seminars in I M M U N OL OG Y, Vol 12, 2000: pp. 5]21
doi: 10.1006rsmim.1999.0203, available online at http:rrwww.idealibrary.com on
Cytoskeletal polarization and redistribution of cellsurface molecules during T cell antigen recognition
P. Anton van der Merwe 1U , Simon J. Davis 2, Andrey S. Shaw 3 and
Michael L. Dustin 3
lymphocytes by, for example, chemotactic factors or
ligation of antigen receptors results in increased adhesiveness,3 cytoskeletal polarization and the development of morphological asymmetry.4 It has long
been recognized that cytoskeletal changes contribute
to lymphocyte effector functions such as polarized
cytokine secretion and target cell killing.5,6 This review focuses on the role of cytoskeletally-mediated
processes in T cell antigen recognition.
Asymmetry of activated lymphocytes also extends to
the distribution of molecules on their surface. In
migrating lymphocytes, chemokine receptors are
concentrated at the leading edge, whereas molecules
such as CD43, CD44 and CD50 are found at the
trailing edge.4 Dramatic changes in cell-surface
molecule distribution are also observed when the T
lymphocytes recognise antigens on the antigen presenting cells ŽAPCs..5,7 ] 10 In this review we first describe the redistribution that occurs during T cell
antigen recognition, placing it in the context of other
cellular events such as changes in cell motility and
morphology, increases in intracellular calcium, and
polarization of the cytoskeleton. We then discuss in
detail the molecular mechanisms underlying redistribution. Finally, we speculate on the functional significance of redistribution, and attempt to integrate
the experimental data into a general model of T cell
antigen recognition.
T cell antigen recognition is accompanied by cytoskeletal
polarization towards the APC and large-scale redistribution
of cell surface molecules into ‘supramolecular activation
clusters’ (SMACs), forming an organized contact interface
termed the ‘immunological synapse’ (IS). Molecules are
arranged in the IS in a micrometer scale bull’s eye pattern
with a central accumulation of TCR r peptide]MHC (the
cSMAC) surrounded by a peripheral ring of adhesion
molecules (the pSMAC). We propose that segregation of cell
surface molecules on a much smaller scale initiates TCR
triggering, which drives the formation of the IS by active
transport processes. IS formation may function as a checkpoint for full T cell activation, integrating information on
the presence and quality of TCR ligands and the nature
and activation state of the APC.
Key words: T cell receptor r T cell antigen recognition r
SMAC r immunological synapse r segregation
Q2000 Academic Press
Introduction
LYMPHOCYTES ARE PROPELLED through the blood as
relatively undifferentiated spheres uniformly covered
with short microvilli, but without obvious asymmetry
or polarity. These cells also relatively non-adhesive,
partly because integrins such as LFA-1 are immobilized by attachment to the actin cytoskeleton
and maintained in a low avidity state.1,2 Activation of
Observations
Snapshots of cytoskeletal polarization and cell-surface
molecule redistribution
From the 1Sir William Dunn School of Pathology, University of
Oxford, Oxford OX1 3RE, UK, 2Nuffield Department of Clinical
Medicine, John Radcliffe Hospital, University of Oxford, Headington OX3 9DU, UK and 3Centre for Immunology and the Department of Pathology, Washington University School of Medicine,
U
660 South Euclid Avenue, St. Louis, MO 63110, USA. Corresponding author.
Q2000 Academic Press
1044-5323r 00 r 010005q 17 $35.00r 0
Studies performed over a decade ago showed that,
following recognition of the antigen on target cells or
APCs, T cells become polarized with respect to the
target cell or APC Žreviewed in ref 5.. The microtubule organising centre ŽMTOC., which is usually
5
P. Anton van der Merwe et al
6
Redistribution of T cell surface molecules
found in the trailing edge of a migrating T cell,
repositions itself between the T cell nucleus and the
target cell.11 This is accompanied by, and is probably
responsible for, the movement of the Golgi apparatus
to the same side of the cell.11 There is also an
increase in actin polymerization adjacent to the contact interface, and the actin-binding protein talin
accumulates in this area.12 The T cell receptor and
its CD3 signalling module ŽTCRrCD3., the TCR coreceptor CD4, and the adhesion receptorrligand pair
LFA-1rICAM-1 all accumulate at the contact interface between CD4 T cells and APCs presenting specific antigens.5 Much more recently, Wulfing and
Davis13,14 have reported evidence for a more generalized, cytoskeletally-driven movement of cell surface
molecules towards the T cellrAPC contact interface.
Recently the redistribution of molecules within the
contact interface has been visualized both in fixed T
cellrAPC conjugates7,8 as well as in live T cells interacting with planar, glass-supported lipid bilayers into
which fluorescently-labelled, lipid-anchored molecules have been incorporated.10,15 These bilayers are
capable of stimulating T cell proliferation, suggesting
that they can function as surrogate APCs. While less
physiological than APCs, the planar bilayer system
has provided higher resolution images and the first
quantitative and dynamic data on this redistribution.
Both approaches reveal that cell-surface molecules
segregate within T cellrAPC contacts into distinct
regions or clusters, referred to by Monks et al 8 as
supramolecular activation clusters ŽSMACs., thereby
forming an organized interface which we refer to as
an immunological synapse10 ŽFigure 1.. The phrase
immunological synapse ŽIS. was coined several years
ago 6 to draw attention to some of the similarities
between these contact areas and neuronal synapses.16
We have extended the term to describe the charac-
teristic molecular patterns formed in the process of
physiological T cell activation.10
IS formation passes through several stages ŽFigure
2., culminating in the ‘mature’ IS ŽFigure 1.. In the
mature IS the TCRrCD3 and several molecules involved in TCR triggering congregate at the centre of
the interface.7,8,10 These include peptide]MHC, the
accessory molecule CD28 and its ligand CD80, and
cytoplasmic signalling molecules Že.g. lck, fyn, PKC-u ..
This central area has been termed the central cluster
or cSMAC.8 A second group of molecules, including
LFA-1, ICAM-1 and talin, form a ring surrounding
the cSMAC, which has been termed the ‘outer adhesion ring’ or peripheral SMAC ŽpSMAC..7,8,10 In the
planar bilayer system10 the accessory molecule CD2
and its ligand CD48 appear to be confined to an
‘inner adhesion ring’. This surrounds the central
zone but lies within the outer LFA-1 adhesion ring.
