An integrated workflow for phosphopeptide identification in natural killer cells (NK-92MI) and their targets (MDA-MB-231) during immunological synapse formation.

Here, we present a protocol to identify and quantify phosphopeptides during the dynamic formation of an immunological synapse. We describe steps for mixing isotope-labeled immune and target cells, the stabilization of cell-to-cell conjugates by cross-linking, and their isolation by fluorescence-activated cell sorting. We detail the isolation of phosphopeptides by phosphopeptide enrichment and their subsequent measurement by mass spectrometry. Finally, we describe the analysis of the resulting data to separate cell-specific phosphopeptides using the isotope label and label-free quantification.

B cell mechanosensing: A mechanistic overview.

B cells are essential to the adaptive immune system for providing the humoral immunity against cohorts of pathogens. The presentation of antigen to the B cell receptor (BCR) leads to the initiation of B cell activation, which is a process sensitive to the stiffness features of the substrates presenting the antigens. Mechanosensing of the B cells, potentiated through BCR signaling and the adhesion molecules, efficiently regulates B cell activation, proliferation and subsequent antibody responses. Defects in sensing of the antigen-presenting substrates can lead to the activation of autoreactive B cells in autoimmune diseases. The use of high-resolution, high-speed live-cell imaging along with the sophisticated biophysical materials, has uncovered the mechanisms underlying the initiation of B cell activation within seconds of its engagement with the antigen presenting substrates. In this chapter, we reviewed studies that have contributed to uncover the molecular mechanisms of B cell mechanosensing during the initiation of B cell activation.

DNA-Based Probes for Measuring Mechanical Forces in Cell-Cell Contacts: Application to B Cell Antigen Extraction from Immune Synapses.

The production of antibodies requires the expansion and selection of high-affinity B cell clones. This process is initiated by antigen uptake through the B cell receptor (BCR), which recognizes and binds antigen displayed on the surface of an antigen-presenting cell (APC). To acquire the antigen, B cells use myosin contractility to physically pull BCR-antigen clusters from the APC membrane. These mechanical forces influence association and dissociation rates of BCR-antigen bonds, resulting in affinity-dependent acquisition of antigen by B cells. Mechanical regulation of B cell antigen acquisition from APCs remains poorly understood, although the recent development of DNA-based force sensors has enabled the measurement of mechanical forces generated in B cell-APC contacts. In this chapter, we describe a protocol to design, synthesize, and purify DNA-based force sensors to measure B cell antigen extraction forces using fluorescence microscopy.

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.

Despite disorganized synapse structure, Th2 cells maintain directional delivery of CD40L to antigen-presenting B cells.

Upon recognition of peptide displayed on MHC molecules, Th1 and Th2 cells form distinct immunological synapse structures. Th1 cells have a bull's eye synapse structure with TCR/ MHC-peptide interactions occurring central to a ring of adhesion molecules, while Th2 cells have a multifocal synapse with small clusters of TCR/MHC interactions throughout the area of T cell/antigen-presenting cell interaction. In this study, we investigated whether this structural difference in the immunological synapse affects delivery of T cell help. The immunological synapse is thought to ensure antigen-specific delivery of cytolytic granules and killing of target cells by NK cells and cytolytic T cells. In helper T cells, it has been proposed that the immunological synapse may direct delivery of other effector molecules including cytokines. CD40 ligand (CD40L) is a membrane-bound cytokine essential for antigen-specific T cell help for B cells in the antibody response. We incubated Th1 and Th2 cells overnight with a mixture of antigen-presenting and bystander B cells, and the delivery of CD40L to B cells and subsequent B cell responses were compared. Despite distinct immunological synapse structures, Th1 and Th2 cell do not differ in their ability to deliver CD40L and T cell help in an antigen-specific fashion, or in their susceptibility to inhibition of help by a blocking anti-CD40L antibody.

Caveolin-1 Influences LFA-1 Redistribution upon TCR Stimulation in CD8 T Cells.

