the immunological synapse: the more you look the less you know

Review

The immunological synapse: the more you look the less you know...

Nicolas Blanchard, Claire Hivroz *

Inserm U520, Institut Curie, 12, rue Lhomond, 75005 Paris, France

Received 9 September 2002; accepted 11 September 2002

Abstract

Before T cells of the immune system can recognize pathogens, antigen presenting cells (APCs) must process pathogen-derived peptidesand present them together with major histocompatibility complex molecules (MHC) to T lymphocytes. T lymphocytes then scan the surfaceof APCs and antigen-specific activation of the T cell will happen after interaction of T cell antigen receptor (TCR) with MHC–peptidecomplexes expressed at the membrane of APCs. This interaction takes place in a nanometer scale gap between the two cells, referred to asan immunological synapse. Recent three-dimensional fluorescence analysis of this synapse revealed a dynamic spatial organizationof membrane receptors, cytoskeleton and intracellular signaling complexes on the T cell side showing specific patterns, which depend onthe nature of the T cell:APC pair. Although it is obvious that establishment of an intimate contact between T cells and APCs will facilitatecell:cell communication it is not clear what is the role, if any, of this receptors patterning. This molecular reorganization has long been thoughtto enhance and/or sustain TCR signaling and thus T cell activation, but this is now a matter of controversy. Moreover, mechanisms controllingimmunological synapse formation are still unraveled. Segregation of proteins may occur spontaneously as proposed by mathematicalmodeling taking into account membrane fluidity, protein size and receptor/ligand affinity. Alternatively patterning of the molecules atthe cell:cell interface could be driven by active processes involving T cell signaling and/or specific features of the APC. These differentquestions will be discussed herein. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

1. Introduction

T cell activation requires recognition by the T cell receptor(TCR) of major histocompatibility complex–peptide com-plexes (MHC–peptide) present at the surface of antigen pre-senting cells (APCs). This interaction induces different func-tional programs depending on the nature of the Tlymphocytes. In thymocytes, binding of MHC–peptide toTCR will either induce positive selection, resulting in matu-ration of T cells capable of recognizing foreign peptides inthe context of MHCs, or negative selection resulting in apo-ptosis of autoreactive thymocytes. In activated CD8+ cyto-toxic T lymphocytes (CTL) recognition of the MHC–peptidecomplexes on the target cell induces killing of the target. In

CD4+ helper T lymphocytes, binding of the TCR to specificMHC–peptide complexes at the surface of B lymphocytesinduces secretion by T cells of cytokines that allow the inter-acting B cells to differentiate in antibodies producing cells.Finally, TCR recognition of MHC–peptide complexespresent on dendritic cells allows naive T cells (T cells thathave never encountered the antigen for which they are spe-cific) to differentiate, secrete cytokines and proliferate.

More than 10 years ago, pioneering studies showed that Tcell recognition of MHC–peptide complexes at the surfaceof targets or B cells was accompanied by cytoskeletal polar-ization and clustering of receptors at the cell–cell interface(Kupfer et al., 1991) explaining the vectorial secretion of cy-tokines and cytotoxic granules. More recently, it was re-ported that several surface and signaling molecules segregatewith a discrete kinetic pattern at the interface. This unex-pected level of organization has generated a lot of interest inthe functional consequences of immune effector function.

Of note immunological synapses have also been describedon B lymphocytes interacting with antigens bound at the sur-face of a cell (Batista et al., 2001) or natural killer cellsinteracting with their target cells (reviewed in Davis, 2002).The present review will concentrate only on immunologicalsynapses involving T cells.

Abbreviations: TCR, T cell receptor; MHC, major histocompatibilitycomplexes; APC, antigen presenting cell; MTOC, microtubule organizingcenter; CTL, cytotoxic T lymphocyte; c or pSMAC, central or peripheralsupra molecular activation cluster; ERM, ezrin radixin moesin proteins.

* Corresponding author. Tel.: +33-1-42-34-64-33;fax: +33-1-42-34-64-38

E-mail address: [email protected] (C. Hivroz)

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2. Definition of the immunological synapses

The term “immunological synapse” is not being usedconsistently by all investigators, indeed this term can coververy different concepts and thus be confusing. Some authorsuse it to refer to any interface between a lymphocyte and an-other cell. Others refer to an organized pattern of surfacemolecule at the interface, whatever this organization is,whereas others refer to one type of organized pattern firstdescribed between T cell clones and antigen-bearing B lym-phoma cells (Monks et al., 1998) and so called bull’s eyepattern. This concern, although semantic, is of great impor-tance since a confusion is often made between the establish-ment of this bull’s eye pattern and T cell activation. This israther misleading since a causal–effect relationship betweenthe existence of an organized interface and the T cell activa-tion has not been proven yet. Moreover, as stated above, morethan one pattern have been observed.

We propose to call immunological synapse the interfacezone between an APC and a T lymphocyte when reorganiza-tion of membrane molecules and cytoskeleton can be ob-served. We will try to show that in this sense several distinctimmunological synapses exist and that their pattern varyaccording to the T:APC pair involved.

3. More than one immunological synapse

Prior to synapse formation, T cells traffic through lymphnodes and the vasculature. They respond to chemokine sig-nals which attract them and arrest their movement (Campbellet al., 1998). These moving lymphocytes take on a character-istic morphology, with the nucleus pushed into their leadingedge and the cytoplasm largely concentrated in projectionslagging behind and called uropods (McFarland, 1969). Tcells are thus highly polarized cells with biased distributionof many molecules. Integrins (Sanchez-Madrid and del Pozo,1999), TCRs, co-receptors (Krummel et al., 2000) and largermolecules such as CD43 (del Pozo, 1995 #95) are mostlyfound in the uropods, together with most of the cytoplasmicorganelles (endoplasmic reticulum/Golgi, microtubule orga-nizing center (MTOC), secretory vesicles) (Kupfer et al.,1987). Conversely, the chemokine receptors are found inthe leading edge (Nieto et al., 1997). In the first minuteof contact with an APC, T cells adhere temporarily tothe APC and scan it for the presence of the appropriatepeptide–MHC complexes. This first adhesion step is inde-pendent of TCR stimulation since it takes place in the ab-sence of antigen (Donnadieu et al., 2001). Although ill char-acterized, this first step is of great importance since it createsan initial membrane/membrane contact area, which may helpTCR recognition of its ligand at the surface of the APC. Italso induces morphological changes in T cells, which be-come more spherical (Donnadieu et al., 1994).

Presence of the right peptide–MHC combination will leadto further modification of the interface zone.

Monks and Kupfer (Monks et al., 1998) were the first toshow, in a model using murine CD4+ cloned T cells interact-ing with a B lymphoma cell line carrying the right pepti-de–MHC combination, that after 5–20 min of contact, mol-ecules are not merely capped but organized in concentricareas in the T/APC cell contact zone. The authors demon-strated that TCRs accumulate in the central zone of interac-tion also called “central supramolecular activating complex”(c-SMAC), while integrins and their ligands occupy the pe-riphery of this array (called p-SMAC) (see Fig. 1 ). Theseresults have been confirmed in other models. Dustin and co-workers used as a surrogate for APCs, planar bi-layers incor-porating fluorescently labeled, lipid-anchored peptide–MHCand the LFA-1 ligand ICAM-1. They also observed, afterseveral minutes of contact, a distribution of the former inc-SMAC and of the latter in p-SMAC (Grakoui et al., 1999).Other molecules have also been characterized as formingpatterned arrays at the synapse. CD45 (Johnson et al., 2000;Leupin et al., 2000), a highly abundant transmembranetyrosine phosphatase and CD43 (Allenspach et al., 2001;Delon et al., 2001; Roumier et al., 2001), two large mole-cules, are predominantly excluded from the interface.

