Cytotoxic Immunological Synapses Eric O

Michael L. Dustin Cytotoxic immunological synapses Eric O. Long

Authors’ addresses Summary: One of the most fundamental activities of the adaptive 1 2 Michael L. Dustin , Eric O. Long immune system is to kill infected cells and tumor cells. Two distinct path- 1 Helen, Martin Kimmel Center for Biology and Medicine, ways mediate this process, both of which are facilitated by a cytotoxic Skirball Institute of Biomolecular Medicine, New York immunological synapse. While traditionally thought of as innate immune University School of Medicine, New York, NY, USA. cells, natural killer (NK) cells are now appreciated to have the capacity 2 Laboratory of Immunogenetics, National Institute of Allergy for long-term adaptation to chemical and viral insults. These cells inte- and Infectious Diseases, National Institutes of Health, grate multiple positive and negative signals through NK cell cytotoxic or Rockville, MD, USA. inhibitory synapses. The traditional CD8+ ab T-cell receptor-positive cells are among the best models for the concept of an immunological synapse, Correspondence to: in which vectoral signaling is linked to directed secretion in a stable inter- Michael L. Dustin face to induce apoptotic cell death in an infected cell. Large-scale molecu- Helen and Martin Kimmel Center for Biology and Medicine, lar organization in synapses generated a number of hypotheses. Studies in Skirball Institute of Biomolecular Medicine, New York the past 5 years have started to provide clear answers regarding the valid- University School of Medicine, 550 First Avenue, ity of these models. In vivo imaging approaches have provided some hints New York, NY 10016, USA as to the physiologic relevance of these processes with great promise for Tel.: +1 212 263 3207 the future. This review provides an overview of work on cytotoxic Fax: +1 212 263 5711 immunological synapses and suggests pathways forward in applying this e-mail: [email protected] information to the development of therapeutic agents.

Acknowledgements Keywords: activation, cytotoxicity, synapse, actin, microscopy, adhesion This study was supported by NIH grants PN2 EY016586 (M.L.D.), R01 AI052812 (M.L.D.) and the Intramural Research Program of the NIH, NIAID (E.O.L.).

Introduction to the immunological synapse Immunological synapses are antigen-specific cell–cell junctions with a synaptic cleft stabilized by bona fide adhesion molecules for vectoral cell–cell communication between an immune cell and an antigen-presenting cell (APC) (1). We use the term synapse to describe junctions that match these criteria. The cytotoxic synapse is one of the earliest and the best defined of immunological synapse types based on a number of key findings in immunology in the 1970s and early 1980s that exploited this system, and clear functional impor- tance that sustained interest even as other T-cell subsets were described. Zinkernagel and Doherty (2) defined the role of the

Immunological Reviews 2010 major histocompatibility complex (MHC) in killing of virally Vol. 235: 24–34 infected cells by sensitized T lymphocytes, which are Printed in Singapore. All rights reserved described as cytotoxic T lymphocytes (CTLs) to distinguish Published 2010. This article is a them from helper T lymphocytes. The description of adhesion US Government work and is in the molecules like leukocyte function-associated antigen-1 (LFA- public domain in the USA Immunological Reviews 1) by Springer (3), the polarization of cytotoxic T cells (4, 5), 0105-2896 and directed secretion of perforins and granzymes triggered