TCR stimulation by peptide-MHC complexes on APCs requires precise reorganization of molecules into the area of cellular contact to form an immunological synapse from where T cell signaling is initiated. Caveolin (Cav)1, a widely expressed transmembrane protein, is involved in the regulation of membrane composition, cellular polarity and trafficking, and the organization of signal transduction pathways. The presence of Cav1 protein in T cells was identified only recently, and its function in this context is not well understood. We show that Cav1-knockout CD8 T cells have a reduction in membrane cholesterol and sphingomyelin, and upon TCR triggering they exhibit altered morphology and polarity, with reduced effector function compared with Cav1 wild-type CD8 T cells. In particular, redistribution of the ß2 integrin LFA-1 to the immunological synapse is compromised in Cav1-knockout T cells, as is the ability of LFA-1 to form high-avidity interactions with ICAM-1. Our results identify a role for Cav1 in membrane organization and ß2 integrin function in primary CD8 T cells.

Plasma Membrane Sheets for Studies of B Cell Antigen Internalization from Immune Synapses.

Surrogate planar and membrane systems have been employed to study the architecture of immune synapses; however, they often do not recapitulate trans-synaptic extraction and endocytosis of ligands by the immune cells. Transendocytosis (or trogocytosis) of antigen from immune synapses is particularly critical for antigen processing and presentation by B cells. Here we describe a protocol for preparation of plasma membrane sheets (PMSs), which are flexible and fluid membrane substrates that support robust B cell antigen extraction. We show how to attach B cell antigens to the PMSs and how to investigate antigen extraction and endocytosis by fluorescent microscopy and computational image analysis. These techniques should be broadly applicable to studies of transendocytosis in a variety of cellular systems.

Förster Resonance Energy Transfer to Study TCR-pMHC Interactions in the Immunological Synapse.

T-cell antigen recognition is remarkably efficient: when scanning the surface of antigen-presenting cells (APCs), T-cells can detect the presence of just a few single antigenic peptide/MHCs (pMHCs), which are often vastly outnumbered by structurally similar non-stimulatory endogenous pMHCs (Irvine et al., Nature 419(6909):845-849, 2002; Purbhoo et al., Nat Immunol 5(5):524-530, 2004; Huang et al., Immunity 39(5):846-857, 2013). How T-cells achieve this is still enigmatic, in particular in view of the rather moderate affinity that TCRs typically exert for antigenic pMHCs, at least when measured in vitro (Davis et al., Ann Rev Immunol 16:523-544, 1998). To shed light on this in a comprehensive manner, we have developed a microscopy-based assay, which allows us to quantitate TCR-pMHC interactions in situ, i.e., within the special confines of the nascent immunological synapse of a T-cell contacting a planar-supported lipid bilayer functionalized with the costimulatory molecule B7-1, the adhesion molecule ICAM-1, and pMHCs (Huppa et al., Nature 463(7283):963-967, 2010) (Fig. 1). Binding measurements are based on Förster resonance energy transfer (FRET) between site-specifically labeled pMHCs and TCRs, which are decorated with recombinant site-specifically labeled single-chain antibody fragments (scFV) derived from the TCRß-reactive H57-597 antibody (Huppa et al., Nature 463(7283):963-967, 2010). FRET, a quantum-mechanical phenomenon, involves the non-radiative coupling of dipole moments of two adjacent fluorophores, a donor molecule and an acceptor molecule. FRET efficiency is inversely proportional to the sixth power of the inter-dye distance. Hence, it can be employed as a molecular ruler (Stryer and Haugland, Proc Natl Acad Sci, USA 58(2):719-726, 1967) or, as is the case here, to score for interactions of appropriately labeled molecules. To facilitate both quantitative and single-molecule readout, it is important to conjugate donor and acceptor dyes in a site-specific manner.While SLBs mimic some but certainly not all properties of a plasma membrane of a living cell, their use features a number of operational advantages: SLBs can be prepared in a fluid state, thereby facilitating the spatial rearrangements that accompany the formation of an immunological synapse (Grakoui et al., Science 285(5425):221-227, 1999). The imaging of a three-dimensional binding process is reduced to two dimensions, which saves time and fluorophore-emitted photons and allows for fast measurements. Furthermore, images can be acquired in noise-attenuated total internal reflection (TIR) mode, so far a necessity for single-molecule detection within the immunological synapse. Importantly, the stimulatory potency of pMHCs is very well preserved compared to cell surface-embedded pMHCs. Hence, while in principle artificial, SLBs are still a good approximation of the physiologic scenario a T-cell encounters when approaching an APC. Vice versa, the reconstitutive approach offers unique opportunities to interrogate the influence of accessory molecules on T-cell antigen recognition in a highly quantitative manner.In this chapter we will provide recommendations for the production of proteins used for SLB decoration as well as hands-on protocols for the production of SLBs. We will describe in detail how to perform and analyze FRET-based experiments to determine synaptic binding constants. In the "Notes" section, we will provide some information regarding the microscope setup as well as the mathematical and biophysical foundation underlying data analysis.