Finally, CD43, a large, highly-abundant mucin-like
molecule, is mostly excluded from the contact interface.9
Dynamics of polarization and redistribution
Studies in live cells have provided insights into the
timing and the sequence of events described above. T
cell clones and hybridomas are highly mobile in cell
culture, migrating at speeds of 4]10 m mrmin.4,17
When they encounter surrogate APCs Žusually cell
lines transfected with suitable MHC molecules. that
are not presenting specific antigens, they show no
changes in motility or morphology.17,18 In contrast,
when they encounter APCs presenting specific antigens they arrest, undergo shape changes, and expand
their contact with the APC.17,18 T cells also migrate
rapidly on artificial substrates coated with ICAM-1,
but stop migrating when they encounter regions
Figure 1. Structure of the mature immunological synapse. Ža. Schematic view of the mature IS. The position of the IS is
shown schematically above. The structure of the IS is shown below as viewed perpendicular to the contact interface. Not
that some of the molecules are expressed on T cells and others on APCs. CD43 is excluded from the entire interface. ŽB.
Fluorescent images revealing the position of the CD28rCD80 Žleft. and CD2rCD48 Žright. complexes in the IS. The left
image shows the positions of peptide]MHC Žgreen., CD80 Žred. and ICAM-1 Žblue. at the interface between 3A9 T cells
and planar bilayers. The right image shows the positions of peptide]MHC Žgreen., CD48 Žred. and ICAM-1 Žblue. at
interface between 2B4 T cells and planar bilayers. The CD28rCD80 complex colocalizes with the TCRrpeptide]MHC
whereas the CD2rCD48 complex segregates to an inner ring between TCRrpeptide]MHC and LFA-1rICAM-1. Data from
Grakoui et al.10
Figure 2. Steps in the formation of the IS. ŽA. Fluorescent images illustrating the dynamics of IS formation. T cells are
brought into contact with planar lipid bilayers at t s 0. The positions of engaged peptide]MHC Žgreen. and engaged
ICAM-1 Žred. at the indicated times after initial contact are shown superimposed on an image of the contact area Žwhite..
Reproduced with permission from Grakoui et al.10 ŽB. The three stages of IS formation in planar lipid bilayer system.
Reproduced with permission from Grakoui et al.10
7
P. Anton van der Merwe et al
coated with ICAM-1 and specific peptide]MHC complex.19 Together these data suggest that an encounter with specific antigens delivers a stop signal to
migrating T cells, which serves to keep them associated with the APC that is presenting specific antigen.19
Naive T cells are less mobile but also show rapid
changes in morphology and motility upon contact
with B cells presenting specific antigens.20,21 These
changes in motility and morphology are followed in a
variable time Žseconds to minutes. later by increases,
sometimes oscillating, in intracellular calcium concentration.17,18,22
A consistent feature in all the above studies is that
changes in T cell motility, morphology and intracellular calcium concentration are only seen if the APC
or planar bilayers present specific antigens, indicating that they require TCR triggering. In contrast,
when dendritic cells are used as APCs, morphological
changes, conjugate formation, and increases in calcium are seen in the absence of antigens,21 even in
the absence of MHC class II.23 This suggests that,
unlike surrogate APCs or B cells, dendritic cells have
the ability to engage and activate naıve
¨ T cells prior
to any TCR engagement, perhaps through the expression of high levels of the CD28 ligands CD80 and
CD86 or chemokines.24,25
Technical innovations such as the use of green
fluorescent protein chimeras13 and the planar bilayer
system15 have made it possible to visualise the redistribution of cell surface molecules in live T cells. A
recent study 10 using primary T cells in the planar
lipid bilayer system has revealed that the formation of
the IS takes several minutes following initial contact
and TCR triggering, and can be divided into at least
three stages ŽFigure 2..
Following contact with planar bilayers presenting
specific antigens, the T cells arrest and within 30 s a
central zone containing the LFA-1 ligand ICAM-1 is
formed. Analysis by interference reflectance microscopy indicated that an area of close contact surrounds the central zone. An unexpected finding was
that clusters of engaged peptide]MHC first appeared
in this outer close contact ring. Since no other accessory molecules were present in this system we ŽA.S.
and M.L.D.. proposed that the cells were using the
LFA-1rICAM-1 interaction as an ‘anchor’ to force
the membranes into close proximity. This raises the
interesting possibility that active processes contribute
to the membrane approximation that is necessary for
the TCR to engage peptide]MHC complexes Žsee
below..
Over the next 1.5]30 min the clusters of
peptide]MHC in the outer ring moved towards the
centre of the interface, forming a compact central
cluster that is surrounded by a ring of ICAM-1 engaged to LFA-1. This organized interface, which is
termed the ‘mature’ IS,10 is very similar to the pattern of SMACs described by Monks et al.8 The latter
were visualized at the interface of T cells and specific
antigens presenting APCs that had been fixed after
30 min of coculture. This transport of peptide]MHC
is inhibited by cytochalasin D, suggesting that IS
formation is dependent on the actin cytoskeleton.10
Using peptide variants it was shown that the final
density of peptide]MHC complexes in the central
cluster correlated best with the half-life of the
TCRrpeptide]MHC interaction.10
A final stage of IS formation was analyzed by fluorescent photobleaching recovery ŽFPR. experiments.
This revealed that, after formation, the central cluster of the engaged peptide]MHC complexes does
not exchange with free peptide]MHC outside the
bleached area. The reasons for this are currently
unclear, and it is not known whether the TCR is also
stably associated with this central cluster. One possible explanation for this stability is that peptide]MHC
complexes, possibly with bound TCR, oligomerise
directly with each other, forming a two-dimensional
lattice.26 A second possibility is that a complex network of associations between TCRrCD3, the co-receptors CD4 or CD8, accessory molecules, and their
various extracellular and intracellular ligands, is
stabilized through the cooperative effects of multiple
weak interactions. How can this stability be reconciled with other data27,28 suggesting that TCRr
peptide]MHC complexes are continuously forming
and dissociating during T cell antigen recognition?
One proposed explanation10 is that there is a high
turnover of TCRrpeptide]MHC complexes during
the formation of the mature IS in the steps preceding
stabilization of the central cluster.
A key finding to emerge from these dynamic studies is that TCR triggering is a very early event, and
that changes in morphology, motility, calcium concentration and, in particular, IS formation all follow,
and are dependent on initial TCR triggering. This
raises three important questions that will be addressed in the remainder of this review. How does
the initial TCR triggering occur? How does this triggering drive formation of the IS? Given that it is not
required for initial TCR triggering, what is the purpose of IS formation?
8
Redistribution of T cell surface molecules
Mechanisms
ment region’., precise alignment of membranes enables the CD2rCD58 interaction, which has a low
solution affinity Že.g. K d ; 2 m M., to achieve a high
two-dimensional affinity Ž2D K d ; 1 moleculer
m m2 ..15,32
The presence of large molecules at the cell]cell
interface would be expected to act as a constraint on
TCR engagement of peptide]MHC by preventing
sufficiently close membrane approximation and
membrane alignment.37 Indeed, T cell antigen
recognition is enhanced when CD43 is absent from T
cells.38 Notably, however, many T cell ‘accessory’
molecules which contribute to T cell antigen recognition are also small ŽFigure 3.. These observation led
to the suggestion30,39,40 that, in order for the TCR to
engage peptide]MHC, molecules at the interface
between T cells and APCs would have to segregate
according to the size ŽFigure 4., with small accessory
molecules contributing to the formation of ‘closecontact zones’, within which the inter-membrane separation distance Ž; 15 nm. matched the dimensions
of the TCRrpeptide]MHC complex, and from which
large molecules such as CD43, CD45 and integrins
are excluded. Structural41 ] 44 and mutagenesis36 studies of CD2 and its ligands CD48 and CD58 have
shown that these molecules bind in a head-to-head
orientation, forming a complex that spans the same
dimensions Ž; 14 nm. as the TCRrpeptide]MHC
complex. On this basis it was proposed that one
function of CD2 is to facilitate the formation of
close-contact zones, thereby enhancing TCR engagement of peptide]MHC.36,39,40 The importance of
close membrane approximation is strongly supported
by the recent observation that, whereas formation of
the wild-type Ž; 14 nm. CD2rCD48 complex strongly
enhances TCR engagement of peptide]MHC, an
elongated Ž; 21 nm. CD2rCD48 complex is strongly
inhibitory.37
Overview
The processes contributing to the redistribution and
segregation of cell surface molecules fall into two
groups. Firstly there are passive mechanisms which
are dependent on properties intrinsic to the
molecules and their interactions. These include interactions with counter-receptors at the contact interface, physical dimensions, and partition into lipid
rafts. Although not active in the sense of consuming
cellular energy, these mechanisms do produce physical forces. Secondly, redistribution can be driven by
active cellular processes, such as polarized secretion
or remodelling of the cortical actin cytoskeleton.
Biophysical considerations
Molecular dimensions, membrane alignment and two-dimensional affinity
The molecular composition of the T cell surface
has been intensively studied and is now reasonably
well characterized.29 One striking observation30,31 is
that these molecules vary enormously in size ŽFigure
3.. The extracellular portions of the TCR and its
ligand peptide]MHC are small Ž; 7 nm. compared
with several highly-abundant cell surface molecules
such as CD43 Ž; 45 nm. and CD45 Ž; 28]50 nm., as
well as important adhesion molecules such as integrins Ž; 21 nm.. It is conceivable that undulations in
the plasma membrane could accommodate the size
differences of interacting molecules. However, a second striking observation is that interactions between
membrane-tethered adhesion molecules generate
precise alignment of apposing membranes.15,32
Consequently, molecular interactions spanning
different dimensions tend to segregate at the interface into distinct, homogenous zones.33 While the
existence and significance of membrane alignment
had been proposed earlier,34 ] 36 it was demonstrated
for the first time by a comparison of the solution
affinity and the membrane Žor two-dimensional. affinity of the CD2rCD58 interaction, using soluble
and membrane-tethered forms of these molecules,
respectively.15,32 The membrane affinity constant is
two-dimensional because the concentration of membrane-tethered molecules is measured as surface density, a two-dimensional parameter Žmoleculesrmm2 ..
This comparison revealed that, by confining both
interacting binding sites to a small volume Ž; 5 nm
high. between apposing membranes Žthe ‘confine-
Receptor lateral mobility
When two cells make contact, complementary surface receptors will usually need to move laterally
within the plane of the membrane in order to encounter each other. This movement will depend on
their lateral mobility,45 a term which refers collectively to two distinct properties; the proportion of the
molecule that is mobile Žthe mobile fraction., and the
diffusion rates of the mobile molecules within the
plane of the membrane Žthe lateral diffusion coefficient..
There is currently only limited information on the
lateral mobility of T cell surface molecules. Approxi9
P. Anton van der Merwe et al
mately 75% of the CD2 molecules expressed in Jurkat
cells were shown to be mobile,46 similar to observations with other molecules.47 This agreed with the
observation that a similar proportion of the CD2
accumulates in contact areas with CD58-bearing planar bilayers.15 In contrast, only 10]20% of the TCR
10
Redistribution of T cell surface molecules
on mature T cells is mobile.10 The reasons for this
low mobility are not clear but may include cytoskeletal associations48,49 or diffusion barriers.50 The latter
may be in the cytoplasm Že.g. cytoskeletal ‘fences’. or
in the membrane itself Že.g. other membrane proteins or lipid rafts.. This low mobility needs to be
reconciled with experiments suggesting that up to
80% of cell surface TCRrCD3 engages peptide]MHC
during antigen recognition.27 One possible explanation is that the immobile TCRrCD3 48,49 is actively
transported to the contact interface over a long period. Alternatively, the T cell may form multiple
contacts Žsimultaneous or sequential. with several
APCs.
Two distinct transport processes have been implicated in the redistribution of cell-surface molecules;
polarized secretion of cytoplasmic vesicles containing
the molecules, and redistribution of molecules already at the surface.
Polarized secretion
Both CD8 T cells 5,53 and CD4 T cells 5,54,55 are
capable of polarized secretion, in which cytoplasmic
vesicles fuse to the plasma membrane at the contact
interface with target cells or APCs. This allows the
contents of these vesicles Že.g. perforin, granzymes,
and cytokines. as well as the vesicle membranes to be
directed to the interface.5,53 ] 55 Recent data suggest
that the T cell surface molecules FasL56 and CTLA-4 52
reside predominantly in cytoplasmic vesicular stores,
and that these molecules are targeted to the site of
TCR ligation by polarized secretion.
There is some evidence that lipid rafts and their
associated molecules Žsee below. are concentrated in
intracellular, presumably vesicular, stores in naıve
¨ T
cells.57,58 Polarized secretion is known to target rafts
to the apical membrane of epithelial cells.59 This
raises the question as to whether lipid rafts and
associated molecules are transported to the IS by
polarized secretion.
Active processes
There is compelling evidence that active processes
contribute to the redistribution of cell surface
molecules during T cell antigen recognition. Firstly,
much of the observed redistribution requires and
follows TCR triggering.5,8 ] 10,13,14,51 Secondly, cytoskeletal inhibitors inhibit redistribution.10,13,14
Thirdly, a number of studies Žsee, e.g. refs 5,52. have
shown that cross-linking of the TCRrCD3 complex
with antibodies can trigger redistribution of
TCRrCD3 Ž‘capping’. as well as other molecules
Ž‘co-capping’. to one region Žthe ‘cap’. of the T cell
surface. And finally, a cytoplasmic protein has been
identified that interacts with the cytoplasmic domain
of CD2 ŽCD2AP. and influences its distribution within
a T cellrmembrane interface.33
Movement of molecules on the cell surface
Several lines of evidence implicate the cortical actin
cytoskeleton ŽCAC. in the redistribution of surface
molecules to the contact interface. Firstly, treatment
of T cells with cytoskeletal inhibitors abrogates the
Figure 3. Dimensions of molecules involved in T cell antigen recognition. A schematic view of the dimensions of cell-surface
molecules involved in T cell antigen recognition. Sizes are estimated from structural studies of these or related molecules29
with the exception of CD148. The size of CD148 Ž; 55 nm. is estimated by adding 10 fibronectin type III domains strung
end-to-end Ž; 40 nm. to an ; 80 amino acid mucin-like region Ž; 15 nm..
Figure 4. The kinetic]segregation model of TCR triggering and immunological synapse formation. The redistribution of
cell surface molecules Ždepicted as in Figure 3. in T cell activation. The green and red boxes on the left are enlarged views
of the boxes on the right. Ži. Before cell]cell contact the TCRrCD3 complex is subject to constitutive tyrosine
phosphorylation and dephosphorylation. Dephosphorylation dominates, leading to a low steady-state level of tyrosine
phosphorylation. Žii. Initial T cell contact with an APC leads the formation of multiple close-contact zones within the
contact area as a result of small-scale segregation of molecules. This is a passive process driven by size and ligand binding.
Within these close-contact zones tyrosine phosphorylation is favoured because tyrosine phosphatases Že.g. CD45. are
excluded, and tyrosine kinases are concentrated. Žiii. Engagement by TCR of specific-peptide]MHC occurs within these
close-contact zones and results in the trapping of the TCRrCD3 complex within a zone for a period, the length of which is
determined primarily by the stability of the TCRrpeptide]MHC complex. While in the zone, TCRrCD3 is tyrosinephosphorylated, initiating the multi-step signalling process required for triggering. Živ. A sufficiently stable TCRrpeptide]MHC
interaction Žcomplex II. allows these signalling steps to be completed, leading to triggering. Unstable complexes ŽI and III.
lead to no or partial triggering because, on leaving the close-contact zone, TCRrCD3, and any recruited signalling
molecules, are re-exposed to high levels of tyrosine-phosphatase activity. Žv. TCR triggering initiates active large-scale
segregation of molecules, culminating in the formation of the mature IS. Žvi. Steps Žii. to Žv. are enhanced by
‘co-stimulatory signals’ such as chemokines or the expression of CD28 ligands on APCs. Žvii. Sustained signalling through
the IS results in full T cell activation.
11
P. Anton van der Merwe et al
redistribution of ICAM-1 and other molecules to the
interface13,14 and inhibits transport of peptide]MHC
within the interface.10 Secondly, a large number of
cell surface molecules which migrate towards and
away from the interface have been shown to associate
with the cytoskeleton or with linker molecules which
themselves associate with the cytoskeleton Žsee below..
Finally, ligation of the TCR leads to localized talin
accumulation5 and actin polymerization.12,60,61
How might the CAC organise the redistribution of
cell surface molecules upon T cell activation? The
CAC moves in a centripetal direction within the
cellular processes that extend from cells, as a result of
a combination of actin filament remodelling and
myosin motor proteins.62 Following recognition, T
cells extend analogous processes around the APC,
forming a cup-shape interface. Centripetal movement
of the CAC within this ‘cup’ might move molecules
coupled to the CAC, such as TCRrCD3,48,49 radially
towards the centre of the contact area. How are some
molecules Že.g. CD2 and LFA-1. prevented from moving into the centre and others Že.g. CD43. actively
excluded? Possible explanations include the absence
of links to the CAC, physical exclusion on the basis of
size, and attachment to other cytoskeletal structures.
are discussed in detail in the accompanying reviews
by Janes et al and Zhang and Samelson, respectively.
Linking TCR triggering to cytoskeletal polarization and
receptor redistribution
Role of TCR triggering
Anti-TCR antibodies can induce both re-orientation of the MTOC,61,70 polarized exocytosis52 and
localized actin polymerization,61,70 suggesting that
TCR triggering may be sufficient for these processes.
However, an important caveat is that these experiments were performed in the presence of serum Ža
source of extracellular matrix proteins. or intact target cells, leaving open the possibility that integrin
engagement is an additional requirement.71 As far as
is known, TCR triggering is necessary for the re-orientation of the MTOC and Golgi apparatus towards the
T cellrAPC contact interface.5,19,70 In contrast, localized actin polymerization does not require TCR triggering.70 An interaction between LFA-1 on T cells
and ICAM-1 on APCs is sufficient to induce cortical
actin polymerization and talin accumulation at the
contact site.70
Anti-TCR antibodies can also induce redistribution
of cell surface molecules, suggesting that TCR triggering is also sufficient for this to occur.5,52 It is
unclear whether TCR triggering is absolutely necessary for active redistribution of cell surface molecules.
The active redistribution described to date at T cell
contact interfaces has always been preceded by TCR
ligation.5,7,8,10 However, the various antigen-independent responses seen when T cells contact dendritic
cells raise the possibility that TCR triggering may not
be essential for redistribution at T cellrdendritic cell
interfaces.21,23,24
Lipid rafts
The plasma membrane of many cells,63 including T
cells,64,65 contains microdomains which are enriched
in glycosphingolipids and cholesterol, termed lipid
rafts. Some T cell surface molecules important in
TCR triggering Že.g. lck, fyn, LAT, CD48 and CD4.
are preferentially associated with these structures64 ] 66 .
Others, most notably CD45 64,67 appear to be depleted
from lipid rafts. This being so, the redistribution of
lipid rafts could contribute to the segregation of cell
surface molecules at the T cellrAPC interface. Recent data suggest that triggering of the TCR can
induce active redistribution of lipid rafts towards the
site of TCR triggering.57 If cell-surface molecules that
associate with lipid rafts, such as the GPI-anchored
molecule CD48, bind to a ligand on APCs, this could
provide a passive mechanism for the accumulation of
rafts at the interface.66 The importance of lipid rafts
in TCR triggering is highlighted by the finding that
chemical disruption of rafts abrogates TCR triggering.64,68 Furthermore, mutations which disrupt the
association with rafts of the essential signalling
molecules LAT 69 or lck 51 also disrupt TCR triggering. The role of lipid rafts and LAT in TCR signalling
Role of accessory molecules: a mechanism of co-stimulation?
Recent studies have implicated accessory molecules
in the active redistribution of T cell surface molecules.
Wulfing and Davis showed that bulk redistribution of
T cell surface molecules to the T cellrAPC interface
is inhibited when CD28rCD80 or LFA-1rICAM-1 interactions are blocked.14 Viola et al found that the
redistribution of lipid rafts to the area in contact with
anti-CD3 antibody-coated beads is dramatically enhanced when the beads are also coated with anti-CD28
antibodies.57 These findings suggest that accessory
molecules may enhance T cell activation by contributing to this redistribution process. The ability of
the CD28rligand interaction to activate Rho family
12
Redistribution of T cell surface molecules
GTPases Žsee below. independently of TCR triggering support this notion.72 As noted above, the LFA1rICAM-1 interaction can trigger changes in the
CAC at the contact site.70
The finding that cytoskeletal polarization and
large-scale receptor redistribution depend on TCR
triggering, and are enhanced by engagement of accessory molecules such as CD28, suggests that IS
formation is the point at which the T cell integrates
information about the presence of the antigen on the
APC and its activation state Že.g. expression of
chemokines or CD28 ligands.. Or, to rephrase in the
terminology of the two-signal hypothesis of T cell
activation, IS formation may integrate signal 1
Žstimulus from antigen. with signal 2 Žco-stimulus
from the APC.. This is supported by the finding that
dendritic cells, the most effective APCs, are uniquely
capable of adhering to and inducing antigen-independent responses in T cells.21,23 ] 25
Thus, the picture emerging is that TCR triggering
activates Rho family GTPases through activation of
proteins such as vav, and that Rho GTPases regulate
various effector proteins, including protein kinases
and WASp, which regulate in turn actin binding
proteins. Direct links between vav1 and the actin73
and microtubule80,81 cytoskeleton have also been
identified. Since the cytoskeleton and associated motor proteins control the active transport processes
implicated in receptor redistribution, the vavrRho
pathway is likely to couple TCR triggering to cytoskeletal polarization and the redistribution of cellsurface molecules.
Linking the cytoskeleton to T cell surface molecules
For TCR-induced changes in the CAC to contribute the redistribution of cell-surface molecules,
these molecules need to associate with the CAC.
Linkages have been reported for LFA-1, which associates via talin,82 CD2, which associates with CD2AP 33
and tubulin,83 the TCR, which associates via the
CD3z chain,48,49 and CD45, which associates with the
linker protein fodrin.84 Furthermore, members of
the ezrin-radixin-moesin ŽERM. family of proteins
couple CD43, CD44, and ICAM-1, -2, and -3 to the
CAC.85 With the exception of CD2AP Žsee below.,
the role of these linker molecules in surface molecule
redistribution is not known. However, their diversity
provides a possible explanation for the distinct localization of cell-surface molecules within the IS.
Signalling pathways
Recently a tentative outline has emerged of the
signal transduction pathways that couple TCR triggering to cytoskeletal polarization and receptor redistribution.72,73 Attention has focused on the Rho family
of small GTP binding proteins Žespecially RhoA,
Cdc42Hs and Rac1. since they have been shown to
regulate cytoskeletal and vesicle transport processes
in other cells.72,74 Using a T cell hybridoma, Stower et
al 60 have shown that a dominant-negative mutant of
Cdc42Hs inhibits actin and microtubule polarization
towards the T cellrAPC interface.
How does TCR triggering activate rho GTPases?
The most likely candidate is the GTPrGDP exchange
factor vav1, which activates Rac1 and has been reported to be essential for TCR-induced actin polymerization and capping.75 ] 77 Vav1 is activated by
tyrosine phosphorylation andror by binding to polyphosphoinositide products generated following activation of phosphatidylinositol ŽPI. 3-kinase.73
The mechanisms by which the rho GTPases regulate the cytoskeleton is not well-understood but is
likely to involve effector proteins which regulate
actin-binding proteins, such as the protein kinases
ROCK and LIM-kinase.72,78 An effector protein already implicated in coupling Rho GTPases to the
cytoskeleton in T cells is WASp, so named because it
is defective in patients with Wiscott-Aldrich immunodeficiency syndrome.79 WASp interacts with
Cdc42Hs and is required for capping induced by
anti-CD3 antibodies, suggesting that it couples
Cdc42Hs activation to the actin cytoskeleton.
CD2AP
CD2AP was first identified as a molecule that binds to
the cytoplasmic domain of CD2 and influences its
distribution at the contact interface between a T cell
and planar lipid bilayers.33 The finding that
CD2rCD48 complexes occupy a special position in
the IS10 is further evidence that CD2 is being actively
moved at the interface, and it seems likely that this
movement is also mediated by CD2AP.
A recent study on CD2AP-deficient mice suggests
that this molecule has a wider role in lymphoid and
non-lymphoid tissues.86 These mice die from complications of excessive leakage of serum proteins into
the urine Žnephrotic syndrome..86 This leakage appears to result from defects in the epithelial cell
Žpodocyte. processes that contribute to the glomerular filtration barrier. Interestingly, CD2AP-deficient T
cells show a substantial reduction in response to
anti-CD3 antibodies,86 whereas CD2-deficient mice
13
P. Anton van der Merwe et al
triggering can be divided into aggregation,88,89 conformational change,90,91 or segregation39,40,92,93 models. Segregation models have in common the proposal that peptide]MHC binding leads to triggering
by holding the TCRrCD3 complex in a membrane
environment in which tyrosine kinases have been
segregated from tyrosine-phosphatases. Kinetic
proof-reading,94 kinetic discrimination95 and serial
triggering 27 models do not fall neatly into the above
classification because they do not address the central
question of how binding transduces a signal. Instead
they try to account for particular features of TCR
triggering, such as the ability of the TCR to discriminate between very similar ligands94,95 and the ability
of a few peptide]MHC complexes to engage many
TCRs.27 Consequently they can and have been incorporated into aggregation, conformational change, or
segregation models.
are normal in this respect, suggesting that CD2AP
plays a broader role in T cell function than CD2.
What might be the purpose of locating CD2rCD48
complexes in an inner ring surrounding the
TCRrpeptide]MHC-containing central zone? One
intriguing possibility, consistent with a generalized
role for CD2AP in forming filtration junctions, is that
this ring of CD2rCD48 functions as a molecular filter
within the IS keeping larger molecules out of the
central zone in the face of centripetal actinomyosinbased transport processes.10,14
Functions
What is the purpose of cytoskeletal polarization and
redistribution of cell surface molecules? One
probable role for cytoskeletal polarization is to facilitate the targeting by T cells of cytokines and the
contents of lytic granules to the site of contact with
APCs or target cells, respectively.5,55 As argued above,
cytoskeletal polarization is also likely to contribute to
the large-scale redistribution of cell surface molecules
leading to the formation of the IS. Here we speculate
on the role of IS formation in T cell activation,
placing it in the context of a recently-proposed model
of TCR triggering.39,40 It is important to re-emphasise
here that the formation of the IS follows and depends on
TCR triggering, indicating that it cannot be the mechanism
of initial TCR triggering. Nevertheless IS formation
does appear to be required for full T cell activation.
What is the evidence for this? Firstly, in the few
studies performed to date, IS formation correlated
perfectly with full T cell activation.8,10 Secondly, disruption of the actin cytoskeleton20,87 or the regulation thereof,75 ] 77 manipulations which would be expected to disrupt IS formation, leads to disruption of
full T cell activation Žproliferation andror cytokine
production. and negative selection. While not conclusive, these findings are consistent with an essential
role for the IS in full T cell activation.
Problems with aggregation and conformational change
models
Aggregation models postulate that peptide]MHC
engagement leads to clustering or aggregation of
TCRrCD3 complexes. At first glance this is an attractive notion since dimerizationraggregation is a wellestablished mechanism of signal transduction,96 and
it is well known that aggregation of TCRrCD3, with
either antibodies or multivalent peptide]MHC,97 will
trigger TCRs. However, there are reasons for doubting that this is the physiological mechanism for TCR
triggering.92 In systems in which dimerizationraggregation lead to signalling, the mechanism of multimerization is usually clear;96 a multivalent ligand Žeither
a monomer with two binding sites or a multimer.
binds two or more receptors, thereby bringing them
into close proximity. In the case of the TCR the
ligand, peptide]MHC, is monovalent, and present at
an exceptionally low density, with as few as 10]100
molecules of a specific peptide]MHC on an entire
cell. Thus, only a very small number of TCRs will be
bound at any one time. Furthermore, each interaction is likely to be very short-lived, with a half-time of
a few seconds.89 This suggests that simultaneous engagement of adjacent TCRs Ži.e. aggregation. will be
exceedingly rare. Furthermore, if TCR triggering depended on aggregation it should be very sensitive to
the surface density of specific peptide]MHC, whereas
the converse is true Žsee, e.g. refs 10,98.. A refinement of the aggregation model, which attempts to
address these problems, proposes that the binding of
TCR to peptide]MHC generates a new binding site
that enables TCRrpeptide]MHC complexes to self-
Small-scale segregation provides a novel mechanism for
signal transduction across membranes
TCR triggering requires binding of the TCR to specific peptide presented on MHC class I or II molecules.
One of the key, unresolved questions in T cell biology is how this binding generates a signal. In other
words how does the T cell ‘know’ that a particular
TCR has bound peptide]MHC? Based on the way
they address this question, current models of TCR
14
Redistribution of T cell surface molecules
associate.26 However, given the huge diversity in TCR
and peptide]MHC structures, and the resulting diversity in the structure of TCRrpeptide]MHC complexes,99 it is difficult to envisage how such binding
sites could be conserved in all TCRrpeptide]MHC
complexes. A further difficulty is understanding how
direct self-association occurs on the cell surface when
each TCR is surrounded by several other molecules
Že.g. CD3 subunits and a co-receptor., all of which
are heavily glycosylated.
A variant of the aggregation model is that triggering is a consequence of heterodimerization of the
TCR with its co-receptor ŽCD4 or CD8.. While this
may be sufficient for TCR triggering in certain circumstances,100 it cannot be the universal mechanism
of triggering, since TCR triggering in vitro and in
vivo 101,102 can occur in the complete absence of CD4
or CD8.
Conformational change models propose that peptide]MHC binding leads to a conformational change
in the TCR, which is somehow transduced into the
cell interior. This model requires that every TCR is
able to undergo the same binding-induced conformational change in the face of huge diversity in the
structure of the antigen binding sites and
peptide]MHC, which would seem highly implausible.
Furthermore, structural data available to date show
that there are no conformational changes in the TCR
induced by binding peptide]MHC except for adjustments within the antigen-binding site.91,99 Taking the
latter data into account, Wiley et al 91 recently proposed a variant of the conformational change model
based on studies of signalling through the erythropoetin receptor.103 In this model the TCR exists as a
preformed dimer. When both TCRs in the dimer
bind to peptide]MHC the two TCRs, reposition with
respect to each other, and this repositioning is detected in some way. Unfortunately this model has the
same drawback as the aggregation models when applied to TCR triggering Žsee above.. A further difficulty is envisaging how the independent binding of
two specific peptide]MHC complexes could change
the relative positions of the two TCRs?
the same mechanism.40 These models emerged from
several key observations described below.39,40
Firstly, the TCR and T cell accessory molecules are
much smaller than other abundant cell surface
molecules, including CD43, CD45, and integrins30,31
ŽFigure 3.. Taken together with the observation that
membrane alignment is important for optimal binding of membrane-tethered molecules,15,32 this suggested that these molecules must segregate according
to size at the contact interface.36,39,40
Secondly, tyrosine phosphatase inhibitors can activate T cells,104,105 producing tyrosine phosphorylation patterns very similar to those seen during TCR
triggering.106,107 Furthermore, an inhibitor of src kinases ŽPP1. rapidly and dramatically decreases the
level of basal tyrosine phosphorylation in T cells.57
These findings show that the tyrosine kinases implicated in TCR triggering are constitutively active and
under tonic inhibitory control by tyrosine phosphatases, and imply that altering the local balance
between these two competing, constitutive processes
would be sufficient to trigger intracellular signals.
Thirdly, one of the most active and abundant tyrosine phosphatases is the large cell surface molecule
CD45.108 Furthermore, CD45 may associate directly
or indirectly Žvia CD45-associated protein. with the
TCR and its signalling apparatus.109 ] 112 This suggested that, in addition to its generally accepted role
maintaining lck in a primed state,93 CD45 may also
tonically inhibit TCR triggering by dephosphorylating key tyrosine kinase substrates such as CD3z ,109
ZAP-70 106 and lck itself.113 There is clear evidence
that membrane-associated tyrosine phosphatases
other than CD45 also reverse constitutively active
tyrosine kinases.107 One possible candidate is CD148,
which has recently been shown to inhibit TCR triggering.114 Interestingly, like CD45, CD148 is predicted to have a very large extracellular domain ŽFigure 3..
Finally, the TCR is able to discriminate between,
and produce very different responses to, very similar
ligands.115 This is difficult to explain purely on the
basis of differences in affinity; all else being equal, a
twofold change in affinity will produce a less than
twofold change in the total number of engaged TCRs.
In contrast, small kinetic differences can readily be
amplified to produce very different biochemical responses.94,95
These observations were synthesized into a new
model of TCR triggering,39,40 which we now refer to
as the kinetic]segregation model to emphasise its two
key elements, molecular segregation and binding ki-
The kinetic-segregation model of TCR triggering
The arguments outlined above against conformational change or dimerizationraggregation models
led to the proposal that an entirely novel mechanism,
based on molecular segregation in contact areas, was
responsible for TCR triggering.39 A related ‘topological model’, based on the implications of new data on
two-dimensional affinity,32 subsequently converged on
15
P. Anton van der Merwe et al
netics. The model postulates the following sequence
of events in TCR triggering ŽFigure 4..
When T cells encounter target cells or APCs,
molecules at the contact interface will segregate according to size. Small molecules such as CD2, CD4,
CD8 and CD28 will contribute to the formation of
multiple, small close-contact zones, from which large
molecules such as CD45, CD43, CD148 and LFA-1
are excluded.
As a result of this segregation, close-contact zones
will be a ‘pro-signalling’ environment, relatively depleted of tyrosine phosphatases Žsuch as CD45 and
perhaps CD148. and enriched in tyrosine kinases
such as lck. The close membrane approximation
within the close-contact zone will facilitate TCR encounters with specific peptide]MHC. The binding of
TCR to specific peptide]MHC will result in the TCR
and its associated CD3 complex being held within a
close contact zone, where they show a net increase in
phosphorylation as a result of exposure to tyrosine
kinases such as lck in the absence of tyrosine phosphatases such as CD45. It is important to emphasise
here that ligation of a single TCRrCD3 can generate
a signal Žno dimerization or aggregation is required..
Furthermore, tyrosine phosphorylation of TCRrCD3
is not strictly dependent on its localization to a ‘prosignalling’ close contact zone, given that it occurs
constitutively Žsee above.. It is the downstream signalling events that are crucially dependent on localization to close-contact zones because this prolongs
the half-life of tyrosine phosphorylated species by
segregating the TCRrCD3 and associated molecules
from tyrosine phosphatases. This tyrosine phosphorylation initiates a cascade of events including recruitment and activation of the tyrosine kinase ZAP-70
and phosphorylation and activation of ZAP-70 substrates.116 This sequence will be interrupted when the
TCRrCD3 complex and associated molecules leaves
such a close-contact zone. Because the TCRrCD3 is
held within the contact zone through binding to
peptide]MHC, tyrosine phosphorylation and TCR
triggering will depend crucially on the half-life of the
TCRrpeptide]MHC interaction117 and a decrease in
the half-life will lead to interruption of the cascade of
signalling events.118,119
To provide kinetic discrimination the model requires that initial close-contact zones be relatively
small or short-lived so that inappropriately phosphorylated TCRrCD3 complexes do not have to diffuse
far prior to their dephosphorylation by CD45 or
other excluded phosphatases. This may be a key
difference between the respective signalling roles of
the initial close-contact zones and the IS, which forms
over a much larger area. It also seems likely that
there will be many such close-contact zones forming
within the contact area between T cells and APCs.
Unfortunately, given the limited resolution of the
light microscope, the existence of such small closecontact zones remains speculative.
Recent studies relevant to the kinetic]segregation model
Since the kinetic]segregation model was first proposed a number of findings have provided support
for key elements of the model. First, mice with reduced levels of CD45 expression show evidence of
enhanced signalling through the TCR,120 consistent
with an inhibitory effect of CD45. Second, CD45 has
been shown to inhibit the function of constitutivelyactive lck by dephosphorylating its substrates.121
Third, studies on planar bilayer systems have shown
that engaged CD2 and LFA-1, which differ considerably in size ŽFigure 3., segregate in the contact interface independent of cytoskeletal interactions.33 This
strongly supports the proposal that size differences
drive the segregation of molecules within the contact
interface. Fourth, it has been demonstrated that increasing the dimensions of an accessory molecule
strongly inhibits TCR engagement.37 Fifth, a form of
CD45 with a truncated extracellular domain has been
shown to inhibit TCR triggering by antigens ŽC. Irles,
A. Symons, F. Michel, P.A. van der Merwe, and O.
Acuto, unpublished observations.. Sixth, T cells are
able to discriminate between peptide]MHC ligands
with similar affinities but with differences in half-life
for TCR binding.89,117 Finally, as with early TCR
signalling events,117 the number of peptide]MHC
complexes that accumulated in the central region of
the IS correlated strongly with the half-life of the
TCR binding.10 This provided the first direct evidence that IS formation is dictated by kinetics of the
TCRrpeptide]MHC interaction,10 most likely
through the dependence of large-scale segregation
on early TCR signalling events that are triggered, we
suggest, by small-scale segregation.
TCR triggering is inhibited by cell-surface receptors such as CTLA-4 and Killer cell Immunoglobulin
Receptors ŽKIRs., which associate with the cytoplasmic tyrosine phosphatases SHP-1 and SHP-2.122,123
Interestingly, the complexes formed by CTLA-4 and
KIRs and their ligands ŽCD80rCD86 and MHC class
I, respectively. are predicted to have dimensions
compatible with their co-localization with the TCR in
contact areas ŽFigure 3.. Furthermore, CTLA-4 inhibits the earliest tyrosine phosphorylation events fol16
Redistribution of T cell surface molecules
lowing TCR triggering.124,125 This suggests that inhibitory receptors may function by altering the
balance between tyrosine phosphorylation and dephosphorylation in the immediate vicinity of the
TCRrCD3. As noted earlier, CTLA-4 is found predominantly in vesicular stores and is directed to the
surface at the site of TCR triggering by polarized
secretion.52 This suggests that CTLA-4 may be targeted to the centre of the IS as a means of modulating or terminating TCR signalling.
The kinetic]segregation model is compatible with
evidence that lipid rafts play a crucial role in TCR
triggering Žsee above.. Lipid rafts may accumulate in
or adjacent to the close-contact zones through either
active 57 or passive mechanisms,66 helping to contribute to the ‘pro-signalling’ environment by recruiting tyrosine kinases and other pro-signalling
molecules and excluding CD45 Žsee accompanying
review by Janes et al.67 Based on these recent data,
new models of TCR triggering have been proposed
which postulate that TCR binding to peptide]MHC
brings the TCR and lipid rafts together.92,93 These
‘lipid raft models’ are similar in principle to the
kinetic]segregation model in that they also propose
that triggering is a consequence of the redistribution
of the TCR into an environment enriched in tyrosine
kinases and depleted of tyrosine phosphatases. These
models may, however, need to be adjusted since
recent evidence suggests that TCRs may be constitutively associated with rafts Žsee Janes et al .. The main
drawback of lipid raft models is that they do not
suggest a clear mechanism, independent of aggregation or conformational change, by which engagement of the TCR with peptide]MHC either enhances
its association with lipid rafts or promotes their aggregation.
viewed in ref 126; see also refs 127,128.. These same
peripheral T cells require a much higher affinity
TCR ligand in order to become fully activated. How
is the TCR able to respond to ligands with such a
wide range of affinities and yet retain the ability to
discriminate between ligand which differ only slightly
in their binding properties?
The kinetic]segregation model would allow very
weak interactions to be detected because TCRrCD3
complexes need only be transiently detained in
close-contact zones to become phosphorylated. Thus,
tyrosine phosphorylation could occur even with very
weak TCR binding to peptide]MHC. Perhaps this is
sufficient to generate the signals required for positive
selection in the thymus or survival in the periphery.
The finding that ZAP-70 is constitutively associated
with partially-phosphorylated TCR-z in peripheral T
cells, but that this is reduced in T cells from mice
lacking self-peptide]MHC and absent in T cells grown
in culture, is consistent with continuous weak signalling through the TCR.126
Because it would be inappropriate for peripheral T
cells to be fully-activated by self-peptide]MHC, a
mechanism must exist that enables a T cell to clearly
distinguish between the signals resulting from weak
versus strong self-peptide]MHC engagement. We
suggest that the IS provides such a mechanism because it appears only to form when TCR binds peptide]MHC above a threshold affinity or half-life.10
Nevertheless the mechanisms that leads to IS formation are exquisitely sensitive since these structures
form at very low surface densities of peptide]MHC.10
Taken together, these findings suggest that IS formation may function as a checkpoint for full T cell
activation, facilitating discrimination between low
affinity Žpresumably self-. peptide]MHC engagement
and high affinity Žpresumably foreign-. peptide]MHC
engagement.
Why might IS formation be required for full T cell
activation? There are at least two possibilities. IS
formation may lead to the generation of a unique
signal. A candidate for such a signal is activated
protein]kinase C-u , which selectively accumulates in
the central portion of the IS or cSMAC 7 Žsee accompanying review by Kativar and Mochly-Rosen.. A second possibility is that IS formation may be necessary
for the prolonged Ž) 1 h. triggering necessary for
full T cell activation.129
If the IS is required for full T cell activation, it
follows that any factor that contributes to IS formation will contribute to full T cell activation. Like full
T cell activation, IS formation requires that the
The immunological synapse as an activation checkpoint
and signal integrator
In addition to being structurally plausible, any model
of TCR triggering has to explain a central paradox.
The TCR detects peptide]MHC ligands with a wide
range of affinities and surface densities but can nevertheless discriminate between peptide]MHC complexes with very subtle differences in binding properties.115,117,126 The ability of a given TCR to respond to
a wide range of affinities is clearly illustrated by the
ability of thymocytes to undergo positive and negative
selection using the same TCR. More recently it has
been shown that mature peripheral T cells also
recognise and respond to self-peptide]MHC Žre17
P. Anton van der Merwe et al
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23. Montes M, McIlroy D, Hosmalin A, Trautmann A Ž1999.
TCRrpeptide]MHC interaction affinity or half-life is
above a threshold value.10 There is also intriguing
evidence that ligation of accessory molecules such as
CD28 and LFA-1 enhances some of the transport
processes that lead to IS formation.14,57 Interactions
with these molecules are regulated by soluble mediators Že.g. chemokines will activate LFA-1. and will
depend on the activation or maturation state of the
APC.24,25 Finally, there is evidence that dendritic cells
are distinct from other APCs in their ability adhere to
and evoke changes in naıve
¨ T cells in the absence of
and prior to antigen recognition.21,23,24 These data
suggest that the formation of the IS serves to integrate information relating to the presence and quality of the antigen Že.g. surface density and binding
properties. and information on the nature and activation state of the APC.24,130
Based on these considerations we suggest the following integrated model of full T cell activation ŽFigure 4.. Small scale-segregation, driven primarily by
ligand binding and steric factors, leads to initial TCR
triggering, by the mechanism outlined in the kinetic]segregation model. This is sufficient to stimulate certain T cell functions, the nature of which
Žpositive selection or survival. depends on the developmental state of the T cell. Full activation, however,
requires the formation of the IS, which involves active transport processes. IS formation requires a
threshold level of triggering through the TCR and is
influenced by the nature and activation state of the
APC.
Acknowledgements
P.A.V. is supported by the Medical Research Council and
S.J.D. by the Wellcome Trust. M.L.D. is supported by the
Arthritis Foundation, The Whitaker Foundation and The
National Institutes of Health. A.S. is supported by the
National Institutes of Health. We thank Don Mason, Neil
Barclay, Marion Brown, Matt Thomas, and Talitha Bakker
for critical comments on the manuscript. We thank S.
Bromley and C. Sumen for the use of images in Figure 2.
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