This article introduces a series of reviews covering Cell:Cell Contacts appearing in Volume 251 of Immunological Reviews.
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The cells of the immune system are continually on the move! Naive T cells in a human collectively crawl over 10 000 miles a day in search of antigens . Each dendritic cell (DC) touches 5000 T cells an hour to scan repertoires over a million deep [2, 3]. The adaptive immune system functions through a vast network of provisional cell–cell interactions that are continuously changing. When this frenetic scanning finds a fit between T-cell antigen receptor (TCR) and DC antigen, the interaction can be prolonged with a pronounced deceleration of the T cell and finally a long-lived interaction that has been described as an immunological synapse [2, 4]. In vitro studies on the immunological synapse suggest a profound supramolecular organization that serves focused trans-synaptic and juxtracrine information transfer between cells [5-8]. Immunological synapses are not restricted to T cells but are also formed by natural killer (NK) cells, B cells, granulocytes, and phagocytes using related immunotyrosine-based activation motif-containing receptors [9, 10]. These interactions are profoundly influenced by the three-dimensional (3D) tissue environment . This volume of Immunological Reviews takes a broader look at the cell–cell interactions in the immune system from the molecular organization of antigen receptors during surveillance to the organization of stromal cell networks in lymph nodes, bone marrow, and the lung (Fig. 1).
The organization of the plasma membrane is a fundamental issue in biology that sets the stage for cell–cell interaction and communication. The classical fluid mosaic model suggested membrane proteins diffusing in a lipid sea with potential for interactions with cytoplasmic and extra-cytoplasmic binding partners that account for regulation and processes like adhesion and receptor capping. Observations on lipid phases in model systems with disordered, liquid-ordered, and gel phases in order of increasing viscosity suggested that such phases could also exist in biological membranes on some scale . Biophysical observations of liquid-ordered domains enriched in various lipid-modified proteins have led to models in which lipids have a role in providing a higher level of organization . Most recently, evidence for protein-driven domains, referred to as tetraspannin domains and protein islands, have been presented with the suggestion that protein–protein interaction may dominate organization of biological membranes, with the lipid rafts being a subtype of protein island [14-16]. Articles from Schamel and Alarcón , Sherman et al. , and Purbhoo  explore issues of antigen receptor organization and how membrane structures, including cytoplasmic vesicles, set the stage for and modulate signaling.
Cell–cell interactions are mediated by receptor–ligand interactions at membrane interfaces. It has been argued that it is often hard to define receptors and ligands in this context, as both molecules can signal, leading to proposals to refer to receptors and counter-receptors. Regardless, this is a unique chemical environment, and despite our success in cataloging hundreds of cell surface receptors and dozens of relevant molecular interactions, the precise chemistry of these interactions has been difficult to quantify and model. Bell  generated a theoretical framework for interactions at cell–cell interfaces and postulated that interactions would have low affinity but could function due to operation in a confinement zone of approximately 1 nm in the interface between cells, such that the interactions would be highly efficient due to pre-positioning for binding and re-binding when released. Quantitative measurements of such two-dimensional (2D) affinities (units of molecules−1 μm2) were consistent with this theory for the CD2–CD58 and related adhesive interactions of the immune system [21-23]. These measurements were based on interaction of live cells with supported planar bilayers containing fluorescent ligands as a model for a cell–cell interface. The application of fluorescence recovery after photobleaching in this system suggested that the low-affinity interactions were short lived in the interface but that the highly ordered contact area might effectively favor relatively long interactions compared to interactions of the same receptor and ligand measured in solution [24-26]. Zhu et al.  and Xie et al.  focus in part on the different approaches to measuring 2D affinity and kinetic rates.
The approach described by Xie et al.  uses a single molecule fluorescence resonance energy transfer approach in the supported planar bilayer model and directly measures 2D off rates with exciting results. The 2D on rates and affinity estimates assume steady-state conditions and a model for receptor organization, such that these questions also become involved with issues discussed by Alarcon  and Davis . Like the CD2–CD58 system, the TCR seems to make up for low affinity in solution by taking advantage of pre-positioning in the immunological synapse. However, the TCR system is much more complex than the early measurement on CD2.