Revealing the Role of Microscale Architecture in Immune Synapse Function Through Surface Micropatterning.

The immune synapse has emerged as a compelling example of structural complexity within cell-cell interfaces. This chapter focuses on the use of microcontact printing to isolate and investigate how spatial organization of signaling molecules drives the function of immune cells. In the process detailed here, multiple rounds of microcontact printing are combined to create patterned surfaces that control the relative spatial localization of CD3 and CD28 signaling in T cells, effectively replacing an antigen presenting cell with an engineered surface. A set of approaches used to address key issues of T cell activation are described and discussed.

Probing Synaptic Biomechanics Using Micropillar Arrays.

Recent insights into the importance of mechanosensing and force transmission at the immune synapse have spurred increased interest in the mechanical properties of leukocyte cell-cell interactions. In this chapter, we describe an imaging-based strategy for measuring cellular forces that utilizes optically transparent arrays of flexible micropillars. This approach has several distinct advantages over standard traction force microscopy, and we anticipate that it will prove very useful for investigators who wish not only to quantify ligand-induced forces with high spatiotemporal resolution but also to place those forces within the context of a broader cell biological response.

Comprehensive Analysis of Immunological Synapse Phenotypes Using Supported Lipid Bilayers.

Supported lipid bilayers (SLB) formed on glass substrates have been a useful tool for study of immune cell signaling since the early 1980s. The mobility of lipid-anchored proteins in the system, first described for antibodies binding to synthetic phospholipid head groups, allows for the measurement of two-dimensional binding reactions and signaling processes in a single imaging plane over time or for fixed samples. The fragility of SLB and the challenges of building and validating individual substrates limit most experimenters to ~10 samples per day, perhaps increasing this few-fold when examining fixed samples. Successful experiments might then require further days to fully analyze. We present methods for automation of many steps in SLB formation, imaging in 96-well glass bottom plates, and analysis that enables >100-fold increase in throughput for fixed samples and wide-field fluorescence. This increased throughput will allow better coverage of relevant parameters and more comprehensive analysis of aspects of the immunological synapse that are well reconstituted by SLB.

L-plastin regulates the stability of the immune synapse of naive and effector T-cells.

T-cells need to be tightly regulated during their activation and effector phase to assure an appropriate defence against cancer or pathogens and - vice versa - to avoid autoimmune reactions. Regulatory signals are provided via the immune synapse between T-cells and antigen-presenting cells (APCs) or target cells. The stability and kinetics of immune synapse formation is critical for proper T-cell functions. It requires dynamic rearrangements of the actin cytoskeleton necessary for organized spatio-temporal redistribution of receptors and adhesion molecules. We identified glucocorticoid-sensitive phosphorylation of serine 5 on the actin-bundling protein L-plastin as one important signalling event for this regulation. Using imaging flow cytometry as well as confocal and super-resolution microscopy we showed that L-plastin relocalizes to the immune synapse upon antigen encounter, where it associates with the ß2-subunit of LFA-1 (CD11a/CD18). Interfering with L-plastin expression or activation leads to a defective LFA-1 recruitment and unstable T-cell/APC contacts. Consequently, the lack of L-plastin diminishes T-cell activation, proliferation and proximal effector responses such as cytokine production. On the other hand, a pro-oxidative milieu leads to prolonged activation of L-plastin resulting in a stronger enrichment of LFA-1 in the cytolytic immune synapse. Concomitant stabilization of conjugates formed by cytotoxic T-cells (CTLs) and their target cells impairs the ability of CTLs to kill more than one target cells (serial killing), which de facto leads to a downregulation of T-cell cytotoxicity. Together, we demonstrate that activation and spacial distribution of L-plastin regulates the maturation and stability of activating and cytolytic immune synapses important for T-cell activation and effector functions.

