Integrin inside-out signaling and the immunological synapse

Integrins dynamically equilibrate between three conformational states on cell surfaces. A bent conformation has a closed headpiece. Two extended conformations contain either a closed or an open headpiece. Headpiece opening involves hybrid domain swing-out and a 70 ? separation at the integrin knees, which is conveyed by allostery from the hybrid-proximal end of the βI domain to a 3 ? rearrangement of the ligand-binding site at the opposite end of the βI domain. Both bent-closed and extended-closed integrins have low affinity, whereas extended-open integrin affinity is 10(3) to 10(4) higher. Integrin-mediated adhesion requires the extended-open conformation, which in physiological contexts is stabilized by post-ligand binding events. Integrins thus discriminate between substrate-bound and soluble ligands. Analysis of LFA-1-ICAM-1 interactions in the immunological synapse suggests that bond lifetimes are on the order of seconds, which is consistent with high affinity interactions subjected to cytoskeletal forces that increase the dissociation rate. LFA-1 βI domain antagonists abrogate function in the immunological synapse, further supporting a critical role for high affinity LFA-1.     

Information transfer at the immunological synapse

Antigen-specific activation of T lymphocytes requires the interaction of their clonally distributed T-cell receptors with plasma membrane ligands composed of foreign peptide antigens bound to major histocompatibility complex molecules. For proliferation and differentiation to ensue, a variety of other adhesive and accessory proteins must also interact with their counter-receptors on the antigen-presenting cell to facilitate and complement the T-cell receptor-antigen recognition event. Recent studies have revealed that these various proteins show an unexpected degree of spatial organization in the zone of cell-cell contact. This region of membrane approximation is now referred to as the "immunological synapse" because of its functional analogy to the site of intercellular information transfer between neurons. Here, we review the evidence for signaling-dependent control of the dynamic changes in protein distribution that gives rise to the synapse and try to relate the emerging spatio-temporal information on synapse formation to T-cell biology.     

Communication,the centrosome and the immunological synapse

Recent findings on the behaviour of the centrosome at the immunological synapse suggest a critical role for centrosome polarization in controlling the communication between immune cells required to generate an effective immune response. The features observed at the immunological synapse show parallels to centrosome (basal body) polarization seen in cilia and flagella, and the cellular communication that is now known to occur at all of these sites.     

Spatial mutation of the T cell immunological synapse

The hardware for intracellular signaling networks consists of cascades of chemical reactions. It is becoming increasingly apparent that the large-scale spatial organization of molecules in these networks can lead to differential outcomes from otherwise chemically equivalent systems. This has amplified interest in controlled spatial organization as a regulator of cellular signal transduction. In response, a new category of experimentation is developing, in which the spatial positions of signaling molecules in living cells are directly manipulated through mechanical means. These methodologies complement conventional genetic and pharmacological approaches, both of which are chemical in nature, by perturbing the system through exclusively physical mechanisms.     

Diversity in immune-cell interactions: states and functions of the immunological synapse

The contact-dependent exchange of signals between epithelial and neuronal cells results from close membrane-membrane appositions, which are stabilized for years by polarized adhesion, cytoskeletal assemblies and extracellular scaffold proteins. By contrast, owing to a lack of scaffold proteins, interactions between immune cells such as T lymphocytes and antigen-presenting cells (APCs) comprise a spectrum of structurally diverse and short-lived interaction modes that last from minutes to hours. Signals exchanged between T cells and APCs are generated in a specific contact region, termed the "immunological synapse", that coordinates cytoskeletal dynamics with the T-cell receptor (TCR), the engagement of accessory receptors and membrane-proximal signaling. Recent data shed light on the different physical and molecular interaction modes that occur between T cells and APCs, including their dynamics and transition stages, and their consequences for signaling, activation and T-cell effector function.     

HIV-1 at the immunological and T-lymphocytic virological synapse

Cell-cell transmission of human immunodeficiency virus type 1 (HIV-1) is considered the most effective mode of viral spread in T-lymphocyte cultures. Evidence has accumulated that HIV-1 assembles polarized synaptic-like structures, referred to as virological synapses, as specialized sites of viral transfer. Interestingly, it was recently also discovered that HIV-1 impairs the formation of the structurally similar immunological synapse, thereby modulating exogenous T-lymphocyte stimulation to yield an optimal activation state for productive HIV-1 infection. The careful dissection of these opposing effects will contribute to our understanding of retroviral spread and cellular signal transduction machineries.     

CD28 signals in the immature immunological synapse

T cell recognition of peptide-MHC complexes on APCs results in the aggregation of TCRs at a central supramolecular activation complex (c-SMAC) within a mature immunological synapse. T cells require a second "costimulatory" signal for activation, the most important of which, for naive T cells, is from CD28. However the time at which CD28-derived signals are induced relative to c-SMAC formation is not well understood. In this study, we have assessed the kinetics of CD28 localization and function relative to well-established aspects of c-SMAC formation. CD28 accumulates at the immature synapse alongside the TCR and is likewise enriched at the synapse at the onset of the calcium signal. In addition, using CD28 deficient or reconstituted murine cells in a single-cell recording approach shows that CD28 regulates this signal within seconds of a TCR-mediated rise in intracellular calcium levels. Finally, CD28 exerts effects on both the initiation and stabilization of the synapse in parallel with its effects on the downstream proliferation of T cells. Together, the data show that CD28 functions in the immunological synapse before the formation of the c-SMAC.     

