Immunological Synapse Assembly Research Articles

T cell activation and effector function in the human Jurkat T cell model.

In order to understand T cell function, it is necessary to completely decipher the molecular dynamics underlying T cell activation and effector function. In vitro easy-to-handle cellular models are valuable tools to study intracellular molecular mechanisms in live cells. The CD4 T cell line Jurkat (JK) has been widely employed to investigate intracellular signaling leading to T cell activation in response to T cell receptor (TCR) triggering. Here, we describe diverse, complementary protocols to evaluate the TCR- and costimulation-mediated T cell activation, as well as the immunological synapse assembly and cytokine production occurring as a consequence of successful early activation events. This in vitro model is extremely useful to address molecular mechanisms operating during T cell activation and effector function acting in diverse pathophysiological scenarios.

Learning from TCR Signaling and Immunological Synapse Assembly to Build New Chimeric Antigen Receptors (CARs).

Chimeric antigen receptor (CAR) T cell immunotherapy is a revolutionary pillar in cancer treatment. Clinical experience has shown remarkable successes in the treatment of certain hematological malignancies but only limited efficacy against B cell chronic lymphocytic leukemia (CLL) and other cancer types, especially solid tumors. A wide range of engineering strategies have been employed to overcome the limitations of CAR T cell therapy. However, it has become increasingly clear that CARs have unique, unexpected features; hence, a deep understanding of how CARs signal and trigger the formation of a non-conventional immunological synapse (IS), the signaling platform required for T cell activation and execution of effector functions, would lead a shift from empirical testing to the rational design of new CAR constructs. Here, we review current knowledge of CARs, focusing on their structure, signaling and role in CAR T cell IS assembly. We, moreover, discuss the molecular features accounting for poor responses in CLL patients treated with anti-CD19 CAR T cells and propose CLL as a paradigm for diseases connected to IS dysfunctions that could significantly benefit from the development of novel CARs to generate a productive anti-tumor response.

The Immunological Synapse: An Emerging Target for Immune Evasion by Bacterial Pathogens.

Similar to other pathogens, bacteria have developed during their evolution a variety of mechanisms to overcome both innate and acquired immunity, accounting for their ability to cause disease or chronic infections. The mechanisms exploited for this critical function act by targeting conserved structures or pathways that regulate the host immune response. A strategic potential target is the immunological synapse (IS), a highly specialized structure that forms at the interface between antigen presenting cells (APC) and T lymphocytes and is required for the establishment of an effective T cell response to the infectious agent and for the development of long-lasting T cell memory. While a variety of bacterial pathogens are known to impair or subvert cellular processes essential for antigen processing and presentation, on which IS assembly depends, it is only recently that the possibility that IS may be a direct target of bacterial virulence factors has been considered. Emerging evidence strongly supports this notion, highlighting IS targeting as a powerful, novel means of immune evasion by bacterial pathogens. In this review we will present a brief overview of the mechanisms used by bacteria to affect IS assembly by targeting APCs. We will then summarize what has emerged from the current handful of studies that have addressed the direct impact of bacterial virulence factors on IS assembly in T cells and, based on the strategic cellular processes targeted by these factors in other cell types, highlight potential IS-related vulnerabilities that could be exploited by these pathogens to evade T cell mediated immunity.

Lymphocyte Polarization During Immune Synapse Assembly: Centrosomal Actin Joins the Game.