Geometry Dynamics of α -Helices in Different Class I Major Histocompatibility Complexes.

MHC α-helices form the antigen-binding cleft and are of particular interest for immunological reactions. To monitor these helices in molecular dynamics simulations, we applied a parsimonious fragment-fitting method to trace the axes of the α-helices. Each resulting axis was fitted by polynomials in a least-squares sense and the curvature integral was computed. To find the appropriate polynomial degree, the method was tested on two artificially modelled helices, one performing a bending movement and another a hinge movement. We found that second-order polynomials retrieve predefined parameters of helical motion with minimal relative error. From MD simulations we selected those parts of α-helices that were stable and also close to the TCR/MHC interface. We monitored the curvature integral, generated a ruled surface between the two MHC α-helices, and computed interhelical area and surface torsion, as they changed over time. We found that MHC α-helices undergo rapid but small changes in conformation. The curvature integral of helices proved to be a sensitive measure, which was closely related to changes in shape over time as confirmed by RMSD analysis. We speculate that small changes in the conformation of individual MHC α-helices are part of the intrinsic dynamics induced by engagement with the TCR.

Neonatal regulatory T cells have reduced capacity to suppress dendritic cell function.

Regulatory T cells (Treg cells) limit contact between dendritic cells (DCs) and conventional T cells (Tcons), decreasing the formation of aggregates as well as down-modulating the expression of co-stimulatory molecules by DCs, thus decreasing DC immunogenicity and abrogating T-cell activation. Despite the importance of this Treg-cell function, the capacity of Treg cells from term and preterm neonates to suppress DCs, and the suppressive mechanisms they use, are still undefined. We found that, relative to adult Treg cells, activated Treg cells from human neonates expressed lower FOXP3 and CTLA-4, but contained higher levels of cAMP. We developed an in vitro model in which Treg function was measured at a physiological ratio of 1 Treg for 10 Tcon and 1 monocyte-derived DC, as Treg target. Term and preterm Treg cells failed to suppress the formation of DC-Tcon aggregates, in contrast to naïve and memory Treg cells from adults. However, neonatal Treg cells diminished DC and Tcon activation as well as actin polymerization at the immunological synapses. In addition, CTLA-4 and cAMP were the main suppressive molecules used by neonatal Treg. Altogether, both preterm and term neonatal Treg cells appear less functional than adult Treg cells, and this defect is consistent with the general impairment of CD4 cell function in newborns.

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.

The synaptic recruitment of lipid rafts is dependent on CD19-PI3K module and cytoskeleton remodeling molecules.

Sphingolipid- and cholesterol-rich lipid raft microdomains are important in the initiation of BCR signaling. Although it is known that lipid rafts promote the coclustering of BCR and Lyn kinase microclusters within the B cell IS, the molecular mechanism of the recruitment of lipid rafts into the B cell IS is not understood completely. Here, we report that the synaptic recruitment of lipid rafts is dependent on the cytoskeleton-remodeling proteins, RhoA and Vav. Such an event is also efficiently regulated by motor proteins, myosin IIA and dynein. Further evidence suggests the synaptic recruitment of lipid rafts is, by principle, an event triggered by BCR signaling molecules and second messenger molecules. BCR-activating coreceptor CD19 potently enhances such an event depending on its cytoplasmic Tyr421 and Tyr482 residues. The enhancing function of the CD19-PI3K module in synaptic recruitment of lipid rafts is also confirmed in human peripheral blood B cells. Thus, these results improve our understanding of the molecular mechanism of the recruitment of lipid raft microdomains in B cell IS.

Three-Dimensional Gradients of Cytokine Signaling between T Cells.