The approach to 2D affinity utilized by Zhu et al.  is completely different and based on capturing the earliest events in TCR interaction with major histocompatibility complex (MHC)–peptide complexes as single tethers at the interface between a live T cell and a swelled erythrocyte coated with MHC–peptide complexes. The red blood cell (RBC) is manipulated into a contact with the T cell of defined area and duration and then withdrawn to determine if a tether has formed with a digital outcome for each test. The MHC–peptide density is set empirically to yield ~50% probability of tether formation with short contacts of less than a second to a couple of seconds. This process is then scored based on an equilibrium model for bond formation in the environment of the T–RBC contact. The results from these analyses have been intriguing, as this measurement is the most successful to date in being able to use a kinetic parameter (the 2D on rate in this case) to rank MHC–peptide complexes by biological potency. Using an equilibrium model to assess an intermediate in forming a signaling cluster in a live T cell complicates the interpretation of these results. In fact, other studies with this system reveal complex time-dependent phenomena related to the role of co-receptors like CD8. This is an exciting area, and the reviews by Xie et al.  and Zhu et al.  provide important perspectives on the current state of the art.
The immune system provides outstanding models for studying cell biology of cell–cell interactions and cell polarization . There is a wealth of information about powerful signals that acutely control the polarization and activation of a variety of immune cell types with clearly defined functions. The only rub is that the cells are smaller than in the most popular cell biology model systems, and thus imaging can be a challenge. The Krummel  and Baldari  groups have risen to this challenge in different ways. Gérard et al.  present a discussion of immunological synapse dynamics and definitions in the context of a larger model for how sequential immunological synapses may be formed by naive T cells on the way to becoming an effector cell. The emphasis is on the necessity of transient and sequential interactions for differentiation to take place. The work builds on earlier concepts about immunological synapse periodicity  and extends this to consideration of T–T synapses , which is particularly attractive given that lymphocyte aggregates are correlates of successful activation of T cells in vitro.
Most mammalian cells have a hair-like projection on their surface, which is non-motile . This primary cilium works as a sensor that helps cells to react to environmental signals. This is achieved by a specialized mechanism that specifically delivers receptors responsible for performing this function at this location. This complex dedicated machinery, known as intraflagellar transport, has recently been identified in T cells, though they do not appear to be ciliated cells. Finett and Baldari  provide a new spin on the immunological synapse by relating it to the primary cilium of other polarized cells. They provide a model for the function of intraflagellar transport proteins in the formation and function of the immunological synapse. The theme is that immune cells have taken many elements that evolved earlier in polarized epithelial cells and have modified these pathways to suit the function of more mobile cells that communicate by provisional synaptic interactions. Finding the lymphocyte-specific modifications in these evolutionarily and more widely used pathways may provide a next generation of immunomodulatory drug candidates.
The classical communication media in the immunological synapse are the signals generated by the receptor–ligand interactions (e.g. TCR–pMHC, CD28–CD80, CD40L–CD40, etc.) and the secreted components such as perforins and granzymes from cytotoxic T lymphocytes and cytokines like interferon-γ from helper T cells. There are exciting new findings related to larger structures that can be transferred through the immunological synapse and related junctions. The first realization that large particles may also be transferred through the immunological synapse came from studies of viral spread. Human T-lymphotrophic virus-1 generates particles that cannot survive in the extracellular milieu and thus can only be spread between T cells through a virological synapse: a direct cell–cell transfer . It has been proposed that both the initial introduction of human immunodeficiency virus (HIV) from DCs to T cells and then spread from T cells to other T cells utilize a virological synapse . Dale et al.  review the literature on cell to cell spread of HIV and the structure of the virological synapse. In this area, it is still not clear how antigen recognition impacts HIV spread and vice versa. Recent work from the Sanchez-Madrid laboratory points to a role of exosomes in transfer of microRNAs from T cells to antigen-presenting B cells via the immunological synapse. Exosomes are small vesicles generated in late endosomes by action of the endosomal sorting complexes required for transport (ESCRT). Gutiérrez-Vázque et al.  present a comprehensive review of not only exosomes but also other types of microparticles and how they could be involved throughout the immune response. The ESCRT pathway appears to play a key role in the formation of the immunological synapse, and this connection is yet to be fully explored.