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by cytoplasmic Ca2+ elevation (6, 7) led to the proposal of a convincing of important underlying mechanism that Janeway synaptic basis for T-cell killing (8). The cloning of the T-cell introduced them into his Immunobiology textbook (28) as early receptor (TCR) (9) and the definition of peptides bound to as 1997, a year prior to peer-reviewed publication. The origi- the groove of MHC molecules as TCR ligands (10, 11) nal publication in 1998 introduced the term supramolecular allowed the generation of monoclonal T-cell mice and the activation cluster (SMAC) into the immunology vocabulary to biochemical preparation of defined TCR ligands that set the describe two distinct micron scale domains formed in a bull’s stage for further molecular dissection of the immunological eye pattern: a central (c)SMAC rich in TCR and a peripheral synapse. (p)SMAC configured as a ring of LFA-1 adhesion receptors The study of natural killer (NK) cell synapses was on a par- (26). Lck and protein kinase C-h (PKCh), a novel PKC isoform allel track to CTL. Natural killing was described in the mid that is uniquely recruited to the T–B interface (29), were 1970s (12). Early studies on the cell biology of NK-mediated co-localized with TCR in the cSMAC. The widely expressed killing noted dramatic secretory and cytoskeletal polarization integrin-cytoskeletal linking protein talin was co-localized that accompanied the cytotoxic process (13–16). The inverse with LFA-1. This structure was observed under conditions of relationship between natural killing and MHC class I expres- T-cell activation, and the organized SMAC were not observed sion was noted in 1986 by Ka¨rre (17). Yokoyama described when antagonistic MHC–peptide ligands were presented on MHC class I binding inhibitory receptor Ly49 as a prototype the B-cell tumors used as APC. In the same time frame, we molecular basis for ‘missing self’ recognition (18). Identifica- were utilizing the supported planar bilayer system to investi- tion of the structurally unrelated but functionally equivalent gate the organization of adhesive contacts formed by LFA-1 MHC class I inhibitory receptors in human NK cells, the killer- and CD2, a second important adhesion receptor utilized by cell Ig-like receptors (KIR), led to the definition of the immu- human CTL. Using a supported planar bilayer model, we notyrosine inhibition motif (ITIM) sequence as V ⁄ IxYxxL, demonstrated segregation of LFA-1 from CD2 and further and the finding that tyrosine phosphatase Src homology (SH) demonstrated active concentration of CD2 through its interac- domain-containing phosphatase 1 (SHP-1) is recruited by tion with the adapter CD2AP (27). In this paper and without phosphorylated ITIM to turn off activation signals (19). The knowledge of the SMAC nomenclature, we proposed the use description of many activating NK cell receptors and associ- of the term immunological synapse to describe the bull’s ated signal transduction modules suggested additional modes eye pattern of integrins around TCR and isometric adhesion of positive signaling that are integrated with the negative sig- systems like CD2 and CD28. That summer we succeeded in nals in the NK synapse (20, 21). NK cells could also link into reconstituting T-cell activation by supported planar bilayers adaptive immunity via FcR but were initially thought of as presenting ICAM-1 and MHC–peptide complexes. We again innate effector cells. Recently, NK cell ‘memory’ responses found the same end point as Kupfer but could watch the were described, which blur the line between adaptive CTL evolution of the patterns from inverted nascent structures in and innate NK cells (22–24). While both the CTL and NK which TCR were engaged in peripheral clusters that translocat- synapses can be cytotoxic in nature, the distinct triggering ed to the center of the interface to generate the SMAC (30). mechanisms and checkpoints make the two cells synapse These results suggested that the immunological synapse func- with potential targets quite differently. The analysis of the NK tioned as a molecular machine to convert early TCR signals cell synapse includes both a cytotoxic synapse and an inhibi- into a stable structure that would sustain signaling to achieve tory synapse in which the negative regulatory receptors are full activation (30). Thus, the mature immunological synapse dominant. was provisionally defined as a stable and antigen-specific While the ‘synaptic basis of T-cell killing’ was first noted in T-cell-APC junction composed of SMACs. 1984 (8) with a prominent review adopting the term in 1994 The concept that actinomyosin-mediated transport plays an (25), this concept remained mostly latent until work by important role in forming the SMAC has been supported by Kupfer on organization of molecules in the helper T–B cell studies in primary helper T cells and the Jurkat T-lymphoma interface and work from our group on the dynamics of pattern model system. Supramolecular topology and membrane fluc- formation provided a molecular signature for an immunologi- tuations can drive segregation of receptor-ligand interactions cal synapse (26, 27). Studies by Kupfer revealed a striking into microclusters, and this process may be particularly segregation of adhesion receptors in the interface between important in defining adhesion domains with the large T cells and antigen-presenting cells. These reconstructed integrin and small immunoglobulin superfamilly receptors images were presented at meetings in 1996, and they were so (27, 31). These domains are typically sub-micron and can be

