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Micron-scale assemblies of molecules are thematic in biology, although their mechanism of formation and exact functional role are oftentimes unknown. The immunological synapse (IS)--the gateway event to the body's initiation of an immune response against infection--is a hallmark example. T cell detection of pathogenic invasion on an antigen-presenting cell leads to the arrangement of receptor-ligand pairs into well-defined concentric zones referred to as supramolecular activation clusters (SMACs). The main signaling molecule, the T cell receptor (TCR), binds its specific foreign peptide-presenting ligand, major histocompatibility complex (pMHC). These complexes form a central cluster in the central SMAC (cSMAC) at the center of the intermembrane junction. Immediately surrounding the cSMAC is the peripheral SMAC (pSMAC), populated by a ring of leukocyte function associated antigen-1 (LFA-1) bound to intercellular adhesion molecule-1 (ICAM-1). In this dissertation, we determine how the final IS pattern emerges from a uniform distribution of receptor-ligand pairs. It is known that the actin cytoskeleton drives the centripetal transport of these proteins, but it is unclear how actin sorts them into their final destinations. We postulate that the large-scale sorting of proteins into the different SMACs is a natural consequence of smaller scale protein sorting into microclusters, which may contain hundreds of molecules. To test this, we increase the LFA-1 cluster size two additional degrees beyond its native state with antibody crosslinkers. We either crosslink LFA-1 directly or indirectly with antibodies against its ICAM-1 ligand, which is presented on a supported membrane with the activating pMHC. Progressively more central localization of LFA-1 proportional to the degree of crosslinking results until LFA-1 occupies the cSMAC with TCR. Based on these results, we propose a sorting mechanism based on frictional protein coupling to actin. In the frictional force coupling model, the extent of radial protein transport by actin is determined by the specific coupling chemistry and the protein cluster size. This model predicts cluster size-based protein sorting across the IS. Using fluorescence fluctuation measurements and a small illumination area, we detect a gradient of LFA-1:ICAM-1 cluster sizes across the pSMAC in the native IS, as predicted by our model. Thus, we demonstrate that the well-regulated event of protein clustering is a critical parameter in regulating spatial patterning in the IS.
This new collection features the most up-to-date essential protocols that are currently being used to study the immune synapse. Beginning with methods for making biophysical measurements, the volume continues by covering the cell biology of synapses, methods for advanced substrate engineering, mechanobiology topics, new technologies to describe and manipulate synaptic components, as well as methods related to sites of action and immunotherapy. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and fully updated, The Immune Synapse: Methods and Protocols, Second Edition serves as an ideal practical guide for researchers working in this dynamic field. Chapters 5, 11,18, 27, 30, and 32 are available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
The immunological synapse (IS) is a specialised cell-cell adhesion that mediates antigen acquisition and regulates the activation of lymphocytes. Initial studies of the IS showed a structure composed of stable supra-molecular activation clusters (SMAC) organised during the interaction of helper T lymphocytes with B lymphocytes, working as antigen presenting cells. A central SMAC of coalesced T cell receptors (TCRs) and a peripheral SMAC for cell-cell adhesion were observed. IS with similar structure was later described during antigen acquisition by B cells and during the interaction of NK cells with target and healthy cells. More recent research developed with microscopy systems that improve the spatial and temporal resolution has showed the complex molecular dynamics at the IS that governs lymphocyte activation. Currently, the IS is seen as a three-dimensional structure where signalling networks for lymphocyte activation and endosomal and cytoskeleton machinery are polarised. A view has emerged in which dynamic microclusters of signalling complexes are composed of molecular components attached to the plasma membrane and other components conveyed on sub-synaptic vesicles transported to the membrane by cytoskeletal fibers and motor proteins. Much information is nonetheless missing about how the dynamics of the endosomal compartment, the cytoskeleton, and signalling complexes are reciprocally regulated to achieve the function of lymphocytes. Experimental evidence also suggests that the environment surrounding lymphocytes exposed to different antigenic challenge regulates IS assembly and functional output, making an even more complex scenario still far from being completely understood. Also, although some signalling molecular components for lymphocyte activation have been identified and thoroughly studied, the function of other molecules has not been yet uncovered or deeply characterised. This research topic aims to provide the reader with the latest information about the molecular dynamics governing lymphocyte activation. These molecular dynamics dictate cell decisions. Thus, we expect that understanding them will provide new avenues for cell manipulation in therapies to treat different immune-related pathologies.
