Correlative light and electron microscopy to explore the lytic immunological synapse between natural killer cells and cancer cells.

Cytotoxic lymphocytes, such as natural killer (NK) cells and cytotoxic T cells, can recognize and kill tumor cells by establishing a highly specialized cell-cell contact called the immunological synapse. The formation and lytic activity of the immunological synapse are accompanied by local changes in the organization, dynamics and molecular composition of the cell membrane, as well as the polarization of various cellular components, such as the cytoskeleton, vesicles and organelles. Characterization and understanding of the molecular and cellular processes underlying immunological synapse formation and activity requires the combination of complementary types of information provided by different imaging modalities, the correlation of which can be difficult. Correlative light and electron microscopy (CLEM) allows for the accurate correlation of functional information provided by fluorescent light microscopy with ultrastructural features provided by high-resolution electron microscopy. In this chapter, we present a detailed protocol describing each step to generate cell-cell conjugates between NK cells and cancer cells, and to analyze these conjugates by CLEM using separate confocal laser-scanning and transmission electron microscopes.

Ectocytosis renders T cell receptor signaling self-limiting at the immune synapse.

Cytotoxic T lymphocytes (CTLs) kill virus-infected and cancer cells through T cell receptor (TCR) recognition. How CTLs terminate signaling and disengage to allow serial killing has remained a mystery. TCR activation triggers membrane specialization within the immune synapse, including the production of diacylglycerol (DAG), a lipid that can induce negative membrane curvature. We found that activated TCRs were shed into DAG-enriched ectosomes at the immune synapse rather than internalized through endocytosis, suggesting that DAG may contribute to the outward budding required for ectocytosis. Budding ectosomes were endocytosed directly by target cells, thereby terminating TCR signaling and simultaneously disengaging the CTL from the target cell to allow serial killing. Thus, ectocytosis renders TCR signaling self-limiting.

Cell-to-Cell Spread of Dengue Viral RNA in Mosquito Cells.

Dengue virus (DENV) is an important mosquito-borne arbovirus that is particularly prevalent in tropical and subtropical areas of the world. The virus is generally ingested with a blood meal, replicates in host tissues, and disseminates into salivary glands for transmission to the next host. Membrane-bound vacuoles carrying DENV particles have been documented in mosquito cells and play a role in the cell-to-cell transmission of DENV2. C189 is one member of the tetraspanin family and generally increases its expression as one component of the vacuoles (C189-VCs) within C6/36 cells infected with DENV2. In the present study, we have further demonstrated via sucrose gradient centrifugation as well as magnetic immune isolation (MI) that the RNA of DENV2 was eventually carried by C189-VCs. In addition, viral RNA was shown to spread from donor to recipient cells in a coculture assay even when 20 mM NH4Cl was added to inhibit virus replication in the culture. In an alternate assay using the transwell system, viral RNA was only detected in recipient cells in the absence of 40 mM NH4Cl, suggesting that cell-cell contact is required for the intercellular spread of DENV2. In turn, the formation of viral synapse (VS) derived from aggregates of viral particles was frequently observed at sites of cell contact. Taken together, the formation of C189-VCs in C6/36 cells is induced by DENV2 infection, which may serve as a vehicle for transferring virions and also viral RNA to neighboring cells by cell-to-cell transmission after cell-cell contact. This finding provides insight into the understanding of viral spread between mosquito cells. It may also elucidate the benign persistent infection in mosquito cells and efficient dissemination of DENV infection within a mosquito vector.

T Cell Activation through Isolated Tight Contacts.

T cells engage antigen-presenting cells in search for cognate antigens via dynamic cell protrusions before forming a tight immune synapse. The spatiotemporal events that may lead to rapid TCR triggering and signal amplification in microvilli-driven isolated contacts, and in subsequent, more uniform contacts, remain poorly understood. Here, we combined interference-reflectance microscopy and single-molecule localization microscopy in live cells to resolve TCR-dependent signaling at tight cell contacts. We show that early contacts are sufficient for robust TCR triggering and ZAP-70 recruitment. With cell spreading, TCR activation and ZAP-70 recruitment increase and shift to the edges of the growing tight contacts. CD45 segregates from TCR at tight contacts and is enriched at high local curvature membrane. Surprisingly, cortical actin and LFA localized at contact regions of intermediate tightness. Our results show in molecular detail the roles of early and tight T cell contacts in T cell activation, as both sensing and decision-making entities.