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organized into larger domains when coupled to cortical actin A B (32–34). Comparative studies demonstrate that the immunological synapse has parallels to integrin-mediated tissue cell spreading on planar substrates (35). One of the signatures of this process is membrane extension and retraction cycles (contractile oscillations) driven by actin polymerization and myosin II mediated contraction in the lamellipodium. This is considered a sensory process related to the ability of tissue cells to measure and eventually influence mechanical properties of the three dimensional (3D) tissue environment. These experiments demonstrated that the CD45-rich immunological synapse compartment defined by Kupfer as a distal (d)SMAC C (36) is a radial lamellipodium that bestows the immune cells with the ability to sense both chemical and mechanical properties of the antigen-presenting cell (37). The predicted retrograde F-actin flow has been imaged directly in Jurkat cells (33) (Fig. 1A). Submicron TCR and LFA-1 microclusters that form in the dSMAC are transported through the pSMAC at approximately 40% of the rate of the actin flow. The actin flow dissipates at the inside edge of the pSMAC. Incorporation of TCR into the core of the cSMAC is dependent upon expression of Tsg101 (38), a protein that recognizes ubiquitinated Fig. 1. Schematic of cytotoxic T lymphocytes (CTL) synapse and cargo and mediates transport into small vesicle into the kinapse. (A). CTL synapse. Strong T-cell receptor (TCR) signal and CD8. interior of endosomes (multivesicular bodies) or into the The key feature is the symmetric actin pattern with centripetal flow (white arrows). This forms the pSMAC (red, ICAM-1) and positions the extracellular space in the context of retroviral budding (39). TCR for Tsg101 dependent movement in the cSMAC (green, TCR). In this Small, dynamic microclusters have been observed exclusively efficient system the granules (purple) are targeted to the microtubule- with the planar bilayer system using total internal reflection organizing center (MTOC) along microtubules (heavy black lines) prior to movement of the MTOC and Golgi to the cSMAC secretory domain. fluorescence microscopy because of sensitivity and contrast Some of the cSMAC-associated TCR is in multivesicular bodies (blue with issues. Methods to improve imaging at cell–cell interfaces to green dots). (B). CTL kinapse. Weak TCR signal or CD4. The key feature is resolve such faint structures do not currently exist, but some an asymmetric actin pattern with net retrograde actin flow (white arrow) inducing forward motion of the cell (black arrow). This forms the asym- promising ideas are in development (40). metric focal zone (red, ICAM-1), whereas TCR microclusters do not accu- The adhesion ring of the immunological synapse is estab- mulate in a cSMAC. The MTOC moves to the actin-depleted secretory lished based on centripetal actin flow. This radial symmetry domain, but the granules reach the domain more slowly. (C). Relative efficiency of the two configurations. The presence of an intact cSMAC allows the T cell to dramatically slow or stop its motility with- gains approximately 6· increase in killing efficiency. The tight granule out losing the sensory advantages of the lamellipodium in packing around the MTOC results in a approximately 30· increase in detection of MHC–peptide complexes (41, 42) and the inter- killing efficiency compared with the loose granule distribution. pretation of mechanical cues (43). This symmetric actin flow the functional implications of signal integration and effector creates the pSMAC. Breaking the symmetry of the synapse functions executed while migrating (45). Similar symmetry restores motility and this process has been directly observed breaking and kinaptic behavior have been observed in NK cells during T-cell priming on supported planar bilayers (37). This receiving a combination of activating and inhibitory signals asymmetric retrograde actin flow creates an LFA-1 focal zone (46). that drives motility (44). It was surprising that even during The implications of the immunological synapse and SMAC antigen recognition T cells use the PKCh signaling pathway to for cytotoxic cells were immediately evident. It was already induce symmetry breaking and bursts of migration followed known that cytolytic granules of CTL move to the interface by Wiskott Aldrich syndrome protein (WASp)-dependent with the microtubule-organizing center (MTOC) prior to re-establishment of the symmetric synapse. We have proposed lethal hit delivery by the CTL. The bull’s eye-like pattern sug- that the motile phases be referred to as a kinapse (Fig. 1B), gested a central secretory target with a ring of adhesion mole- with the distinct, but etymologically related name to reflect cule to prevent leakage of cytolytic cargo and spare bystander

