Seminars in Immunology 17 (2005) 400–410

Review A dynamic view of the immunological synapse

Michael L. Dustin ∗ Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine, and Department of Pathology, New York University School of Medicine, 540 1st Ave, New York, NY 10016, USA

Abstract T cell activation requires interactions of T cell antigen receptors (TCR) and peptides presented by major histocompatibility complex molecules (MHCp) in an adhesive junction between the T cell and antigen-presenting cell. Stable junctions with bull’s eye supramolecular activation clusters (SMACs) have been defined as immunological synapses (IS). These structures maintain T cell-APC interaction and allow directed secretion. T cells can also be activated by asymmetric hemi-synapses (HS) that allow migration during signal integration. IS and HS operate in different stages of T cell priming. Optimal effector functions may also depend upon cyclical use of IS and HS. © 2005 Published by Elsevier Ltd.

Keywords: Intravital microscopy; Immunological synapse; Total internal reflection fluorescence microscopy; T cell receptor; Signalling

1. Introduction in vitro and in vivo systems have emphasized the ability of T cells to integrate signals without the formation of a long-lived The partnership between dendritic cells (DC) and T lym- IS. Therefore, a challenge for in vitro molecular imaging is to phocytes (T cells) defends the body against microbes, parasites, provide insight into how T cells integrate signals from IS and abnormal cells and environmental toxins that breach the barrier more dynamic interactions. function of skin and epithelial surfaces [1]. Diverse tools includ- One of the basic biological questions in immunology is what ing those of biochemistry, cell biology, genetics and imaging distinguishes T cell responses to DC that lead to tolerance or have been employed to understand the mechanistic basis of this priming. One concept is that the outcome of antigen presentation partnership. In recent years imaging approaches have become depends upon the activation status of the DC [4]. Immature DCs increasingly useful as molecular technologies for labeling cells patrol the tissue spaces and boundaries of the body and gather and proteins and imaging hardware and software have improved. antigenic structures, both self and foreign. Induced or sponta- In vitro imaging led to the initial definition of the immunological neous maturation of DC triggers their migration to the lymph synapse (IS) based on cell polarity and segregation of adhesion node and concurrent processing of antigens to generate pep- molecules and T cell antigen receptors (TCR) in the interface tides that bind to major histocompatibility complex molecules between T cells and antigen presenting cells [2]. In vivo imaging (MHCp) that are then presented at the cell surface. DC migrate has led to a basic understanding of the dynamics of T cell-APC to the lymph node via the lymphatics and then migrate in the interactions in the lymph node and the effect of antigen, which parenchyma and join DC networks in the T cell zones where they causes formation of long lived T cell-APC contacts that may be encounter many T cells. The level of costimulatory molecules the in vivo counterparts of immunological synapses [3]. Both expressed by the DC is determined by the level of cytokines like TNF produced in response to various endogenous or exogenous activators of innate immunity [5]. This level of innate stimu- Abbreviations: APC, antigen presenting cell; CFP, cyan fluorescent protein; lation appears to control whether the antigen dependent T-DC DC, dendritic cell; eGFP, enhanced green fluorescent protein; HS, hemisynapse; interactions lead to tolerance or priming of an immune response ICAM, intercellular adhesion molecule; IS, immunological synapse; LFA, lym- over a period of 5–7 days [6]. phocyte function associated; MHCp, major histocompatibility complex-peptide Once T cells are primed they can either exit the lymph node complex; SMAC, supra molecular activation cluster; TCR, T cell receptor; to migrate to sites of inflammation or remain within the lymph TIRFM, total internal reflection fluorescence microscopy; YFP, yellow fluo- rescent protein node to help B cells. While there has been no work to date on ∗ Tel.: +1 212 263 3207; fax: +1 212 263 5711. antigen specific effector T cells in peripheral effector sites, we E-mail address: [email protected]. will discuss a recent paper on initial stages of T cell help. There

1044-5323/$ – see front matter © 2005 Published by Elsevier Ltd. doi:10.1016/j.smim.2005.09.002 M.L. Dustin / Seminars in Immunology 17 (2005) 400–410 401 has also been little work on memory T cell interactions with DC ment [16]. It was later found that the primary role of perforin during secondary stimulation, however, the availability of mice was to induce the target cell to take a “poison pill” by intro- with stably integrated fluorescent proteins that do not dilute out ducing granzyme A or B into the cytoplasm, which initiates a during cell division should enable such studies. pro-apoptotic caspase cascade [17]. Evidence that cell T cell Peripheral tissue scanning by DC is only one mode of innate polarity was related to directed secretion was provided by sem- immune surveillance of tissues. Two striking examples are the inal studies of Geiger and Kupfer showing that the microtubule surveillance of the brain by the dynamic processes of microglial organizing center and Golgi apparatus reorients toward the target cells [7] and the active patrolling of liver sinusoids by natural cell for killing [18,19]. Kupfer continued with a series of studies killer T cells, an innate like T cell [8]. on molecular makeup of the T cell-B cell interface with the first In this review we will summarize a new view of sustained description of CD4, LFA-1, IL-4, talin and protein kinase C-␪ T cells activation through the IS and the implications of how polarization to the interface between T cells and B cells [20–24]. migrating cells integrate signals through a “hemi-synapse” (HS). All of these studies were performed with fixed cells so temporal Then how the IS and HS work together in T cell tolerance and information was deduced from populations of images for cells immune surveillance will be discussed. fixed at different times. In 1998 Kupfer published a paper on the organization of LFA- 2. New model for sustained signaling through the IS 1, talin, TCR and