The Immunological Synapse, Part A, Volume 173 in the Methods in Cell Biology series provides state-of-the-art methods for the study of the immunological synapse. Sections cover Imaging polarized granule release at the cytotoxic T cell immunological synapse using TIRF microscopy: control by polarity regulators, Analysis of centrosomal area actin reorganization and centrosome polarization upon lymphocyte activation at the immunological synapse, P815-based redirected degranulation assay to study human NK cell effector functions, Cytotoxic and Chemotactic Dynamics of Natural Killer Cells Quantified by Live-cell Imaging, Quantification of interaction frequency between antigen-presenting cells and T cells by conjugation assay, and more. Other chapters focus on the Study of the Effects of NK-Tumor Cell Interaction by Proteomic Analysis and Imaging, Quantification of lymphocytic choriomeningitis virus specific T cells and LCMV viral titers, Quantification of lymphocytic choriomeningitis virus specific T cells and LCMV viral titers, An in vitro model to monitor natural killer cell effector functions against primary breast cancer, and Standardized Protocol for the Evaluation of Chimeric Antigen Receptor (CAR)-modified Cell Immunological Synapse Quality using the Glass-supported Planar L. Covers various methods related to the study of the immunological synapse Includes detailed, point-by-point, methods as well as various important notes Provides the authority and expertise from an international board of leading scientists
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Structural Biology in Immunology, Structure/Function of Novel Molecules of Immunologic Importance delivers important information on the structure and functional relationships in novel molecules of immunologic interest. Due to an increasingly sophisticated understanding of the immune system, the approach to the treatment of many immune-mediated diseases, including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease has been dramatically altered. Furthermore, there is an increasing awareness of the critical role of the immune system in cancer biology. The improved central structure function relationships presented in this book will further enhance our ability to understand what defects in normal individuals can lead to disease. Describes novel/recently discovered immunomodulatory proteins, including antibodies and co-stimulatory or co-inhibitory molecules Emphasizes new biologic and small molecule drug design through the exploration of structure-function relationship Features a collaborative editorial effort, involving clinical immunologists and structural biologists Provides useful and practical insights on developing the necessary links between basic science and clinical therapy in immunology Gives interested parties a bridge to learn about computer modeling and structure based design principles
The proper physiological functioning of most eukaryotic cells requires their assembly into multi-cellular tissues that form organized organ systems. Cells of the immune system develop in bone marrow and lymphoid organs, but as the cells mature they leave these organs and circulate as single cells. Antigen receptors (TCRs) of T cells search for membrane MHC proteins that are bound to peptides derived from infectious pathogens or cellular transformations. The detection of such speci?c peptide–MHC antigens initiates T cell activation, adhesion, and immune-effectors functions. Studies of normal and transformed T cell lines and of T cells from transgenic mice led to comprehensive understanding of the mole- lar basis of antigen-receptor recognition and signaling. In spite of these remarkable genetic and biochemical advances, other key physiological mechanisms that par- cipate in sensing and decoding the immune context to induce the appropriate cellular immune responses remain unresolved. TCR recognition is tightly regulated to trigger sensitive but balanced T cell responses that result in the effective elimination of the pathogens while minimizing collateral damage to the host. The sensitivity of TCR recognition has to be properly tempered to prevent unintended activation by self-peptide–MHC complexes that cause autoimmune diseases. It is likely that once the TCR is engaged by a peptide– MHC and TCR signaling begins, additional regulatory mechanisms, involving other receptors, would increase the ?delity of the response.