Spatial and Temporal Dynamics
of T Cell Receptor Signaling
with a Photoactivatable Agonist
Morgan Huse,1,5,6Lawrence O. Klein,2,5Andrew T. Girvin,3Joycelyn M. Faraj,1Qi-Jing Li,1,4Michael S. Kuhns,1
and Mark M. Davis1,4,*
1Department of Microbiology and Immunology
2Graduate Program in Biophysics
3Graduate Program in Immunology
4The Howard Hughes Medical Institute
Stanford University School of Medicine, Stanford, CA 94305, USA
5These authors contributed equally to this work.
6Present address: Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
T cell receptor (TCR) is poorly understood. To
address this problem, we prepared major histo-
compatibility complexes containing an anti-
genic peptide that is biologically inert until
exposed to ultraviolet (UV) light. UV irradiation
of these complexes in contact with cognate T
ysis of signaling. Phosphorylation of the LAT
adaptor molecule was observed in 4 s, and di-
acylglycerol production and calcium flux was
observed in 6–7 s. TCR activation also induced
cytoskeletal polarization within 2 min. Antibody
blockade of CD4 reduced the intensity of LAT
phosphorylation and the speed of calcium
flux. Furthermore, strong desensitization of di-
acylglycerol production, but not LAT phosphor-
ylation, occurred shortly after TCR activation,
suggesting that different molecular events
play distinct signal-processing roles. These re-
sults establish the speed and localization of
early signaling steps, and have important impli-
cations regarding the overall structure of the
The recognition of antigen by T cells on the surface of an-
tigen-presenting cells (APCs) induces a profound set of
physiological changes, culminating in T cell proliferation,
cytokine secretion, and in the case of cytotoxic T cells,
the lysis of the cell being recognized (Janeway and Bot-
tomly, 1994). FormostT cells,specific antigen recognition
is governed by the ab T cell receptor (TCR), which binds to
antigenic peptides presented by class I or class II major
et al., 1998; Rudolph et al., 2006).
The signaling events that link peptide MHC (pMHC) rec-
proximal adaptormolecules. Amongtheseisthe Linkerfor
the Activation of T cells (LAT) (Sommers et al., 2004),
which contains multiple tyrosine phosphorylation sites
that recruit a diverse set of downstream proteins. The re-
cruitment of the Grb2 adaptor and the associated SOS
exchange factor to LAT stimulates MAP kinase signaling.
Phospho-LAT also binds to phospholipase C-g (PLC-g),
which hydrolyzes phosphatidylinositol bis-phosphate to
generate inositol trisphosphate (IP3) and diacylglycerol
whereas DAG recruits RasGRP and protein kinase C-q
(PKCq) to the cell membrane. Phospho-LAT also induces
dramatic changes in the T cell cytoskeleton; these
changes include the reorientation of the microtubule
organizing center (MTOC) to the T cell-APC contact site
(Geiger et al., 1982; Kupfer and Dennert, 1984). This is
accompanied by the organization of the cell-cell junction
et al., 1999; Monks et al., 1998).
T cells can initiate intracellular signaling responses to as
few as one agonist pMHC (Irvine et al., 2002). These re-
sponses are remarkably fast, occurring within seconds
muth, 2002; Houtman et al., 2006; Patrick et al., 2000; Wei
et al., 1999). They also display exquisite specificity for
high-affinity agonist pMHC. Indeed, modest changes in
the lifetime of a pMHC-TCR interaction can lead to pro-
found differences in biological responses (Kersh et al.,
1998). A number of theoretical models have been pro-
posed that attempt to explain how the TCR signaling net-
work can combine sensitivity, speed, and discriminatory
power in this manner (Altan-Bonnet and Germain, 2005;
Chan et al., 2001; Chan et al., 2004; McKeithan, 1995).
It is difficult, however, to evaluate any of these hypoth-
eses without a better understanding of the spatial and
ular, it is unclear exactly how quickly signaling occurs.
76 Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc.
Furthermore, our knowledge of how TCR-induced signals
change over time is limited. Theoretical studies have de-
scribed how quickly positive and negative feedback can
contribute to ligand discrimination and sensitivity (Altan-
Bonnet and Germain, 2005; Chan et al., 2004; Li et al.,
2004), and indeed, the TCR signaling network contains
a number of distinct feedback loops (Egen et al., 2002;
Naramura et al., 2002; Sanjuan et al., 2001; Stefanova
et al., 2003; Wang et al., 2000; Zhong et al., 2002). How-
ever, little is known about the timescale over which these
interactions occur and whether certain branches of the
network are affected more than others.
Also unclear is the precise role played by subcellular lo-
calization. Imaging experiments have revealed that the
TCR and several other signaling proteins colocalize in dis-
tinct microclusters within the plasma membrane (Bunnell
et al., 2002; Campi et al., 2005; Douglass and Vale,
2005; Yokosuka et al., 2005). It is thought that these mi-
croclusters are important sites of early TCR signaling,
but it is not clear whether the kinetics of microcluster
formation is consistent with such a role. Furthermore,
how signals propagate from microclusters to induce
more generalized T cell activation is not known.
Localized signaling is also thought to play a crucial role
in cell polarization. In T cells, the movement of the MTOC
toward the immunological synapse occurs within minutes
of TCR triggering (Kupfer and Dennert, 1984). A recent
study demonstrated that T cells selectively polarize to-
ward APCs bearing greater amounts of cognate antigen,
suggesting that TCR signals are locally integrated (Depoil
et al., 2005). The mechanism of local signal integration is
not known, nor is it clear how precise comparisons of
TCR signaling strength are made as the T cell encounters
additional antigen over time.
ics on the basis of the UV activation of pMHC. We have
used this system to study signaling kinetics, propagation,
and localization downstream of the TCR. Photoactivation
of pMHC stimulated LAT phosphorylation, DAG produc-
tion, and calcium flux within seconds and stimulated
MTOC translocation in less than 2 min. Blocking the core-
ceptor CD4 reduced the magnitude of LAT phosphoryla-
tion and the speed of calcium flux. We also found that
whereas DAG production desensitized quickly after initial
TCR triggering, other responses, such as LAT phosphory-
lation and MTOC reorientation, remained sensitive to re-
like incidence detectors, whereas others acted more like
signal integrators. These results provide insights into the
dynamics and the structure of the TCR signaling network.
A Photoactivatable Agonist Peptide
for the 5C.C7 TCR
To achieve precise spatiotemporal control of T cell stimu-
lation, we modified a well-characterized antigenic peptide
from moth cytochrome c (residues 88–103, hereafter
called MCC) so that its ability to bind the TCR could be
controlled by UV light. MCC triggers T cell activation
through the 5C.C7 and 2B4 TCRs when presented by
the mouse class II MHC I-Ek. Previous studies had indi-
cated that Lys 99 of MCC is critical for biological activity
hydrophobic group attached at this position would block
TCR binding. Thus, we prepared an MCC variant bearing
a photocleavable 1-ortho-nitrophenyl-ethyl urethane (NPE)
moiety on the 3-amino group of Lys 99 (NPE-MCC,
Figure 1A). Irradiation with UV light cleaves off the NPE
group to yield native MCC peptide (data not shown). In
a recent report, an alternative synthetic approach was
used to prepare a photoactivatable MCC peptide with
similar properties (DeMond et al., 2006).
On the basis of previous studies involving similar photo-
release strategies, we expect that the photoactivation
reaction occurs in less than 1 ms (McCray and Trentham,
1989). Importantly, NPE-MCC is nonstimulatory to 5C.C7
T cells until it is exposed to UV light (Figure S1 in the
Supplemental Data available online). Thus, it displays the
expected properties of a photoactivatable agonist.
Calcium Signaling Kinetics in T Cell-APC Conjugates
a fluorescence videomicroscope such that UV light from
a 337 nm laser could be used to photoactivate NPE-
MCC peptide within the imaging sample (Figure S2). This
configuration enabled both the controlled photoactivation
of NPE-MCC and the imaging of subsequent intracellular
We first measured calcium signaling dynamics in pre-
formed T cell-APC conjugates. APCs pulsed with NPE-
MCC were mixed with 5C.C7 T cell blasts loaded with
the Fluo-4 calcium indicator. Under these conditions, the
cells form contacts, but there is no detectable calcium
flux (L.O.K. and M.M.D., unpublished data). Exposing
a T cell-APC couple to UV laser light triggered an increase
in Fluo-4 fluorescence within the T cell, but only after
a characteristic delay of 6.5 ± 0.5 s (Figures 1B and 1C;
Movie S1). We refer to the delay between agonist activa-
tion and the subsequent response as the ‘‘offset time,’’
which can be interpreted as a measurement of signaling
speed. These results demonstrate that NPE-MCC can
be used to measure signaling kinetics with high temporal
precision in the context of an imaging experiment.
Photoactivation Induces Rapid
rated two fluorescent probes for earlier steps in the TCR
signaling cascade (Figure S3). The adaptor protein Grb2,
which is recruited to the plasma membrane by phospo-
LAT, was used as a reporter for LAT phosphorylation.
Similarly, the tandem C1 domains from PKCq were used
to monitor DAG production by PLC-g. Fluorescently la-
it displays substantial colocalization with LAT (Figure S4).
The PKCq C1 domains are also recruited to the immuno-
logical synapse upon TCR activation (Figure S5; Monks
Photoactivation of T Cell Signaling Responses
Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc. 77
et al.,1997)and behavesimilarly to a previouslydescribed
reporter for DAG (Spitaler et al., 2006).
The translocation of fluorescent probes like these to the
cell membrane is monitored most efficiently and at the
highest resolution by TIRF microscopy (Codazzi et al.,
rectly above the imaging surface. Accordingly, we per-
formed subsequent experiments by using glass surfaces
coated with I-Ekcontaining NPE-MCC. Agonist pMHC
on these surfaces triggers the tight adhesion of the T cell
to the glass, MTOC polarization toward the surface, and
the secretion of cytokines. Surfaces containing photoacti-
vatable pMHC, however, induce only weak adherence by
5C.C7 T cells, and thus do not allow for a precise UV-trig-
gered ‘‘start’’ time for pMHC-TCR interaction. To circum-
tration of antibody against the class I MHC protein H2-Kk.
5C.C7 T cells, which express H2-Kk, adhere tightly to
surfaces containing this antibody without becoming
activated (Movies S2 and S3), and this adhesion enables
a uniform initiation of pMHC-TCR contact with UV light.
