Characterization of molecular immunological interactions using biosensors and whole cells

Molecular interactions that govern cell?cell interactions are mediated by molecules embedded in the cell membrane, are very common in the immune system and are distinct from interactions between soluble molecules. Assays that can detect these interactions in a non?invasive way are particularly useful. Work was focused towards the development of such assays using biosensors. A well?defined cellular model system was used to study the responses of biosensor systems during the interaction of cell membrane receptors, class I MHC HLA?A2 molecules on the surface of cells, bound to ligands, anti?HLA?A2 monoclonal antibodies, immobilized on the sensor devices. The acoustic biosensor systems used, a Love?wave sensor and a quartz crystal microbalance (QCM) sensor detected the molecular interaction quantitatively but showed different sensitivity to different cell treatments: the QCM sensor detected cytoskeleton perturbations by cytochalasin whereas the Love?wave sensor distinguished cell types on the basis of cell surface HLA densities. For the Love?wave sensor, acoustic energy dissipation was found to correlate directly with the number of cell?membrane receptors specifically attached to immobilized antibodies, which enabled the characterization of the interaction via the calculation of the 2D kinetic and affinity parameters. Also, the dependence of cell adhesion on glycocalyx condition was demonstrated for HLA/anti?HLA binding. In order to move towards the characterization of a more biologically significant interaction pair, membrane models on biosensor surfaces were developed for the attachment of proteins of interest. Two types of lipid membranes were prepared, lipid monolayers on self?assembled thiol layers and lipid bilayers on hydrophilic silica surfaces. A real?time biosensing technique to study the binding of T cells to a pMHC?modified surface was proposed. As a TCR system, the JM22 TCR, which recognizes the influenza matrix peptide MP(58?66) presented on HLA?A2, was selected. The biosensor surface was prepared using a lipid monolayer presenting HLA?A2/MP(58?66) complexes. Two T cell systems were prepared for the biosensor technique, a cell line transfected with JM22 TCR and primary T cell clones isolated from peripheral blood. These cells were characterized for TCR expression and cytotoxicity. The long?term goal is to measure the 2D binding parameters of the TCR?pMHC interaction and correlate these with the physiological response of cells to antigen recognition.Οι μοριακές αλληλεπιδράσεις που καθορίζουν τις αλληλεπιδράσεις κυττάρου/κυττάρου μεσολαβούνται από μόρια ενσωματωμένα στην κυτταρική μεμβράνη, είναι άφθονες στο ανοσοποιητικό σύστημα και είναι διαφορετικές από ότι ανάμεσα σε διαλυτά μόρια. Εστίαση έγινε στην ανάπτυξη τεχνικών μελέτης τέτοιων αλληλεπιδράσεων χρησιμοποιώντας βιοαισθητήρες. Ένα καλά ορισμένο μοντέλο κυτταρικό σύστημα χρησιμοποιήθηκε για τη μελέτη των αποκρίσεων βιοαισθητήρων κατά την αλληλεπίδραση μεμβρανικών υποδοχέων, τάξης Ι MHC μορίων HLA?A2 στην επιφάνεια κυττάρων, με προσδέτες τους, μονοκλωνικά αντισώματα ειδικά έναντι του HLA?A2, ακινητοποιημένους στην επιφάνεια των αισθητήρων. Οι βιοαισθητήρες που χρησιμοποιήθηκαν, ένας αισθητήρας τύπου Love και ένας αισθητήρας?μικροζυγός κρυσταλλικού χαλαζία (QCM), ανίχνευσαν ποσοτικά τη μοριακή αλληλεπίδραση. Επίσης, εμφάνισαν διαφορετική ευαισθησία σε διαφορετικούς χειρισμούς των κυττάρων, όπως διατάραξη του κυτταροσκελετού από κυτοχαλασίνη (στον QCM), ή διαχωρισμός των κυτταρικών τύπων με βάση την επιφανειακή πυκνότητα των HLA μορίων (στον αισθητήρα Love). Για τον αισθητήρα Love, η απώλεια ενέργειας του ακουστικού κύματος έδειξε γραμμική συσχέτιση με τον αριθμό των μεμβρανικών υποδοχέων που προσδένονται ειδικά στα ακινητοποιημένα αντισώματα και χρησιμοποιήθηκε για το χαρακτηρισμό της αλληλεπίδρασης υπολογίζοντας τις κινητικές παραμέτρους και τη συγγένεια σε 2D. Επιπλέον, φανερώθηκε η εξάρτηση της κυτταρικής προσκόλλησης από την κατάσταση του γλυκοκάλυκα των κυττάρων. Για τη μετάβαση σε ένα περισσότερο βιολογικά σημαντικό ζεύγος αλληλεπίδρασης, αναπτύχθηκαν μοντέλα μεμβρανών σε επιφάνειες βιοαισθητήρων για την πρόσδεση πρωτεϊνών ενδιαφέροντος. Δυο τύποι λιπιδικών μεμβρανών προετοιμάστηκαν, λιπιδικές μονοστοιβάδες σε μονοστοιβάδες θειολών και λιπιδικές διπλοστοιβάδες σε υδρόφιλες επιφάνειες silica. Επίσης, προτάθηκε μια τεχνική βιοαισθητήρα για τη μελέτη της πρόσδεσης Τ κυττάρων σε επιφάνεια με pMHCs. Ως σύστημα TCR, επιλέχθηκε o JM22 TCR, ο οποίος αναγνωρίζει το πεπτίδιο MP(58?66) από τον ιό της γρίπης παρουσιασμένο στο HLA?A2. Η επιφάνεια του βιοαισθητήρα προετοιμάστηκε με λιπιδική μονοστοιβάδα που παρουσίαζε σύμπλοκα HLA?A2/MP(58?66). Δυο συστήματα Τ κυττάρων προετοιμάστηκαν, μια μεταμολυσμένη με JM22 TCR κυτταρική σειρά και πρωτογενείς Τ κυτταρικοί κλώνοι απομονωμένοι από περιφερικό αίμα. Αυτά τα κύτταρα χαρακτηρίστηκαν για έκφραση TCR και κυτταροτοξικότητα. Ο μακροπρόθεσμος στόχος είναι η μέτρηση παραμέτρων πρόσδεσης σε 2D για την αλληλεπίδραση TCR/pMHC και η συσχέτισή τους με την φυσιολογική απόκριση των κυττάρων στην αναγνώριση αντιγόνου

