The Immunological Synapse Balances T Cell Receptor Signaling ...

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  • 1. REPORTS that is compatible with the b haplotype. The Immunological Synapse Therefore, we crossed the CD2AP / ani- mals to the AND TCR transgenic mouse (8). Balances T Cell Receptor This TCR recognizes a cytochrome c peptide bound to the MHC class II molecule, I-Ek, Signaling and Degradation but it can develop in the thymus of a b haplotype mouse. CD4 and CD8 profiles from young animals, as well as bone marrow Kyeong-Hee Lee,1,2* Aaron R. Dinner,3*‡ Chun Tu,1 chimeras, showed normal distributions of Gabriele Campi,5 Subhadip Raychaudhuri,4 Rajat Varma,5 CD4- and CD8-positive cells, suggesting that Tasha N. Sims,5 W. Richard Burack,1 Hui Wu,1 Julia Wang,1 thymocyte development is not grossly im- Osami Kanagawa,1 Mary Markiewicz,1 Paul M. Allen,1 paired in CD2AP / mice (9). Naıve T cells ¨ were isolated from mice at 3 weeks of age Michael L. Dustin,5† Arup K. Chakraborty,3,4,6† Andrey S. Shaw1† (the time of kidney disease initiation) and were used immediately or established as T The immunological synapse is a specialized cell-cell junction between T cell and cell lines. antigen-presenting cell surfaces. It is characterized by a central cluster of Images of the interface between antigen- antigen receptors, a ring of integrin family adhesion molecules, and temporal bearing APCs and T cells were reconstructed stability over hours. The role of this specific organization in signaling for T cell with confocal microscopy and staining with activation has been controversial. We use in vitro and in silico experiments to antibodies to the TCR and LFA-1. As reported determine that the immunological synapse acts as a type of adaptive controller previously (4), by 30 min, WT T cells form a that both boosts T cell receptor triggering and attenuates strong signals. “mature” synapse: a ring of LFA-1, marking the P-SMAC, surrounding a region enriched in The mature immunological synapse is char- (WT) cells. Our attempts were initially com- TCR, marking the C-SMAC [fig. S9A (10)]. acterized by a reorganization of membrane plicated by lethal nephrotic syndrome in Consistent with previous work (6), CD2AP proteins, resulting in a stable central cluster CD2AP / mice by 6 to 7 weeks of age (7). deficiency profoundly altered these morpholog- of T cell receptors (TCRs) (the C-SMAC) The location of the CD2AP gene on mouse ical features [fig. S9B (10)]. At no time were surrounded by a ring of adhesion molecules chromosome 17, close to the major histocom- clearly defined C- and P-SMACs visualized, (the P-SMAC) (1–3). Although it was origi- patibility complex (MHC) class II locus, ne- and TCRs were homogeneously distributed nally proposed that the immunological syn- cessitated the use of a TCR transgenic mouse with LFA-1 throughout the synapse. apse serves to enhance and sustain signaling through the TCR for long periods of time (3), the paucity of active signaling molecules in the C-SMAC after a few minutes suggests that it may not be involved in signaling (4, 5). We combined in vitro experiments with CD2AP / T cells and simulations of a com- putational (or in silico) model to address this controversy. Our results demonstrate that the C-SMAC is a site for both strong receptor triggering and increased TCR degradation. Because CD2AP is required for receptor segregation into the C-SMAC in a model immunological synapse (6), we compared synapse formation and signaling in Fig. 1. Hypersensitivi- CD2AP / cells with those in wild-type ty of CD2AP-deficient T cells to antigen. Pro- liferative responses of 1 Department of Pathology and Immunology, Wash- (A) freshly isolated ington University School of Medicine, Box 8118, 660 naıve splenic T cells ¨ South Euclid, Saint Louis, MO 63110, USA. 2Depart- and (B) long-term cul- ment of Immunology, Genentech, 530 Forbes Boule- vard, One DNA Way, South San Francisco, CA 94080, tured primary T cell USA. 3Department of Chemistry, 4Department of lines from AND / Chemical Engineering, University of California, Berke- CD2AP / or AND / ley, CA 94720, USA. 5Program in Molecular Pathogen- CD2AP / mice were esis, Skirball Institute of Biomolecular Medicine and stimulated by anti- Department of Pathology, New York University, New genic peptide–pulsed York, NY 10016, USA. 6Physical Biosciences and Ma- APCs at the indicated terials Sciences Division, Lawrence Berkeley National doses of MCC peptide Laboratory, University of California, Berkeley, CA (88 –108) for 48 hours. (A) A representative result from nine independent experiments. The T cell 94720, USA. line in (B) was cultured for 6 weeks in vitro and rested for 2 weeks before antigenic stimulation. *These authors contributed equally to this work. Mean 3H-thymidine uptakes SD are shown. (C) Delayed and sustained tyrosine phosphorylation †To whom correspondence should be addressed: E- in CD2AP-deficient T cells. T cells from AND /CD2AP / or AND /CD2AP / primary cell lines mail: (A.S.S.), arup@uclink. were stimulated with the B cell hybridoma TA3 prepulsed with 100 mM MCC peptide. At the (A.K.C.), and indicated time points, cells were lysed and postnuclear lysates were immunoprecipitated with (M.L.D.) anti-ZAP70 or anti-TCR polyclonal rabbit sera. Samples were resolved by SDS–polyacrylamide gel ‡Present address: Department of Chemistry, Univer- electrophoresis and immunoblotted with anti-phosphotyrosine (4G10). As a protein-loading con- sity of Chicago, Chicago, IL 60637, USA. trol, the same blots were stripped and reblotted with ZAP70- or TCR -specific antibodies. 1218 14 NOVEMBER 2003 VOL 302 SCIENCE
  • 2. REPORTS To assess T cell activation, we incubated The enhanced proliferation associated 10 min, peaked at 20 min, and was main- cells with peptide-pulsed splenocytes. Both with CD2AP deficiency correlated with tained at this level for at least 60 min. naıve and cultured T cell lines from ¨ prolonged tyrosine phosphorylation of the Thus, CD2AP / cells did not form a CD2AP / T cells showed an increased sen- TCR chain and of ZAP70 (Fig. 1C), pa- C-SMAC with APC and exhibited sustained sitivity to antigen and augmented cell prolif- rameters that reflect TCR signaling. TCR tyrosine phosphorylation, which correlates eration compared with WT cells (Fig. 1, A and ZAP70 immunoprecipitates, prepared with increased proliferation. These findings and B). Some of the enhanced proliferation from T cells stimulated with APC-peptide, appear to support the view that the C-SMAC may be attributed to increased levels of se- were immunoblotted with an antibody to does not potentiate TCR signaling. However, creted interleukin-2 (IL-2) because naıve ¨ phosphotyrosine. In WT cells, tyrosine phos- the veracity of this interpretation is compli- CD2AP / T cells secreted increased IL-2 at phorylation of ZAP70 and TCR was de- cated by the many factors that affect ampli- all peptide concentrations tested [fig. S6A (9, tectable at 10 min, peaked at 20 min, and fication and termination of signaling (e.g., 10)]. CD2AP / T cells also exhibited an returned to base line by 60 min. In contrast, receptor-ligand binding, formation of intra- increased number of cell divisions and in- CD2AP deficiency delayed and markedly cellular signaling complexes, kinetic proof- creased apoptosis compared with WT cells prolonged the detectable response; tyrosine reading, serial triggering, receptor endocyto- [fig. S6B (9, 10)]. phosphorylation was barely detectable at sis and degradation, and receptor clustering). To delineate the roles played by each of these factors and to understand how their interplay and coordination affect signal transduction, we developed a computational model in which proteins are represented by particles on a lattice. A kinetic Monte Carlo (MC) algo- rithm (10) simulated the dynamics of recep- tor-ligand binding, signal transduction, and protein movement. Particles diffuse, form complexes, catalyze phosphorylation and nu- cleotide exchange, and undergo phospho- transfer. Each attempt of one such event cor- responds to an increment in time of one MC step (the time unit in the simulations). The TCR signaling cascade (Fig. 2) was deter- mined by a specific set of allowed reactions (11–13). Signaling is initiated by binding of peptide MHC ( pMHC) to the TCR. This allows Lck to sequentially phosphorylate two sites on the TCR (first TCR 1 and then TCR 2 ). These sites are a simplified repre- sentation (Fig. 2A, i) of the immunoreceptor tyrosine-based activation motifs (ITAMs). A TCR with only TCR 1 phosphorylated corre- sponds to the p21 form, whereas a TCR phosphorylated at both TCR 1 and TCR 2 corresponds to the p23 form (14). The extent to which the TCR becomes phosphorylated is dependent on the half-life of the interaction between pMHC and TCR. The phosphoryl- ated TCR 1 and TCR 2 can then be bound by one or more ZAP70s, which in turn can be phosphorylated and activated by Lck Fig. 2. Schematic representation of the signaling network. (A) Basic network used in the simula- (Fig. 2A, ii). When ZAP70 is phosphoryl- tions. Each box corresponds to a particle on the lattice; molecules composed of more than one particle are indicated by larger boxes, some of which are subdivided to indicate multiple internal- ated, it can recruit adapter molecules like state variables (e.g., TCR , TCR 1, and TCR 2 ). Colored boxes represent active states and gray LAT, GADS (or Grb2), Itk, SLP-76, and boxes represent inactive ones; white boxes represent internal states that do not change. Yellow SLAP-130, as well as the signaling mole- boxes represent phosphorylation, blue boxes represent conformational changes, and green boxes cules that bind to them (Fig. 2A, iv to vi). represent GTPase. Species pairs that form complexes are indicated by dashed lines. Thin single- The various downstream intracellular sig- headed arrows indicate activation (and deactivation) events, and wide double-headed arrows naling molecules in the model (such as Ras) indicate equilibria between states. Thin T-shaped lines indicate inhibition. Two additional transfer reactions, corresponding to generic phosphatases (Pase1 and Pase2 ) that dephosphorylate TCR 1 serve merely as “counters” that give us and TCR 2, are not shown. These reactions were included to allow signaling TCR to revert in the potential readouts and measure the signal absence of pMHC interactions. (B) Additional interactions and reactions included in the full network strength. Lck interactions with SHP-1 and used in the simulations. The symbols are the same as in (A). LckY reflects the phosphorylation state ERK (Fig. 2B) provided negative and pos- of residue Y394 and is taken to be partially active even when not phosphorylated. We neglect itive feedback, respectively, to the cascade regulation at the Y505 site of Lck (by CD45 and Csk), which corresponds to assuming that it is (13, 15, 16 ). always dephosphorylated (not inhibitory). Ignoring CD45 prevents our model from exhibiting the brief reduction in tyrosine phosphorylation observed in the first few minutes of signaling (5). LckY To model formation of the C-SMAC, we activates its own inhibitor, the phosphatase SHP-1. When active, SHP-1 dephosphorylates LckY, as introduced a force that biases TCR motion shown in (ii). This last reaction is prevented by phosphorylation of residue S59 (LckS) by ERK (a toward the center of the interface with the positive-feedback loop). SHP-1 also inhibits signaling by competing with ZAP70 to bind TCR 1. APC. The central accumulation of TCR de- SCIENCE VOL 302 14 NOVEMBER 2003 1219
  • 3. REPORTS pends on cytoskeletal and membrane forces ated signaling, thus allowing us to assess how loop associated with SHP-1 (Fig. 2B) in- regulated by the Rho family of GTPases (17). various factors interact to influence signal creased specificity by inhibiting TCR-based This was modeled by linking the biasing transduction. As an example of the time signaling for shorter TCR-pMHC half-lives force to active ZAP70-mediated recruitment course of signaling obtained with our model (13, 15, 16). of an adapter that can activate a heterotrimer- (Fig. 3A), we used phosphorylation of Eliminating centrally biased TCR move- ic GTP-binding protein (G protein) (referred ZAP70 as the readout because its activation is ment from the model allowed us to ask how to in Fig. 2 as a GTPase that could be Rac). a critical intermediate in the signaling pro- the lack of an organized C-SMAC (as seen Specifically, receptor movement was stimu- cess. Other readouts downstream of active with conjugates of CD2AP / cells with lated when the number of these activated G ZAP70 are also accessible in our simulations, APC) affected signaling. When the C-SMAC proteins exceeded a threshold (10). and in all cases, exhibited the same qualita- did not form, the strength of the signal at TCR internalization and degradation were tive behavior (10). early times was somewhat lower than when also included in the model as these processes The simulations showed that the magni- the C-SMAC did form, but the signal was are thought to play critical roles in turning off tude of signaling increases and then decreases sustained for a much longer period of time TCR signaling. On the basis of existing data, over time. Analysis of these results demon- (Fig. 3A). This qualitative difference resem- our model constitutively internalizes TCRs at strated that the initial rise reflects the time it bles the difference in the time course of a fixed rate (18). If a receptor is not phos- takes for the signal to propagate through the signaling between CD2AP / and WT cells phorylated or only singly phosphorylated network of biochemical events, and the de- (Fig. 1C), except that the in silico experi- ( p21), it is returned to the surface. If a TCR cline of signaling occurs because of receptor ments without C-SMAC formation did not is fully phosphorylated ( p23), it is degraded; degradation. When the model was tested over exhibit a marked delay in the onset of signal- i.e., it is removed from the system (18). Both a range of different TCR-pMHC half-lives, ing. The qualitative results in Figure 3A are bound and unbound TCRs in the p23 form the magnitude of signaling was maximized at robust to 20-fold variations in the kinetics of were subject to degradation (19). The quali- an intermediate TCR-pMHC half-life (Fig. the reactions that constitute the signaling tative results of our simulations were insen- 3B). Half-lives longer than the optimum val- pathways in our simulations. sitive to whether internalized TCRs were re- ue impaired the ability of pMHC to engage Our analysis of the simulation results turned to the same spot on the membrane or many TCRs (serial triggering) (20). In con- demonstrates that the C-SMAC enhances sig- whether they were randomly returned to any trast, short half-lives did not allow sufficient naling by concentrating TCR, pMHC, and spot on the surface. time for receptor triggering (kinetic proof- kinases like Lck into a small area. Clustering Although this model (Fig. 2) is clearly a reading) (21). This competition results in an TCR and pMHC allows for more frequent simplified representation of signaling in T optimal half-life (19–21). In addition, the TCR-pMHC binding. When the C-SMAC cells, it includes key features of TCR-medi- simulations showed that a negative-feedback does not form, the median time for a pMHC to rebind a TCR after dissociation is 20.3 Fig. 3. (A) Time course of 106 MC steps. In contrast, the corresponding ZAP70 activity from simula- time is only 2.7 106 MC steps when TCRs tions of the full model: No cluster in the C-SMAC. Receptor clustering C-SMAC formation (blue in the C-SMAC should therefore directly en- line; ps 1.00, where ps is hance the frequency of TCR-pMHC complex the probability of accepting formation without any change in the TCR- a displacement away from the center of the junction) pMHC half-life. Our analysis also showed and C-SMAC formation (red that the C-SMAC facilitates TCR phospho- line; ps 0.85). Error bars rylation by Lck because Lck can act on clus- indicate the standard error tered TCRs rather than single TCRs. These of the mean for 40 trials. factors combine to result in a much higher The panels on the right rate of production of fully phosphorylated show the spatial distribu- tion of active ZAP70 at the TCRs ( p23) when the C-SMAC does form. approximate peak in signal- Why then do we observe a paucity of phos- ing activity (20 109 MC phorylated molecules in the C-SMAC over steps). On average, TCRs long times in the in vitro and in silico exper- were internalized every 1 iments (Figs. 1C and 3A)? 109 MC steps. The basic re- Because only fully phosphorylated recep- sults shown here are repro- duced by a simpler field tors are subject to degradation, the higher rate model (10). (B) The average of production of fully phosphorylated TCRs number of active ZAP70 as in the C-SMAC enhances receptor degrada- a function of TCR-pMHC off tion. Thus, counterintuitively, enhanced re- rate at 10 109 MC steps. ceptor triggering in the C-SMAC also serves Blue line: basic network to limit sustained tyrosine kinase activity in shown in Fig. 3B; red line: full network (includes the the C-SMAC over long times. The higher the interactions in Fig. 3C). Er- rate of receptor degradation, the shorter will ror bars indicate the SEM for be the time period over which phosphorylated 10 trials. At the slowest off molecules are observed in the C-SMAC. In- rate, each pMHC interacts deed, the model suggests that the only way to with only 1.3 TCRs on aver- sustain TCR signaling over longer periods age within the simulation time. As the off rate increases, each pMHC triggers a larger number of TCR until Lck can no would be to have TCR replenishment from longer phosphorylate TCR 2 within the time a TCR is bound to a pMHC. On average, at the new synthesis (22, 23). The persistent high fastest off rate, the time for p23 TCR to form once pMHC is bound is about 13 times as long level of signaling in the absence of CD2AP as the lifetime of the TCR-pMHC complex. may therefore stem from defects that lower 1220 14 NOVEMBER 2003 VOL 302 SCIENCE
  • 4. REPORTS / the rate of TCR triggering, and/or concomi- examine whether CD2AP cells could C-SMAC facilitates and enhances signaling tant degradation of activated TCRs. form a C-SMAC with planar bilayers. by the TCR and that the absence of signaling To directly assess whether CD2AP defi- Despite some defects in the fine structure, intermediates at later time points is due to ciency affects TCR down-regulation, we C- and P-SMACs were readily formed in the concomitantly higher receptor degradation. measured TCR expression levels before and absence of CD2AP (Fig. 5A), demonstrating The observation that CD2AP / cells in the after T cell activation. Antigen-pulsed APCs that CD2AP is not absolutely required for bilayer system exhibit discernable C-SMACs induced WT T cells to down-regulate TCR C-SMAC formation. This experimental sys- that are the site of the strongest signaling also expression (Fig. 4A) (24). CD2AP / T cells tem allowed us to measure signaling in a provides evidence against a role for CD2AP only minimally down-regulated TCR expres- C-SMAC in the absence of receptor degrada- only in formation of the C-SMAC. If sion (Fig. 4A). This was due directly to the tion. The in silico model was used to predict CD2AP’s only function was to promote C- absence of CD2AP because reconstitution of the outcome of signaling in the C-SMAC SMAC formation, WT cells and CD2AP / CD2AP / cells with a CD2AP-expressing with (WT cells) and without (CD2AP / cells should exhibit nearly identical behavior retrovirus restored TCR down-regulation cells) receptor degradation. Based on the in the bilayer experiments. Indeed, (Fig. 4B). To assess the effect of CD2AP presence of phosphotyrosine as the readout, CD2AP / cells exhibited the strongest deficiency on degradation of TCR (22), we the model predicts sustained and strong phos- phosphotyrosine levels, and WT cells, the measured TCR expression before and after photyrosine staining in the C-SMAC, assum- weakest phosphotyrosine levels, in the C- stimulation with peptide-pulsed APCs. ing formation of the synapse in the absence of SMAC (Fig. 5). Whereas T cell activation greatly reduced the receptor degradation (Fig. 5B). Consistent Combining experiments with CD2AP / level of TCR in WT T cells, there was no with this prediction, anti-phosphotyrosine cells and simulations of a computational effect on TCR chain levels in CD2AP / T stained the P-SMAC but not the C-SMAC in model reveals an unsuspected function of the cells (Fig. 4C). This could be due to a role for WT cells (Fig. 5C). In contrast, when C-SMAC and resolves the controversy re- CD2AP in intracellular trafficking because CD2AP / T cells were stained for phospho- garding the role of the C-SMAC in propagat- CD2AP deficiency impaired delivery of the tyrosine, the greatest staining was observed ing and attenuating signals. Concentrating TCR to lysosomes (fig. S7) (10, 25, 26). in the C-SMAC (Fig. 5C). These experiments antigen, TCR, and kinases in the C-SMAC To determine whether the defect in down- confirm that concentrating receptors in the enhances signaling by decreasing the amount regulation also involved changes in TCR in- ternalization, we measured the basal rate of TCR internalization using an inhibitor of an- terograde transport, brefeldin A. In the pres- ence of brefeldin A, TCRs were steadily lost from the plasma membrane because internal- ized TCRs are unable to recycle (18). CD2AP deficiency did not alter this basal rate of internalization (fig. S5) (9). Furthermore, an- tibody-mediated TCR internalization at early time points was similar between WT and CD2AP / T cells (9). Therefore, the failure to down-regulate TCR in the CD2AP / T cells appears to be due to a defect in TCR degradation, not internalization. A counterintuitive prediction of the com- putational model is that the attenuation of phosphotyrosine levels in the C-SMAC is due to enhanced receptor triggering, which results in increased receptor degradation. Because CD2AP / cells cannot mediate receptor Fig. 4. Defective TCR degradation, we reasoned that, if these cells down-regulation and could be induced to form a C-SMAC, we degradation in CD2AP- could directly assess whether there is en- deficient T cells. (A) Naıve T cells from ei- ¨ hanced receptor triggering in the C-SMAC. A ther AND/CD2AP / mathematical model analyzing the thermody- or AND/CD2AP / namics of synapse formation predicts that a mice were stimulated C-SMAC should form more readily with pla- with splenic APCs nar lipid bilayers containing intercellular ad- from B10.BR mice in hesion molecule 1 (ICAM-1) and pMHC than the presence or absence of 10 M MCC peptide. After 2 hours, cell conjugates were disrupted by treatment with EDTA-trypsin. The surface expression of AND TCRs was analyzed by flow cytometry with APCs because of higher ligand mobility with the TCR Vb3-specific antibody KJ25 and gating on Thy1.2-positive cells. (B) Retroviral in the planar bilayer and because only one transfection of CD2AP reconstitutes TCR down-regulation in CD2AP / T cells. (Left) CD2AP / deformable membrane is involved (27); both T cell lines were transduced with CD2AP–green fluorescent protein (GFP) retrovirus and stimulated factors make reorganization of receptors and as described in (A). The surface expression of AND TCRs was analyzed in the GFP-positive (R1) or ligands easier. Furthermore, CD2AP may be -negative (R2) T cell population. (Right) CD2AP / T cells were transduced with a retrovirus required for C-SMAC formation only when expressing GFP alone. (C) TCR degradation in CD2AP / and CD2AP / T cells. Lymph node T cells from either AND/CD2AP / or AND/CD2AP / mice were stimulated for 4 hours with the B cell there are large numbers of CD2-CD48 inter- hybridoma CH27 prepulsed with 100 M MCC peptide. Cells were lysed in RIPA lysis buffer, and actions that require organization, which are postnuclear lysates were immunoprecipitated and immunoblotted with anti-TCR polyclonal rabbit present in the cell-cell system but absent in sera. To block synthesis of TCRs, T cells were pretreated with cycloheximide for 1 hour before the bilayer system. These reasons led us to antigenic stimulation. SCIENCE VOL 302 14 NOVEMBER 2003 1221
  • 5. REPORTS Fig. 5. CD2AP / cells form a C-SMAC with planar bilayers and exhibit strong signaling. (A) Immune synapse formation with planar lipid bilayers. T cell blasts from either CD2AP / or CD2AP / mice were incubated on a supported planar lipid bilayer containing Oregon Green- I-Ek ( prepulsed with 100 M peptide) at 166 molecules/ m2 and Cy5–ICAM-1 at 266 molecules/ m2. Cells were imaged at 37°C with a Zeiss Confocal LSM510 microscope in real time. (B) Time course of phosphorylated TCR, LckY, and ZAP70 ( pY ) obtained from the com- putational model for cases that form the C-SMAC with (red curve) and without (blue curve) TCR degradation. Error bars indicate the SEM for 40 trials. The panels show the levels of phosphotyrosine at 60 109 MC steps in the C-SMAC only. Brighter shades of red correspond to higher phosphotyrosine levels. (C) Tyrosine phosphorylation patterns in CD2AP / and CD2AP / T cells on planar lipid bilayer. T cell blasts were plated on supported planar lipid bilayer containing I-EK ( pre- (Right) Quantitation of these patterns was obtained by calculating the ratio pulsed with peptide) and ICAM-1, as in (A). After 1 hour, cell-bilayer of C-SMAC fluorescence divided by the P-SMAC fluorescence in over 15 cells conjugates were fixed and permeabilized. Tyrosine phosphorylation was per experiment. Data are the average of three independent experiments. visualized by staining with a phosphotyrosine-specific antibody (4G10). Error bars denote standard deviations. of time required for antigenic ligand to search ments that will result in a deeper under- 16. I. Stefanova et al., Nature Immunol. 4, 248 (2003). and find TCR and for subsequent receptor standing of the complex issues underlying 17. C. Wulfing, M. M. Davis, Science 282, 2266 (1998). 18. H. Liu, M. Rhodes, D. L. Wiest, D. A. Vignali, Immunity phosphorylation. Because of these factors, it T cell activation. 