Dynamic regulation of T cell activation and co-stimulation through TCR-microclusters.
ABSTRACT TCR-microclusters (MC) are generated upon TCR stimulation prior to the immune synapse formation independently of lipid rafts. TCR-MCs contain receptors, kinases and adaptors, and function as the signaling unit for T cell activation. The TCR complex, but not the signaling molecules, is transported to the center to form cSMAC. The co-stimulation receptor CD28 joins the signaling region of cSMAC and recruits PKCθ and Carma1. CTLA-4 accumulates in the same region and competes with CD28 for negative regulation of T cell activation. T cell activation is therefore mediated by two spatially distinct signaling compartments: TCR signaling by the peripheral TCR-MC and co-stimulation signal by the central signaling cSMAC.
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ABSTRACT: Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.Nucleic acid therapeutics. 02/2014;
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ABSTRACT: T cell signaling is triggered through stimulation of the T cell receptor and costimulatory receptors. Receptor activation leads to the formation of membrane-proximal protein microclusters. These clusters undergo tyrosine phosphorylation and organize multiprotein complexes thereby acting as molecular signaling platforms. Little is known about how the quantity and phosphorylation levels of microclusters are affected by costimulatory signals and the activity of specific signaling proteins. We combined micrometer-sized, microcontact printed, striped patterns of different stimuli and simultaneous analysis of different cell strains with image processing protocols to address this problem. First, we validated the stimulation protocol by showing that high expression levels CD28 result in increased cell spreading. Subsequently, we addressed the role of costimulation and a specific phosphotyrosine phosphatase in cluster formation by including a SHP2 knock-down strain in our system. Distinguishing cell strains using carboxyfluorescein succinimidyl ester enabled a comparison within single samples. SHP2 exerted its effect by lowering phosphorylation levels of individual clusters while CD28 costimulation mainly increased the number of signaling clusters and cell spreading. These effects were observed for general tyrosine phosphorylation of clusters and for phosphorylated PLCγ1. Our analysis enables a clear distinction between factors determining the number of microclusters and those that act on these signaling platforms.PLoS ONE 01/2013; 8(10):e79277. · 3.53 Impact Factor
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ABSTRACT: Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis, and its capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. We formulated a novel nanovaccine containing meningococcal CPS as an antigen encapsulated in albumin-based nanoparticles (NPs) that does not require chemical conjugation to a protein carrier. These nanoparticles are taken up by antigen-presenting cells and act as antigen depot by slowly releasing the antigen. In this study, we determined the ability of CPS-loaded vaccine nanoparticles to induce co-stimulatory molecules, namely CD80, CD86, and CD95 that impact effective antigen presentation. Co-stimulatory molecule gene induction and surface expression on macrophages and dendritic cells pulsed with meningococcal CPS-loaded nanoparticles were investigated using gene array and flow cytometry methods. Meningococcal CPS-loaded NP significantly induced the surface protein expression of CD80 and CD86, markers of dendritic cell maturation, in human THP-1 macrophages and in murine dendritic cells DC2.4 in a dose-dependent manner. The massive upregulation was also observed at the gene expression. However, high dose of CPS-loaded NP, but not empty NP, induced the expression of death receptor CD95 (Fas) leading to reduced TNF-α release and reduction in cell viability. The data suggest that high expression of CD95 may lead to death of antigen-presenting cells and consequently suboptimal immune responses to vaccine. The CPS-loaded NP induces the expression of co-stimulatory molecules and acts as antigen depot and can spare antigen dose, highly desirable criteria for vaccine formulations.The AAPS Journal 07/2014; · 4.39 Impact Factor
Dynamic regulation of T cell activation and co-stimulation through
Takashi Saito⇑, Tadashi Yokosuka, Akiko Hashimoto-Tane
Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan
a r t i c l e i n f o
Received 1 November 2010
Revised 17 November 2010
Accepted 18 November 2010
Available online 24 November 2010
Edited by Israel Pecht
T cell activation
a b s t r a c t
TCR-microclusters (MC) are generated upon TCR stimulation prior to the immune synapse formation
independently of lipid rafts. TCR-MCs contain receptors, kinases and adaptors, and function as the
signaling unit for T cell activation. The TCR complex, but not the signaling molecules, is transported
to the center to form cSMAC. The co-stimulation receptor CD28 joins the signaling region of cSMAC
and recruits PKCh and Carma1. CTLA-4 accumulates in the same region and competes with CD28 for
negative regulation of T cell activation. T cell activation is therefore mediated by two spatially dis-
tinct signaling compartments: TCR signaling by the peripheral TCR-MC and co-stimulation signal by
the central signaling cSMAC.
? ? 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
T cells recognize antigen (Ag) as a peptide–MHC complex on Ag-
presenting cells (APC) such as dendritic cells (DC) through direct
cell–cell interactions. The T cell antigen receptor (TCR) binds to
the Ag peptide–MHC complex and triggers T cell activation by
recruiting various signaling molecules including the src-family ki-
nase Lck and the syk-family kinase ZAP-70. Upon formation of the
T cell–APC conjugate, T cells become polarized towards the APC
and create a unique structure at the interface between the two
cells, called the immune synapse (IS). Upon Ag recognition/activa-
tion, the TCR–CD3 complex accumulates at the center of the IS as
the central (c-) supramolecular activation complex (cSMAC) and
is surrounded by the integrin LFA1 as the peripheral (p-) SMAC
[1,2]. When originally characterized, this structure appeared to
support a model in which the cSMAC contained the TCR as the
Ag recognition structure and the pSMAC contained the integrin
to promote cell–cell adhesion. Together with accumulated evi-
dence that various signaling molecules are recruited to the IS, this
structure appeared to be the site of Ag recognition and signal trans-
duction for T cell activation. However, the generation of the cSMAC
and pSMAC of the IS takes 5–10 min after T cell-APC or T cell-MHC-
containing planar bilayer interaction, kinetics that did not corre-
spond at all to those of early activation events such as tyrosine
phosphorylation, and intracellular Ca flux .
In addition, early analysis of the signaling complex responsible
for T cell activation using Jurkat cells stimulated with immobilized
anti-CD3 antibody (Ab) revealed that this intracellular complex,
which includes adaptor proteins LAT and SLP-76/Gads as well as
effector molecules such as PLCc , is formed immediately upon
stimulation. These imaging analyses using Jurkat cells were consis-
tent with previous biochemical analyses of the well-established
proximal signal transduction events upon TCR stimulation [5–7];
phosphorylation of the ITAMs of CD3 chains by Lck recruits ZAP-
70 which induces phosphorylation of adaptor proteins LAT and
SLP-76 followed by activation of downstream effector molecules.
In normal T cells, Davis and Krummel first observed small cluster
of CD3f at the interface between T cells and B cell lines prior to
cSMAC formation .
To precisely and dynamically analyze membrane proximal
events upon stimulation of normal T cells with Ag peptide–MHC
complexes, we have utilized the combination of Total Internal
Reflection Fluorescence (TIRF) microscopy and a supported planar
bilayer membrane containing mobile peptide-MHC and ICAM-1 as
a pseudo-APC membrane for high-resolution imaging. Using this
system, we found that the TCR complexes accumulate to form
small clusters, termed TCR-microclusters (TCR-MC), immediately
after the T cells attached to the membrane, which is much earlier
than IS formation [9–11]. The analysis of generation and regulation
0014-5793/$36.00 ? 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
⇑Corresponding author. Address: Laboratory for Cell Signaling, RIKEN Research
Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama
230-0045, Japan. Fax: +81 45 503 7036.
E-mail address: email@example.com (T. Saito).
FEBS Letters 584 (2010) 4865–4871
journal homepage: www.FEBSLetters.org
of TCR-MCs has provided new insights into the molecular mecha-
nism of T cell activation. In this article, we highlight our current
understanding of the spatiotemporal regulation of T cell activation
and co-stimulation through TCR- and CD28-MCs.
2. TCR-microclusters upon initial activation
When T cells become attached to the planar membrane contain-
ing peptide-MHC and ICAM-1, the TCR-MCs are generated first at
the contact site. As T cells start spreading on the membrane,
TCR-MCs are formed over the entire interface. After maximum
spreading, T cells then start to contract and all of the TCR-MCs
move towards the center of the interface to form the cSMAC. Quan-
titative analysis of the fluorescence intensity of individual TCR-MC
revealed that a single TCR-MC contains approximately 50–300
CD3f chains . Some of the MCs fuse with each other as they
move to the center from the periphery. By tracking the movement
of TCR signaling molecules ZAP-70 and SLP-76 as representative ki-
nase and adaptor proteins, respectively, we made two critical
observations; first, a single TCR-MC contains both ZAP-70 and
SLP-76, indicating that a TCR-MC represents a functional signal-
some containing TCR, kinases and adaptors which is responsible
for transducing T cell activation signals. Secondly, although
ZAP-70 and SLP-76 accumulate within the same TCR-MCs, unlike
TCR-CD3 they do not move to the center of the interface but in-
stead disappear on the way to the center. Thus, we noted that only
the TCR complex is transported to the center to form the cSMAC.
TCR-MCs are continuously generated at the very edge of the
periphery of the contact site, where lamelipodia-like structures
may be newly associated with peptide-MHC complexes. TCR-MC
generation is not restricted to the artificial bilayer system, but
has also been confirmed at the T cell-APC interface. Thus, it appears
to reflect the physiological state, although the detection of TCR-MC
required further technical advances in microscopic analysis in vivo.
Whether the TCR-MCs detected in the planar bilayer are really
the minimal unit of Ag recognition and cellular activation is a crit-
ical issue and remains to be resolved using various approaches. For
example, the Davis group has shown that recognition of a single
agonistic peptide-MHC by TCR induces Ca2+flux and several pep-
tide-MHCs are even sufficient to trigger effector function .
However, it is still possible that single agonist peptide with many
other peptides including self-peptides are recognized by clustered
TCR. Before Ag stimulation, it has been shown by electron micros-
copy that some small clusters of TCR or LAT were pre-formed and
the TCR clusters are different from those of LAT, and that these two
kinds of clusters will merge together upon Ag stimulation, proba-
bly to form TCR-MCs , suggesting that the clusters competent
for signaling will be TCR-MCs.
3. Signaling through TCR-MC
The idea that the TCR-MC is the unit responsible for generating
TCR activation signals came from the finding that every TCR-MC is
stained with anti-phospho-tyrosine Ab and anti-phospho-ZAP-70
upon stimulation. Analysis of individual T cells revealed that the
Ca2+influx is induced in parallel with TCR-MC generation, which
is a much earlier event than cSMAC formation. Thus, TCR-MCs rep-
resent a unit to transduce TCR recognition signals for activation. To
compare kinetics of the known biochemical events of the TCR prox-
imal signaling cascade, T cells were treated with the src-kinase
inhibitor PP2. Although TCR-MCs were still induced even in the ab-
sence of Lck function, PP2 blocked the recruitment of ZAP-70 to the
TCR-MCs and generation of the cSMAC.
To even more rigorously test the idea that the TCR-MC is a min-
imal unit of TCR activation, we analyzed the components of
TCR-MCs using individual GFP-fused signaling molecules. Using
this approach, we found that proximal signaling molecules such
as CD3s, Lck, ZAP-70, LAT, SLP-76, Gads, Grb2, Itk, PLCc, PI3K, Car-
ma1, IKK, Vav, Nck, and WASP, are contained within TCR-MCs, but
other molecules such as Ras, Rac, Sos, Erk, Akt, Pdk-1, and CD45 are
not present. Therefore, consistent with the proposed role of TCR-
MCs, the proximal signaling molecules to induce initial activation
signals are the main components. These findings are almost en-
tirely consistent with the signaling complex analyzed in Jurkat
cells [4,14] except that the TCR-CD3 complex is not involved with-
in the signaling complex since the TCR complex on the cell surface
is fixed and does not move nor form the cSMAC in Jurkat cells stim-
ulated with immobilized Ab.
4. TCR-MCs for sustained T cell activation
It is well established that full activation of T cells, leading to
cytokine production or cell proliferation, requires continuous
stimulation for several hours . In the planar bilayer model,
TCR-MCs are continuously generated for hours at the periphery
of the contact interface even after cSMAC formation. Newly gen-
erated TCR-MCs at the periphery also contain phosphorylated
signaling molecules, including ZAP-70, which suggests that the
peripheral TCR-MCs also induce signals for sustained activation.
Phosphorylation of MCs in the periphery is in great contrast to
the situation in the cSMAC, where very little tyrosine phosphor-
ylation can be detected, suggesting that the sustained signal for
T cell activation is induced through the peripheral TCR-MCs but
not through the cSMAC. When Jurkat cells are stimulated with
immobilized anti-TCR Ab, a SLP-76-nucleated signaling complex
was formed and induced signals intracellularly at pre-Golgi, sug-
gesting that sustained activation signal maybe induced in intra-
cellular compartments [14,16]. By contrast, in normal T cells,
this phosphorylated intracellular protein complex has not been
observed, and the peripheral TCR-MCs are likely to mediate sus-
tained as well as initial signaling.
Whereas the planar bilayer contains abundant Ag peptide-MHC,
Ag concentration in vivo would obviously be more limited. Under
stimulation condition with limited amounts of Ag peptide (under
0.1 lM) in the planar bilayer, T cells exhibit detectable TCR-MCs
but no cSMAC. Thus, under conditions where peptide is limited
such as in vivo, both the initial and sustained T cell activation will
be induced via TCR-MCs in the absence of cSMAC formation or
translocation of TCR-MCs to the center of the IS. The cSMAC will
be formed and play a significant functional role only upon strong
stimulation; either where there is high concentration of peptide,
or strong co-stimulation signals. Such strong stimulation induces
formation of the cSMAC, which then negatively regulates excessive
T cell responses.
5. Lipid raft and TCR-MCs
Lipid rafts are specialized liquid-ordered membrane microdo-
mains that are enriched in cholesterol and sphingolipids. Numer-
ous studies have revealed that lipid rafts exist as small leaflets
and float on the plasma membrane [17–19]. They are thought to
function in protein sorting and cell activation as a platform for
recruiting various signaling molecules such as src-family kinases,
G proteins and adaptor proteins mainly through lipid-anchors such
as palmitoylation. The functional significance of lipid rafts in signal
transduction has been particularly well demonstrated for T cell
activation [20,21]. Early studies indicated that crosslinking of the
raft-associated ganglioside GM1 induced T cell activation ,
and that LAT with mutant palmitoylation sites, which no longer
localized to lipid, rafts, failed to induce an activation signal .
T. Saito et al./FEBS Letters 584 (2010) 4865–4871
Currently, lipid rafts are thought to serve as a platform for signal-
ing molecules involved in T cell activation [22,23]. Since the most
commonly used ‘‘raft marker’’ GM1 was found to accumulate at the
IS upon stimulation, lipid rafts have been thought to accumulate at
the cSMAC and serve as signaling platforms. However, TCR-MCs
also recruit various signaling molecules, thus they too function as
a kind of platform for T cell activation. Thus, the relationship be-
tween lipid raft clusters and TCR-MCs was addressed from the
point of view of a signaling platform for T cell activation.
We performed imaging analysis to look for the co-localization
of TCR-MCs and lipid rafts . Lipid rafts were visualized by
fluorescent raft probes defective in signaling capacity such as a
truncated form of Lck as an inner membrane probe, LAT as a
transmembrane probe and GM1 as an extracellular probe. No
co-localization of these lipid raft probes with any TCR-MC mole-
cules such as CD3f, LAT or ZAP-70 was observed in either resting
or stimulated cells. Considering that the lipid raft cluster is small
(<100 nm) probably even after clustering, we used more sensi-
tive FRET techniques to analyze molecular association between
TCR-MCs and lipid raft probes. However, no increase of specific
FRET between CD3f and Lck- or LAT-based raft probes was ob-
served in the region of TCR-MCs upon stimulation . There-
fore, our data indicate that lipid raft clusters are not involved
in either the generation or signaling function of TCR-MCs but
they are rather involved in intracellular protein trafficking, such
as the transport of LAT to the plasma membrane. Lipid rafts
therefore do not serve as the signaling platform to recruit signal-
ing molecules. TCR-MCs accumulate mainly through protein
interactions, consistent with recent report  suggesting that
the plasma membrane is composed of protein-based islands that
are formed independently of lipid raft clusters.
6. IS in migrating cells and cytoskeleton regulation
TCR activation through TCR-MCs induces the ‘‘stop signal’’ in T
However, T cells can also interact with APC during migration condi-
tion. Such dynamic interactions are driven by chemokines either on
cell surface of APC or stroma cells. Often in vivo, chemokine-medi-
ated migration dominates over the stop signals . Therefore,
tion signals mediated by chemokines in order to form IS, and their
balance determines the fate of T cells, either movement or stable IS
formation. When migration signals dominate, the interaction be-
tween migrating T cells and APC becomes short and T cells are not
tin proposed that moving T cells form ‘‘immune kinapse’’  to in-
duce activation signal during the unstable and unpolarized
interaction. TCR accumulates at uropod while TCR-MCs is formed
at leading edges as lamellipodium and LFA-1 at the lamella. In the
and microtubule dynamics regulates the expansion and contraction
at the leading edge and uropod of T cells.
Such dynamic regulation of TCR-MC movement in T cells either
with synapse or kinapse is supported both by actin and microtu-
bule cytoskeleton network. The targeting of vesicles containing
LFA-1 integrin and GTPase Rap-1 together with RapL  and Talin
induces translocation and activation of integrin, regulating T cell-
APC adhesion during kinapse and synapse formation. The interplay
between cortical actin and microtubules are critical for both stabil-
ity and polarity of IS. The nucleation of TCR-MCs at IS relies on
functional actin cytoskeleton. TCR-MCs appear to move towards
the center of the interface by the force of actin retrograde flow
. In addition, the centripetal movement of the microclusters
requires organized microtubules with motor protein dynein-med-
iated movement .
7. Co-stimulation and TCR-MCs
7.1. Positive regulation by CD28-MC
T cell activation and the consequent fate of T cells are posi-
tively and negatively regulated by several co-stimulation signals.
The major co-stimulation receptor is CD28, whose ligands on
APC are CD80 and CD86 [26,27]. It has been known for some
time that in the absence of CD28-mediated co-stimulation, T
cells become unresponsive, a status termed ‘‘anergy’’ [28,29].
The co-stimulation signal appears to be independent of TCR sig-
nals since independent stimulation, with no co-ligation of these
receptors, significantly enhances T cell activation. In spite of
extensive analysis of CD28-mediated signaling pathways, which
have suggested a critical role of PI3K to mediate co-stimulation
signals [30,31], the molecular nature and spatial relationship be-
tween CD28 and TCR remain elusive.
We analyzed the dynamics of CD28 and related signaling mole-
cules, particularly to understand the spatial and signaling relation-
ship between TCR-MCs and co-stimulation . CD28 was found
to be co-localized with TCR-MCs upon initial Ag stimulation.
CD28-MCs moved to the center of the interface, similar to TCR-
CD3, and when the cSMAC was formed, CD28 accumulated at the
periphery of the cSMAC, but was still within the cSMAC since it
was surrounded by the ring of LFA-1 representing the pSMAC. To
identify molecule(s) mediating downstream events in CD28-medi-
ated co-stimulation that move and function together with CD28
this analysis we chose molecules that are thought to be involved in
CD28-mediated co-stimulation, including PI3K, Grb2, Gads, Itk,
Vav, PP2A and PKCh [33–36], although most of these molecules are
also involved in the TCR-downstream signaling pathway. None of
them except for PKCh accumulated into the cSMAC upon stimula-
tion. Indeed, PKCh was co-localized in TCR-MCs upon initial activa-
tion, and then moved together with the TCR and then accumulated
in the same region as CD28. CD28 and PKCh not only move together,
but they are also physically associated since we could co-immuno-
precipitate them from normal T cells upon stimulation. The specific
CD28 region that located at the periphery of the cSMAC is main-
tained dynamically because both the CD28 and PKCh accumulated
In addition, blocking of the CD28-CD80 interaction by CTLA-4-Ig
abrogated the accumulation of not only CD28 but also PKCh in that
region, indicating that CD28 recruits PKCh to this particular region
of the cSMAC probably to mediate sustained co-stimulatory signals
such as NF-jB activation  (Fig. 1). Indeed, we have now shown
that Carma1, which forms the Carma1-Bcl10-Malt1(CBM) complex
to induce NF-jB activation, also accumulates in the same region as
CD28 and PKCh . We have begun to analyze CD28 mutants to
mation and PKCh recruitment. Mutant CD28 lacking CD80-binding
capacity cannot make clusters, whereas a tail-less CD28 mutant
defective in signaling could induce clustering but failed to recruit
and accumulate PKCh, a defect that was correlated with defective
co-stimulatory function .
Therefore, our analysis on CD28-mediated co-stimulation re-
vealed an important mode of signal regulation during co-stimu-
lation; the CD28 co-stimulatory receptor is initially accumulated
in the TCR-MCs and then in the special region of the cSMAC.
PKCh is associated with and co-translocates with CD28 and
T. Saito et al./FEBS Letters 584 (2010) 4865–4871
7.2. Negative regulation by CTLA-4-MC
After analyzing the dynamic regulation of CD28-mediated posi-
tive co-stimulation, we investigated the negative regulation medi-
ated by CTLA-4, a major negative co-stimulatory receptor. CTLA-4
has the same ligands, CD80 and CD86, as CD28 but, whereas
CD28 is constitutively expressed on the cell surface, CTLA-4 is
not until its expression is induced by TCR stimulation [38,39].
The CTLA4 gene is transcribed and translated upon stimulation,
but the protein is retained and degraded within lysosomes in the
absence of further stimulation through endocytosis by assembly
with the AP2 complex [40–42]. Since CTLA-4 has a much higher
affinity than CD28 for the same ligands, even low level expression
of CTLA-4 on the cell surface can compete for ligand binding with
CD28, which is thought to be the main mechanism of CTLA-4-med-
iated inhibition of T cell activation. Previous studies revealed that
CTLA-4 is transported from storage in endosome/lysosomes to
the plasma membrane upon further T cell stimulation, and that
the induction of cell surface expression is only induced by strong
activation whereas weak activation only leads to translocation of
these vesicles to the vicinity of the plasma membrane . How-
ever, how CTLA-4 blocks T cell activation has remained unclear.
When we analyzed spatiotemporal regulation of CTLA-4 expres-
sion, we found that CTLA-4 also forms microclusters, but these are
not initially co-localized in TCR-MCs and instead directly accumu-
late in cSMAC . The region of the cSMAC where the CTLA-4-
MCs accumulate is exactly the same region where CD28 and PKCh
accumulate. Thus, CTLA-4-MCs, once translocated to the cSMAC,
push CD28 and PKCh away from a specific region of cSMAC, which
results in the blockade of CD28-mediated co-stimulation (Fig. 1).
To determine whether the accumulation of CTLA-4 in the cSMAC
is required for CTLA-4-mediated inhibition of activation, we pro-
duced CTLA-4 chimeras with CD22 possessing various sizes of
the CD22 ectodomain and the first ligand-binding Ig domain of
CTLA4. The idea behind this experiment is that only short mole-
cules with one or two Ig domains can accumulate in cSMAC
whereas large molecules would be excluded and accumulate out-
side of the cSMAC. As expected, we found that the short ectodo-
main (one or two Ig domains)-bearing CTLA-4 co-localized with
the TCR-MCs and accumulated later in the cSMAC whereas mole-
cules with longer ectodomains failed to localize in either the
TCR-MCs or the cSMAC. Functionally, only CTLA-4 with short ecto-
domains could inhibit T cell activation. These results proved the
importance of its localization in the cSMAC for CTLA-4 to mediate
inhibitory function .
8. Signaling at cSMAC
8.1. Bi-function of cSMAC
The cSMAC contains the concentrated TCR complex and this
representative structure of the IS has been thought to be responsi-
ble for inducing activation signals. However, since tyrosine phos-
phorylation in the cSMAC was barely detectable, while abundant
tyrosine phosphorylation was observed in individual TCR-MCs at
the periphery, it has become clear that the cSMAC is not responsi-
ble for signal transduction. This concept was further supported by
the finding that the cSMAC contains negatively regulating phos-
phatases and degradation machinery-related molecules [44,45]
and that T cells from CD2AP-deficient mice, which have defective
cSMAC formation, show enhanced TCR expression and cellular acti-
vation . These findings all indicate that the cSMAC plays a role in
negative regulation of T cell activation by promoting endocytosis
and degradation of the TCR complex. However, recently several
lines of analyses have provided evidence that the cSMAC also has
signaling function, but in a way distinct from TCR-MCs. One, as de-
scribed above, is in CD28-mediated co-stimulatory signaling, prob-
ably including NF-kB activation, and is mediated through the
special region of cSMAC [32,46]. The other stems from the observa-
tion that TCR signals including phopho-CD3f may be actively in-
duced under certain restricted conditions, particularly with weak
Fig. 1. Spatiotemporal regulation of formation of TCR-microcluster and cSMAC and signal regulation through them. Upon recognition of antigen-MHC, a T cell exhibits
sequential processes; spreading, contraction and immune synapse formation. As soon as a T cell attaches, TCR-MCs containing TCR, ZAP-70, SLP76 as well as CD28 and PKCh
are formed which induce initial activation signals. TCR-MCs then move towards the center of the interface to generate cSMAC while ZAP-70 and SLP-76 do not move and
accumulate at the center. In contrast, both CD28 and PKCh are accumulated in the specific region within cSMAC (CD3losignaling cSMAC) to positively induce sustained co-
stimulation signals. At late phase of activation, CTLA-4 is induced upon activation and accumulated not to TCR-MCs but directly to the same region of cSMAC as CD28
accumulated where CTLA-4 competes the ligand-binding with CD28. CD28-PKCh is pushed out from the signaling cSMAC and consequently activation is inhibited.
