Impact of TCR-ligand avidity for viral and tumor antigens on human CD8 T cell potency and long-term persistence

The development of immunotherapies against viral infections and cancer requires a better understanding of the key parameters that control T cell-mediated immune responses, such as TCR-ligand binding avidity. The overall aim of this thesis was to improve our knowledge regarding the contribution of TCR binding avidity in mediating the functional potency and maintaining the long-term memory of antigen-specific CD8 T cells. We first performed a comprehensive study of TCR-pMHC binding avidity (i.e. off-rates) combined with various functional assays on large libraries of tumor- and virus-specific CD8 T cell clones from melanoma patients and healthy donors. We demonstrated that TCR-pMHC off-rates accurately predicted the functional potential of antigen-specific CD8 T cells. Our data also confirmed the superior binding avidities of virus-specific compared with tumor–specific T cell clonotypes. The TCR-pMHC off-rate is a more stable and robust biomarker of CD8 T cell potency than frequently used functional assays that depend on multiple parameters, including T cell activation state. Together, our data show that the TCR-pMHC binding avidity is a reliable biophysical parameter for patient monitoring during immunotherapy. In the second part of this thesis, we investigated whether TCR-ligand avidity is a determining factor for the clonal selection and evolution of antigen-specific T cells over time. We studied TCRαβ clonotype composition and persistence over a period of 15 years combined with TCR-pMHC binding avidity analyses on large repertoires of cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)- specific CD8 T cell clones from healthy donors. Within CMV-specific T cell repertoires, we observed the progressive contraction of clonotypes of higher TCR-pMHC avidity and lower CD8 binding dependency during chronic antigen exposure. Strikingly, we identified a unique transcriptional signature preferentially expressed by high-avidity T cell clonotypes, including elevated expression of the inhibitory receptor LILRB1. Enhanced proliferative capacity was also observed upon LILRB1 blockade. This was not the case for the EBV-specific T cell clonal composition and distribution that, once established, displayed an unprecedented stability for at least 15 years, independently of TCR-pMHC avidity. Our findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell responses in humans are distinct. -- Le développement des immunothérapies ciblant les infections virales et les cancers requiert une meilleure compréhension des paramètres-clés qui contrôlent les réponses cellulaires T, tel que l’avidité des TCRs pour leur ligand. L’objectif global de cette thèse était d’améliorer nos connaissances sur la contribution de l’avidité du TCR à la médiation des fonctions cellulaires et au maintien de la mémoire à long terme des cellules T CD8. Nous avons initialement mené une étude analytique sur l’avidité du TCR combinée à divers essais fonctionnels sur des lymphocytes T CD8 dirigés contre des antigènes viraux et tumoraux chez des donneurs sains et des patients atteints de mélanome. Nous avons démontré que l’avidité du TCR prédisait avec précision les fonctions cellulaires des cellules T CD8. Nos résultats confirment également que les cellules T CD8 spécifiques pour les antigènes viraux sont de plus haute avidité que celles spécifiques pour les antigènes tumoraux. De plus, l’avidité du TCR est un biomarqueur de la capacité fonctionnelle des cellules T, qui est plus stable et robuste que les tests fonctionnels habituellement utilisés. Dans l’ensemble, nos résultats montrent que l’avidité du TCR est un paramètre biophysique fiable pour le suivi des patients traités par immunothérapie. Par la suite, nous avons évalué si l’avidité du TCR était un facteur déterminant pour la sélection clonale des cellules T CD8 au cours du temps. Sur une période de 15 ans, nous avons étudié la composition et la persistance des répertoires clonotypiques dirigés contre le cytomégalovirus (CMV) et l’Epstein-Barr virus (EBV) ainsi que l’avidité du TCR chez des donneurs sains. Dans le cas de la réponse lymphocytaire T contre le CMV, nous avons observé la contraction progressive des clonotypes de plus haute avidité et peu dépendants de l’interaction avec le corécepteur CD8, au cours du temps. Nous avons identifié une signature transcriptionnelle distincte chez les clonotypes de plus haute avidité, avec notamment de l’expression élevée du récepteur inhibiteur LILRB1. Une augmentation de la capacité proliférative des cellules T a également été observée lors du blocage de LILRB1. Cela n’était pas le cas des répertoires des cellules T CD8 dirigées contre l’EBV qui, une fois établis, sont maintenus de façon stable pendant au moins 15 ans, indépendamment de l’avidité du TCR. Nos résultats révèlent un déclin global à long terme de l'avidité des répertoires clonaux de lymphocytes T spécifiques du CMV, mais non de l'EBV, soulignant le rôle différent joué par l'avidité des TCRs au cours des infections latentes induites par ces deux virus. Nous suggérons que des mécanismes distincts régulent l’évolution à long terme des réponses lymphocytaires mémoires T CD8 spécifiques du CMV et de l’EBV chez l'homme

