Sophie V. Pageon

Peptide antagonism as a mechanism for NK cell activation

Inhibition of natural killer (NK) cells is mediated by MHC class I receptors including the killer cell Ig-like receptor (KIR). We demonstrate that HLA-C binding peptides can function as altered peptide ligands for KIR and antagonize the inhibition mediated by KIR2DL2/KIR2DL3. Antagonistic peptides promote clustering of KIR at the interface of effector and target cells, but do not result in inhibition of NK cells. Our data show that, as for T cells, small changes in the peptide content of MHC class I can regulate NK cell activity.

Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse.

Vesicles dynamically deliver the T cell adaptor protein LAT to sites of T cell signalosomes. Submariner Adaptor Engagement of the T cell antigen receptor (TCR) on the surface of a T cell with peptide-loaded major histocompatibility complex on the surface of an antigen-presenting cell occurs at a specialized contact point known as the immunological synapse (IS). Stimulation of the TCR triggers the activation of the proximal kinases Lck and ZAP-70, which leads to the formation at the IS of microclusters of kinases and adaptor molecules that are required for T cell signaling. Two of these adaptor molecules are the cytosolic protein SLP-76 and the plasma membrane–associated protein LAT. SLP-76 and LAT form distinct microclusters, but how they interact to propagate T cell signals is unclear (see the Perspective by Billadeau). Purbhoo et al. used imaging techniques to show that a fraction of LAT was enriched in intracellular vesicles that resided below the IS and that these vesicles moved to the IS, where they interacted with SLP76-containing microclusters. LAT-containing vesicles were corralled by TCR–ZAP-70 microclusters in the IS and were more mobile than the SLP-76 microclusters, but they slowed when they interacted with SLP-76. Cells that contained a mutant LAT that could not interact with SLP-76 (through the associated protein GADS) contained fewer SLP-76 microclusters, and LAT-containing vesicles interacted less with them. Together, these data suggest that vesicular LAT interacts with protein microclusters at the IS and contributes to the propagation of T cell signaling. Imaging studies have identified clusters of kinases and adaptor proteins that serve as centers of signaling at the contact points between T cells and antigen-presenting cells (APCs). Here, we report that the kinase ZAP-70 and the adaptor proteins LAT and SLP-76 accumulated in separate clusters at the interface between T cells and coverslips coated with a stimulatory antibody against CD3, a component of the T cell antigen receptor complex. A fraction of LAT was detected in motile vesicles that repeatedly moved to surface microclusters of SLP-76 and the adaptor protein GADS (growth factor receptor–bound protein–related adaptor downstream of Shc), where they exhibited decreased motility. LAT molecules in which the residues tyrosine 171 and tyrosine 191 (which are required for the binding of LAT to GADS) were mutated to phenylalanine did not dwell at clusters of SLP-76. At immunological synapses, LAT-containing vesicles also colocalized with microclusters of SLP-76, as detected in experiments in which laser tweezers were used to position T cell–APC conjugates vertically for high-resolution imaging. Phosphorylation of LAT was most prominent when vesicular LAT colocalized with SLP-76. Indeed, the abundance of phosphorylated LAT within a microcluster of SLP-76 was greatest in those clusters that had more recent interactions with LAT-containing vesicles. Finally, negative signals by the inhibitory receptor ILT2 disrupted the assembly of SLP-76–containing microclusters. Together, these data show that the movement of LAT-containing vesicles is linked to the organization of protein microclusters and suggest an important role for vesicular LAT in the SLP-76 signalosome.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Significance T-cell activation requires the translation of antigen binding to the T-cell receptor (TCR) into intracellular signaling. However, how antigen recognition and signal transduction are mechanistically linked is poorly understood. Here, we used single-molecule localization microscopy to link TCR clustering to signaling. We found that the likelihood of a single receptor to initiate signaling upon ligand binding depended on receptor-to-receptor spacing, with TCRs in dense clusters having the highest signaling efficiency. This means that antigen recognition must first be translated into a spatial reorganization of receptors into dense, signaling-competent clusters before signaling can begin. Thus, the quality of an antigen in terms of signaling is given by its ability to densely cluster receptors. Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Superresolution Microscopy Reveals Nanometer-Scale Reorganization of Inhibitory Natural Killer Cell Receptors upon Activation of NKG2D

