppe nucléaire des cellules dendritiques et des macrophages. Son expression est associée à l'état d'inactivation de ces cellules. L'ensemble des ces résultats contribuent à une meilleure compréhension des mécanismes gouvernant le rejet ou l'induction de tolérance dans ce modèle d'allogreffeUnderstanding of immune mechanisms leading to allograft rejection or tolerance is essential to improve organ transplantation in human, with the aim to avoid immunosuppressive treatments. The first part of this thesis is devoted to the study of the role of a new TNF family member, TRANCE, in allograft rejection in the rat. We show that the TRANCE-RANK pathway participates in acute rejection processes as well as in the development of CD40L-independent chronic rejection. The second part, representing the main objective of this thesis, consists in isolating genes specifically expressed in cardiac allografts in a rat model of tolerance induction by donor-specific blood transfusions (DST). We have set up a methodology of gene searching using suppressive subtractive hybridization and real-time quantitative PCR. We show that the chemokine Fractalkine and its receptor CX3CR1 are strongly expressed in tolerated grafts, and that host CD8+ cells, which are required for tolerance, control this induction. Moreover, we have identified a molecule of unknown function which we named TORID (TOlerance-Related and InduceD transcript). We show that TORID belongs to the CD20 family and is localized at the nuclear envelope of dendritic cells and macrophages. TORID expression is associated with the inactivation state of these cells. Taken together, these results contribute to a better understanding of the mechanisms governing rejection or tolerance induction in this modelNANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Etude des mécanismes d'induction et de maintien de la tolérance dans des modèles d'allogreffe et de maladie auto-immune chez le rat

La connaissance approfondie des mécanismes immunologiques impliqués dans la tolérance représente un enjeu capital pour améliorer le succès de la transplantation d'organes et le traitement des maladies auto-immunes. L'objet de cette thèse a consisté en l'étude des mécanismes responsables de la tolérance dans deux modèles expérimentaux chez le rat. Dans un modèle d'allotransplantation cardiaque, nous montrons qu'un nouvel immunosuppresseur, le LF15-0195, permet d'induire une tolérance spécifique du donneur et sans signe de rejet chronique, en favorisant notamment la génération de cellules T CD4+ régulatrices spécifiques d'antigène. Nous montrons ensuite que l'injection de cellules dendritiques immatures autologues en combinaison avec un traitement sous-optimal avec le LF15-0195 permet également d'induire une tolérance spécifique du donneur. Enfin, dans un modèle d'encéphalomyélite auto-immune expérimentale, nous montrons que le LF15-0195 induit une tolérance durable en favorisant la génération de cellules T CD4+ régulatrices.The comprehension of the immunological mechanisms leading to allograft- and self- tolerance represents a major issue to improve the success of organ transplantation and the treatment of autoimmune diseases in humans. During this Ph.D, we investigated the regulatory mechanisms responsible for tolerance in two experimental rat models. First, in a cardiac allotransplantation model, we demonstrate that a new immunosuppressant, LF15-0195, induces donor specific tolerance without any sign of chronic rejection, notably by favoring the generation of antigen-specific CD4 regulatory T cells. We then demonstrate that the injection of autologous immature dendritic cells in combination with a suboptimal treatment with LF15-0195 also induces donor-specific tolerance. Second, in an experimental model of multiple sclerosis, the experimental autoimmune encephalomyelitis, we demonstrate that LF15-0195 induces long term protection by favoring the generation of CD4 regulatory T cells.NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Etude des mécanismes régulateurs d'induction et de maintien de la tolérance aux allogreffes cardiaques chez le rat

La compréhension des mécanismes régulateurs d'induction et de maintien de la tolérance à une greffe demeure à l'heure actuelle un objectif primordial en transplantation. L'objet du travail de thèse a été d'étudier ces mécanismes dans deux modèles différents de tolérance à une allogreffe cardiaque chez le rat. Le premier modèle de tolérance est induit par deux transfusions de sang du donneur 14 et 7 jours avant l'allogreffe. Nous avons mis en évidence le rôle des cellule CD8+....Dans un second modèle de tolérance induit par un traitement de vingt jours avec le LF15-0195, un analogue de la Deoxyspergualine, nous avons observé chez les receveurs une inhibition de la production des anticorps anti-donneurs, de l'infiltrat leucocytaire, de l'expression d'IFN7 et de cytokines inflammatoires dans les allogreffes...The understanding of the regulatory mechanisms of induction and maintenance of graft tolerance currently remains an elusive goal in transplantation. The work of my thesis was to study these mechanisms in two different MHC mismatched heart allografts models in rats. In the first model, tolerance is induced by donor specific blood transfusion prior to transplantation. We demonstrated the involvment of CD8+....In the seçond model, tolerance is induced by a twenty day treatment with LF15-0195, a Deoxyspergualin analog. In this case, we demonstrated an inhibition of anti-donor alloantibody production, leukocyte infiltration, IFNg and inflammatory cytokine expression in allografis. Moreover, we revealed a direct inhibitory effect of LF 15-0195 on IFNg and 1L2 production by activated CD4+ T cells. This strong immunosuppression induced donor-specific CD4+ regulatory T cells, which are CD25+ in the thymus and CD25+/- in the periphery....NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF