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Department of Molecular Microbiology and Immunology University of Southern California/Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA 90033
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We report in this study that CD4+CD25+ Treg cells undergo death by necrotic lysis within seconds when brought into contact with low concentrations of the common metabolites, NAD and ATP. Conventional T cells are more resistant and undergo slower death, associated with annexin V staining. We also show that the two metabolites act by providing ligands for the purinergic receptor P2X7 (P2X7R) and that cells lacking the receptor are resistant to rapid death induction. Moreover, by demonstrating that mice with deleted P2X7Rs possess increased numbers of CD4+CD25+ cells expressing forkhead/winged helix transcription factor gene (Foxp3), we suggest a role for this receptor in the homeostasis of CD4+CD25+ cells. It is proposed that P2X7R provides a signaling structure by which intracellular components NAD and ATP regulate CD4+CD25+ Treg cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Pathogen-free female C57BL/6 mice, 6–8 wk of age, were obtained from The Jackson Laboratory. C57BL/6 P2X7R gene-deleted mice (P2X7R–/–) were provided by Dr. C. Gabel (Pfizer, Inc., Ann Arbor, MI) (5) and were bred at the University of Southern California animal facility. Mice were injected i.v. with 1 or 10 mg of NAD or benzoylbenzoyl-ATP (Bz-ATP; Sigma-Aldrich) dissolved in 300 µl of PBS either once or three times at 30-min intervals. All mice were killed 2 h after the first injection.
Cell isolation, cell culture, and cell death assays
Spleen, cervical, and inguinal lymph nodes or peripheral blood cells were used in all experiments as indicated. Erythrocytes were removed before analysis or culture by treatment for 5 min on ice with 155 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA, pH 7.3.
To purify CD4+CD25+ and CD4+CD25– cells, spleen cells were incubated with IMag anti-mouse CD4 magnetic particles (BD Biosciences) in 1x IMag Buffer (BD Biosciences) for 30 min at 4°C and then separated by IMagnet (BD Biosciences). The enriched CD4+ population was then incubated with FITC-conjugated anti-mouse CD4 Ab and PE-conjugated anti-mouse CD25 Ab for 30 min at 4°C. CD4+CD25+ and CD4+CD25– cells were separated by FACSVantage SE (BD Biosciences). Purity was verified by fluorometry to be >95%. The CD4– cell population was used to provide APCs to cultures.
To assay the function of CD4+CD25+ T cells, purified CD4+CD25– T cells (5 x 104/well) were cultured with 10 µg/ml anti-CD3 mAb (eBioscience) in the presence of CD4–, APC-containing cells and varying numbers of CD4+CD25+ T cells for 3 days in complete RPMI 1640 medium containing 10% FBS (6). The APC-containing CD4– population (2 x 105 cells/well) was irradiated with 3000 rad. [3H]thymidine (Amersham Biosciences; 0.5 µCi/well) was added during the last 16 h of culture.
To assay induction of cell death, spleen cells were incubated with or without NAD, ATP (Sigma-Aldrich), or Bz-ATP in RPMI 1640 for various times and assayed for annexin V staining or by microscopic counting in the presence of trypan blue. To assay the short-term effects of NAD (
5 min), a 10-fold excess of ice-cold PBS was added to the cell suspension to dilute out NAD (7). After centrifugation, cells were washed twice in ice-cold PBS before culture in complete RPMI 1640 medium. To inhibit P2X7 receptors, 20 µM KN-62 (Sigma-Aldrich) dissolved in 0.01% DMSO was added 10 min before addition of NAD or ATP (7, 8).
Flow cytometric analysis and assay for Foxp3 expression
For FACS analysis, cells were preincubated with anti-mouse CD16/CD32 (2.4G2) mAb (BD Biosciences) to block Fc
Rs, followed by incubation with mAbs for 30 min at 4°C. The following mAbs were used: PerCP-conjugated anti-mouse CD4 (L3T4), PE-conjugated anti-mouse CD25 (PC61), and allophycocyanin-conjugated anti-mouse CD8 (Ly-2; BD Biosciences). To monitor induction of death, cells were stained with the Annexin VFITC Apoptosis Detection Kit I (BD Biosciences). To detect Foxp3 protein expression, cells stained with anti-CD4 and anti-CD25 Abs were fixed using Fix/Perm Buffer (eBioscience) for 24 h, then incubated with FITC-conjugated anti-mouse Foxp3 Ab (FJK-16; eBioscience) for 30 min at 4°C. For quantification of cell surface ADP-ribosylation, cells (4 x 106 cells/ml in RPMI 1640) were incubated with 300 µM etheno-NAD (Sigma-Aldrich) for 30 min at 37°C, followed by incubation with etheno-ADP-ribose-specific Ab 1G4, provided by Dr. R. Santella (Mailman School of Public Health of Columbia University, New York, NY) (9) and FITC-conjugated goat anti-mouse Ig (BD Biosciences). FACS analysis was performed on a FACSCalibur (BD Biosciences).