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cells. However, the next synapse to be described was not the KIR recruitment to the synapse, it is not absolutely required cytotoxic synapse but the inhibitory NK cell synapse. We (57). This observation suggested that the interactions driving review the data on NK and CTL synapses and particularly focus inhibition would operate under a broader range of physical on the role of synapse organization in function where there conditions than activating systems like the TCR and thus are mechanistic insights. would dominantly inhibit NK killing under any condition where the NK cells contact another cell expressing the appro- priate ligand. Rather than targeting activation receptors and Inhibitory NK cell synapses their associated signaling subunits for dephosphorylation, NK cells look similar to T lymphocytes but lack antigen recep- ITIM-containing receptors appear to block activation by differ- tors and instead express an array of activating and inhibitory ent types of receptors and signaling pathways through actin- receptors and the Fc receptor CD16. Most host cells are pro- independent inactivation of the Vav1-Rac1 pathway (20). tected from NK cells by expressing MHC–peptide complexes Furthermore, inhibitory KIRs do not block the actin-depen- on their surface. In mice, these molecules are recognized by dent accumulation of activation receptors but promote an members of the Ly49 family, a group of dimeric type 2 trans- actin-independent accumulation of activation receptors at membrane proteins with C-type lectin domains, which none- inhibitory synapses, where they prevent their phosphorylation theless recognize protein determinants of MHC class I (47). (56). This actin-independence is a unique situation in Just how rapidly this system is evolving is underscored by the immune synapse formation. Surprisingly, phosphorylated KIR fact that humans have a completely different family of immu- is not evenly distributed at inhibitory synapses but is concen- noglobulin superfamily receptors, the KIR, to serve the same trated in a few microclusters (58). Live imaging of the function (48). In both cases, the inhibitory receptors have dynamics of NK cell inhibitory synapses should provide cytoplasmic domains with ITIM. Mice and men express also insights into the unique and unusual mechanism of inhibition the more conserved lectin-like inhibitory receptor CD94- by ITIM-containing receptors. It remains to be determined if NKG2A, which binds to the non-classical MHC class I mole- inhibitory receptors expressed on T cells, such as programmed cule Qa1 and HLA-E, respectively. ITIM are phosphorylated by death-1 (PD-1), and other cells behave similarly or have dis- Src family kinases, recruit the phosphatase SHP-1 and termi- tinct biophysical mechanisms. nate signaling at a proximal step through dephosphorylation NK cell effector functions are controlled by a balance – or of Vav1 (19, 49–51). There are activating members of all rather an integration – of multiple activating and inhibitory three NK cell inhibitory receptor families. The ability of the signals. A recent study examined in detail how different sig- activating receptors to bind host MHC class I is crippled by nals control NK cell motility and shape (46). NKL cells stimu- mutations in the binding sites, but it seems that they are lated by glass slides coated with the NKG2D ligand MICA evolved for recognition of MHC class I-like viral antigens that spread and contracted, and a symmetric ring of F-actin may have initially evolved to engage inhibitory receptors formed. In contrast, NKL cells spread asymmetrically and (52). It is likely that such ‘arms races’ with viruses drive the moved over the LFA-1 ligand ICAM-1. In the presence of both rapid evolution of NK cell receptor families (53). ligands, NKG2D engagement imposed a stop signal and a Model systems with NK cell lines and target cells transfected symmetric synapse with peripheral F-actin formed. Interest- to express green ﬂuorescence protein (GFP)-tagged forms of ingly, addition of HLA-E, the ligand of inhibitory receptor human leukocyte antigen C (HLA-C) and KIR were developed CD94-NKG2A, reversed the stop signal. This migratory behav- to visualize NK cell inhibitory synapses (53). When NK cell ior of NK cells under conditions where inhibitory signals lines contact cells expressing the MHC class I (HLA-C) recog- dominate, which is reminiscent of the T-cell kinapses nized by a KIR, these molecules undergo dramatic accumula- described above (45), may facilitate disengagement from cells tion in the contact area, and the NK cell migrates past the that have to be spared, thus allowing NK cells to sample target putative target (54). Typically, KIR and HLA-C molecules cells more rapidly. accumulate in a central area surrounded by LFA-1 and ICAM-1 (55, 56). Interestingly, the interaction of KIR with HLA-C and their central accumulation was F-actin, temperature, and Cytotoxic NK cell synapse energy independent, in striking contrast to the F-actin, NK cell-mediated killing can be triggered by a number of temperature, and energy-dependent interactions in the helper pathways, and in most cases, activation actually requires inte- T-cell immunological synapse. Although F-actin can accelerate gration of multiple signals (59). The most potent mechanism

