A Natural Variant of the Signaling Molecule Vav1 Enhances Susceptibility to Myasthenia Gravis and Influences the T Cell Receptor Repertoire

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell receptor (TCR) signals and plays an important role in T cell development and activation. Previous genetic studies identified a natural variant of Vav1 characterized by the substitution of an arginine (R) residue by a tryptophane (W) at position 63 (Vav1R63W). This variant impacts Vav1 adaptor functions and controls susceptibility to T cell-mediated neuroinflammation. To assess the implication of this Vav1 variant on the susceptibility to antibody-mediated diseases, we used the animal model of myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). To this end, we generated a knock-in (KI) mouse model bearing a R to W substitution in the Vav1 gene (Vav1R63W) and immunized it with either torpedo acetylcholine receptor (tAChR) or the α146-162 immunodominant peptide. We observed that the Vav1R63W conferred increased susceptibility to EAMG, revealed by a higher AChR loss together with an increased production of effector cytokines (IFN-γ, IL-17A, GM-CSF) by antigen-specific CD4+ T cells, as well as an increased frequency of antigen-specific CD4+ T cells. This correlated with the emergence of a dominant antigen-specific T cell clone in KI mice that was not present in wild-type mice, suggesting an impact on thymic selection and/or a different clonal selection threshold following antigen encounter. Our results highlight the key role of Vav1 in the pathophysiology of EAMG and this was associated with an impact on the TCR repertoire of AChR reactive T lymphocytes

Dendritic Cell-Mediated-Immunization with Xenogenic PrP and Adenoviral Vectors Breaks Tolerance and Prolongs Mice Survival against Experimental Scrapie

In prion diseases, PrPc, a widely expressed protein, is transformed into a pathogenic form called PrPSc, which is in itself infectious. Antibodies directed against PrPc have been shown to inhibit PrPc to PrPSc conversion in vitro and protect in vivo from disease. Other effectors with potential to eliminate PrPSc-producing cells are cytotoxic T cells directed against PrP-derived peptides but their ability to protect or to induce deleterious autoimmune reactions is not known. The natural tolerance to PrPc makes difficult to raise efficient adaptive responses. To break tolerance, adenovirus (Ad) encoding human PrP (hPrP) or control Ad were administered to wild-type mice by direct injection or by transfer of Ad-transduced dendritic cells (DCs). Control Ad-transduced DCs from Tg650 mice overexpressing hPrP were also used for immunization. DC-mediated but not direct administration of AdhPrP elicited antibodies that bound to murine native PrPc. Frequencies of PrP-specific IFNγ-secreting T cells were low and in vivo lytic activity only targeted cells strongly expressing hPrP. Immunohistochemical analysis revealed that CD3+ T cell infiltration was similar in the brain of vaccinated and unvaccinated 139A-infected mice suggesting the absence of autoimmune reactions. Early splenic PrPSc replication was strongly inhibited ten weeks post infection and mean survival time prolonged from 209 days in untreated 139A-infected mice to 246 days in mice vaccinated with DCs expressing the hPrP. The efficacy appeared to be associated with antibody but not with cytotoxic cell-mediated PrP-specific responses

Approches vaccinales des maladies neurodégénératives (applications aux maladies à prions)

Les maladies à prions sont des maladies neurodégénératives fatales causées par la transconformation d une protéine endogène, la PrPc, en une forme pathogène, la PrPsc et pour lesquelles il n existe aucune thérapie efficace. Le traitement de ces maladies par immunothérapie active se heurte à la forte tolérance à la PrPc. L objectif de ce travail a été de développer des stratégies de stimulation des réponses T CD4+ et CD8+ spécifiques de la PrPc afin d évaluer leur activité protectrice ou auto-immune chez des souris infectées par la PrPsc.La stimulation avant infection des cellules T CD4+ spécifiques des épitopes immunogènes de la PrPc a permis la production d anticorps et de cytokines ainsi que l allongement de la phase d incubation de la maladie.Les réponses T CD8+ anti-PrP pourraient éliminer des cellules accumulant la PrPSc. Nous avons caractérisé le répertoire T CD8+ et les épitopes cibles de la PrPc en utilisant différentes stratégies. L induction avant infection d une réponse T CD8+ cytotoxique spécifique d un peptide immunogène a augmenté significativement la phase clinique.Les immunisations appliquées après infection et/ou répétées ont entrainé la perte de fonction des T CD4+ et CD8+ et de l effet protecteur. La perte des réponses spécifiques est due à des T régulateurs (Tregs). De plus, ces Tregs modulent l accumulation périphérique de la PrPsc.En conclusions, en fonction du mécanisme mis en jeu par stimulation spécifique, les cellules T peuvent contrôler les différentes phases de l infection à prions sans atteintes auto-immunes délétères. Les T CD4+, CD8+ et Tregs doivent être pris en compte pour le développement d immunothérapies anti-prions efficaces.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Best Practices of Using AI-Based Models in Crystallography and Their Impact in Structural Biology

