Impact of Plasma Membrane Domains on IgG Fc Receptor Function.

Lipid cell membranes not only represent the physical boundaries of cells. They also actively participate in many cellular processes. This contribution is facilitated by highly complex mixtures of different lipids and incorporation of various membrane proteins. One group of membrane-associated receptors are Fc receptors (FcRs). These cell-surface receptors are crucial for the activity of most immune cells as they bind immunoglobulins such as immunoglobulin G (IgG). Based on distinct mechanisms of IgG binding, two classes of Fc receptors are now recognized: the canonical type I FcγRs and select C-type lectin receptors newly referred to as type II FcRs. Upon IgG immune complex induced cross-linking, these receptors are known to induce a multitude of cellular effector responses in a cell-type dependent manner, including internalization, antigen processing, and presentation as well as production of cytokines. The response is also determined by specific intracellular signaling domains, allowing FcRs to either positively or negatively modulate immune cell activity. Expression of cell-type specific combinations and numbers of receptors therefore ultimately sets a threshold for induction of effector responses. Mechanistically, receptor cross-linking and localization to lipid rafts, i.e., organized membrane microdomains enriched in intracellular signaling proteins, were proposed as major determinants of initial FcR activation. Given that immune cell membranes might also vary in their lipid compositions, it is reasonable to speculate, that the cell membrane and especially lipid rafts serve as an additional regulator of FcR activity. In this article, we aim to summarize the current knowledge on the interplay of lipid rafts and IgG binding FcRs with a focus on the plasma membrane composition and receptor localization in immune cells, the proposed mechanisms underlying this localization and consequences for FcR function with respect to their immunoregulatory capacity.

Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state.

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

Bacterial membrane vesicles as promising vaccine candidates.

Both Gram-positive and Gram-negative bacteria can release nano-sized lipid bilayered structures, known as membrane vesicles (MVs). These MVs play an important role in bacterial survival by orchestrating interactions between bacteria and between bacteria and host. The major constituents of MVs are proteins, lipids and nucleic acids. Due to the immunogenicity of the membrane lipids and/or proteins of the MVs, in combination with adjuvant danger signals and the repeating patterns on the nanosized surface, MVs can effectively stimulate the innate and adaptive immune system. Since they are non-replicating, they are safer than attenuated vaccines. In addition, by genetic engineering of the donor cells, further improvements to their safety profile, immunogenicity and yield can be achieved. To date, one MV-based vaccine against Neisseria meningitidis (N. meningitidis) serogroup B was approved. Other (engineered) MVs in the pipeline study are mostly in the preclinical phase.

Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses.

A missense C1858T single nucleotide polymorphism within PTPN22 is a strong genetic risk factor for the development of multiple autoimmune diseases. PTPN22 encodes a protein tyrosine phosphatase that negatively regulates immuno-receptor proximal Src and Syk family kinases. Notably, PTPN22 negatively regulates kinases downstream of T-cell receptor (TCR) and LFA-1, thereby setting thresholds for T-cell activation. Alterations to the quality of TCR and LFA-1 engagement at the immune synapse and the regulation of downstream signals can have profound effects on the type of effector T-cell response induced. Here we describe how IFNγ+ Th1 responses are potentiated in Ptpn22-/- T-cells and in T-cells from mice expressing Ptpn22R619W (the mouse orthologue of the human genetic variant) as they age, or following repeated immune challenge, and explore the mechanisms contributing to the expansion of Th1 cells. Specifically, we uncover two LFA-1-ICAM dependent mechanisms; one T-cell intrinsic, and one T-cell extrinsic. Firstly, we found that in vitro anti-CD3/LFA-1 induced Th1 responses were enhanced in Ptpn22-/- T-cells compared to WT, whereas anti-CD3/anti-CD28 induced IFNy responses were similar. These data were associated with an enhanced ability of Ptpn22-/- T-cells to engage ICAM-1 at the immune synapse when incubated on planar lipid bilayers, and to form conjugates with dendritic cells. Secondly, we observed a T-cell extrinsic mechanism whereby repeated stimulation of WT OT-II T-cells with LPS and OVA323-339 pulsed Ptpn22-/- bone marrow derived dendritic cells (BMDCs) was sufficient to enhance Th1 cell development compared to WT BMDCs. Furthermore, this response could be reversed by LFA-1 blockade. Our data point to two related but distinct mechanisms by which PTPN22 regulates LFA-1 dependent signals to enhance Th1 development, highlighting how perturbations to PTPN22 function over time to regulate the balance of the immune response.

Pneumolysin-damaged cells benefit from non-homogeneous toxin binding to cholesterol-rich membrane domains.