In the lipid bi-layers models, kinetic analysis of the mol-ecules have shown that TCR ligands are engaged in an out-ermost ring at the beginning of the contact and that theirtransport into a central cluster takes at least 5 min of contactand is dependent of the presence of the right peptide–MHCcombination (Grakoui et al., 1999). Studies performed byhigh speed microscopy in a murine T cell clone expressinga green fluorescent chimera of the TCR associated f chainand as APC, a murine B cell lymphoma, also demonstratedthat the T/APC interface is highly dynamic (Krummel et al.,2000). In this study Krummel and Davis show, at the begin-ning of the contact, transient formation of very small(< 1 µm) clusters of TCR, which move over time. After5–10 min, these clusters coalesce to give rise to the cSMAC.Both the initial clustering and the following coalescencewere shown to be dependent on the presence of the rightpeptide–MHC combination (Krummel et al., 2000). Morerecently, studies performed on CD8+ T cells have shown thatcytotoxic T cells also form SMACs after engagementof the TCR by APCs (Potter et al., 2001; Stinchcombe et al.,2001) and that the pattern of surface molecules at the T/APCinteraction zone is very similar to the one observed withhelper CD4+ T cells (see Fig. 1).

In human models, concentric organization of the TCR inthe central interaction zone, of integrins in a peripheral zoneand exclusion of large molecules such as CD43 and CD45have also been observed. We and others have described sucha pattern in the widely used Jurkat leukemic CD4+ T cellsinteracting with a B lymphoma pulsed with a superantigen(Blanchard et al., 2002a; Roumier et al., 2001), and in humanT cell blasts prepared from peripheral blood T cells fromcontrol donors (our unpublished data). However, this patternis not always observed. In a human CD4+ T cell clone, we didnot observe a central clustering of the TCR (Fig. 2 ). In these

346 N. Blanchard, C. Hivroz / Biology of the Cell 94 (2002)

T cells, the interaction zone with an autologous EBV B cellline carrying the right MHC–peptide combination (Fig. 2)was characterized by the presence of several small clustersof the TCR/CD3 complexes. Although no central SMAC wasformed, the interaction was “productive” since the T cellclones produced lymphokines (data not shown). These re-sults strongly suggest that formation of the bull’s eye patternis not required for T cell response.

This lack of cSMAC formation has also been reported inmodels wherein immature murine CD4+ CD8+ thymocytesspecific of a given peptide–MHC combination were activatedin a lipid bilayer system or with real APCs (Hailman et al.,2002; Richie et al., 2002). In both these studies, the authorsshow that TCR/CD3 did not accumulate at the centerof the synapse, but either form small clusters spread all overthe T/APC interaction zone (Hailman et al., 2002) or areexcluded from the central zone (Richie et al., 2002). Theseclusters of TCR correspond to areas of exclusion of LFA-1and are mobile and transient (Hailman et al., 2002). Againthese interactions were “productive” since they resulted inpositive or negative selection of the thymocytes.

Visualization of the immunological synapses describedso far was limited to imaging sectioned tissue (Reichert et al.,2001) or cultured cells. Recently, the advent of two-photonlaser scanning microscopy has allowed to dynamically fol-low T cell–dendritic cell interactions in intact explantedlymph nodes (Miller et al., 2002; Stoll et al., 2002) or thy-mocyte–stromal interactions in a three-dimensional thymicorgan culture (Bousso et al., 2002). These studies and anotherrealized by confocal microscopy on an explanted lymph node(Stoll et al., 2002) suggest that immunological synapse takesplace in vivo. However, in these models the exact patterningof molecules at the interface has not yet been determined.

4. Signaling and synapse formation: an “hen and eggstory”

A considerable amount of research has focused on a pos-sible relationship between immunological synapse formationand signaling through the TCR. Initially, it was thought thatformation of the immunological synapse by concentrating

Fig. 1. Synaptic patterns at the equilibrium (“mature synapse”) in different T cell types. For each panel, localization of cell surface molecules is shown onthe left-hand side of the dotted line whereas position of signaling and/or intra-cellular molecules is depicted on the right-hand side. (A) Synaptic pattern ina CD4+ T lymphocyte interacting with an agonist-loaded B lymphoma cell is characterized by the assembly of proteins into concentric zones: a central SMAC,a peripheral SMAC and a more distal zone of exclusion (TCR, LFA, PKC, Lck, ZAP (Lee et al., 2002; Monks et al., 1998), f (Blanchard et al., 2002a; Krummelet al., 2000), CD45 (Leupin et al., 2000), CD43 and ERM (Allenspach et al., 2001; Blanchard et al., 2002a; Delon et al., 2001; Roumier et al., 2001). (B) Synapticpattern in CD4+ CD8+ thymocytes negatively selected by thymic stromal cells bearing peptide (upper scheme) or an artificial lipid bilayer containing pMHCcomplexes and ICAM-1 molecules (lower scheme). In both cases, the patterns observed are distinct from the one depicted in (A). In thymocytes interacting withthymic stromal cells, TCR, f chain and Lck preferentially segregate within an outer ring surrounding the central zone of contact (Richie et al., 2002). Inthe artificial bilayer model, pMHC complexes/TCR and tyrosine phosphorylated proteins accumulate into multi-focal clusters, from where integrin molecules,such as ICAM-1/LFA-1, are largely excluded (Hailman et al., 2002). (C) Synaptic pattern in a CD8+ CTL interacting with a lysis-susceptible target cell. Like in(A), molecules are arranged within concentric areas; in addition to the signaling-specialized module, a secretion-specialized zone is present in the cSMAC (pinkcolor). Enzymes, such as granzyme A and cathepsin D, which participate to the target cell lysis, are present in this zone (Potter et al., 2001; Stinchcombe et al.,2001).

347N. Blanchard, C. Hivroz / Biology of the Cell 94 (2002)

TCRs and co-stimulatory receptors in a confine area and seg-regating molecules, which could negatively regulate TCRsignaling, from this area would create the ideal environmentfor signaling to take place. TCR engagement is followed byphosphorylation on tyrosine residues of specialized domainscalled ITAMs via the Src family kinases Lck or Fyn. Thesedomains are present in the CD3/f chains associated tothe TCR and constitute, when phosphorylated, docking sitesfor the tandem SH2 domains of the Syk family kinaseZAP-70 (Chan et al., 1994; Hatada et al., 1995; Hivroz

and Fischer, 1994). Recruitment of ZAP-70 leads to its phos-phorylation and enzymatic activation, allowing phosphoryla-tion of the integral membrane adapter protein LAT (Zhangand Samelson, 2000). Phosphorylated LAT allows in turnthe recruitment of several enzymes playing key role in sig-naling pathways such as increase of the intracytoplasmicCa2+ concentration ([Ca2+]i), PKC and small GTP bindingproteins activation (Samelson, 2002). The idea that synapseformation played a role in TCR signaling was reinforced bythe fact that surface molecules patterning is accompanied at

Fig. 2. Synaptic patterns vary according to the human CD4+ T cell type. (A) Immunological synapse between HA-specific human T cell clones and autologousimmortalized B cells loaded with HA peptide (obtained from F. Faure, INSERM U520, Institut Curie). Small clusters of f accumulate at the contact zone withthe B cell only in the presence of peptide, as shown by the green immunofluorescent labeling in the lower image (our unpublished results). (B) Synaptic patternbetween human SEE-specific T cell blasts and SEE-pulsed B lymphoma cells. SEE-specific blasts were obtained by expanding T cells from a healthy donor inthe presence of SEE and IL-2. After 7–9 days, the majority of T cells were specific of SEE (70–80%). In contrast to panel A wherein small clusters of f wereobserved, a massive recruitment of f was detected in the synapse between SEE-specific T cell blasts interacting with SEE-pulsed B cells, as shown by the greenlabeling on the left-hand side pictures. CD43 was excluded from this zone, as shown by the red immuno-labeling on the right-hand side images. LAT did notaccumulate at the synapse, as assessed by the green labeling on the right-hand side images (our unpublished results). (C) Synaptic pattern in a human leukemicT cell line (Jurkat) interacting with a SEE-pulsed B lymphoma cell. On the left-hand side panel, immunofluorescence images show that a f-GFP chimeraaccumulated in the central zone of the synapse (green fluorescence). LAT (red immuno-fluorescence) co-distributed with f-GFP, in contrast to what is observedin panel B (see on the bottom images, the “en face” view of the synapse obtained by projection of the cropped contact zone along the Y axis). As shown bythe right-hand side panel, CD43 (red immuno-labeling) is excluded from the cSMAC wherein f-GFP (green fluorescence) clustered (also reported in Blanchardet al., 2002a; Roumier et al., 2001).