Visualization of perforin/gasdermin/complement-formed pores in real cell membranes using atomic force microscopy.

Different types of pores ubiquitously form in cell membranes, leading to various types of cell death that profoundly influence the fate of inflammation and the disease status. However, these pores have never truly been visualized to date. Atomic force microscopy (AFM), which is emerging as a powerful tool to analyze the mechanical properties of biomolecules and cells, is actually an excellent imaging platform that allows biological samples to be visualized by probing surface roughness at the level of atomic resolution. Here, membrane pore structures were clearly visualized using AFM. This visualization not only describes the aperture and depth of the pore complexes but also highlights differences among the pores formed by perforin and gasdermins in tumor cell membranes and by complement in immune cell membranes. Additionally, this type of visualization also reveals the dynamic process of pore formation, fusion, and repair.

Membrane Ultrastructure and T Cell Activation.

The immune system serves as a crucial line of defense from infection and cancer, while also contributing to tissue homeostasis. Communication between immune cells is mediated by small soluble factors called cytokines, and also by direct cellular interactions. Cell-cell interactions are particularly important for T cell activation. T cells direct the adaptive immune response and therefore need to distinguish between self and foreign antigens. Even though decades have passed since the discovery of T cells, exactly why and how they are able to recognize and discriminate between antigens is still not fully understood. Early imaging of T cells was very successful in capturing the early stages of conjugate formation of T cells with antigen-presenting cells upon recognition of peptide-loaded major histocompatibility complexes by the T cell receptor (TCR). These studies lead to the discovery of a "supramolecular activation cluster" now known as the immunological synapse, followed by the identification of microclusters of TCRs formed upon receptor triggering, that eventually coalesce at the center of the synapse. New developments in light microscopy have since allowed attention to turn to the very earliest stages of T cell activation, and to resting cells, at high resolution. This includes single-molecule localization microscopy, which has been applied to the question of whether TCRs are pre-clustered on resting T cells, and lattice light-sheet microscopy that has enabled imaging of whole cells interacting with antigen-presenting cells. The utilization of lattice light-sheet microscopy has yielded important insights into structures called microvilli, which are small membrane protrusions on T cells that seem likely to have a large impact on T cell recognition and activation. Here we consider how imaging has shaped our thinking about T cell activation. We summarize recent findings obtained by applying more advanced microscopy techniques and discuss some of the limitations of these methods.

Nonstimulatory peptide-MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling.

Foreign antigens are presented by antigen-presenting cells in the presence of abundant endogenous peptides that are nonstimulatory to the T cell. In mouse T cells, endogenous, nonstimulatory peptides have been shown to enhance responses to specific peptide antigens, a phenomenon termed coagonism. However, whether coagonism also occurs in human T cells is unclear, and the molecular mechanism of coagonism is still under debate since CD4 and CD8 coagonism requires different interactions. Here we show that the nonstimulatory, HIV-derived peptide GAG enhances a specific human cytotoxic T lymphocyte response to HBV-derived epitopes presented by HLA-A*02:01. Coagonism in human T cells requires the CD8 coreceptor, but not T-cell receptor (TCR) binding to the nonstimulatory peptide-MHC. Coagonists enhance the phosphorylation and recruitment of several molecules involved in the TCR-proximal signaling pathway, suggesting that coagonists promote T-cell responses to antigenic pMHC by amplifying TCR-proximal signaling.

Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation.

The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT microcluster formation and vesicle delivery, using lattice light sheet microscopy to image a T cell from the earliest point of activation. A kinetic lag occurs between LAT microcluster formation and vesicular pool recruitment to the synapse. Correlative 3D light and electron microscopy show an absence of vesicles at microclusters at early times, but an abundance of vesicles as activation proceeds. Using TIRF-SIM to look at the activated T-cell surface with high resolution, we capture directed vesicle movement between microclusters on microtubules. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters.

Fas Ligand localizes to intraluminal vesicles within NK cell cytolytic granules and is enriched at the immune synapse.