13, 665 (2000). is predicted that fully phosphorylated recep- The function of the immunological syn- 19. D. Coombs, A. M. Kalergis, S. G. Nathenson, C. Wofsy, tors ( p23) form in the absence of C-SMAC apse is an emergent property involving many B. Goldstein, Nature Immunol. 3, 926 (2002). 20. S. Valitutti, S. Muller, M. Cella, E. Padovan, A. Lanza- formation only with high-affinity agonists; inextricably linked variables, and our work vecchia, Nature 375, 148 (1995). p23 generation is greatly facilitated by the illustrates how the analysis of such complex 21. T. W. McKeithan, Proc. Natl. Acad. Sci. U.S.A. 92, C-SMAC when ligand quality is weaker. biological systems benefits greatly from syn- 5042 (1995). 22. A. G. Schrum, L. A. Turka, J. Exp. Med. 196, 793 However, enhanced receptor triggering in the ergistic experimental and computational stud- (2002). C-SMAC results in increased receptor degra- ies. Without the computational model, for 23. A. Lanzavecchia, F. Sallusto, Curr. Opin. Immunol. 12, dation, which limits sustained tyrosine phos- example, we might have wrongly concluded 92 (2000). phorylation within the C-SMAC. The model that the C-SMAC is not involved in TCR 24. S. Valitutti, S. Muller, M. Salio, A. Lanzavecchia, J. Exp. Med. 185, 1859 (1997). therefore predicts that the C-SMAC is re- triggering and that it functions only to atten- 25. J. Kim et al., Science 300, 1298 (2003). sponsible for intense but self-limited signal- uate signaling. The surprising interpretation 26. I. Dikic, S. Giordano, Curr. Opin. Cell. Biol. 15, 128 ing. Thus, the synapse functions as an adap- that the C-SMAC balances TCR signaling (2003). 27. S. Y. Qi, J. T. Groves, A. K. Chakraborty, Proc. Natl. tive controller. Limiting strong signaling over and degradation emerged from the computa- Acad. Sci. U.S.A. 98, 6548 (2001). long times may serve to protect against cell tional model and the cellular experiments it 28. J. Sloan-Lancaster, P. M. Allen, Annu. Rev. Immunol. death caused by overstimulation. This is con- suggested. 14, 1 (1996). sistent with our observation of enhanced ap- 29. Y. Itoh, B. Hemmer, R. Martin, R. N. Germain, J. Immunol. 162, 2073 (1999). optosis in CD2AP / cells (9). References and Notes 30. S. Martin, M. J. Bevan, Eur. J. Immunol. 28, 2991 The adaptive control function of the C- 1. M. F. Krummel, M. M. Davis, Curr. Opin. Immunol. 14, (1998). 66 (2002). 31. This research is supported by the NIH. We thank D. SMAC revealed by our work touches on 2. C. R. Monks et al., Nature 395, 82 (1998). Wiley, D. Chandler, E. Unanue, E. Hailman, and T. many complex issues including ligand 3. A. Grakoui et al., Science 285, 221 (1999). Starr for discussions or assistance. T.N.S. was also quality, TCR down-modulation, and partial 4. K. H. Lee et al., Science 295, 1539 (2002). supported by the National Psoriasis Foundation. 5. B. A. Freiberg et al., Nature Immunol. 3, 911 M.L.D. was also supported by the Irene Diamond TCR signaling. For example, our model has (2002). Foundation. A.R.D. was also supported by a Bur- implications for the biology of signaling by 6. M. L. Dustin et al., Cell 94, 667 (1998). roughs Wellcome Fund Hitchings–Elion Fellowship altered peptide ligands (APLs) (28). Our 7. N. Y. Shih et al., Science 286, 312 (1999). and the NSF (grant CHE-0078458). 8. J. Kaye et al., Nature 341, 746 (1989). model suggests that the critical link be- 9. K.-H. Lee et al., data not shown. Supporting Online Material tween partial chain phosphorylation and 10. Supporting material is available on Science Online. inefficient TCR down-modulation of APLs 11. G. A. Koretzky, P. S. Myung, Nature Rev. Immunol. 1, SOM Text 95 (2001). Fig. S1 to S9 (29, 30) is their inability to form the im- 12. A. Weiss, D. R. Littman, Cell 76, 263 (1994). Table S1 to S4 munological synapse (3), which both en- 13. R. N. Germain, I. Stefanova, Annu. Rev. Immunol. 17, References hances and limits signaling. These and oth- 467 (1999). er hypotheses emerging from the adaptive 14. J. Sloan-Lancaster, A. S. Shaw, J. B. Rothbard, P. M. 6 May 2003; accepted 26 August 2003 Allen, Cell 79, 913 (1994). Published online 25 September 2003; control function of the C-SMAC are test- 15. C. Chan, A. J. George, J. Stark, Proc. Natl. Acad. Sci. 10.1126/science.1086507 able and should motivate further experi- U.S.A. 98, 5758 (2001). Include this information when citing this paper. 1222 14 NOVEMBER 2003 VOL 302 SCIENCE