For assay of Foxp3 expression by RT-PCR, spleen cells were incubated in normal medium or in the presence of NAD for 15 min at 37°C. CD4+ cells were then purified using CD4 magnetic particles (BD Biosciences) as described above. Total RNA from CD4+ cells was extracted using TRIzol reagent (Invitrogen Life Technologies). RNA was reverse transcribed into cDNA using the Omniscript RT Kit (Qiagen). PCR was conducted using the Taq PCR MasterMix Kit (Qiagen) with the following primers synthesized at the University of Southern California Microchemical Core Facility: Foxp3, 5'-CAG CTG CCT ACA GTG CCC CTA-3' and 5'-CAT TTG CCA GCA GTG GGT AG-3'; and GAPDH, 5'-TGA AGG TCG GTG TGA ACG GAT-3' and 5'-CAG GGG GGC TAA GCA GTT GGT-3'. PCRs consisted of an initial 5-min 94°C denaturing step, followed by 35 cycles of 45 s at 94°C, 45 s at 57°C, and 60 s at 72°C (10). PCR was performed using a DNA Thermal Cycler 480 (PerkinElmer).
Statistical analysis
Results are expressed as the mean ± SD. Statistical significance of differences between experimental groups was calculated by Student’s t test.
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Results |
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We had previously shown that NAD, when added to cultures of B6 spleen cells, induces death in a small proportion of T cells (11), raising the question of whether a defined T cell subset expresses preferential sensitivity to the death-inducing signal. Subsequent experiments demonstrated that rapid death induction requires the expression of ADP-ribosyltransferase 2 (ART-2), an NAD-consuming cell surface enzyme that attaches ADP-ribosyl groups to arginines of cell surface proteins (7, 12, 13), prompting the hypothesis that it is this reaction that induces the death signal. Therefore, to assess effects of NAD, we assayed ART-2 activity on T cell subsets.
B6 spleen cells were incubated with the ART-2 substrate etheno-NAD (7, 9) and assayed for cell surface etheno-APP-ribosylation by labeling with etheno-ADP-ribose-specific Ab 1G4. Fig. 1 shows that CD4+CD25– and CD4+CD25+ cells undergo comparable labeling, whereas CD8+CD25– cells show somewhat lower labeling. An expectation, therefore, is that all three T cell subsets should be sensitive to NAD-induced death. To test this, spleen cells were incubated with NAD and assayed for the recovery of CD4+CD25+ T cells. Fig. 2A shows the percentages of CD4+ CD25+, CD4+CD25–, and CD8+CD25– T cells in spleen cells incubated with 500 µM NAD for 30 min; Fig. 2B shows the complete time kinetics. As early as 30 s after addition of NAD, a 40% decrease in CD4+CD25+ cells was seen, followed by only a minor decrease over the next 30 min. In contrast, CD4+CD25– and CD8+CD25– cells decreased only slightly during this period. To determine the NAD concentrations required for this effect, spleen cells were incubated with increasing NAD concentrations for 30 min. Fig. 2C shows that addition of 1 µM NAD induced a substantial decrease in CD4+CD25+ cells. In contrast, there was almost no effect on CD4+CD25– and CD8+CD25– cells.
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50% of CD4+CD25+ cells disappeared from NAD-incubated cultures (Fig. 2, B and C). Therefore, it is likely that the remaining CD4+CD25+ cells were also undergoing cell death.
To further examine this, cells from NAD-incubated cultures were stained for annexin V. Fig. 3, A and B, shows that contact with 500 µM NAD for only 30 s led to 80–90% annexin V staining in the CD4+CD25+ T cell population. Moreover, the addition of only 10 µM NAD sufficed to induce annexin V staining to a similar degree after a 30-min incubation (Fig. 3C). In contrast, induction of annexin V staining in CD4+CD25– and CD8+CD25– cells was much lower and increased gradually with time and NAD concentrations. These results show that NAD induces cell death in CD4+CD25+ Treg cells at a much faster rate and at lower NAD concentrations than in conventional T cells.
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Recent experiments had shown that P2X7Rs play a pivotal role in NAD-induced death of conventional T cells (7, 16), which suggests that these receptors play a similar role in CD4+CD25+ cells. To examine this, the ability of P2X7R inhibitor KN-62 (8, 17) to interfere with cell death induction was assayed at a maximal NAD concentration of 500 µM. Fig. 4shows that in the presence of NAD, KN-62 inhibited the induction of annexin V staining in CD4+CD25+ T cells. Moreover, KN-62 increased cell recoveries of CD4+CD25+ T cells cultured without addition of NAD. The effects of KN-62 on conventional CD4 and CD8 cells were similar. Addition of KN-62 to cultures of B6 cells incubated with lower concentrations of NAD showed concordant results (data not shown).
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The finding that NAD-induced death in CD4+CD25+ T cells involved P2X7Rs, predicts that ATP, the well-established ligand of this receptor (18, 19), should exert effects similar to those of NAD. To examine this, spleen cells were incubated with increasing concentrations of ATP and assayed for cell recovery and annexin V staining. Fig. 6A shows that 300 µM ATP caused a significant decrease in CD4+CD25+ cells and an increase in annexin V staining in the remaining cells, effects that further increase at 1000 µM ATP. Effects on conventional CD4 and CD8 cells were concordant, but much less pronounced.
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Bz-ATP is a high affinity, nonhydrolysable ligand, of the P2X7R (18). It was therefore interesting to test the effects of this ligand on CD4+CD25+ cells. Fig. 7A shows that Bz-ATP concentrations one-third or less than those of ATP induced a decrease in CD4+CD25+ cells and an increase in annexin V staining. As expected, much smaller effects were elicited in conventional CD4 and CD8 cells (Fig. 7A). Importantly, no effects of Bz-ATP were demonstrable in CD4+CD25+ cells from P2X7R–/– mice (Fig. 7B). These results demonstrate that a nonhydrolysable derivative of ATP induces effects in CD4+CD25+ cells similar to those of ATP and that these effects are mediated by P2X7Rs.
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The results presented in Figs. 2 and 6 show that a large percentage of CD4+CD25+ cells disappear within 30 s of NAD or ATP contact. To test whether this rapid disappearance is due to cell death, rather than a loss of cell surface CD4 or CD25 expression, CD4+CD25+ cells were isolated by magnetic bead adsorption and cell sorting. FACS analysis revealed >95% homogeneity (Fig. 8A), but no expression of ART-2 activity (data not shown), consistent with our previous observation that stimulation of T cells causes the release of cell surface ART-2 (20). Therefore, the effects of P2X7R stimulation were tested with ATP. The results presented in Fig. 8B show that addition of 500 µM ATP for 15 min led to 80% lysis of purified CD4+CD25+ cells. We conclude, the observed disappearance of CD4+CD25+ cells incubated with NAD or ATP is due to cell lysis and not loss of cell surface CD4 or CD25 Ags.
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The finding that efficient death is induced within seconds of NAD contact in CD4+CD25+ cells raises the possibility that this is also demonstrable in intact animals. To examine this, B6 mice were injected with increasing doses of NAD, and spleen cells were assayed for annexin V staining 2 h later. The results revealed that a single injection of 1 mg of NAD had no demonstrable effect (data not shown); however, a dose 10 times higher, injected once or three times, induced a 50% decrease in recovered CD4+CD25+ cells and a significant increase in annexin V staining of the remaining CD4+CD25+ cell population (Fig. 9A). Much smaller, if any, effects were seen in conventional CD4 and CD8 cells (Fig. 9A and data not shown). Because NAD action requires functional P2X7Rs, it is predicted that there should be no effects of NAD in P2X7R–/– mice. Fig. 9A shows that at the highest NAD dose there was neither a decrease nor annexin V staining of CD4+CD25+ cells in P2X7R–/– mice.
