The first observations of the immunological synapse have demonstrated that immune-cell signalling in situ does not simply depend on protein structures and signalling pathways but also on temporal and spatial coordinates. With the advent of new live-cell, three-dimensional fluorescence microscopy techniques our understanding of the relationship between the formation of the immunological synapse and the development of an immune response has been greatly improved.
Using artificial activating substrates as surrogate target cells or antigen presenting cells in conventional microscopes has so far been the state-of-the-art to obtain high-resolution images of immunological synapses. However, such artificial substrates may not fully recapitulate the complexity of intercellular interactions. Newly developed super-resolution techniques are very promising, but they remain inadequate for live-cell imaging. Technical improvements are therefore crucially needed to address these bottlenecks and improve our understanding of immune-cell signalling.
In this report we achieve high-speed high-resolution imaging of live intercellular immunological synapses by combining confocal microscopy with optical tweezers. We design, build and demonstrate the performance and flexibility of the instrument by imaging a variety of molecules at T cell and NK cell synapses.
NKG2D is an important receptor that allows NK cells to recognise and kill tumour cells. Due to the lack of suitable imaging technology, NKG2D signalling at the synapse remains unclear. We specifically use our new instrument to gain further understanding of NKG2D signalling, signal integration, and NKG2D-mediated cytotoxicity. For the first time at live intercellular NK-cell immunological synapses, we describe the formation and the dynamics of NKG2D microclusters. We show that these microclusters actively signal and that they coalesce around a secretory domain through which lytic secretions are delivered. Importantly, these results suggest that the physical distribution of NKG2D at the immunological synapse may play an important role in directing lytic-secretion delivery at the NK cell synapse.