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Published online: May 19, 2014
Article
Adenosine triphosphate acts as a paracrine
signaling molecule to reduce the motility of T cells
Chiuhui Mary Wang1,2,*, Cristina Ploia1, Fabio Anselmi1,†, Adelaida Sarukhan3 & Antonella Viola1,2,**
Abstract
Organization of immune responses requires exchange of information between cells. This is achieved through either direct cell–cell
contacts and establishment of temporary synapses or the release
of soluble factors, such as cytokines and chemokines. Here we
show a novel form of cell-to-cell communication based on adenosine triphosphate (ATP). ATP released by stimulated T cells induces
P2X4/P2X7-mediated calcium waves in the neighboring lymphocytes. Our data obtained in lymph node slices suggest that, during
T-cell priming, ATP acts as a paracrine messenger to reduce the
motility of lymphocytes and that this may be relevant to allow
optimal tissue scanning by T cells.
Keywords adenosine triphosphate; calcium wave; cell migration; imaging; T
cells
Subject Categories Immunology; Signal Transduction
DOI 10.15252/embj.201386666 | Received 23 August 2013 | Revised 11 April
2014 | Accepted 16 April 2014
Introduction
Extracellular adenosine-50 -triphosphate (ATP) is an important
secondary messenger with roles in many cellular processes including
cell proliferation, apoptosis and differentiation (Popper & Batra, 1993;
Vandewalle et al, 1994; Atarashi et al, 2008). ATP mediates its signaling activities through activation of the P2 purinergic family of receptors (Burnstock, 2006). Based on pharmacological and functional
data, protein topology and gating properties, P2 purinergic receptors
are divided into P2X and P2Y subfamilies. Stimulation of both of these
receptors leads to an increase in intracellular calcium although via
two distinct mechanisms. Ionotropic P2X receptors (P2X1—7) are
ligand-gated cation channels that, upon ATP binding, form non-selective pores allowing ion influx from the extracellular space (Burnstock,
2006). In contrast, metabotropic P2Y receptors (P2Y1, 2, 4, 6, 11–14)
are coupled to G-proteins and ATP binding to these receptors results
in phospholipase C–b (PLCb) activation, IP3 production and the
consequent release of intracellular calcium from ER stores (Burnstock, 2006).
In T cells, ATP is released through gap junction pannexin-1 pores
during lymphocyte activation (Schenk et al, 2008; Woehrle et al,
2010) and it enhances interleukin-2 (IL-2) production by binding to
purinergic receptors P2X1, P2X4 and P2X7 in an autocrine manner
(Yip et al, 2009; Woehrle et al, 2010). The binding of ATP results in
calcium entry, which subsequently activates the nuclear factor of activated T cells (NFAT), the mitogen-activated protein kinase (MAPK)
signaling and IL-2 expression, providing feedback regulation to T-cell
activation (Schenk et al, 2008; Yip et al, 2009). Extracellular ATP was
also shown to have synergistic effect on the proliferation of peripheral
blood lymphocytes stimulated with phytohemagglutinin (Baricordi
et al, 1996). On the other hand, antagonists to purinergic receptors or
ATP degradation were shown to inhibit T-cell capacitative calcium entry
and favor T-cell anergy (Schenk et al, 2008; Yip et al, 2009). More
recently, ATP was shown to inhibit immunosuppressive functions of
regulatory T cell and to promote their conversion to T helper 17 cells in a
murine model of inflammatory bowel disease (Schenk et al, 2011).
Adenosine triphosphate released by stimulated cells may also act
as a paracrine messenger involved in intracellular calcium mobilization activated by purinergic receptors (Osipchuk & Cahalan, 1992;
Zsembery et al, 2003; Yegutkin, 2008). Thus, ATP-induced intercellular spreading of calcium signals—the so called calcium waves—has
been observed in many cell types such as mast cells, epithelial cells,
astrocytes and hepatocytes (Cotrina et al, 1998; Guthrie et al, 1999).
Calcium waves represent a dynamic intercellular signaling mechanism that allows spatiotemporal information to be rapidly propagated
in tissues (Arcuino et al, 2002; Weissman et al, 2004; Dupont et al,
2007). For example, the propagation of signals between brain cells
depends on the formation of calcium waves (Nedergaard, 1994),
whereas normal hearing function depends on calcium waves in the
cochlear of the ear (Zhang et al, 2005b; Anselmi et al, 2008).
Although the autocrine signaling of ATP has been extensively
investigated in lymphocytes, it is not known whether ATP is used
by T cells to communicate with neighboring lymphocytes in
lymphoid tissues. Thus, the aim of our study was to investigate ATP
paracrine signaling in T lymphocytes and to determine its impact on
T-cell physiology.
1 Humanitas Clinical and Research Center, Rozzano, Italy
2 Department of Translational Medicine, University of Milan, Rozzano, Italy
3 INSERM, Paris, France
*Corresponding author. Tel: +39 02 82245252; Fax: +39 02 82245101; E-mail: [email protected]
**Corresponding author. Tel: +39 02 82245252; Fax: +39 02 82245101; E-mail: [email protected]
†Present address: Laboratory for Computational and Statistical learning, MIT, Cambridge, MA, USA
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Extracellular ATP alters T-cell motility
Results
Identification of an ATP-induced paracrine calcium signaling
among lymphocytes
In T cells, the interaction of the T-cell receptor (TCR) with cognate
antigen results in the activation of phospholipase C–c, which generates inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG)
from phosphatidylinositol 4,5 bisphosphate (PIP2) in the plasma
membrane (Lewis, 2001). IP3 induces Ca2+ release from the endoplasmic reticulum (ER), which, in turn, stimulates activation of the
Ca2+-permeable calcium release activated channels (CRAC) Orai1/
CRACM1 in the plasma membrane, resulting in a sustained influx of
Ca2+ from the extracellular space into the cytosol, termed capacitative Ca2+ entry (CCE) (Putney & Bird, 1993; Zhang et al, 2005a;
Feske et al, 2006; Prakriya et al, 2006; Vig et al, 2006; Yeromin
et al, 2006).
Using a cell membrane permeable caged-IP3 (Li et al, 1998), we
triggered single-cell Ca2+ influx in human peripheral blood CD4+ T
lymphocytes using a short UV laser pulse (Fig. 1). Interestingly, we
clearly observed calcium signal propagation to bystander T cells that
were not in contact with the triggered one (Fig 1 A and Supplemen-
Chiuhui Mary Wang et al
tary Video S1), suggesting that calcium waves were propagated
through a soluble mediator (Suadicani et al, 2006). In T cells, Ca2+
influx induces ATP synthesis and release (Schenk et al, 2008), and,
on the other hand, extracellular ATP is known to induce Ca2+ influx
in several cell types (Osipchuk & Cahalan, 1992; Schlosser et al,
1996; Guthrie et al, 1999; Gomes et al, 2006). Thus, we speculated
that the calcium waves observed in T cells might be caused by paracrine ATP signaling. To verify the involvement of extracellular ATP
in this process, we repeated the experiment in the presence of the
ATPase enzyme apyrase (Fig 1 B and Supplementary Video S2), and
we found that in the absence of extracellular ATP, the percentage of
calcium-influxing T cells was significantly reduced (Fig 1C). The
analysis of the kinetic of the calcium waves indicated that it was a
function of the distance from the triggered cell, suggesting that it
depended on ATP diffusion (Fig 1D). Limited calcium waves were
obtained using the Jurkat T-cell line, too (not shown).
The effects of apyrase on changes in single-cell intracellular
calcium concentration [Ca2+]i were also analyzed (Fig. 1E and F).
Apyrase reduced calcium influx in the cell stimulated by UV exposure (Fig 1E), indicating that extracellular ATP sustains calcium
signaling in an autocrine manner (Yegutkin et al, 2006; Yip et al,
2009). Notably, in agreement with data shown in Fig 1B and C, the
A
B
C
D
E
F
Figure 1. Identification of adenosine triphosphate (ATP) paracrine signaling.
A An intercellular Ca2+ wave initiated by flash photolysis of caged-IP3 of ROI in one human CD4+ T cell (arrowhead) propagates to non-adhering bystander CD4+ T cells.
Increase in cytosolic calcium was indicated by the increase in green fluorescence intensity (Fluo-4). The time-lapse video is illustrated in Supplementary Video S1.
B Calcium waves were inhibited by the ATPase apyrase (5 U/ml). Arrowhead marks the uncaged cell. Scale bar 10 lm, time is indicated in seconds. See also
Supplementary Video S2.
C The number of bystander cells showing calcium influx after the uncaging of one central cell was quantified from each imaging frame. In the control condition,
approximately 65% of the bystander cells would respond to extracellular ATP released within the 3-min time-lapse videos, and apyrase significantly reduced this
percentage (n = 6, ***P < 0.001).
D The relationship between the distance of bystander cells from the uncaged one versus the time of calcium level increase.
E Graph showing ratiometric measurements of cytosolic calcium levels in the uncaged cells in control and in the presence of 5 U/ml active or heat-inactivated (HI)
apyrase. The control cells responded to UV uncaging with increased cytosolic calcium. In the presence of apyrase, the increase was markedly reduced (n = 11,
Wilcoxon signed rank test P < 0.001). Heat-inactivated apyrase did not significantly alter the cytosolic calcium increase after uncaging (n = 4, P > 0.05).
F The ratiometric measurements of cytosolic calcium levels showed that in the bystandered cells, the intracellular calcium oscillated significantly and increased with
time in the control condition (n = 48 cells), whereas in the presence of apyrase, the intracellular calcium level remained flat in the bystander cells (n = 20, Wilcoxon
signed rank test P < 0.001). Heat-inactivated apyrase did not prevent the calcium responses in the bystander cells (n = 14).
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Extracellular ATP alters T-cell motility
cells nearby the uncaged one no longer responded respond in the
presence of apyrase (Fig 1F). Individual traces of calcium influx in
response to ATP for each cell are shown in Supplementary Fig S1.
To estimate the level of ATP released by T cells in our experimental conditions, we stimulated T cells with anti-CD3 plus antiCD28 antibodies, a condition known to induce ATP release (Schenk
et al, 2008; Yip et al, 2009), or UV photolysis of caged-IP3 loaded
cells and quantified the ATP released by the cells. The results indicate that the level of ATP released in response to the two stimuli is
not significantly different (Supplementary Fig S2A). The level of
intracellular calcium increase was also similar when the T cells were
stimulated either by anti-CD3 and anti-CD28 antibodies, or extracellular ATP (100 lM, Supplementary Fig S2B).
To verify the existence of T-cell calcium waves in a more physiological environment, we performed experiments similar to those
described above using lymph node (LN) slices that had been previously loaded with the calcium indicator Fluo-4 and caged-IP3 (Fig 2,
Supplementary Video S3–S5). Calcium waves were clearly detected
in the LN microenvironment (Fig 2A–B and Supplementary Video
S3) and required the presence of extracellular ATP (Fig 2 C and
Supplementary Video S4).
Altogether, these data demonstrate the existence of an ATPinduced, Ca2+-mediated paracrine signaling among lymphocytes.
Identification of the receptors involved in lymphocyte paracrine
ATP signaling
Adenosine triphosphate mediates its signaling activities through
activation of the P2X and P2Y purinergic receptors (Burnstock,
2006). On the basis of the distinctive properties of the P2X and P2Y
receptors, we tried to identify the subfamily of receptors responsible
for paracrine ATP binding by repeating the in vitro IP3-uncaging
experiment in the presence (Fig 3A) or in the absence (Fig 3B) of
extracellular Ca2+. Indeed, in the absence of extracellular Ca2+,
P2X receptors would be unable to signal, whereas P2Y-mediated
A
[Ca2+]i would still be detected. To further confirm the involvement
of P2X receptors, T cells were pre-incubated with the P2X antagonist
suramin before UV uncaging. Suramin inhibited the bystander cell
calcium increase, similarly to apyrase (Fig 3E). Our experiments
demonstrated that extracellular Ca2+ is required for ATP-mediated
T-cell paracrine signaling (Fig 3), suggesting the involvement of the
purinergic receptors belonging to the P2X subfamily.
The P2X family comprises seven receptor subunits (P2X1—7).
We analyzed the expression of the seven subunits in Jurkat and
human peripheral blood CD4+ T cells by reverse transcriptase PCR
(Fig 4A). P2X2 and P2X3 mRNAs were not detectable in both CD4+
and Jurkat T cells (not shown). The only P2X receptors expressed in
both cell types were P2X1, P2X4 and P2X7, although they were
expressed at very low levels in Jurkat cells as compared to the
human peripheral CD4+ T cells (Fig 4A). To further characterize
the contribution of these three receptors to ATP paracrine signaling
in human T cells, we separated peripheral blood CD4+ T cells in
three distinct populations—T naive, central memory and effector
memory (Sallusto et al, 2004)—and quantified the expression of
each receptor subunit (Fig 4C), as well as the calcium response
induced by paracrine ATP in the different cell populations (Fig 4B).
For this, we performed experiments using extracellular ATP to stimulate T-cell calcium influx, detected by flow cytometry. The analysis
demonstrated that P2X1 is not expressed in memory T cells, that
are, however, characterized by a strong ATP-induced calcium
signaling, suggesting that P2X1 is not involved in the bystander activation of T cells. This conclusion was supported by experiments in
which pharmacological blockers for P2X1, P2X4 or P2X7 were used
to interfere with ATP signaling and that demonstrated that blockage
of P2X4 and P2X7, but not of P2X1, inhibited ATP-induced calcium
influx in T cells (Fig 4D and E). To provide further support in favor
of a role for P2X4 and P2X7, we knocked down in T cells the expression of the two receptors using siRNA technology (Fig 4F) and
analyzed the calcium response induced by extracellular ATP
(Fig 4G). The results indicated that the two receptors are involved
B
C
Figure 2. Calcium waves in lymph node slices.
A Fresh mouse lymph nodes were cut into slices and loaded with caged-IP3 and calcium indicator Fluo-4 for ex vivo calcium wave imaging. Calcium wave rapidly
formed upon uncaging of IP3 at ROI (the small, green circle) in mouse lymph node slices. Cytosolic calcium is pseudocolored with the ‘Fire’ spectrum, where basal
calcium level is in blue and the increase is indicated in red and yellow colors. White lines demarcate the expansion of the calcium wave. These data are
representative of six independent experiments; scale bar, 30 lm; time is indicated in seconds. See also Supplementary Video S3.
B The spread of calcium waves is represented as a function distance from the uncaged ROI and time.
C No calcium waves were detected in lymph node slices incubated in apyrase (n = 4). Related time-lapse illustration is shown in Supplementary Video S4. Basal
calcium oscillation in resting mouse ex vivo lymph node slices is shown in Supplementary Video S5.
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Extracellular ATP alters T-cell motility
A
B
C
D
E
Figure 3. Identification of purinergic receptors responsible for calcium
waves in T cells.
A–D IP3 was uncaged in human CD4+ T cells by UV exposure in buffers
containing or not Ca2+, in order to distinguish the functional role of P2X
or P2Y families in bystander adenosine triphosphate (ATP) signaling.
(A, B) Representative images of calcium responses in the presence (A) or
absence (B) or extracellular Ca2+. Arrowhead marks the uncaged cell.
Scale bar 10 lm, time is indicated in seconds. (C, D) Analyses of three
independent experiments showing calcium influx in uncaged (C) or
bystander (D) cells. In calcium-free PBS, bystander cells did not
demonstrate any calcium response (B, D), indicating that extracellular
calcium influx was responsible for the observed increase in cytosolic
calcium and that the ATP receptors involved in paracrine signaling
belong to the P2X family. Cytosolic calcium level increased in the
uncaged cell both in the presence and absence of extracellular calcium
(A, C), although in this last condition, calcium influx was not sustained,
as expected. (n = 3, lines represent the mean; Wilcoxon matched pairs
test P < 0.0001).
E
UV uncaging experiments were repeated with CD4+ T pre-incubated
with suramin, a P2X receptor antagonist. Similarly to apyrase, suramin
inhibited the bystander cell calcium increase (n = 24 cells).
Chiuhui Mary Wang et al
ATP-induced effects on T-cell motility. Importantly, addition of ATP
did not alter the migration velocity in the absence of extracellular
calcium (Fig 5E). The projected migration tracks of the T cells from
the time-lapse videos are shown in Supplementary Fig S3. In these
experiments, when the ATP-induced intracellular calcium increase
was buffered by the calcium chelator BAPTA, we no longer
observed the slowing down of T-cell chemotactic migration velocity
(Supplementary Fig S4).
Altogether, the data allow us to propose a scenario in which ATP
released by antigen-triggered T cells reduces the motility of the
unstimulated T cells that are in close proximity. To verify this
hypothesis, the motility of T cells in LN slices was analyzed by twophoton microscopy. In these experiments, two different types of
mouse T cells, expressing either a TCR specific for the OVA peptide
(OT-II T cells) or wild-type T cells (WT T cells), were overlaid and
allowed to penetrate into LN slices containing dendritic cells (DCs)
loaded with the OVA peptide. In these conditions, only OT-II T cells
can be triggered by OVA-DCs, whereas the large majority of WT
T cells would remain unstimulated. As expected, OT-II T cells
reduced their motility in the presence of the specific antigen, indicating
establishment of contacts (Miller et al, 2004b) (Fig 6 and Supplementary Videos S6). In agreement with recent findings that
observed a general reduction of T-cell motility—independently of
antigenic specificities—in regions of LN where there are prolonged
interactions between antigen-specific T cells and antigen-presenting
ones (McKee et al, 2013; Salgado-Pabon et al, 2013), in the presence
of the OVA peptide, also the WT T cells displayed reduced motility
(Fig 6A). On the basis of the data presented above in the manuscript, we speculated that the reduced motility of the untriggered
WT T cells depended on the ATP released by triggered OT-II T cells,
and to prove this hypothesis, we performed the same experiments
in the presence of apyrase. Notably, the velocity of WT T cells
recovered to the control condition in the presence of the ATPase
apyrase (Fig 6A and Supplementary Video S7), whereas OT-II
T cells remained unaffected (Fig 6B).
Discussion
in ATP-induced calcium signaling in T cells and that when both
receptors are knocked down, T cells are unresponsive to extracellular ATP.
Functional significance of calcium waves in the lymphoid tissue
Our data suggest that T-cell activation in lymph nodes would result
in ATP release and raise of [Ca2+]i in neighboring, unstimulated T
cells. Robust evidence indicates that an increase in [Ca2+]i reduces
T-cell motility in tissues (Bhakta et al, 2005). Thus, we speculated
that ATP-dependent calcium waves may reduce bystander cell
motility to create a zone of lymphocyte swarming and clustering as
observed in antigen challenged lymph nodes (Miller et al, 2002) and
to favor T-cell scanning of antigen-loaded dendritic cells. Accordingly, in vitro experiments with human CD4+ T cells showed that
addition of extracellular ATP significantly reduced the mean migration speed toward the chemokine CXCL12, and it disrupted the
straight chemotactic migration and the final cell displacement
(Fig 5A–D). The P2X receptor antagonist suramin prevented these
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Extracellular ATP is an important modulator during inflammation
and immunity, regulating several aspects of immune cell biology
such as the shedding of CD21 and CD62L (L-selectin) in neutrophils
(Sengstake et al, 2006; Scheuplein et al, 2009), the differentiation of
Th17 cells in the lamina propria (Atarashi et al, 2008) and the
generation of regulatory T cells (Schenk et al, 2011). ATP was first
described to have mitogenic effect on human T lymphocytes stimulated by PHA or anti-CD3 antibodies (Baricordi et al, 1996) and,
more recently, it was demonstrated that ATP is released during
T-cell activation via gap junction pannexin-1 hemichannels (Schenk
et al, 2008; Yip et al, 2009; Woehrle et al, 2010) and that it acts as
autocrine costimulator for IL-2 production (Schenk et al, 2008; Yip
et al, 2009).
Our data indicate that, in addition to these autocrine effects, ATP
may act as a paracrine signaling molecule by inducing calcium
waves that regulate T-cell motility during immune responses.
We show that extracellular ATP released by triggered T cells is
sensed by resting, bystander cells through P2X4 and P2X7 receptors,
since pharmacological inhibition as well as silencing of P2X4 and
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◂
B
C
D
E
F
G
P2X7 receptors abolished ATP-induced calcium signaling in human
T cells. Interestingly, during T-cell priming, P2X1 and P2X4 receptors are recruited into the immunological synapse, whereas P2X7
receptors remain diffused in the plasma membrane (Woehrle et al,
2010). This suggests that different P2X receptor subtypes have
A
B
Figure 4. P2X4 and P2X7 receptors are both involved in paracrine
adenosine triphosphate (ATP) signaling.
A Reverse transcriptase PCR analysis of P2X receptors expression in Jurkat
and human CD4+ T cells.
B All human CD4+ T cells (naïve, central and effector memory) show calcium
influx in response to paracrine ATP signals, with effector/memory T cells
showing higher percentages of fluxing cells than naïve ones. Jurkat T cells
demonstrated very low levels of bystander cell calcium responses
(***P < 0.001).
C Real-Time PCR analysis of P2X1, X4 and X7 in naïve, central memory,
effector memory CD4+ T (four donors) and Jurkat T cells (P2X2 and P2X3 are
not expressed in lymphocytes, not shown). The expression of P2X1 was
barely detectable, whereas P2X4 and P2X7 are highly expressed in effector/
memory cells. Jurkat cells expressed low levels of the P2X4 and P2X7
receptors.
D Human CD4+ T cells were treated with pharmacological blockers of
purinergic receptors P2X1, P2X4 or P2X7 (n = 56 cells) and subjected to
single-cell IP3 uncaging using the UV laser as the experiment described in
Fig. 1. NF023 (P2X1 blocker) did not affect the percentage of bystander cells
showing calcium influx in the imaging frame. 5-BDBD (P2X4 blocker)
reduced the percentage of responding bystander cells, although not
significantly. P2X7 blockers KN62 and A438079 both significantly reduced
the percentage of responding bystander cells (*P < 0.05). Inhibition of both
P2X4 and P2X7 receptors significantly prevented bystander cell calcium
influx (***P < 0.001).
E Calcium influx in response to addition of extracellular ATP is prevented by
A438079 and 5-BDBD.
F To reconfirm the data, we employed siRNA to knocked down P2X4 and
P2X7 in primary human CD4+ T cells. Successful knock down of mRNA
expression was confirmed with real-time PCR analysis.
G FACs analysis of calcium influx in human CD4+ T cells in response to
extracellular ATP (paracrine signaling). Single knock down of P2X4 or P2X7
(dotted lines) did not alter the magnitude of calcium influx, but double
knock down (gray solid line) rendered T cells unresponsive to extracellular
ATP (two-way ANOVA P < 0.001).
different functions during T-cell activation and that P2X7 receptors
may allow T cells to sense ATP present in the microenvironment
(Junger, 2011). Our data indicate that P2X1 is not expressed in
memory T cells. It would be therefore interesting to analyze how
ATP signaling at the immunological synapse is orchestrated in these
cells lacking P2X1. As for paracrine signaling, the fact that memory
T cells do not express P2X1 but are characterized by a strong
ATP-induced calcium signaling suggests that P2X1 is not involved in
C
D
E
Figure 5. Adenosine triphosphate (ATP) signaling reduces T-cell chemotactic migration.
A–D Human CD4+ T cells were seeded onto chemotaxis chambers and bright field time-lapse images were recorded to analyze migratory responses to CXCL12 (2.5 nM).
Addition of extracellular ATP (100 lM) significantly modified T-cell chemotaxis, in term of (A) migratory tracks, (B) migration speed, (C) straightness and (D) final
displacement (n = 3 experiments; control 79 cells, ATP 176 cells *** P < 0.001). The P2X receptor antagonist suramin blocked the effect of ATP on migration speed
and final displacement of T cells, but it only inhibited ATP effect on directionality (n = 3; 36 cells).
E
Chemotactic T-cell migration toward CXCL12 in the absence of extracellular calcium (calcium-free PBS). The addition of ATP did not alter the migration velocity in
the absence of extracellular calcium. ns, not significant
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Extracellular ATP alters T-cell motility
B
Figure 6. Adenosine triphosphate (ATP) signaling inhibits bystander cell
motility during T-cell activation in LNs.
A, B Multiphoton analyses of T-cell motility in LN slices. Mouse LN slices
containing either OVA-pulsed or unpulsed DCs were overlaid with murine
CD4+ T cells with different antigen specificity (OVA or wild-type). The
graph shows the motility of T cells from WT mice (WT T bystander cells)
(A) or from OT-II mice (triggered T cells) (B). Control condition was LN
slices containing unpulsed DC with both OT-II and WT T cells (nDC). The
migration speed of bystander WT T cells (A) decreased significantly when
OT-II T cells were stimulated by pulsed DCs (OVA-DC), but migration
speed returned to control conditions with the addition of apyrase (OVADC + apy) or pre-treatment of WT T cells with suramin, confirming that
ATP signaling modulated the motility of bystander T cells within the LN
microenvironment. Interestingly, the motility of OT-II T cells was not
affected by apyrase or suramin (B). (*P < 0.05, **P < 0.01; each dot
represents one cell in the LN slice; n = 4 independent experiments;
representative graphs from three experiments are shown here; ns, not
significant). Related time-lapse recordings are shown in Supplementary
Videos S6–S8.
the bystander stimulation of T cells. However, we cannot exclude
that in distinct T-cell subpopulations P2X1 may also play a role in
bystander signaling.
We propose that ATP released by T cells during priming not only
exerts autocrine effects that may potentiate activation, but also
paracrine effects on the other lymphocytes present in the LN microenvironment. Indeed, in agreement with previous studies (Miller
et al, 2004a,b), we observed that OVA-specific T cells slowed down
their velocity in lymph node slices in the presence of OVA-pulsed
DCs. Interestingly, in the same slices, unstimulated wild-type T cells
also reduced their motility, although the great majority of them
could not form conjugates with OVA-presenting DCs. The slowing
down of wild-type T cells was clearly due to the simultaneous triggering of OT-II T cells, as demonstrated by the control experiments
without the OVA peptide, and this depended on the release of ATP
in the LN microenvironment, as demonstrated by the apyrase
condition. These results are in line with recent studies of T-cell
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Chiuhui Mary Wang et al
motility in the LN microenvironment that have demonstrated a
significant drop in the velocity of polyclonal T cells during antigenic
stimulation of TCR-specific cells (McKee et al, 2013; Salgado-Pabon
et al, 2013). The reduced motility of T lymphocytes in a tissue
where antigenic recognition is occurring may be strategic for a
better scanning of resident DCs and, in this perspective, our data
provide an additional and novel mechanism to the emerging
concept that the extracellular ATP is an ‘alert signal’ for the
immune system. We have recently demonstrated the existence of a
delicate equilibrium between adhesive and chemoattractant forces
operating in lymph nodes during T-cell priming, allowing enough
motility for T-cell repertoire scanning while ensuring the formation
of long-lasting conjugates, once a cognate T-APC pair is formed
(Viola et al, 2006; Kallikourdis et al, 2013). Variations in this
equilibrium, such as those due to an hyperfunctional mutant
CXCR4, leads to impaired stability of the immunological synapse
and consequently contribute to an aberrant adaptive immune
response (Kallikourdis et al, 2013). ATP may thus alter this equilibrium and modify T-cell priming. Interestingly, extracellular ATP can
be modulated by CD39 and CD73, two cell surface ecto-enzymes
that hydrolyze extracellular ATP to ADP, AMP and adenosine
(Yegutkin, 2008). These ecto-enzymes are expressed at high levels
by CD4+FoxP3+ regulatory T cells (Borsellino et al, 2007; Deaglio
et al, 2007) but are also upregulated by intratumour CD8+ regulatory T cells (Parodi et al, 2013), and other immune cells when activated in the presence of TGFb (Regateiro et al, 2011), as well as by
neoplastic vascular cells (Feng et al, 2011). The immune suppressive role of CD39 and CD73 has been attributed to the production of
adenosine, that, acting through the A2A receptor, prevents activation and proliferation of CD4+ T cells and exerts anti-inflammatory
effects on dendritic cells, macrophages, neutrophils and other
immune cells (Regateiro et al, 2013). Importantly, our data suggest
that CD39 and CD73 may also contribute to the suppression of
immune responses by interfering with the paracrine effect of extracellular ATP and may further explain how regulatory T cells destabilize T-dendritic cell conjugates in vivo (Tadokoro et al, 2006;
Tang et al, 2006).
Using an approach similar to the one described in our study
(two-photon microscopy in thymus slices), it was demonstrated
that Ca2+ signaling during positive selection inhibits motility and
prolongs interactions with stromal cells, reinforcing the interaction
that is essential during differentiation from double-positive thymocytes to mature T cells (Bhakta & Lewis, 2005; Cahalan, 2005). In
this study, we confirm the role of Ca2+ in tuning lymphocyte
motility in tissues and propose that ATP is responsible for modulation of motility in unstimulated T cells in a reactive lymph
node. Thus, the ‘stop’ signal mediated by Ca2+ influx involves
not only cells that have already found their antigenic partners,
but also lymphocytes that may be potentially triggered within the
tissue.
The mechanism by which increases in [Ca2+]i inhibit lymphocyte motility is not entirely understood. In Dictyostelium discoideum,
increases in [Ca2+]i induce phosphorylation of myosin heavy chain
IIA at a site (Thr1939) that disrupts myosin bundling, thus affecting
the cell’s traction force (Jacobelli et al, 2004). Although this may be
relevant for mouse cells (Jacobelli et al, 2004), the human isoform
contains an alanine substitution at this site (Buxton & Adelstein,
2000), suggesting that Ca2+ signaling inhibits motility through
ª 2014 The Authors
Published online: May 19, 2014
Chiuhui Mary Wang et al
additional mechanisms. Increase in [Ca2+]i is known to trap mitochondria at specific subcellular sites of high Ca2+ microdomains
(Rizzuto et al, 2004; Yi et al, 2004). We have previously shown that
accumulation of mitochondria at the uropode of migrating cells is
required to sustain phosphorylation of the myosin light chain
(MLC), a key step in high-speed moving cells (Campello et al,
2006). Trapping of mitochondria is therefore a possible alternative
mechanism responsible for motility arrest upon increases in [Ca2+]i
in lymphocytes.
In addition to our data, recent evidence strengthens the
concept of ATP as a key player in the inflammatory microenvironment, regulating the release of cytokines and chemokines (Rayah
et al, 2012) and triggering the activation of the Nrlp3 inflammasome (Iyer et al, 2009). Interestingly, in tumors, ATP appears to
be crucial for infiltration, differentiation and maintenance of
myeloid cells in the tumor upon chemotherapy (Chen et al, 2006;
Ma et al, 2013). Our data propose a new role for ATP in immunity through the paracrine regulation of lymphocyte motility
during antigen recognition. In future studies, it would be important to evaluate the consequences of these findings in the context
of anti-tumor immunity.
Materials and Methods
Cells
Human peripheral CD4+ cells were isolated from whole human
blood collected by Desio Hospital, Milan, in accordance to institutional guidelines with patient consent for research purposes. The
CD4+ lymphocytes were isolated using RosetteSep CD4+ Enrichment Cocktail (Stemcell) and cultured in RPMI supplemented with
L-glutamine, non-essential amino acids, sodium pyruvate and
10% FBS. Cells were used for experiments 1 or 2 days after isolation. The purity of the CD4+ was confirmed with FACs staining
and analysis. The Jurkat clone E6.1 was maintained in the same
medium.
Mice
Wild-type C57BL/6J (H-2b) and OT-II TCR transgenic mice were
purchased from Charles River Laboratories (Italy). Mice were kept
in regular light and dark cycles, with unrestricted access to water
and food. Mice were kept under Italian national and EU directives
(2010/63/EU) for animal research with protocols approved by
institute Ethical Committee and the Italian Ministry of Health
(162/2011-B).
Cell sorting
Total peripheral CD4+ lymphocytes were sorted with BD FACsAria
cell sorter to T naive (CD45ROCD45RA+CD62L+), T central
memory (CD45RO+CD45RACD62L+) and T effector memory
(CD45RO+CD45RACD62L) subpopulations (Sallusto et al, 2004).
Briefly, total CD4+ T cells were stained with conjugated antibodies
against CD4, CD45RO, CD45RA and CD62L (BD Biosciences) in PBS
containing 1% of FBS for 30 min in 4°C. The stained cells were
washed twice and sorted straight away.
ª 2014 The Authors
The EMBO Journal
Extracellular ATP alters T-cell motility
Confocal calcium imaging
The cell membrane permeable ester of caged-IP3-PM (2 lM,
SiChem, Bremen, Germany) was loaded into total CD4+ T cells, or
for some experiments in CD4+ subpopulations or Jurkat T cells,
simultaneously with calcium indicators Fluo-4-AM (3.5 lM) and
FuraRed-AM (6 lM, Molecular Probes) for 30 min at 37°C (Li et al,
1998). After washing, the loaded T cells were seeded on coverslips
coated with poly-L-lysine (Sigma-Aldrich) and allowed to settle for
10 min in imaging buffer (RPMI without phenol red with 1% FBS).
An Olympus FluoView FV1000 laser scanning confocal microscope
fitted with two scanning lasers was used to acquire time-lapse
images of calcium change. The SIM UV laser (405 nm, 45% power,
80 ms pulse) was used to release active IP3 within a region of interest (ROI), and time-lapse images were acquired every 3 s for 3 or
5 min in a 37°C heated imaging chamber. The images were acquired
with a 60× oil immersion objective with numerical aperture 1.35
(Plan-Apochromat, Olympus). Fluo-4 and FuraRed fluorophores
were excited simultaneously with a 488-nm laser, and the emissions
were collected with 500- to 550-nm and 620- to 680-nm band-pass
filters. Differential interference contrast (Nomarski technique) was
also used. In some experiments ATPase apyrase (5 U/ml, SigmaAldrich) was added to the imaging buffer. Additional control experiments using heat-inactivated apyrase (10 min in 100°C; De Miranda
et al, 2002), PBS vehicle or T cells loaded with calcium indicators
alone were performed. To confirm the role of P2X receptors, experiments were performed on T cells pre-incubated with suramin
(45-min pre-incubation, 100 lM). Fluorescent intensities of Fluo-4
and FuraRed were used for ratiometric calcium-level calculations in
NIH ImageJ (n = 6 independent experiments).
ATP quantification
The amount of ATP released by CD4+ T cells after UV photolysis of
caged-IP3 and stimulation with anti-CD3 (2 lg/ml) and anti-CD28
(1 lg/ml) antibodies was quantified using ATP Bioluminescence
Assay Kit HS II (Roche). 106 CD4+ T cells were loaded with cagedIP3, resuspended in 100 ll HBSS and plated in a 96-well plate
(n = 7 donors). Caged-IP3 was released by photolysis (100 ms,
DAPI filter) using an epi-fluorescence microscope (Olympus IX81
CellR microscope, UPLFLN 4× objective numerical aperture 0.13).
106 unmanipulated, resting T cells were placed in Eppendorf tubes
and stimulated with anti-CD3 and anti-CD28 antibodies (n = 4
donors). The plate/samples were kept in 37°C for 10 min, after
which the samples were collected and spun down for 5 min in a 4°C
microfuge. For each sample, 50 ll of supernatant was collected and
transferred into a black microplate for ATP quantification. Controls
were T cells that were not loaded with caged-IP3 and that underwent
the same flash photolysis or unstimulated T cells. ATP measurement
was carried out using manufacturer’s protocol. Bioluminescence
signals were detected using the Synergy H4 microplate reader (BioTek). Data were exported and analyzed with Prism 4 (GraphPad).
RNA and Real-Time PCR
Peripheral CD4+ lymphocytes and Jurkat T cells were centrifuged and washed once with PBS without calcium or magnesium
(Biowhittaker, Lonza). The pellets were used for RNA isolation with
The EMBO Journal
7
Published online: May 19, 2014
The EMBO Journal
RNeasy mini kit (Qiagen), and the RNA is reverse-transcribed into a
cDNA library using First Strand Synthesis Kit (Sigma). Purinergic
receptor expressions in the different cell populations were detected
by reverse transcriptase PCR with a panel of primers (Ihara et al,
2005) and quantified with SYBR green-based quantitative real-time
PCR systems (Applied Biosystems; n = 4 donors). Actin was used as
internal reaction control.
Identification of purinergic receptors
To distinguish the involvement of P2X or P2Y families of receptors,
the same confocal calcium imaging experiment using human CD4+
T cells was repeated but using phosphate-buffered saline with or
without calcium or magnesium (Biowhittaker, Lonza) as the imaging buffer. Pharmacological purinergic receptor blockers NF023
(10 lM), 5-BDBD (100 lM), KN62 (1 lM) and A438073 (5 lM)
were used to identify the receptors involved in paracrine ATP signaling (TOCRIS, Bristol, UK). Alternatively, two pairs of siRNAs to
P2X4 and P2X7 were used to knockdown gene expression (s9955,
s9956 and s9959, s9960, respectively, Applied Biosciences), and a
scrambled siRNA was included as control. Primary CD4+ lymphocytes were transfected with 3 lg of siRNA using the Amaxa Human
T Cell Nucleofector kit (Lonza). Expression knockdown was
confirmed with real-time PCR in duplicates. CD4+ T cells pretreated with purinergic receptors blockers for 40 min prior to ROI
uncaging and time-lapse imaging as described previously (n = 4).
Calcium influx in response to extracellular ATP (100 lM) or soluble
anti-CD3 (2 lg/ml, OKT3 clone) and anti-CD28 (1 lg/ml) antibodies (eBiosciences) was also analyzed with FAC Canto flow cytometry (BD Biosciences). CD4+ T cells were loaded with calcium
indicators Fluo-4-AM and FuraRed-AM as described previously
(Contento et al, 2010). Basal intracellular calcium level was
acquired for 45 or 60 s before addition of extracellular ATP or the
antibodies. Samples were acquired for a further 3 or 5 min, and
ionomycin (1 lg/ml, Sigma) was added at the end as positive
control for maximum calcium influx. Data were analyzed with
FlowJo Software version 7.6.4 (Tree Star, Inc.) and normalized to
the mean basal intracellular calcium level.
Ex vivo lymph node (LN) preparation and calcium wave imaging
Fresh inguinal LNs were collected from 10-week-old adult C57BL/6J
mice, embedded in low-melt agarose (Sigma) and cut with a vibratome to 300-lm slices (Asperti-Boursin et al, 2007; Salmon et al,
2011). The slices then were loaded with calcium indicator Fluo-4
(7 lM, Molecular Probes) and caged-IP3 (3 lM, SiChem, Bremen,
Germany) in RPMI containing 1% FBS and 0.2% pluronic acid
(Sigma) in a 37°C incubator for 45 min. The slices were rinsed
gently in fresh RPMI without phenol red (Lonza) and imaged on the
confocal microscope with heated imaging chamber (Olympus FluoView FV1000, n = 5 independent experiments). A ROI was selected
for uncaging IP3 with the SIM laser (80% laser power, 80 ms pulse),
and time-lapse images of Fluo-4 intensity changes were acquired
over a period of 3 min at 3 s per frame with a 40× numerical aperture 1.30 objective (excitation 488 nm laser and emission 500- to
550-nm band-pass filter). Apyrase (5 U/ml, Sigma) was added to
the imaging buffer in some of the experiments. We have observed
calcium waves in C57BL/6J murine LNs despite the known DNA
8
The EMBO Journal
Extracellular ATP alters T-cell motility
Chiuhui Mary Wang et al
polymorphism in the p2x7 gene that reduces receptor function. LN
slides loaded with Fluo-4 only (no caged-IP3) were negative controls
for calcium wave.
In vitro migration assay
Human peripheral CD4+ T cells were seeded in a cell microscopy
l-slide coated with fibronectin (ibidi, GmBH, Germany). Cells were
allowed to migrate toward a CXCL12 gradient (2.5 nM, R&D
Systems). Differential interference contrast images were acquired
every 30 s for 20 min using an inverted Olympus IX81 CellR microscope with a 20× (0.5 numerical aperture) objective and Olympus
IX70 FluoView Camera. Depending on the experiment, T cells were
imaged in RPMI without phenol red plus 1% FBS or phosphatebuffered saline with or without calcium and magnesium (Biowhittaker, Lonza) in a 37°C chamber buffered with 5% carbon dioxide
and 40% humidity (n = 3). Extracellular ATP (100 lM) was added
to the cells in some of the experimental conditions. Suramin
(100 lM, Sigma) was used as a pan-P2X receptor antagonist. Cell
migration tracks were analyzed and plotted using ibidi Chemotaxis
and Migration Tool (version 2.0).
T-cell migration in lymph node slices (OT-II, WT)
Wild-type C57Bl/6 and OT-II transgenic mice were obtained from
Charles River Laboratories (Calco, Italy). OT-II transgenic mice
express a TCRab transgene that is specific for the OVA peptide 323–
339 (TCR-OVA). Wild-type CD4+ lymphocytes do not express TCRs
that significantly recognize the OVA peptide and are therefore used
as bystander T cells. Ex vivo T-cell activation visualization using
multiphoton microscopy has been described elsewhere (AspertiBoursin et al, 2007; Kallikourdis et al, 2013). Briefly, dendritic cells
were matured from bone marrow (Dal Secco et al, 2009). The day
before the experiment, DCs were labeled with CellTracker Orange
CMTMR (5 lM, Molecular Probes, Invitrogen) and pulsed for 2 h
with OVA323-339 peptide (10 lg/ml, Anaspec) or PBS vehicle. The
DCs were injected intraperitoneally near the inguinal LNs with lipopolysaccharide (40 ng/mouse) in PBS, at a concentration of 4
million cells per mouse. On the day of the experiment, lymph nodes
containing labeled DCs were cut into slices as described previously
for the calcium wave experiments. CD4+ T cells from OT-II and
wild-type mice were negatively selected from LNs using the mouse
CD4+ T-cell isolation kit II (MACS Miltenyl Biotec) and labeled with
the CellTracker Blue CMAC (40 lM, Molecular Probes) or CFSE
(5 lM, Molecular Probes), respectively, following manufacturer’s
protocols. Labeled OT-II and wild-type T cells were overlaid on to
the slices and allowed to integrate for 60 min in a 37°C incubator.
Cell interactions within a tissue volume 300 × 300 × 50 lm at 5 lm
z-steps was visualized using a two-photon microscope (LaVision
TrimScope) fitted with a 20× water immersion objective (Olympus
XLUMPFL, numerical aperture 1.0). The slices were imaged in
oxygenated phenol red free RPMI supplemented with L-glutamine,
pen-strep and HEPES. In some experiments, apyrase 2 U/ml was
added to the running buffer. In the suramin condition, WT T cells
were pre-incubated with 100 lM suramin (irreversible) for 1 h and
washed before overlaying onto the slices. Time-lapse images were
acquired every 30 s for 30 min and exported to Imaris (version
7.3.2, Bitplane) for analysis (n = 6).
ª 2014 The Authors
Published online: May 19, 2014
Chiuhui Mary Wang et al
The EMBO Journal
Extracellular ATP alters T-cell motility
Statistical analysis
Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune
suppression. Blood 110: 1225 – 1232
Data analysis was performed using Prism 4 (GraphPad, USA).
Student’s t-test or ANOVA followed by Bonferroni post hoc test were
used to compare groups against the control condition. Wilcoxon
matched pairs test was used to compare the calcium response in T
cells over the course of time-lapse video in control or presence of
apyrase. Statistical significance was taken at P < 0.05; n value represents the number of independent experiments.
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Acknowledgements
The work was supported by grants from EC FP7 Program SYBILLA (grant agreement HEALTH-F4-2008-201106) and MIUR—PRIN 2009 (Protocol 2009NREAT2) to AV. The authors wish to thank R. L. Contento and P. Pinton for
essential advice, D. Morone and J. Cibella for technical assistance, A. Anselmo
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Author contributions
Sarukhan A (2009) Tunable chemokine production by antigen presenting
CMW performed the majority of the experiments and prepared the figures; FA
dendritic cells in response to changes in regulatory T cell frequency in
and CP prepared samples for the multiphoton experiment; CMW, AS and AV
wrote the paper. CMW and AV conceived the experiments AV provided
funding.
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Conflict of interest
The authors declare that they have no conflict of interest.
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