protein kinase C-␪ in the interface between antigen specific T cells and antigen presenting B cells [25]. Studies on the IS bring together three parallel lines of experi- LFA-1 and talin were shown to form a ring in the interface and mentation in immunology through high-resolution fluorescence TCR and protein kinase C-␪ were shown to cluster in the mid- microscopy: TCR signal transduction, T cell adhesion and dle. These structures were defined as supramolecular activation directed secretion mechanisms involved in T cell effector func- clusters (SMAC). The TCR cluster marked the cSMAC, while tion. TCR signaling is based on a tyrosine kinase cascades the LFA-1 ring marked the pSMAC. It was implied that TCR that leads to rapid activation of phospholipase C ␥ [9]. The signaling was initiated and sustained by the cSMAC. My col- key tyrosine kinases are Lck, which initiates phosphorylation laborators and I published a paper in parallel in which live T of immunotyrosine activation motifs (ITAMs) in the cytoplas- cells interacting with supported planar bilayers were imaged in mic domain of the TCR, ZAP-70, which is recruited to phos- real time to visualize segregated adhesive domains composed phorylated ITAMs and phosphorylates LAT and ITK, which of LFA-1-ICAM-1 and CD2–CD58 interactions [26]. It was phosphorylates phospholipase C ␥ that is recruited to phospho- posited that the segregation of the adhesion molecules was driven rylated LAT. Phospholipase C ␥ activation leads to generation by the different topology of the LFA-1-ICAM-1 (40 nm domain) of inositol-1,4,5-triphosphate leading to Ca2+ mobilization and and CD2–CD58 (15 nm domain) interactions [27]. The antigen diacylglycerol leading to activation of protein kinase C and Ras dependent organization of these domains into a bull’s eye pat- exchange factors [10]. The triggering of the cascade is based on tern, similar to that reported in several international meetings recruitment of Lck associated co-receptors to the TCR and on by Kupfer, was an active process. We proposed the definition of TCR oligomer formation. “immunological synapse” for the bull’s eye pattern described by Members of the integrin and immunoglobulin families medi- Kupfer and colleagues and our studies with adhesion molecules, ate T cell adhesion to APCs. These interactions greatly extend linking a specific molecular pattern to the widely discussed con- the sensitivity of TCR to small numbers of MHCp bearing cept [28,29]. Taking these two studies together the IS was defined agonist peptides [11]. Costimulatory molecules also are con- as a specialized cell–cell junction composed of a cSMAC and a figured as adhesion molecules, but have regulatory features that pSMAC [26]. IS has subsequently been applied to a more diverse make them less effective as adhesion molecules [12]. By def- array of structures, but here we will adhere to this original defi- inition, adhesion enhances the physical interaction of T cells nition. with APC and the interaction of TCR and MHCp, while cos- The formation of the cSMAC was first evaluated in live T cell- timulation enhances TCR signaling or produces independent supported planar bilayer models [30]. It was shown that TCR are signals that integrate with the TCR signal to influence T cell engaged first in the periphery within 30 s and then these TCR activation. However, the major T cell adhesion molecules have clusters translocate to the center of the IS to form the cSMAC some co-stimulatory activity. For example, LFA-1 contributes by 5 min. Examination of cell–cell systems showed a similar to the adhesion of T cells in many contexts, contributes to TCR- pattern with peripheral TCR clusters merging in the center to MHCp interactions and provides signals that enhance Ca2+, form the cSMAC [31,32]. This process could take up to 30 min phosphatidylinositol-3-kinase and MAPK pathway activation with naive T cells. IS formation is enhanced by CD28–CD80 [13]. There are also negative costimulators. For example, CTLA- mediated co-stimulation [33], but CD28–CD80 interactions are 4 and PD-1 negatively regulate T cell expansion at intermediate dependent upon TCR-MHCp interactions [12,34,35], perhaps and late periods of activation [14,15]. due to induce local rearrangements of actin that are compati- Directed secretion is a hallmark of the neural synapses and is ble with enhanced CD28–CD80 interaction. Thus, costimulation one of the most compelling parallels between the IS and neural mediated by CD28 interaction with CD80/86 is a positive feed- synapse [2]. Early studies on the mechanism of T cell mediated back loop in IS formation and function. killing suggested that killing worked by exocytosis of preformed The IS pattern was highly correlated with full T cell activation granules containing lytic molecules with activity like comple- in vitro in multiple studies using both T cell-B cell, T cell-MHCp 402 M.L. Dustin / Seminars in Immunology 17 (2005) 400–410 and ICAM-1 bearing planar bilayer and NK cell-target cell IS [25,30,36]. While it was recognized early on TCR signaling was initiated well before the cSMAC was formed, it was still posited that the cSMAC might be involved in sustained signaling. While kinases can be localized to the cSMAC at 1–5 min, there is a consensus that the cSMAC has relatively low levels of phospho- tyrosine, activated phospho-Lck or activated phospho-ZAP-70 at later times [32,37,38]. Phosphotyrosine staining was retained in the cSMAC at 1 h in CD2AP deficient T cells. Since CD2AP regulates TCR degradation it was argued that the cSMAC is engaged in continuous signaling, which is made occult by TCR degradation processes [38]. A striking property of the cSMAC is that TCR-MHCp inter- actions in the cSMAC are stable as measured by fluorescence photobleaching recovery [30]. Since each TCR-agonist MHCp interaction has a half-life of 5–30 s it would be expected that half of the TCR-MHCp interactions in the interface would exchange Fig. 1. Modes of T cell interaction deduced from in vitro and in vivo studies. with free MHCp over a period of several minutes. In fact the (A) En face view of the IS with cSMAC, pSMAC and dSMAC as defined by TCR-MHCp interactions in the cSMAC do not exchange with Kupfer [25,37]. TCR nanoclusters as defined by Varma et al. [43] are indicated. MHCp in the bilayer over a period of 1 h. The basis of this sta- Varma et al. proposes that these structures mediate sustained signaling. Arrows bilization is