In a typical experiment, glass coverslips were coated
with NPE-MCCI–Ekmolecules diluted 10-fold into I-Ek
containing a null peptide derived from murine hemoglobin
(residues 64–76, hereafter called Hb) (Evavold et al.,
1992). Dilution of photoactivatable pMHC into null pMHC
was performed in order to more closely mimic physiolog-
ical conditions, where genuine agonist ligands are typi-
cally encountered in the context of an excess of nonagon-
ist pMHC containing endogenous peptides. 5C.C7 T cell
blasts were attached to these surfaces and subjected to
brief UV laser exposures. Subsequent signaling re-
sponses were monitored with TIRF microscopy (C1-YFP
and Grb2-GFP probes) or epifluorescence (Fluo-4).
The Fluo-4 responses on glass surfaces (Figure 2 and
Movie S2) were similar to those observed in T cell-APC
conjugates (Figures 1B and 1C), indicating that the glass
surfaces effectively mimic an APC for early signaling ki-
netics. Photoactivation induced the relatively uniform in-
crease of C1-YFP fluorescence across the membrane in
contact with the glass, with an offset time similar to that
of the Fluo-4 response (Figure 2). The more TCR proximal
Figure 1. Photoactivation of T Cell Signaling
(A) The NPE-MCC peptide. K99 modified with NPE is indicated in red, and unmodified K99 in green.
(B and C) Calcium responses to NPE-MCC photoactivation in T cell-APC conjugates. APCs (CH27 B cells) pulsed with NPE-MCC were mixed with
T cell blasts loaded with Fluo-4. (B) shows a time-lapse montage of a representative T cell-APC conjugate shown in both monochrome (top) and
pseudocolor (bottom), with UV irradiation at 2.5 s. (C) shows normalized fluorescence intensity of the T cell in (B) graphed as a function of time.
Red lines denote offset time. The average offset time ± SEM is derived from 11 cell couples.
78 Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc.
Photoactivation of T Cell Signaling Responses
Grb2-GFP probe, however, displayed qualitatively differ-
ent behavior (Figure 2A), forming fluorescent puncta that
were similar in appearance to the microclusters observed
in previous studies (Bunnell et al., 2002; Campi et al.,
2005; Douglass and Vale, 2005; Yokosuka et al., 2005).
Quantification of this microcluster formation (see Experi-
mental Procedures) revealed that the offset time between
photoactivation and Grb2-GFP recruitment was signifi-
cantly shorter than the delays for both C1-YFP transloca-
tion and calcium flux (Figures 2B and 2C, p < 0.0001,
t test), consistent with the fact that LAT phosphorylation
occurs further upstream. The average offset times pre-
sented in Figure 2C were each derived from data col-
lected from one preparation of 5C.C7 blasts on a single
day. Although these values varied somewhat for different
preparations of blasts (4–6 s for Grb2-GFP and 6–9 s for
C1-YFP and Fluo-4), the order of responses (i.e., Grb2-
GFP before C1-YFP and Fluo-4) remained consistent.
Importantly, signaling responses were not observed after
UV exposure on surfaces lacking NPE-MCC–I-Ek(Fig-
ure S6). Thus, the photoactivation of cognate pMHC
results in a substantial progression through the TCR
signaling cascade in less than 10 s.
Offset Times Represent Signaling Delays
In order to verify that these offset times reflected the rates
of intracellular signaling, it was necessary to determine
whether an appreciable amount of time was needed for
TCR binding to pMHC. If it were, then lower densities of
photoactivatable agonist would produce longer offset
times. Accordingly, we measured offset times on surfaces
of varying NPE-MCC–I-Ekdensity. The C1-YFP and Grb2-
GFP delays did not change over an order of magnitude
range of NPE-MCC–I-Ekconcentrations (Figure 3A), sug-
gesting that pMHC-TCR binding occurred very quickly af-
ter UV irradiation and that the offset times we measured
can be attributed predominantly to intracellular signaling.
Interestingly, the offset time for calcium responses did
show a weak inverse correlation with agonist concentra-
tion (Figure 3B). Given that the delays for LAT phosphory-
lation and DAG production, which both occur upstream of
calcium flux, did not change with agonist density, the con-
centration dependence for calcium signals cannot be at-
tributed to an increased time for pMHC binding. Rather,
these results imply that there is a signaling step whose
rate is sensitive to the density of activated pMHC-TCR
complexes between PLC-g and calcium elevation.
CD4-Blocking Antibodies Inhibit LAT
Phosphorylation and Delay Calcium Flux
Having established the basic kinetic parameters of early
TCR signaling steps, we next performed perturbation
studies in order to shed light on the mechanism. The cor-
eceptor CD4 enhances T cell responses to antigen 10- to
100-fold (Hampl et al., 1997; Vidal et al., 1999) both by re-
cruiting the Src kinase Lck into the TCR complex (Shaw
et al., 1989; Turner et al., 1990) and by contributing to re-
ceptor clustering (Balamuth et al., 2004). CD4-blocking
Figure 2. Kinetics of Early TCR Signaling
5C.C7 T cell blasts were stimulated by UV
irradiation on glass surfaces containing NPE-
MCC–I-Ekand Hb–I-Ek(1:10 ratio).
(A) Time-lapse montages of representative
Grb2-GFP, C1-YFP, and Fluo-4 responses
(UV irradiation at 0 s). The Fluo-4 response
was imaged in epifluorescence mode, and
the others were imaged in TIRF mode.
by normalized fluorescence intensity for Fluo-4
(green) and C1-YFP (pink) or by the number of
fluorescent microclusters for Grb2-GFP (blue).
(C) Average offset times for Grb2-GFP, C1-
YFP, and Fluo-4 responses. Each bar repre-
sents data collected from more than ten cells.
Error bars denote SEM.
Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc. 79
Photoactivation of T Cell Signaling Responses
antibodies have also been shown to increase the number
of antigenic peptides required to stimulate T cell blasts
(Irvine et al., 2002).
Consistent with this previous data, the magnitude of
Grb2-GFP responses after UV irradiation was strongly re-
duced in the presence of 20 mg/mL CD4-blocking anti-
body (Figure 4A), a concentration that effectively blocks
CD4-pMHC interactions in cell-cell conjugates (Irvine
et al., 2002; Li et al., 2004). However, the offset time for
the Grb2-GFP response did not change significantly rela-
tive to untreated controls (Figure 4B, p = 0.68, t test). In
contrast, the offset time for calcium flux increased 4- to
5-fold in the presence of CD4 antibodies (Figure 4C).
This effect was specific for CD4 because antibodies
against other T cell surface proteins had no effect on the
calcium response (data not shown). These results demon-
strate that blocking CD4 reduces the intensity of early LAT
phosphorylation and augments the delay prior to calcium
flux, and such findings establish a correlation between the
magnitude of upstream events and the speed of down-
Localized Signaling and Cytoskeletal Polarization
at the Region of Photoactivation
By altering the microscope optics (Figure S2), we can
confine most of the intensity of the photoactivating laser
to areas as small as 3 mm in diameter (Figure S7). This
has allowed us to examine the dynamics of localized
UV irradiation of a subcellular region beneath a T cell led
to an increase in the fluorescence intensity of Grb2-GFP
microclusters within the membrane apposed to that re-
gion, with little fluorescence increase elsewhere in the
cell (Figures 5A and 5D; Movie S7). Similarly, the C1-
YFP probe also translocated preferentially to the exposed
region (Figures 5B and 5D; Movie S8). In contrast, Fluo-4
fluorescence increased uniformly throughout the cell after
subcellular UV irradiation, indicating that calcium flux was
not localized (Figures 5C and 5D; Movie S9).
Localized TCR signaling is important for immunological
synapse formation and cytoskeletal polarization (Davis
mine whether the local signaling induced by photoactiva-
tion could drive cell polarization, we prepared T cell blasts
expressing a-tubulin fused to YFP. The MTOC was ap-
parent in the vast majority of these T cells and could be
imaged with TIRF illumination after cell attachment to
NPE-MCC-I-Ek-containing surfaces, suggesting that cell
spreading onto the glass surface brought the MTOC
close to the cell membrane.
location to the activated zone in greater than 80% of cells
tested (Figure 6A; Movie S10). This movement was typi-
62 ± 9 s, 19 cells) during which the MTOC remained static;
this was followed by the translocation of the MTOC to the
irradiated region (average duration 51 ± 7 s, 19 cells). This
biphasic pattern suggests that an obligatory delay exists
during which the T cell membrane and cytoskeleton are
primed for MTOC polarization.
Recent studies have demonstrated that T cell polariza-
tion can distinguish between APCs bearing different
amounts of antigen (Depoil et al., 2005). The ability to
trigger MTOC reorientation to localized agonist pMHC
provided us with an opportunity to confirm and extend
these results. Accordingly, two subcellular UV-laser
Figure 3. Effects of Agonist Density on
the Speed of Early Responses
5C.C7 T cell blasts were UV irradiated on glass
surfaces containing NPE-MCC–I-Ekdiluted
into increasing amounts of Hb–I-Ek. Average
offset times for Grb2-GFP ([A], blue diamonds),
C1-YFP ([A], pink squares), and Fluo-4 (B) re-
sponses are plotted as a function of fold dilu-
nine cells. Error bars denote SEM.
Figure 4. Early Responses Are Inhibited
by CD4 Blockade
5C.C7 T cell blasts were UV irradiated on glass
surfaces containing NPE-MCC–I-Ekand Hb–
(A and B) The average magnitude (A) and offset
time (B) of Grb2-GFP responses in the pres-
ence or absence of 20 mg/mL CD4-blocking
either in the presence or in the absence of 20
mg/mL GK1.5. Graphs show representative
experiments where each value was derived
from at least nine cells. Error bars denote SEM.
80 Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc.
Photoactivation of T Cell Signaling Responses
pulses of different intensity were used to establish com-
peting zones of agonist pMHC beneath each T cell. A
lower-intensity UV pulse would produce fewer agonist
pMHC molecules because of the low quantum yield ex-
pected for NPE-MCC photoactivation (McCray and Tren-
tham, 1989). In a first set of experiments, both UV pulses
were applied before any movement took place (typically
20–30 s between pulses), and subsequent MTOC translo-
cation to either irradiated region was monitored over
5 min (Figure 6B, top). In almost every case (14 out of
15 cells), the MTOC moved to the region that received
the higher-intensity UV pulse, irrespective of whether the
Figure 5. Localization of Grb2-GFP and C1-YFP Responses, but Not Calcium Flux
5C.C7 T cell blasts attached to glass surfaces containing NPE-MCC–I-Ekand Hb–I-Ek(1:10 ratio) were UV irradiated in a subcellular region. Repre-
sentative Grb2-GFP (A), C1-YFP (B), and Fluo-4 (C) responses are shown. Top portions of (A)–(C) show timelapse montages of individual responses.
Yellow ovals denote the UV irradiated region and are shown at the approximate moment of agonist activation and also shown at the final time point.
Bottomportionsshow normalized fluorescenceintensity inside(blue) and outside(pink)theirradiated region graphed asafunction oftime. Grb2-GFP
and C1-YFP responses were imaged in TIRF mode, and Fluo-4 was imaged in epifluorescence mode. (D) shows average maximal Grb2-GFP,
C1-YFP, and Fluo-4 responses from inside and outside the irradiated region quantified by fluorescence intensity. Each value is the average from
at least ten cells. Error bars denote SEM.
Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc. 81
Photoactivation of T Cell Signaling Responses
high- or low-intensity UV region was established first (Ta-
ble 1). If two UV pulses of equal intensity were used, the
MTOC either polarized toward one of the two irradiated
regions or did not reorient at all.
We performed a second set of experiments to deter-
mine whether the ability of T cells to discriminate between
different amounts oflocalizedTCRsignaling changesover
time. T cells were first allowed to polarize toward a single
photoactivated region for up to 5 min. Then, a second
pulse of higher, lower, or equivalent intensity was applied,
and MTOC reorientation to the second region was as-
sessed over a 5 min time span (Figure 6B, bottom). Con-
sistent translocation to the second region was observed
only if it received a higher intensity UV laser pulse than
the first region (Table 2). Thus, T cells can accurately inte-
grate localized responses for a substantial period of time
after the initial TCR engagement and use this information
to compare regions of activated membrane.
Grb2, C1, and Fluo-4 Responses Display Different
The ability to trigger TCR activation in subcellular regions
provided an opportunity to probe the levels at which TCR
Figure 6. MTOC Translocation to the
Region of TCR Activation
5C.C7 blasts expressing a-tubulin-YFP were
attached to glass surfaces containing NPE-
MCC–I-Ekand Hb–I-Ek(1:10 ratio) and irradi-
ated in a subcellular region.
(A) The top portion shows a time-lapse mon-
tage of a representative polarization response.
The red arrow indicates the initial position of
the MTOC. The bottom portion shows the
root mean square distance (RMSD) of the
MTOC from the center of the irradiated region
plotted as a function of time. The yellow line
denotes the UV exposure.
(B) MTOC polarization in response to two
subcellular stimulations. The top series show
a time-lapse montage of a representative T
cell that received a low-intensity UV pulse
and a high-intensity UV pulse before MTOC
translocation. The bottom two series show
time-lapse montages of a representative T
cell that was allowed to undergo MTOC polar-
ization toward a low-intensity stimulus (top)
and that was then subjected to high-intensity
irradiation (bottom) in a different region. Irradi-
ated regions are indicated by yellow or pink
circles at the approximate moment of agonist
activation and also at the final time point.
Complete results are shown in Table 1.
Table 1. MTOC Translocation to Photoactivated Regions: Competing Regions Established prior to Movement
MTOC Translocation to .
Intensity Region (%)First Pulse Second Pulse First Region Second RegionNeither
Low High0 100 100
82 Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc.
Photoactivation of T Cell Signaling Responses
signaling is subject to feedback inhibition and to deter-
mine the speed of this densensitization process.
We used a restimulation protocol to investigate feed-
back inhibition of early signals. T cell blasts attached to
surfaces containing NPE-MCC–I-Ekwere subjected to
subcellular irradiation in a small region and then restimu-
lated with a UV pulse of equal intensity at a distal location.
The time gap between the first and second UV pulses was
typically between 1 and 2 min. Technical constraints pre-
vented us from shortening this gap to less than 30 s,
whereas extending the gap to 5 min did not alter the de-
sensitization properties of any of the observed reporter
The recruitment of the C1-YFP reporter to the mem-
brane was dramatically reduced upon secondary stimula-
tion (Figures 7A and 7C, blue bars; Movies S11 and S12).
This desensitization was fast, occurring in less than 2 min,
and acted at a distance, downregulating distal parts of
the plasma membrane. The desensitization of calcium
signals was even more profound, with little or no observ-
able secondary responses (Figure S8; Movies S13 and
S14). In contrast, secondary Grb2-GFP responses were
comparable in intensity to their primary counterparts,
consistent with an absence of feedback inhibition (Fig-
ures 7B and 7D, blue bars; Movies S15 and S16). Occa-
sionally, a modest degree of Grb2-GFP desensitization
was observed, but this was always substantially weaker
than the corresponding inhibition of the C1-YFP and
To determine whether the desensitization properties of
either response could be modulated by the intensity of
stimulation, we next examined C1-YFP and Grb2-GFP
recruitment in cells subjected to two sequential UV-laser
pulses of different intensities (either low intensity fol-
lowed by high intensity or vice versa). The amount of
Grb2-GFP recruitment to the membrane in these experi-
ments predominantly reflected the intensity of UV irradi-
ation, with little dependence upon the order of stimula-
tion (Figure 7D, yellow and red bars). In contrast, the
order of stimulation had a strong effect on the intensity
of C1-YFP responses (Figure 7C, yellow and red bars).
High-intensity primary stimulation profoundly inhibited
secondary responses to a low-intensity UV pulse. More
surprisingly, however, low-intensity primary stimulation
was as effective as high-intensity primary stimulation at
inhibiting secondary C1-YFP responses to a high-intensity
UV pulse (compare second blue bar to second yellow
bar in Figure 7C). This suggested that the degree of
C1-YFP desensitization was not directly proportional
to the strength of the primary stimulus, but rather de-
pended on the presence or absence of the primary
Thus, DAG production and calcium flux were strongly
desensitized by prior stimulation, whereas the more TCR
proximal LAT-phosphorylation step remained close to
full strength. Taken together, the data suggest that these
three events play distinct roles within the TCR signaling
network, with DAG and calcium acting as an incidence
detector for antigen and LAT phosphorylation involved in
Thedynamicproperties ofintracellularsignaling pathways
have been difficult to define with standard approaches. In
this study, we used a photoactivatable pMHC reagent to
investigate TCR signaling dynamics. This work has pro-
vided new insights into signaling kinetics, localization,
The ability to define the moment of agonist engage-
early TCR signaling events with unprecedented precision.
We consistently observed an offset time of approximately
4 s for LAT phosphorylation and 6–7 s for DAG production
and calcium flux. Previous videomicroscopy studies have
noted the rapidity of TCR signaling (Bunnell et al., 2002;
Delon et al., 1998; Harriague and Bismuth, 2002; Negu-
lescu et al., 1996; Wulfing et al., 1997) but were limited
in their ability to accurately measure offset times because
of ambiguities about precisely when antigen recognition
occurred as well as much slower sampling rates (typically
15–30 s intervals).
Other studies attempted to establish the moment of
TCR triggering by forcing together a T cell and a stimula-
tory glass surface at a specified time (Patrick et al.,
2000; Wei et al., 1999). We feel that this approach overes-
timates signaling delays by incorporating into the mea-
surement the additional time required for the close appo-
sition of the T cell with the activating surface. Indeed, the
offset times reported in this previous work are substan-
tially longer than what we observed here. In our system,
the agonist is photoactivated only after T cells have ad-
hered and spread onto the glass, thereby allowing us to
separate the time interval required for cell-surface contact
formation from the interval that encompasses the
Table 2. MTOC Translocation to Photoactivated Regions: Movement to First Region before Second Photoactivation
Laser Intensity MTOC Translocation to Second Region
Chooses Higher Intensity Region (%)First Pulse Second PulseYes No
High Low05 100
Low High83 73
Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc. 83
Photoactivation of T Cell Signaling Responses
signaling response. Furthermore, the fact that the offset
times for the Grb2-GFP and C1-YFP responses did not
change with ligand density suggests that TCR-pMHC en-
gagement is relatively rapid and that our measurements
predominantly reflect intracellular signaling times.
T cells are able to identify small numbers of agonist
ligands in the context of a vast excess of endogenous
pMHC molecules. This remarkable quality ispoorly under-
stood, and it has been difficult to evaluate the predomi-
nantly theoretical models proposed to explain it. An accu-
rate knowledge of the temporal gaps between steps in the
TCR signaling cascade is important because it puts
constraints on these models. For instance, the kinetic-
proofreading hypothesis suggests that the time delays
imposed by a multistep signaling pathway can be used
to identify rare agonist ligands in the presence of excess
endogenous pMHC (McKeithan, 1995). For achieving op-
timal discrimination, the length of this ‘‘proofreading
delay’’ would need to be several times longer than the
half-life of a typical agonist ligand (?1 s at 37?C;
Figure 7. Desensitization of the C1-YFP and Grb2-GFP Responses
5C.C7 blasts attached to glass surfaces containing NPE-MCC–I-Ekand Hb–I-Ek(1:10 ratio) were irradiated in a subcellular region and then irradiated
again in a different region.
(A and B) Representative desensitization experiments for C1-YFP (A) and Grb2-GFP (B). On the left are time-lapse montages of responses to each of
two successive high-intensity UV pulses. Yellow ovals denote the UV-irradiated region and are shown at the approximate moment of agonist acti-
vation and also shown at the final time point. On the right, normalized fluorescence intensity inside the irradiated region is graphed as a function
of time for both the first (blue) and second (pink) stimulations.
(C andD)Averagemaximal C1-YFP(C)and Grb2-GFP(D)responses fromdesensitizationexperimentsquantifiedbyfluorescence intensity. Thethree
bars), and (3) high followed by low (red bars). Red bars are averages from at least five cells, whereas yellow and blue bars are averages from at least
ten cells. Error bars denote SEM.
84 Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc.
Photoactivation of T Cell Signaling Responses
Krogsgaard et al., 2003) and would also need to be inde-
pendent of ligand density in order to preclude T cell re-
sponses to high concentrations of nonspecific ligands. It
is intriguing that the offset times we measured for LAT
phosphorylation and DAG production both satisfy these
criteria. Although it is unclear which delay plays the
proofreading role in this system, or whether they both
participate, our results suggest that T cells discriminate
between genuine agonists and low-affinity binders within
the first 6–7 s, by using signaling steps upstream of
The ability to photoactivate micron-scale regions of
pMHC beneath T cells allowed us to investigate localized
signaling with high spatial resolution. The tight restriction
of LAT phosphorylation and DAG production to the acti-
vated region is most likely to result from the rapid metab-
olism of phospho-LAT and DAG by cellular phosphatases
and diacyglycerol kinases, respectively (Schlessinger,
2000; Sanjuan et al., 2001; Zhong et al., 2002). TCR acti-
vation is also known to restrict the diffusion of LAT within
the membrane (Douglass and Vale, 2005), and this restric-
tion presumably enhanced the localization of phospho-
LAT that we observed. The fact that calcium responses
iments indicates that localized signals propagate away
from regions of stimulated membrane downstream of
PLC-g, either as IP3or perhaps as Ca2+itself.