Nucleated cells eliminate lesions induced by bacterial pore-forming toxins, such as pneumolysin via shedding patches of damaged plasmalemma into the extracellular milieu. Recently, we have shown that the majority of shed pneumolysin is present in the form of inactive pre-pores. This finding is surprising considering that shedding is triggered by Ca2+-influx following membrane perforation and therefore is expected to positively discriminate for active pores versus inactive pre-pores. Here we provide evidence for the existence of plasmalemmal domains that are able to attract pneumolysin at high local concentrations. Within such a domain an immediate plasmalemmal perforation induced by a small number of pneumolysin pores would be capable of triggering the elimination of a large number of not yet active pre-pores/monomers and thus pre-empt more frequent and perilous perforation events. Our findings provide further insights into the functioning of the cellular repair machinery which benefits from an inhomogeneous plasmalemmal distribution of pneumolysin.

Understanding of B Cell Receptor Signaling Through a Photo-Activatable Antigen Presentation System.

Antibody responses are initiated by the binding of antigens to cell surface expressed B cell receptors (BCRs) that trigger signaling cascades resulting in the activation of B cells. However, it has been difficult to study these cascades due to their fast, dynamic, and transient nature. Using a conventional antigen-presenting system, such as planar lipid bilayers (PLBs), the initial events during B cell activation have been difficult to be captured. Here, we describe the general procedures for the utilization of a photoactivatable antigen-presenting system in the combination with a high speed live cell imaging method to investigate the early activation events in the same B cells in response to antigen stimulation.

Scaffolds that mimic antigen-presenting cells enable ex vivo expansion of primary T cells.

Therapeutic ex vivo T-cell expansion is limited by low rates and T-cell products of limited functionality. Here we describe a system that mimics natural antigen-presenting cells (APCs) and consists of a fluid lipid bilayer supported by mesoporous silica micro-rods. The lipid bilayer presents membrane-bound cues for T-cell receptor stimulation and costimulation, while the micro-rods enable sustained release of soluble paracrine cues. Using anti-CD3, anti-CD28, and interleukin-2, we show that the APC-mimetic scaffolds (APC-ms) promote two- to tenfold greater polyclonal expansion of primary mouse and human T cells compared with commercial expansion beads (Dynabeads). The efficiency of expansion depends on the density of stimulatory cues and the amount of material in the starting culture. Following a single stimulation, APC-ms enables antigen-specific expansion of rare cytotoxic T-cell subpopulations at a greater magnitude than autologous monocyte-derived dendritic cells after 2 weeks. APC-ms support over fivefold greater expansion of restimulated CD19 CAR-T cells than Dynabeads, with similar efficacy in a xenograft lymphoma model.

Human in vitro-induced regulatory T cells display Dlgh1dependent and PKC-θ restrained suppressive activity.

In vitro induced human regulatory T cells (iTregs) have demonstrated in vivo therapeutic utility, but pathways regulating their function have not been elucidated. Here, we report that human iTregs generated in vitro from naïve cord blood cells preferentially recruit Disc large homolog 1 (Dlgh1) and exclude protein kinase C (PKC)-θ from immunological synapses formed on supported lipid bilayers with laterally mobile ICAM-1 and anti-CD3 mAb. Also, iTregs display elevated Dlgh1 overall and Dlgh1-dependent p38 phosphorylation, higher levels of phosphatase and tensin homolog (PTEN), and diminished Akt phosphorylation. Pharmacological interruption of PKC-θ increases and Dlgh1 silencing decreases the ability of iTregs to suppress interferon-γ production by CD4+CD25- effector T cells (Teff). Comparison with expanded cord blood-derived CD4+CD25hi tTreg and expanded Teffs from the same donors indicate that iTreg are intermediate between expanded CD4+CD25hi tTregs and Teffs, whereas modulation of suppressive activities by PKC-θ and Dlgh1 signaling pathways are shared.

Reconstitution of TCR Signaling Using Supported Lipid Bilayers.

Biochemical reconstitution has served as an important tool for understanding the mechanisms of many cellular processes including DNA replication, transcription, translation, vesicle trafficking, and ubiquitin-mediated proteolysis. Here, we demonstrate that biochemical reconstitution can be applied to studying a complex signaling pathway involving as many as 12 proteins or protein complexes acting at the surface of model membranes. We show that a temporal sequence of events in activated T cells beginning with phosphorylation of the T cell receptor and culminating in the activation of actin polymerization can be replicated in vitro. Our reconstitution demonstrates the sufficiency of these proteins in producing many of the complex behaviors observed during T cell activation. The ability to manipulate all of the components, measure reaction rates, and observe molecular behaviors, including at single molecule resolution, has enabled us to gain insight into some of the important biochemical features of this signaling pathway such as microcluster formation. The same system could be adapted to study other membrane-proximal signaling pathways, including growth factor receptors, death receptors, and Eph receptors.

