Coordination of and Migration through Formation of the Immunological Synapse

MICHAEL L. DUSTIN Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine and the Department of Pathology, New York University School of Medicine, New York, New York 10016, USA

ABSTRACT: T cell activation is based on interactions of T cell antigen receptors with MHC-peptide complexes in a specialized cell–cell junction between the T cell and antigen-presenting cell—the immunological synapse. The immunolog- ical synapse coordinates naïve T cell activation and migration by stopping T cell migration with antigen-presenting cells bearing appropriate major histo- compatibility complex (MHC) peptide complexes. At the same time, the immu- nological synapse allows full T cell activation through sustained signaling over a period of several hours. The immunological synapse supports activation in the absence of continued T cell migration, which is required for T cell activa- tion through serial encounters. Src and Syk family kinases are activated early in immunological synapse formation, but this signaling process returns to the basal level after 30 min; at the same time, the interactions between T cell re- ceptors (TCRs) and MHC peptides are stabilized within the immunological synapse. The molecular pattern of the mature synapse in helper T cells is a self- stabilized structure that is correlated with production and prolifera- tion. I propose that this molecular pattern and its specific biochemical constit- uents are necessary to amplify signals from the partially desensitized TCR.

KEYWORDS: adhesion; migration; activation; antigen; receptors; kinases; phosphorylation; signaling


Naïve T cells recirculate between the blood and secondary lymphoid tissues com- pleting an average of two traversals per day.1,2 Naïve T cell activation in vivo results in the removal of antigen-reactive cells from the recirculating pool for approximate- ly two days.3,4 During this time, the antigen-specific cells are concentrated in drain- ing lymph nodes or the spleen, depending upon the route of antigen entry. After this two-day period, the daughter cells from proliferation of the primary naïve cells em-

Address for correspondence: Michael L. Dustin, Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine and the Department of Pathology, New York University School of Medicine, 540 First Avenue, New York, NY 10016.Voice: 212-263-3207; fax 212-263- 5711. [email protected]

Ann. N.Y. Acad. Sci. 987: 51–59 (2003). © 2003 New York Academy of Sciences.

51 52 ANNALS NEW YORK ACADEMY OF SCIENCES igrate from the secondary lymphoid tissues and migrate either to secondary lym- phoid tissues or to distinct peripheral tissues. This retention of antigen-specific cells is selective and may be explained by the tendency of antigen-specific T cells to stop migrating following T cell receptor engagement.5 The stop signal is based on both the Ca2+-dependent suspension of motility in the short term (minutes) and the main- tenance of T cell polarity towards the antigen-presenting cells (APCs) in the long term (hours).5,6 The stop signal was further demonstrated to correlate with a specific molecular pattern in the interface between T cells and APCs.7 This pattern has been revealed through studies of fixed cell–cell conjugates, live cell–cell conjugates, and interactions between T cells and supported planar bilayers containing major histo- compatibility complex (MHC) peptides and adhesion (FIG. 1).7–12 The initial molecular pattern can be described as a nascent or immature immunological synapse in which T cell receptors (TCRs) are engaged in the periphery of the exten- sive contact area.7 This pattern is followed by formation of a mature immunological synapse in which the TCRs are clustered in the center of the contact area.7,8 The pat- tern in which the TCRs and MHC peptides are centrally clustered and stabilized over a period of hours is correlated with full T cell activation when both the dose and quality of MHC-peptide complexes is varied. In the mature immunological synapse, the central cluster is also referred to as a central supramolecular activation cluster (cSMAC), and the LFA-1 ring, as a peripheral supramolecular activation cluster (pSMAC).8 T cells deficient in specific signaling molecules in this pathway, such as WASP and Vav, which are involved in TCR movement on the surface following activation, have defects in T cell activation, suggesting that active T cell movements are important for activation and immunological synapse formation.13–15

FIGURE 1. Location of signaling in the immunological synapse. Two domains are shown: central and peripheral. Abundance is represented as gray scale from dark (= low) to light (= high). Based on data in Lee et al.12 DUSTIN: SIGNALING IN THE IMMUNOLOGICAL SYNAPSE 53

While mature immunological synapse formation is a common mode of interac- tion during T cell signaling in vitro, certain environmental conditions, or APCs trig- ger a different mode of T cell interaction characterized by continued T cell migration.16,17 This mode of activation is more similar to the classical concept of se- rial triggering in which a few MHC-peptide complexes engaged a large number of TCRs to sustain signaling over a prolonged period as the T cells continually encoun- ter new and recycled MHC-peptide complexes.18,19 The concept that environmental signals can be dominant over TCR-mediated polarization signals has been described for certain chemokines,20 but is likely to extend to other microenvironmental sig- nals. Given that the T cell can respond in different ways to similar stimuli, it’s im- portant to understand the function of the immunological synapse and the conditions under which it forms as the primary mode of T cell activation.


Supported planar bilayers containing MHC-peptide complexes and the adhesion ICAM-1 have provided a surprisingly effective surrogate APC allowing dynamic imaging of molecular patterns during T cell activation.7 ICAM-1 is a ligand for the integrin LFA-1, perhaps the most ubiquitously employed adhe- sion molecule on T cells. The supported planar bilayers containing only an agonist MHC-peptide complex and ICAM-1 can trigger proliferation of previously activated T cells, and bilayers containing agonist MHC-peptide complexes,7 ICAM-1, and CD80 can trigger proliferation of naïve T cells.21 The advantage of the bilayer sys- tem is that the ligands can be fluorescently labeled, and fluorescence imaging can then be used to identify sites of interaction between receptors on the T cell and the fluorescent ligands in the bilayer. Since the ligands in the bilayer are initially uni- formly distributed and can only interact with the receptors or each other, the inter- pretation of the images is greatly simplified compared to the situation with cell–cell interaction in which cytoskeletal interactions and lateral heterogeneity of mem- branes greatly complicates interpretation.22 For example, MHC-peptide accumula- tion in the bilayer can be interpreted in terms of TCR–MHC-peptide interactions with the only complexity arising from co-receptors and oligomerization. On the oth- er hand, MHC-peptide accumulation in a cell–cell interface could be due to associ- ation with liquid ordered membrane domains (rafts), the cytoskeleton, and vesicular stores of MHC-peptide complexes near the interface in addition to interactions with the T cell receptor and co-receptors. The clarity of interpretation provided by the bi- layer system comes at the expense of loss of the potentially important topology, cy- toskeletal associations, and membrane heterogeneity present in the APC. Therefore, it is important to use both approaches in a complementary fashion, since neither sys- tem alone can address all the important questions. In fact, an important technology for future experiments will be to take these studies into the in vivo environment through intravital imaging. The planar bilayer system and cell–cell systems both concur on the following mo- lecular choreography for immunological synapse formation.7,23 The LFA-1–ICAM- 1 interaction is initiated in the central region of the nascent immunological synapse. In the first moments of contact (<30 s), it is likely that membrane projections bearing 54 ANNALS NEW YORK ACADEMY OF SCIENCES

TCRs at the periphery of the LFA-1-dependent contact come close enough to the APC surface to interact with MHC-peptide complexes. This peripheral TCR engage- ment initiates signals that trigger the tyrosine kinase cascade. The initial evidence for this early signaling came from studies on the Ca2+ signal that show that this sig- nal is initiated very early6,7,10 and in specific situations where peripheral TCR en- gagement was imaged in parallel.7,23 The influx of cytoplasmic Ca2+ is downstream of activation of phopholipase Cγ through the tyrosine kinase cascade requiring , ZAP-70, and ITK.24 Thus, early (<30 s) Ca2+ signaling strongly implied the Lck is activated within seconds of contact, well before formation of the mature immuno- logical synapse with its focused, central TCR engagement. The mature immunological synapse is characterized by stabilization of the TCR– MHC-peptide interaction in the central cluster. This was deduced by fluorescence photobleaching recovery experiments.7,23 In this experiment, the fluorescence of MHC-peptide complexes interacting in the central region of the mature synapse is photobleached with a brief flash from an appropriately focused laser beam, render- ing these molecules non-fluorescent, but functionally intact. The fluorescence is then monitored over time to determine if the bleached molecules accumulated in the central cluster will exchange with the fluorescent molecules that diffuse in from out- side the small bleached area. While fluorescence in the bilayer did recover, the ac- cumulated fluorescence in the central cluster did not, suggesting that the interaction in this region had become stable. It is possible that this stabilization is based on oli- gomerization of the TCR-MHC-peptide complex as detected in solution for the 2B4 TCR-Ek-MCC88-103 system.25 This stabilization process does not appear to be re- versible, since migrating T cells were observed to shed TCRs from their surface dur- ing disengagement from the central cluster. This stabilization of TCRs may provide a focal point for polarization of T cells in stable synapses and may represent a form of self-catalyzed refolding to form these oligomers.