the inner face of the T/APC contact by an organized pattern-ing of signaling molecules. Lck, ZAP-70 (Holdorf et al.,2002; Lee et al., 2002), and PKCh (Monks et al., 1998) allknown key players of T cell activation, are concentrated atthe synapse and we have shown that the adaptor moleculeLAT is also recruited at the center of the synapse after TCRactivation (Blanchard et al., 2002a). However, detailed com-parisons of the kinetics of signaling versus synapse forma-tion in T cells ruled out the hypothesis of a role for synapse inearly TCR signaling (Davis and van der Merwe, 2001; Delonand Germain, 2000). Recently compelling evidence havebeen provided that early TCR signaling does not requireimmunological synapse formation. Indeed, it has been shownthat TCR-mediated tyrosine kinase signaling in naive murineT cells occurred primarily at the periphery of the synapseand was largely abated before the organization in cSMACand pSMAC was observed (Lee et al., 2002). These resultsclearly show that synapse formation is neither required forinitiating nor for stabilizing TCR signaling, at least in termsof tyrosine phosphorylation and activation of Lck and ZAP-70. However, it does not completely rule out a role forthe synapse in late signaling events. For example, the in-

crease in intracellular free Ca2+ concentration has beenshown to last for more than an hour and this has also beenshown for serine threonine kinases activation (Matthews etal., 2000), see Fig. 3 for kinetic comparisons. We have shownwith others that duration of the signal is important since inimmunodeficient patients presenting only a short increaseof intracellular Ca2+ concentration, T cell activation wasdefective (Feske et al., 2001; Le Deist et al., 1995).

However, the question of a potential role for the synapse isstill open.

One of the key role for synapse may be for T cells topolarize secretion of cytokines towards the B cell or cyto-toxic granules towards the target. As stated in the introduc-tion, helper CD4+ T cells interacting with B cells secretecytokines that instruct B cells to mature and produce anti-body. Likewise, cytotoxic CD8+ T cells secrete lytic granulesin a polarized fashion. This polarization may ensure that Tcells communicate with or lyse only the appropriate cells,avoiding bystander effects due to a secretion that will not beconfined at the T cell/target cell interface. Indeed, Stinch-combe et al. have recently shown that lytic granule secretionoccurs in a separate domain of the synapse, surrounded by

Fig. 3. Schematic time-dependent evolution of the immunological synapse between a CD4+ T lymphocyte and a B lymphoma cell. Biological processesdepicted here are restricted to the first hour of contact between both cells since most phenomena involved in synapse formation take place within this periodand most studies done so far have focused on this time window. While the upper part of the figure synthesizes the dynamical patterning of several preponderantcell surface molecules, the lower part deals with the time-dependent localization and activation status of intra-cellular signaling molecules, as well asthe evolution of cytoskeleton-based phenomena.


the adhesion molecule ring, maintaining signaling proteinorganization during exocytosis (Stinchcombe et al., 2001)see Fig. 1.

The immune synapse formation may not only serve topolarize cytokines and granules secretion towards the targetcells but may also control the arrival of intracellular poolsof molecules, which regulate T cell activation. We and othershave shown that polarization of the T cell towards the APC isaccompanied by the delivery of an intracellular poolof TCR/CD3/f complexes (Blanchard et al., 2002a) at the siteof interaction as well as an intracellular pool of the adaptorprotein LAT (Montoya et al., 2002). This recruitment of LATand of the intracellular pool of TCR was impaired in the ab-sence of ZAP-70, showing that if synapse formation does notcontrol signaling, signaling controls recruitment of transduc-tion molecules at the synapse. It is worth noting at this pointthat signal transduction in T cells involved the formationof a TCR associated macromolecular complex or signalo-some, whose formation is essential for quantitative and quali-tative control of TCR signaling (for review see Werlenand Palmer, 2002), and is regulated (Jabado et al., 1997).LAT plays a key role since it serves as a scaffold protein byinteracting after phosphorylation with numerous signalingcomponents (for review see Wange, 2000). A polarized deliv-ery of signaling molecules such as LAT may thus controlthe coordinate formation of the signalosome.