L’image et le contexte : nouvelle étude des panneaux figuratifs incrustés de Mari

Mari a fourni le plus grand nombre de fragments d’incrustations figurées au Proche-Orient. Ces incrustations en coquille formaient des compositions à thématique militaire ou cérémonielle. Les panneaux ne sont plus conservés et seuls des fragments d’incrustations nous sont parvenus, nous privant de l’intégralité des scènes. À partir du corpus de Mari, nous proposons d’étudier à nouveau ces panneaux figuratifs incrustés et leur place au sein de la culture matérielle des sociétés proche-orientales du Bronze ancien, sur la base d’une analyse iconographique et archéologique. De là émerge une nouvelle définition de ces objets : mettant en scène la classe dominante de la cité, garante de l’ordre social, ces panneaux sont destinés à évoquer bien plus qu’à narrer des événements. À travers la représentation de cérémonies sociales, cultuelles ou de scènes de bataille, ils deviennent alors un vecteur par lequel cette classe assure sa légitimité, dans les lieux mêmes où elle exerce son pouvoir, palais ou temples de la ville.Mari has provided the largest number of inlays in the ancient Near East. These shell inlays were composing figurative scenes, mainly military and ceremonial. Nowadays, the panels are no longer preserved and only small fragments of inlays remain, depriving us of the entire compositions. From the study on the corpus of Mari, we propose here a new interpretation of the figurative inlaid panels and of their position within the material culture of the ancient near eastern Bronze age societies, based on an iconographical analysis and on an archaeological study of the discovery contexts. Thus emerges a new definition of the figurative inlaid panels: by staging the ruling class of the city, who guarantees the social order, the panels are meant to evoke much more than to narrate events. Through representation of social or worship ceremonies and war scenes, the panels become a vehicle through which this class ensures its legitimacy in the places where it exerts its power, namely the palaces and the temples.موقع ماري الآثاري هو حتّی اليوم الموقع الأكثر توفيرًا فيما يخصّ قطع الترصيعات ذات الصور في الشرق الأوسط. كانت هذه الترصيعات المحارية، وهي مدخلة جنبًا إلی جنب في طبقة زفت علی ألواح خشبية، تكوّن تأليفات ذات المواضيع العسكريّة أو الطقوسية. لم تعد هذه الألواح محفوظة اليوم ولم يصل إلينا إلّا بعض قطع ترصيعات، فبالتالي يمنعنا هذا من كمال المشاهد. بالفعل لم تكن هذه المواد مدروسة إلّا دراسات قليلة ويستمرّون حتّی اليوم في نقل نظريّات وظيفية أو تأويلات أعرب عنها في الماضي المنقّبون الأوّليّون، ولا تقوم هذه ابتداءً من مجموعة وثائق ماري، نقترح أن ننظر إلی هذه الألواح الشكلية المرصّعة بنظر جديد وأن نعيد نأمّل في مكانها ضمن الثقافة الماديّة والمجتمعات الشرقوسطية في العصر الربونزي القديم، ونحن نعتمد علی تحليل إيقونوغرافيّ وآثاريّ. فالتحليل الإيقونوغرافيّ يميل إلی الرصد الشامل لهذه الصور من مكوّناتها الصغری إلی مواضيعها المتناولة العامّة بغية فهم أحسن لخصوصية هذه التأليفات الفنّيّة. أمّا التحليل الآثاريّ فتقوم علی فحص إطار الاكتشافات وفحص تعيين الموضع وتعيين التنضّد لهذه الترصيعات. فبفضل هذا يظهر تحديد جديد لهذه المواد : إنّ هذه الألواح، وهي تمثّل طبقة المدينة الحاكمة والضامنة للنظام الاجتماعيّ فإن هذه الألواح موجّهة للتذكير أكثر مما هي موجّهة لرواية الأحداث. بالفعل، عبر تمثيل رتب اجتماعيّة أو ثقافية أو عبر مشاهد معارك، تصبح هذه الألواح وسيلة تضمن بها هذه الطبقة شرعيتها في الأماكن نفسها تمارس فيها سلطتها، أي القصر أو معابد المدينة.النظريّات والتأويلات علی أيّ تحليل

Quantum Hall resistance standards from graphene grown by chemical vapor deposition on silicon carbide

Replacing GaAs by graphene to realize more practical quantum Hall resistance standards (QHRS), accurate to within $10^{-9}$ in relative value, but operating at lower magnetic fields than 10 T, is an ongoing goal in metrology. To date, the required accuracy has been reported, only few times, in graphene grown on SiC by sublimation of Si, under higher magnetic fields. Here, we report on a device made of graphene grown by chemical vapour deposition on SiC which demonstrates such accuracies of the Hall resistance from 10 T up to 19 T at 1.4 K. This is explained by a quantum Hall effect with low dissipation, resulting from strongly localized bulk states at the magnetic length scale, over a wide magnetic field range. Our results show that graphene-based QHRS can replace their GaAs counterparts by operating in as-convenient cryomagnetic conditions, but over an extended magnetic field range. They rely on a promising hybrid and scalable growth method and a fabrication process achieving low-electron density devices.Comment: 12 pages, 8 figure

High‐Throughput Miniaturized Screening of Nanoparticle Formation via Inkjet Printing

This is the peer reviewed version of the following article:Ioanna D. Styliari, et al, ‘High‐Throughput Miniaturized Screening of Nanoparticle Formation via Inkjet Printing’, Macromolecular Materials and Engineering, (2018), which has been published in final form at https://doi.org/10.1002/mame.201800146. Under embargo until 27 May 2019. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.The self‐assembly of specific polymers into well‐defined nanoparticles (NPs) is of great interest to the pharmaceutical industry as the resultant materials can act as drug delivery vehicles. In this work, a high‐throughput method to screen the ability of polymers to self‐assemble into NPs using a picoliter inkjet printer is presented. By dispensing polymer solutions in dimethyl sulfoxide (DMSO) from the printer into the wells of a 96‐well plate, containing water as an antisolvent, 50 suspensions are screened for nanoparticle formation rapidly using only nanoliters to microliters. A variety of polymer classes are used and in situ characterization of the submicroliter nanosuspensions shows that the particle size distributions match those of nanoparticles made from bulk suspensions. Dispensing organic polymer solutions into well plates via the printer is thus shown to be a reproducible and fast method for screening nanoparticle formation which uses two to three orders of magnitude less material than conventional techniques. Finally, a pilot study for a high‐throughput pipeline of nanoparticle production, physical property characterization, and cytocompatibility demonstrates the feasibility of the printing approach for screening of nanodrug delivery formulations. Nanoparticles are produced in the well plates, characterized for size and evaluated for effects on metabolic activity of lung cancer cells.Peer reviewe