Classical biochemical studies using antibody cross-linking or APCs suggested that Lck activation, which includes the tyrosine kinase cascade, is transient, but thinking about the newly described immunological synapse and the stable central cluster raised the possibility that in situ phosphorylation could be more sustained (FIG. 2).26,27 Recently, this question was directly addressed using phosphotyrosine specific antibodies to the activation loop of Lck and a key autophosphorylation site of ZAP-70, two key kinases whose activities are central to the classical definition of TCR signaling, to determine the timing and location of tyrosine kinase activation in T cell–splenic APC immunological synapses.12 Consistent with the classical para- digm, it was found that both Lck and ZAP-70 were activated early in the nascent syn- apse and were no longer active by the time the mature immunological synapse had formed. The mechanisms that return tyrosine phosphorylation to the basal level are not known, but it was noted that the splenic APC induced dramatic TCR down-reg- ulation just following the formation of the central TCR cluster. This down-regulation may be related to the role of ubiquitin ligases of the cbl family. Interestingly, cbl may be recruited to the central TCR cluster by the CIN85-related protein CD2AP.28,29 CD2AP binds the cytoplasmic domain of CD2 in a T cell activation dependent man- DUSTIN: SIGNALING IN THE IMMUNOLOGICAL SYNAPSE 55

FIGURE 2. Model for signaling kinetics in serial encounters versus the stable synapse. The lines in the top and bottom panels reflect tyrosine kinase signaling (dashed lines) and a hypothetical signaling process required for cytokine transcription (solid line). This signaling to the nucleus is downstream of activation and of the tyrosine kinase cascade and has a half- life that is determined by the structure of the interface—short in an unstructured interface and longer in a structured interface (immunological synapse). ner and brings CD2 to the center of the immunological synapse.9 In addition, CD2AP, like CIN85, may recruit cbl and facilitate TCR degradation. This process may contribute to the rapid return of the TCR signal to basal levels. The results on tyrosine phosphorylation stimulated an experiment to determine how long the T cell–APC conjugate had to persist to program T cells for full activa- tion.12 The result was that the immunological synapse had to persist for 2–3 h to commit the T cell to one division and for 3–6 h to commit the T cell to multiple cell divisions. This creates a paradox that the tyrosine phosphorylation of the TCR re- turns to basal levels by 1 h, but signal integration continues for up to 6 h and likely longer.30 The events that constitute signaling in this time frame are of great interest. One possibility is that the efficacy of tyrosine kinases for activation of downstream transcription systems is greatly enhanced in the organized immunological synapse. Support for this comes from the observation that Ca2+ mobilization is sustained in the immunological synapse over a period of hours.7,10 This suggests an ongoing pro- cess that stimulates Ca2+ mobilization, which would be consistent with some level of effective phospholipase Cγ activation through a tyrosine phosphorylation depen- dent process. Alternatively, the maintenance of transcription factor translocation may be tyrosine kinase independent in the mature synapse and may rely on distinct signaling mechanisms that are set up in the early stages of immunological synapse formation. Why form stable synapses when repeated early signaling can trigger T cells? As mentioned above, T cells can signal without forming a stable immunological syn- apse. This alternative mode of activation requires crawling of the T cell over the APC and between APCs, and the signaling is immediately blocked by addition of cytoch- 56 ANNALS NEW YORK ACADEMY OF SCIENCES alasin D.16,18 Naïve murine T cells interacting with dendritic cells in a collagen gel are stimulated to proliferate through a series of transient interactions with APCs that were each on the order of 12 min, but were repeated many times as the T cells mi- grated through the three-dimensional collagen gel.17 With every new interaction be- tween T cells and antigen-positive APCs, the T cells underwent a robust Ca2+ flux, suggesting repeated rounds of early TCR signaling that broke off just as the Lck and ZAP-70 activation would be expected to return to baseline, although tyrosine kinase activity was not directly examined. In fact, it may be important that these T cells break contact with the APCs to reset the tyrosine kinases, so that they can undergo repeated rounds of signaling. In vivo, it is most likely that stable immunological syn- apse formation would be used to coordinate T cell activation with migration so that the antigen-specific T cells can be focused in one compartment to contribute to the collective process of making an appropriate response to the pathogen.31,32 However, this serial encounter mode may be important in different in vivo settings, such as the movement of effector cells in the skin and other collagen-rich peripheral tissues. Relevant to this argument is the observation of Anderson and Shaw that collagen in lymph nodes is sequestered in reticular fibers; thus, T cells in lymph nodes are not exposed to collagen.33,34 I propose that the process of effective activation through formation of a stable immunological synapse evolved to focus antigen-specific T cells on the appropriate secondary lymphoid tissue and further on the appropriate APCs.


Self-assembly processes have captured the imagination of many biologists be- cause such processes posit the ability to build large-scale biological devices from less complex building blocks. The prevailing view of immunological synapse forma- tion has been based on the paradigm of topological segregation combined with anti- body-mediated capping.35,36 In the capping process, the signaling triggered by cross-linking surface initiates a process of polymerization and myosin activation leading to contraction of the actin gel and collection of the actin-associat- ed, cross-linked receptors into a cap at one pole of the cell.37 Chakraborty and col- leagues have employed partial differential equations describing molecular segregation by size, reaction-diffusion processes, and membrane fluctuations to model immunological synapse formation as a self-assembly process that does not re- quire actin-myosin transport.38 Remarkably, the model can be run with measured pa- rameters and predicts stable immunolologcal synapse formation through the same series of intermediates that are observed experimentally. While the model is in the early stages of development, it raises the important possibility that synapse forma- tion or stability may be based on a self-assembly process that does not require trans- port or may operate in parallel with cytoskeletal transport mechanisms. While the synapse self-assembly model does not require transport, an intact actin cytoskeleton is required to set the appropriate membrane-bending properties to generate synaptic patterns. Therefore, we can consider two alternatives for synapse formation that should be distinguishable at the single-molecule level. The capping model predicted that molecules are transported to the center of the synapse along straight-line paths, DUSTIN: SIGNALING IN THE IMMUNOLOGICAL SYNAPSE 57 whereas the self-assembly model predicts a biased random walk. If there is a signif- icant element of membrane-driven self-assembly, then some of the genetic experi- ments may require reinterpretation. It is possible that rather than setting up a capping- like phenomenon, molecules such as Vav and WASP may have a role in adjusting the physical properties of the membrane to favor self-assembly of the synapse.


There is still much to learn about the function of the immunological synapse. For helper T cells, both naïve and effector, the synapse formation process is related to antigen dose and strength. Formation of helper T cell immunological synapses ap- pears to be very sensitive to the kinetics of the TCR–MHC-peptide interaction. This may be related to the self-assembly process identified by Qi et al.38 Despite the in- trinsic tendency of T cells to form stable synapses when triggered by agonist MHC- peptide complexes and adhesion molecules, the environment can have a strong im- pact on signaling. If the environment is dominant for control of T cell polarity, the T cell does not stop migration on the APC, but can still be activated through a process of serial encounters with multiple APCs involving a resetting of the tyrosine kinase cascade to allow repeated firing. When the TCR signal is dominant for control the T cell polarity, the T cell stops and uses one round of transient tyrosine kinase signal- ing to set up a stable synapse. The stable immunological synapse is then required to generate signals that maintain transcription factor activation. The specific nature of these signals is not known and represents an important area for future research.