not clear, but may be as simple as the very high on schematic indicate direction of TCR nanocluster movement. (B) En face view of hemi-synapse formed by migrating T cell on antigen positive APC. In local density of TCR and MHCp creating diffusion barriers that migrating cells the leading edge is a lamellipodium, followed by the lamella and exclude free MHCp or favor rebinding of the same MHCp to trailed by the uropod. Sims et al. [51] and Varma et al. present data that equates the TCR following spontaneous dissociation [38]. In contrast, structures of the IS with structures of the HS. (C) Antigen specific T cells push sustained signaling by T cells appears to be maintained by new antigen bearing small resting B cells through the 3D tissue environment [93]. TCR-MHCp interactions since it can be acutely inhibited by This suggests that IS and HS character can coexist in the same cell with the dSMAC/lamellipodium edge as a line of symmetry. We propose that under these antibodies to MHCp that compete for TCR binding. This acute conditions a distinct uropod like structure will form at the distal pole to the IS. inhibition by anti-MHCp appears to be inconsistent with a cen- This may be the condition for formation of the distal pole complex discovered by tral role of stable TCR-MHCp interactions in the cSMAC with Burkhardt and co-workers [98]. For simplicity the 3D tissue/matrix environment TCR-MHCp interactions, however, in the absence of other TCR against which the lateral surfaces of the migrating T cells are engaged is not containing structures after 5–30 min attention has continued to depicted. focus on the cSMAC as a signaling structure, even though the for- mation of a cSMAC is not required for T cell activation [38,39]. of TCR-MHCp movement from the periphery to the cSMAC, To search for other TCR containing structures in the IS my lab TIRFM revealed continued formation of TCR-MHCp clusters in has employed total internal reflection fluorescence microscopy the periphery of the IS [43]. TCR clustering had been recognized (TIRFM) with the live T cell-supported planar bilayer system. as an important concept on theoretical grounds based on work This is a uniquely advantageous combination for increasing with growth factor receptors [44,45] and the Fc␧RI receptor sensitivity to small, low contrast structures. Through the lens [46], but the minimal clusters sufficient to sustain TCR signal- total internal reflection fluorescence microscopy is based on ing had been assumed to be too small for direct visualization using very high-resolution oil immersion objectives with a laser [26,47]. Initially, TCR microcluster can contain up to 150 TCR focused at the outer edge of the back aperture to steer the shaft each at which point they are readily detectable by con∼ventional of illumination to the sample at an angle that exceeds the “criti- methods, but by 60 min of sustained signaling the TCR clusters cal angle” [40]. Under these conditions all the light is reflected contain only 10 TCR per cluster and could only be detected off of the interface between the coverglass and the cell, but an by TIRFM [43]∼. These later clusters can be defined as nanoclus- evanescent wave is generated that can excite fluorescence within ters (10 TCR) to distinguish them from order of magnitude larger 200 nm of the interface (Fig. 1). This method is generally not microclusters (>100 TCR) formed during early responses to sur- useful in cell–cell interfaces because these structures are many faces with high densities of MHCp [31,43,48,49]. Nanoclusters microns away from the interface, but it can be very effective are detected over the entire range of MHCp densities leading for examination of the interface with cells and the supported to cSMAC formation [30,43]. Levels of MHCp below the limit planar bilayer, which is only 2 nm off the surface of the cover- required to form an IS appear to trigger signaling in transient glass [41]. Thus, the entire IS can be illumination with lateral peripheral foci [43]. The role of TCR nanoclusters in signaling and axial resolution of 200 nm, a uniquely optimal situation in was supported by the demonstration that peripheral nanoclus- light microscopy. TIRFM∼ is used for single fluorophore imaging ters were stained with anti-phosphotyrosine antibodies, they so as a long as contrast exists a small fluorescent structure can recruit ZAP-70-GFP and anti-MHCp Ab that block Ca2+ sig- be detected [41,42]. naling within 2 min disperse the TCR nanoclusters in the same Application of TIRFM to the IS led to a striking discovery. time frame, but have no effect on the cSMAC [43,49]. Therefore, While the field had seen the IS formation process as a single wave contrary to initial perceptions that small TCR clusters were only M.L. Dustin / Seminars in Immunology 17 (2005) 400–410 403 formed early in IS formation, we could demonstrate the small gration of signals. The uropod and cSMAC may differ in that a TCR clusters are newly formed in the periphery of the IS dur- cSMAC receives and can preserve, to some degree, TCR-MHCp ing sustained signaling. These structures are co-localized with complexes and associated molecules, although there is no evi- peripheral sites of tyrosine phosphorylation, phospho-ZAP-70 dence that the molecules accumulated in the cSMAC continue and GFP-ZAP-70. The small TCR clusters are rapidly elimi- to signal in normal T cells, while the uropod may only maintain nated by blocking TCR interactions with MHCp [43,49]. This long term connections using membrane nanotubes, which may finding redirects our attention from the cSMAC to the more prolong signaling connections between immune cells, but are dynamic nanostructures in the periphery of the IS. likely to break beyond a few 10’s of micrometers [60,61]. The The TCR nanoclusters are formed in the periphery of the IS- major differences between IS and HS is that IS form a cSMAC, a structure that Kupfer described as a distal SMAC (dSMAC) while HS form nanotubes and T cells forming IS spend more [37]. The dynamics of the dSMAC are very similar to the time with the APC than cells forming HS, which move serially lamellipodium of a spreading or migrating fibroblasts in that from APC to APC. it displays cycles of extension and retraction referred to as con- tractile oscillations [50,51]. The dSMAC is also rich in CD45, 3. In vivo functions of IS and HS the transmembrane tyrosine phosphatase the primes activation of Src family kinases