Interestingly, it has recently been shown that CTLA-4,a transmembrane receptor retained in intracellular compart-ments in resting T cells, accumulates at the immunologicalsynapse upon TCR triggering (Egen and Allison, 2002).Moreover, depending on the TCR signal strength, the intrac-ellular pool of CTLA-4 is either polarized towards the APCstaying intracellular or expressed at the T cell membrane inthe central SMAC (Egen and Allison, 2002). This is of par-ticular relevance, since CTLA-4 has been shown to nega-tively regulate T cell activation. Activation of T cells by TCRengaging peptide–MHC is dramatically enhanced by inter-action of the CD28 co-stimulatory receptor with its ligandson the APC surface and CD28 has been shown to be recruitedto the center of the immunological synapse (Bromley et al.,2001). CTLA-4 by binding to the same ligands as CD28but with a higher avidity, compete with CD28 and thus inhibitits co-stimulatory effect (Greene et al., 1996; van der Merweet al., 1997). CTLA-4 being transported, upon certain condi-tions, in the very place where CD28 interact with its ligandswill thus efficiently inhibit CD28 co-stimulatory effects.

Another possibility is that the organized bull’s eye struc-ture of the synapse will facilitate material transfer betweenthe interacting cells. Indeed, uptake of molecules by cellsintimately interacting in a synapse has been shown in severalmodels. T cells have been shown to internalize pepti-de–MHC complexes after peptide specific interaction withAPCs (Huang et al., 1999), CTLs have been shown to inte-grate membrane markers from their targets (Stinchcombe etal., 2001). NK cells acquire transmembrane molecules fromthe target cells (Carlin et al., 2001) and B cells interacting

with target cells also acquire membrane-integral Ag fromtheir targets (Batista et al., 2001). These intercellular trans-fers may have some physiological consequences. T cellsabsorbing peptide–MHC-complexes can become targets forCTLs, leading to a fratricidal killing of T cells (Huang et al.,1999). Acquisition by B cells of Ag may enhance the extentto which cognate B cells will present peptides that are de-rived from proteins recognized by the BCR to T cells. Themechanisms underlying intercellular transfer of integralmembrane proteins are unknown. It may be due to secretionof vesicles of endosomal origin such as exosomes (Blanchardet al., 2002b; Thery et al., 2002), or pinching of membranemicrodomains from the target cells. Alternatively, intercellu-lar transfer could be mediated by formation of membranebridges such as those reported between the cytotoxic T celland the target membranes (Stinchcombe et al., 2001). Re-gardless of the mechanisms implicated, organization of re-ceptors and lipid microdomains at the synapse may providean adequate environment for this to happen.

5. How does the synaptic pattern formation take place?

As discussed above, the role, if any, of the synapse isunknown. This state of ignorance also concerns the mecha-nisms underlying formation of immune synapses.

The pattern organization of molecules at the cell–cellinterface may be driven by passive mechanisms driven them-selves by receptor–ligand binding or active mechanisms,driven by cytoskeletal movement and polarized secretion.

Davis and van der Merwe (1996) and Shaw and Dustin(1997) have argued that the dimensions of receptor:ligandinteraction will drive the mechanism of receptor organiza-tion. According to this view the smaller molecules in the in-terface will cluster together at closely apposed membraneregions and larger molecules will also cluster together inseparate regions wherein the membranes are more distant.The relative size of the TCR–MHC ligand pair (15 nm)within the interface will drive this receptor into the centerof the interface whereas molecules with longer dimensions,such as ICAM-LFA-1, CD43 and CD45 would be segregatedoutside of the central region by purely steric mechanism. Insupport of this theory, it has been shown that increasingthe interaction size between CD2 and CD48, a pair of mol-ecules which co-stimulates T-cell response, will lead to its“mis-placing” in the interface and prevent T cell activation(Wild et al., 1999). Recently, a mathematical model takinginto account membrane fluidity, protein size and receptor:li-gand affinity has shown that segregation of proteins atthe cell:cell interface may occur spontaneously (Qi et al.,2001). However, experimental studies using inhibitors of ac-tin polymerization (Krummel et al., 2000; Wulfingand Davis, 1998) or myosin motors (Wulfing and Davis,1998) have shown that active, cytoskeletally driven processesare involved in the synaptic pattern formation (for review seeAnton van der Merwe et al., 2000).