International audienceThe recent breakthrough made in the field of 3D structure prediction by artificial intelligence softwares such as initially AlphaFold2 1 (AF2) and RosettaFold 2 (RF) and more recently large Language Models 3 (LLM), has revolutionized the field of structural biology in particular but also biology as a whole. These models have clearly generated a great enthusiasm within the scientific community and different applications of these 3D predictions are regularly described in scientific articles demonstrating the impact of these high quality models. Despite the acknowledged high accuracy of these models in general, it seems important to make users of these models aware of the wealth of information they offer and to encourage them to make the best use of them. Here, we focus on the impact of these models in a specific application by structural biologists using X-ray crystallography. We propose guidelines to prepare models to be used for molecular replacement trials to solve the phase problem. We also encourage colleagues to share as much detail as possible about how they use these models in their research, where the models did not yield correct molecular replacement solutions, and how these predictions fit with their experimental 3D structure. We feel this is important to improve the pipelines using these models and also to get feedback on their overall quality

Contribution of antibody and T cell-specific responses to the progression of 139A-scrapie in C57BL/6 mice immunized with prion protein peptides

Prion diseases are associated with the conversion of the normal host cellular prion protein to an abnormal protease-resistant (PrPres) associated with infectivity. No specific immune response against prions develops during infection due to the strong tolerance to cellular prion protein. We examined the protective potential on prion diseases of immune responses elicited in C57BL/6 mice with PrP peptides 98-127 (P5) or 158-187 (P9) with CpG. After immunization, P5-treated mice developed high titer and long-lasting Abs, and P9-treated mice developed transient IFN-γ secreting T cells and poor and variable Ab responses. Both treatments impaired early accumulation of PrPres in the spleen and prolonged survival of mice infected with 139A scrapie. Additional P9 boosts after 139A infection sustained the T cell response and partially inhibited PrPres early accumulation but did not improve the survival. Surprisingly, when P9 injections were started 1 mo after infection and repeated subsequently, specific T cell and Ab responses were impaired and no beneficial effect on prion disease was observed. After a single injection of P9, the number of IFN-γ secreting CD4 T cells was also reduced in mice 8- to 10-wk postinfection compared with healthy mice. In vivo and in vitro removal of CD4CD25 T cells restored the T cell response to P9 in infected mice. In conclusion, CD4 T cells as well as Abs might participate to the protection against scrapie. Of importance, the peripheral accumulation of PrPres during infection negatively interferes with the development of T and B cell responses to PrP and regulatory T cells might contribute to this phenomenon

Efficient Plasma Cell Differentiation and Trafficking Require Cxcr4 Desensitization

CXCR4 plays a central role in B cell immune response, notably by promoting plasma cell (PC) migration and maintenance in the bone marrow (BM). Gain-of-function mutations in CXCR4 affecting receptor desensitization have been reported in the rare immunodeficiency called WHIM syndrome (WS). Despite lymphopenia, patients mount an immune response but fail to maintain it over time. Using a knockin mouse model phenocopying WS, we showed that, counter-intuitively, a gain of Cxcr4 function inhibited the maintenance of antibody titers after immunization. Although the Cxcr4 mutation intrinsically and locally promoted germinal center response and PC differentiation, antigen-specific PCs were barely detected in the BM, a defect mirrored by early accumulation of immature plasmablasts potentially occupying the survival niches for long-lived PCs. Therefore, fine-tuning of Cxcr4 desensitization is critically required for efficient PC differentiation and maintenance, and absence of such a regulatory process may account for the defective humoral immunity observed in WS patients

Perspective sur l'utilisation prospective de l'IA dans la prédiction de la structure protéique