including Lck and Fyn [37,48]. Therefore, TCR signal integration through nanoclusters formed in IS or actin dependent TCR clustering by agonist ligands will exclude HS provides a framework for thinking about results from recent CD45, setting conditions for rapid tyrosine phosphorylation of in vivo studies. Since we can now understand the ability of T partially activated Lck and TCR signaling. cells to signal while migrating we can consider regulation of T The TCR nanoclusters traverse the pSMAC, a domain rich in cell migration not in terms of signaling changes, but in terms on the larger LFA-1-ICAM-1 interaction. The pSMAC is not a solid controlling the network of spatiotemporal cell–cell interactions ring, but a meshwork of micron scale LFA-1 rich domains with that recruit different cells into the immune response or allow interspersed holes lacking LFA-1-ICAM-1 interaction [30]. The them to execute effector functions. Another consideration is nanoclusters appear to navigate these holes in a tortuous fash- directed secretion, which is likely best sustained through a stable ion [43]. Thus, the TCR nanoclusters do not take a straight-line IS, but may also be executed from a HS for shorter periods. path to the cSMAC, but zigzag through the pSMAC, perhaps Naive T cell priming is a central process in initiation of because of the obstacles formed by the dynamic LFA-1 clusters. immune responses. Priming of naive T cells requires interac- It is notable that ZAP-70 recruitment and activation appears to be tions with dendritic cells in vivo [62]. However, not all antigen maximal as the TCR nanoclusters traverse the pSMAC [43,49]. specific T-DC interactions will lead to priming. When antigen Thus, the close juxtaposition of TCR nanoclusters and surround- is presented by dendritic cells in the steady state (absence of ing integrin microclusters appears to be an optimal condition for inflammation) the result is induction of peripheral tolerance TCR signaling, but signaling turns off as or shortly after the TCR [63,64]. Presentation under steady state conditions leads to T clusters join the cSMAC. cell proliferation followed by induction of antigen specific non- It has been observed in many studies that when T cells responsiveness in the effector cells or their deletion [65]. A migrate on ICAM-1 containing surfaces they produce “focal second mechanism of peripheral tolerance is the induction of zones” of LFA-1-ICAM-1 interaction that accumulate in the antigen specific regulatory T cells [66]. The steady state process lamella, the force generating structure in amoeboid cell loco- of inducing tolerance preconditions the active peripheral T cell motion [30,52–57]. These focal zones can vary in shape from repertoire to react selectively with pathogen associated foreign crescents to wedges that are analogous to a half or quarter IS. antigens under conditions of infection or tissue damage since T Hypothetically, in a complete IS the inward directed forces gen- cells specific for self antigens and benign foreign antigens are erated in the pSMAC are balanced and the cell moves slowly or deleted or anergic [4]. The dynamics of priming or priming ver- not at all [58]. If half or more of the IS is eliminated then the sus tolerance has been the subject of many of the initial wave of remaining portion mediates rapid cell movement because the intravital microscopy studies. forces generated by the lamella are unbalanced. Thus, I propose Stand alone studies on priming of naive T cells have been that the contact structure used by a migrating T cell to integrate carried out using three different TCR transgenic models, two signal from an APC can be defined as a hemi-synapse (HS). different adjuvant systems and both explanted intact lymph node HS signal by forming TCR nanoclusters in the leading lamel- and intravital imaging approaches. Consistent themes are emerg- lipodia, the nanoclusters signal as they translocate through the ing and hypotheses can be developed regarding the basis for LFA-1 rich focal zone and are inactivated in the uropod, the differences between studies. trailing structure that is analogous to the cSMAC (R. Varma and M.L.D., unpublished observation). This model suggests a way 4. In vivo analysis of CD4+ T cell priming and tolerance to resolve the controversy regarding IS formation and signal- induction ing between groups that typically observe IS formation during T cell activation and those who observe migration (HS) only Stoll et al. [67] examined dynamics of T cell priming by [59]. Since TCR signaling is initiated and sustained in lead- transferred mature DC. CFSE labeled 5C.C7 TCR Tg T cells ing lamellipodium by TCR nanoclusters, the formation of these were introduced into the lymph nodes by intravenous adoptive structures by migrating T cells is fully compatible with inte- transfer and DiI or DiD labeled DC were introduced to spe- 404 M.L. Dustin / Seminars in Immunology 17 (2005) 400–410 cific draining lymph nodes by subcutaneous injection. 5C.C7 is tive the Alum is at preventing drainage of soluble antigen to the an I-Ek restricted TCR that binds moth cytochrome C (MCC) lymph node ahead of immigrant DC, which may have an impact peptide 91–103 as an agonist so DC were either pulsed with on early priming [75]. Nonetheless, this was a highly effective this peptide or a control peptide. The lymph nodes were then method for examining T cell interactions with DC that migrated explanted, embedded and imaged with a conventional confocal from the injection site. microscope. This is the only study of lymph node T cell dynam- The fundamental findings from this series of experiments ics by conventional confocal microscopy, which limits depth of were (1) T cells move rapidly and, to a first approximation, ran- penetration to tens of micrometers. T cells displayed low basal domly in the T cell zones [68], (2) similar movement of T cells motility in the absence of antigen, which may be attributed to is observe in vivo using intravital microscopy of the inguinal low perfusion (media movement over the surface of the lymph lymph node [69], (3) DC move slowly, but contact many T cells node). Nonetheless, many more 5C.C7 T cell interacted with by probing with many veil-like processes such that each DC can antigen pulsed DC compared to unpulsed DC. The antigen spe- contact up to 5000 T cell per hour [70], (4) antigen specific inter- cific interactions appeared to be stable in that T cells and DC actions