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.

Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs.

The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context.

Elastohydrodynamics and Kinetics of Protein Patterning in the Immunological Synapse.

We propose a minimal mathematical model for the physical basis of membrane protein patterning in the immunological synapse (IS), which encompass membrane mechanics, protein binding kinetics and motion, and fluid flow in the synaptic cleft. Our theory leads to simple predictions for the spatial and temporal scales of protein cluster formation, growth and arrest as a function of membrane stiffness, rigidity and kinetics of the adhesive proteins, and the fluid flow in the synaptic cleft. Numerical simulations complement these scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment shows that passive elastohydrodynamics and kinetics of protein binding in the synaptic cleft can describe the short-time formation and organization of protein clusters, without evoking any active processes in the cytoskeleton. Despite the apparent complexity of the process, our analysis shows that just two dimensionless parameters characterize the spatial and temporal evolution of the protein pattern: a ratio of membrane elasticity to protein stiffness, and the ratio of a hydrodynamic time scale for fluid flow relative to the protein binding rate. A simple phase diagram encompasses the variety of patterns that can arise.

Super-resolution imaging of the natural killer cell immunological synapse on a glass-supported planar lipid bilayer.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells ('APCs') referred to as 'immunological synapses'. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of 'semi-professional APCs'. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic 'planar cellular APC model' for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Skin barrier defects in atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin condition with complex etiology that is dependent upon interactions between the host and the environment. Acute skin lesions exhibit the features of a Th2-driven inflammatory disorder, and many patients are highly atopic. The skin barrier plays key roles in immune surveillance and homeostasis, and in preventing penetration of microbial products and allergens. Defects that compromise the structural integrity or else the immune function of the skin barrier play a pivotal role in the pathogenesis of AD. This article provides an overview of the array of molecular building blocks that are essential to maintaining healthy skin. The basis for structural defects in the skin is discussed in relation to AD, with an emphasis on filaggrin and its genetic underpinnings. Aspects of innate immunity, including the role of antimicrobial peptides and proteases, are also discussed.

A new and robust method of tethering IgG surrogate antigens on lipid bilayer membranes to facilitate the TIRFM based live cell and single molecule imaging experiments.

Our understanding of cell-cell interactions has been significantly improved in the past years with the help of Total Internal Reflection Fluorescence Microscope (TIRFM) in combination with an antigen presenting system supported by planar lipid bilayer (PLB) membranes, which are used to mimic the extensive receptor and ligand interactions within cell-cell contact interface. In TIRFM experiments, it is a challenge to uniformly present ligand molecules in monomeric format on the surface of PLB membranes. Here, we introduce a new and robust method of tethering IgG surrogate antigen ligands on the surface of Ni(2+)-containing PLB membranes. In this method, we use a modified D domain from staphylococcal protein A molecule that is fused with an N-terminus polyhistidine tag (H12-D-domain) to tether IgG surrogate antigens on Ni(2+)-containing PLB membranes. We systematically assessed the specificity and capability of H12-D-domain construct to capture IgG molecules from different species through live cell and single molecule TIRFM imaging. We find that these IgG surrogate antigens tethered by H12-D-domain show better lateral mobility and are more uniformly distributed on PLB membranes than the ones tethered by streptavidin. Neither IgM molecules, nor Fab or F(ab')2 fragments of IgG molecules can be tethered on PLB membranes by H12-D-domain construct. These tethered IgG surrogate antigens strongly induce the formation and accumulation of signaling active antigen receptor microclusters within the immunological synapse in B or T lymphocyte cells. Thus our method provides a new and robust method to tether IgG surrogate antigens or other molecules fused with IgG Fc portion on PLB membranes for TIRFM based molecule imaging experiments.

Insights from in situ analysis of TCR-pMHC recognition: response of an interaction network.