International audienceAlphaFold2 (AF2) and RoseTTaFold (RF) have revolutionized structural biology, serving as highly reliable and effective methods for predicting protein structures. This article explores their impact and limitations, focusing on their integration into experimental pipelines and their application in diverse protein classes, including membrane proteins, intrinsically disordered proteins (IDPs), and oligomers.In experimental pipelines, AF2 models aid X-ray crystallography in resolving the phase problem, while complementarity with Mass Spectrometry and NMR data enhances structure determination and protein flexibility prediction. Predicting the structure of membrane proteins remains challenging for both AF2 and RF due to difficulties in capturing conformational ensembles and interactions with the membrane. Improvements in incorporating membrane-specific features and predicting the structural effect of mutations are crucial. For Intrinsically Disordered Proteins, AF2's confidence score (pLDDT) serves as a competitive disorder predictor, but integrative approaches with molecular dynamics simulations or hydrophobic cluster analyses are advocated for accurate dynamics representation. AF2 and RF show promising results for oligomeric models, outperforming traditional docking methods, with AlphaFold-Multimer showing improved performance, however, somes caveats remain in particular for membrane proteins. Real-life examples demonstrate AF2's predictive capabilities in unknown protein structures, but models should be evaluated for their agreement with experimental data. Furthermore, combining AF2 models with molecular dynamics simulations can be used complementarily. In this perspective we propose a "wish list" for improving deep learning-based protein folding prediction models, including using experimental data as constraints and modifying models with binding partners or post-translational modifications. Additionally, a meta-tool for ranking and suggesting composite models is suggested, driving future advancements in this rapidly evolving field.AlphaFold2 (AF2) et RoseTTaFold (RF) ont révolutionné la biologie structurale, servant de méthodes très fiables et efficaces pour prédire les structures protéiques. Cet article explore leur impact et leurs limites, en se concentrant sur leur intégration dans des pipelines expérimentaux et leur application dans diverses classes de protéines, y compris les protéines membranaires, les protéines intrinsèquement désordonnées (IDP) et les oligomères.Dans les pipelines expérimentaux, les modèles AF2 aident la cristallographie radiographique à résoudre le problème de phase, tandis que la complémentarité avec les données de spectrométrie de masse et RMN améliore la détermination de la structure et la prédiction de la flexibilité des protéines. Prédire la structure des protéines membranaires reste difficile pour AF2 et RF en raison de difficultés à capturer des ensembles conformationnels et des interactions avec la membrane. Des améliorations dans l'incorporation de caractéristiques spécifiques à la membrane et la prédiction de l'effet structurel des mutations sont cruciales. Pour les Protéines Intrinsèquement Désordonnées, le score de confiance d'AF2 (pLDDT) sert de prédicteur de désordre compétitif, mais des approches intégratives avec des simulations de dynamique moléculaire ou des analyses de cluster hydrophobes sont préconisées pour une représentation dynamique précise. AF2 et RF montrent des résultats prometteurs pour les modèles oligomériques, surpassant les méthodes d'amarrage traditionnelles, avec AlphaFold-Multimer montrant des performances améliorées, cependant, certaines mises en garde subsistent en particulier pour les protéines membranaires. Des exemples réels démontrent les capacités prédictives de l'AF2 dans des structures protéiques inconnues, mais les modèles devraient être évalués pour leur accord avec les données expérimentales. De plus, la combinaison de modèles AF2 avec des simulations de dynamique moléculaire peut être utilisée de manière complémentaire.Dans cette perspective, nous proposons une "liste de souhaits" pour améliorer les modèles de prédiction du repliement des protéines basés sur l'apprentissage profond, y compris l'utilisation de données expérimentales comme contraintes et la modification de modèles avec des partenaires de liaison ou des modifications post-traductionnelles. De plus, un méta-outil pour classer et suggérer des modèles composites est suggéré, ce qui conduira à de futures avancées dans ce domaine en évolution rapide

A pulsed high-voltage decelerator system to deliver low-energy antiprotons

The GBAR (Gravitational Behaviour of Antihydrogen at Rest) experiment at CERN requires efficient deceleration of 100 keV antiprotons provided by the new ELENA synchrotron ring to synthesize antihydrogen. This is accomplished using electrostatic deceleration optics and a drift tube that is designed to switch from -99 kV to ground when the antiproton bunch is inside - essentially a charged-particle “elevator” - producing a 1 keV pulse. We describe the simulation, design, construction and successful testing of the decelerator device at -92 kV on-line with ELENA