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Immunological synapse

Christiane S Heilingloh, Stefan Klingl, Claudia Egerer-Sieber, Benedikt Schmid, Sigrid Weiler, Petra Mühl-Zürbes, Jörg Hofmann, Joachim D Stump, Heinrich Sticht, Mirko Kummer, Alexander Steinkasserer, Yves A Muller
CD83 is a type-I membrane protein and an efficient marker for identifying mature dendritic cells. Whereas membrane-bound full-length CD83 co-stimulates the immune system, a soluble variant (sCD83), consisting of the extracellular domain, only, displays strong immune-suppressive activities. Besides a prediction that sCD83 adopts a V-set Ig-like fold, however, little is known about the molecular architecture of CD83 and the mechanism by which CD83 exerts is function on dendritic cells and additional immune cells...
March 14, 2017: Journal of Molecular Biology
Audrey Le Floc'h, Yoshihiko Tanaka, Niels S Bantilan, Guillaume Voisinne, Grégoire Altan-Bonnet, Yoshinori Fukui, Morgan Huse
No abstract text is available yet for this article.
March 14, 2017: Journal of Experimental Medicine
Yuanqing Ma, Yui Yamamoto, Philip R Nicovich, Jesse Goyette, Jérémie Rossy, J Justin Gooding, Katharina Gaus
Membrane charge has a critical role in protein trafficking and signaling. However, quantification of the effective electrostatic potential of cellular membranes has remained challenging. We developed a fluorescence membrane charge sensor (MCS) that reports changes in the membrane charge of live cells via Förster resonance energy transfer (FRET). MCS is permanently attached to the inner leaflet of the plasma membrane and shows a linear, reversible and fast response to changes of the electrostatic potential...
March 13, 2017: Nature Biotechnology
Hélène D Moreau, Philippe Bousso
T cells can become activated in lymph nodes following a diverse set of interactions with antigen-presenting cells. These cellular contacts range from short and dynamic to stable and long-lasting interactions, termed kinapses and synapses, respectively. Here, we describe a methodology to generate naïve T cells expressing a fluorescent probe of interest through the generation of bone marrow chimeras and to image T cell dynamics using intravital two-photon microscopy. In these settings, the formation of kinapses and synapses can be triggered by the administration of low and high affinity peptides, respectively...
2017: Methods in Molecular Biology
Iratxe Del Río-Iñiguez, Jérôme Bouchet, Andrés Alcover
T cells are the main cellular targets of the human immunodeficiency virus 1 (HIV-1). HIV-1 infection induces pleiotropic effects on the infected T cell that modify the T cell capacity to respond to antigen and facilitates virus replication. HIV-1 infection subverts the formation and function of the immunological synapse altering both actin cytoskeleton remodeling and intracellular vesicle traffic. We describe here our methods to unveil how HIV-1 and in particular its protein Nef modify vesicle traffic to the immunological synapse, perturbing the synapse activation capacity...
2017: Methods in Molecular Biology
Fabienne McClanahan Lucas, John G Gribben
Aberrant immune synapse formation between antigen-presenting and immune effector cells is a central mediator of immune dysfunction and can be observed across several haematologic malignancies. Here, we describe the cell preparation, conjugation and immune synapse quantification of B and T cells obtained from patients with leukaemia and the adaptions required when using cells from murine models of disease.
2017: Methods in Molecular Biology
George P Cribaro, Elena Saavedra-López, Paola V Casanova, Laura Rodríguez, Carlos Barcia
In this chapter, we describe the technical details to visualize and analyze effector immunological synapses between T cells and astrocytes in the brain with high-resolution confocal imaging. This procedure is critical for the optimal and even penetration of labeling antibodies within the nerve tissue to obtain accurate staining and allow a uniform three-dimensional analysis of the T cell-astrocyte interactions. We emphasize here the comprehensive exploration of the tissue and analysis with confocal microscope as well as the display of microanatomical details of the three-dimensional reconstruction for interface visualization (including peripheral and central supramolecular activation clusters, effector molecules, and other organelles such as microtubule organizing centers (MTOCs) and Golgi apparatus)...
