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Accumulation of Dynamic Catch Bonds Betwwen TCR and Agonist Peptide-MHC Triggers T Cell Signaling
Accumulation of Dynamic Catch Bonds Betwwen TCR and Agonist Peptide-MHC Triggers T Cell Signaling
*Correspondence: cheng.zhu@bme.gatech.edu
http://dx.doi.org/10.1016/j.cell.2014.02.053
Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc. 357
concurrent Ca2+ imaging, we simultaneously observed Ca2+
signals in live T cells induced by force on the TCR. This allowed
us to delineate the relationships among force attributes, bond
characteristics, and signaling outcomes, thereby defining the
role of force at the earliest protein interaction to provide insights
on T cell antigen recognition, discrimination, activation, and
antagonism.
RESULTS
358 Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc.
Figure 2. TCR Forms an Agonist-Specific Catch-Slip Bond
(A) Lifetimes of bonds of OT1 T cells with probe beads coated with indicated pMHCs at 0 (white) and 10 (black) pN.
(B and C) Lifetime versus force curves showing that OT1 TCR formed catch-slip bonds with progressively weaker agonists OVA (red open square), A2 (light blue
open triangle), and G4 (inverted magenta open triangle) (B) but slip-only bonds with antagonists R4 (blue open circle) and E1 (green open diamond) (C). Error bars
in (A)–(C) represent SEM.
(D) Force regulation of antigen discrimination, measured by the average lifetime ratio of TCR bonds with OVA to another peptide.
See also Figure S2.
soluble proteins by SPR (Alam et al., 1996) and with the predic- the fura-2 ratio rapidly increased to >150% of the initial baseline
tion of the original kinetic proofreading model (McKeithan, 1995). followed by decay (Figures 3A and 3C and Movie S4), and type b
Remarkably, this trend was reversed by a 10 pN force on the curves stayed near the baseline or gradually increased to
TCR-pMHC bond, such that the more stimulatory the peptide, modest levels (Figures 3B and 3D and Movie S5). To examine
the longer the bond lifetime (Figure 2A, close bar). This occurs how various attributes of force impact T cell triggering, we
because agonist pMHCs form catch-slip bonds (Marshall et al., analyzed their correlations with the maximal percent Ca2+ in-
2003) with the TCR with biphasic bond lifetimes that first in- crease. The area under the Ca2+ curve provided another metric
crease, reach a maximum, and then decrease with increasing that correlated well with the maximal response (Figure S3B).
force (Figures 2B and S2A–S2C). Interestingly, the more stimula- Note that our experiments differ from previous studies (Huse
tory the peptide, the more pronounced the catch bond, which re- et al., 2007; Purbhoo et al., 2004) in that Ca2+ was induced by
quires a greater force to produce the maximal lifetime that also is intermittent single TCR-pMHC bonds formed infrequently and
longer (Figure 2B). By comparison, antagonist pMHCs form slip sequentially (Figures 3C and 3D) in the absence of any other
bonds (Marshall et al., 2003) with monotonically decreased bond receptor-ligand engagement. Although Ca2+ can certainly be
lifetimes as force increases (Figures 2C, S2D, and S2E). Thus induced under more favorable conditions (Kim et al., 2009; Li
at >5 pN force, TCR bonds with the most stimulatory agonist et al., 2010), our rigorous conditions allowed determination of
are the longest, whereas those with progressively less biologi- the minimum requirements for Ca2+ induction.
cally active ligands are progressively shorter (Figures 2B and Clamped cycles of forces on TCR via OVA resulted in robust
2C). Catch bonds are not isolated characteristics of the OT1 Ca2+ (Figure 4A, red open square). In contrast, force-ramp cycles
system; the 2C TCR also formed catch bonds with its agonist generated only baseline Ca2+ (Figure 4A, red solid square),
pMHC (Figure S2F). similar to the null pMHC control (Figure 4A, green open dia-
Our results reveal catch bonds as a mechanism for mechani- mond). Ca2+ triggering was peptide dependent; pulling via the
cal regulation of antigen discrimination by the TCR. The ratios weaker ligand G4 using the same force-clamp cycles failed to
of OT1 TCR-pMHC bond lifetime for the agonist OVA to those induce Ca2+ (Figure 4A, inverted light blue open triangle). The
for other peptides depend on force biphasically, with maxima triggering was also TCR specific; pulling another T cell surface
at 10 pN (Figure 2D). At this force level, the ratio for the strongest molecule (LFA-1) via an antibody did not trigger Ca2+, despite
(OVA) to the weakest (R4) pMHC becomes 57-fold greater than much longer bond lifetimes (Figure 4A, orange open circle, and
its value at zero force. Thus, force substantially increases the Figure S2G).
power of antigen discrimination. The triggering of Ca2+ by OVA, but not G4 (Figure 4A), might
be due to the longer lifetimes of TCR bonds with OVA than G4
Optimal Force Triggers Ca2+ by Prolonging TCR-pMHC under force. We therefore examined the correlation of signaling
Lifetime via Catch Bond and lifetime by measuring Ca2+ at 5, 10, and 20 pN with force-
To evaluate how the force-regulated pMHC dissociation from clamp cycles and at 0 pN with the thermal fluctuation cycles
and discrimination by the TCR relates to T cell signaling, we using either OVA or anti-TCR to form serial bonds with OT1
added a fluorescence optical path to our BFP (fBFP, Figure S3A) TCRs. The biphasic pattern of calcium versus force matched
and simultaneously measured bond lifetime and Ca2+ flux (Fig- the catch-slip bond lifetime versus force pattern for OVA (Fig-
ure 3). For each T cell, the force-clamp cycle was repeated for ure 4B), suggesting that the duration of force is important for
10 min; concurrently, intracellular Ca2+ was observed using T cell signaling. By comparison, the TCR-antibody interaction
fura-2 ratiometric imaging. Two types of Ca2+ signals were iden- behaved as a slip-only bond, not matching the calcium pattern
tified: type a curves have an initial latent phase of 1–3 min before that was also biphasic (Figure 4C). It is remarkable that similar
Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc. 359
Figure 3. Single-Cell Concurrent Measure-
ment of Ca2+ Flux and In Situ TCR-pMHC
Bond Kinetics
(A and B) Representative wide-field pseudocol-
ored images of two types of intracellular Ca2+
signals.
(C and D) Representative time courses of relative
fura-2 ratio of type a (C, magenta curve, left ordi-
nate) or type b (D, green curve, left ordinate)
intracellular Ca2+ signal synchronized with con-
current measurement of rupture (red open circle)
and lifetime (blue solid triangle) events and the
cumulative lifetime (blue dashed curve, right ordi-
nate). Note that there are no rupture events for (C).
A dashed-dot horizontal line indicates a 10 s
threshold of cumulative lifetime.
See also Figure S3 and Movies S4 and S5.
Ca2+ Correlates with Duration of pMHC Binding Events Ca2+ Best Correlates with Total Lifetime Accumulated in
We asked whether the variable calcium responses observed the First Minute
under identical experimental conditions were due to the hetero- Early binding events likely contributed most to Ca2+ onset, which
geneity in the cells or in the ways they were triggered (e.g., Fig- appeared mostly after 1 min of the 10 min experimental period.
ure 4B, red open square). Although the monoclonal OT1 TCR We therefore tested whether excluding the later events from
was used to reduce population heterogeneity, individual T cells the parameter estimations would improve their correlations.
were still likely triggered differently because TCR-pMHC interac- Reducing the window size (TL, Figure 6A), which excludes pro-
tions are stochastic at the single-bond level (see Figures 3C and gressively later events from the 10 min experiment, did not affect
3D). For any contact between a T cell and a pMHC-coated bead, the qualitative observations of the preceding section. Event-
it is impossible to know a priori whether a bond would form and counting parameters still correlated poorly with Ca2+, whereas
whether it would survive force ramping to the clamped level or its event duration parameters performed much better (Figures 6B
lifetime. Nevertheless, the probabilistic characteristics of a sto- and S4), with all Pearson coefficients reaching their maxima at
chastic sequence of such random events are governed by the TL < 100 s (Figure 6D). Interestingly, the cumulative bond lifetime
kinetic properties of the TCR-pMHC interaction, which can be now displays the best relationship to Ca2+ (Figures 6B, 6D, S4D,
extracted by analysis of an ensemble of these events (Figure 2). and S4E). This suggests that a long-lasting TCR-pMHC bond
The correlation between calcium and the force-regulated TCR- may be unnecessary for inducing calcium, as proposed by the
360 Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc.
tion parameters first increased, reached maxima at 60 s, and
then decreased (Figures 6E, S5D, and S5E). In the 60 s window,
all 13 cells with a cumulative lifetime >10 s fluxed type a Ca2+
(Figure 6B, highlighted center panel, C); these all occurred with
10 pN force clamp to generate the longest average bond lifetime
(Figure 2B). By comparison, only type b Ca2+ was observed in 34
T cells that were also engaged with OVA pMHC by 10 pN force
clamp but accumulated <10 s bond lifetime (Figure 6B, high-
lighted center panel, green open triangle). Thus, single-cell anal-
ysis reveals that Ca2+ response is digital (Altan-Bonnet and
Germain, 2005; Mukhopadhyay et al., 2013) with a threshold of
10 s total lifetime, an optimal 60 s window, and zero time delay
of accumulation for Ca2+ triggering. The optimal initial 60 s win-
dow suggests a kinetic mechanism for efficient antigen scan-
ning: T cells in vivo would lose interest in an APC after short initial
contact times (presumably from endogenous pMHCs on the
APC), whereas effective serial engagement with cognate antigen
that generated longer cumulative bond lifetimes would deliver a
timely stop signal for the T cell to appropriately act upon.
Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc. 361
Figure 5. Correlating Ca2+ Signals with Ki-
netic-Associated Statistics from Each Cell’s
600 s Event Sequence
(A–F) Percent increase of fura-2 ratios versus
number of adhesions Na (A), number of lifetimes
Nlt (B), adhesion frequency Pa (C), average life-
time < t > (D), longest lifetime tmax (E), and cumu-
lative lifetime Sti (F) of OT1 TCR bonds with
OVA at 0 (brown solid square), 5 (orange open
square), 10 (magenta open circle or green open
triangle for type a or b Ca2+, respectively) or 20
(inverted open red triangle) pN, or with G4 at 0
(yellow solid diamond) or 10 (light blue open dia-
mond) pN for each T cell calculated from the
binding events in the entire 10 min experimental
period. Dashed lines are linear fits to data. The
Pearson coefficient of the correlation (R) is indi-
cated in each subpanel.
(G) Summary of Pearson coefficients.
362 Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc.
Figure 6. Ca2+ Best Correlates with TCR-pMHC Bond Lifetimes Accumulated in the First Minute of Successive Force Applications
(A) Schematic of a sliding window (highlighted), starting at T0 with length TL and containing different binding events (red, no adhesion; green, rupture force; blue,
lifetime).
(B and C) Percent increase of fura-2 ratio versus cumulative lifetime of OT1 TCR bonds with OVA at 0 (brown solid square), 5 (orange open square), 10 (magenta
open circle or green open triangle, for type a or b, respectively), or 20 (inverted open red triangle) pN, or with G4 at 0 (yellow solid diamond) or 10 (light blue open
diamond) pN for each T cell accumulated in windows of indicated lengths and starting times. Dashed lines are linear fits to data, and the Pearson coefficients (R)
are indicated. The horizontal and vertical dotted lines in the subpanel with the best correlation (highlighted) denote the demarcation of types a (magenta open
circle) and b (brown solid square, orange open square, green open triangle, inverted open red triangle, yellow solid diamond, light blue open diamond) Ca2+ and
the 10 s threshold of cumulative lifetime for triggering type a Ca2+.
(D–F) Pearson coefficient analysis to search for the window for the kinetic parameters to achieve the best correlation with Ca2+.
(D) Maximal Pearson coefficients Rmax versus TL of the initial window (T0 = 0) within which the kinetic parameters best correlate with Ca2+.
(E) Pearson coefficient for cumulative lifetime versus sliding window size TL for the indicated starting times T0.
(F) Pearson coefficients for indicated kinetic parameters calculated in a 60 s sliding window versus its starting time T0. Different symbols in (D) and (F)
denote number of adhesions Na (purple open circle), number of lifetimes Nlt (magenta open square), adhesion frequency Pa (blue open triangle), average
lifetime < t > (green solid square), longest lifetime tmax (orange solid circle), and cumulative lifetime Sti (inverted red solid triangle).
See also Figures S4, S5, and S6.
Wang and Reinherz, 2012), receptor deformation/conforma- (Huang et al., 2010). The mechanosensor model (Wang and
tional changes (Ma et al., 2008), kinetic segregation (Davis and Reinherz, 2012) proposes that the ab TCR acts as a mechanical
van der Merwe, 2006), kinetic proofreading (Dushek et al., lever to tilt the Cb FG loop to push down on the CD3ε, causing a
2009; McKeithan, 1995), digital triggering (Altan-Bonnet and piston-like movement of the rigid CD3gε heterodimer (Sun et al.,
Germain, 2005), and serial engagement as recently modified 2001) for phosphorylation of the ITAMs by Lck (Xu et al., 2008;
Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc. 363
Figure 7. Long TCR-pMHC Bond Lifetime
Accumulated after Many Short Ones Fails
to Induce Ca2+
(A) Maximal cumulative lifetime max{Sti} of TCR
bonds with OVA at 0 (brown solid square), 5
(orange open square), 10 (magenta open circle or
green open triangle, for type a or b Ca2+, respec-
tively), or 20 (inverted red open triangle) pN or with
G4 at 0 (yellow solid diamond) or 10 pN (light blue
open diamond) for each T cell accumulated in a
60 s window versus its starting time T0.
(B) Percent increase of fura-2 ratio versus maximal
cumulative lifetime max{Sti} calculated for each cell
in its own 60 s window with starting time adjusted
to allow Sti to achieve maximum. Demarcation of
50% increase of fura-2 ratio and threshold of 10 s
cumulative lifetime (horizontal and vertical red
dotted lines) are used to segregate cells into three
groups. Group A cells accumulated >10 s lifetime in
the initial 60 s window and generated type a Ca2+
(magenta open circle). Group B cells accumulated
>10 s lifetime in later 60 s windows but generated
type b Ca2+ (light blue open square). Group C cells
accumulated <10 s lifetime and generated type b
Ca2+ (green open triangle). Dashed lines in (A) and
(B) are linear fits to data with R values indicated.
(C–F) Normalized lifetime histogram (C and E) and
number (left ordinate) and fraction (right ordinate) of
short lifetimes (D and F) before Ca2+ peak pooled
from cells in groups A (magenta) and B (cyan).
(E and F) are similar to (C) and (D) except that life-
times were collected from an initial 60 s window for
group A; for group B, the starting time was adjusted
for each cell to allow Sti to achieve maximum.
Error bars in (D) and (F) represent SEM. See also
Figure S7.
364 Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc.
or thermodynamic characteristics obtained under force-free of antigen pMHC also supports the extended serial engagement
conditions may or may not correspond to the triggered state. model (Huang et al., 2010). Our data suggest that the concentra-
Our data highlight the importance of force for TCR triggering tions of signaling intermediates may be key determinants. They
and suggest that signaling competent conformational and/or may build up progressively by sequential engagements of
flexibility changes may depend on force magnitude, particularly different TCRs and their gradual decay during intermission be-
TCR complex quaternary change as previously suggested tween two successive TCR-pMHC bonds may provide a mem-
(Wang and Reinherz, 2012). ory mechanism (Zarnitsyna et al., 2007). The observation that
The kinetic segregation model proposes that formation of Ca2+ signaling was digital—i.e., whether a cell fluxed Type a
small-sized TCR-pMHC bonds excludes large-sized phospha- Ca2+ depended on whether the cumulative lifetime reached the
tases (e.g., CD45), which tilts the homeostatic balance between 10 s threshold in the first minute of contacts—further suggests
kinase and phosphatase in favor of the already activated kinases that certain reaction steps are highly cooperative, possibly via
(e.g., Lck) to phosphorylate TCR/CD3 and initiates signaling a digital positive feedback mechanism (Altan-Bonnet and Ger-
(Davis and van der Merwe, 2006; van der Merwe and Dushek, main, 2005).
2011). The segregation of phosphatases from kinases is largely Based on previous in situ kinetic studies (Huang et al., 2010;
based on differential extracellular domain sizes of the molecular Huppa et al., 2010) and the present work, we propose the
species (Davis and van der Merwe, 2006). However, a recent following model for antigen discrimination and T cell activation.
study using a reconstituted system revealed that molecules During antigen recognition, the initial TCR-pMHC bond forma-
whose extracellular domains have similar sizes as the TCR tion is governed by cellular environment-dependent 2D on rates
were also excluded from the contact zone (James and Vale, with a broad range, providing the first-level antigen discrimina-
2012). It was suggested that the TCR-pMHC binding energy tion (Huang et al., 2010). The rapid zero-force 2D off rates allow
alone was sufficient to exclude other membrane proteins. Our TCRs to quickly scan various pMHC species displayed by an
finding of the TCR-pMHC catch bond may offer a biophysical APC. The pMHCs with high 2D affinities as measured at zero
explanation for these observations. As the T cell and APC are force will engage TCRs and pull on them. At this point, force
brought into close proximity by membrane fluctuation or active elicits agonist-specific catch bonds, which selectively prolong
motion, TCR-pMHC binding would prevent membrane separa- lifetimes. The high on rates allow the dissociated agonist pMHCs
tion by the same processes. This would result in a tensile force to quickly rebind and form serial bonds over time, thereby accu-
on the TCR and a compressive force on the nonbinding mole- mulating long bond lifetimes to induce Ca2+. In contrast, force in-
cules, including, but not limited to, bulky phosphatases. The duces slip bonds in response to less biologically active ligands,
compressive force would exclude nonengaged molecules from which further shorten lifetimes. The low on rates decrease the
the contact zone. The tensile force would elicit TCR catch bonds weak ligands’ chance to rebind or form serial bonds over time,
for antigen pMHC, which would increase bond lifetime and in thereby failing to accumulate sufficiently long lifetimes to acti-
turn strengthen the T cell-APC linkage. The first TCR-pMHC vate. Thus, force acts as an additional checkpoint to further
bond could then serve as a nucleation site for rapid serial forma- discriminate agonists from less biologically active ligands by ex-
tion of additional TCR-pMHC bonds enhanced by exclusion of panding the dynamic range of 2D off rates. As antagonism acti-
other molecules that do not bind. Therefore, the antigen-specific vates negative signals (Altan-Bonnet and Germain, 2005; Kilgore
catch bond may create a kinetic trap for accelerated engage- et al., 2003), future work could find that short-lived antagonist-
ment of additional TCRs with a built-in positive feedback for specific slip bonds favor phosphatase activity, which may corre-
segregation by ‘‘catchy’’ TCR-pMHC interactions. spond to different phosphorylation patterns identified for the
Our data are also relevant to the kinetic proofreading model, CD3 cytoplasmic tails (Sloan-Lancaster et al., 1994). The core-
which proposes that T cell activation requires completion of a ceptor CD8 or CD4 is also likely affected by force. Thus, our
series of reaction steps that proceed only when the TCR is data integrate force as a key concept in the paradigm of T cell
engaged by a pMHC but would be immediately and completely triggering.
reversed upon bond dissociation (McKeithan, 1995). The original
model has subsequently been refined and extended to accom- EXPERIMENTAL PROCEDURES
modate new experimental results (Dushek et al., 2009; Jansson,
2011). One recent extension allows the intermediate signaling Standard experimental procedures, including cells and proteins, preparation
of RBCs and beads, and measurement of molecular densities on the surfaces
states to persist when the pMHC dissociates and the proof-
of T cells and BFP beads are detailed in the Extended Experimental Proce-
reading steps to resume upon pMHC rebinding, thus incorpo- dures. New experimental methods and analyses are briefly summarized
rating the TCR-pMHC on rate as another important parameter below, with more details described in the Extended Experimental Procedures.