can be more dynamic that those observed by Stoll et al. formed extensive interfaces that excluded CD43, a characteris- with stable interaction between 3 and 16 h, with resumption of tic of in vitro IS. Naive 5C.C7 T cells expressing CD43-GFP antigen specific swarming and rapid migration by 16–24 h and were prepared by retroviral transduction of bone marrow stem beyond [71]. Even at the most stable phases of interaction the cells followed by reconstitution of irradiated mice. A remarkable apparent contact area size and the changes over time suggested transformation took place at 36 h when the activated antigen spe- a more dynamic situation than in vitro IS analysis, however, no cific blasts initiated rapid migration. This 36 h period took place molecular imaging was performed. The same model was also in vivo, not in the organ culture, but nonetheless the imaging used to study oral tolerance in mesenteric and peripheral lymph conditions were the same so it is apparent that recently activated nodes [76]. In conditions of priming and tolerance T cell clusters blasts “break” IS like interactions after 36 h when they initiate were formed and it was assumed based on the similarity of T cell rapid migration under conditions where naive T cells were inca- patterns to those in Miller et al. [71] that these clusters formed pable of migration. Subsequent encounters with antigen positive around DC. Under conditions of priming the clusters were larger DC did not lead to IS. This study suggested that CD4 T cells and longer-lived, but stable, IS-like, interactions were observed remain in IS until they start to proliferate such that all commu- in both priming and tolerance induction. The prevalence of a nication might take place through IS like structures. spectrum of interaction types from rapid migration to swarming A series of studies by Miller et al. using two-photon laser in addition to stable interactions suggest that HS-like interac- scanning microscopy (TPLSM) from 2002 to 2004 [68–71] shed tions also have a role in in vivo priming and tolerance. light on repertoire scanning and priming in a similar hybrid Steady state DC have been visualized in vivo using CD11c in vivo/explant system with some modifications compared to promoter YFP transgenic (CD11c-YFP) mice [77]. Lymph node Stoll et al. For information on TPLSM, which allows imag- DCs include immigrants from the tissues and resident cells that ing of cells at depths of up to 500 ␮m in tissues, I refer the may enter directly from the blood. While tissue inflammation reader to two outstanding reviews [72,73]. Subsequently in this stimulates synchronous migration of many DC to the draining review, all studies use TPLSM unless otherwise noted. Adop- lymph node, there is a poorly understood steady state migration tively transferred DO11.10 TCR Tg T cells were activated with of DC from the tissues to the lymph nodes that is likely to be ovalbumin in alum [68,71]. Alum is an adjuvant that primes critical for peripheral tolerance to tissue antigens. These immi- Th2 responses perhaps due to the activation of IL-4 producing grant DC are mature in that they express high levels of MHC APC [74]. Unlike the Stoll et al. system where the node was class II on their surfaces, but they are not activated since they immobilized by implanting it in a drop of agarose and imag- only express low to intermediate levels of CD80 and CD86. The ing it from the immobilized side [67], Miller et al. attached the CD11c promoter was used to generate a number of transgenic lymph node to the bottom surface of a dish via the hillus and strains that were tested for bright enough fluorescence to be use- then imaged from above with a continuous perfusion of highly ful in two-photon intravital microscopy in up to 300 ␮m depth. oxygenated media [68]. These conditions, and perfusion may be One founder of several tested had high expression in CD11chi the key difference, resulted in the first observations of dramatic myeloid DC. In the T cell zones of inguinal lymph nodes of motility of naive T cells in the lymph node, which his similar live mice, steady-state DC form extensive sessile networks [77]. to that observed by intravital microscopy [69], and established Distinct DC behaviors were described in the subcapsular sinus the current paradigm for repertoire scanning [70]. In studies on (migrating DC), superficial surface of B cell follicles (layer of priming purified DO11.10 T cells were labeled with CMTMR dim stationary DC), interfollicular zones (clusters of DC trap- and adoptively transferred i.v., while DC were labeled in situ ping T and B cells) and T cell zones (DC networks). Immigrant by subcutaneous injection of CFSE with the Alum/antigen mix- DC migrated from the subcapsular sinus to the T cell zones ture. Therefore, all DC that were present at the injection site and rapidly and joined the T cell zone networks. Thus, rapid T cell them migrated to the lymph node would be labeled. Resident migration through the DC networks and DC outreach to passing DC in the lymph node that were exposed to antigen draining T cells through formation of long membrane processes are the via the lymph or immigrant DC that entered the tissue hours mechanisms that drive repertoire scanning. after the injection would not be labeled because the reactive dye CD4+ T cell priming and tolerance was studied in vivo using would be hydrolyzed in a few minute. It is not clear how effec- the CD11c-YFP Tg mice and a strategy for targeting antigen M.L. Dustin / Seminars in Immunology 17 (2005) 400–410 405 to DC by attaching antigenic peptides to mAb specific for expansion [79,83]. However, induction of antigen dependent tol- the scavenger receptor DEC-205 [63,78]. Under these condi- erance of CD8+ T cells appears to require sustained contact with tions all of the DEC-205 positive DC, which include CD11c antigen positive DC after 72 h [6]. Thus, while priming of CD8+ high and low DC types, will present antigen. In the absence of T cell responses may be imprinted by early interactions with innate immune stimulation presentation of antigen to three dif- mature, activated DC, the peripheral deletion of auto-reactive ferent MHC class II restricted TCR tested thus far (3A9/HEL, CD8+ T cells appears not to be imprinted, but to require sustained 2D2/MOG, OTII/OVA) leads to tolerance [63,64,78]. Tolerance interactions with DC for longer than 3 days. These biological involves initial activation and expansion of