2017: Methods in Molecular Biology
Hsiang-Ting Hsu, Alexandre F Carisey, Jordan S Orange
Natural killer (NK) cells contain specialized lysosome-related organelles termed lytic granules allowing them to mediate cytotoxicity against tumorigenic or virally infected target cells. NK cells polarize their lytic granules toward a target cell via the microtubule-organizing center (MTOC). Prior to that, however, lytic granules converge to the MTOC along microtubules utilizing minus-end-directed microtubule motors. Herein we describe how to visualize and quantify lytic granule convergence using confocal microscopy to gain quantitative insight into NK cell cytotoxicity and its regulation...
2017: Methods in Molecular Biology
Ana Mafalda Santos, Jiandong Huo, Deborah Hatherley, Mami Chirifu, Simon J Davis
Innate and adaptive cellular immunity is dependent on interactions of cell surface receptors that initiate signaling, resulting in the formation of the immunological synapse and targeted delivery of effector functions. There has been considerable interest over the past 30 years in methods for isolating the extracellular regions of these receptors and components of the cytoplasmic signaling networks. This chapter describes our current protein expression toolkit used for structural studies of signaling proteins and the functional reconstitution of model cell surfaces, which comprises both bacterial and mammalian cell-based protein expression methodologies...
2017: Methods in Molecular Biology
Salvatore Valvo, Viveka Mayya, Elena Seraia, Jehan Afrose, Hila Novak-Kotzer, Daniel Ebner, Michael L Dustin
Supported lipid bilayers (SLB) formed on glass substrates have been a useful tool for study of immune cell signaling since the early 1980s. The mobility of lipid-anchored proteins in the system, first described for antibodies binding to synthetic phospholipid head groups, allows for the measurement of two-dimensional binding reactions and signaling processes in a single imaging plane over time or for fixed samples. The fragility of SLB and the challenges of building and validating individual substrates limit most experimenters to ~10 samples per day, perhaps increasing this few-fold when examining fixed samples...
2017: Methods in Molecular Biology
Jaime Llodrá
The immunological synapse is a critical event for immune response development. The use of planar supported bilayers as surrogate antigen-presenting cells is a useful tool to study this phenomenon. Here we describe electron microscopy methods and approaches to expand our knowledge of the events taking place during the initial phases of T cell activation after antigen recognition at the nanometer scale.
2017: Methods in Molecular Biology
Hélène D Moreau, Philippe Bousso, Ana-Maria Lennon-Duménil
T Cells can form very stable (synapses) or very transient and migratory (kinapses) contacts with antigen-presenting cells. Here, we describe how microchannels can be used to conveniently study the distinct dynamics of T cells during antigen recognition. Microchannels provide a controlled confined environment that promotes T cell migration and recapitulates kinapse and synapse behaviors when coated with appropriate pMHC molecules. We also depict the advantages of this in vitro approach for addressing mechanistic issues and for analysis...
2017: Methods in Molecular Biology
Etienne Gagnon, Audrey Connolly, Jessica Dobbins, Kai W Wucherpfennig
Over the last decade, advancements in the time and space resolution of microscopy technologies have enabled dissection of the molecular events involved in T cell Immunological Synapse (IS) formation. Using a combination of Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imagining Microscopy (FLIM), we have demonstrated dynamic plasma membrane binding by cytoplasmic domains of T cell receptor (TCR)-associated CD3 chains and other T cell transmembrane receptors. We have developed methods for imaging such membrane binding both at steady state and during receptor triggering at the IS...
