9 research outputs found
Etude des phénomÚnes d'adhésion entre des cellules B et des gouttes d'huile fonctionnalisées par des anticorps à l'aide de piÚges microfluidiques
In the immune system, B cell activation is triggered by the specific binding of the B cell receptors with the antigens present on antigen-presenting cells like macrophage, dendritic cells, ... This recognition leads to an accumulation of adhesive proteins at the contact. This molecular structure is called immune synapse, from which the antigens are extracted by the B cell. The precise biological mechanisms implied in the extraction of antigens are still unknown. In our work, we propose to use antibody-coated oil droplets as substrates for the activation of B cells. These droplets have the advantage to have a liquid surface, allowing the antibodies-coated diffusion at their surface. Moreover, these droplets are potentially deformable, so they behave as cellular force probes. Lastly, the droplets have the same size of the B cells, so they mimic the antigen-presenting cells. In order to put in contact the B cells with the droplets, we fabricated a regular network of microfluic traps with U-shape in a microfluic chamber. The shape of the chamber has been optimized to guaranty a good trapping efficiency. Using the traps, we have been able to put into contact one antibody-coated droplet with one B cell in several traps in parallel, and to observe the contact in function of time by fluorescence microscopy. We observed an accumulation of the antibodies coated on the droplet at the surface of contact. The kinetics of accumulation has been measured and the time scale of accumulation has been deduced. Also, observations with fluorescence showed a polarization of the lysosomes near the contact, suggesting a B cell response.Dans le systĂšme immunitaire, lâactivation des cellules B nĂ©cessite la reconnaissance spĂ©cifique par les rĂ©cepteurs membranaires des cellules B des antigĂšnes portĂ©s par cellules prĂ©sentatrices dâantigĂšnes (macrophages, cellules dendritiques, âŠ). Cette reconnaissance se traduit par une accumulation de protĂ©ines adhĂ©sives constituant une structure molĂ©culaire appelĂ©e synapse immunologique, Ă partir de laquelle sont extraits les antigĂšnes. Les mĂ©canismes biologiques impliquĂ©s dans lâextraction dâantigĂšnes par les cellules B sont encore mal connus. Dans notre Ă©tude, nous proposons dâutiliser des gouttes dâhuile dont la surface est fonctionnalisĂ©e par des anticorps, comme substrat pour lâactivation des cellules B. Ces gouttes prĂ©sentent lâintĂ©rĂȘt de possĂ©der une interface liquide et d'ĂȘtre dĂ©formables (mesure de forces possible). Afin de mettre en contact les gouttes fonctionnalisĂ©es avec les cellules B, nous avons fabriquĂ© un rĂ©seau de piĂšges ayant la forme dâun U rĂ©partis dans une chambre microfluidique. La forme de cette chambre a Ă©tĂ© optimisĂ©e de maniĂšre Ă garantir un remplissage efficace des piĂšges. Le dispositif expĂ©rimental mis au point permet de mettre en contact une goutte fonctionnalisĂ©e par des anticorps et une cellule B dans plusieurs piĂšges en parallĂšle, et dâobserver lâĂ©volution du contact dans le temps par microscopie. Nous avons observĂ© que le contact goutte/cellule induisait une accumulation dâanticorps greffĂ©s Ă la goutte au niveau de la zone de contact. La cinĂ©tique dâaccumulation est mesurĂ©e. Des observations en fluorescence des lysosomes des cellules B montrent une polarisation vers la zone de contact, suggĂ©rant une rĂ©ponse de la part de la cellule
Study of the adhesion phenomena between B cells and antibody-functionalized oil droplets using microfluidic traps
Dans le systĂšme immunitaire, lâactivation des cellules B nĂ©cessite la reconnaissance spĂ©cifique par les rĂ©cepteurs membranaires des cellules B des antigĂšnes portĂ©s par cellules prĂ©sentatrices dâantigĂšnes (macrophages, cellules dendritiques, âŠ). Cette reconnaissance se traduit par une accumulation de protĂ©ines adhĂ©sives constituant une structure molĂ©culaire appelĂ©e synapse immunologique, Ă partir de laquelle sont extraits les antigĂšnes. Les mĂ©canismes biologiques impliquĂ©s dans lâextraction dâantigĂšnes par les cellules B sont encore mal connus. Dans notre Ă©tude, nous proposons dâutiliser des gouttes dâhuile dont la surface est fonctionnalisĂ©e par des anticorps, comme substrat pour lâactivation des cellules B. Ces gouttes prĂ©sentent lâintĂ©rĂȘt de possĂ©der une interface liquide et d'ĂȘtre dĂ©formables (mesure de forces possible). Afin de mettre en contact les gouttes fonctionnalisĂ©es avec les cellules B, nous avons fabriquĂ© un rĂ©seau de piĂšges ayant la forme dâun U rĂ©partis dans une chambre microfluidique. La forme de cette chambre a Ă©tĂ© optimisĂ©e de maniĂšre Ă garantir un remplissage efficace des piĂšges. Le dispositif expĂ©rimental mis au point permet de mettre en contact une goutte fonctionnalisĂ©e par des anticorps et une cellule B dans plusieurs piĂšges en parallĂšle, et dâobserver lâĂ©volution du contact dans le temps par microscopie. Nous avons observĂ© que le contact goutte/cellule induisait une accumulation dâanticorps greffĂ©s Ă la goutte au niveau de la zone de contact. La cinĂ©tique dâaccumulation est mesurĂ©e. Des observations en fluorescence des lysosomes des cellules B montrent une polarisation vers la zone de contact, suggĂ©rant une rĂ©ponse de la part de la cellule.In the immune system, B cell activation is triggered by the specific binding of the B cell receptors with the antigens present on antigen-presenting cells like macrophage, dendritic cells, ... This recognition leads to an accumulation of adhesive proteins at the contact. This molecular structure is called immune synapse, from which the antigens are extracted by the B cell. The precise biological mechanisms implied in the extraction of antigens are still unknown. In our work, we propose to use antibody-coated oil droplets as substrates for the activation of B cells. These droplets have the advantage to have a liquid surface, allowing the antibodies-coated diffusion at their surface. Moreover, these droplets are potentially deformable, so they behave as cellular force probes. Lastly, the droplets have the same size of the B cells, so they mimic the antigen-presenting cells. In order to put in contact the B cells with the droplets, we fabricated a regular network of microfluic traps with U-shape in a microfluic chamber. The shape of the chamber has been optimized to guaranty a good trapping efficiency. Using the traps, we have been able to put into contact one antibody-coated droplet with one B cell in several traps in parallel, and to observe the contact in function of time by fluorescence microscopy. We observed an accumulation of the antibodies coated on the droplet at the surface of contact. The kinetics of accumulation has been measured and the time scale of accumulation has been deduced. Also, observations with fluorescence showed a polarization of the lysosomes near the contact, suggesting a B cell response
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 actinâmicrotubule 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 lymphocyteâantigen contact site, thereby allowing the formation and maintenance of a unique competent immune synapse
Longitudinal to Transverse Metachronal Wave Transitions in an In Vitro Model of Ciliated Bronchial Epithelium
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Phenotyping polarization dynamics of immune cells using a lipid droplet-cell pairing microfluidic platform
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