Imaging T‐cell antigen recognition and comparing immunological and neuronal synapses

In the process of antigen recognition, the molecular structure detected by a T cell is never a soluble ligand. Recognition always requires the association between an antigenic peptide and a MHC molecule, present either on the surface of dedicated antigen-presenting cells (APCs, bearing class I and class II MHC) or on the surface of any other type of cell (bearing class I MHC). Thus, antigen recognition starts with a cell–cell interaction. At the interface between a T cell and an APC, concentrations of membrane receptors, intracellular adaptors, cytoskeletal components and enzymes form a structure controlling the communication between the two cells. In particular, this structure controls activation of the T cell. The contact is reminiscent of that observed at synapses of the nervous system, and hence the term ‘immunological synapse’ has been proposed.1,2 The main properties of this structure, which we will call ‘I-synapse’, have already been discussed in excellent reviews.3–5 In the present review, we will focus on how our present knowledge of this synapse is limited by the cells and the methods used to visualize this structure. We will then present a current view of the synapses formed by neurons (N-synapses) and, finally, examine how the more detailed information available for N-synapses can guide our studies of I-synapses. Let us first examine the different experimental models which have been used to characterize the I-synapse and the specific information obtained from each of them. We define an I-synapse as a region of membrane apposition between two cells of the immune system. This usage is more restrictive and appropriate than that found in some of the literature, where the term ‘synapse’ is sometimes employed to describe receptor clustering, or capping, induced by cross-linking antibodies. More than 15 years ago, Abraham Kupfer and colleagues pioneered the study of T-cell–APC (T–APC) interactions at the single-cell level. Immunofluorescence of fixed cellular conjugates was used to identify the molecules and structures concentrated at the T–APC interface. The first molecular reorganization described was the polarization of the microtubule organizing centre (MTOC) in CD8+ T-cell clones, the MTOC turning towards the target cell.6 Similar observations were then made during antigen-specific interactions between CD4+ T-cell clones and APCs, i.e. B-cell hybridomas or lymphomas7,8 or MHC II-transfected fibroblasts.9 This MTOC polarization was observed only in the T cells but not in the APCs. These initial observations were followed by several reports describing the redistribution of various proteins, such as the coreceptor CD4, the integrin LFA-1 and the cytoskeletal adaptor talin, which all colocalized with the T-cell receptor (TCR) at the T–APC contact zone.10,11 More recently, Kupfer, Monks and collaborators showed that PKC-θ was also recruited at this antigen-specific interface, and that its serine/theonine kinase activity was necessary for TCR-induced NF-κB activation and T-cell proliferation.12,13 The most recent experiments took advantage of an improvement of the imaging system, deblurring after deconvolution of a Z-series of images, giving access to a third dimension of the I-synapse and to further details of its structure. After about 30 min of antigen-specific interaction between a T-cell clone and a B-cell lymphoma, concentrations of several molecules (PKC-θ, CD3, TCR, and two tyrosine kinases, Fyn and Lck) were found at the centre of the I-synapse. This cluster was called the cSMAC (central supra-molecular activation cluster). Talin and LFA-1 surrounded this cSMAC to form the pSMAC ( peripheral supra-molecular activation cluster).14 This molecular organization was antigen-specific, since it was not found when B-cell lymphomas did not present the specific antigen peptides to T-cell clones. However, I-synapse formation did not appear to depend solely upon a TCR-specific signal. Indeed, PKC-θ clustering was observed in T-cell clones but not in T-cell hybridomas.12 Thus, immunofluorescence experiments show the accumulation of a large number of molecules at the I-synapse. This accumulation is probably facilitated by the formation of a lamellipodium protruding from the T cell towards the APC, most clearly seen in scanning electron microscopy.15 However, one should not conclude that all types of cell surface protein accumulate at this interface. Indeed, the specific exclusion from the I-synapse of CD43 has recently been reported,4,16 while the exclusion of CD45 from the I-synapse is still a matter of controversy.16,17 The monitoring in living cells of fluorescent, GFP-tagged proteins expressed in either APCs or T-cell clones has provided another set of interesting data on the I-synapse. This method was first used to visualize the rearrangement of ICAM-1 during antigen-specific T–APC interaction.18 ICAM-1 accumulation in the presynaptic element, i.e. in a B-cell hybridoma, was detectable a few minutes after the intracellular Ca2+ rise. This study confirmed that polarization of LFA-1 molecules occurring in the postsynaptic (T cell) element was a very early event, probably necessary to sustain Ca2+ signalling.18 The same type of approach allowed Monks and colleagues to show that translocation of T-cell MEK kinase 2 (MEKK2-GFP) occurred within 10 s after exposure of the D10 T-cell line to an antigen-pulsed B-cell line.19 In another study, the movement of CD3ζ-GFP or CD4-GFP molecules was followed during antigen-specific T–APC interactions.20 During the first few minutes, both CD3ζ and CD4 accumulated at the centre of the T-cell–B-cell (T–B) interface, but shortly afterwards, while CD3ζ remained in the central zone, CD4 migrated away from this central spot, suggesting that CD4 was only necessary to trigger initial signalling. Using a slightly different approach, in which beads were attached non-specifically to T cells, Wulfing and Davis provided evidence that the molecular accumulation at the I-synapse was driven by an active cytoskeletal mechanism requiring costimulatory molecules and an intracellular Ca2+ increase.21 However, they did not observe a general movement of proteins on the B-cell surface towards the T–B interface, despite the ICAM-1 accumulation in the B cell described above. A face-on view of an artificial synapse with an outstanding spatial resolution has been obtained by observing with interference-reflection microscopy (IRM) the contact formed between T cells and a planar bilayer containing glycosylphosphatidylinositol-anchored fluorescent MHC, ICAM-1, CD48 or CD80 molecules.2 In this system, surprisingly, it was LFA-1 which initially accumulated at the centre of the contact and was surrounded by a ring of TCRs. Then, within a few minutes, TCRs and LFA-1 exchanged their respective positions in the centre and periphery, to end up in an arrangement strikingly similar to that of the SMACs described by Kupfer's group. This configuration, which was called a ‘mature immunological synapse’, was stable for at least 1 hr. The addition to the planar bilayer of either CD48 or CD80 molecules did not modify the behaviour of LFA-1. Similar experiments demonstrated that CD2 accumulated at the centre of the interface, inside the LFA-1 ring. The same approach has been used to analyse the dynamic redistribution of the membrane phosphatase CD45 at the T-cell surface, simultaneously with those of MHC and ICAM-1 in the lipid bilayer.22 Johnson et al. described an initial exclusion of CD45 which subsequently migrated back into the central zone. Their interpretation is that initial CD45 exclusion from the synapse would permit integrin activation, whereas the return of CD45 to the vicinity of the TCR would maintain Src kinase activity for the duration of the TCR engagement. Although our knowledge of the molecules that accumulate at the I-synapse has improved considerably, simple questions related to the structure of the I-synapse are still unresolved. In particular, what is the distance between the membranes of the T cell and those of the APC at the I-synapse? The direct answer to this question will come from electron microscopy experiments (Fig. 1). Despite this lack of direct information, immunologists tend to favour a scheme in which the synaptic cleft is approximately 15 nm at the centre of the synapse, where the TCR and MHC are assumed to interact with each other, the size of these molecules being around 7 nm. In the immediate vicinity of these molecules, one could easily imagine the presence of molecules of the same size, i.e. CD2, CD28 and their ligands. In contrast, large molecules such as LFA-1 and ICAMs and the even larger CD45 and CD43 could hardly be the immediate neighbours of TCRs at the I-synapse. All these predictions fit neatly with the observed exclusion of CD43 from the synapse, and with the detrimental effects on synapse formation of the expression in a T cell of large chimeric CD2 molecules.23 Mike Dustin and Anton van der Merwe have thoroughly discussed these points and even proposed that formation of the synapse could be driven to a large extent by size-based segregation of receptors and adhesion molecules. They have discussed an additional point, related to the low affinity of CD2 for its ligand when measured in solution. When CD2 (for example) is restricted to a surface, it is automatically concentrated and dissociation reactions are less favoured. Thus, when expressed at the cell membrane, even low-affinity receptors like CD2 might well play an important role in synapse formation/stabilization when their surface expression is large enough to result in a significant 2D affinity.5,24 This is probably the case for CD2, the density of which has been calculated to range between 200 and 300 molecules/µm2 in Jurkat cells.25 Comparison between N- and I-synapses. Top: electron micrographs of glycinergic synapse (left, courtesy of Dr A. Triller) and of a T–DC synapse (right, courtesy of Dr G. Raposo). Scale: 0·5 µm (0·25 µm for the insets). The inset of the N-synapse highlights the presence of a PSD. Bottom: schematic summary of molecules involved at the N- and I-synapses (left and right). R, neurotransmitter receptor; Nx, neurexin; Ng, neuroligin; Cad, cadherin; PA, first row adaptors; PSD, postsynaptic density; TCR, T-cell receptor; MHC, major histocompatibility complex. Thus, strong predictions about the structure of the I-synapse have been derived from theoretical arguments based on the size, ligand binding affinity and abundance of a number of molecules. However, we have seen that only some of them have been tested experimentally, most often indirectly. Immunofuorescence studies of fixed cell-conjugates have provided important information on the identity of several molecules included within or excluded from the I-synapse. An increased spatial resolution was achieved after image deconvolution. Initial studies provided information on the state of the synapse after 15–30 min of interaction, but more recently briefer interactions have been examined, and, in principle, kinetic information could be obtained from the statistical analysis of sets of data obtained at different times after the initiation of the T–APC contact. In addition, the use of GFP-tagged proteins has given access to direct dynamic information and shown some subtle movements, for instance that of CD4, which is only transiently in a position to interact with TCR–CD3, in a T/B interaction. Finally, by far the best spatio-temporal information is obtained in the IRM–lipid bilayer system, but at the expense of using a surrogate APC quite different from true APCs. The fact that studies have been on synapses is often and the to all I-synapses. However, some important I-synapses have not been A significant is the synapse formed between T cells and cells appear to to the of to T cells. In an initial antigen in the of the the or various They then the antigen to to the T of the a that is by T cells. have their to and can present to T cells the in the at this are the only cells to conjugates with T cells in an a which very probably antigen recognition when only specific are available at the APC We use the term for the structure that at the T–DC interface either in the of antigen the antigen is Despite the large number of adhesion and molecules expressed on and T cells, the of the surface molecules that the contact between T and molecules two the molecules this interaction have a or 2D for their ligand. they have the appropriate adhesion by electron microscopy that the initial contact between T cells and This is reminiscent of the initial interaction between and cells during the an interaction by and expressed at the of a is to contact between the two already the TCR is to the initial T–DC evidence for this is that the initial T–DC adhesion is from a theoretical the of of specific to an surface density of not the described above. 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