The parallel analysis of the immunological synapse in vitro and in vivo has been invaluable for our appreciation of the diversity of immune responses. Early studies on the T-cell immunological synapse suggested that in vitro conditions may be established to result in antigen recognition through stable immunological synapses  or highly motile interactions, which may be defined as kinapses [39, 40]. Early in vivo imaging provided a win-win answer, as T-cell priming in lymph nodes takes place through dynamic and stable phases . Parallel in vitro and in vivo imaging continues to be of great value, as in vitro systems still offer unparalleled resolution and contrast, whereas in vivo systems offer insight into cellular interactions and signaling in a more physiological setting. Huse et al.  and Deguine and Bousso  provide excellent examples of the value of in vitro and in vivo approaches applied to NK cell studies. Cell–cell interactions play an important role for these frontline immune effectors.
Huse and colleagues [43, 44] have made extensive use of photoactivation as a tool to dissect the order of events in T cells. Photoactivation can be applied to the rapid generation of new chemical signals (also called uncaging) and the generation of fluorescent signals to follow changes in localization over time. In this volume, Huse et al.  applies photoactivation of inhibitory ligands of NK cells and the disruption of the NK cell immunological synapse. This is a valuable approach in this context, because it allows the study of how the inhibitory ligands impact an existing NK cell immunological synapse, as described in the article.
While the greater focus of in vivo imaging approaches has initially been on B and T cells, as suggested by Deguine and Bousso , the lack of tracking tools has delayed NK cell studies until very recently. As discussed in their review, cell–cell interactions are important in every stage of NK cell life. They regulate NK cell survival, modulate their responsiveness in the periphery, and are involved in their activation. Given the dynamic nature of these interactions, they are mainly studied in vivo. Deguine and Bousso  review their recent understanding of NK cell interactions in vivo at steady state, during inflammation, and during tumor elimination, emphasizing the similarities and differences with T cells. Recent application of photoactivation methods in vivo may allow the approaches of Huse and Bousso to be combined to gain additional insights .
Tissue niches are critical to function of the immune system and establish environments for unique cell–cell interactions. The niches are defined largely by the most understudied cells of the immune system, the stromal cells that provide a scaffold for the tissues, but also much more . Three reviews focus on lymph node stroma, the bone marrow niches of plasma cells, and the unique characteristics of airways.
Cells of the immune system are able to respond to a large variety of pathogenic challenges. Secondary lymphoid organs (SLOs) have evolved to maximize the chances of lymphocytes encountering their cognate antigen as well as to find the right partners to mount an adequate immune response. SLOs are highly organized structures that establish the right collaborative environment for numerous cellular interactions to occur. Contractility of stromal cells may also provide a critical mechanical environment for immune cell interactions . Recent studies suggest that stromal cells also influence the chemical environment in ways that profoundly influence adaptive immune responses [47, 48]. Turley's group  has done pioneering work on this topic, as discussed in their review herein.
Cell–cell interactions are key in the generation of humoral response as well as maintaining memory . In recent years, progress has been made in understanding how B cells are activated in secondary lymphoid tissues. This is mediated by B-cell interaction with antigen-presenting cells, such as DCs, macrophages, and follicular DCs [51-53]. However, we still know very little about how B cells, once activated, are able to produce antibodies for years, in some cases for a lifetime. One possibility is that antigen-specific B cells are continuously differentiated into plasmablasts, which in turn continuously secrete antibodies. Another more likely possibility is that once activated, B cells differentiate into plasma cells that will live for many years. This raises interesting questions regarding what type of environment these plasma cells live in and what requirements they have to survive for decades. Part of the answer to this question relies on their survival niche in the bone marrow. Chu and Berek  discuss here what is known about the cell–cell interactions needed to support the long-term survival of plasma cells in the bone marrow. A particular emphasis is put on the role of eosinophils, which have been shown to be key providers of plasma cell survival factors. This novel example of cell–cell interaction is where attention will be focused on over the next few years.
The airway system is a highly vulnerable site both for microbial infection and immunopathology related to inappropriate immune responses to benign environmental antigens. Hasenberg et al.  provide an overview of the anatomy of airways and the unique physical and physiological issues that face the innate and adaptive immune cell–cell interaction networks. The alveoli are among the most fascinating microenvironments, displaying a remarkable combination of fragility and the crushing pressure of the surfactant layer.
We very much hope that readers will enjoy this collection of exciting articles on cell–cell interactions. We anticipate that the studies discussed here will inspire even more exciting new investigations.