Interactions among immune cells are essential for the development of adaptive immune responses. The immunological synapse (IS) provides a specialized platform for integration of signals and intercellular communication between T lymphocytes and antigen presenting cells (APCs). In the T cell the reorganization of surface molecules at the synaptic interface is initiated by T cell receptor binding to a cognate peptide-major histocompatibility complex on the APC surface and is accompanied by a polarized remodelling of the cytoskeleton and centrosome reorientation to a subsynaptic position. Although there is a general agreement on polarizing signals and mechanisms driving centrosome reorientation during IS assembly, the primary events that prepare for centrosome repositioning remain largely unexplored. It has been recently shown that in resting lymphocytes a local polymerization of filamentous actin (F-actin) at the centrosome contributes to anchoring this organelle to the nucleus. During early stages of IS formation centrosomal F-actin undergoes depletion, allowing for centrosome detachment from the nucleus and its polarization towards the synaptic membrane. We recently demonstrated that in CD4+ T cells the reduction in centrosomal F-actin relies on the activity of a centrosome-associated proteasome and implicated the ciliopathy-related Bardet-Biedl syndrome 1 protein in the dynein-dependent recruitment of the proteasome 19S regulatory subunit to the centrosome. In this short review we will feature our recent findings that collectively provide a new function for BBS proteins and the proteasome in actin dynamics, centrosome polarization and T cell activation.

Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ Tcells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.

The Role of the Cytoskeleton in Regulating the Natural Killer Cell Immune Response in Health and Disease: From Signaling Dynamics to Function.

Natural killer (NK) cells are innate lymphoid cells, which play key roles in elimination of virally infected and malignant cells. The balance between activating and inhibitory signals derived from NK surface receptors govern the NK cell immune response. The cytoskeleton facilitates most NK cell effector functions, such as motility, infiltration, conjugation with target cells, immunological synapse assembly, and cytotoxicity. Though many studies have characterized signaling pathways that promote actin reorganization in immune cells, it is not completely clear how particular cytoskeletal architectures at the immunological synapse promote effector functions, and how cytoskeletal dynamics impact downstream signaling pathways and activation. Moreover, pioneering studies employing advanced imaging techniques have only begun to uncover the architectural complexity dictating the NK cell activation threshold; it is becoming clear that a distinct organization of the cytoskeleton and signaling receptors at the NK immunological synapse plays a decisive role in activation and tolerance. Here, we review the roles of the actin cytoskeleton in NK cells. We focus on how actin dynamics impact cytolytic granule secretion, NK cell motility, and NK cell infiltration through tissues into inflammatory sites. We will also describe the additional cytoskeletal components, non-muscle Myosin II and microtubules that play pivotal roles in NK cell activity. Furthermore, special emphasis will be placed on the role of the cytoskeleton in assembly of immunological synapses, and how mutations or downregulation of cytoskeletal accessory proteins impact NK cell function in health and disease.

Orchestration of Immunological Synapse Assembly by Vesicular Trafficking.

Ligation of the T-cell antigen receptor (TCR) by cognate peptide bound to the Major Histocompatibility Complex on the surface of an antigen-presenting cell (APC) leads to the spatial reorganization of the TCR and accessory receptors to form a specialized area of intimate contact between T cell and APC, known as the immunological synapse (IS), where signals are deciphered, coordinated, and integrated to promote T cell activation. With the discovery that an endosomal TCR pool contributes to IS assembly and function by undergoing polarized recycling to the IS, recent years have witnessed a shift from a plasma membrane-centric view of the IS to the vesicular trafficking events that occur at this location following the TCR-dependent translocation of the centrosome toward the synaptic membrane. Here we will summarize our current understanding of the trafficking pathways that are responsible for the steady delivery of endosomal TCRs, kinases, and adapters to the IS to sustain signaling, as well as of the endocytic pathways responsible for signal termination. We will also discuss recent evidence highlighting a role for endosomes in sustaining TCR signaling after its internalization at the IS and identifying the IS as a site of formation and release of extracellular vesicles that allow for transcellular communication with the APC.

Regulation of CD4+ T Cell Signaling and Immunological Synapse by Protein Tyrosine Phosphatases: Molecular Mechanisms in Autoimmunity.

