Autocrine regulation of T-cell activation by ATP release and P2X₇ receptors

Abstract

T-cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X₇ receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real-time PCR and immunohistochemistry, we find that Jurkat T cells and human CD4⁺ T cells express abundant P2X₇ receptors. We show, using a novel fluorescent microscopy technique, that T-cell receptor (TCR) stimulation triggers the rapid release of ATP (<100 μM). This release of ATP is required for TCR-mediated calcium influx, NFAT activation, and interleukin-2 (IL-2) production. TCR activation up-regulates P2X₇ receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi-anion channel blocker gadolinium chloride, or siRNA silencing of P2X₇ receptors blocks calcium entry and inhibits T-cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X₇ receptors, compared to control BALB/c mice, which express fully functional P2X₇ receptors. We conclude that ATP release and autocrine, positive feedback through P2X₇ receptors is required for the effective activation of T cells.—Yip, L., Woehrle, T., Corriden, R., Hirsh, M., Chen, Y., Inoue, Y., Ferrari, V., Insel, P. A., Junger, W. G. Autocrine regulation of T-cell activation by ATP release and P2X₇ receptors.

T cells play a central role in controlling adaptive immune responses. Stimulation of T cells triggers a rapid rise in cytosolic Ca²⁺ that is required for activation of nuclear factor of activated T cells (NFAT), which leads to the expression of interleukin-2 (IL-2) and ultimately T-cell proliferation (1, 2). Previous work has demonstrated that osmotic stress and mechanical stimulation of T cells induce the release of cellular ATP, which regulates T-cell function (3,4,5). In addition, activated cytotoxic T cells can release ATP as a means of killing target cells (6,7,8).

The binding of extracellular ATP to P2X receptors, which are ATP-gated ion channels, induces the influx of extracellular Ca²⁺ (9, 10). Among the seven known P2X receptor subtypes (P2X_1–7), P2X₇ receptors are particularly highly expressed in immune tissues (9,10,11,12,13). In T cells, P2X₇ receptors initiate Ca²⁺-dependent downstream signals that lead to T-cell activation and proliferation (4, 14). Activation of P2X₇ receptors has also been implicated in the production of proinflammatory cytokines (e.g., IL-2, IL-1β, and IL-18) and in the regulation of cell proliferation and cell death (14,15,16,17,18). In human peripheral blood mononuclear cells (PBMCs) and T cells, P2X₇ receptor stimulation induces proliferation and IL-2 synthesis (4, 14, 19), while pharmacological blockade of P2X₇ receptors inhibits TCR-stimulated T-cell activation (18). Stimulation of P2X₇ receptors also induces the shedding of L-selectin (CD62L), a process associated with T-cell activation (20,21,22).

While previous studies have shown that prolonged stimulation of P2X₇ receptors with high concentrations of ATP induces cellular apoptosis (15, 23,24,25), transient stimulation of T cells with lower ATP concentrations, such as those secreted in an autocrine or paracrine fashion, can stimulate T-cell proliferation (23). Under such conditions, overexpression of P2X₇ receptors can further promote T-cell proliferation (23, 26). Our previous work using Jurkat T cells has demonstrated that osmotic stress induces the release of endogenous ATP, which achieves extracellular concentrations of ∼10 μM (3). In contrast, apoptosis induced by P2X₇ receptor in CD4⁺ and CD8⁺ T cells occurs only when extracellular ATP concentrations exceed 100 μM (15). Thus, we hypothesized that stimulation of P2X₇ receptors by ATP concentrations that are released endogenously contribute to T-cell activation. The objective of the present study was to examine whether activation of T cells involves the release of endogenous ATP and if so, whether the released ATP acts via P2X₇ receptors to regulate early T-cell activation processes, such as Ca²⁺ influx, NFAT activation, and IL-2 production.

