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Enhanced T Lymphocyte Migration
Signaling Sets a Temporal Threshold for
Cross-Talk Between TCR and CCR7
Groettrup and Daniel F. Legler
Karin Schaeuble, Mark A. Hauser, Eva Singer, Marcus
2011; 187:5645-5652; Prepublished online 31
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The Journal of Immunology
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The Journal of Immunology
Cross-Talk Between TCR and CCR7 Signaling Sets a
Temporal Threshold for Enhanced T Lymphocyte
Karin Schaeuble,* Mark A. Hauser,* Eva Singer,†Marcus Groettrup,*,‡and
Daniel F. Legler*
Lymphocyte homing to, and motility within, lymph nodes is regulated by the chemokine receptor CCR7 and its two ligands
CCL19 and CCL21. There, lymphocytes are exposed to a number of extracellular stimuli that influence cellular functions and
determine the cell fate. In this study, we assessed the effect of TCR engagement on CCR7-mediated cell migration. We found that
long-term TCR triggering of freshly isolated human T cells through CD3/CD28 attenuated CCR7-driven chemotaxis, whereas
short-term activation significantly enhanced CCR7-mediated, but not CXCR4-mediated, migration efficiency. Short-term activa-
tion most prominently enhanced the migratory response of naive T cells of both CD4 and CD8 subsets. We identified distinct roles
for Src family kinases in modulating CCR7-mediated T cell migration. We provide evidence that Fyn, together with Ca2+-inde-
pendent protein kinase C isoforms, kept the migratory response of naive T cells toward CCL21 at a low level. In nonactivated
T cells, CCR7 triggering induced a Fyn-dependent phosphorylation of the inhibitory Tyr505 of Lck. Inhibiting Fyn in these
nonactivated T cells prevented the negative regulation of Lck and facilitated high CCR7-driven T cell chemotaxis. Moreover, we
found that the enhanced migration of short-term activated T cells was accompanied by a synergistic, Src-dependent activation of
the adaptor molecule linker for activation of T cells. Collectively, we characterize a cross-talk between the TCR and CCR7 and
provide mechanistic evidence that the activation status of T cells controls lymphocyte motility and sets a threshold for their
migratory response. The Journal of Immunology, 2011, 187: 5645–5652.
expression of diverse chemokines, which guide lymphocytes
through the lymphatic system by activating the corresponding
chemokine receptors (1). Under homeostatic conditions, naive
T cells circulate continuously between the blood and lymphoid
tissues, where they scan dendritic cells for their cognate Ag. T cell
homing to lymph nodes is mainly enabled by the chemokine re-
ceptor CCR7 and its two ligands CCL19 and CCL21 (2). CCR7
belongs to the family of G-protein coupled, seven-transmembrane
domain receptors and is expressed on thymocytes, naive lym-
phocytes, central memory T cells, regulatory T cells, NK cells,
and mature dendritic cells (2). Triggering of CCR7 with one of its
irected migration of lymphocytes is a prerequisite to
maintain the immune surveillance and to acquire an ef-
ficient immune response. Chemotaxis depends on local
ligands induces the onset of various signaling cascades, leading
to cell polarization and directional migration toward the ligand
source. Both CCR7 ligands are constitutively expressed mainly by
fibroblastic reticular cells distributed throughout the T cell zone
of secondary lymphoid organs (3, 4). Although CCR7 ligands are
also produced by human DCs, murine CCL21 is additionally
produced in high endothelial venules (3, 5, 6). CCR7-deficient
mice show strongly impaired lymphocyte homing accompanied
by severe alterations in the architecture of lymphoid organs,
manifested by a delayed induction of an adaptive immune re-
sponse (7). In addition to its ability to recruit lymphocytes into
lymphoid organs, CCR7 enables their intranodal motility (8, 9).
Because the number of naive T cells specific for a given Ag is
extremely low, naive T cells must scan large areas for the ap-
propriate APC to permit the initiation of an effective immune
response (10–12). Upon TCR engagement by its cognate peptide/
MHC complex expressed on APCs, T cells become activated and
start to proliferate, resulting in clonal expansion of Ag-specific
Before a T cell starts to proliferate, it enters a complex priming
phase, in which the T cell consistently interacts for short intervals
with several APCs and begins to upregulate early activation
markers, such as CD69 (13). Within this initial priming phase,
T cells stay highly motile. In a second step, the interaction of the
APC with the cognate T cell becomes prolonged, leading to the
production of IL-2 and IFN-g (13). During this priming phase,
naive T cells are exposed simultaneously to a number of different
stimuli, including signals arising from chemokine receptors and
the TCR, which together finally build up and tune a specific cel-
lular response. Hitherto, only a few studies have investigated the
interplay between chemokine receptor and TCR signaling. For
instance, TCR triggering was demonstrated to downregulate the
*Biotechnology Institute Thurgau, University of Konstanz, CH-8280 Kreuzlingen,
Switzerland;†Klinikum Konstanz, D-78464 Konstanz, Germany; and‡Division of
Immunology, Department of Biology, University of Konstanz, D-78457 Konstanz,
Received for publication June 22, 2011. Accepted for publication September 30,
This work was supported in part by research funding from the Swiss National Science
Foundation (SNF 31003A-127474/1), the Thurgauische Stiftung fu ¨r Wissenschaft
und Forschung, the Swiss State Secretariat for Education and Research, and the
Thurgauische Krebsliga. D.F.L. is a recipient of a career development award from the
Max Cloe ¨tta Foundation. M.A.H. is a scholarship holder of Research Training Group
1331 founded by the German Research Foundation.
Address correspondence and reprint requests to Prof. Daniel F. Legler, Biotechnology
Institute Thurgau, University of Konstanz, Unterseestrasse 47, CH-8280 Kreuzlingen,
Switzerland. E-mail address: firstname.lastname@example.org
Abbreviations used in this article: BimI, bisindolylmaleimide I; LAT, linker for
activation of T cells; PKC, protein kinase C isoform; SFK, Src family kinase; siRNA,
small interfering RNA.
by guest on June 18, 2013
surface expression of CXCR4, leading to a reduced migratory
response toward CXCL12 (14), and to attenuate CXCR3-in-
duced phosphorylation of ZAP70 and linker for activation of
T cells (LAT), resulting in reduced chemotaxis (15). These
phenomena might contribute to the stop-and-go model in which
TCR engagements during synapse formation deliver stop signals
to chemokine receptors, facilitating prolonged T cell–APC in-
teraction and efficient T cell activation (16). However, there is
recent evidence that TCR stimulation acts via Rac1 to reduce the
phosphorylation of ERM proteins required for cell polarization
and chemokine-independent random motility (17). Moreover,
the TCR-mediated stop signal can be reverted by CTLA-4 to
enhance random motility (18). These two studies suggested that
the proposed TCR stop signal does not directly interfere with
chemokine receptor-mediated migration. In contrast, CXCR4
was also shown to directly interact with the TCR upon chemo-
kine stimulation (19). The physical interaction between TCR
and CXCR4 resulted in prolonged ERK-1/2 phosphorylation,
strong transcriptional activity of AP-1, and enhanced cytokine
secretion. Furthermore, CXCR4 was shown to use ZAP70 to
enhance the migratory response of T cells (19–21). These find-
ings are in line with in vivo observations demonstrating that
tonic TCR signaling is required for normal naive CD4 T cell
motility (22), as well as for a sustained active migration of
tumor-infiltrating effector T cells through the tumor microen-
In this study, we investigated the effect of TCR engagement
through CD3/CD28 activation on CCR7-mediated cellular re-
sponse offreshly isolated primary human T cells. We demonstrated
that short-term activation of human naive T cells modified CCR7
signaling manifested by an enhanced chemotactic response toward
low concentrations of the CCR7 ligands CCL19 and CCL21. We
family kinases (SFKs). Altogether, our data propose a mechanism
that permits short-term activated T cells to move in a CCR7-
dependent manner within the lymph nodes to scan APCs.
Materials and Methods
Isolation and activation of primary human T cells
Human PBMCs were isolated from blood samples of healthy donors by
Ficoll density-gradient centrifugation. PBLs were obtained by privation of
monocytes with CD14+magnetic sorting (Miltenyi Biotec). CD3+T cells
were isolated from PBLs by negative selection using the Pan T Cell Iso-
lation Kit II (Miltenyi Biotec), yielding a purity of 90–97%. T cells were
permitted to recover from the isolation procedure by maintaining the cells
in RPMI 1640 medium supplemented with 0.5% human AB serum for 1 h.
For longer cultivation and migration assays, T cells were maintained in
RPMI 1640 medium supplemented with 10% FCS, 1% 2-ME, and 1%
nonessential amino acids. A total of 200 U/ml human IL-2 was added
at day 3 of cultivation. For short-term activation, T cells were plated on
dishes precoated with 5 mg/ml anti-CD3 (clone OKT3; Janssen-Cilag) and
2 mg/ml anti-CD28 Abs (clone CD28.2; eBioscience), for indicated time
points. A fraction of cells was kept under the same condition but on un-
Cell surface expression of proteins was determined by flow cytometry
(LSRII; BD Biosciences) using the following Abs: PE- or fluorescein-
labeled anti-CCR7 mAb (Clone 150503) and PE-labeled anti-CXCR4 mAb
(clone 12G5) from R&D Systems and PE-labeled anti-CD62L (clone
FMC46) and PE-labeled anti-CD69 (clone FN50) from AbD Serotec.
In several experiments, cells were treated with the inhibitory com-
pounds MG132 (Calbiochem), LY294002 (Cell Signaling), Y-27632 (Sigma-
Aldrich), SU6656 (Biaffin), PP2 (Calbiochem), bisindolylmaleimide I
(Sigma-Aldrich), or Go ¨6976 (Calbiochem).
Human CD3+T cells (1 3 105) were placed in Transwell filters (Corning
Costar), with 5 mm pore size, and allowed to migrate to the lower com-
partment containing the indicated concentrations of recombinant human
CCL19, CCL21, or CXCL12 (PeproTech) for 3 h. The numbers of input
and migrated cells were determined by flow cytometry (LSRII; BD Bio-
sciences) by gating on live, TOPRO-32(Molecular Probes) cells and
acquiring events for 90 s. Specific cell migration was calculated by
subtracting the number of cells migrating in the absence of chemokines.
Results are shown as percentage of specifically migrated cells relative to
the number of input cells.
T cell stimulation by chemokines and Western blot analysis
PBLs (1 3 106cells/assay point) were stimulated with 1–2 mg/ml che-
mokine for the indicated times at 37˚C and immediately lysed on ice in 1%
Nonidet P-40, 0.25% sodium desoxycholate, 0.4% N-dodecyl maltoside,
50 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, and 1 mM
NaF (pH 7.4), supplemented with protease- and phosphatase-inhibitor mix
(Roche). Total-cell lysates were separated on 10% SDS-PAGE and trans-
ferred to nitrocellulose membranes by wet blotting. Western blot analysis
was performed using the following Abs: anti–phospho-p44/42 MAPK
(pERK-1/2, Thr202/Tyr204), anti-p42 MAPK (ERK-2), anti-pLck (Y505),
anti-Fyn, anti-LAT, and anti-pLAT (Y191) (all from Cell Signaling
Technology). Anti-Lck (clone 3A5) was purchased from Millipore, and
anti-pFyn (pY528/c-Src pY530) was purchased from BD Bioscience. To
investigate the role of SFKs, cells were either preincubated for 30 min with
10 mM PP2 before TCR engagement or for 1 h with 5 mM SU6656 before
chemokine triggering, respectively.
Statistical analysis was performed using GraphPad InStat (GraphPad
Software, La Jolla, CA), applying one of the following algorithms as
specified in the figure legends: paired t test, repeated-measures ANOVA
with Bonferroni multiple comparison posttest, or one-way ANOVA with
Bonferroni multiple comparison posttest. Data displaying statistical anal-
yses were collected at least five times independently using lymphocytes
from different donors.
Modulation of CCR7-driven migration of primary human
T cells upon TCR triggering
The chemokines CCL19 and CCL21 recruit CCR7+naive and
central memory T cells to lymph nodes. Within this chemokine-
rich area of lymph nodes, naive T cells scan dendritic cells
searching for cognate Ags. It is poorly understood how T cells
integrate diverse signals derived from CCR7 and the TCR. To
gain new insights about the influence of T cell activation on the
chemotactic response toward lymphoid chemokines, we first ex-
amined CCR7-mediated migration of T cells at different activa-
tion stages. Therefore, we isolated CD3+human T cells from
healthy donors, cultured them for increasing periods of time on
anti-CD3/CD28 Ab-coated plates in the presence of human IL-2,
and assessed the migratory capacity in Transwell-migration
assays. Freshly isolated CD3+T cells migrated readily toward
CCL19, and CD3/CD28 triggering steadily attenuated the mi-
gration efficiency between days 2 and 6 (Fig. 1A), similar to a
reduced CXCL10/CXCL12-dependent (14, 15) and chemokine-
independent (17) T cell motility observed after TCR engagement.
In contrast, CCL21-mediated chemotactic responses were rather
poor in freshly isolated CD3+T cells, but they transiently in-
creased upon CD3/CD28 triggering, reaching the greatest
responses at day 2 before progressively declining down to base-
line levels (Fig. 1A). Similar results were observed in PBLs
cultured in the presence of IL-2 alone (5). Next, we determined
surface expression levels of CCR7 by flow cytometry. CCR7
expression increased slightly at day 2 after TCR stimulation, but
it declined at day 3 and, more profound, at day 6 (Fig. 1B). This
result is in line with previous data showing that TCR ligation led
to an enhanced CCR7 surface expression that was accompanied
by increased responsiveness (24). Supposing that small differ-
ences in receptor surface expression influence the migratory re-
5646TCR–CCR7 CROSS-TALK ENHANCES T CELL MIGRATION
by guest on June 18, 2013
sponse, observed changes in CCR7 expression could explain the
chemotaxis results for one of the ligands but not the other. To
investigate the consequence of T cell activation on CCR7 func-
tions other than migration, we examined the activation of the
MAPKs ERK-1/2 in nonactivated and CD3/CD28-activated hu-
man T cells. Strikingly, CCR7-mediated ERK-1/2 activation was
profoundly enhanced by both ligands at day 2 after T cell acti-
vation compared with nonactivated T cells (Fig. 1C). At day 6,
the ability of CCR7 to trigger ERK-1/2 phosphorylation was
much lower compared with day 2, but it was still greater than
in freshly isolated CD3+T cells. Given that receptor expression
at day 6 was significantly reduced, the enhanced chemokine-
mediated ERK-1/2 phosphorylation did not correlate with the
expression levels of CCR7. These data suggested that T cell ac-
tivation via CD3/CD28 modifies CCR7 signaling and cellular
Short-term TCR triggering is sufficient for enhanced
To assess whether early TCR signals account for the enhanced
CCR7 signaling, we performed Transwell-migration assays with
short-term activated and nonactivated T cells. To this end, we
isolated primary human CD3+T cells from blood samples and
activated them on anti-CD3/CD28–coated plates. After 2 h of
incubation, short-term CD3/CD28-activated and nonactivated
CD3+T cells were allowed to migrate toward CCL19, CCL21,
and CXCL12. Interestingly, short-term CD3/CD28-activated CD3+
T cells migrated significantly better toward CCL19 and CCL21 at
concentrations near the Kdvalues (∼0.01–0.1 mg/ml) (5, 25, 26) of
the two chemokines (Fig. 2A). Maximal migration was observed
at 0.5 mg/ml of CCR7 ligands, and it decreased again at greater
chemokine concentrations, independently of whether T cells were
activated. Remarkably, enhanced CCL21-induced migration upon
sponse of short-term activated human
CD3+T cells is specific for CCR7. A,
Migratory responses of freshly isolated,
nonactivated or 2-h short-term CD3/
CD28-activated human CD3+T cells to-
ward graded concentrations of CCL19,
CCL21, and CXCL12 were determined
by Transwell-migration assays. Cells
were allowed to migrate toward the li-
gand source for 3 h at 37˚C, and the
number of migrated cells was measured
by flow cytometry. Mean values 6 SEM
derived from six independent experi-
ments are presented. *p , 0.05, **p ,
0.01, ***p , 0.001 (paired t test). B,
Surface expression of the chemokine
receptors CCR7 and CXCR4, the T cell
activation marker CD69, as well as the
lymphocyte homing marker CD62L, was
determined by flow cytometry. A repre-
sentative result from the above-men-
tioned experiment is depicted.
Enhanced migratory re-
anti-CD3/CD28 Ab-coated dishes and cultured for up to 6 d. T cell migration toward 1 mg/ml CCL19 and CCL21 was determined by Transwell-migration
assay. The percentage of cells migrated to the lower compartment within 3 h was determined by flow cytometry. Cells migrated in the absence of che-
mokines were subtracted. Mean values 6 SEM derived from four independent experiments are shown. B, Simultaneously, cell surface expression of CCR7
was assessed by flow cytometry using an FITC-labeled CCR7-specific Ab. C, Freshly isolated CD3+T cells and CD3/CD28-stimulated cells were
stimulated for 2 min with 2 mg/ml CCL19 or CCL21 at 37˚C. Chemokine-induced ERK-1/2 activation was determined by Western blotting using
a phospho-specific anti–ERK-1/2 Ab. Reprobing the same blot with a total ERK-2 Ab served as protein loading control. A representative of three inde-
pendent experiments is shown.
Temporarily modified CCR7 signaling and migration upon T cell activation. A, Freshly isolated human CD3+T cells were activated or not on
The Journal of Immunology 5647
by guest on June 18, 2013
short-term T cell activation was observed at any chemokine con-
centration tested, whereas more efficient CCL19-mediated chemo-
taxis was only noted at concentrations close to the Kdvalue
(Fig. 2A). In contrast, CXCL12-mediated migration was slightly
attenuated in activated T cells (Fig. 2A), confirming previous
observations for CXCR4 (14). As depicted in Figure 2B, short-
term activation of T cells strongly induced CD69 expression, a
marker for early T cell activation, but it did not significantly alter
the expression levels of the homing receptors CCR7, CXCR4, and
CD62L. These data clearly indicated that the enhanced T cell
migration mediated by TCR triggering is restricted to CCR7, is
most prominently observed at chemokine concentrations near the
Kdvalues, and cannot be attributed to changes in chemokine re-
ceptor surface expression. These observations also suggested that
the TCR-signaling pathway differentially cooperates with distinct
modules of chemokine receptor signaling. All subsequent che-
motaxis assays were performed at 0.2 mg/ml of CCR7 ligands, for
which differences in cell-migration efficiency between nonacti-
vated and TCR-triggered T cells are most prominent.
Next, we tested whether even shorter TCR triggering was suf-
ficient for inducing an enhanced T cell-migratory phenotype.
Therefore, we activated primary human CD3+T cells for 45 min
on anti-CD3/CD28–coated plates, transferred the cells to uncoated
dishes, and cultured them for an additional 75 min before the
migratory response was analyzed. Even this shortened period of
time was sufficient to provoke a significantly enhanced CCR7-
dependent T cell-migration phenotype (Fig. 3A). TCR triggering
for 45 min seemed to be the minimal activation time, because
a further reduction in the duration of TCR stimulation no longer
induced the enhanced migratory phenotype (data not shown). In
addition, this result also demonstrated that the promigratory effect
remained after TCR ligation for $1 h and did not require si-
multaneous stimulation of the TCR and the chemokine receptor.
Enhanced migration by short-term TCR triggering is most
effective for naive T cells
Next, we aimed to determine whether different T cell sub-
populations are more prone to develop an enhanced migratory
phenotype. To this end, we stained CD3+T cells for CD45RO,
CD45RA, CD4, and CD8 before and after migration toward CCR7
ligands and determined the percentage of specifically migrated
T cell subsets relative to the input of cells. As depicted in Figure
3B, all T cell subpopulations showed an enhanced migratory
phenotype induced by short-term CD3/CD28 activation, but the
most prominent effect was observed for CD45RA+naive T cells,
irrespective of whether they were CD4+Th cells or CD8+cyto-
toxic T cells.
SFKs are important for the enhanced CCR7-mediated
migration of short-term activated T cells
Because we obtained no evidence for the regulation of CCR7 ex-
pression by short-term activation of T cells that could account for
the enhanced migratory capacity, we next focused on potential
signaling cross-talks between the TCR and CCR7. First, we used
pharmacological inhibitors and assessed the migratory capacity of
drug-treated T cells. Importantly, we first stimulated CD3+T cells
with anti-CD3/CD28 for 1 h (which was sufficient for inducing
the enhanced migratory phenotype, as shown in Fig. 3A). Cells
were then further incubated in the presence of inhibitors or sol-
vent controls for another hour prior to assessing their migratory
capacities. Inhibiting PI3K by LY294002 or the Rho pathway
using the ROCK inhibitor Y-27632 partially diminished the
overall migratory response but did not abrogate the enhanced
migration of short-term activated T cells toward CCL19 and
CCL21 (data not shown). T cells treated with the proteasome
inhibitor MG132 prior to T cell activation to prevent degradation
of potential signaling proteins hampering CCR7-mediated mi-
gration still displayed enhanced migration upon TCR triggering
(data not shown). Inhibiting SFKs using PP2 led to an overall
attenuation of CCR7-mediated migration, but it additionally sig-
nificantly reduced the enhanced migratory capacity of short-term
activated CD3+T cells (Fig. 4A). The SFKs Fyn and Lck are well-
known tyrosine kinases downstream of the TCR that play crucial
roles in T cell activation (27). To investigate whether one of these
SFKs accounts for the enhanced T cell migration, we performed
small interfering RNA (siRNA) experiments in primary human
T cells to specifically knockdown individual kinases. Unfortu-
nately, siRNA transfection itself, whether specific or nonspecific,
resulted in T cell activation manifested by enhanced CD69 and
reduced CD62L expression. Despite multiple approaches, we
were unable to establish conditions to introduce siRNA in primary
T cells while keeping the enhanced CCR7-driven migration phe-
notype upon CD3/CD28 triggering (data not shown). As an al-
ternative strategy, we exploited the pharmacological compound
SU6656, which selectively inhibits the SFK members Fyn and Src
with only minor or no effect on Lck activity (28). Primary human
CD3+T cells were pretreated for 2 h with SU6656 before being
subjected to migration assays. Surprisingly, SU6656 treatment
significantly enhanced migration of nonactivated T cells toward
CCL19 and CCL21, but it did not influence the migratory re-
anti-CD3/CD28 Ab-coated dishes, followed by further incubation for 75 min on uncoated dishes. Cells were subsequently subjected to Transwell-migration
assays toward 0.2 mg/ml CCL19 and CCL21. Mean values 6 SEM derived from six independent experiments are shown. **p , 0.01 (paired t test). B,
Human CD3+T cells were activated or not for 2 h on anti-CD3/CD28–coated dishes and subjected to Transwell-chemotaxis assays. Input and migrated T
cell populations were assessed for the expression of CD45RO, CD45RA, CD4, and CD8 by flow cytometry using specific Abs. The percentage of individual
T cell subsets was calculated relative to the initial population. Mean values 6 SEM derived from four independent experiments are presented.
Enhanced migration by short-term TCR triggering is most effective for naive T cells. A, Human CD3+T cells were activated for 45 min on
5648TCR–CCR7 CROSS-TALK ENHANCES T CELL MIGRATION
by guest on June 18, 2013
sponse of CD3/CD28-activated T cells (Fig. 4B). Interestingly,
cell-migration efficiency was similar in SU6656-treated nonacti-
vated cells and in short-term activated T cells without inhibitor
treatment. SU6656 treatment did not affect CXCR4-mediated
migration of activated or nonactivated T cells (Fig. 4B), demon-
strating again a specificity for CCR7. These data indicated that
Fyn plays a role in the TCR–CCR7 cross-talk leading to enhanced
migration of short-termed TCR-triggered T cells.
To substantiate this finding, we inhibited another protein kinase
family, the protein kinase C isoforms (PKCs), which were shown to
serine phosphorylate Fyn in platelets (29). To investigate the role
of PKC and, indirectly, Fyn on CCR7-mediated T cell migration,
we pretreated nonactivated and short-term activated CD3+T cells
with the PKC inhibitor bisindolylmaleimide I (BimI). Strikingly,
we observed an enhanced CCR7-mediated migration, especially
toward CCL21, in BimI-treated nonactivated T cells, whereas in
short-term activated T cells PKC inhibition diminished migratory
responses (Fig. 5A). Similarly to SU6656 treatment, cell migration
did not increase upon TCR ligation in BimI-treated cells. Because
BimI inhibits several PKC isoforms, including PKCa, b1, b2, g, d,
and ε, we used a second, more specific, PKC inhibitor (Go ¨6976),
which selectively inhibits the Ca2+-dependent a and b1 isozymes.
Pretreatment of nonactivated CD3+T cells with Go ¨6976, in con-
trast to BimI, attenuated CCL19- and CCL21-mediated cell mi-
gration. Based on these observations, we assumed that the SFK
Fyn, together with Ca2+-independent PKC isoforms, keeps the
migratory response of naive T cells toward CCL21 at a low level.
The activation status of Lck and Fyn is reflected in their
phosphorylation pattern. Thereby, phosphorylation of Tyr394 of
Lck results in activation of the kinase, whereas Tyr505 is the
regulatory tyrosine residue, and its phosphorylation leads to an
inactive conformation of Lck. For Fyn, Tyr419 is the activating,
whereas Tyr528 is the negative regulating, tyrosine phosphory-
lation site. To assess the involvement of these two kinases in the
negativeregulationof CCR7-mediated human Tcellmigration,we
determined their phosphorylation pattern after chemokine trig-
gering. To this end, we challenged freshly isolated human PBLs
with CCL21 for different times and determined the phosphory-
lation status of the regulatory tyrosines by immunoblotting.
CCL21 induced a strong Tyr505 phosphorylation of Lck in
nonactivated PBLs, which was maximal after 30 min of stimu-
lation (Fig. 6A). In contrast, the phosphorylation status of Tyr528
of activated primary human T cells. Freshly isolated human CD3+T cells
were either activated for 2 h by CD3/CD28 or left untreated. A, After the
first hour of activation, 10 mM PP2 and equivalent amounts of its solvent
control, DMSO, was added. B, Cells were treated for 2 h with the Fyn and
Src inhibitor SU6656 (5 mM) prior to the chemotactic response being
determined. Chemotaxis toward 0.2 mg/ml CCL19, 0.2 mg/ml CCL21, or
50 ng/ml CXCL12 was assessed by Transwell-migration assays. The
numbers of migrated cells were determined by flow cytometry. Simulta-
neous TOPRO-3 staining was included to exclude that inhibitor treatment
affected cell viability. Mean values 6 SEM derived from seven (A) or eight
(B) independent experiments are shown. *p , 0.05, **p , 0.01, ***p ,
0.001 (repeated-measures ANOVA, with Bonferroni multiple comparison
SFKs contribute to the enhanced CCR7-mediated migration
Nonactivated and short-term activated human CD3+T cells were treated
with 10 mM of the PKC inhibitors BimI (A) or Go ¨6976 (B) 1 h before
T cells were subjected to migration assays in response to 0.2 mg/ml CCL19
or CCL21. Cells were allowed to migrate for 3 h before the numbers of
migrated cells were determined by flow cytometry. Cell viability after
inhibitor treatment was assessed by TOPRO-3 staining. Mean values 6
SEM derived from six (A) or seven (B) independent experiments are
shown. *p , 0.05, **p , 0.01, ***p , 0.001 (one-way ANOVA with
Bonferroni multiple comparison posttest).
Contribution of PKCs to CCR7-mediated T cell migration.
The Journal of Immunology5649
by guest on June 18, 2013
of Fyn did not change upon CCR7 triggering (Fig. 6B). Because
Fyn was postulated to act as a negative-feedback loop regulator of
Lck (30), we investigated its role in CCR7-mediated phosphoryla-
tion of the regulatory Tyr505 of Lck. To this end, we treated human
PBLs with SU6656 prior to CCR7 stimulation. Indeed, we found
that SU6656 inhibited CCL21-mediated Tyr505 phosphorylation of
Lck (Fig. 6A), suggesting a negative regulation of Lck by Fyn.
CCR7 triggering leads to LAT phosphorylation in activated
TCR triggering is known to induce phosphorylation of LAT by
ZAP70, which is itself activated by Lck (31). To gain further in-
formation about the activation state of Lck and Fyn, we tested
whether CCR7 triggering induced LAT phosphorylation. In non-
activated PBLs, CCL19 and CCL21 barely induced LAT phos-
phorylation (Fig. 7A). In contrast, in short-term TCR-engaged
PBLs, LAT phosphorylation was strongly enhanced upon CCR7
triggering. Of note, at day 6 of PBL activation, CCL19 and CCL21
no longer induced LAT phosphorylation (data not shown). Treating
of CD3/CD28 short-term activated PBLs with PP2 completely
blocked CCR7-triggered LAT phosphorylation (Fig. 7B), demon-
strating that an Src kinase member, most likely Lck, is responsible
for CCL19/CCL21-mediated LAT phosphorylation.
Taken together, we found that short-term activation of (naive)
T cells resulted in a significantly enhanced migratory response to
that SFKs, presumably Lck, are responsible for these increased
migratory responses. Moreover, CCR7 triggering induces LAT
phosphorylation by SFKs in synergy with TCR engagement. This
study demonstrated a profound signaling cross-talk between CCR7
and the TCR.
Within lymph nodes, naive T cells are very motile and scan a large
number of Ag-presenting dendritic cells in search of presented
cognate Ags. Upon Ag recognition, T cells are activated and start to
proliferate, resulting in clonal expansion and differentiation. How-
ever, TCR triggering alone is not sufficient for a full-blown T cell
activation. It is now well established that the affinity of the Ag for
the TCR, as well as T cell costimulation, is critical for the T cell-
activation outcome (32, 33). Intravital microscopy revealed that
naive T cells initially interact only temporarily with dendritic cells
presenting cognate Ags (13). During this early priming phase, naive
T cells upregulate the activation marker CD69 and stay highly
motile. Of note, tonic TCR signaling in secondary lymphoid tissues
was shown to be required for maintaining a basal Rac1 and Rap1
activation, which is critical for normal T cell motility and Ag re-
ceptivity of naive CD4 T cells (22). The high motility of T cells
during the early priming phase is mediated by the chemokine recep-
tor CCR7 (8, 9). It is poorly understood how the migratory behavior
of T cells is regulated during early- and late-priming phases. In the
current study, we investigated the influence of TCR ligation on the
chemotactic response toward the lymph node chemokines CCL19
and CCL21 using human peripheral blood-derived T cells as a
model system. We demonstrated that mainly CD45RA+naive
T cells, but also CD45RO+memory T cells, migrate more readily
toward CCL19 and CCL21 upon short-term triggering of the TCR.
This promigratory effect was specific for the chemokine receptor
CCR7 and did not occur for CXCR4. This is in line with the sole
previous finding that CCL21, but not CXCL12, could override
the TCR-mediated stop signal in a two-dimensional system where
T cells were subjected to opposing gradients of coated Ags and
chemokines (34). Moreover, our findings are also supported by
intravital imaging studies describing a promigratory role for TCR
signals in naive (22), as well as in effector T cells (23), without
specifically investigating involved chemokines/chemokine recep-
tors. Substantially more information is available about the interplay
between CXCR4 and the TCR. In fact, CXCR4 was shown to
physically interact with the TCR after chemokine binding (19). On
a Fyn-dependent manner in nonactivated PBLs. Human PBLs were stim-
ulated for the indicated times with 1 mg/ml CCL21. Cell lysates were
separated on SDS-PAGE and immunoblotted for the inhibitory tyrosine of
Lck, pTyr505 (A) or Fyn, pTyr528 (B), respectively. Blots were stripped
and reblotted with Abs against total Lck and Fyn to assure equal protein
loading. Where indicated, PBLs were pretreated with 5 mM of the Fyn
inhibitor SU6656 for 2 h prior to chemokine stimulation.
CCL21 induces inhibitory Tyr505 phosphorylation of Lck in
activated for 2 h with Abs against CD3 and CD28 (B), were stimulated for the indicated times with 1 mg/ml CCL19 or CCL21. Chemokine-induced LAT
phosphorylation at Tyr191 was determined by Western blotting. Cells were lysed and immunoblotted for the detection of total and phosphorylated LAT.
Where indicated, 10 mM PP2 was added 30 min prior to chemokine (A) or TCR (B) triggering. A representative Western blot of four derived from in-
dividual blood donors is depicted.
CCR7 stimulation synergistically induces LAT phosphorylation exclusively in TCR-triggered PBLs. Human PBLs, either untreated (A) or
5650 TCR–CCR7 CROSS-TALK ENHANCES T CELL MIGRATION
by guest on June 18, 2013
one hand, this heterodimer between CXCR4 and the TCR enabled
the chemokine receptor to signal via the TCR ITAM motifs in a
ZAP70-dependent manner, resulting in sustained ERK activation,
altered transcriptional activity, and increased intracellular Ca2+
concentration mobilization (19, 21). On the other hand, CXCR4
triggering diminished TCR activation by lowering the phosphory-
lation of ZAP70, LAT, and SLP76 (14). The same investigators
reported that TCR activation inhibited T cell migration toward
CXCL12 and caused augmented CXCR4 internalization (14). Our
observations confirmed the reduced CXCR4-mediated migratory
response after TCR engagement in human T cells; however, this
effect did not correlate with a decreased surface chemokine receptor
expression (Fig. 2).
Using a panel of pharmacological inhibitors, we determined that
migration of T cells upon short-term triggering of the TCR. In our
experimental set-up, inhibition of SFKs by PP2 led to an overall
reduced lymphocyte migration, as well as abrogated the TCR-
mediated promigratory phenotype. Surprisingly, we observed a
significantly enhanced migration of nonactivated human T cells if
the SFK Fyn was blocked by SU6656. This enhanced migration
induced by Fyn inhibition could not be augmented by TCR trigger-
ing, strengthening the assumption that different members of SFK
opposingly effect CCR7-driven T cell migration. Lck and Fyn are
the major SFKs in T cells and are among the first downstream-
signaling molecules of the TCR. Activation of these tyrosine
kinases is, on the one hand, regulated by conformational changes
that are due to the binding of target proteins to the SH3 and/or SH2
domains of the kinase and, on the other hand, by the phosphoryla-
tion status of two critical tyrosine residues (27). The activating ty-
rosine is located within the kinase domain, whereas the inhibitory/
regulatory tyrosine is positioned in the C terminus (Tyr505 for Lck
and Tyr528 for Fyn). After TCR engagement, SFKs, essentially
Lck, phosphorylate the ITAM motifs within the g-, d-, ε-, and
z-chains of CD3. Subsequently, ZAP70 is recruited to phosphory-
lated CD3 andisitselfactivated byLck (31).Phosphorylation ofthe
adaptor molecules LAT and SLP76 by activated ZAP70 is critical
for an effective TCR triggering resulting in a fully activated T cell
activation of Lck and Fyn, differences in the phosphorylation status
of these two kinases before and after TCR engagement were re-
ported. It is well established that tyrosine residues within the kinase
domain of Lck are phosphorylated after efficient T cell activation
(36, 37). However, a recent study described that, in naive T cells, a
significant proportion of Lck was constitutively active and, strik-
ingly, partially also phosphorylated at the inhibitory Tyr505 (38).
Moreover, in these cells, the amount of active Lck did not increase
upon TCR/CD3 engagement but determined the extent of CD3z
phosphorylation (38). Few studies have investigated the role of Lck
and Fyn in chemokine receptor signaling and function: CXCR4
stimulation was reported to enhance Lck activity, which was impor-
tant for the onset of different downstream pathways involving
ZAP70,Dok-1 (39,40), andFyn(41). Lck was also reported toplay
a crucial role in CXCR4-mediated lymphocyte homing (42). Fur-
thermore, a negative effect on TCR-mediated Lck activation was
described for CXCL12-treated cells (43), suggesting an interplay
between the chemokine receptor and the TCR signaling via the
action of SFKs. In the current study, we identified a crucial role for
SFKs in T cell migration toward the CCR7 ligands CCL19 and
CCL21. Furthermore, we demonstrated that, in naive T cells, the
regulatory Tyr505 of Lck was phosphorylated in a Fyn-dependent
manner upon CCR7 triggering. Together with the finding that in-
hibition of Fyn led to an enhanced migratory response in nonacti-
vated human T cells, our results provided evidence for a negative
role for Fyn in CCR7-mediated migration, which presumably
depended on the inhibition of Lck. Fyn was recently shown to
control the time of TCR engagement, thereby regulating the
threshold of T cell activation (30). Further, this study dem-
onstrated a reduced phosphorylation of the regulatory Tyr505 of
Lck in the absence of Fyn (30), assuming a negative-feedback
loop, similar to our observation. The assumption that Lck is acti-
vated by CCR7 in activated T cells and, thereby, positively influ-
ences chemokine-mediated migration, is supported by the fact that
we detected a profound SFK-dependent phosphorylation of LAT
upon CCR7 triggering, which is in line with Lck-mediated ZAP70
activation upon TCR ligation (44).
In addition to SFK, we identified PKCs as a kinase regulating
CCR7-mediated T cell migration. In fact, pharmacological inhi-
bition of several PKC isoforms by BimI led to an increased mi-
gration of nonactivated human T cells toward CCL21. The CCR7-
mediated migratory response of BimI-treated naive T cells re-
sembles that of SU6656-treated cells. Again, TCR ligation could
not augment the migratory response. No enhanced T cell migration
was observed in cells in which Ca2+-dependent PKC isoforms, like
PKCa and PKCb1, were inhibited, pointing to a major role for
Ca2+-independent PKC isoforms in naive T cell migration. Dis-
crepant results have been reported for different PKC isoforms with
regard to cell migration. For instance, T cell migration was re-
ported to be resistant to PKC inhibitors (45). In contrast, PKCd
was required for CCR4-mediated chemotaxis (46, 47). Interest-
ingly, it was noted that, upon culturing, T cells switch from a
PI3K-dependent to a PKC-dependent chemotactic response to
CXCL12, although the mechanism remains elusive (48). It was
also speculated that different PKC isoforms differentially regulate
actin reorganization and integrin activation and, hence, account
for different mechanisms of migration-signaling paths (49, 50).
From this perspective, it is worth noting that Fyn activity can be
regulated by PKC (29), which could explain our migration data.
In summary, our data revealed that the migratory response of
human primary T cells toward the lymph node chemokines CCL19
and CCL21 is linked to the activation status of the cells. We ob-
served a significantly enhanced T cell migration upon short-term
TCR activation that was independent of CCR7 surface expres-
sion. Moreover, we identified SFKs as integral signaling molecules
between the TCR and CCR7. We provided evidence that, in naive
human T cells, CCR7 activation led to Fyn-dependent Lck inhi-
bition, whereas in short-term activated T cells, CCR7 triggering
induced a strong chemokine-dependent LAT phosphorylation,
presumably through Lck. Moreover, we determined a negative role
for Ca2+-independent PKC isoforms in CCR7-mediated migration,
which faded away in activated T cells. These data clearly indicated
that TCR engagement results in modified CCR7 signaling, leading
to an altered migratory T cell phenotype. Our results provided
mechanistic insights into how T cells acquire a highly motile
phenotype during early-priming phases in lymph nodes, despite
opposing stop signals provided by the TCR.
We thank Dr. Eva Boneberg for initial assistance with flow cytometry.
The authors have no financial conflicts of interests.
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