T cells followed by issues need to be considered in interpretation of the imaging death of the expanded cells between days 3 and 7 after initial acti- data. vation. Induction of tolerance requires both the early exposure Bousso and Robey studied the priming of LCMV gp33 spe- to antigen, which induces activation and proliferation and late cific P14 TCR Tg T cells using transferred dendritic cells [84] exposure to antigen after day 3 to induce deletion (M. Nussen- and a similar explanted lymph node imaging approach as Miller zweig, personal communication). et al. [68]. They focused on one time point that was 24 h after To perform these experiments OTII TCR Tg/chicken ␤ actin transfer. It is assumed that DC migrated rapidly to the lymph promoter-GFP (GFP) Tg T cells (specific) and non-TCR Tg/CFP node since they were injected into very proximal subcutaneous Tg T cells (non-specific) were transferred into CD11c-YFP Tg sites. Bousso et al. provided the first estimate of DC repertoire hosts that had been injected 4 h prior with DEC-205-Ova peptide scanning rate at 500 T cells per DC per hour. This is 10-fold such that the specific and non-specific T cells enter lymph nodes lower than the subsequent estimate by Miller et al. of 5000 T that already contain antigen presenting DC. Blood flow in the cells per DC per hour, and the major difference may be in the intravital inguinal lymph node preparation was followed using presence of fine dendritic processes that greatly increase the red quantum dots, which excite well at the same TPLSM wave- effective target size of the DC in the latter study. This may be an length. Tolerance was induced with the DEC-205-Ova alone and imaging issue or an issue related to handling of the DC altering priming was induced by addition of anti-CD40 mAb to induce their morphology. Like Stoll et al., Bousso et al. emphasize the activation of DC. This study is distinguished from other stud- formation of stable IS like interactions, but only at this one time ies on CD4+ T cells and other studies on tolerance by using point. A unique aspect of this study is that it is the only intact intravital microscopy in which blood and lymph flow are intact. lymph node study where antigen dose was varied. They found Imaging was initiated in three time frames from 0 to 6 h, 6 to that stable IS like interactions were formed over the entire range 12 h and 12 to 18 h after T cell transfer. Specific T cells showed that would lead to priming. rapid arrest near high endothelial venules (HEV) within an hour The first kinetic study of T cell-DC interaction in vivo was of injection regardless of whether the conditions favored toler- Mempel et al. [85]. Mempel et al. performed extensive studies ance or priming. Over the next 18 h the specific T cell regained with the P14 line also used before by Bousso et al. [84]. Unlike rapid motility in the DC networks and did not systemically form Bousso, Mempel et al. imaged the popliteal lymph node of live stable interactions at later times. Some statistically significant anesthetized mice with intact blood and lymph flow. While stud- differences were detected between tolerance and priming in that ies were performed with the DO11.10 CD4+ T cells this data set under conditions of priming the rate of return to control migra- was very limited compared to the more complete study by Miller tion velocity was slower than for tolerizing conditions. Since et al. [71]. Mempel et al. used LPS-treated mature CMTMR activation and proliferation are induced under both tolerizing labeled DC that were injected into the foot pad, which then and priming conditions there was a positive correlation between drained uniquely to the ipsilateral popliteal lymph node. They IS like stable interactions and induction of T cell activation. It injected CFSE labeled P14 T cells, allowed the cells an hour to appeared that critical signals for tolerance were integrated dur- enter the lymph node via the HEV and then injected mAb to ing later, dynamic HS-like T-DC interactions. In fact, continued L-selectin throughout the rest of the experiment to block further interaction of T cells with antigen positive DC appears to be crit- entry. This ensured that the one temporally defined cohort of T ical for full expansion of CD4+ T cells [79]. Since all 3 studies cells is followed. Mempel et al. is the first study to clearly demon- on CD4+ T cells, Stoll et al., Miller et al. and Shakhar et al., all strate 3 phases of interaction during priming. Antigen specific concur that T-DC interactions are dynamic after proliferation is T cells began to encounter DC soon after entering the lymph initiated it seems very likely that both IS-like stable and HS-like node, but for the first 8 h the encounters were short lived. These migrating interactions play a key role in CD4+ T cells priming short-lived encounters did activate the T cells since CD69 was and tolerance. upregulated in this time frame. Signal integration in this period (phase 1) suggests HS-like interactions that might involve TCR 5. In vivo analysis of CD8+ T cell priming and tolerance nanocluster formation. In the 8–12 h time period the contact were induction all long lived IS-like interactions. After this period (phase 2) cytokine production was initiated. At 24–26 h there was a mixed The priming of CD8 T cells has been shown to require on a picture of stable, intermediate and short-lived interaction and short time of interaction with antigen presenting cells followed by 44–48 h all interactions were again short lived. This period by a long antigen independent expansion process [80–82]. As (phase 3) was characterized by proliferation. Thus, although it mentioned in the previous section this is very different from the was been found that CD8+ T cells only require a few hours of current understanding of antigen requirements for CD4+ T cell stimulation to fully commit to many rounds of cells division, 406 M.L. Dustin / Seminars in Immunology 17 (2005) 400–410 the in vivo profile is of T-DC interaction is very similar to what T cells undergoing tolerance induction all integrate signals at was reported subsequently for CD4+ T cells, which integrate late time (>24 h) and all show rapid migration with short dura- signals over longer periods. It is possible that CD8+ T cells in tion interactions with DC in this time frame. Thus, migratory HS vitro immediately form IS like interactions with antigen rich like signal integration is likely to be