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for triggering degranulation is linked to antibody recognition dependent transport. While actin rearrangement is required through the Fc receptor CD16, but this mechanism does not for polarization of NK cells, cortical F-actin forms a barrier induce polarity of granule release on its own. The transmem- that lytic granules must traverse to reach and fuse with the brane isoform of CD16 that mediates killing is a classical plasma membrane. Recent studies have reported that myosin immunoreceptor in which signal transduction is accomplished IIA is not required for the formation of an organized and by forming a complex between the ligand binding transmem- polarized NK cell synapse but is essential for the final step of brane receptor with an ITAM-containing signal transduction lytic granule exocytosis (66, 67). Myosin IIA is associated module on a separate transmembrane protein, most signifi- with lytic granules and promotes their transport through the cantly FcR-c, which is non-covalently associated through a final layer of F-actin at the cytotoxic synapse (67). process requiring charged residues in the transmembrane Several groups working on NK cell synapses have converged domain (60). CD16 signals can trigger killing by human NK on the conclusion that classical NK-mediated killing results cells without other signals and have the potential to overcome from a multistage process with respect to patterning, cytoskel- inhibition by KIR despite the advantages of KIR noted above. etal polarization and killing (68, 69). Early studies suggested Other NK cell-activating receptors must be engaged in combi- that NK cells sustained nascent immunological synapses over nations to trigger cytotoxicity and the most synergistic combi- longer periods compared with T cells (54). This prolonged nations are activating receptors with different types of motifs nascent synapse, although not observed by all, suggested that (61). For example, several activating receptors associate with NK cells might use the nascent synapse over time to test the DAP12, which has an ITAM, while NKG2D associates with ratio of activating to inhibitory inputs prior to commitment. DAP10, which has a YINM motif shared with costimulatory The relationship between receptor accumulation, actin polari- receptors like CD28. Activating receptors in the signaling lym- zation, and killing suggested that NK cells use formation of a phocytic activating molecule (SLAM) family possess phosp- mature synapse as a checkpoint, the passage of which is hotyrosine motifs that link to the small adapter SAP (SLAM- dependent upon the ratio of activating and inhibitory signals associated protein) to deliver activating signals through (70, 71). While a few NK cells rapidly committed to the recruitment of Fyn. Several of the activating NK cell receptors mature synapse and killed the target cell, as many as half of have unknown but widely expressed ligands, such that not all the cells took many minutes after contact to form a synapse of the receptors engaged in an activating NK synapses can be and kill the target, if the target would be killed at all (70). This known at present. finding suggests that with a population of normal NK cells Recruitment of signal transduction molecules to cytotoxic and nominally susceptible targets, there is a probability that and inhibitory synapses were compared by Vyas and Dupont the balance of activating and inhibitory synapses will fail to using NK cell lines, NK clones, and primary NK cells (62–65). pass checkpoints for synapse formation and cytotoxic trigger- NK cells readily formed cSMAC and pSMAC-like compart- ing or may be delayed in passing these checkpoints. Thus, ments in 1–10 min, regardless of whether the synapses would compared with CTL synapses described below, the NK cyto- lead to cytotoxicity or inhibition. Thus, in these studies, the toxicity commitment process is prolonged. LFA-1-talin system seemed to be uniformly activated in this Two recent concepts in NK cell development have not been time frame to allow for sampling of signals present on extensively studied with respect to synapse formation. NK apposed cells. The significant difference between cytotoxic cells must be ‘licensed’ by interactions with MHC class I (72). and inhibitory synapses was the ratio of activating tyrosine This process, which may occur at an early developmental stage kinases, like Syk, ZAP70, and Lck, to the tyrosine phosphatase to generate mature NK cells, appears to be akin to positive SHP-1 in the cSMAC. This ratio was high in cytotoxic synapses selection in T cells. An alternative hypothesis to explain this and low in inhibitory synapses. This finding is consistent with phenomenon of NK cell ‘tolerance’ is that NK cells that do not the model that SHP-1 recruitment by inhibitory receptors receive inhibitory signals through MHC class I receptors must act locally on key phosphorylated tyrosines generated by become desensitized, or ‘disarmed’, through persistent activa- activating receptors to prevent the tyrosine kinase cascade tion signals (73). Epigenetic factors that are not well under- from propagating. Cytotoxic NK synapses are similar in many stood define the array of activating and inhibitory receptors respects to T-cell synapses in that the active signaling mole- that are expressed in any particular NK cell. Once these cells cules are recruited to the synapse and a well-defined pSMAC is develop, only the NK cells that express sufficient levels of formed. Delivery of lytic granules to the cytotoxic synapse inhibitory receptors that recognize host MHC class I gain func- requires actin cytoskeleton remodeling and microtubule- tional competence and are considered licensed, self-tolerant