T cell activation and effector function is mediated by the formation of a long-lasting interaction established between T cells and antigen-presenting cells (APCs) called immunological synapse (IS). During T cell activation, different signaling molecules as well as the cytoskeleton and the endosomal compartment are polarized to the IS. This molecular dynamics is tightly regulated by phosphorylation networks, which are controlled by protein tyrosine phosphatases (PTPs). While some PTPs are known to be important regulators of adhesion, ligand discrimination or the stimulation threshold, there is still little information about the regulatory role of PTPs in cytoskeleton rearrangements and endosomal compartment dynamics. Besides, spatial and temporal regulation of PTPs and substrates at the IS is only barely known. Consistent with an important role of PTPs in T cell activation, multiple mutations as well as altered expression levels or dynamic behaviors have been associated with autoimmune diseases. However, the precise mechanism for the regulation of T cell activation and effector function by PTPs in health and autoimmunity is not fully understood. Herein, we review the current knowledge about the regulatory role of PTPs in CD4+ T cell activation, IS assembly and effector function. The potential molecular mechanisms mediating the action of these enzymes in autoimmune disorders are discussed.

Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function.

The regulatory role of most dual specific phosphatases during T cell activation remains unknown. Here, we have studied the expression and function of phosphatases of regenerating liver (PRLs: PRL-1, PRL-2, and PRL-3) during T cell activation, as well as, the dynamic delivery of PRL-1 to the Immunological Synapse (IS). We found that T cell activation downregulates the expression of PRL-2, resulting in an increased PRL-1/PRL-2 ratio. PRL-1 redistributed at the IS in two stages: Initially, it was transiently accumulated at scanning membranes enriched in CD3 and actin, and at later times, it was delivered at the contact site from pericentriolar, CD3ζ-containing, vesicles. Once at the established IS, PRL-1 distributed to LFA-1 and CD3ε sites. Remarkably, PRL-1 was found to regulate actin dynamics during IS assembly and the secretion of IL-2. Moreover, pharmacological inhibition of the catalytic activity of the three PRLs reduced the secretion of IL-2. These results provide evidence indicating a regulatory role of PRL-1 during IS assembly and highlight the involvement of PRLs in immune responses by mature T cells.

Transcellular communication at the immunological synapse: a vesicular traffic-mediated mutual exchange.

The cell’s ability to communicate with the extracellular environment, with other cells, and with itself is a crucial feature of eukaryotic organisms. In the immune system, T lymphocytes assemble a specialized structure upon contact with antigen-presenting cells bearing a peptide-major histocompatibility complex ligand, known as the immunological synapse (IS). The IS has been extensively characterized as a signaling platform essential for T-cell activation. Moreover, emerging evidence identifies the IS as a device for vesicular traffic-mediated cell-to-cell communication as well as an active release site of soluble molecules. Here, we will review recent advances in the role of vesicular trafficking in IS assembly and focused secretion of microvesicles at the synaptic area in naïve T cells and discuss the role of the IS in transcellular communication.

Signaling and Polarized Communication Across the T Cell Immunological Synapse.

T cells express a somatically recombined antigen receptor (αβTCR) that is calibrated during development to respond to changes in peptides displayed by major histocompatibility complex proteins (pMHC) on the surface of antigen-presenting cells (APC). A key characteristic of pMHC for adaptive immunity is the ability to sample internal states of cells and tissues to sensitively detect changes associated with infection, cell derangement, or tissue injury. Physical T cell-APC contact sets up an axis for polarization of TCR, adhesion molecules, kinases, cytoskeletal elements, and organelles inherent in this mode of juxtacrine signaling. The discovery of further lateral organization of the TCR and adhesion molecules into radially symmetric compartments, the immunological synapse, revealed an intersecting plane of symmetry and potential for regulated symmetry breaking to control duration of T cell-APC interactions. In addition to organizing signaling machinery, the immunological synapse directs the polarized transport and secretion of cytokines and cytolytic agents across the synaptic cleft and is a site for the generation and exocytic release of bioactive microvesicles that can functionally affect recipient APC and other cells in the environment. This machinery is coopted by retroviruses, and human immune deficiency virus-1 may even use antigen-specific synapses for infection of healthy T cells. Here, we discuss recent advances in the molecular and cell biological mechanisms of immunological synapse assembly and signaling and its role in intercellular communication across the synaptic cleft.

