NK cells integrate signals over large areas when building immune synapses but require local stimuli for degranulation

Immune synapses are large-scale, transient molecular assemblies that serve as platforms for antigen presentation to B and T cells, and target recognition by cytotoxic T cells and natural killer (NK) cells. The formation of an immune synapse is a tightly regulated, stepwise process where the cytoskeleton, cell-surface receptors and signaling proteins rearrange into supramolecular activation clusters (SMACs). Here we use a reductionist system of microcontact-printed artificial immune synapses (AIS) shaped as hallmark SMAC structures to show that the spatial distribution of activating ligands influences the formation, stability and outcome of NK cell synapses. Organizing ligands into donut-shaped AIS resulted in fewer long-lasting, symmetrical synapses compared to dot-shaped AIS. NK cells spreading evenly over either AIS exhibited similar arrangement of the lytic machinery, however degranulation was only possible in regions allowing local signaling. Our results demonstrate that the macroscopic organization of ligands in the synapse can affect its outcome, which could be exploited by target cells as an escape mechanism.