Gouttes d’émulsions et pièges microfluidiques pour l’étude de la polarisation et des mécanismes des lymphocytes B

Right after a pathogen invasion, an organism deploys two lines of defense: the innate and adaptive immunities. The first one is a short-term response providing pathogen destruction then antigen presentation promoted by Antigen Presenting Cells (APC). The latest ensures an important long-term response mostly thanks to B lymphocytes which promote high-affinity antibodies secretion and memory B cells differentiation. B cells either catch soluble antigens or extract them onto different APC surfaces such as macrophages, dendritic, and follicular dendritic cells. In the latest case, APC and B lymphocytes ensure the antigen transfer via a crucial highly organized contact: the immunological synapse. To precisely understand the physicochemical mechanics involved upon the synapse, we first model the cell-cell contact by creating a new APC-like substrate respecting crucial features such as antigens concentration, mobility and surface deformability. Emulsion droplets properly mimic antigen mobility at the oil/water interface as observed on the cellular membrane; and due to low and controllable surface tension, droplets are as deformable as real APC. We also use microfluidic-trap devices to ease the observation of several isolated synapses simultaneously and follow their formation over time. Consequently, we temporally and spatially control the APC-like droplet/B cell synapse. By combining emulsion and microfluidic tools, we quantify the kinetic of lysosomes moving to the synapse area as a cell polarization readout. We analyze both polarization triggering and kinetics when B cells interact with different conditions. We demonstrate that (i) the polarization events are triggered when more than 75 antigens/µm2 are involved at the synapse, (ii) the antigens mobility only helps trigger such events when antigens are lacking, and (iii) high stiffnesses both trigger polarization and modulate their kinetics. We also observe anti-polarization events, i.e. lysosomes move to the back of cells, which whose kinetics only depends on stiffness however their triggering depends on both antigens concentration and mobility. Altogether, these results clearly bring a quantification of APC membrane properties affecting the B cells behavior. Besides, in a few preliminary results, we quantify the force applied by B cells on soft droplets. We find out softest droplets we formulate are deformed by lymphocytes during the synapse formation. Such results prove droplets are promising tools to dynamically measure the cellular force applied by non-cytotoxic or non-phagocytic immune cells as B lymphocytes are. We determine the viscoelastic modulus of droplets and B cells thanks to glass microplates experiments. We confirm that B cells are respectively 3-fold and 9-fold softer than dendritic cells and macrophages, therefore, they face to a large range of APC stiffnesses in vivo. Unexpectedly, microplates experiments also reveal the high stiffness of droplets having a low surface tension: such droplets exhibit stiffer membrane properties than most rigid APCs (macrophages) although these droplets trigger a B cell behavior comparable to soft live APC (dendritic cells) during in vitro experiments. Therefore, we inquire what/which property does induce such difference. We hypothesize that an adhesion force is the key difference between microplates experiments where droplets non-specifically adhere to glass surface, and droplets-cell contact which specifically adhere thanks to the strong antigen/antibody interaction and the presence of adhesion molecules (integrins). Altogether, these results and suggestions point out the crucial role of adhesion into cell-cell contact which seems to be the next property to explore and quantify during the B cell synapse. As a conclusion, this interdisciplinary Ph.D. project brings a novel tool to quantify the B cell synapse and mechanics. We think such droplets can be now used both to explore directly in lymph nodes (the place where [...]Juste après l'invasion d'un agent pathogène, un organisme déploie deux lignes de défense : l'immunité innée et l'immunité adaptative. La première est une réponse à court terme qui prévoit la destruction de l'agent pathogène puis la présentation de l'antigène par des cellules présentatrices d'antigènes (CPA). La dernière assure une importante réponse à long terme principalement grâce aux lymphocytes B qui sécrètent des anticorps à haute affinité et se différencient en lymphocytes B mémoires. Pour ce faire, les cellules B capturent soient des petits antigènes solubles ( 70 kDa) sur différentes CPA telles que les macrophages, les cellules dendritiques et les cellules dendritiques folliculaires. Dans le dernier cas, les CPA et les lymphocytes B assurent le transfert d'antigènes via un contact hautement organisé : la synapse immunologique. Pour comprendre précisément les mécanismes physico-chimiques impliqués dans cette synapse, nous avons d'abord modélisé le contact APC-lymphocyte par nouveau substrat imitant les propriétés membranaires des CPA, et respectant des caractéristiques cruciales telles que la concentration d'antigènes, la mobilité et la déformabilité de la surface. Les gouttes de l'émulsion huile dans eau imitent correctement la mobilité des antigènes à l'interface huile/eau telle qu'elle est observée sur la membrane cellulaire ; et en raison d'une tension superficielle faible et contrôlable, les gouttes sont aussi déformables qu'un véritable APC. Nous avons également utilisé des dispositifs de piégeage microfluidiques pour faciliter l'observation de plusieurs synapses isolées simultanément et suivre leur formation dans le temps. Par conséquent, nous contrôlons temporellement et spatialement la synapse de entre la goutte de type APC et le lymphocyte B. En combinant ces outils, gouttes et pièges microfluidiques, nous avons quantifié la cinétique des lysosomes au cours du temps. En se déplaçant vers la zone de la synapse, ils participent à la polarisation des lymphocytes. Nous avons donc choisi de suivre ces lysosomes afin de quantifier la polarisation cellulaire. Nous avons analysé à la fois le déclenchement de la polarisation et sa cinétique lorsque les cellules B interagissent avec différentes conditions de gouttes. Nous démontrons que (i) les événements de polarisation sont déclenchés lorsque plus de 75 antigènes/µm2 sont impliqués au niveau de la synapse, (ii) la mobilité des antigènes ne contribue à déclencher de tels événements que lorsque les antigènes viennent à manquer (< 75 antigens/µm2), et (iii) les rigidités élevées des gouttes déclenchent la polarisation et modulent leur cinétique. Nous observons également des événements d’anti-polarisation, c'est-à-dire que les lysosomes se déplacent vers l'arrière des cellules, dont la cinétique ne dépend que de la rigidité ; cependant, leur déclenchement dépend à la fois de la concentration et de la mobilité des antigènes. En conclusion, ces résultats apportent clairement une quantification des propriétés de la membrane APC affectant le comportement des lymphocytes B lors de la formation de la synapse immune. En outre, dans quelques résultats préliminaires nous avons quantifié la force appliquée par les cellules B sur les gouttes les plus déformables. Nous découvrons que celles-ci sont en effet déformées par les lymphocytes B lors de la formation de la synapse. De tels résultats prouvent que les gouttelettes que nous avons formulées sont des outils prometteurs pour mesurer dynamiquement la force cellulaire appliquée par les cellules immunitaires non cytotoxiques ou non phagocytaires comme le sont les lymphocytes B. Nous avons déterminé le module viscoélastique des gouttes et des lymphocytes B grâce à des expériences sur microplaques de verre. Premièrement, nous avons confirmé que les cellules B sont 3 fois à 9 fois plus souples que respectivement les cellules dendritiques et les macrophages, et sont donc confrontées [...