Versatile routes to functional RAFT chain transfer agents through the Passerini multicomponent reaction

The widespread adoption of RAFT polymerization stems partly from the ease and utility of installing a functional chain transfer agent onto the ends of the generated polymer chains. In parallel, the Passerini multicomponent reaction offers great versatility in converting a wide range of easily accessible building blocks to functional materials. In this work, we have combined the two approaches such that a single, commonly available, RAFT agent is used in Passerini reactions to generate a variety of multifunctional RAFT chain transfer agents containing ester linkages. Reactions to generate the multifunctional RAFT agents took place under mild conditions and in good yields. The resulting Passerini-RAFT agents were able to exert control over radical polymerization to generate materials of well-defined molecular weights and dispersity. Furthermore, the presence in these polymer cores of ester and amide functionality through the Passerini chemistries, provided regions in the materials which are inherently biodegradable, facilitating any subsequent biomedical applications. The work overall thus demonstrates a versatile and facile synthetic route to multi functional RAFT chain transfer agents and biodegradable polymers

Starch/Poly(glycerol-adipate) Nanocomposites: A Novel Oral Drug Delivery Device

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Biocompatible and bio-based materials are an appealing resource for the pharmaceutical industry. Poly(glycerol-adipate) (PGA) is a biocompatible and biodegradable polymer that can be used to produce self-assembled nanoparticles (NPs) able to encapsulate active ingredients, with encouraging perspectives for drug delivery purposes. Starch is a versatile, inexpensive, and abundant polysaccharide that can be effectively applied as a bio-scaffold for other molecules in order to enrich it with new appealing properties. In this work, the combination of PGA NPs and starch films proved to be a suitable biopolymeric matrix carrier for the controlled release preparation of hydrophobic drugs. Dynamic Light Scattering (DLS) was used to determine the size of drug-loaded PGA NPs, while the improvement of the apparent drug water solubility was assessed by UV-vis spectroscopy. In vitro biological assays were performed against cancer cell lines and bacteria strains to confirm that drug-loaded PGA NPs maintained the effective activity of the therapeutic agents. Dye-conjugated PGA was then exploited to track the NP release profile during the starch/PGA nanocomposite film digestion, which was assessed using digestion models mimicking physiological conditions. The collected data provide a clear indication of the suitability of our biodegradable carrier system for oral drug delivery.Peer reviewedFinal Published versio

The rationale, design, and methods of a randomized, controlled trial to evaluate the efficacy and safety of an active strategy for the diagnosis and treatment of acute pulmonary embolism during exacerbations of chronic obstructive pulmonary disease

Introduction: Some previous studies have suggested a high prevalence of pulmonary embolism (PE) during exacerbations of chronic obstructive pulmonary disease (ECOPD). The SLICE trial aims to assess the efficacy and safety of an active strategy for the diagnosis and treatment of PE (vs usual care) in patients hospitalized because of ECOPD. Methods: SLICE is a phase III, prospective, international, multicenter, randomized, open-label, and parallel-group trial. A total of 746 patients hospitalized because of ECOPD will be randomized in a 1:1 fashion to receive either an active strategy for the diagnosis and anticoagulant treatment of PE or usual care (ie, standard care without any diagnostic test for diagnosing PE). The primary outcome is a composite of all-cause death, non-fatal (recurrent) venous thromboembolism (VTE), or readmission for ECOPD within 90 days after enrollment. Secondary outcomes are (a) death from any cause within 90 days after enrollment, (b) non-fatal (recurrent) VTE within 90 days after enrollment, (c) readmission within 90 days after enrollment, and (d) length of hospital stay. Results: Enrollment started in September 2014 and is expected to proceed until 2020. Median age of the first 443 patients was 71 years (interquartile range, 64-78), and 26% were female. Conclusions: This multicenter trial will determine the value of detecting PEs in patients with ECOPD. This has implications for COPD patient morbidity and mortality