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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
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: firstname.lastname@example.org (A.S.S.), arup@uclink. were stimulated with the B cell hybridoma TA3 prepulsed with 100 mM MCC peptide. At the
berkeley.edu (A.K.C.), and email@example.com 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 www.sciencemag.org
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-
www.sciencemag.org SCIENCE VOL 302 14 NOVEMBER 2003 1219
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 www.sciencemag.org
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.
www.sciencemag.org SCIENCE VOL 302 14 NOVEMBER 2003 1221
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
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1222 14 NOVEMBER 2003 VOL 302 SCIENCE www.sciencemag.org