T. Saito et al./FEBS Letters 584 (2010) 4865–4871
8.2. Subregions of the cSMAC
Regarding the spatial regulation of signals in the cSMAC, we de-
fined two distinct sub-regions based on our high-resolution micro-
scopic analysis. The distinction between the two is based on the
density of CD3; CD3hiand CD3loregions (Fig. 2) [32,46]. CD3hirep-
resents the well-known standard cSMAC in which TCR-CD3 is
highly accumulated. When analyzed using fluorescently labeled
probes for MHC class II, we found that there was no overlap be-
tween the CD3hiregion and the pMHC, however the CD3loregion
correlated well with pMHC. This analysis indicates that TCR in
the CD3loregion are associated with peptide-MHC on the cell sur-
face, whereas TCR in the CD3hiregion appear to be no longer
assembled with pMHC, and are probably destined for internaliza-
tion and degradation. Photobleaching experiments indicated that
the CD3loregion is dynamically regulated whereas CD3hiis very ri-
gid and did not recover after bleaching. Importantly, since CD28
and PKCh as well as Carma-1 all accumulate in this CD3loregion,
CD3lois the region mediating signal transduction as the ‘‘signaling
The relative dimensions of the CD3loand CD3hiregions are reg-
ulated by the strength of the TCR and co-stimulation signals as
shown in Fig. 2. A high dose of Ag induces a larger proportion of
CD3hi, which will be internalized/degraded, whereas a low dose
of Ag results in a larger CD3loregion, which induces active
Fig. 2. Schematic model of functionally distinct subregions of cSMAC. cSMAC is segregated into two regions according to the density of CD3; CD3-high (CD3hi) and CD3-low
(CD3lo). The size of cSMAC is determined by TCR signal strength. In the absence of co-stimulation, the majority of cSMAC is occupied CD3hiregion where extensive
internalization and degradation of TCR takes place. In contrast, in the presence of strong co-stimulation, CD3loregion as dominate within cSMAC as ‘‘signaling cSMAC which is
responsible for co-stimulation signaling. Thus, the proportion of CD3hivs. CD3lois determined by the signal strength of TCR signal and co-stimulatory signal. TCR signal
becomes strong by high dose of antigen or strong agonist while become weak with low concentration of antigen or weak agonist. Co-stimulatory signal becomes strong with
high expression of CD28 on T cells orCD80/86 on APC, while become weak with low level of CD28 or CD80/86.
Fig. 3. Possible implication of spatial and differential regulation of TCR- and co-
stimulation signals through TCR-MCs and signaling cSMAC. Whereas the peripheral
TCR-microclusters induce Ag recognition signal through activation of ZAP-70, LAT,
SLP-76, co-stimulation signals are induced in the localized specialized cSMAC
region ‘‘signaling cSMAC’’ through recruiting CD28, PKCh and Carma-1. From the
point of 2 signal model of T cell activation, these signals may represent ‘‘signal 1’’
and ‘‘signal 2’’, respectively, which are therefore generated by spatially distinct
T. Saito et al./FEBS Letters 584 (2010) 4865–4871
signaling. Conversely, strong co-stimulation increases the CD3lo
and weak co-stimulation increases the CD3hiregions, respectively.
These studies lead to a new global and testable model for TCR
signaling. Spatially differential regulation through peripheral
TCR-MC and signaling cSMAC represent the site for ‘‘signal 1’’ and
‘‘signal 2’’ in classic co-stimulation models [29,48]. TCR-MCs in-
duce Ag recognition signals through TCR as ‘‘signal 1’’ and signaling
cSMAC (CD3lo cSMAC) induce co-stimulatory signals as ‘‘signal 2’’
9. Concluding remarks
In this review, we describe dynamic feature of antigen recogni-
tion and activation of T cells from the analysis of newly defined
microclusters. TCR-MC is generated, translocated and regulated
dynamically by assembly and disassembly with various molecules.
Since the regulation of cell activation by microcluster is similarly
observed in B cells and NK cells, the dynamic regulation of recep-
tor-mediated activation through microclusters is common mecha-
nism among all lymphocytes. Probably not only lymphocytes but
also other type of cells such as nerve cells may have similar regu-
latory system. Molecular imaging of single T cell made advance to
understand molecular dynamics of signaling event. Many ques-
tions remain to be solved; where do signaling molecule such as
ZAP-70 and SLP-76 disappeared, how sustained activation is regu-
lated with very low concentrations of antigen, how much extent do
TCR-MC regulation reflect in vivo, and so on. Further analysis of
spatiotemporal regulation of TCR-MCs within a cell and in vivo will
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