These two models can be reconciled if it is assumed thatcytoskeletal changes are essential to fulfill some of the as-sumptions of the mathematical modeling, or alternativelypassive mechanisms are sufficient to drive the initial redistri-bution and that cytoskeletal-dependent processes relay tomaintain the large-scale clusters of the synapse.

Studies performed for over a decade have shown that TCRtriggering leads to cytoskeletal remodeling and MTOC polar-ization. Some of the mechanisms underlying these phenom-ena have been unraveled. TCR activation leads to activationof the small GTPase cdc42, which binds and activate knownregulator of actin cytoskeleton such as WASP (Cannon et al.,2001). Evidence that actin cytoskeletal activity feeds backinto receptor positioning at the T cell:APC interface zone issuggested by the fact that T cells deficient for WASP givesless robust formation of clusters at the site of TCR stimula-tion (Snapper et al., 1998) and that a similar defect is ob-served in T cells lacking the expression of the GTP exchangefactor Vav (Holsinger et al., 1998). Moreover a role forthe cortical actin cytoskeleton has also been shown forthe exclusion of CD43 from the T cell:APC interaction zone.A cluster of positively charged amino acids in the conservedcytoplasmic domain of CD43 interacts with membersof the ezrin radixin moesin (ERM) family of proteins, whichcouple many surface receptors to the cortical actin. Experi-ments using mutant forms of CD43 and ERM proteins haveshown that the stable exclusion of CD43 is driven by the ex-clusion of ERM proteins (Allenspach et al., 2001; Delon etal., 2001).

The pathways that couple TCR triggering to changes inthe microtubules cytoskeleton are less well understood.Again rho-GTPases appear to play a key role, since introduc-ing a dominant-negative form of cdc42 into a T cell hybri-doma inhibits polarization of the MTOC towards the APC(Stowers et al., 1995). A recent study has shown using videoimaging that in a CTL the MTOC is drawn vectorially tothe contact site with theAPC by a sliding mechanism and thatmicrotubules loop through and anchor to the peripheralSMAC defined by the dense clustering of LFA-1 (Kuhnand Poenie, 2002). However, the motors controlling the vec-torial organization of microtubules and molecules, whichenable microtubules anchoring to the T cells cortex are stillunknown. We have recently shown that the tyrosine kinaseZAP-70 plays a crucial role in the TCR-dependent reorienta-tion of the MTOC since ZAP-70 deficient T cells or T cellsexpressing a dominant-negative form of ZAP-70 displayan altered ability to polarize their MTOC towards an APC(Blanchard et al., 2002a). In this model we were able to showthat central clustering of CD3 and CD2 as well as exclusionof CD45 and CD43 did not require MTOC polarization sincethese events occurred in ZAP-70 deficient T cells. In con-trast, LAT and PKCh recruitment at the inner face of the cen-tral SMAC did not happen in ZAP-70 deficient cells. It istantalizing to propose that the absence of signaling moleculesrecruitment is at least partially due to MTOC mispolariza-tion. Indeed, it has been shown that signaling molecules bind

to microtubules, which then may even play the role of scaf-fold bringing together components of a signaling pathway(Nagata et al., 1998).

Another possible mechanism of surface molecules redis-tribution at the synapse has been suggested by a study fromKhan et al. (2001). In this study the authors showed thatthe aggregating protein agrin, a secreted proteoglycan thataggregates receptors at the neuromuscular junction, accumu-lates at the synapse and is expressed by T cells (Khan et al.,2001). Furthermore, treatment of T cells with the formof agrin purified from activated T cells induced the aggrega-tion of TCR and enhanced T cell activation (Khan et al.,2001). This study shows that immunological and neurologi-cal synapses may share some common features. The identi-fication of neuropilin-1, a neuronal receptor, as participatingin naive T cells:APC contact (Tordjman et al., 2002) empha-sizes the molecular similarities between these systems.