Recognition of peptide presented by the major histocompatibility complex (pMHC) molecule by the T-cell receptor (TCR) determines T-cell selection, development, differentiation, fate, and function. Despite intensive studies on the structures, thermodynamic properties, kinetic rates, and affinities of TCR-pMHC interactions in the past two decades, questions regarding the functional outcome of these interactions, i.e. how binding of the αß TCR heterodimer with distinct pMHCs triggers different intracellular signals via the adjacent CD3 components to produce different T-cell responses, remain unclear. Most kinetic measurements have used surface plasmon resonance, a three-dimensional (3D) technique in which fluid-phase receptors and ligands are removed from their cellular environment. Recently, several two-dimensional (2D) techniques have been developed to analyze molecular interactions on live T cells with pMHCs presented by surrogate antigen-presenting cells or supported planar lipid bilayers. The insights from these in situ analyses have provided a sharp contrast of the 2D network biology approach to the 3D reductionist approach and prompted rethinking of our current views of T-cell triggering. Based on these insights, we propose a mechanochemical coupled triggering hypothesis to explain why the in situ kinetic parameters differ so much from their 3D counterparts, yet correlate so much better with T-cell functional responses.

Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells.

Supported lipid bilayers are an important biomolecular tool for characterizing immunological synapses. Immobilized bilayers presenting tethered ligands on planar substrates have yielded both spatio-temporal and structural insights into how T cell receptors (TCRs) reorganize during the initial formation of synapses upon recognition of peptide antigens bound to major histocompatibility complex (MHC) molecules. The prototypical configuration of these assays, however, limits the extent to which the kinetics and structure of the supramolecular activation clusters of the synapse (that occur in seconds or minutes) can be related to subsequent complex cellular responses, such as cytokine secretion and proliferation, occurring over hours to days. Here we describe a new method that allows correlative measures of both attributes with single-cell resolution by using immobilized lipid bilayers and tethered ligands on the surface of dense arrays of subnanoliter wells. This modification allows each nanowell to function as an artificial antigen-presenting cell (APC), and the synapses formed upon contact can be imaged by fluorescence microscopy. We show that the lipid bilayers remain stable and mobile on the surface of the PDMS, and that modifying the ligands tethered to the bilayer alters the structure of the resulting synapses in expected ways. Finally, we demonstrate that this approach allows the subsequent characterization of secreted cytokines from the activated human T cell clones by microengraving in both antigen- and pan-specific manners. This new technique should allow detailed investigations on how biophysical and structural aspects of the synapse influence the activation of individual T cells and their complex functional responses.

Lepromatous leprosy patients produce antibodies that recognise non-bilayer lipid arrangements containing mycolic acids

Non-bilayer phospholipid arrangements are three-dimensional structures that form when anionic phospholipids with an intermediate structure of the tubular hexagonal phase II are present in a bilayer of lipids. Antibodies that recognise these arrangements have been described in patients with antiphospholipid syndrome and/or systemic lupus erythematosus and in those with preeclampsia; these antibodies have also been documented in an experimental murine model of lupus, in which they are associated with immunopathology. Here, we demonstrate the presence of antibodies against non-bilayer phospholipid arrangements containing mycolic acids in the sera of lepromatous leprosy (LL) patients, but not those of healthy volunteers. The presence of antibodies that recognise these non-bilayer lipid arrangements may contribute to the hypergammaglobulinaemia observed in LL patients. We also found IgM and IgG anti-cardiolipin antibodies in 77% of the patients. This positive correlation between the anti-mycolic-non-bilayer arrangements and anti-cardiolipin antibodies suggests that both types of antibodies are produced by a common mechanism, as was demonstrated in the experimental murine model of lupus, in which there was a correlation between the anti-non-bilayer phospholipid arrangements and anti-cardiolipin antibodies. Antibodies to non-bilayer lipid arrangements may represent a previously unrecognised pathogenic mechanism in LL and the detection of these antibodies may be a tool for the early diagnosis of LL patients.

The scaffolding protein synapse-associated protein 97 is required for enhanced signaling through isotype-switched IgG memory B cell receptors.

After their first encounter with a foreign antigen, naïve B cells that have immunoglobulin M (IgM) B cell receptors (BCRs) trigger the primary antibody response and the generation of memory B cells with IgG BCRs. When these memory B cells reencounter the same antigen, the cell surface IgG BCRs stimulate their rapid differentiation into plasma cells that release large amounts of IgG antibodies. We showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ (postsynaptic density 95/disc large/zona occludens 1)-binding motif, associated with synapse-associated protein 97 (SAP97), a PDZ domain-containing scaffolding molecule that is involved in controlling receptor density and signal strength at neuronal synapses. SAP97 accumulated and bound to IgG BCRs in the immunological synapses that formed in response to B cell engagement with antigen. Knocking down SAP97 in IgG⁺ B cells or mutating the putative PDZ-binding motif in the BCR tail impaired formation of the immunological synapse, initiation of IgG BCR signaling, and downstream activation of the mitogen-activated protein kinase p38. Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immunological synapse.