INTRODUCTION: T cell and NK cell cytotoxicity can be mediated via the perforin/granzyme system and Fas Ligand (FasL, CD178). FasL is synthesized as a type II transmembrane protein that binds its cognate receptor Fas (CD95). Membrane-bound FasL is expressed on the plasma membrane of activated lymphocytes and is the main form of FasL with cytotoxic activity, but whether FasL is delivered to the immune synapse along with granzyme and perforin-containing granules is unclear. METHODS: We stably expressed FasL-fluorescent fusion proteins into human NK cells and examined the localization of FasL relative to other intracellular markers by confocal and immunoelectron microscopy, and examined the trafficking of FasL during formation of immune synapses with HLA-deficient B cells. RESULTS: FasL co-localized with CD63 more strongly than perforin or Lamp1+ in cytolytic granules. Electron microscopy revealed that FasL is enriched on intraluminal vesicles (ILVs) adjacent to the dense-core within cytolytic granules. In NK cells forming immune synapses with HLA-deficient B cells, a portion of FasL-containing granules re-localize toward the immune synapse, while a distinct pool of FasL remains at the distal pole of the cell. CONCLUSIONS: Localization of FasL to intra-luminal vesicles within cytolytic granules facilitates FasL trafficking to immune synapses and cytotoxic function in NK cells.

Membrane lipid order of sub-synaptic T cell vesicles correlates with their dynamics and function.

During an immune response, T cells survey antigen presenting cells for antigenic peptides via the formation of an interface known as an immunological synapse. Among the complex and dynamic biophysical phenomena occurring at this interface is the trafficking of sub-synaptic vesicles carrying a variety of proximal signalling molecules. Here, we show that rather than being a homogeneous population, these vesicles display a diversity of membrane lipid order profiles, as measured using the environmentally sensitive dye di-4-ANEPPDHQ and multi-spectral TIRF microscopy. Using live-cell imaging, vesicle tracking and a variety of small molecule drugs to manipulate components of the actin and tubulin cytoskeleton, we show that the membrane lipid order of these vesicles correlate with their dynamics. Furthermore, we show that the key proximal signalling molecule Linker for Activation of T cells (LAT) is enriched in specific vesicle populations as defined by their higher membrane order. These results imply that vesicle lipid order may represent a novel regulatory mechanism for the sorting and trafficking of signalling molecules at the immunological synapse, and, potentially, other cellular structures.

Human profilin 1 is a negative regulator of CTL mediated cell-killing and migration.

The actin-binding protein profilin1 (PFN1) plays a central role in actin dynamics, which is essential for cytotoxic T lymphocyte (CTL) functions. The functional role of PFN1 in CTLs, however still remains elusive. Here, we identify PFN1 as the only member of the profilin family expressed in primary human CD8+ T cells. Using in vitro assays, we find that PFN1 is a negative regulator of CTL-mediated elimination of target cells. Furthermore, PFN1 is involved in activation-induced lytic granule (LG) release, CTL migration and modulation of actin structures at the immunological synapse (IS). During CTL migration, PFN1 modulates the velocity, protrusion formation patterns and protrusion sustainability. In contrast, PFN1 does not significantly affect migration persistence and the rates of protrusion emergence and retraction. Under in vitro conditions mimicking a tumor microenvironment, we show that PFN1 downregulation promotes CTL invasion into a 3D matrix, without affecting the viability of CTLs in a hydrogen peroxide-enriched microenvironment. Highlighting its potential relevance in cancer, we find that in pancreatic cancer patients, PFN1 expression is substantially decreased in peripheral CD8+ T cells. Taken together, we conclude that PFN1 is a negative regulator for CTL-mediated cytotoxicity and may have an impact on CTL functionality in a tumor-related context.

Ultrastructure of Immune Synapses.

The immunological synapse is a critical event for immune response development. The use of planar supported bilayers as surrogate antigen-presenting cells is a useful tool to study this phenomenon. Here we describe electron microscopy methods and approaches to expand our knowledge of the events taking place during the initial phases of T cell activation after antigen recognition at the nanometer scale.

Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy.

Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apart from these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiate and upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave as antigen presenting cells (APC). The initial step for activating T-cells is the formation of an immune synapse between T-cells and antigen-presenting cells. However, the immune synapse that develops at the PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods for analysis of large number of PMN interactions. In order to overcome these obstacles, we introduce here a workflow to analyse the immune synapse of primary human PMN and T-cells using multispectral imaging flow cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose, we used CD3 and CD66b as the lineage markers for T-cells and PMN, respectively. Thereafter, we applied and critically discussed various "masks" for identification of T-cell PMN interactions. Using this approach, we found that a small fraction of transdifferentiated PMN (CD66b+CD86high) formed stable PMN/T-cell conjugates. Interestingly, while both CD3 and CD66b accumulation in the immune synapse was dependent on the maturation state of the PMN, only CD3 accumulation was greatly enhanced by the presence of superantigen. The actin cytoskeleton was weakly rearranged at the PMN side on the immune synapse upon contact with a T-cell in the presence of superantigen. A more detailed analysis using super-resolution microscopy (structured-illumination microscopy, SIM) confirmed this finding. Together, we present an InFlow microscopy based approach for the large scale analysis of PMN/T-cell interactions and - combined with SIM - a possibility for an in-depth analysis of protein translocation at the site of interactions.