This work was supported by grants from the National Institutes of Health and the Irene Diamond Professorships in .


1. SPRINGER, T.A. 1995. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu. Rev. Physiol. 57: 827. 2. JENKINS, M.K., A. KHORUTS, E. INGULLI, et al. 2001. In vivo activation of antigen-spe- cific CD4 T cells. Annu. Rev. Immunol. 19: 23. 3. SPRENT, J., J.F.A.P. MILLER & G.F. MITCHELL. 1971. Antigen-induced selective recruit- ment of circulating . Cell. Immunol. 2: 171. 4. SPRENT, J. & J.F.A.P. MILLER. 1972. Interaction of thymus lymphocytes with histoin- compatible cells. II. Recirculating lymphocytes derived from antigen-activated thy- mus cells. Cell. Immunol. 3: 385. 5. DUSTIN, M.L., S.K. BROMELY, Z. KAN, et al. 1997. Antigen receptor engagement deliv- ers a stop signal to migrating T lymphocytes. Proc. Natl. Acad. Sci. USA 94: 3909. 6. NEGULESCU, P.A., T. KRAIEVA, A. KHAN, et al. 1996. Polarity of T cell shape, motility, and sensitivity to antigen. Immunity 4: 421. 7. GRAKOUI, A., S.K. BROMLEY, C. SUMEN, et al. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285: 221. 8. MONKS, C.R., B.A. FREIBERG, H. KUPFER, et al. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395: 82. 58 ANNALS NEW YORK ACADEMY OF SCIENCES