critical for full activation DC and integrate signals quickly to commit fully to an effector of CD4+ T cells and tolerance induction in CD4+ and CD8+ T program. In contrast, sparse antigen positive DC in vivo may cells. require a longer period of signal integration requiring phase 1 and 2 before becoming antigen independent in phase 3. More 7. Effector sites work would be required to determine if CD8+ T cells do or do not integrate meaningful signals through HS-like interactions in At the time of writing of this review there are only a few phase 3. papers on intravital microscopy of T cell-APC interactions at Hugue et al. studied tolerance versus priming of CD8+ OTI effector sites. While there are a number of other studies that TCR Tg T cells [86] using an explant system very similar to report on various lymphoid cells in the intestine and in tumors I that of Miller et al. [68]. They use the DEC-205 antigen deliv- have not included these because there was not concept of antigen ery approach after CFSE labeled OTI T cells were transferred specific interaction, rather the movement of cell types was doc- and labeled all DC in the explanted lymph nodes by inject- umented often with only a limited understanding of the cell type ing fluorescently labeled anti-CD11c IgG into the parenchyma involved. The primary studies that I will discuss have focused on prior to imaging. Thus, unlike Shakhar et al. Hugues et al. natural killer T (NKT) cells in the liver [8], helper T cells in the delivered antigen to DC after T cells were equilibrated in the lymph node [87] and activated effector cells in the central ner- lymph nodes, rather than injecting T cells into mice that had vous system (CNS) [88]. These studies all suggest that effector been equilibrated with the antigen, and performed the imaging cells form stable IS like interactions with antigen positive APC. with explanted lymph nodes in which DC had CD11c and per- Geissmann et al. [8] took advantage of the expression pattern haps FcR engaged by anti-CD11c mAb, rather than performing of the chemokine receptor CXCR6 in the liver to follow NKT intravital microscopy in a mouse in which DC express YFP. cells in vivo. CXCR6 is highly expressed on activated T cells In this study OTI T cells were found to engage in a spec- and NKT cells. In the liver of healthy mice NKT cells represent trum of interactions with DC that were biased toward stable 30% of the mononuclear cells and 70% of the CXCR6+ cells. interactions in priming conditions and biased to dynamic inter- Unutmaz et al. replaced the major coding exon of CXCR6 with actions in conditions of tolerance. Hugues et al. interpreted this GFP by homologous recombination [89]. The CXCR6gfp/+ mice set of experiments to indicate priming involved stable IS-like have normal number of NKT cells in the liver, which are GFPhi, interactions, whereas tolerance induction involves HS-like inter- whereas the CXCR6gfp/gfp mice have 3–5 fold reduced numbers actions. It is not clear if the differences between Hugues et al. of NKT cells in the liver, which remain GFPhi [8]. The rea- and Shakhar et al. are due to differences between CD4+ and son for the reduced numbers of NKT cells in the CXCR6gfp/gfp CD8+ systems or due to technical differences in the way the mice appears to be reduced NKT cell survival in the absence experiments were performed. In Shakhar et al. the area around of CXCR6. The high levels of GFP expressed in the NKT cells the HEV played a key role in early IS like interactions and afforded the opportunity to track these cells in the liver of mice it might be argued this region may not function in the same by intravital microscopy and to determine the effect of antigen. way in the absence of blood flow. Zinselmeyer et al. used a It had been reported that a population of leukocytes identified totally different antigen deliver route and also found that both as Kupffer cells migrated within the sinusoids of the liver [90]. priming and tolerance involved stable, IS-like T cell migration Geissmann et al. discovered that Kupffer cells stained by high patterns. It will be important to revisit the issue of T-DC dynam- molecular weight rhodamine dextran are stationary cells, while ics during CD8+ T cell tolerance induction in light of these NKT cells migrate rapidly within the sinusoids with or against discrepancies. blood flow. This rapid migration within the fenestrated sinu- soids allowed WT NKT cells to visit each hepatocyte in the 6. Priming versus tolerance liver on average every 15 min. Geissmann et al. never observed GFP+ cells extravasating. NKT cells have a dominant V␣14 Priming and tolerance require T cell activation. Tolerance rearrangement that produces a high affinity TCR for CD1d with induction by deletion of effector cells appears to require pro- ␣-galactosylceramide (AGC), which corresponds structurally to longed TCR signaling beyond 3 days. Thus far, activation is a class of bacterial lipids [91,92]. When AGC was injected i.v. associated with stable, IS like interaction in 9 of 10 data sets. most of the NKT cells become activated within 2 h based on Only one data set on tolerance induction is associated with acti- cytokine production and 60% of the GFP+ cells stopped migrat- vation, but no stable, IS-like interactions and this is contradicted ing and formed long lived IS like interactions within 20 min [8], by two other studies that readily detected these interactions under apparently with Kupffer cells (Supplemental movie 1). NKT conditions of tolerance induction following primary activation. cells have a previously activated memory/effector phenotype in In most cases the formation of stable IS like interaction is highly vivo, such that the rapid IS-like response provides the first evi- correlated to T cell activation, but not to the generation of effec- dence of interactions with IS-like stability in the effector phase tor cells or memory, which is only correlated with more subtle of an immune response. We have also found that conventional changes in the dynamics of interaction. CD4+ T cells and CD8+ effector T cells patrol liver sinusoids (Supplemental movie 2). M.L. Dustin / Seminars in Immunology 17 (2005) 400–410 407

