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Lecture by Michael Dustin (Kennedy Institute of Rheumatology, UK)
Use of multiparameter flow cytometry to quantify the output of the immunological synapse
Kennedy Institute of Rheumatology, United Kingdom
The Immunological Synapse is a pivotal hub for the regulation of adaptive immunity. The identity of receptor and ligands interacting and clustering at the cell-cell interface is crucial in defining the global features of the triggered immune response. Significant efforts have been invested in developing multiparametric analyses deciphering T cell transcriptional phenotypes driving disease, however scalable methods allowing their effector characterization are still to be developed. Here, we harness Bead Supported Lipid Bilayers (BSLB) and multiparameter flow cytometry as a general method for the “enhanced functional interrogation” of the effector phase of T-cell immune synapses allowing the identification of protein and RNA species packed into synaptic vesicles (SV). SV can come from at least two sources- exosomes released from multivesicular bodies (intracellular stores) by directed exocytosis or ectosomes that bud directly from the pre-synaptic plasma membrane. Antigen, co-stimulation, co-repression, adhesion molecules and viral proteins are loaded on the surface of BSLB to generate tailored, synthetic antigen presenting cells. By providing these signals, BSLB instigate synapse formation and the release of patches of SV containing a variety of effectors, including CD40L, Perforin, ectonucleotidases, tetraspanins and small RNA species. Using CD40L as a model helper T cell effector we then explored the dynamics influencing the release of CD40L+ SV, and demonstrate the role of T cell phenotypes, antigen and CD40 density, antigen potency, co-stimulation, co-repression, enzymatic processing by ADAM10 and the facilitating role of structural proteins including bone marrow stromal cell Bone Marrow Stromal Cell Antigen 2 (BST2, CD317) and CD81. Altogether, we demonstrate the broad applicability of BSLB for the dissection of T cell effectors delivered in the form of synaptic vesicles born either from the plasma membrane or intracellular stores.