Finally, vesicle cargo was shown to correlate with different vesicle populations, based on their membrane order, demonstrating vesicle order may mirror the heterogeneous nature of the PM. These results demonstrate the complex biophysical processes that control the T cell immunological synapse are an amalgamation of cytoskeletal organisation, vesicle dynamics and membrane polarity.

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 - Part B, Volume 178 in the Methods in Cell Biology series provides state-of-the-art methods for the study of the immunological synapse. This first volume covers various aspects on T cell and natural killer (NK) cell synapses, including imaging polarized granule release using TIRF microscopy, analysis of actin reorganization and centrosome polarization, redirected degranulation, live cell-imaging to quantify cytotoxic and chemotactic dynamics, quantification of interactions between APCs and T cells, assessment of membrane lipid state at the immunological synapse, proteomic analysis and imaging of NK-tumor cell interaction, evaluating natural killer cell effector functions against breast cancer cells derived from human tumor tissue, evaluation of chimeric antigen receptor (CAR)-modified cell immunological synapse quality using the glass-supported planar lipid bilayer, and monitoring potency of therapeutic CAR T cells. It also includes one chapter on quantification of lymphocytic choriomeningitis virus specific T cells and LCMV viral titers, and one chapter on the murine antibody-dependent cellular phagocytosis assay. - 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

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

This volume gives an overview on the progress in immune synapse research, from basic science to clinical trials, and the major mechanisms involved. It discusses how interfering with T cell activation may lead to immune tolerance, immune modulation, and the recruitment of regulatory T cells; the role of monoclonal antibodies in tolerance induction; and mechanisms maintaining dominant tolerance.

T cell activation entails a complex series of signal transduction events that begin with ligation of the T cell receptor by a cognate antigen, displayed on the surface of an antigen presenting cell. During T cell signaling proteins in the cell surface partition from one another into large, stereotyped molecular pattern that is known as the immunological synapse. This thesis attempts to understand the mechanisms by which this segregation occurs, and uses a number of fluorescence imaging techniques---in particular, single molecule microscopy---to this end. Contrary to reports that suggest a role for lipid raft domains in patterning the synapse, we find that protein-protein interactions, and not putative lipid raft associations, are a strong driving force in synapse formation. We also develop a method for reconstituting synapse formation in an immortalized T cell line that allows us to perform single molecule imaging on a fluid substrate of a defined composition. A common finding is that both passive mechanisms, involving diffusional trapping and exclusion, as well as active mechanisms, involving actin-driven transport, can act in concert to shape the immunological synapse.