HIV-1 Nef Impairs the Formation of Calcium Membrane Territories Controlling the Signaling Nanoarchitecture at the Immunological Synapse.

The ability of HIV-1 to replicate and to establish long-term reservoirs is strongly influenced by T cell activation. Through the use of membrane-tethered, genetically encoded calcium (Ca2+) indicators, we were able to detect for the first time, to our knowledge, the formation of Ca2+ territories and determine their role in coordinating the functional signaling nanostructure of the synaptic membrane. Consequently, we report a previously unknown immune subversion mechanism involving HIV-1 exploitation, through its Nef accessory protein, of the interconnectivity among three evolutionarily conserved cellular processes: vesicle traffic, signaling compartmentalization, and the second messenger Ca2+ We found that HIV-1 Nef specifically associates with the traffic regulators MAL and Rab11b compelling the vesicular accumulation of Lck. Through its association with MAL and Rab11b, Nef co-opts Lck switchlike function driving the formation Ca2+ membrane territories, which, in turn, control the fusion of LAT-transporting Rab27 and Rab37 vesicles and the formation of LAT nanoclusters at the immunological synapse. Consequently, HIV-1 Nef disengages TCR triggering from the generation of p-LAT and p-SLP nanoclusters driving TCR signal amplification and diversification. Altogether our results indicate that HIV-1 exploits the interconnectivity among vesicle traffic, Ca2+ membrane territories, and signaling nanoclusters to modulate T cell signaling and function.

Origins of the cytolytic synapse.

Cytotoxic T lymphocytes (CTLs) kill virus-infected and tumour cells with remarkable specificity. Upon recognition, CTLs form a cytolytic immune synapse with their target cell, and marked reorganization of both the actin and the microtubule cytoskeletons brings the centrosome up to the plasma membrane to the point of T cell receptor signalling. Secretory granules move towards the centrosome and are delivered to this focal point of secretion. Such centrosomal docking at the plasma membrane also occurs during ciliogenesis; indeed, striking similarities exist between the cytolytic synapse and the primary cilium that throw light on the possible origins of immune synapses.

Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images.

The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.

High- and Super-Resolution Microscopy Imaging of the NK Cell Immunological Synapse.

Recent advances in imaging technology have enabled significant advances in the study of NK cell cytotoxic effector function through quantitative analysis of the NK cell immunological synapse. This can include the use of high- and super-resolution microscopy to quantify dynamics of cytoskeletal elements and the role they play in the regulation and execution of NK cell directed secretion. Here we describe a protocol for the recapitulation of the NK cell lytic synapse on glass, the acquisition of microscopy images, and suggested approaches for the processing and analysis of microscopy data.

Human Cytotoxic T Lymphocytes Form Dysfunctional Immune Synapses with B Cells Characterized by Non-Polarized Lytic Granule Release.

Suppression of the cytotoxic T cell (CTL) immune response has been proposed as one mechanism for immune evasion in cancer. In this study, we have explored the underlying basis for CTL suppression in the context of B cell malignancies. We document that human B cells have an intrinsic ability to resist killing by freshly isolated cytotoxic T cells (CTLs), but are susceptible to lysis by IL-2 activated CTL blasts and CTLs isolated from immunotherapy-treated patients with chronic lymphocytic leukemia (CLL). Impaired killing was associated with the formation of dysfunctional non-lytic immune synapses characterized by the presence of defective linker for activation of T cells (LAT) signaling and non-polarized release of the lytic granules transported by ADP-ribosylation factor-like protein 8 (Arl8). We propose that non-lytic degranulation of CTLs are a key regulatory mechanism of evasion through which B cells may interfere with the formation of functional immune synapses by CTLs.

Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes.

Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca2+] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting.

Protein clustering and spatial organization in T-cells.

T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.