The repositioning of inhibitory receptors on natural killer cells by an activating receptor is revealed by superresolution microscopy. Imaging Receptor Crosstalk Natural killer (NK) cells mediate the immune response to virally infected cells and tumor cells, and their activation state depends on the integration of signals from multiple activating and inhibitory cell-surface receptors. The limitations of light microscopy have prevented a clear understanding of the distribution of these receptors, so Pageon et al. used superresolution fluorescence microscopy to view the organization of the inhibitory receptor KIR2DL1 on the surface of human NK cells. KIR2DL1 was constitutively organized in nanometer-scale clusters that became smaller and denser when cells were treated with a KIR2DL1-specific antibody; unexpectedly however, a similar redistribution of KIR2DL1 occurred in response to engagement of the activating receptor NKG2D. Together, these data reveal crosstalk between activating and inhibitory receptors that may have important effects on NK cell function. Natural killer (NK) cell responses are regulated by a dynamic equilibrium between activating and inhibitory receptor signals at the immune synapse (or interface) with target cells. Although the organization of receptors at the immune synapse is important for appropriate integration of these signals, there is little understanding of this in detail, because research has been hampered by the limited resolution of light microscopy. Through the use of superresolution single-molecule fluorescence microscopy to reveal the organization of the NK cell surface at the single-protein level, we report that the inhibitory receptor KIR2DL1 is organized in nanometer-scale clusters at the surface of human resting NK cells. Nanoclusters of KIR2DL1 became smaller and denser upon engagement of the activating receptor NKG2D, establishing an unexpected crosstalk between activating receptor signals and the positioning of inhibitory receptors. These rearrangements in the nanoscale organization of surface NK cell receptors were dependent on the actin cytoskeleton. Together, these data establish that NK cell activation involves a nanometer-scale reorganization of surface receptors, which in turn affects models for signal integration and thresholds that control NK cell effector functions and NK cell development.

Super-resolution microscopy of the immunological synapse

Deciphering the spatial organisation of signalling proteins is the key to understanding the mechanisms underlying immune cell activation. Every advance in imaging technology has led to major breakthroughs in unravelling how receptor and signalling proteins are distributed within the plasma membrane and how membrane signalling is integrated with endosomes and vesicular trafficking. Recently, super-resolution fluorescence microscopy has been applied to immunological synapses, gaining new insights into the nanoscale organisation of signalling processes. Here, we review the advantages and potential of super-resolution microscopy for elucidating the regulation of many aspects of immune signalling.

Distinct Mechanisms Regulate Lck Spatial Organization in Activated T Cells

Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein–protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation.

Insights into Adhesion Biology Using Single‐Molecule Localization Microscopy

Focal adhesions are complex multi-protein structures that mediate cell adhesion and cell migration in multicellular organisms. Most of the protein components involved in focal adhesion formation have been identified, but a major challenge remains: determination of the spatial and temporal dynamics of adhesion proteins in order to understand the molecular mechanisms of adhesion assembly, maturation, signal regulation, and disassembly. Progress in this field has been hampered by the limited resolution of fluorescence microscopy. Recent advances have led to the development of super-resolution techniques including single-molecule localization microscopy (SMLM). Here, we discuss how the application of these techniques has revealed important new insights into focal adhesion structure and dynamics, including the first description of the three-dimensional nano-architecture of focal adhesions and of the dynamic exchange of integrins in focal adhesions. Hence, SMLM has contributed to the refinement of existing models of adhesions as well as the establishment of novel models, thereby opening new research directions. With current improvements in SMLM instrumentation and analysis, it has become possible to study cellular adhesions at the single-molecule level.

An intermolecular FRET sensor detects the dynamics of T cell receptor clustering

Clustering of the T-cell receptor (TCR) is thought to initiate downstream signalling. However, the detection of protein clustering with high spatial and temporal resolution remains challenging. Here we establish a Förster resonance energy transfer (FRET) sensor, named CliF, which reports intermolecular associations of neighbouring proteins in live cells. A key advantage of the single-chain FRET sensor is that it can be combined with image correlation spectroscopy (ICS), single-particle tracking (SPT) and fluorescence lifetime imaging microscopy (FLIM). We test the sensor with a light-sensitive actuator that induces protein aggregation upon radiation with blue light. When applied to T cells, the sensor reveals that TCR triggering increases the number of dense TCR–CD3 clusters. Further, we find a correlation between cluster movement within the immunological synapse and cluster density. In conclusion, we develop a sensor that allows us to map the dynamics of protein clustering in live T cells.

Dynamics of natural killer cell receptor revealed by quantitative analysis of photoswitchable protein.

Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies that rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report an apparently novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, after a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 ± 0.06 μm(2) s(-1)) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse, and remains surprisingly stable there. Unexpectedly, however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected intersynaptic exchange of protein.

Illuminating the dynamics of signal integration in Natural Killer cells

Natural Killer (NK) cell responses are shaped by the integration of signals transduced from multiple activating and inhibitory receptors at their surface. Biochemical and genetic approaches have identified most of the key proteins involved in signal integration but a major challenge remains in understanding how the spatial and temporal dynamics of their interactions lead to NK cells responding appropriately when encountering ligands on target cells. Well over a decade of research using fluorescence microscopy has revealed much about the architecture of the NK cell immune synapse – the structured interface between NK cells and target cells – and how it varies when inhibition or activation is the outcome of signal integration. However, key questions – such as the proximity of individual activating and inhibitory receptors – have remained unanswered because the resolution of optical microscopy has been insufficient, being limited by diffraction. Recent developments in fluorescence microscopy have broken this limit, seeding new opportunities for studying the nanometer-scale organization of the NK cell immune synapse. Here, we discuss how these new technologies, super-resolution imaging and other novel light-based methods, can illuminate our understanding of NK cell biology.