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effector cells. Mouse models and human systems in which synapse and retrieval of LAMP-1 at the center are absolutely inhibitory receptor expression can be linked to specific MHC dependent on LFA-1 interaction with ICAM-1 on the bilayer. class I alleles demonstrate that licensed and unlicensed cells Thus, an unanticipated function of an LFA-1 pSMAC in the both express perforin and granzymes, but only licensed cells cytotoxic NK synapse may be for the efficient recycling of can engage in missing self recognition. The epigenetic mecha- granule membrane to form new cytolytic granules – a process nisms that control expression of inhibitory receptor also gen- that may be critical for serial killing. Another surprise in this erate NK cells (10–15%) that lack MHC class I-binding study was that although both 2B4-CD48 and NKG2D-ULBP inhibitory receptors, and these cells are hypoesponsive (74– interactions span 15 nm between membranes and might be 77). The licensing interactions and cytotoxic synapse forma- expected to co-cluster based on size rules for microcluster for- tion have not been examined in models where licensing can mation (79), these molecules were dramatically segregated. be controlled, and the signaling differences between licensed The 2B4-CD48 interactions were positioned at the center, per- and unlicensed cells are not well defined. For example, it is haps as expected, whereas the NKG2D-ULBP-1 interactions not known if unlicensed cells form a nascent synapse similar were co-localized with the LFA-1-ICAM-1 interactions in a to the bulk of primary NK cells, which are licensed. peripheral region. Thus, NK cells integrate NKG2D and 2B4 A second process that is of great interest in NK cells is based signals from different compartments of the synapse to trigger on recent observations that virus-specific NK cells can engage degranulation into the cSMAC. in adaptive responses with primary expansion, contraction, memory, and recall phases (23). While populations of mouse cytomegalovirus (mCMV)-specific NK cells expressing the Cytotoxic T cells Ly49H activating receptor are much more abundant than Cajal described the neural synapse as ‘The protoplasmic oscu- mCMV-specific naive T cells, the Ly49H+ NK cells nonetheless lation (kiss)... the final saga in an epic love story.’ Following undergo a 100-fold expansion during infection. These cells on this precedent, the cytotoxic synapse of CTL has been aptly then contract back to near pre-infection levels to initiate a described as a kiss of death (80), certainly the final saga for memory phase. Recall responses are more efficient, as mem- the target. There are two modes of CTL-mediated killing: ory Ly49H+ NK cells elaborate a 10-fold enhanced ability Ca2+-dependent killing by perforin and granzymes and Ca2+- to protect NK cell-deficient mice from mCMV infection com- independent killing mediated by Fas ligand (FasL) binding to pared with naı¨ve Ly49H+ NK cells. The characteristics of syn- Fas (CD95) on target cells. Perforin-mediated killing can also apse formation by memory and naive NK cells are not known, be diagnosed with the vacuolar acidification inhibitor con- but this could certainly be addressed in the mouse models. canamycin A (80). Both pathways trigger death by apoptosis, While most mice are raised in specific pathogen-free condi- but the perforin pathway is typically faster. Perforin-mediated tions, humans experience many viral infections, and it is likely killing is more general, since it is based on a highly conserved that peripheral blood NK populations contain both naive and membrane injury response and endosomal lysis leading to memory NK cells. It remains to be determined how much introduction of granzymes into the target cell cytoplasm (82), of the heterogeneity in human NK cell behavior is related to and thus, no specific receptor is needed. As perforin and gran- differences in these subsets. zymes are released from the cell, the synaptic cleft has been The cytotoxic NK cell synapse has been modeled using assumed to enhance function through high local concentra- supported planar bilayers (78). Bilayers containing CD48 and tion and also to prevent bystander exposure to active perforin. ULBP1 trigger synergistic granule release in primary human Fas must also function in a cell-cell contact, since FasL and Fas NK cells. In human NK cells, the receptor for CD48 is 2B4, a are membrane proteins with a limited reach (approximately member of the SLAM family that associates with signaling 15 nm). FasL has been reported to be present in the granules adapter SAP, and ULBP1 is a ligand of NKG2D. Whereas both of CTL that co-express perforin and granzymes and to be exo- CD48 and ULBP-1 are needed to trigger degranulation, CD16 cytosed into the synaptic cleft in response to TCR triggering engagement with immunoglobulin G alone is sufficient for (83). This is in part explained by the sorting of FasL into mul- degranulation. Lysosomal membrane glycoprotein-1 (LAMP- tivesicular body core vesicles that are released from cells dur- 1) (CD107a) molecules that have been delivered to the cell ing degranulation (84). It has been observed that in transplant surface upon degranulation are not allowed to diffuse at the rejection, early CTL express perforin and FasL, while during plasma membrane but are retrieved into a stable and central the retraction phase the CTL lose the perforin pathway, but endocytic compartment. Formation of an organized cytotoxic continue to be active killers using FasL. In CTL utilizing only

