High-Resolution Imaging of Natural Killer Cell Immunological Synapses

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

Matched sizes of activating and inhibitory receptor/ligand pairs are required for optimal signal integration by human Natural Killer cells

It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions

Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy

Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules

High-resolution imaging of natural killer cell immunological synapses

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.EThOS - Electronic Theses Online ServiceEPSRC, CRUK and ICR via ICBGBUnited Kingdo

High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells

Cholesterol- and glycosphingolipid-enriched membrane lipid microdomains, frequently called lipid rafts, are thought to play an important role in the spatial and temporal organization of immunological synapses. Higher ordering of lipid acyl chains was suggested for these entities and imaging of membrane order in living cells during activation can therefore help to understand the mechanisms responsible for the supramolecular organization of molecules involved in the activation of T cells. Here, we employ the phase-sensitive membrane dye di-4-ANEPPDHQ together with a variety of spectrally-resolved microscopy techniques, including 2-channel ratiometric TIRF microscopy and fluorescence lifetime imaging, to characterize membrane order at the T cell immunological synapse at high spatial and temporal resolution in live cells at physiological temperature. We find that higher membrane order resides at the immunological synapse periphery where proximal signalling through the immunoreceptors and accessory proteins in microclusters has previously been shown to take place. The observed spatial patterning of membrane order in the immunological synapse depends on active receptor signalling

High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells

Cholesterol- and glycosphingolipid-enriched membrane lipid microdomains, frequently called lipid rafts, are thought to play an important role in the spatial and temporal organisation of immunological synapses. Higher ordering of lipid acyl chains was suggested for these entities and imaging of membrane order in living cells during activation can therefore help to understand the mechanisms responsible for the supramolecular organisation of molecules involved in the activation of T cells. Here, we employ the phase-sensitive membrane dye di-4-ANEPPDHQ together with a variety of spectrally-resolved microscopy techniques, including 2-channel ratiometric TIRF microscopy and fluorescence lifetime imaging, to characterize membrane order at the T cell immunological synapse at high spatial and temporal resolution in live cells at physiological temperature. We find that higher membrane order resides at the immunological synapse periphery where proximal signalling through the immunoreceptors and accessory proteins in microclusters has previously been shown to take place. Observed spatial patterning of membrane order in the immunological synapse depends on active receptor signalling

High-Speed High-Resolution Imaging of Intercellular Immune Synapses Using Optical Tweezers

Imaging in any plane other than horizontal in a microscope typically requires a reconstruction from multiple optical slices that significantly decreases the spatial and temporal resolution that can be achieved. This can limit the precision with which molecular events can be detected, for example, at intercellular contacts. This has been a major issue for the imaging of immune synapses between live cells, which has generally required the reconstruction of en face intercellular synapses, yielding spatial resolution significantly above the diffraction limit and updating at only a few frames per minute. Strategies to address this issue have usually involved using artificial activating substrates such as antibody-coated slides or supported planar lipid bilayers, but synapses with these surrogate stimuli may not wholly resemble immune synapses between two cells. Here, we combine optical tweezers and confocal microscopy to realize generally applicable, high-speed, high-resolution imaging of almost any arbitrary plane of interest. Applied to imaging immune synapses in live-cell conjugates, this has enabled the characterization of complex behavior of highly dynamic clusters of T cell receptors at the T cell/antigen-presenting cell intercellular immune synapse and revealed the presence of numerous, highly dynamic long receptor-rich filopodial structures within inhibitory Natural Killer cell immune synapses