to the model (Dushek et al., 2009). This is supported by our
finding that cumulative, but not individual, bond lifetime best cor- Fluorescence Biomembrane Force Probe
relates Ca2+. Our data further indicate that rebinding of the disso- Our fBFPs were developed to simultaneously measure in situ kinetics of recep-
ciated pMHC to the same TCR, which the model assumes, may tor-ligand binding and intracellular signaling with an added fluorescence mod-
not be required, as in our experiment the serial bonds that trig- ule (Figure S3A) to the previous BFP (Chen et al., 2008a; Evans et al., 1995).
Briefly, the BFP used a pMHC- or antibody-coupled glass bead attached to
gered Ca2+ were likely formed between different TCRs due to
a micropipette-aspirated RBC as a pico-force probe (Figure 1A) whose posi-
the stochastic nature of single-bond formation with excessive tion was tracked by a high-speed camera at 1,600 frames per s and 3 nm
TCRs. Our observation that T cell signaling was induced by a displacement precision. To simultaneously record the fluorescent image, a
series of contacts with a surrogate APC expressing a low density fluorescence module was added with redesigned light paths (Figure S3A).
Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc. 365
BFP Single-Bond Kinetic Measurement and plotted versus their timings for all the cells to examine their correlation
Three types of BFP assays were used: force-ramp, force-clamp, and thermal with T0 (Figures 7A and S7B–S7F). Based on the thresholds of 50% fura-2 ratio
fluctuation assays. Briefly, in the force-ramp assay, TCR-pMHC bond was increase and 10 s cumulative lifetime (Figure 7B), the cells were classified into
loaded by retracting the T cell at a constant speed until rupture (Figure 1D). three categories: group A cells with both >50% fura-2 ratio increase and >10 s
In the force-clamp assay, the retracting T cell was clamped at a preset force cumulative lifetime, group B cells with <50% fura-2 ratio increase and >10 s
until bond rupture (Figure 1C). Bond lifetime was the duration of the clamped cumulative lifetime, and group C cells with <50% fura-2 ratio increase
phase. In the thermal fluctuation assay, the retraction stopped at zero force. and <10 s cumulative lifetime (Figure 7B). For both group A and B cells, we
Bond association and dissociation events were identified from reduction pooled lifetime events before the Ca2+ peak and analyzed them by histograms
and resumption, respectively, of bead fluctuations (Figures S1C and S1D). on a per cell basis (Figure 7C). Using the peak lifetime on the catch bond curve
Bond lifetime was measured as the duration from fluctuation reduction to (Figure 2B) for OT1 TCR interacting with OVA as the threshold, we counted the
resumption. numbers of short (<0.8 s) and long (>0.8 s) lifetimes for each cell in groups A
and B and presented them as mean ± SEM (Figure 7D).
Simultaneous 2D Kinetic Analysis and Ca2+ Imaging Alternatively, we collected and pooled lifetime events in the initial (i.e.,
To relate TCR-pMHC interaction characteristics to intracellular signaling at the T0 = 0 s) 60 s window for group A cells and in the 60 s window with maximal
single-cell level, we used our newly developed fBFP to simultaneously mea- cumulative lifetime for each cell in group B. These were analyzed by histogram
sure 2D kinetics and observe Ca2+ signaling in the same T cells loaded with on a per cell basis (Figure 7E), which were fitted with a double log-Gaussian
fura-2 by ratiometric imaging. The T cell was excited at 340 and 380 nm kernel function (Equation 1):
(two excitations separated by 200 ms in that order) every second, and the fluo-
X ðlntmi Þ2
rescent images were recorded. The experiment was performed at 37 C and
2
wi
N= pffiffiffiffiffiffi e 2s2
i (Equation 1)
continued for 10 min for each cell, and the kinetic-associated statistics were i=1 si 2p
analyzed.
where N is the number of lifetimes per cell and t is lifetime (Figure 7E).