One aspect of synapse formation that have often beenneglected is the role of the APC. This is probably due tothe fact that formation of the central and peripheral SMACwere observed in models using planar lipid bi-layers contain-ing only ICAM-1 and peptide–MHC (Grakoui et al., 1999). Itwas also long thought that the APC cytoskeleton did not playany role in the formation of the bull’s eye pattern. This wasshown by Wülfing et al., who demonstrated that cytochalasinD treatment of a B lymphoma cell line, used as APC, did notaffect formation of the bull’s eye pattern suggesting thatthe APC was rather passive (Wulfing and Davis, 1998).However, these results have been challenged by recent stud-ies showing that dendritic cells, which are the onlyAPCs ableto prime naive T cells, may actively contribute to synapseformation. Disruption of their cytoskeleton dramatically de-creased the formation of the central TCR cluster (Al-Alwanet al., 2001). Furthermore, functional antigen-independentsynapses have been reported between T cells and dendriticcells, this is unique to dendritic cells since B cells cannotinduce surface molecules redistribution in the absence of Ag(Revy et al., 2001). Using human dendritic cells obtained bydifferentiation of peripheral monocytes with GM-CSFand IL-4, we have shown that dendritic cells at differentstages of maturation differentially control the formationof the immune synapse (our unpublished data). As shown inFig. 4 , morphology of mature and immature dendritic cellsare very different leading to a very different “quality” of con-tact with a T cell. It remains to determine why the dendriticcells have such unique feature, several explanations can beproposed. Dendritic cells express specific combinationsof surface molecules that are ligands for receptors on T cells,this combination varying when dendritic cells mature (Mell-man and Steinman, 2001). Some of these molecules control Tcell adhesion to the dendritic cell and others are co-stimulators of T cell activation. Dendritic cells also secretechemokines that stimulate T cell adhesion and may modifysynapse formation (Bromley and Dustin, 2002). Finally,the mere structure and organization of the dendritic cellsmembrane may contribute to its ability to induce synapse


formation. In mature dendritic cells, it has been shown thatpeptide–MHC class II complexes are transported atthe plasma membrane together with co-stimulatory mol-ecules where they remain clustered (Turley et al., 2000).These clusters may increase the avidity of the receptors on Tcells for their ligands and according to the “passive model”facilitate formation of a synapse. Studying the mechanismsby which dendritic cells control their interaction with T cellswill help understanding the unique feature of this cell asAPC.

6. Concluding remarks

Although a considerable amount of studies have dealt withthe immunological synapse, a lot remain to be discovered. Aparticular attention should be devoted to the role of the syn-apse and the mechanisms involved in its formation.

Technical advances in imaging now allow to followthe movement of molecules in living cells and even in intacttissues, this should help to elucidate the molecular mecha-nisms underlying and physiological function of the immuno-logical synapse.

Acknowledgements

We thank S. Amigorena and F. Benvenuti for helpfuldiscussions, K. Yang for experimental help, F. Faure for kindgift of cells and reagents and INSERM and ARC for financialsupport.

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Fig. 4. Importance of the APC in synapse formation: maturation state of dendritic cells determines the nature of contacts with CD4+ T cells. Dendritic cells wereobtained by differentiation of monocytes from peripheral blood of a healthy donor with IL4 and GM/CSF. Maturation of dendritic cells was induced byover-night treatment with TNF-a and prostaglandin-E2. Dendritic cells were then pulsed with SEE and mixed with Jurkat CD4+ T cells at a 1:1 ratio. Whilecontacts between T cells and immature dendritic cells remained loose (A), they seemed to be closer and tighter with mature dendritic cells (B). Furthermore, aswitnessed by these two representative images, the number of T cells per dendritic cell was consistently higher with mature dendritic cells.


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the immunological synapse: the more you look the less you know

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