MATERIALS AND METHODS

Cells and cell stimulation

Jurkat cells (E6–1 clone) from American Type Culture Collection (ATCC; Manassas, VA, USA) were maintained in complete RPMI 1640 medium (Irvine Scientific; Santa Ana, CA, USA) containing 10% heat-inactivated fetal calf serum (Omega Scientific; Tarzana, CA, USA), 50 μg/ml gentamicin (Life Technologies, Inc.; Grand Island, NY, USA), 10 mM HEPES (Fisher Scientific; Waltham, MA, USA), 1 mM sodium pyruvate (Fisher Scientific), 0.1 mM MEM nonessential amino acids (Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate (Invitrogen, Carlsbad, CA, USA). Human PBMCs and purified CD4⁺ T cells were prepared and maintained as described previously (3). Cells were stimulated with anti-CD3 (OKT-3; obtained from ATCC clone CRL8001) and anti-CD28 antibodies (BD Pharmingen; San Jose, CA, USA) as described previously (4). Mouse splenocytes (10⁶) were isolated from the spleens of BALB/c or C57BL/6 mice (Charles River Labs, Wilmington, MA, USA), stimulated with phytohemagglutinin (PHA; Sigma-Aldrich, St. Louis, MO, USA) for 2 h, and IL-2 production of lymphocytes was assessed as described below. Unstimulated cells or cells stimulated with 50 ng/ml phorbol myristate acetate and 5 μM ionomycin served as negative or positive controls, respectively.

ATP release

Jurkat cells were stimulated by ligation of CD3/CD28 using MACSibeads coated with anti-CD3 and anti-CD28 antibodies (1 bead/cell; Miltenyi Biotec; Auburn, CA, USA) and extracellular ATP levels near the cell surface were measured using fluorescence microscopy or HPLC analysis (3, 5, 17) or by use of an ATP Bioluminescence Assay Kit HSII from Roche (Palo Alto, CA, USA) and a Luminoskan chemiluminometer (Labsystems, Helsinki, Finland), according to the manufacturer’s instructions. The specificity of the fluorescence microscopy assay has previously been described (5). ADP, AMP, and adenosine levels in the bulk medium of anti-CD3- and anti-CD28-stimulated cells were also measured by HPLC (3, 5, 17).

Intracellular Ca²⁺ measurements

Cells were loaded with Fura-2-AM (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions, and changes in intracellular Ca²⁺ levels in response to cell stimulation were measured (4). To assess the role of P2 receptors and ATP in Ca²⁺ signaling, Fura-2 loaded cells were treated with suramin, NF023, or o-ATP (Sigma-Aldrich) for 1 h before stimulation or with apyrase (Sigma-Aldrich) or alkaline phosphatase (ALP; New England Biolabs; Ipswich, MA, USA) for 15 min prior to stimulation.

IL-2 measurements

IL-2 expression was measured in cell supernatants of CD3-stimulated PBMCs (2×10⁵) or CD3/CD28-stimulated Jurkat cells (5×10⁴) using an ELISA assay (3, 4). IL-2 expression in mouse lymphocytes was measured with an intracellular cytokine staining assay using flow cytometry (27). Mouse splenocytes were isolated and stimulated as described above, and then they were incubated in the presence of 2 μg/ml brefeldin A for 20 h, treated with 0.1% saponin in PBS containing 1% FCS and 2 mM EDTA, washed, and stained with PE-labeled anti-mouse IL-2 antibody (BD Pharmingen) or isotype controls. Cells were washed, fixed, and analyzed by flow cytometry. Lymphocytes were identified according to forward- and side-scatter properties, and data from 10⁴ gated cells were acquired for each sample.

P2X₇ receptor and IL-2 mRNA expression

We assayed P2X₇ receptor mRNA transcript levels in Jurkat cells or human CD4⁺ T cells before or 1 h after stimulation with CD3/CD28-loaded beads using real-time RT-PCR (3, 17). The comparative Ct method for relative quantification of gene expression (ΔΔCt method) was used, and gene expression levels were normalized to gene expression of 18S rRNA (as a housekeeping gene control) using the following equation: normalized expression = 2^{[Ct(18S rRNA) − Ct(P2X7 receptor)]}. P2X₇ mRNA and 18S rRNA were assessed using the following primer sets: P2X₇: 5′-ATC GGC TCA ACC CTC TCC TAC-3′, 5′-CTG GAG TAA GTG TCG ATG AGG AAG-3′; and 18S rRNA: 5′-CCG CAG CTA GGA ATA ATG GA-3′, 5′-CCC TCT TAA TCA TGG CCT CA-3′ (3). IL-2 mRNA expression was determined in anti-CD3/CD28-stimulated Jurkat cells in the presence or absence of 100 μM ATP using a primer set purchased from Qiagen (Germantown, MD, USA). This ATP concentration was chosen because it significantly enhances IL-2 secretion in stimulated Jurkat cells (Supplemental Fig. 1).

Plasmids, cell transfection, and NFAT-luciferase reporter gene assay

Plasmids containing wild-type or mutant (T1729A) human P2X₇ receptor were kindly provided by Dr. J. Wiley (University of Sydney, Sydney, Australia). The NFAT-luciferase reporter plasmid was a gift from Dr. A. Altman (La Jolla Institute of Allergy and Immunology, San Diego, CA, USA), and the β-galactosidase control plasmid was purchased from Roche (Pleasanton, CA, USA). Jurkat cells were transiently transfected by electroporation using a Bio-Rad GenePulser X-cell (Bio-Rad, Hercules, CA, USA) set at 260 V and 950 μF with a 4-mm path length (28). After electroporation, cells were incubated for 24–72 h in complete culture medium. For reporter assays, Jurkat cells (10⁷ in 0.4 ml RPMI) were transfected with 6 μg NFAT-luciferase reporter plasmid, 8 μg β-galactosidase reporter plasmid, and 5 μg wild-type P2X₇ receptor plasmid or appropriate empty control. After incubation for 72 h, cells were stimulated with beads loaded with antibodies to CD3/CD28, as described above and incubated for another 8 h. Luciferase activity was measured using the Luciferase Substrate kit (BD Pharmingen); β-galactosidase activity was used to normalize for transfection efficiency (28).

P2X₇ receptor silencing using siRNA

Three different siRNA constructs targeting the P2X₇ receptor, and a Cy3-labeled nonsense siRNA construct were purchased from Ambion (Austin, TX, USA). siRNA transfection was performed by electroporation as described above. To determine the transfection efficiency, Jurkat cells were transfected with the Cy3-labeled nonsense siRNA construct. Cells were washed and visualized by microscopy 24 h posttransfection; >95% of cells contained Cy3-labeled siRNA (Fig. 6A). We selected the most effective of the three different siRNA constructs for further experiments. This siRNA silenced P2X₇ mRNA levels in Jurkat cells by 90% (Fig. 6B). Ca²⁺ signaling, after silencing P2X₇ receptors, was assessed using flow cytometry and Fluo-3-AM as a calcium probe (Molecular Probes). Briefly, cells were transfected with 3 μg siRNA and 3 μg empty DsRed-N1 plasmid to distinguish transfected from nontransfected cells. After 72 h, cells were loaded with Fluo-3-AM in the presence of Pluronic (Molecular Probes), according to the manufacturer’s instructions; changes in Fluo-3 fluorescence in response to cell stimulation were assessed by flow cytometry. Viable cells were selected based on their forward- and side-scatter properties, and changes in fluorescence were assessed in DsRed-positive cells. Cell responses were normalized using 5 μM ionomycin to account for differences in Fluo-3 loading between individual cell preparations. Results are expressed as percentage maximal response relative to stimulation of nonsilenced control cells.

Figure 1.

Stimulation of Jurkat cells releases cellular ATP. A) Jurkat cells were stimulated by ligation of CD3/CD28 using MACSibeads coated with anti-CD3 and anti-CD28 antibodies (arrowheads), and extracellular ATP levels near the cell surface were visualized with a fluorescence microscope assay and estimated using ATP standards. B) Extracellular ATP concentrations determined from the indicated areas of interest shown in A. C) Changes in ATP, ADP, AMP, and adenosine concentrations as a function of time in the bulk cell supernatant of Jurkat cells (5×10⁶) treated with anti-CD3 and anti-CD28 antibodies (1 μg/ml each) were measured with HPLC. Data shown in A and B are representative of 3 and 2 similar experiments, respectively, performed in separate experiments.

Figure 2.

Extracellular ATP is required for T-cell activation. A) IL-2 mRNA levels of Jurkat cells (10⁷) stimulated for 1 h with anti-CD3/CD28 antibody-coated beads (1 bead/cell) in the presence or absence of 100 μM ATP. B) Inhibition of ATP release from Jurkat cells (10⁷) treated with the maxi-anion channel blocker, GdCl₃ (100 μM). Cells were treated in HBSS for 20 min at 37°C and stimulated with anti-CD3/CD28 antibodies (1 μg/ml each) for 1 min, and ATP release was determined using the ATP Bioluminescence Assay Kit HSII. C) GdCl₃ (100 μM) and apyrase (20 U/ml) inhibit IL-2 mRNA expression of stimulated Jurkat cells and IL-2 production of stimulated human PBMCs. IL-2 mRNA expression was determined by real-time RT-PCR analysis, and IL-2 production of PBMCs was determined with ELISA. Data are means ± se. *P ≤ 0.05; n = 3; two-tailed unpaired Student’s t test.

Figure 3.

Scavenging of released ATP inhibits calcium signaling and IL-2 expression. A, B) Scavenging extracellular ATP with 10 U/ml of apyrase (A) or alkaline phosphatase (ALP; B) inhibits intracellular Ca²⁺ signaling of Jurkat cells stimulated with anti-CD3 antibodies. C) Apyrase and ALP suppressed IL-2 mRNA transcription in response to stimulation of Jurkat cells by ligation of CD3 and CD28 for 4 h. IL-2 mRNA levels were determined by real-time RT-PCR, and intracellular Ca²⁺ mobilization was measured using Fura-2. Data shown in A and B are representative of 3 similar experiments performed separately. Data shown in C represent means ± se. *P ≤ 0.05; n = 3; two-tailed unpaired Student’s t test.

Figure 4.

P2X receptor antagonists inhibit Ca²⁺ signaling and IL-2 production. A, B) The nonselective P2X receptor antagonists suramin (100 μM) and NF023 (10 μM) and the P2X₇-selective antagonist o-ATP (10 μM) blocked Ca²⁺ signaling in Jurkat cells stimulated with 2 mM ATP (A) or 1 μg/ml anti-CD3 antibodies (B). C) Suramin and o-ATP inhibited IL-2 production of stimulated PBMCs. Cells were treated with antagonists for 1 h prior to cell stimulation. Ca²⁺ mobilization was measured using Fura-2, and IL-2 production of PBMCs was determined by ELISA. Data in A and B are representative of 3 separate experiments; data in C represent means ± se. *P ≤ 0.05; n = 3; two-tailed unpaired Student’s t test.

Figure 5.

P2X₇ receptors are involved in T-cell activation. A) P2X₇ receptor mRNA expression in Jurkat cells and human peripheral CD4⁺ T cells was quantified using real-time RT-PCR analysis. B) Immuncytochemical assessment of P2X₇ receptor expression of Jurkat cells and human CD4⁺ T cells was evaluated using a fluorescence microscope. Controls were probed with goat anti-rabbit Alexa 555 secondary antibody only. C) P2X₇ receptor mRNA expression of Jurkat cells was determined with real-time RT-PCR 4 h after stimulation with or without anti-CD3/CD28 antibody-coated beads. D) NFAT activation of Jurkat cells overexpressing wild-type (WT) or mutated (T1729A) P2X₇ receptor or empty expression vector (control) was assessed using a NFAT-luciferase reporter assay. Cells were stimulated with anti-CD3/CD28 antibody-coated beads. NFAT-luciferase activity was normalized using a β-galactosidase control reporter coexpressed in the cells. Transfected cells were cultured for 72 h, stimulated with anti-CD3/CD28 antibody-coated beads for 8 h, and luciferase activity was measured. Data represent means ± se of triplicates. *P ≤ 0.05; n ≥ 3; two-tailed unpaired Student’s t test.

Figure 6.

P2X₇ receptors are involved in Ca²⁺ signaling and IL-2 expression. A) Jurkat cells electroporated with a Cy3-labeled nonsense siRNA construct were examined using a microscope to verify siRNA uptake. B) Jurkat cells were transfected with 3 μg siRNA targeting P2X₇ or nonsense siRNA, and P2X₇ gene expression was examined at the indicated time points by assessing mRNA levels using real-time RT-PCR. C) Jurkat cells were treated with nonsense control siRNA or with siRNA targeting P2X₇ receptors for 72 h, then stimulated with anti-CD3/CD28 antibody-loaded beads, and NFAT-luciferase activation,and IL-2 transcription were determined. Data represent means ± se. *P ≤ 0.05; n = 3; two-tailed unpaired Student’s t test. D, E) Jurkat cells were treated with siRNA to silence P2X₇ receptors or with nonsense control siRNA, and Ca²⁺ signaling in response to stimulation with 0.5 μg/ml anti-CD3 antibody (D) or 2 mM ATP (E) was determined by flow cytometry using Fluo-3 as a Ca²⁺ indicator. Data are representative of similar results obtained in 3 separate experiments.

Immunocytochemistry

Jurkat cells and human CD4⁺ T cells were fixed in 4% paraformaldehyde in PBS for 10 min on ice, blocked with 1% BSA in PBS, and stained with rabbit anti-P2X₇ receptor antibody overnight at 4°C (1:100 in blocking buffer; Alomone Labs, Jerusalem, Israel). Washed cells were then incubated with goat anti-rabbit Alexa 555 antibody (Molecular Probes) for 1 h at room temperature (diluted 1:2000 in blocking buffer), washed again, and placed on cover slips in mounting medium (Vectashield, Vector Laboratories, Burlingame, CA, USA). As a control, cells were stained in the absence of primary antibody. Immunofluorescence and bright field images were captured using a Leica DMIRB fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Hamamatsu ORCA II camera (Hamamatsu Photonics, Hamamatsu, Japan).

Statistical analyses

Unless otherwise stated, data are expressed as means ± se of experiments performed in triplicate. For comparisons between groups, two-tailed unpaired Student’s t-tests were used. A value of P ≤ 0.05 was considered significant.

RESULTS

TCR stimulation induces ATP release

Released cellular ATP can serve as a paracrine signaling molecule for intercellular communication, or it can act in an autocrine manner to regulate cellular functions, including those of leukocytes (17, 29). We found that the ligation of the TCR/CD3 complex and CD28 coreceptor induces the release of ATP from Jurkat T cells (Fig. 1). ATP concentrations in the bulk medium reached levels of 3 μM in 30 s and remained elevated for >5 min (Fig. 1C), but extracellular ATP concentrations in close proximity to the cell surface achieved concentrations of >60 μM within 2 min after cell stimulation (Fig. 1A, B). These ATP concentrations are sufficient to stimulate P2X receptors (9, 30, 31) but are lower than the concentrations of ATP that induce apoptosis in CD4⁺ and CD8⁺ T cells (15). Low levels of ADP, AMP, and adenosine, which are likely generated from the breakdown of ATP, were also detected in the bulk medium during the 5-min stimulation period (Fig. 1C).

Stimulation of Jurkat cells in the presence of ATP at concentrations similar to those released endogenously in response to TCR/CD28 stimulation (100 μM) enhanced IL-2 transcription by >47-fold compared to controls stimulated in the absence of additional ATP (Fig. 2A). Treatment of Jurkat cells with 50 μM and 100 μM ATP also enhanced IL-2 release in stimulated Jurkat cells (Supplemental Fig. 1). Thus, ATP concentrations of ∼100 μM promote T-cell activation rather than apoptosis in Jurkat cells.

ATP release from Jurkat cells may occur through the stimulation of stretch-activated maxi-anion channels (32, 33). During T-cell activation, mechanical perturbations of the cell membrane by interactions of T cells with antigen-presenting cells may lead to ATP release through similar mechanisms. To examine this possibility, we treated Jurkat cells with the maxi-anion channel blocker GdCl₃ (100 μM). We found that GdCl₃ significantly inhibits ATP release and IL-2 expression of Jurkat cells and human PBMCs (Fig. 2B, C). Because GdCl₃ does not completely abolish ATP release, other mechanisms besides Gd³⁺-sensitive channels may also contribute to the release of ATP from stimulated T cells. Endogenously released ATP plays an important role in mediating T-cell activation, as treatment of stimulated PBMCs with apyrase (20 U/ml) suppressed IL-2 production (Fig. 2C). This is consistent with our previous findings demonstrating that ATP, released endogenously in response to hypertonic saline treatment, enhances IL-2 release in human PBMCs (4).

TCR-induced ATP release and P2X receptors are required for Ca²⁺ mobilization and IL-2 synthesis

Treatment of peripheral T cells with exogenous ATP induces the influx of Ca²⁺ (14, 20). T-cell stimulation elicits the release of endogenous ATP (Fig. 1), but it is unclear whether released ATP contributes to the influx of Ca²⁺ that occurs in response to TCR stimulation (34). To test this possibility, we stimulated Jurkat cells in the presence or absence of the nucleotide-hydrolyzing enzymes apyrase or alkaline phosphatase and found that both enzymes inhibited TCR-stimulated Ca²⁺ influx and IL-2 expression (Fig. 3). Higher concentrations of apyrase and alkaline phosphatase (20 to 40 U/ml) did not result in a further inhibition of Ca²⁺ influx (data not shown). The doses of apyrase and alkaline phosphatase (>10 U/ml) required to inhibit Ca²⁺ influx and IL-2 expression are consistent with previous observations demonstrating the presence of an apyrase-resistant pericellular ATP pool that surrounds T cells (35). Our results imply that ATP released into the extracellular milieu is essential for Ca²⁺ influx and subsequent T-cell activation processes that result in IL-2 expression.

To evaluate the role of P2X receptors in the influx of Ca²⁺ and activation of IL-2 expression, we used pharmacological inhibitors to block P2X receptors. The P2X receptor antagonists suramin and NF023 block a number of P2X receptor subtypes, while o-ATP is a more selective inhibitor of P2X₇ receptors (11, 36, 37). We found that suramin (100 μM), NF023 (10 μM), and o-ATP (10 μM) inhibited Ca²⁺ mobilization of Jurkat cells stimulated with anti-CD3 antibodies (1 μg/ml) or exogenous ATP (2 mM; Fig. 4A, B). Treatment of human PBMCs with suramin (100 and 500 μM) or o-ATP (10 and 100 μM) inhibited IL-2 production (Fig. 4C). These results are akin to findings that have demonstrated the ability of o-ATP to reduce IL-2 production and proliferation of CD3/CD28-stimulated naive CD4⁺ T cells (38). Taken together, the results support the conclusion that ATP acts on P2 receptors to mediate Ca²⁺ influx and IL-2 expression in response to T-cell stimulation. To examine the role of P2X₇ receptors in T-cell activation, we performed experiments using immunohistochemistry and assessment of cells with decreased P2X₇ receptor expression.

P2X₇ receptor expression and function in T lymphocytes

Real-time RT-PCR analysis and immunocytochemical examination revealed that Jurkat cells and peripheral human CD4⁺ T cells express P2X₇ receptors (Fig. 5A, B) and that stimulation of Jurkat cells by ligation of CD3/CD28 up-regulates P2X₇ receptor expression (Fig. 5C). Together with the findings above, these data imply that changes in P2X₇ receptor expression levels may serve to regulate T-cell activation processes. To test whether increases in P2X₇ receptor expression promote T-cell activation, we utilized a NFAT-luciferase reporter assay (28). NFAT, which is activated in response to increases in intracellular calcium concentrations, is required for the induction of IL-2 gene transcription (2). We found that overexpression of human wild-type P2X₇ receptors more than doubled NFAT activation in TCR/CD28-stimulated Jurkat cells compared to control cells that were transfected with an empty expression vector (Fig. 5D). A T1729A mutation occurs as a single nucleotide polymorphism in the human P2X₇ gene, conferring a loss of receptor function due to the failure of the mutant receptors to traffic to the cell surface (39). Overexpression of the nonfunctional T1729A mutant P2X₇ receptor resulted in suppressed NFAT activation in response to TCR/CD28 stimulation (Fig. 5D).

To examine the role of P2X₇ receptors in Ca²⁺ influx, NFAT activation, and IL-2 expression in more detail, we silenced the expression of P2X₇ receptors using siRNA. Using Cy3-labeled nonsense siRNA, we found that >95% of treated Jurkat cells incorporated the siRNA constructs (Fig. 6A). Treatment with a P2X₇ receptor siRNA construct reduced P2X₇ receptor mRNA levels in a time-dependent manner, with a ∼90% decrease in receptor transcripts after 72 h of siRNA treatment (Fig. 6B). The decrease in P2X₇ receptor expression resulted in the inhibition of NFAT activation and IL-2 mRNA synthesis in response to TCR/CD28 stimulation (Fig. 6C). NFAT activation and IL-2 mRNA synthesis require Ca²⁺ signaling (2). Consistent with evidence that P2X₇ receptor stimulation involves Ca²⁺ influx, we found that silencing of P2X₇ receptors inhibits Ca²⁺ signaling in response to cell stimulation by TCR or exogenous ATP (Fig. 6D, E). Intracellular Ca²⁺ concentrations decreased below baseline levels when cells with silenced P2X₇ receptors were stimulated with ATP, suggesting that the lack of P2X₇ receptors impairs the ability of the cells to replenish cytosolic Ca²⁺ sequestered in response to cell stimulation. Thus, P2X₇ receptors may contribute more generally to Ca²⁺ homeostasis in T cells (40, 41).

Lack of functional P2X₇ receptors impairs T-cell function

C57BL/6 mice have a mutation in the P2X₇ receptor gene that impairs the function of the receptor; by contrast, BALB/c mice express fully functional P2X₇ receptors (42). Consequently, T cells of BALB/c mice are more sensitive to ATP and other P2X₇ receptor agonists compared to T cells of C57BL/6 mice (15, 42). We thus used splenocytes from these two mouse strains as a further means to examine the role of P2X₇ receptors in T-cell activation. We found that lymphocytes of BALB/c mice showed robust IL-2 production in response to stimulation with PHA while lymphocytes of C57BL/6 mice failed to induce IL-2 production, even at the highest PHA concentration tested (Fig. 7A). Studies comparing ATP-induced responses in T-cell subsets of C57BL/6, BALB/c, and P2X₇ receptor knockout mice have previously been reported (15). Of particular interest are data demonstrating increased proliferation of CD4⁺ T cells from BALB/c, but not C57BL/6 and P2X₇ knockout mice in response to stimulation with 100 μM ATP. The differences in the sensitivity of CD4⁺ T cells to ATP was found to correlate with different levels of P2X₇ receptor that are expressed by the T cells of these strains (15). Our findings are consistent with these data and, together with the siRNA silencing data above, provide further support for the notion that P2X₇ receptors play an essential role in the activation of T cells.

Figure 7.

P2X₇ receptors regulate IL-2 production in mouse splenocytes and a model for the role of ATP release and P2X₇ receptors in T-cell activation. A) IL-2 expression of splenocytes from C57BL/6 mice lacking functional P2X₇ receptors or BALB/c mice with functional P2X₇ receptors was assessed using intracellular cytokine staining and flow cytometry. Splenocytes were stimulated overnight with the indicated concentrations of phytohemagglutinin (PHA), and IL-2 expression of lymphocytes was assessed by intracellular staining. Data are representative of 3 experiments; values are means ± se of triplicate measurements. B) Schematic depiction of the role of ATP release and P2X₇ receptors in T-cell activation. TCR activation by antigen presenting cells (APC) induces ATP release, resulting in the activation of P2X₇ receptors that facilitate Ca²⁺ entry, which, in turn, induces T-cell activation events, through the activation of NFAT and IL-2 transcription.

DISCUSSION

The current findings provide evidence that release of cellular ATP by T cells and stimulation of P2X₇ receptors regulate T-cell activation. We found that T-cell stimulation up-regulates P2X₇ receptor expression, while silencing of P2X₇ receptors suppresses T-cell activation events that include Ca²⁺ influx, NFAT activation, and IL-2 expression. We and others have shown that the release of cellular ATP from leukocytes and other cells occurs in a controlled manner and that released ATP serves as a local autocrine or paracrine signaling molecule that regulates the functions of numerous cell types (17, 29, 43, 44).

We found that Jurkat T cells release ATP within seconds after TCR stimulation and that the extracellular ATP concentrations generated near the cell surface are sufficient to activate P2X₇ receptors of the cells (45). Released ATP can be converted to ADP, AMP, and adenosine by Jurkat cells (3), but ATP levels measured in the bulk medium of stimulated cells are highest, with adenosine levels remaining relatively low (∼0.2 μM) throughout the 5-min stimulation period (Fig. 1C). Adenosine can inhibit IL-2 release of stimulated Jurkat cells; however, this requires treatment with adenosine concentrations of ≥1 μM (3).

We found that Jurkat cells release more cellular ATP in response to TCR stimulation than with other stimuli, such as osmotic stress and local mechanical stimulation (3,4,5). We speculate that this greater release of ATP may be a consequence of sustained local perturbations of the plasma membrane caused by interaction of cells with the beads used for activation, thus perhaps modeling the interaction of T cells with antigen-presenting cells.

These close intercellular interactions may induce greater concentrations of extracellular ATP within the immune synapse that forms between T cells and antigen-presenting cells (APC), thereby facilitating the initiation of events that are critical for T-cell activation (29). This suggestion is consistent with the slightly higher level of ATP measured near the beads than at the rear of the cell. However, ATP was detected throughout the cell surface on cell activation and thus may mediate signal transduction events outside the T-cell:APC contact site. This idea is consistent with previous findings, indicating that signal transduction in activated T cells can occur outside of the immunological synapse (46).

Our results obtained with GdCl₃ suggest that T cells release ATP via stretch-activated anion channels. However, because GdCl₃ did not completely block ATP release or IL-2 production in response to T-cell stimulation, other mechanisms of ATP release, such as vesicular release or release from pannexin-1 or connexin hemichannels, may also be involved in the release of ATP from stimulated T cells (38, 47, 48). A recent study has shown that ATP release from naive CD4⁺ T cells can occur through pannexin-1 hemichannels (38). De novo synthesis of extracellular ATP through sequential phosphor-transfer reactions by ecto-adenylate kinase and NDP kinase expressed on the T-cell surface may also contribute to the accumulation of extracellular ATP (43, 49). In addition to ATP that is released by stimulation of T cells, Yegutkin et al. (35) have shown that a pericellular “ATP halo” envelopes resting T cells, which may mediate various autocrine and paracrine signaling events.

Although ATP concentrations of >300 μM can induce apoptosis in mouse peripheral T cells, Jurkat cells are comparatively insensitive to ATP-induced apoptosis; instead, they proliferate in the presence of ATP at concentrations as high as 3 mM (14, 15). We find that exogenous ATP concentrations similar to those released in response to the stimulation of Jurkat cells enhance IL-2 mRNA expression, consistent with evidence that exogenous ATP in this concentration range augments IL-2 production of activated Jurkat cells (Supplemental Fig. 1) (4).

Differences in the abundance of P2X₇ receptors in Jurkat cells vs. CD4⁺ cells may explain why Jurkat cells are less sensitive to ATP-induced apoptosis than are CD4⁺ cells. Human CD4⁺ T cells have a ∼6-fold greater copy number of P2X₇ mRNA than do Jurkat cells (Fig. 5A). By comparing the responses of T cells isolated from P2X₇ receptor knockout mice with T cells from strains of wild-type mice, Aswad et al. (15) showed that the abundance of functional P2X₇ receptors expressed by T cells correlates with the degree of apoptosis that is induced in response to ATP stimulation. Thus, the lower P2X₇ receptor expression in Jurkat cells, a cell line originally derived from a patient with acute T-cell leukemia, may protect the cells from apoptosis and thereby contribute to their growth. P2X₇ receptor expression correlates with cell proliferation (23, 26), implying that the apoptotic vs. proliferative roles of P2X₇ receptors differ among different types of T cells.

Influx of extracellular Ca²⁺ is an early signaling event that is critical for T-cell activation (50), including, as our data suggest, in response to stimulation by P2X₇ receptors. We found that stimulation of Jurkat cells with exogenous ATP induces rapid Ca²⁺ influx, while the removal of endogenous ATP that is released in response to cell stimulation or elimination of P2X₇ receptors with antagonists or siRNA inhibits Ca²⁺ signaling and downstream events that lead to IL-2 production (Figs. 3 and 4). Inhibition of P2X₇ receptors in other cell types, (i.e., human and mouse peripheral T cells) also suppresses cell activation (18, 38).

A defect in P2X₇ receptor signaling or polymorphisms in the P2X₇ receptor gene that result in a loss of receptor function (T1729A, A1513C, and G496A) can lead to decreased immune cell function (39, 51, 52). Here, we show that C57BL/6 mice, which carry an allelic mutation that impairs P2X₇ receptor function (42), are unable to produce IL-2 in response to cell stimulation. Experiments using P2X₇ receptor knockout mice or leukocytes isolated from patients with genetic defects in P2X₇ receptors support their importance in T-cell function (53). In addition, single nucleotide polymorphisms that lead to the gain of function of P2X₇ receptors increase Ca²⁺ influx in lymphocytes of patients with chronic lymphocytic leukemia (CLL) (54). Lymphocytes of patients with the aggressive variant of CLL can have up-regulated P2X₇ receptor expression that enhances Ca²⁺ influx and cell proliferation (23, 55).

Overall, the current findings imply an essential role for ATP release and autocrine or paracrine feedback through P2X₇ receptors in T-cell activation (Fig. 7B). Such localized feedback processes may be important for the induction of T-cell proliferation and for the amplification and modulation of T-cell responses to cues such as those provided by antigen-presenting cells. Future studies that explore the role of P2X₇ receptor splice variants and other P2X receptor subtypes should aid in defining the precise role of ATP and P2X receptors in T-cell activation.