9. DUSTIN, M.L., M.W. OLSZOWY, A.D. HOLDORF, et al. 1998. A novel adapter protein orchestrates receptor patterning and cytoskeletal polarity in T cell contacts. Cell 94: 667. 10. WÜLFING, C., M.D. SJAASTAD & M.M. DAVIS. 1998. Visualizing the dynamics of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels. Proc. Natl. Acad. Sci. USA 95: 6302. 11. KRUMMEL, M.F., M.D. SJAASTAD, C. WULFING & M.M. DAVIS. 2000. Differential clus- tering of CD4 and CD3zeta during T cell recognition. Science 289: 1349. 12. LEE, K.-H., A.D. HOLDORF, M.L. DUSTIN, et al. 2002. Tyrosine kinase signaling pre- cedes formation of the immunological synapse. Science 295: 1539. 13. FISCHER, K.D., Y.Y. KONG, H. NISHINA, et al. 1998. Vav is a regulator of cytoskeletal reorganization mediated by the T- cell receptor. Curr. Biol. 8: 554. 14. SNAPPER, S.B., F.S. ROSEN, E. MIZOGUCHI, et al. 1998. Wiskott-Aldrich syndrome pro- tein-deficient mice reveal a role for WASP in T but not B cell activation. Immunity 9: 81. 15. WÜLFING, C., A. BAUCH, G.R. CRABTREE & M.M. DAVIS. 2000. The Vav exchange fac- tor is an essential regulator in actin-dependent receptor translocation to the lympho- cyte-antigen-presenting cell interface. Proc. Natl. Acad. Sci. USA 97: 10150. 16. UNDERHILL, D.M., M. BASSETTI, A. RUDENSKY & A. ADEREM. 1999. Dynamic interac- tions of macrophages with T cells during . J. Exp. Med. 190: 1909. 17. GUNZER, M., A. SCHAFER, S. BORGMANN, et al. 2000. Antigen presentation in extracel- lular matrix: interactions of T cells with dendritic cells are dynamic, short lived, and sequential. Immunity 13: 323. 18. VALITUTTI, S., M. DESSING, K. AKTORIES, et al. 1995. Sustained signalling leading to T cell activation results from prolonged T cell receptor occupancy: role of T cell actin cytoskeleton. J. Exp. Med. 181: 577. 19. VALITUTTI, S., S. MÜLLER, M. CELLA, et al. 1995. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature 375: 148. 20. BROMLEY, S.K., D.A. PETERSON, M.D. GUNN & M.L. DUSTIN. 2000. Cutting edge: hier- archy of chemokine receptor and TCR signals regulating T cell migration and prolif- eration. J. Immunol. 165: 15. 21. BROMLEY, S.K., A. IABONI, S.J. DAVIS, et al. 2001. The immunological synapse and CD28-CD80 interactions. Nat. Immunol. 2: 1159. 22. DUSTIN, M.L., L.M. FERGUSON, P.Y. CHAN, et al. 1996. Visualization of CD2 interac- tion with LFA-3 and determination of the two-dimensional dissociation constant for adhesion receptors in a contact area. J. Cell Biol. 132: 465. 23. JOHNSON, K.G., S.K. BROMLEY, M.L. DUSTIN & M.L. THOMAS. 2000. A supramolecular basis for CD45 regulation during T cell activation. Proc. Natl. Acad. Sci. USA 97: 10138. 24. SCHAEFFER, E.M., J. DEBNATH, G. YAP, et al. 1999. Requirement for Tec kinases Rlk and Itk in T cell receptor signaling and immunity. Science 284: 638. 25. REICH, Z., J.J. BONIFACE, D.S. LYONS, et al. 1997. Ligand-specific oligomerization of T cell-receptor moleucles. Nature 387: 617. 26. DUSTIN, M.L. & A.C. CHAN. 2000. Signaling takes shape in the . Cell 103: 283. 27. BROMLEY, S.K., W.R. BURACK, K.G. JOHNSON, et al. 2001. The immunological syn- apse. Annu. Rev. Immunol. 19: 375. 28. PETRELLI, A., G.F. GILESTRO, S. LANZARDO, et al. 2002. The endophilin-CIN85-Cbl complex mediates ligand-dependent downregulation of c-Met. Nature 416: 187. 29. SOUBEYRAN, P., K. KOWANETZ, I. SZYMKIEWICZ, et al. 2002. Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors. Nature 416: 183. 30. IEZZI, G., K. KARJALAINEN & A. LANZAVECCHIA. 1998. The duration of antigenic stim- ulation determines the fate of naive and effector T cells. Immunity 8: 89. 31. DUSTIN, M.L. & A.R. DE FOUGEROLLES. 2001. Reprograming T cells: the role of extra- cellular matrix in coordination of T cell activation and migration. Curr. Opin. Immu- nol. 13: 286. DUSTIN: SIGNALING IN THE IMMUNOLOGICAL SYNAPSE 59

32. DUSTIN, M.L., P.M. ALLEN & A.S. SHAW. 2001. Environmental control of immunologi- cal synapse formation and duration. Trends Immunol. 22: 192. 33. EBNET, K., E.P. KALDJIAN, A.O. ANDERSON & S. SHAW. 1996. Orchestrated information transfer underlying leukocyte endothelial interactions. Annu. Rev. Immunol. 14: 155. 34. GRETZ, J.E., C.C. NORBURY, A.O. ANDERSON, et al. 2000. Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via spe- cialized conduits while a functional barrier limits access to the lymphocyte microen- vironments in lymph node cortex. J. Exp. Med. 192: 1425. 35. HOLSINGER, L.J., I.A. GRAEF, W. SWAT, et al. 1998. Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduc- tion. Curr. Biol. 8: 563. 36. DUSTIN, M.L. & J.A. COOPER. 2000. The immunological synapse and the actin cytosk- eleton: molecular hardware for T cell signaling. Nat. Immunol. 1: 23. 37. BRAUN, J., K. FUJIWARA, T.D. POLLARD & E.R. UNANUE. 1978. Two distinct mecha- nisms for redistribution of lymphocyte surface macromolecules. I. Relationship to cytoplasmic myosin. J.Cell Biol. 79: 409. 38. QI, S.Y., J.T. GROVES & A.K. CHAKRABORTY. 2001. Synaptic pattern formation during cellular recognition. Proc. Natl. Acad. Sci. USA 98: 6548.