MOG Antibody Disease with Hearing Loss

MOG antibody disease is an autoimmune disease of the central nervous system associated with antibodies against myelin outer membrane oligodendrocyte glycoprotein. MOG antibody disease commonly presents with episodes of optic neuritis, transverse myelitis and/or encephaliti

T-lymphocyte passive deformation is controlled by unfolding of membrane surface reservoirs

International audienceT-lymphocytes in the human body routinely undergo large deformations, both passively, when going through narrow capillaries, and actively, when transmigrating across endothelial cells or squeezing through tissue. We investigate physical factors that enable and limit such deformations and explore how passive and active deformations may differ. Employing micropipette aspiration to mimic squeezing through narrow capillaries, we find that T-lymphocytes maintain a constant volume while they increase their apparent membrane surface area upon aspiration. Human resting T-lymphocytes, T-lymphoblasts, and the leukemic Jurkat T-cells all exhibit membrane rupture above a critical membrane area expansion that is independent of either micropipette size or aspiration pressure. The unfolded membrane matches the excess membrane contained in microvilli and membrane folds, as determined using scanning electron microscopy. In contrast, during transendothelial migration, a form of active deformation, we find that the membrane surface exceeds by a factor of two the amount of membrane stored in microvilli and folds. These results suggest that internal membrane reservoirs need to be recruited, possibly through exocytosis, for large active deformations to occur

Different TCR-induced T lymphocyte responses are potentiated by stiffness with variable sensitivity

International audienceT cells are mechanosensitive but the effect of stiffness on their functions is still debated. We characterize herein how human primary CD4 + T cell functions are affected by stiffness within the physiological Young's modulus range of 0.5 kPa to 100 kPa. Stiffness modulates T lymphocyte migration and morphological changes induced by TCR/CD3 triggering. Stiffness also increases TCR-induced immune system, metabolism and cell-cycle-related genes. Yet, upon TCR/ CD3 stimulation, while cytokine production increases within a wide range of stiffness, from hundreds of Pa to hundreds of kPa, T cell metabolic properties and cell cycle progression are only increased by the highest stiffness tested (100 kPa). Finally, mechanical properties of adherent antigen-presenting cells modulate cytokine production by T cells. Together, these results reveal that T cells discriminate between the wide range of stiffness values found in the body and adapt their responses accordingly

Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes

International audienceExosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles

In vivo genome-wide CRISPR screens identify SOCS1 as intrinsic checkpoint of CD4 + T H 1 cell response

International audienceInactivation of SOCS1 optimizes adoptive T cell therapy including human CAR-T cell composition and efficacy

Measurement of Two-Dimensional Binding Constants between Cell-Bound Major Histocompatibility Complex and Immobilized Antibodies with an Acoustic Biosensor

Gaining insights into the dynamic processes of molecular interactions that mediate cell-substrate and cell-cell adhesion is of great significance in the understanding of numerous physiological processes driven by intercellular communication. Here, an acoustic-wave biosensor is used to study and characterize specific interactions between cell-bound membrane proteins and surface-immobilized ligands, using as a model system the binding of major histocompatibility complex class I HLA-A2 proteins to anti-HLA-A2 monoclonal antibodies. The energy of the acoustic signal, measured as amplitude change, was found to depend directly on the number of HLA-A2/antibody complexes formed on the device surface. Real-time acoustic data were used to monitor the surface binding of cell suspensions at a range of 6.0 × 104 to 6.0 × 105 cells mL−1. Membrane interactions are governed by two-dimensional chemistry because of the molecules' confinement to the lipid bilayer. The two-dimensional kinetics and affinity constant of the HLA-A2/antibody interaction were calculated (ka = 1.15 × 10−5 μm2 s−1 per molecule, kd = 2.07 × 10−5 s−1, and KA = 0.556 μm2 per molecule, at 25°C), based on a detailed acoustic data analysis. Results indicate that acoustic biosensors can emerge as a significant tool for probing and characterizing cell-membrane interactions in the immune system, and for fast and label-free screening of membrane molecules using whole cells

T-lymphocyte passive deformation is controlled by unfolding of membrane surface reservoirs

T-lymphocytes in the human body routinely undergo large deformations, both passively, when going through narrow capillaries, and actively, when transmigrating across endothelial cells or squeezing through tissue. We investigate physical factors that enable and limit such deformations and explore how passive and active deformations may differ. Employing micropipette aspiration to mimic squeezing through narrow capillaries, we find that T-lymphocytes maintain a constant volume while they increase their apparent membrane surface area upon aspiration. Human resting T-lymphocytes, T-lymphoblasts, and the leukemic Jurkat T-cells all exhibit membrane rupture above a critical membrane area expansion that is independent of either micropipette size or aspiration pressure. The unfolded membrane matches the excess membrane contained in microvilli and membrane folds, as determined using scanning electron microscopy. In contrast, during transendothelial migration, a form of active deformation, we find that the membrane surface exceeds by a factor of two the amount of membrane stored in microvilli and folds. These results suggest that internal membrane reservoirs need to be recruited, possibly through exocytosis, for large active deformations to occur