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Abstract The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. It is important, therefore, to be able to elucidate molecular processes occurring at these interfaces. However, the detailed investigation of each component’s contribution to the formation and regulation of the contact is hampered by the complexity of cellular composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially for using advanced imaging technology. One approach to circumventing these problems is to establish in vitro systems that faithfully mimic immune cell interactions, incorporating complexity that can be ‘dialled-in’ as needed. Here, we present an in vitro system making use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we begin to investigate the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and the intracellular rearrangements that accompany the initiation of signalling in T cells. The model system presented here is expected to be widely applicable. Summary Statement Immune cell-cell interactions are reconstituted in free-standing vesicles wherein spatiotemporal aspects of immune synapse formation can be investigated.

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