Immune responses are regulated by diffusible mediators, the cytokines, which act at sub-nanomolar concentrations. The spatial range of cytokine communication is a crucial, yet poorly understood, functional property. Both containment of cytokine action in narrow junctions between immune cells (immunological synapses) and global signaling throughout entire lymph nodes have been proposed, but the conditions under which they might occur are not clear. Here we analyze spatially three-dimensional reaction-diffusion models for the dynamics of cytokine signaling at two successive scales: in immunological synapses and in dense multicellular environments. For realistic parameter values, we observe local spatial gradients, with the cytokine concentration around secreting cells decaying sharply across only a few cell diameters. Focusing on the well-characterized T-cell cytokine interleukin-2, we show how cytokine secretion and competitive uptake determine this signaling range. Uptake is shaped locally by the geometry of the immunological synapse. However, even for narrow synapses, which favor intrasynaptic cytokine consumption, escape fluxes into the extrasynaptic space are expected to be substantial (≥20% of secretion). Hence paracrine signaling will generally extend beyond the synapse but can be limited to cellular microenvironments through uptake by target cells or strong competitors, such as regulatory T cells. By contrast, long-range cytokine signaling requires a high density of cytokine producers or weak consumption (e.g., by sparsely distributed target cells). Thus in a physiological setting, cytokine gradients between cells, and not bulk-phase concentrations, are crucial for cell-to-cell communication, emphasizing the need for spatially resolved data on cytokine signaling.

Nanodomains in biological membranes.

Lipid rafts are defined as cholesterol- and sphingomyelin-enriched membrane domains in the plasma membrane of cells that are highly dynamic and cannot be resolved with conventional light microscopy. Membrane proteins that are embedded in the phospholipid matrix can be grouped into raft and non-raft proteins based on their association with detergent-resistant membranes in biochemical assays. Selective lipid-protein interactions not only produce heterogeneity in the membrane, but also cause the spatial compartmentalization of membrane reactions. It has been proposed that lipid rafts function as platforms during cell signalling transduction processes such as T-cell activation (see Chapter 13 (pages 165-175)). It has been proposed that raft association co-localizes specific signalling proteins that may yield the formation of the observed signalling microclusters at the immunological synapses. However, because of the nanometre size and high dynamics of lipid rafts, direct observations have been technically challenging, leading to an ongoing discussion of the lipid raft model and its alternatives. Recent developments in fluorescence imaging techniques have provided new opportunities to investigate the organization of cell membranes with unprecedented spatial resolution. In this chapter, we describe the concept of the lipid raft and alternative models and how new imaging technologies have advanced these concepts.

Kupfer-type immunological synapses in vivo: Raison D'être of SMAC.

T cells engage with antigen-presenting cells to form immunological synapses. These intimate contacts are characterized by the complex arrangement of molecules at the intercellular interface, which has been described as the supramolecular activation cluster (SMAC). However, due to T cells functioning without SMAC formation and the difficulties of studying these complex arrangements in vivo, its biological importance has been questioned. In light of recent data, we focus this review on the putative functionality of SMACs in T-cell synaptic contacts in vivo and emphasize the therapeutic potential of SMAC manipulation in immune-driven diseases.

The bullseye synapse formed between CD4+ T-cell and staphylococcal enterotoxin B-pulsed dendritic cell is a suppressive synapse in T-cell response.

The immunological synapse (IS) is a supermolecular activation cluster formed between T cells and antigen-presenting cells. Although diverse IS structures have been reported, the function of the IS in T-cell activation remains unclear. Here, we found that the bullseye IS, one of IS types at the interface of CD4(+) T cells and staphylococcal enterotoxin B-pulsed dendritic cells, suppressed CD4(+) T-cell activation, whereas multifocal IS, another synapse type, stimulated CD4(+) T-cell activation. Consistent with these results, bullseye IS formation was accompanied by a low-level calcium response in T cells and a loss of T-cell receptor signalling molecules from the synapse, whereas multifocal IS exhibited the opposite. Furthermore, we found that CD4(+)CD25(+) regulatory T cells (T(regs)) more efficiently formed bullseye IS and promoted bullseye IS formation in CD4(+) CD25(-) T cells. Cytotoxic T-lymphocyte antigen-4 (CTLA-4), an inhibitory molecule expressed continuously on T(regs), was localised in bullseye IS. Moreover, blocking CTLA-4 reduced the percentage of bullseye IS formation and promoted T-cell activation. Our data thus indicate that bullseye IS formation is mediated by CTLA-4, and may negatively control T-cell activation as a suppressive synapse.