The offset times for LAT phosphorylation and DAG
production did not change over a 10-fold range of NPE-
MCC–I-Ekdensity. In contrast, the delay prior to calcium
flux increased with agonist dilution; this relationship has
been observed for calcium signaling in other cell types
(Randriamampita and Trautmann, 1989; Wang et al.,
1995). Interestingly, CD4 blockade also increased the off-
set time for calcium flux and altered the magnitude but not
the speed of LAT phosphorylation. These results, when
taken together with our studies of signal localization, sug-
gest that TCR signaling upstream of calcium flux takes
place in localized units whose kinetics are independent
of the density of activated TCRs. Subsequently, signals
from each of these upstream modules act cooperatively
to elicit a generalized calcium response. If calcium flux
requires a threshold level of a second messenger like
IP3, one would expect that changing the number of active
pMHC-TCR complexes (e.g., by dilution) or changing the
activity of each pMHC-TCR complex (e.g., by CD4 block-
ade) would modulate the offset time for the response; this
modulation is precisely what we observed.
The accumulation of Grb2-GFP into microclusters
within seconds of TCR triggering is consistent with the
idea that they play a central role in early TCR signaling
steps. Interestingly, a small number of Grb2-GFP micro-
clusters show no overlap with fluorescently labeled LAT
(Figure S4) and are present in T cells attached to surfaces
containing null pMHC alone or even no pMHC (data not
shown). We have attributed the formation of non-LAT-
associated microclusters to the involvement of Grb2 in
cellular-adhesion pathways (e.g., focal adhesions). It will
be interesting to determine whether the speed of TCR
signaling isenhanced inany way bypreexisting adhesions
of this kind.
Negative feedback is thought to play an important role
in modulating TCR-mediated signals. By sequentially acti-
vating small, distinct regions of pMHC beneath an individ-
ual T cell, we were able to examine the desensitization of
early signaling responses within minutes of primary stimu-
tion and calcium flux were strongly inhibited after primary
stimulation, whereas LAT phosphorylation was not. At this
with the developmental stage of the T cell or with the
context of antigen presentation. Nevertheless, our results
suggest that DAG production and calcium flux play differ-
ent signal-processing roles than LAT phosphorylation:
DAGand calcium actasanincidence detector for antigen,
whereas phospho-LAT appears to be involved in signal
integration over time.
It is likely that both incidence detection and signal inte-
gration are important for shaping T cell responses. Inci-
dence detectors may be required to delineate the primary
encounter with antigen; this encounter differs from subse-
quent recognition events by inducing a ‘‘stop signal’’ that
arrests T cell motility and triggers the formation of a tight
T cell-APC contact (Dustin et al., 1997). Calcium elevation
and presumably PLC-g activity are required for the mor-
phological changes that stabilize the cell-cell interface,
facilitating the comprehensive scanning of the APC sur-
face (Kuhne et al., 2003; Negulescu et al., 1996). Likewise,
previous work has demonstrated that signal integration
over a period of hours is required for optimal T cell activa-
tion in vitro (Huppa et al., 2003). Furthermore, the ability of
T cells to interact sequentially with multiple DCs in vivo
(Mempel et al., 2004) implies that they are capable of an-
tigen recognition well after initial TCR engagement. Our
results provide an explanation for how T cells continue
to receive and integrate additional signals well after their
initial encounter with antigen. The selective desensitiza-
tion of a branch of the signaling network allows the T cell
to qualitatively change the signaling output of the TCR.
Ouranalysesof MTOCmovement inresponseto photo-
activation show that signal integration also plays a critical
role in shaping cell polarity. When presented with two
toward the region of higher agonist density, in accord with
previous observations (Depoil et al., 2005). These results
demonstrate that T cells compare locally integrated TCR
signals in order to establish the appropriate cellular orien-
tation. They also provide insight into the mechanism of
MTOC translocation, especially in light of the C1-YFP-
and Grb2-GFP-desensitization experiments. The obser-
vation of MTOC movement from a region of low agonist
density to a new region of high agonist density, a situation
where a robust C1-YFP response is lacking, suggests that
strong DAG production is not essential for polarization. In
contrast, MTOC movement does correlate well with Grb2-
GFP responses, consistent with the notion that LAT phos-
phorylation is involved in the integration of TCR signaling.
However, given the rapid dephosphorylation of LAT after
Immunity 27, 76–88, July 2007 ª2007 Elsevier Inc. 85
Photoactivation of T Cell Signaling Responses
TCR activation (Zhang et al., 1998), it is likely that any sig-
nal integration represented by phospho-LAT would need
to be translated quickly into a more stable protein or lipid
modification that could direct the MTOC polarization
response over a longer timescale.
The temporal control afforded by photoactivation has
been exploited in the past to study the kinetics of sec-
larization, and nuclear translocation (Adams and Tsien,
subsecond time resolution with micron-scale spatial reso-
lution. In principle, this approach can be applied to study
a variety of transmembrane-receptor signaling networks.
The spatial and temporal parameters that characterize
these systems remain, for the most part, unknown, and
pathways work but also how they interact with each other.
Stimulatory Glass Surfaces
The incorporation of NPE-MCC and Hb peptides into I-Ekand the sub-
sequent biotinylation of pMHC complexes was performed as previ-
ously described (Boniface et al., 1999). The synthesis of NPE-MCC
is described in Figure S9.
Stimulatory surfaces were prepared with a protocol adapted from
Lillemeier et al. (2006). Eight-well chamber slides (Nalge Nunc Int.)
were coated with biotinylated poly-L-lysine (produced with NHS-bio-
tin, Pierce Biotechnology), washed with H2O, and allowed to dry.