A Multiparametric and High-Throughput Assay to Quantify the Influence of Target Size on Phagocytosis

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Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes

International audienceImmune synapse formation is a key step for lymphocyte activation. In B lymphocytes, the immune synapse controls the production of high-affinity antibodies, thereby defining the efficiency of humoral immune responses. While the key roles played by both the actin and microtubule cytoskeletons in the formation and function of the immune synapse have become increasingly clear, how the different events involved in synapse formation are coordinated in space and time by actin–microtubule interactions is not understood. Using a microfluidic pairing device, we studied with unprecedented resolution the dynamics of the various events leading to immune synapse formation and maintenance in murine B cells. Our results identify two groups of events, local and global, dominated by actin and microtubules dynamics, respectively. They further highlight an unexpected role for microtubules and the GEF-H1-RhoA axis in restricting F-actin polymerization at the lymphocyte–antigen contact site, thereby allowing the formation and maintenance of a unique competent immune synapse

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International audienceIn this work we report on the development of mannose-coated fluorescent lipid microparticles to study the role of C-type lectin membrane receptor in phagocytosis. The micrometric droplets of soybean oil in water emulsion were functionalized with a tailor-made fluorescent mannolipid. The amphiphilic ligand was built from a mannose unit, a lipid C11 spacer and a naphthalimide fluorophore. The droplets functionalization was monitored by fluorescence microscopy as well as the interaction with concanavalin A which was used as a model lectin in vitro. The use of a monovalent ligand on the surface of emulsion droplets yielded particles with an affinity approximately 40 times higher than that of free mannose. In cellulo, the coated droplets were shown to be specifically internalized by macrophages in a receptor-dependent phagocytic pathway. The naked droplets on the other hand display very little internalization due to their low immunogenicity. This work thus brings evidence that C-type lectin membrane receptors may act as phagocytic receptors. The droplets functionalization with the tailored amphiphilic fluorescent ligand and the droplets functionalization also provides insights into the development of organic fluorescent particles that may prove useful for developing targeted imaging and delivery tools

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International audienceThe immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intracellular components necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied. Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how ligand concentration, surface fluidity, and substrate rigidity impact lysosome po-larization, we show that its onset and kinetics depend on the local antigen concentration at the synapse and on substrate rigidity. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout