Research ArticleImmunology

Purinergic Control of T Cell Activation by ATP Released Through Pannexin-1 Hemichannels

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Science Signaling  30 Sep 2008:
Vol. 1, Issue 39, pp. ra6
DOI: 10.1126/scisignal.1160583


T cell receptor (TCR) stimulation results in the influx of Ca2+, which is buffered by mitochondria and promotes adenosine triphosphate (ATP) synthesis. We found that ATP released from activated T cells through pannexin-1 hemichannels activated purinergic P2X receptors (P2XRs) to sustain mitogen-activated protein kinase (MAPK) signaling. P2XR antagonists, such as oxidized ATP (oATP), blunted MAPK activation in stimulated T cells, but did not affect the nuclear translocation of the transcription factor nuclear factor of activated T cells, thus promoting T cell anergy. In vivo administration of oATP blocked the onset of diabetes mediated by anti-islet TCR transgenic T cells and impaired the development of colitogenic T cells in inflammatory bowel disease. Thus, pharmacological inhibition of ATP release and signaling could be beneficial in treating T cell–mediated inflammatory diseases.


Elevation in the concentration of cytosolic Ca2+ represents an essential component of the regulation of the adaptive immune response. The interaction of the αβ T cell receptor (TCR) with cognate antigen results in the activation of phospholipase C–γ in the T cell, which generates inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) from phosphatidylinositol 4,5 bisphosphate (PIP2) in the plasma membrane, resulting in IP3-induced Ca2+ release from the endoplasmic reticulum (ER) (1). Depletion of ER stores of Ca2+ stimulates stromal interaction molecule–mediated activation of the Ca2+-permeable Ca2+ release–activated Ca2+ (CRAC) channels, which were recently identified as Orai1/CRACM1, in the plasma membrane (26). This results in a sustained influx of Ca2+ from the extracellular space into the cytosol, termed capacitative Ca2+ entry (CCE) (7), which constitutes an indispensable event for efficient T cell activation (8).

The increase in the concentration of cytosolic Ca2+ is accompanied by active mitochondrial uptake of Ca2+. Mitochondria serve as a high-capacity Ca2+ sink, thus avoiding cellular Ca2+ overload, and contribute to a rapid clearing of Ca2+ in spatially restricted areas, such as near Ca2+ channels in the plasma membrane or the ER (9). Moreover, mitochondrial uptake of Ca2+ stimulates the aerobic synthesis of adenosine triphosphate (ATP) through the activation of the pyruvate, α-ketoglutarate, and isocitrate dehydrogenases (10, 11). Ca2+-dependent synthesis of ATP in mitochondria meets the higher energy demands of stimulated T cells compared with those of resting T cells (12). Sustained saturation of the mitochondrial electron transport chain by Ca2+ also generates reactive oxygen species, which are required for T cell proliferation (13). Finally, activation of costimulatory receptors, such as CD28, increases glucose uptake and induces a metabolic change from the use of oxidative phosphorylation to high-throughput glycolysis, which results in faster production of ATP and generation of metabolic intermediates critical for cell growth (14, 15). ATP synthesis, either through oxidative phosphorylation or through glycolysis, usually exceeds the cellular energy demand (12, 15).

Environmental signals induce the release of ATP from cells either through secretory vesicles (16, 17) or through mechano- or osmosensitive nonjunctional (hemi-) channels, which allow the diffusion of ions and small molecules across the plasma membrane (18, 19). Under normal conditions, immune cells are exposed to negligibly low concentrations of extracellular ATP. Moreover, extracellular ATP is readily degraded by the combined actions of the ubiquitous ectonucleotidases ectoapyrase (CD39) and ecto-5′-nucleotidase (CD73) (20). In the immune system, ATP is recognized as a damage-associated molecular pattern (DAMP); once released from dying or activated cells, it contributes to the efficient priming of the innate immune system in inflammatory settings together with pathogen-associated molecular patterns (PAMPs). Plasma membrane receptors for extracellular nucleotides, termed P2 receptors, which are divided into two families, P2X and P2Y, are found on almost all cell types (21, 22). P2X1 to P2X7 receptors are permeable to monovalent cations, and some P2X receptors (P2XRs) exhibit substantial permeability to Ca2+ or anions, whereas P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 to P2Y14 receptors are guanine nucleotide–binding protein–coupled receptors, which bind preferentially to adenosine diphosphate, uridine diphosphate, uridine triphosphate, or uridine diphosphate glucose. Activation of P2 receptors on T cells was suggested to contribute to the outcome of TCR stimulation (2325). However, it is not clear how extracellular ATP at sites of injury and inflammation might influence T cell function.

The chaperone protein calreticulin (CRT) is the most important buffer of Ca2+ in the ER (26). Lymphopenic mice that receive fetal liver hematopoietic progenitors from CRT-deficient (crt−/−) embryos by adoptive transfer display severe T cell–dependent immunopathological traits including alopecia, blepharitis, and wasting syndrome (27). In crt−/− T cells, altered regulation of Ca2+ influx after TCR stimulation causes pulsatile elevations in cytosolic Ca2+, protracted nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT), and sustained activation of mitogen-activated protein kinase (MAPK) signaling, which result in exaggerated T cell responsiveness (27). In this study, we show that increased ATP production and release from activated crt−/− T cells is a critical pathogenic determinant in these animals. ATP released from activated T cells through pannexin-1 (panx1) hemichannels acts as an essential autocrine costimulatory signal through P2X purinergic receptors. The inhibition of this signaling pathway in vitro resulted in diminished interleukin-2 (IL-2) secretion and T cell proliferation leading to T cell anergy. In addition, inhibition of P2XR signaling substantially rescued T cell–mediated inflammation in vivo.


Increased mitochondrial uptake of Ca2+ in crt−/− T cells

Mitochondrial buffering of cytosolic Ca2+ influences Ca2+-dependent inactivation of CRAC channels by removing Ca2+ ions from the proximity of the channel mouth (2830). Because the abundance of CRT in the ER is inversely correlated with Ca2+ uptake by mitochondria (31), we investigated whether mitochondrial uptake of Ca2+ in crt−/− T cells might be higher than that in wild-type cells. Thapsigargin is a cell-permeable inhibitor of the sarcoendoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase), which pumps Ca2+ into the ER; as such, thapsigargin causes depletion of ER Ca2+ stores, which results in the activation of CRAC channels in the plasma membrane. To assess mitochondrial buffering of Ca2+ in crt−/− T cells, we depleted thapsigargin-sensitive stores of Fura-2–loaded T cell clones in Ca2+-free medium and subjected the cells to two sequential additions of 0.5 mM Ca2+ (fig. S1A). Because the rate of increase in cytosolic Ca2+ depends on the activity of CRAC channels, quantification of this parameter at the first and the second addition of extracellular Ca2+ enabled the estimation of the degree of Ca2+-dependent inactivation of CRAC channels (32). We measured a 57% reduction in CRAC activity at the second addition of extracellular Ca2+ in crt+/+ cells, which is in agreement with previous reports (28, 32). However, in crt−/− cells, only 14% inactivation of CRAC channels was observed at the second addition of extracellular Ca2+ (Fig. 1A). Inhibition of mitochondrial uptake of Ca2+ by CCCP, a protonophore and uncoupler of electron transport from oxidative phosphorylation in mitochondria, led to comparable Ca2+-dependent inactivation of CRAC channels in both crt−/− and crt+/+ cells (Fig. 1B), thus confirming the contribution of mitochondria to this phenomenon. Western blotting analysis of the abundance of cytochrome c oxidase (COX) subunit IV protein and flow cytometric analysis of nonyl acridine orange staining (not shown), both measurements of mitochondrial mass, and flow cytometric analysis of mitochondrial membrane potential (fig. S1B) revealed no differences between crt−/− and crt+/+ cells.

Fig. 1

Increased mitochondrial buffering of Ca2+ in crt−/− T cells. (A) crt−/− T cell clones were treated with thapsigargin followed by two separate additions of Ca2+ to the extracellular medium (see also fig. S1A). The rate of rise in the concentration of cytosolic Ca2+ was calculated at the first and second addition of Ca2+. A significant reduction in Ca2+ influx was observed in crt+/+ but not crt−/− T cells. (B) When the same experiment was repeated in the presence of a mitochondrial uncoupler (CCCP), CRAC inactivation was comparable between crt+/+ and crt−/− T cells. (C) After depletion of Ca2+ from the ER with thapsigargin (TG), CCE was induced in crt−/− and crt+/+ T cell clones by the addition of 0.5 mM Ca2+ to the extracellular medium (black bar). After complete washout of extracellular Ca2+, the mitochondrial uptake of Ca2+ was visualized by the addition of ionomycin. (D) Histograms represent ionomycin-stimulated release of Ca2+ from mitochondria after CCE with 0.5, 1, or 2 mM extracellular Ca2+ in crt−/− and crt+/+ T cell clones. *P < 0.05, ***P < 0.0001.

We investigated the mitochondrial uptake of Ca2+ in Fura-2–loaded crt−/− and crt+/+ T cells in which Ca2+ stores were depleted with thapsigargin in Ca2+-free medium followed by addition of 0.5 mM Ca2+ to induce CCE. After complete washout of extracellular Ca2+, ionomycin was added to release Ca2+ that had accumulated in mitochondria during CCE (32). Mitochondrial buffering of Ca2+ was higher in crt−/− cells than in crt+/+ cells, although the amplitude of the elevation in cytosolic Ca2+ during CCE was similar in both cell types (Fig. 1C). If ionomycin was added before 0.5 mM Ca2+, no differences were observed between crt−/− and crt+/+ T cells (not shown), thereby confirming that Ca2+ had accumulated in mitochondria during CCE. This also excludes constitutive mitochondrial Ca2+ overload in crt−/− cells as an explanation for the difference in Ca2+ buffering between crt+/+ and crt−/− cells. The increase in extracellular Ca2+ concentrations after Ca2+ store depletion was paralleled by an increase in mitochondrial uptake of Ca2+ in crt+/+, but not crt−/−, T cells (Fig. 1D). Indeed, mitochondrial buffering remained constant in crt−/− T cells in a range of 0.5 to 2 mM extracellular Ca2+. Together, these results indicate that crt−/− CD4+ T cells have an enhanced mitochondrial Ca2+-buffering potential, which results in a slower Ca2+-dependent inactivation of CRAC channels than is observed in crt+/+ CD4+ T cells.

TCR-induced synthesis of ATP and its release through panx1 hemichannels

The uptake of Ca2+ by mitochondria stimulates their metabolic activity and the oxidative synthesis of ATP (10, 11). Capacitative Ca2+ entry induced by antigenic activation of crt−/− CD4+ T cells might therefore be linked to higher mitochondrial production of ATP. Indeed, after stimulation of naïve CD4+ T cells with anti-CD3 antibodies, we detected higher concentrations of intracellular ATP in crt−/− cells than in crt+/+ cells (Fig. 2A). Treatment with the mitochondrial ATP synthase inhibitor oligomycin abolished activation-induced synthesis of ATP, thus showing its dependence on mitochondrial respiration (Fig. 2B).

Fig. 2

Mitochondrial uptake of Ca2+ during T cell activation leads to the synthesis and release of ATP. (A) Fold increase in the concentrations of cytosolic ATP in sorted naïve CD4+ T cells from crt−/− and crt+/+ mice at different time points after T cell activation with anti-CD3 antibodies. (B) ATP production upon T cell activation in the absence (control) or presence of the ATP synthetase inhibitor oligomycin. (C) Naïve CD4+ T cells were stained with the nucleotide-binding compound quinacrine. The homogenous cytosolic staining indicates the absence of secretory vesicles containing ATP. (D) Subcellular fractionation of nonstimulated (black trace) and activated (red trace) T cell clones on a continuous sucrose gradient. ATP was detected only in fractions that also contained the cytosolic protein ZAP-70 and not in those fractions characterized by the small vesicular marker cellubrevin. Western blots incubated with anti-CD3 and anti–COX subunit 4 (COX4) antibodies revealed fractions containing plasma membranes and mitochondria, respectively. (E) Preincubation of naïve CD4+ T cells with the pannexin-blocking peptide 10panx1 resulted in higher increases in intracellular ATP concentrations compared to untreated cells after treatment with anti-CD3 antibodies. (F) T cells stimulated with anti-CD3 antibody–coated beads released ATP through a carbenoxolone (cbx)- but not α18-glycyrrhetinic acid (α18GA)–sensitive pathway (see also fig. S2, A and B). ctrl, control.

Recently, elegant experiments have shown that ATP is released into the extracellular space upon TCR stimulation (33, 34). The release of ATP through vesicles (16, 17, 34) and plasma membrane–localized hemichannels (18, 19) has been reported. We determined the subcellular distribution of ATP with the fluorescent, nucleotide-binding compound quinacrine (35). Quinacrine staining of ex vivo purified naïve CD4+ T cells showed a homogenous cytosolic distribution of ATP without any evidence of the vesicular staining that is reminiscent of secretory granules (Fig. 2C). As a further confirmation of the cytosolic localization of ATP, we separated T cell homogenates on sucrose gradients and determined that ATP was found exclusively in cytosolic and not membrane fractions. Moreover, T cell stimulation with anti-CD3 antibodies before fractionation resulted in the increased recovery of ATP in cytosolic fractions compared to that in cytosolic fractions from untreated cells (Fig. 2D). These results exclude a mechanism in which release of ATP from activated T cells is vesicular and suggest that ATP may be released through either connexin (19) or pannexin (18, 36) hemichannels.

Pannexin-1 allows the passage of small molecules between the cytoplasm and the extracellular space; its messenger RNA is highly expressed in T cells (not shown), and it was recently identified as a plasma membrane hemichannel sensitive to cytoplasmic Ca2+ (18, 37, 38). We hypothesized that stimulated T cells would accumulate higher concentrations of intracellular ATP if they were in the presence of connexin or pannexin hemichannel–blocking peptides. Incubation of naïve T cells with the panx1-blocking peptide 10panx1 (39) led to a faster accumulation and higher concentration of intracellular ATP compared to that in untreated cells (Fig. 2E). In contrast, the connexin-inhibitory peptide Gap26 (40) did not substantially affect the intracellular ATP concentration (not shown), thus pointing to a role for panx1 hemichannels in ATP release from T cells. We measured ATP secretion with a recently described two-enzyme assay (33). Micromolar concentrations of ATP were detected in the pericellular area of sorted naïve CD4+ T cells after synapse formation with anti-CD3 antibody–coated beads (Fig. 2F and fig. S2, A and B). Carbenoxolone is a licorice derivative that preferentially (albeit nonselectively) inhibits panx1 rather than connexin hemichannels in the concentration range of 1 to 20 μM (41, 42). ATP release was significantly reduced when cells were incubated with 5 μM carbenoxolone before and during TCR stimulation compared to that of control cells, thus supporting a role for panx1 hemichannels in the release of ATP upon T cell activation. In contrast, ATP release was modestly reduced by treatment with α18-glycyrrhetinic acid at doses that partially inhibit pannexin hemichannels but potently block Cx43 hemichannel permeability (43) (Fig. 2F).

Elevations in cytosolic Ca2+ induce the opening of panx1 hemichannels, which renders the cell permeable to fluorescent dyes such as carboxyfluorescein. To verify that TCR-stimulated CCE induced the opening of panx1 hemichannels, we stimulated naïve CD4+ T cells in the presence of carboxyfluorescein. Higher amounts of the dye were taken up by anti-CD3 antibody–stimulated cells than were taken up by unstimulated cells. In agreement with the results obtained by measuring release of ATP, we detected 77% inhibition of carboxyfluorescein uptake by treatment with 5 μM carbenoxolone, whereas 50 μM α18-glycyrrhetinic acid resulted in only a 26% reduction in carboxyfluorescein uptake (fig. S2C). Because stimulation of P2X7 receptors promotes the activation of panx1 (39), we tested the effect of oxidized ATP (oATP), which preferentially inhibits P2XRs (44), on the opening of panx1 hemichannels after triggering of the TCR. The release of ATP (fig. S2E) and the uptake of carboxyfluorescein (fig. S2C), induced by stimulation with anti-CD3 antibodies, were both significantly inhibited by oATP, indicating that autocrine P2X7 stimulation exerts a positive feedback on the permeability of panx1 hemichannels.

Protracted MAPK activation in crt−/− T cell by autocrine activation of P2XRs

Extracellular ATP has been suggested to act as a costimulator in the mitogenic response of T cells (24). We hypothesized that the hyperresponsiveness of crt−/− T cells could be a result of the increased production and release of ATP upon antigen encounter, leading to the protracted activation of MAPK that we observed in crt−/− cells (27). Messenger RNAs for P2X1, P2X4, and P2X7 as well as for P2Y1, P2Y12, P2Y13, and P2Y14 were detected in T cells by reverse transcription polymerase chain reaction (RT-PCR) assays (fig. S4A). The functional competence of these receptors was confirmed in Ca2+-imaging experiments performed with preferential agonists (fig. S4, C and D). To verify whether autocrine activation of P2 receptors might play a role in crt−/− T cell hyperresponsiveness, crt−/− CD4+ T cells were stimulated with anti-CD3 antibodies for 16 hours in the presence of oligomycin, PPADS (a nonspecific P2 receptor antagonist), or oATP. The prolonged activation of extracellular signal–regulated kinase (ERK) and p38 MAPK detected in untreated cells was almost completely abolished by the various pharmacological agents (Fig. 3A). The decreased MAPK activation after treatment with oligomycin, PPADS, or oATP was not caused by cellular damage as a result of toxicity, because propidium iodide staining of treated cells (an indicator of cell death) was not different from that of untreated cells (not shown). Despite prominent inhibition of MAPK signaling, both P2 receptor antagonists did not affect the nuclear translocation of NFAT, which was instead inhibited by treatment with oligomycin (Fig. 3A). These results are in accordance with an earlier report that showed that treatment with oATP does not influence TCR-dependent elevations in cytosolic Ca2+ (24), which is necessary for the nuclear translocation of NFAT. Our results suggest an autocrine costimulatory role for extracellular ATP released on T cell activation.

Fig. 3

Role of pericellular ATP in protracted MAPK activation after TCR stimulation. (A) crt−/− T cell clones were stimulated with anti-CD3 antibodies (αCD3) for 16 hours (h). Western blotting analyses shown in the panels on the left show the characteristic protracted phosphorylation of p38 MAPK (P-p38) and ERK (P-ERK) and the nuclear translocation of NFAT after 16 hours of stimulation (27). Activation of these molecules was abolished by oligomycin, an inhibitor of mitochondrial ATP synthesis, whereas the P2 receptor antagonists oATP and PPADS inhibited MAPK activation but not nuclear translocation of NFAT. ctrl, control. (B) Inhibition of TCR signaling by the Src-like kinase inhibitor PP2 after 30 min of T cell stimulation did not affect the synthesis of ATP. (C) crt−/− T cell clones were stimulated with biotinylated anti-CD3 antibodies that were cross-linked by treatment with avidin. After 30 min of stimulation, cells were either left untreated (first panel on the left) or treated with PP2 either alone (second panel) or in combination with oATP (third panel) or ARL67156 (fourth panel). PP2 efficiently inhibited TCR signaling, as shown by dephosphorylation of ZAP-70, whereas protracted ERK activation was less affected. The combination of PP2 with oATP almost completely abolished ERK phosphorylation, whereas the combination of PP2 and the ectonuclease inhibitor ARL67156 increased ERK activation at late time points.

To further confirm this hypothesis, we monitored ERK activation at early time points after stimulation of cells with anti-CD3 antibodies. ERK activation peaked at around 1.5 hours, transiently decreased at 3.5 hours, and peaked again between 5.5 and 7.5 hours after stimulation (Fig. 3C). To distinguish between TCR-dependent and -independent activation of ERK, TCR signaling was blocked with PP2, a pharmacological inhibitor of Src-like kinases, 30 min after the addition of anti-CD3 antibodies. Whereas PP2 affected the TCR-dependent phosphorylation of the proximal kinase ζ chain–associated protein kinase 70 (ZAP-70), it had less effect on ERK phosphorylation at later time points, thus suggesting that ERK activation was maintained independently of TCR signaling (Fig. 3C). However, when PP2 was added in combination with either PPADS (not shown) or oATP, substantial inhibition of ERK phosphorylation was observed. Conversely, an increase in ERK activation was detected when PP2 was combined with ARL67156, an inhibitor of ecto-ATPases, which prolongs the half-life of ATP in the extracellular medium (Fig. 3C). In agreement with these results, we found that the addition of PP2 30 min after T cell activation did not affect the mitochondrial production of ATP (Fig. 3B). These data are consistent with transient elevations in the concentration of mitochondrial Ca2+ determining long-lasting metabolic “priming” that ensures ATP production after the elimination of agonist (10). Analysis of Ca2+ signaling as well as ATP production and release upon TCR stimulation of purified naive, effector/memory, and CD4+CD25+ cells, which include regulatory T cells (Treg), revealed striking reductions of these responses in the Treg subset, which displays poor proliferative potential (fig. S3).

Autocrine activation of P2XRs stimulates IL-2 expression and T cell proliferation

Phosphorylation of ERK during the late phase of T cell activation plays a crucial role in the production of IL-2 (45). We inferred that T cell stimulation in the presence of oATP might inhibit IL-2 production in normal cells. Indeed, stimulation of ex vivo purified naïve and effector/memory (not shown) CD4+ T cells with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of oATP led to a substantial reduction in the amount of secreted IL-2 compared with that of untreated cells. Similar inhibition was observed after treatment with oATP, PPADS (Fig 4B), or 10panx1, but not GAP26 peptide (fig. S2D). Protein kinase C– and RasGRP1 [a guanine nucleotide exchange factor (GEF) for Ras]–mediated activation of ERK by phorbol 12-myristate 13-acetate (PMA) overcame oATP- and PPADS-dependent inhibition of IL-2 production (Fig. 4B). PMA is a DAG mimetic, which in T cells activates RasGRP1 (46), thus triggering ERK phosphorylation and IL-2 secretion (47). Because the increased abundance of CD69 is caused mainly by activation of the RasGRP1–ERK pathway (48), we tested whether the abundance of CD69 in CD4+ naïve T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies was sensitive to oATP. This was indeed the case, and the reduced abundance of CD69 was rescued by PMA (fig. S5A). We concluded that the RasGRP1–ERK–CD69 axis and the secretion of IL-2 were both inhibited by oATP and that both could be rescued by PMA-mediated activation of RasGRP1.

Fig. 4

Pharmacological inhibition of P2 receptors impairs T cell proliferation and IL-2 secretion and implements anergy. (A) Flow cytometric profiles showing dilution of CFSE fluorescence in sorted naïve CD4+ T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the absence (upper panels) or presence (lower panels) of oATP. Addition of IL-2 (middle panels) or PMA (right panels) is indicated. Numbers inside the graph indicate the numbers of cells that had diluted CFSE (proliferating cells) in timed acquisitions. (B) A representative experiment showing IL-2 secretion as measured by ELISA in culture supernatants of naïve CD4+ cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 48 hours alone, in the presence of oATP, or in the presence of PPADS. IL-2 measured in each sample is expressed as a percentage of that measured in the control sample, which was set at 100%. IL-2 secretion in the absence (black bars) or presence (gray bars) of PMA is shown. (C) Quantification of Egr2 and Egr3 transcripts by real time RT-PCR at 2 and 16 hours (h) after stimulation of sorted naïve CD4+ T cells with plate-bound anti-CD3 and anti-CD28 antibodies either in the absence or in the presence of oATP. *P < 0.05. (D) Cytosolic Ca2+ profiles after anti-CD3 stimulation of T cell clones preincubated for 16 hours as indicated.

Baricordi et al. have described an inhibitory effect of oATP on mitogen-induced T cell proliferation (24). We found that oATP inhibited T cell proliferation as measured by the dilution of 5,6-carboxyfluorescein diacetate succinimyl ester (CFSE) in naïve CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies (Fig. 4A). This inhibition was partially rescued by the addition of IL-2 and was completely restored by the addition of PMA. We therefore hypothesize that ATP released from activated T cells is part of an autocrine loop, which exerts positive feedback on TCR-mediated MAPK activation. The hemichannel inhibitors carbenoxolone and 10panx1, but not GAP26, efficiently inhibited T cell proliferation, whereas α18-glycyrrhetinic acid had a moderate effect, consistent with a role for panx1 hemichannels in ATP release (42, 49) (fig. S5, B and C). In addition, PMA overcame the inhibition of T cell proliferation by the panx1 hemichannel blocker 10panx1 (fig. S5C).

T cell activation in the absence of purinergic costimulation induces T cell anergy

Nuclear translocation of NFAT without concomitant activation of MAPK signaling, which occurs, for example, as a result of treatment with ionomycin, implements a transcriptional program leading to T cell anergy (50). Because oATP did not affect CCE or nuclear translocation of NFAT (Fig. 3A), we hypothesized that activation of T cells in the presence of oATP induces T cell anergy. This prediction was supported by the reduced Ca2+ influx observed on triggering the TCR of Fura-2–loaded T cells previously stimulated in the presence of oATP (Fig. 4D), which is analogous to what occurs in cells pretreated with ionomycin, (Fig. 4D) (51). The effect of oATP was not a result of altered turnover of the TCR–CD3 complex, because we found these proteins to be equally abundant at the cell surface of oATP-treated and untreated cells, as measured by flow cytometry with specific antibodies. To further address whether inhibition of P2XRs during T cell activation led to the induction of anergy, we performed real-time PCR analysis of the expression of the genes early growth response 2 (Egr2) and Egr3, which encode two transcription factors specifically involved in the expression of anergy-associated genes (52). Both transcripts were significantly up-regulated at 2 and 16 hours after T cell activation when oATP was added to the culture medium (Fig. 4C). Addition of PMA together with oATP to overcome the lack of MAPK activation robustly reduced the expression of Egr2 and Egr3 (not shown). These results indicate that lack of P2XR signaling during T cell activation blunts MAPK activation and implements a transcriptional program characteristic of T cell anergy.

Effect of oATP on T cell–mediated inflammation

We next investigated whether the immunopathology characteristic of crt−/− chimeric mice might be ameliorated by inhibitors of P2XR signaling. To test whether systemic administration of oATP was well tolerated in healthy animals, we injected BALB/c mice intravenously each day for 10 days with 100 μl of 3 mM oATP. Treated animals were indistinguishable from their untreated counterparts and displayed no macroscopic abnormalities at autopsy. crt−/− chimeric mice were treated daily by intravenous injection of oATP for 14 days. The percentage of effector/memory CD44+62L CD4+ T cells in lymph nodes was decreased from 33 ± 8.2% in untreated to 23 ± 2.6% in oATP-treated crt−/− chimeric animals, compared with 18 ± 3.2% in untreated crt+/+ mice. Treatment with oATP also substantially improved the skin immunopathology of crt−/− compared with that in untreated mice.

Because crt−/− mice represent a complex model for T cell–mediated inflammation, we further explored the possible use of oATP as a pharmacological agent to limit T cell–mediated inflammation in established experimental models of type 1 diabetes and inflammatory bowel disease (IBD). Adoptive transfer of influenza hemagglutinin (HA)-specific transgenic TCR 6.5 (TCR-HA) CD4+ T cells into RAG-2−/− mice that express HA under the control of the rat insulin promoter (INS-HA) provokes insulitis and the rapid onset of diabetes (53). We treated these mice twice daily with phosphate-buffered saline (PBS) or oATP by intravenous and intraperitoneal injections from days 1 to 10 after adoptive transfer. Mice injected with oATP did not display any pathological traits. The concentration of blood glucose at day 12 was normal in oATP-treated mice, whereas severe hyperglycemia was displayed in untreated and PBS-treated mice (Fig. 5A). No relevant pathological findings were present in the pancreases of oATP-treated mice. In contrast, multifocal to coalescing inflammatory lesions that replaced pancreatic islets were detected in PBS-treated animals (Fig. 5B).

Fig. 5

Prevention of diabetes in INS-HA transgenic RAG-2−/− mice by oATP. (A) Concentrations of blood glucose in RAG-2−/− mice expressing HA under the control of the rat insulin promoter at day 12 after adoptive transfer of TCR 6.5 anti-HA transgenic CD4+ T cells. Mice were either untreated or injected daily (day 1 to 10) after adoptive transfer with two doses, intravenously and intraperitoneally, of PBS or oATP. (B) Histopathological examination of hematoxylin–eosin–stained sections (100 × magnification) shows severe destructive insulitis in the pancreas from a PBS-treated mouse, whereas no relevant pathological findings were observed in the pancreas from an oATP-treated mouse. Pancreatic islets from the PBS-treated mouse were almost completely effaced by dense coalescing lymphohistiocytic and granulocytic infiltrates, which together with fibrosis extended into the surrounding parenchyma dissecting the residual pancreatic acini (arrows). Images shown are representative of five mice per group. (C) Histograms containing the number of transgenic TCR6.5+ cells recovered from the spleen and (D) pancreas of PBS- or oATP-treated mice. Histograms on the right represent the percentage of CD69+ TCR 6.5+ cells recovered from the pancreases of the indicated mice. n.d., not detectable. (E) Splenocytes from PBS- or oATP-treated mice were unstimulated (white bars) or stimulated (black bars) in vitro with HA peptide and the indicated cytokines were measured by cytometric bead arrays. Histograms display the concentrations of TNF-α, IFN-γ, and IL-6 in the supernatants expressed as nanograms per milliliter per 104 TCR6.5+ cells (mean ± SD, n = 5 mice). *P < 0.05, **P < 0.001, ***P < 0.0001.

TCR-HA+ cells of adoptively transferred mice were reduced in number in the spleens (Fig. 5C) and barely detectable in the pancreases of oATP-treated animals (Fig. 5D). In addition, whereas most transgenic CD4+ T cells were activated and expressed CD69 in the pancreases of PBS-treated mice, CD69+ cells were undetectable in the pancreases of oATP-treated mice (Fig. 5D). Ex vivo culture of splenocytes pulsed with HA(110–120) peptide and analysis of their culture supernatants for tumor necrosis factor–α (TNF-α), interferon-γ (IFN-γ), and IL-6 revealed significantly lower production of these cytokines on a per-cell basis in cultures from oATP-treated mice compared to those from PBS-treated mice (Fig. 5E). These results further confirm the induction of T cell unresponsiveness upon treatment with oATP.

To test oATP in a nontransgenic model of T cell–mediated inflammation, we induced IBD in T lymphopenic cd3ε−/− mice by injecting them with naïve CD4+ T cells. We used cd3ε−/− mice injected with naïve CD4+ T cells together with CD4+CD25+ cells (including Tregs) as healthy controls. Indeed, active suppression of locally activated naïve CD4+ T cells by Tregs and immunosuppressive cytokines controls mucosal immunity and organ integrity (54). Starting on day 15 after cell transfer, the group of mice reconstituted with naïve cells only received daily intravenous injections of either PBS or oATP. Macroscopic analysis of the intestine 5 weeks after transfer revealed thickening of the bowel wall and unformed or absent stool in the mice that received naïve CD4+ T cells and were injected with PBS. This response was significantly ameliorated in oATP-treated mice; in addition, their spleens and mesenteric lymph nodes were normal in size (Fig. 6A).

Fig. 6

Amelioration of inflammatory bowel disease by oATP. (A) Representative mesenteric lymph nodes, spleens, and colons from cd3ε−/− mice that received naïve CD4+ T cells and Tregs (CD4 + Treg), naïve CD4+ T cells alone (CD4), or naïve CD4+ T cells and oATP (CD4 + oATP) by adoptive transfer. Scale bar = 1 cm. (B) Histograms representing inflammation scores [mean ± SD; n = 7 mice, CD4 + Treg (healthy control group); n = 7, CD4 (untreated group); n = 8, CD4 + oATP (treated group), **, Mann–Whitney test, P = 0.0083]. (C) Hematoxylin–eosin (upper panels) and Alcian–PAS (lower panels) staining of colon sections (scale bar = 50 μm). In mice reconstituted with naïve CD4+ T cells and Tregs (CD4 + Treg), no inflammatory changes were evident and a large number of goblet cells with voluminous Alcian–PAS–positive droplets lined the colonic crypts (arrowheads). In mice that received naïve CD4+ T cells and were injected with oATP (CD4 + oATP), the lamina propria was focally expanded by an infiltrate of inflammatory cells (arrow) and the epithelia of colonic crypts showed moderate hyperplasia. Partial depletion of goblet cells and a reduction in the size of Alcian–PAS–positive droplets were also noticeable (arrowheads). In mice reconstituted with naïve CD4+ T cells and treated with PBS (CD4), the lamina propria was markedly expanded by infiltrating inflammatory cells with focal findings of crypt abscessation (arrow). Colonic crypts were also severely dysplastic with almost complete loss of goblet cells. (D) Numbers of cells recovered from mesenteric lymph nodes (LN) and spleens of the indicated mice. (E) Absolute numbers of IL-2–, TNF-α–, IFN-γ–, and IL-17–producing CD4+ T cells in mesenteric lymph nodes [mean ± SD; n = 5 mice, CD4 + Treg (healthy group); n = 7, CD4 (untreated group); n = 8, CD4 + oATP (treated group]; *P < 0.05, **P < 0.001, ***P < 0.0001. (F) Absolute numbers of CD44+CD62L effector/memory (E/M) cells and CD4+CD69+ T cells recovered from mesenteric lymph nodes (LN) and spleens of the indicated mice (bars represent mean values). For (D) to (F), Treg indicates mice that received naïve CD4+ T cells and Tregs (healthy group), CD4 indicates mice that received only naïve CD4+ T cells and PBS (untreated group), and oATP indicates mice that received naïve CD4+ T cells and were treated with oATP (treated group).

Histopathological analysis of the colon revealed a milder inflammation in mice treated with oATP compared with untreated mice (Fig. 6C), and the inflammation score of oATP-treated mice was not significantly different from that of animals injected with both naïve CD4+ T cells and Tregs (Fig. 6B). Mice treated with oATP were indistinguishable from healthy control mice in terms of their cellularity and the representation of the CD4+ effector/memory subset (CD44+62L) and CD69+ cells in mesenteric lymph nodes and spleens (Fig. 6, D and F). Moreover, oATP significantly reduced the number of cells that secreted the proinflammatory cytokines IL-2, IFN-γ, and TNF-α. In mesenteric lymph nodes of oATP-treated mice, the number of IL-17–secreting cells, which were recently shown to synergize with IFN-γ–producing cells to provoke severe intestinal inflammation (55), was similar to that of control mice that received naïve CD4+ T cells and Tregs (Fig. 6E). Together, these results strongly support the view that inhibition of P2XRs by oATP dampens T cell activation, proliferation, and effector function and inhibits tissue damage in mouse models of T cell–dependent inflammation.


Productive T cell activation is initiated by the TCR recognizing an immunogenic peptide bound to a major histocompatability complex (MHC) presented by a professional antigen-presenting cell (APC). In addition, PAMPs and DAMPs participate in shaping the immune response. Extracellular ATP released from dying cells is considered a DAMP, which stimulates and modulates lymphocyte function, but may also induce cell death. We show here that ATP is synthesized by T cells and released through panx1 hemichannels after TCR activation. Moreover, we show that autocrine activation of P2XRs on T cells is required for productive T cell activation. P2X7 is the most abundant P2XR on mouse CD4+ T cells. However, much higher concentrations of ATP are needed to activate P2X7 than are normally detected in pericellular regions. Because panx1 hemichannels associate directly with P2X7 (39), we hypothesize that ATP released through this pathway may reach a sufficiently high local concentration in proximity to P2X7 to induce its activation. In agreement with this hypothesis, we found that inhibition of panx1 hemichannel function during in vitro stimulation of naïve T cells strongly reduced IL-2 production and cellular proliferation, thus suggesting that autocrine secretion gives rise to effective concentrations of ATP, which are probably not obtained by ATP released from nearby dying cells. Finally, P2X7 activation increases the opening of panx1 hemichannels and thus contributes through a positive feedback loop to potentiate ATP release.

TCR triggering is followed by CCE and mitochondrial buffering of Ca2+, which stimulates the synthesis of ATP. The efficiency of mitochondrial buffering of Ca2+ varies depending on the amount of Ca2+ that enters the cell and thus depends on the strength of TCR activation. In CD4+ T cells, weak activation of the TCR induces small elevations in the concentration of cytosolic Ca2+, low uptake of Ca2+ by mitochondria, and low quantities of ATP synthesized. Moreover, because the opening of pannexin hemichannels is regulated by elevations in cytosolic Ca2+, it is conceivable that little ATP is released. Our findings suggest that ATP in CD4+ T cells acts as a costimulatory factor whose generation and release depend directly on the strength of T cell activation. Protracted MAPK signaling after TCR triggering and inhibition of such signaling by P2 receptor antagonists supported this hypothesis. In this context, it is noteworthy that inhibition of P2XRs by oATP during T cell activation induced the expression of anergy-associated genes. This mechanism might ensure that weak recognition of self-peptide–MHC complexes does not lead to productive T cell activation, thus preventing expansion of autoreactive cells. On the other hand, higher ATP synthesis and release in T cells isolated from crt−/− FLC mice result in a lowered threshold for activation and T cell–mediated immunopathology (27).

Extracellular ATP activates phospholipase D (PLD) through P2X7 signaling in various cell types, including macrophages (56), lymphocytes (57), astrocytes (58), and osteoblasts (59). In lymphocytes, brief exposure to ATP is sufficient to generate long-lasting activation of PLD (60). PLDs are phosphodiesterases that hydrolyze phosphatidylcholine to form phosphatidic acid (PA) and choline. PLD2-generated PA recruits the Ras guanine nucleotide exchange factor SOS to the plasma membrane through its N-terminal pleckstrin homology domain and induces the recruitment of Ras to the plasma membrane as well as its activation (61). In addition to a direct effect of PA on MAPK signaling, PA is converted to DAG by phosphatidic acid phosphatase. Activation of PLD2 by lymphocyte function–associated antigen–1 (LFA-1) costimulation in T cells increases the pool of DAG at the plasma membrane, thus resulting in selective activation of Ras by RasGRP1 at this site (62). Finally, PLD2 activity promotes sustained ERK activation and IL-2 production in T cells (63), which were both inhibited by treatment with oATP in our study. We are currently addressing the role of purinergic signaling in regulating PLD activity during T cell activation.

The possible beneficial effect of oATP in T cell–mediated inflammation was investigated in murine models of type 1 diabetes and IBD. Repeated administration of oATP to these mice inhibited the development of effector T cells and lessened tissue destruction compared to that observed in untreated mice. That more impressive effects were obtained in the type 1 diabetes mice than in the mice with IBD probably relates to the more aseptic nature of the inflammation in the former mice compared with that in the latter. The intestinal microenvironment probably provides more abundant stimuli for the innate immune system, thus rendering these mice less sensitive to the effects of ATP costimulation. Nevertheless, the possible influence of oATP on other cells, such as natural killer cells, dendritic cells, and macrophages, was not addressed in the present study and needs further analysis.

Pericellular ATP is readily degraded by ectoenzymes expressed on Tregs leading to the generation of adenosine as part of an immunosuppressive circuit (64, 65). Stimulation of adenosine A2a receptors by adenosine promotes T cell anergy and the generation of adaptive Tregs when engaged during antigen recognition (66). The observed unresponsiveness of T cells upon treatment with oATP could be a result of the generation of adenosine rather than the lack of purinergic costimulation. However, oATP reduces the activity of ecto-ATPase and causes the accumulation of extracellular ATP (67, 68), which suggests that the induction of T cell anergy by ATP is unlikely to be a result of the generation of adenosine. Moreover, we showed that treatment with the ecto-ATPase inhibitor ARL67156 increased ERK phosphorylation, thereby showing that blockade of ATP hydrolysis together with an increase in extracellular ATP stimulate the T cell activation machinery. These results point to a selective role of purinergic signaling in sustaining T cell activation and led us to propose a model of T cell activation (Fig. 7). In this model, triggering of the TCR leads to Ca2+ influx through CRAC channels (step 1). Ca2+ buffering by mitochondria (step 2) results in ATP synthesis (step 3) and the release of ATP through panx1 hemichannels. Pericellular ATP activates P2XRs on the T cell in an autocrine fashion (step 4) and perhaps also P2X7 receptors on the APC in a paracrine fashion resulting in IL-1β processing and release (step 5).

Fig. 7

Purinergic control of T cell activation. Schematic representation of a T cell (red) interacting with a professional APC (gray). The early phase of T cell activation, which is triggered by the interaction of the TCR with the MHC–peptide complex, is characterized by CCE (step 1), which is accompanied by mitochondrial uptake of Ca2+ (step 2) and Ca2+-dependent stimulation of ATP synthesis (step 3). ATP is released from activated T cells through pannexin hemichannels, which open when the cytosolic [Ca2+] is elevated. This mechanism guarantees a high concentration of pericellular ATP, which by binding to P2XRs, serves as an indispensable autocrine costimulus for productive T cell activation (step 4). Finally, ATP released from activated T cells might also modulate APC function, such as secretion of IL-1β (step 5).

ATP in bronchoalveolar fluid promotes T helper 2 (Th2)–dependent sensitization in asthmatic subjects and oATP reduces airway inflammation (69). crt−/− FLC mice display phenotypic and cellular characteristics typical of Th2-type disease (27). Nevertheless, the inhibition of T cell activation by oATP in two non-Th2–dependent immunopathological conditions examined in the current study suggests that blockade of purinergic signaling during T cell activation could be generally beneficial in T cell–mediated inflammation rather than strictly during Th2-inducing conditions. Evidence that extracellular ATP influences T cell activation comes from experiments showing that ATP has a synergistic effect on proliferation stimulated by selective T cell mitogens (24) and that IL-2 production and p38 MAPK activation after hypertonic stress involve the release of ATP (25). Our study shows that activity-dependent ATP release upon TCR engagement could maintain MAPK activation through purinergic signaling. Blockade of this signaling pathway reduced the secretion of proinflammatory cytokines, induced T cell unresponsiveness to subsequent TCR stimulation, and dramatically ameliorated the outcome of T cell–mediated inflammation in vivo, thereby suggesting that inhibition of this pathway could have therapeutic potential when dampening T cell activation is desirable.

Materials and Methods


crt+/− (H-2b) mice (70), BALB/c recombinase–activating gene–2 (RAG-2)/common γ chain double-knockout (H-2d) mice obtained from M. Ito (Central Institute for Experimental Animals, Miyamae, Kawasaki, Japan), B6D2F1 mice (BALB/c × C57Bl/6 F1, H2d/b), C57Bl/6 mice from Charles River Germany, C57BL/6 cd3ε−/− mice (71), and transgenic DO11.10 (H-2d) mice from the Jackson Laboratory were bred and treated in accordance with the Swiss Federal Veterinary Office guidelines. Experiments were approved by “Dipartimento della Sanitá e della Socialitá.” crt−/− and crt+/+ FLC mice were generated as described (27). IBD was induced by the adoptive transfer of 400,000 naïve CD4+ T cells purified from C57Bl/6 mice into cd3ε−/− animals. Mice that received 100,000 CD4+CD25+ T cells at the same time as receiving naïve CD4+ T cells served as healthy controls. Two weeks after reconstitution, a group of mice was treated daily intravenously with 100 μl PBS containing 3 mM oATP. At 6 weeks, the animals were killed and lymph nodes, spleen, and bone marrow were analyzed by flow cytometry; colons were analyzed histologically. Diabetes was induced by the adoptive transfer of 250,000 naïve CD4+ T cells purified from TCR-HA/RAG2−/− mice into INS-HA/RAG2−/− animals. RAG2−/− mice that received 250,000 CD4+ T cells served as healthy controls. Starting 1 day after adoptive transfer, mice were treated twice daily intravenously or intraperitoneally with PBS or PBS containing 6 mM oATP for 10 days. The animals were killed on day 12 and spleens and pancreases were analyzed by flow cytometry. Pancreatic biopsies were used for histological analysis.

Antibodies and cells

For flow cytometric analyses, monoclonal antibodies (mAbs) conjugated with either fluorescein isothiocyanate, phycoerythrin, CyChrome, or allophycocyanin (eBioscence) were used. Clonotype-specific anti–KJ1-26 mAb was from Caltag. Cytokine-producing T cells were detected by intracellular staining after incubation with 100 nM PMA (Sigma-Aldrich) and 1 mM ionomycin (Sigma-Aldrich) for 4 hours. Brefeldin A (10 μg/ml; Sigma-Aldrich) was added for the last 2 hours of incubation to prevent cytokine secretion. Cytokines were detected with allophycocyanin-conjugated antibodies to IL-2, IFN-γ, TNF-α, and IL-17 (eBioscence) after cell fixation and permeabilization with Cytofix/Cytoperm (BD Pharmingen). All samples were analyzed with a FACScalibur (Becton Dickinson). Viable cells were electronically gated by exclusion of propidium iodide. Statistical analysis was performed by Student’s t test. Data are reported as mean ± SD. Values of P < 0.05 were considered statistically significant. CD4+ DO11.10tg T cell clones were generated from the spleens and lymph nodes of crt−/− and crt+/+ DO11.10tg chimeras (27). Bone marrow–derived dendritic cells from BALB/c mice generated in the presence of recombinant granulocyte–macrophage colony–stimulating factor (R&D Systems) and loaded with OVA peptide (synthesized at the University Pompeu Fabra Facility, Barcelona, Spain, a member of the ProteoRed network) were used to restimulate T cell clones every 14 days. For all experiments, T cell clones were used 12 to 15 days after restimulation. T cells were isolated from peripheral lymph nodes and spleens by positive selection with anti-CD4 immunomagnetic beads (Miltenyi Biotech). CD4+ naïve (CD4+CD25CD44CD62L+) and effector/memory (CD4+CD25CD44+CD62L) T cell subsets were sorted with a FACSAria (Becton Dickinson). For proliferation assays, cells were labeled with CFSE (Molecular Probes) and stimulated with 10 μg/ml of plastic-bound anti-CD3ε mAb and 5 μg/ml of coimmobilized anti-CD28 mAb (eBioscence). In the appropriate experiments, 10–100 μM oATP (Sigma-Aldrich), 5 μM carbenoxolone (Sigma-Aldrich), 20–50 μM α18-glycyrrhetinic acid, 200 μM GAP26 (Tocris), or 200–500 μM 10panx1 (synthesized at the University Pompeu Fabra Facility) were added. At 16 or 40 hours, cells were transferred to uncoated wells to terminate TCR stimulation and cell culture was continued for 96 hours in medium. Fluorescence-activated cell sorting acquisitions were standardized by fixed numbers of calibration beads (BD Pharmingen). IL-2 was measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instruction (Quantikine, R&D Systems).

Biochemical procedures and measurement of ATP

For in vitro stimulations, T cell clones were incubated for 30 min at room temperature with 1 μg/ml biotinylated CD3ε mAb (clone 145-2C11) in cell culture medium. Unbound antibody was removed by centrifugation and cells were resuspended in medium containing 1 μg/ml avidin (Sigma-Aldrich) for 30 min to 16 hours, as indicated. For biochemical analysis, cells were washed twice in PBS and lysed in a hypoosmotic buffer (10 mM N-2-Hydroxyethylpiperazine-N′-2-Ethanesulfonic Acid, 1.5 mM MgCl2, 10 mM KCl, 0.05% NP-40, containing protease inhibitors) for 10 min on ice. The nuclei were separated from the cytosolic fraction by 10 min centrifugation at 2000g. Samples were resolved by SDS–polyacrylamide gel electrophoresis and analyzed by Western blotting. Rabbit mAbs against phospho-p38 MAPK (3D7) and phospho-ERK (197G2) (Cell Signaling Technology) and a mouse mAb against β-actin (A-5441) (Sigma-Aldrich) were used to analyze Western blots of cytosolic fractions. Antibodies against NFAT1 were kindly provided by N. R. Rice (NCI-Frederick Cancer Research and Development Center, Frederick, MD). For measurement of cellular ATP, cells were lysed in 1% Triton X-100 and frozen on dry ice until analyzed with the ATP determination kit (Molecular Probes). ATP release from naïve T cells stimulated with anti-CD3–coated microbeads (Polysciences Inc.) was performed as described by Corriden et al. (33).

Ca2+ imaging and measurement of carboxyfluorescein uptake

Cultured T cell clones or ex vivo isolated T cells were loaded for 30 min at room temperature with 5 μM Fura-2 pentacetoxy-methylester in RPMI 1640 containing fetal calf serum (FCS), washed in the same solution, and plated on poly-l-lysine–coated coverslips in RPMI 1640 containing FCS and 2.5 μg/ml biotinylated CD3ε mAb for 15 min. Coverslips were then washed and transferred to the recording chamber of an inverted microscope (Axiovert 200, Zeiss) equipped with an Andor 885 JCS iXON classic camera. For Ca2+ measurements, Polychrome V (Till Photonics) was used as a light source. After excitation at 340 and 380 nm, the emitted light was acquired at 505 nm. The sampling rate was 1 Hz. Ca2+ concentrations are expressed as the 340/380 fluorescence ratio. The ratio values in discrete areas of interest were calculated from sequences of images to obtain temporal analyses. The experiments were performed in a static bath (155 mM NaCl, 4.5 mM KCl, 10 mM glucose, 5 mM HEPES, pH 7.4, 1 mM MgCl2, 2 mM CaCl2) at 28°C to 30°C. Cells were stimulated by cross-linking surface-bound anti-CD3ε antibodies by the addition of 2.5 μg/ml avidin. To measure carboxyfluorescein uptake, Fura-2–loaded naïve CD4+ T cells were stimulated by cross-linking of surface-bound anti-CD3 antibodies with avidin for 150 s. Only those cells showing robust Ca2+ entry were considered for further analysis. Carboxyfluorescein (4 mM) was added for 8 min and, after perfusion for 10 min, a picture was acquired with fixed parameters. Image analysis was performed with the use of TILLvisION software.


The large intestine (from the ileocecocolic junction to the anorectal junction) and the pancreas were removed in toto, fixed in 10% buffered formalin solution, and routinely processed for histological examination. For each animal, scoring of colitis was performed on two serial cross sections obtained at three different levels: 1 cm below the ileocecocolic junction, in the middle of the colon–rectal tract, and 1 cm before the anorectal junction. Of the two intestinal cross sections obtained at each level, the one most severely affected was considered in the scoring system. Colitis was scored in a blinded fashion by two pathologists according to Asseman et al. (72) with minimal modifications. Inflammatory changes were scored on a scale of 0 to 4 as follows: grade 0, no pathological changes; grade 1, minimal inflammatory changes consisting of scant luminal catarrhal exudation, scattered infiltrating inflammatory cells (prevalence of lymphocytes and plasma cells) in the lamina propria, submucosa, or both, and occasional hyperplasia of the crypts with minimal depletion of goblet cells; grade 2, mild inflammatory changes consisting of mild luminal catarrhal exudation, locally extensive infiltrate of inflammatory cells (prevalence of plasma cells and histiocytes) in the lamina propria, submucosa, or both, and multifocal hyperplasia of the crypts with mild depletion of goblet cells; grade 3, moderate inflammatory changes consisting of moderate luminal catarrhal exudation, focal erosions, diffuse mural to transmural infiltrate of inflammatory cells (prevalence of granulocytes and histiocytes), occasional crypt abscesses, locally extensive hyperplasia–dysplasia of crypts with moderate depletion of goblet cells and focal subserosal lymphangitis; grade 4, severe inflammatory changes consisting of moderate to abundant luminal catarrhal exudation, multifocal erosions, ulcerations, or both, diffuse transmural infiltrate of inflammatory cells (prevalence of granulocytes and histiocytes), multifocal crypt abscesses, diffuse crypts, hyperplasia–dysplasia of crypts with marked depletion of goblet cells and multifocal subserosal lymphangitis. For each animal, the number of goblet cells was assessed in three randomly chosen microscopic fields at 400× magnification in Alcian–periodic acid–Schiff (PAS)–stained sections. Only those mucosal epithelial cells with a large intracytoplasmatic mucous (Alcian–PAS–positive) droplet measuring more than 10 μm in diameter were counted.


This work was supported by grant 3100A0-104139 of the Swiss National Science Foundation, the Sixth Research Framework Program of the European Union, Project MUGEN (MUGEN LSHG-CT-2005-005203), Fondazione Ticinese per la Ricerca sul Cancro and Fondazione per la Ricerca sulla Trasfusione e sui Trapianti. U.S. was supported by a fellowship from the Roche Research Foundation and A.C. was supported by a fellowship from the Stella Major Foundation.

Supplementary Materials

Fig. S1. Decreased CRAC inactivation and unaltered mitochondrial membrane potential in crt−/− T cells.

Fig. S2. Analysis of ATP release from activated T cells.

Fig. S3. Reduced Ca2+ entry, ATP synthesis, and ATP release in Tregs.

Fig. S4. Analyses of purinergic receptor expression and function in T cells.

Fig. S5. Influence of oATP and pannexin hemichannel inhibitors on productive T cell activation.

References and Notes

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