Measuring Intracellular Ca2+ Level by Ratiometric Images mi ; si ; and wi (i = 1,2) are, respectively, the mean, the SD, and the weight of
Fluorescence images recorded by fBFP were imported into a customized the ith subpopulation. The weight wi represents an approximation to the total
Matlab (The MathWorks, Natick) program for fura-2 ratio analysis. Briefly, number of lifetimes of the ith subpopulation. The fraction of each subpopulation
two adjacent images from the two emission channels were aligned to eliminate is calculated by wi/(w1 + w2).
errors introduced by cyclic T cell movements. Fura-2 ratio was calculated by
dividing the aligned emission intensity excited by 340 nm by that by 380 nm SUPPLEMENTAL INFORMATION
pixel by pixel over the entire cell. A single fura-2 ratio value calculated by the
whole-cell average of fura-2 ratios over all pixels was plotted versus time Supplemental Information includes Extended Experimental Procedures, seven
(cf. Figures 3C and 3D). The percent increase of fura-2 ratio from the value figures, and five movies and can be found with this article online at http://dx.
at the beginning of the experiment to the maximal value was used to represent doi.org/10.1016/j.cell.2014.02.053.
the overall Ca2+ level (Figures 4, 5A–5F, 6B, 6C, and 7B).
AUTHOR CONTRIBUTIONS
Correlative Analysis of Ca2+ Level and Kinetic-Associated Statistics
of TCR-pMHC Interaction
B.L., W.C., and C.Z. designed experiments; B.L. and W.C. performed experi-
To identify the best predictive parameter for Ca2+ signaling, we performed
ments; B.L., W.C., and C.Z. analyzed the data; all authors contributed to
single-cell correlative analysis between the percent increase of fura-2 ratio
writing the paper.
and two groups of kinetic-associated statistics of TCR-pMHC interaction (Fig-
ures 3C and 3D). The first group is event-counting parameters associated with
ACKNOWLEDGMENTS
2D affinity, including the number of adhesions Na (Figure 5A), number of life-
times Nlt (Figure 5B), and adhesion frequency Pa (Figure 5C). The second
We thank the NIH Tetramer Core Facility at Emory University for providing
group is event duration parameters associated with force-regulated 2D off
pMHC monomers, John Altman for providing the H2-Kba3A2 construct,
rate (Figures 3C and 3D), including average lifetime < t > (Figure 5D), longest
Larissa Doudy for purifying T cells, Michelle Krogsgaard for advice on Ca2+
lifetime tmax (Figure 5E), and cumulative lifetime Sti (i.e., sum of all bond life-
imaging, Lining Ju for constructing the BFP-2, Jin-sung Hong for initial design
times, Figure 5F). Each parameter was calculated as a statistic from the bind-
of the Ca2+ imaging system, Yunfeng Chen for designing the temperature con-
ing events observed in a window (specified by the starting time T0 and window
trol unit, Qinghua Ji for designing the cell chamber, Li Gu for help on statistical
length TL) within the total 10 min experiment for each cell (Figure 6A).
analysis, and Rodger P. McEver, Ellis L. Reinherz, Michelle Krogsgaard, Hai-
We plotted the percent increase of fura-2 ratio against each parameter
Tao He, and Jeffrey A. Donnell for helpful comments. This work was supported
defined for a given time window (given T0 and TL) for all individual cells
by NIH grants AI38282 and GM096187 (to C.Z.) and NS071518 and AI096879
(n = 99) as a scattergram and analyzed their correlation with linear regression
(to B.D.E.). The authors declare no competing financial interests.
(Figures 5A–5F, 6B, and 6C). The goodness of fit was accessed with the
Pearson coefficient R (Figure 5G). For each parameter, the optimal window
was identified by selecting the highest R value for a combination of T0 (0, 30, Received: May 10, 2013
45, 60, 90, 120, 150, 240, and 450 s) and TL (30, 45, 60, 90, and 120 s) (Figures Revised: September 27, 2013
6D–6F). The kinetic parameter in the identified optimal window with the highest Accepted: February 21, 2014
R value is considered the best predictor of Ca2+ signaling. Published: April 10, 2014
366 Cell 157, 357–368, April 10, 2014 ª2014 Elsevier Inc.
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