Signal Integration during T Lymphocyte Activation and Function: Lessons from the Wiskott-Aldrich Syndrome.

Over the last decades, research dedicated to the molecular and cellular mechanisms underlying primary immunodeficiencies (PID) has helped to understand the etiology of many of these diseases and to develop novel therapeutic approaches. Beyond these aspects, PID are also studied because they offer invaluable natural genetic tools to dissect the human immune system. In this review, we highlight the research that has focused over the last 20 years on T lymphocytes from Wiskott–Aldrich syndrome (WAS) patients. WAS T lymphocytes are defective for the WAS protein (WASP), a regulator of actin cytoskeleton remodeling. Therefore, study of WAS T lymphocytes has helped to grasp that many steps of T lymphocyte activation and function depend on the crosstalk between membrane receptors and the actin cytoskeleton. These steps include motility, immunological synapse assembly, and signaling, as well as the implementation of helper, regulatory, or cytotoxic effector functions. The recent concept that WASP also works as a regulator of transcription within the nucleus is an illustration of the complexity of signal integration in T lymphocytes. Finally, this review will discuss how further study of WAS may contribute to solve novel challenges of T lymphocyte biology.

Surface expression of human Respiratory Syncytial Virus (hRSV) Nucleoprotein inhibits immunological synapse assembly by T cells

Surface expression of human Respiratory Syncytial Virus (hRSV) Nucleoprotein inhibits immunological synapse assembly by T cells

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells.

Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse- and human cell-based studies have shown that hRSV infection prevents naïve T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface-anchored N protein prevented immunological synapse assembly by naive CD4(+) T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell-bilayer interface, suggesting that N protein interferes with pMHC-TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.

The human respiratory syncytial virus nucleoprotein is expressed on the surface of dendritic cells and inhibits immunological synapse assembly by T cells (VIR2P.1018)

Abstract The human Respiratory Syncytial Virus (hRSV) is the major cause of children severe respiratory tract infections worldwide. Both, the poor activation of T cells and the limited generation of memory T cells following disease resolution are considered key processes favoring hRSV epidemics, which are based on re-infections. It is known that hRSV infects Dendritic cells (DCs) dampening their capacity to prime antigen-specific naïve T cells, a process needed to mount a proper antiviral immune response and eliminate hRSV from the airways. Here we show that the hRSV Nucleoprotein (N) is expressed at the surface of a variety of infected cells, including DCs. After estimating the approximate density of N in the surface of DCs and other cells, we evaluated whether these physiological densities could affect the assembly of the immunological synapse (IS) by naïve CD4+ T cells. We observed that N, but not control proteins inhibited the assembly of IS by interfering with TCR-pMHC interactions, which was accompanied with reduced accumulation of TCR and reduced central clustering of pMHC at the T cell-bilayer interface, as well as with reduced phosphorylation of tyrosine residues in the interacting pole of stimulated T cells. Altogether, our findings propose N-hRSV as a key virulence factor responsible for the suboptimal T cell activation observed in hRSV-infected individuals.

Phosphoinositide binding by the SNX27 FERM domain regulates its localization at the immune synapse of activated T-cells.

Sorting nexin 27 (SNX27) controls the endosomal-to-cell-surface recycling of diverse transmembrane protein cargos. Crucial to this function is the recruitment of SNX27 to endosomes which is mediated by the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by its phox homology (PX) domain. In T-cells, SNX27 localizes to the immunological synapse in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide-lipid-binding capabilities of full-length SNX27, and discovered a new PtdInsP-binding site within the C-terminal 4.1, ezrin, radixin, moesin (FERM) domain. This binding site showed a clear preference for bi- and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the immunological synapse of activated T-cells, cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FERM domain alters the SNX27 distribution in both endosomal recycling compartments and PtdIns(3,4,5)P3-enriched domains of the plasma membrane during synapse formation. Our results suggest that SNX27 undergoes dynamic partitioning between different membrane domains during immunological synapse assembly, and underscore the contribution of unique lipid interactions for SNX27 orchestration of cargo trafficking.