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FasL, the pathway is not dependent upon protein synthesis, nar bilayers (97). Surprisingly, CTL were very efficient at suggesting the FasL is stored in an intracellular compartment forming pSMAC-like adhesion rings and would do so tran- such as cytotoxic granules, but FasL-mediated killing is selec- siently even in the absence of specific MHC–peptide com- tively inhibited by brefeldin A (85, 86). Brefeldin A inhibits plexes (97). Griffiths (96) described the cSMAC as being both endoplasmic reticulum to Golgi and some regulated divided into signaling and secretory domains. Such functional trans-Golgi to plasma membrane secretion pathways, but not compartmentalization of the cSMAC is likely to be important the release of granules (secretory lysosome) or recycling in integration of costimulatory signals with TCR signal and endosomes (87). Thus, FasL-mediated killing can be diag- receptor degradation in helper cells (98). While both LFA-1 nosed by anti-FasL antibodies and by acute treatment with and CD2 adhesion pathways contribute to conjugate forma- brefeldin A (long-term treatment will eventually limit the tion, only the LFA-1 pathway leads to formation of orga- availability of MHC–peptide complexes on the target). nized SMACs (99). Recently, the secretion of granules directly into the model cytotoxic synapse with a planar bilayer has been observed by total internal reflection fluores- The CTL immunological synapse cence microscopy (TIRFM) based on the detection of Studies on the CTL synapse have focused primarily on under- CD107a+ foci at the cSMAC (100, 101). These studies con- standing of the perforin and granzyme-mediated killing. CTL firm that there is a secretory domain in the cSMAC. It has are known to engage in a killing cycle that involves tenacious been reported that target lysis can take place without an adhesion, release of cytotoxic agents, and detachment from immunological synapse (102). The quantitative advantages of the target (80). CTL undergo dramatic polarization changes forming a synapse versus other modes of interaction are only with movement of the CTLs’ MTOC and linked cytotoxic beginning to be understood. granules to the synapse (4). Electron microscopy demonstrated that the MTOC is moved directly to the membrane, much like the basal body of a primary cilium (88). Some of The competitive edge offered by the synapse the mechanisms mediating granule movement in the CTL syn- The model that secretion of granule contents into the immu- apse have been illuminated by study of patients and mouse nological synapse is important for efficiency of killing has models selected based on defects in pigmentation arising from recently been tested and an unexpected new parameter gov- melanocyte granule movement defects, which in some cases erning efficiency has been revealed. Sykulev and colleagues predict CTL granule movement defects (89–91). Molecules (100, 101) initiated studies comparing human cytotoxic T- involved in this process include Rab27a, Munc13-4, and myo- cell clones expressing CD8 and CD4. The CD8+ T cells are sin V. Separating CTL from target cells during the peak of 100-fold more efficient than the CD4+ CTL in terms of the adhesion is difficult and traumatic to both cells suggesting time and granule investment needed to kill targets. Both types strong cell adhesion (92). LFA-1 and CD2 adhesion pathways of CTL kill primarily using the perforin ⁄ granzyme pathway, mediate human CTL adhesion to an array of targets (93). It is and both cell types are equivalently armed at the level of gran- likely that other pathways contribute in different contexts. ule activity. Comparison of synapse formation by both cell How CTL know to let go after targets are programmed for types revealed that the CD8+ CTL form more stable synapses lysis is not clear. It is possible that some chemical or mechani- (80% vs 40% of total conjugates), whereas CD4+ CTL have cal feedback is involved, as membranes of cells undergoing more prevalent kinapses (the asymmetric junctions that lead apoptosis undergo loss of phospholipid asymmetry and dra- to migration and absence of a protected secretory domain). As matic blebbing based on changes in the cortical cytoskeleton described above, the generation of kinapses in CD4+ T cells is (94). promoted by activation of PKCh, such that inhibition of PKCh Molecular patterns in the immunological synapse were first stabilized the synapses of CD4+ CTL. Synapse stabilization by described for CD4+ helper T cells as described above. Naive this mechanism resulted in a threefold increase in killing CD8+ T cells were subsequently found to form similar bull’s efficiency. Since approximately 40% of CD4+ CTL did form eye synapses during priming in vitro (95) and in mature CTL synapses without the inhibitor, it is suggested that stable syn- during target lysis (96). The cytotoxic synapses were charac- apses are sixfold more efficient than kinapses (101). The terized by a well-defined pSMAC (LFA-1 ring) and cSMAC remaining 30-fold difference in efficiency between CD4+ and (TCR, CD8, and Lck). This process could be reconstituted with CD8+ T cells was because of a different, unexpected mecha- ICAM-1 and MHC class I peptide complexes in supported pla- nism (Fig. 1C).