Intercellular transfer of oncogenic H-Ras at the immunological synapse

Immune cells establish dynamic adhesive cell-cell interactions at a specific contact region, termed the immunological synapse (IS). Intriguing features of the IS are the formation of regions of plasma membrane fusion and the intercellular exchange of membrane fragments between the conjugated cells. It is not known whether upon IS formation, intact intracellular proteins can transfer from target cells to lymphocytes to allow the transmission of signals across cell boundaries. Here we show by both FACS and confocal microscopy that human lymphocytes acquire from the cells they scan the inner-membrane protein H-Ras, a G-protein vital for common lymphocyte functions and a prominent participant in human cancer. The transfer was cell contact-dependent and occurred in the context of cell-conjugate formation. Moreover, the acquisition of oncogenic H-RasG12V by natural killer (NK) and T lymphocytes had important biological functions in the adopting lymphocytes: the transferred H-RasG12V induced ERK phosphorylation, increased interferon-gamma and tumor necrosis factor-alpha secretion, enhanced lymphocyte proliferation, and augmented NK-mediated target cell killing. Our findings reveal a novel mode of cell-to-cell communication-allowing lymphocytes to extend the confines of their own proteome-which may moreover play an important role in natural tumor immunity.     

Cutting edge: quantitative imaging of raft accumulation in the immunological synapse

Although the accumulation of lipid rafts at the immunological synapse is now well accepted, the degree of the accumulation, the localization within the fine structure of the immunological synapse, and the region from which lipid rafts are recruited have not been defined. In this work we show that lipid rafts preferentially accumulate in the central zone of the immunological synapse, the central supramolecular activation complex (C-SMAC). However, quantitative analyses indicate that the level of recruitment of lipid rafts to the C-SMAC is relatively small and suggests that rearrangement of lipid rafts from the peripheral zone of the synapse into the C-SMAC can account for this accumulation. We also assessed the effects of CD28 deficiency on lipid raft recruitment to the immunological synapse. The accumulation of lipid occurred independently of the CD28/B7 system and was not measurably altered by CD28.     

Modulation of immunological synapse by membrane-bound and soluble ligands

An efficient adaptive immune response should prevent pathogen infections and tumor growth without causing significant damage to host constituents. A crucial event determining the balance between tolerance and immunity is antigen recognition by T cells on the surface of antigen presenting cells (APC). Several molecular contacts at the interface between T cells and APCs contribute to define the nature of the adaptive immune response against a particular antigen. Upon TCR engagement by a peptide-MHC complex (pMHC) on the surface of an APC, a specialized supra-molecular structure known as immunological synapse (IS) assembles at the interface between these two cells. This structure involves massive re-distribution of membrane proteins, including TCR and pMHC complexes, as well as co-stimulatory and adhesion molecules. Furthermore, IS assembly leads to several important intracellular events necessary for T cell activation, such as recruitment of signaling molecules and cytoskeleton rearrangements. Because IS assembly leads to major consequences on the function of T cells, several studies have attempted to identify both soluble and membrane-bound molecules that could contribute to modulate the IS function. Here we describe recent literature on the regulation of IS assembly and modulation by TCR/pMHC binding kinetics, chemokines and cytokines focusing on their role at controlling the balance between adaptive immunity and tolerance.     

Reciprocal polarization of T and B cells at the immunological synapse

Cognate interactions between T and B lymphocytes lead to the formation of the immunological synapse (IS) where bidirectional activation signals are exchanged. Although the molecular architecture and the function of the IS have been studied extensively on the T cell side, little is known about events occurring during synapse formation in Ag-presenting B cells. We investigated the impact of BCR and TLR signaling on human B cell activation and on the T and B cell side of the IS. On the T cell side, we observed that T cells polarized toward both naive and previously activated B cells. Nevertheless, when T cells interacted with different B cells simultaneously, T cells selectively polarized their secretory machinery toward preactivated B cells. Furthermore, both naive and preactivated B cells reoriented their microtubule-organizing center toward the synaptic T cell during cognate interactions. This phenomenon was rapid and not dependent on T cell secretory activity. Interestingly, not only the microtubule-organizing center but also the Golgi apparatus and Lamp-3(+) and MHC class II(+) vesicles all repositioned beneath the IS, suggesting that the entire endocytic/exocytic B cell compartment was reoriented toward the T cell. Taken together, our results show that the B cell activation status fine-tunes T cell polarization responses and reveal the capacity of naive and activated B cells to polarize toward T cells during cognate interactions.     

Live-cell dynamics and the role of costimulation in immunological synapse formation

Using transfected fibroblasts expressing both wild-type I-E(k) and green fluorescent protein-tagged I-E(k) with covalently attached antigenic peptide, we have monitored movement of specific MHC:peptide complexes during CD4(+) T cell-APC interactions by live-cell video microscopy. Ag recognition occurs within 30 s of T cell-APC contact, as shown by a sharp increase in cytoplasmic calcium ion concentration. Within 1 min, small MHC:peptide clusters form in the contact zone that coalesce into an immunological synapse over 3-20 min. When T cells conjugated to APC move across the APC surface, they appear to drag the synapse with them. This system was used to examine the role of costimulation in the formation of the immunological synapse. Blocking CD80/CD28 or ICAM-1/LFA-1 interactions alters synapse morphology and reduces the area and density of accumulated complexes. These reductions correlate with reduced T cell proliferation, while CD69 and CD25 expression and TCR down-modulation remain unaffected. Thus, costimulation is essential for normal mature immunological synapse formation.