2017: Methods in Molecular Biology
Gerhard J Schütz, Johannes B Huppa
T-cell antigen recognition is remarkably efficient: when scanning the surface of antigen-presenting cells (APCs), T-cells can detect the presence of just a few single antigenic peptide/MHCs (pMHCs), which are often vastly outnumbered by structurally similar non-stimulatory endogenous pMHCs (Irvine et al., Nature 419(6909):845-849, 2002; Purbhoo et al., Nat Immunol 5(5):524-530, 2004; Huang et al., Immunity 39(5):846-857, 2013). How T-cells achieve this is still enigmatic, in particular in view of the rather moderate affinity that TCRs typically exert for antigenic pMHCs, at least when measured in vitro (Davis et al...
2017: Methods in Molecular Biology
Anastasios Siokis, Philippe A Robert, Michael Meyer-Hermann
During antigen recognition by T cells, a specific spatial structure is formed at the contact face to an antigen-presenting cell (APC), called an immunological synapse (IS). The IS supports bidirectional signaling and release of effector molecules and is widely studied both biologically and numerically, in order to understand the process of T cell activation and signaling. This specialized structure harbors a central area (central supramolecular activation cluster, cSMAC) populated by T cell receptor-peptide-major histocompatibility complex (TCR-pMHC ) interactions, hedged by a peripheral ring (peripheral supramolecular activation cluster, pSMAC) of integrin lymphocyte function associated-1 interactions with its immunoglobulin superfamily ligand intercellular adhesion molecule-1 (LFA-1-ICAM-1)...
2017: Methods in Molecular Biology
Jérôme Bouchet, Iratxe Del Río-Iñiguez, Andrés Alcover
Immunological synapse formation is the result of a profound T cell polarization process that involves the coordinated action of the actin and microtubule cytoskeleton, as well as intracellular vesicle traffic. Endosomal vesicle traffic ensures the targeting of the T cell receptor (TCR) and various signaling molecules to the synapse, being necessary for the generation of signaling complexes downstream of the TCR. Here we describe the microscopy imaging methods that we currently use to unveil how TCR and signaling molecules are associated with endosomal compartments and deliver their cargo to the immunological synapse...
2017: Methods in Molecular Biology
Maria K Traver, Suman Paul, Brian C Schaefer
The T cell receptor (TCR) to NF-κB signaling pathway plays a critical role in regulation of proliferation and effector T cell differentiation and function. In naïve T cells, data suggest that most or all key cytoplasmic NF-κB signaling occurs in a TCR-proximal manner at the immunological synapse (IS). However, the subcellular organization of cytoplasmic NF-κB-activating complexes in effector T cells is more complex, involving signaling molecules and regulatory mechanisms beyond those operative in naïve cells...
2017: Methods in Molecular Biology
Katarzyna I Jankowska, Janis K Burkhardt
T cell signaling is inextricably linked to actin cytoskeletal dynamics at the immunological synapse (IS). This process can be imaged in living T cells expressing GFP actin or fluorescent F-actin binding proteins. Because of its planar nature, the IS provides a unique opportunity to image events as they happen, monitoring changes in actin retrograde flow in T cells interacting with different stimulatory surfaces or after pharmacological treatments. Here, we described the imaging methods and analytical procedures used to measure actin velocity across the IS in T cells spreading on planar stimulatory surfaces...
2017: Methods in Molecular Biology
Michael L Dustin, Cosima T Baldari
Immunological synapses are specialized cell-cell junctions characterized by (1) close apposition of the immune cell membrane with the membrane of another cell driven by adaptive or innate immune recognition, (2) adhesion, (3) stability, and (4) directed secretion. This phenomenon was first recognized in the 1970s and the early 1980s through electron microscopy of ex vivo functioning immune cells. Progressive advances in fluorescence microscopy and molecular immunology in the past 20 years have led to rapid progress on understanding the modes of cell-cell interaction and underlying molecular events...
2017: Methods in Molecular Biology
Jérôme Bouchet, Iratxe Del Río-Iñiguez, Elena Vázquez-Chávez, Rémi Lasserre, Sonia Agüera-González, Céline Cuche, Mary W McCaffrey, Vincenzo Di Bartolo, Andrés Alcover
The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3)...
February 24, 2017: Journal of Immunology: Official Journal of the American Association of Immunologists
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