This suggests that patrolling of sinusoids is typical of immune to be able to reference this staining to the dynamics of interaction surveillance by activated T cells in the liver. in ex vivo live tissue. While the use of intact antibodies could Okada et al. [87] studied the T-B interactions during recog- be criticized, for example, Fab fragments with non-blocking Ab nition of hen egg white lysozyme (HEL) or I-Ab-HEL 74–88 would be a better choice, this study provided a step toward imag- complexes by MD4 IgG Tg B cells and TCR7 TCR Tg T cells, ing of SMACs in vivo. respectively. The mice were immunized with HEL in alum and Innate immune surveillance of the CNS was studied by Dava- explanted lymph nodes were imaged exactly as in Miller et al. los et al. [7] and Nimmerjahn et al. [96]. Microglial cells are the [70,71]. T cell proliferation was extensive by day 2, whereas B innate immune cells of the central nervous system in the steady cells did not proliferate extensively until day 3. The B cells state. These cells can be visualized in live mice in which the slowed down in the first 1–3 h and then regained speed and CX3CR1 chemokine receptor’s major coding exon is replaced migrated toward the junction between the T zone-B zone inter- by GFP. Microglial cells are the only cells in the CNS that face. This migration was guided by CCL21 gradients in the express GFP in CX3CR1+/gfp mice. Both of these studies used follicle and required CCR7 expression on the B cells. This is the thinned skull technology developed by Gan and colleagues the first demonstration of chemokine directed migration in an to image [97] microglial cells in the intact brain of anesthetized intact lymph node. By 30 h the B and T cells are activated and mice. Parenchymal microglial cells are referred to as “ramified” long-lived antigen specific T-B conjugates begin to form at the T because each cells projects many long processes in three dimen- zone-B zone interface. The interactions are strikingly different sions to cover a territory of 65,000 ␮m3 (Supplemental movie than priming T-DC interactions in that the B cells continue to 3). The processes extend and retract at a velocity of 1–2 ␮m/min. migrate rapidly and drag the T cells behind them. This could Focal injury in the CNS results in a rapid response by the ram- be explained by the T cell forming an IS-like stable interaction ified microglial cells in which the cell bodies remain in place, with the B cell via the B cell uropod, but the B cell continuing but all the processes within a radius of 75 ␮m from the site of to migrate under the influence of chemokinetic and chemotactic injury, dozens of individual processes, converge on the site while factors in the environment. This is also different from an earlier maintaining tethers to the cell bodies (Supplemental movie 4). study in which activated T cells interacted with naive B cells While the biological function of this response is not know with as viewed by conventional confocal microscopy in the inguinal certainty, the release ATP is necessary and sufficient to trigger lymph node [93]. In this case the activated T cells pushed the B the response and a reasonable biological hypothesis is that the cells, which rounded up and became sessile after T cell contact cells are sealing off local injuries within 30 min. This does not [93]. While Gunzer et al. opens an interesting physical possibil- appear to be a classical phagocytic response, and in fact clas- ity that the lateral surfaces of a T cell can function in migration sical phagocytes like neutrophils and monocytes appear to be while the front part forms an IS, it is less physiological than excluded from small injuries that are completely surrounded by Okada et al., which studied a classical T cell dependent antibody microglial foot processes (J. Kim, unpublished observations). response. Again, effector cells form stable IS-like interactions Whether microglial cells that respond to focal injuries are com- rapidly after encountering APC. petent to present antigen to CD4+ or CD8+ T cells is not known. Kawakami et al. [88] studied the dynamics of T cells in acute spinal cord slice from rats with early experimental aller- 8. Summary gic encephalitis (EAE) lesions. The EAE lesions were induced by injecting 5 million GFP+ myelin basic protein (MBP) spe- Recent evidence suggests that the basic unit of sustained T cific cloned T cells i.v. Acute spinal cord slices were prepared cell receptor signaling are small TCR clusters or nanoclusters 4 days after T cell transfer, at which time antigen specific and dynamically generated near the leading edge of migrating T non-antigen specific ovalbumin specific cloned T cells were also cells forming HS-like structures or in the dSMAC of T cells found in the lesions. This is characteristic of inflamed sites, forming stable IS-like structures. Retention of many TCR in which are equally attractive for extravasation of antigen spe- the cSMAC is only possible when the T cell stays with one cific and non-specific cells. Tracking of MBP specific T cells APC, which requires a stop signal, but even then the peripheral revealed what 40% were immobile over long periods whereas TCR nanoclusters are required to sustain signaling. The specific only 5% of ovalbumin specific cells were similarly stationary. function of the cSMAC is unknown, but it is not sufficient to The remaining cells were migrating in the cultured brain tis- sustain Ca2+ signals in T cells. In vivo, activation of T cells sue. Staining of the live sections in real time with antibodies to associated with activation and tolerance requires both stable IS- LFA-1, TCR and MHC class II revealed polarization of these like and migratory HS-like interactions. IS like interactions are molecules toward the shared interface between MBP specific T a common feature of effector phase and these interactions are cells and class II positive APC, but rarely with Ova specific T initiated rapidly after antigen recognition. cells. While these images were not sufficient resolution, nor were the staining methods really refined enough, to detect SMACs, but nonetheless, evidence or IS like structures in the tissue sections Acknowledgements was compelling. Prior histological analysis in lymph nodes [94] and in the meninges during the CD8+ T cell response to lym- I thank R. Varma, G. Campi, T. Sims, T. Cameron, J. Kim, phocytic choriomeningitis virus [95] have also detected IS like G. Shakhar and M. Nussenzweig for valuable discussions and structures in fixed tissue, but Kawakami et al. was the first study permission to discuss unpublished data. 408 M.L. Dustin / Seminars in Immunology 17 (2005) 400–410

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