Modulation of Tumor Immunity by Soluble and Membrane-Bound Molecules at the Immunological Synapse

To circumvent pathology caused by infectious microbes and tumor growth, the host immune system must constantly clear harmful microorganisms and potentially malignant transformed cells. This task is accomplished in part by T-cells, which can directly kill infected or tumorigenic cells. A crucial event determining the recognition and elimination of detrimental cells is antigen recognition by the T cell receptor (TCR) expressed on the surface of T cells. Upon binding of the TCR to cognate peptide-MHC complexes presented on the surface of antigen presenting cells (APCs), a specialized supramolecular structure known as the immunological synapse (IS) assembles at the T cell-APC interface. Such a structure involves massive redistribution of membrane proteins, including TCR/pMHC complexes, modulatory receptor pairs, and adhesion molecules. Furthermore, assembly of the immunological synapse leads to intracellular events that modulate and define the magnitude and characteristics of the T cell response. Here, we discuss recent literature on the regulation and assembly of IS and the mechanisms evolved by tumors to modulate its function to escape T cell cytotoxicity, as well as novel strategies targeting the IS for therapy.

Microspectroscopy reveals mechanisms of lymphocyte activation

The immunological synapse (IS) regulates immune responses by integrating extracellular stimuli into intracellular signalling networks, which causes leukocyte differentiation and effector functions. The dynamic spatial organisation of molecules at the IS was initially characterised by wide-field fluorescence microscopy of cell conjugates and cells interacting with planar lipid bilayers. These methods showed stable supramolecular clusters of several microns in size, which were proposed to be responsible for sustained signalling and cell-cell adhesion. The recent emergence of microspectroscopy techniques with higher spatial and temporal resolution nonetheless reveals the complex dynamics of molecular reactions that mediate IS assembly and function. This review describes microspectroscopy-based in vitro experimental approaches for imaging the molecular dynamics at the IS, as well as their contributions and open questions in the field. It also describes experimental methods to obtain quantitative parameters of dynamic biochemical reactions in living cells, and discusses about the important role of quantitative imaging and theoretical science in our understanding of molecular mechanisms underlying lymphocyte activation.

Cytofluorimetric evaluation of N-glycolylated GM3 ganglioside expression on murine leukocytes

Gangliosides are considered relevant components of lipid rafts at the plasma membrane. Antigen encounter, immunological synapse assembly and signal transduction modify lipid raft composition and distribution on immune system cells. On the contrary of other gangliosides, differential expression of the N-glycolylated variant of GM3 (NGcGM3) on murine leukocytes has received limited attention. In particular, whether cell activation modulates the expression of NGcGM3 on lymphoid and myeloid cells is still unexplored. Availability of the NGcGM3 specific 14F7 MAb allows us to characterize by cytofluorimetric assays the presence of this molecule on resting and activated immune system cells. On T cells, preferential expression of NGcGM3 was detected on CD4 + single positive thymocytes, peripheral CD4 + lymphocytes and natural occurring regulatory T cells. In comparison with peritoneal B1 cells, reduced expression of NGcGM3 was observed in peritoneal B2 and splenic B cell subpopulations. Of note, activation of CD4 + and NK 1.1 + cells abrogated NGcGM3 expression while LPS-maturated DC increased the ganglioside level at the plasma membrane. Modifications on the presence of NGcGM3 mediated by cell activation did not influence the expression of the N-acetylated variant of GM3 (NAcGM3). In addition to extend previous descriptions of NGcGM3 expression on immunity cell subpopulations, this work highlights the opposite effect of cellular activation over NGcGM3 levels on lymphoid and myeloid cellular series. Obtained results complement the evaluation of a tumor-specific, non-human sialic acid containing ganglioside that has been considered an attractive target for cancer immunotherapy.

Altered Actin Centripetal Retrograde Flow in Physically Restricted Immunological Synapses

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3ε on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network.