They were then incubated in blocking buffer (HEPES-buffered saline
[HBS; pH 7.4], with 2% BSA) for 2 hr at room temperature; this was
followed by streptavidin (100 mg/mL in blocking buffer) for at least 4 hr.
After washing in HBS, the surfaces were incubated in the desired mix-
ture of biotinylated I-EK(typically a 1:10 ratio of NPE-MCC:Hb, total
in blocking buffer. They were washed again and left in HBS until use.
Approximately 8000 biotinylated proteins are immobilized per mm2
(Figure S10 and Table S1). Given the standard 10-fold dilution of
NPE-MCC–I-Ekinto Hb–I-Ek, combined with the low quantum yield
(<5%) expected for the photoactivation reaction, we anticipate that
UV irradiation generates ?80 agonist pMHC molecules per mm2in
a typical experiment.
Fluorescent-Protein Signaling Probes
Full-length murine Grb2 was amplified from a cDNA library derived
vector (kind gift of W. Shaw) in frame with a C-terminal GFP tag. The
tandem C1 domains from murine PKCq (amino acids 160–281) were
amplified from the same cDNA library, fused to the N-terminus of
YFP, and cloned into a pIB2 retroviral expression vector. DNA encod-
ing human a-tubulin fused to YFP was purchased from Clontech and
cloned into pIB2.
The preparation of ecotropic retrovirus and the transduction of
5C.C7 T cell blasts were performed as described previously (Huse
et al., 2006). The Stanford University Committee on Animal Welfare
approved the protocols used for this study.
Imaging was performedwith T cell blasts 6–8 days after extraction. For
calcium-flux experiments, the T cells were loaded with either Fluo-
4AM or Fura-2AM (Molecular Probes) at 5 mM for 30 min. All Fluo-4
RPMI with 5% FCS and 10 mM HEPES) and allowed to adhere to the
coated chamber slides for ?10 min at 37?C before data collection.
Recordings were made with Metamorph software (Universal Imaging).
Fluo-4, Grb2-GFP, and C1-YFP responses were imaged at 100 ms
intervals for 30–60 s with either a 403 objective (for Fluo-4 responses)
or a 1003 TIRF objective (for Fluo-4 responses to subcellular stimula-
tion, and all Grb2-GFP and C1-YFP responses). a-tubulin responses
were imaged at 5 s intervals for 5 min with the 1003 objective. NPE-
MCC photoactivation was typically performed with 10 3 4 ns UV-laser
pulses over a period of 0.5 s. For MTOC polarization and desensitiza-
tion studies, 4 pulses over 0.5 s were used for ‘‘low-intensity’’ irradia-
For T cell-APC-conjugate experiments, CH27 B cells were pulsed
with 1 mM NPE-MCC for 4 hr at 37?C, washed with MIM, and mixed
with T cell blasts (2:1 APC:T). The conjugates were incubated in the
imaging chambers for ?15 min before data collection.
We quantified Fluo-4 responses by determining the average intensity
of a region within each cell as a function of time with Metamorph soft-
ware. We quantified C1-YFP translocation (both whole-cell and local-
ized stimulation) and Grb2-GFP translocation (localized stimulation
only) by recording the average intensity of the irradiated region after
thresholding with the ImageJ program (Wayne Rasband, NIH). The
quantification of Grb2-GFP responses by average intensity at
a whole-cell level was complicated by inhomogeneities in fluores-
cence increase. Thus, we quantified whole-cell Grb2-GFP responses
by counting fluorescent microclusters as a function of time. The iden-
tification and enumeration of microclusters was automated by adapta-
tion of a Matlab script from Blair and Dufresne based on IDL code from
Weeks(PhysicsDepartment,Emory University), Grier(Physics Depart-
Biomolecular Engineering, University of Pennsylvania).
All intensity profiles were background subtracted and normalized to
a set of frames taken just prior to photoactivation. The UV-irradiation
event was visible in the majority of experiments because of its added
excitation of the signaling probe. Offset time was defined as the time
between the start of the UV pulse and the point where the intensity
had increased to 15% of its maximal value in the recording. Maximal
response was defined as the highest average intensity value achieved
after photoactivation. All maximal response values, as well as rate
measurements for Grb2-GFP responses, were derived from data
that had been smoothed with a 1 s time window.
Ten figures, two tables, and sixteen movies are available at http://
We thank M. Bose and A. Sanchez for assistance with chemical syn-
thesis and G. Marriott for advice on photoactivation. We also thank
Q. Hu for help with CD4-blocking studies, J. Huppa for the a-tubulin-
YFP retrovirus, N. Prado for technical support, and other members
of theDavis lab for helpful discussions. M.H. wassupported byaGian-
niniFamily FoundationFellowship,L.O.K. wassupportedbyaNational
Defense Science and Engineering Graduate Fellowship, A.T.G. was
supported by a National Science Foundation Graduate Fellowship,
J.M.F. was supported by the Stanford Summer Research Program in
Biomedical Sciences, and M.S.K. was supported by a senior fellow-
ship from the Irvington Institute for Immunological Research. Addi-
tional support was provided by grants from the National Institutes of
Health and from the Howard Hughes Medical Institute to M.M.D.
Received: February 13, 2007
Revised: April 26, 2007
Accepted: May 15, 2007
Published online: July 12, 2007
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