30 Published 2010. This article is a US Government work and is in the public domain in the USA • Immunological Reviews 235/2010 Dustin & Long Æ NK and CTL synapses

When triggered by agonist MHC–peptide complexes, examined the dynamics of LCMV-specific CTL in the menin- CD8+ CTL release granules faster than CD4+ CTL. Trigger- ges during fatal LCMV meningitis (111). While histologic ing using the same anti-CD3 antibody eliminated the analysis suggested the presence of stable synapses in this kinetic difference, and eliminating CD8–MHC class I inter- model, dynamic imaging revealed that the CTL are con- action slowed granule release from CD8+ CTL. Examination stantly in motion, suggesting that they are forming kinapses. of the movement of the MTOC and the granules to the It is difficult in fixed images to distinguish a synapse with a synapse revealed that strong TCR signals promoted by CD8 transient break in the pSMAC from kinapse (104). It is only led to fast granule recruitment to the MTOC and movement through dynamic imaging that the stability of interactions of that MTOC-granule complex to the secretory domain, can be assessed. The kinapse formation by CTL in the such that the granules were tightly localized in the cSMAC. meninges precedes massive recruitment of myelomonocytic Signals generated by weaker agonist MHC–peptide complex, cells that is fatal to the host (111). It is not clear why more CD8 blockade, or use of CD4 as the coreceptor led to stable synapses leading to efficient killing of virally infected movement of the MTOC to the secretory domain without cells is not observed in this site. Future advances in intravi- the granules, which then needed to slowly make there way tal imaging should also provide more insight into cytotoxic to the secretory domain via the pSMAC such that the gran- synapse functioning in vivo. In vivo imaging methods with the ules appears loosely arrayed in the synapse or kinapse required molecular resolution are already in use in the ner- (100). Similar results were obtained by the Griffith labora- vous system (112) and may soon illuminate immune cell tory utilizing altered peptide ligands in a CD8 system, sug- function in physiologic settings. gesting that both the CD8 and TCR–MHC–peptide interaction strength can determine the early signaling kinetics and pathway for granule delivery (103). Granule exocy- Conclusions tosis rates and the diffusion rate for perforin and The immunological synapse defined by formation of an granzymes from the synaptic cleft will determine the effi- intact adhesion ring and the movement of cytolytic granules ciency of granzyme delivery to the cytoplasm. Therefore, to a secretory domain provides a site for signal integration in the formation of a cytotoxic synapse based on pSMAC NK cells and contributes to the efficiency of CTL-mediated integrity and polarized granule delivery to the secretory killing. Studies of NK cells have revealed a central role of domain are two components that offer a significant com- integrin LFA-1 in the organization and dynamics of NK cyto- petitive advantage in the race with pathogens. toxic synapses. Imaging of T-cell cytotoxic synapses has demonstrated physical differences between CD4+ and CD8+ cells that result in vastly superior cytotoxic activity of CD8+ cells. Cytotoxic synapses in vivo Outstanding problems in the field remain. For example, the In vitro studies have provided insights into the function of permeability of the pSMAC ring is not known, and studies in the immunological synapse, but in vivo studies have the CD4+ T cells suggest that it is surprisingly permeable to anti- potential to put this system in a more physiologic context. bodies and Fab fragments (113). In fact, the anti-LAMP-1 Histologic analysis supports a role of organized immunolog- CD107a Fab-based method used to rapidly detect granule ical synapses, particularly in the central nervous system exocytosis in the synapse with planar bilayers and TIRFM is (CNS) (104, 105). Two photon laser scanning microscopy dependent upon the ability of the labeled Fab to diffuse particularly has enabled imaging of micron scale details freely into the synaptic cleft for both NK cells and CTL (78, hundreds of microns deep in live tissues to capture the 100, 101). Another issue is the minimal signal integration dynamics of interactions (106). Priming of CD8+ T cells time for NK cells versus CTL. It had been argued that CTL involves prolonged interactions with dendritic cells (107, were much more decisive than NK cells, but this apparently 108). The effector phase involves stable interactions, but depends upon the ligand of the CTL. NK cells may make CTL could kill 2.4-specific B-cell targets per hour. Interest- decisions in a more tentative process, but this seems to be ingly, the presence of regulatory T cells reduced the killing followed by an efficient killing process. In contrast, the rate rate to 0.4 ⁄ h, a sixfold decrease in the killing rate (109). of commitment with CTL depends upon ligand strength, The tumor microenvironment further decreases the rate of with lower potency ligands leading to an inefficient killing killing to <0.2 ⁄ h (110). Less has been performed at this process. This factor may be one that leads to slow killing of point with CTL in the context of infection. Recently, we tumor cells.

Published 2010. This article is a US Government work and is in the public domain in the USA • Immunological Reviews 235/2010 31 Dustin & Long Æ NK and CTL synapses

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34 Published 2010. This article is a US Government work and is in the public domain in the USA • Immunological Reviews 235/2010