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P2X7R–/– mice have increased numbers of functional, Foxp3-expressing CD4+CD25+ cells
The demonstration that NAD induces a death signal in CD4+CD25+ T cells of normal, but not P2X7R–/– mice, raises the possibility that P2X7R regulates the homeostasis and/or function of CD4+CD25+ Treg cells in vivo. To examine this, the distributions of CD4+CD25+ cells in normal and P2X7R–/– mice were compared. Table I shows that in peripheral blood, lymph node, and spleen of P2X7R–/– mice, there were significantly more CD4+CD25+ T cells than in normal B6 mice. To test whether this increase in CD4+CD25+ cells reflected an increase in Treg cells, CD4+CD25+ cells were assayed for the expression of Foxp3. The results presented in Fig. 10A demonstrate that spleen, lymph nodes, and PBL from P2X7R–/– mice contained significantly more Foxp3-expressing CD4+CD25+ cells than respective organs from B6 mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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10 times that in the extracellular fluid (22, 23, 24, 25), induces almost instantaneous death. Effects of ATP are concordant, but not as impressive, because concentrations of 300 µM, i.e., 1/10th those inside cells, are required to induce effects comparable to those of NAD. Although these results show that very low concentrations of NAD suffice to induce cell death in Treg cells, our experiments also suggest that this probably only occurs at specific sites at which NAD is released by cell necrosis or other means. Indeed, very high doses of NAD had to be injected i.v. to induce systemic cell death in CD4+CD25+ T cells, and such high doses are not likely to ever be reached by cell lysis. Our data show that P2X7Rs play an obligatory role in rapid NAD- and ATP-induced death of CD4+CD25+ cells. Therefore, we suggest that free ATP as well as ADP-ribosyl groups attached close to the binding site of the P2X7R provide ligands for its activation. Free ADP-ribose, the breakdown product of NAD by action of CD38 (21), induces only minimal, if any, effects in CD4+CD25+ cells (data not shown), which is consistent with our previous demonstration that rapid death of conventional T cells is induced by NAD, but not by ADP-ribose and requires the expression of functional ART-2 (7). Apparently, the affinity of P2X7Rs for free ADP-ribose molecules is too low to induce efficient signaling, a conclusion consistent with previous results in the P2X7R-oocyte expression system (26). It is interesting that even the canonical ligand for P2X7Rs, i.e., ATP, has to be added in substantial concentrations to induce a death signal. Therefore, free ligands appear to be less efficient in receptor signaling than ADP-ribosyl groups attached to proteins. This enables regulation of the P2X7R function on several levels, i.e., release of NAD by dying cells and presence of ART-2 on CD4+CD25+ cells. Shedding of ART-2 induced by cell activation would increase resistance to NAD-induced cell death (20). Consequently, high concentrations of ATP would be required to trigger cell death. In this respect, it is interesting to note that human T cells lack expression of ART-2 (Ref.27 and our unpublished observations), making regulation of human T cells by cell surface ADP-ribosylation unlikely. However, the signaling sensitivity of the human P2X7R appears to be higher than that of the mouse receptor (28), perhaps enabling more efficient regulation of human T cells via soluble ligands, such as ATP. It is also interesting that mouse strains differ in their P2X7Rs. Indeed, the P2X7Rs in B6 and BALB/c mice differ by one amino acid, resulting in a difference in efficiency of receptor signaling in the two mouse strains (29). Despite this difference, Treg cells from BALB/c mice are also more sensitive to death induction by P2X7R signaling than conventional T cells (data not shown).
The P2X7R is a 595-aa polypeptide with two membrane-spanning domains and N- and C-terminal cytoplasmic domains (18). It is predominantly expressed on monocytes, macrophages, and DC’s (30, 31, 32), but, as shown in this study and elsewhere, it is also expressed on other cell types of the hemopoietic cell lineage, including T cells (7, 16, 33, 34). Engagement of the receptor with ATP (18, 19) opens nonselective ion channels, followed by formation of pores that allow passage of 900-Da molecules (35, 36, 37, 38). Although shorter permeabilization periods may be tolerated as cells reseal their membranes, longer periods often induce death (39, 40, 41). We show that engagement of the P2X7R leads to rapid cell death in a large proportion of the CD4+CD25+ population, associated with complete cell disintegration, consistent with the earlier demonstration that P2X7Rs can induce cell death by a necrotic, osmoid colloidal, lysis mechanism (42). In addition, CD4+CD25+ cells as well as conventional CD4 and CD8 cells can undergo much slower death, indicated by annexin V staining and consistent with apoptosis inducible by the receptor (7, 42, 43). The reason why there exist these dramatic differences in sensitivity among T cell populations remains to be investigated. Reasons could be differences in P2X7R structure, densities, or proteins associated with the receptor. Recent gene array analyses have indicated higher expression of P2X7Rs on Treg cells compared with conventional T effector cells (44). It is therefore quite possible that the observed differences in sensitivity are due to differential expression of the P2X7R on T cell subsets.
The finding that CD4+CD25+ cells undergo much faster death than conventional CD4 and CD8 T cells may point to an important role of this pathway in immune regulation. Both NAD and ATP have exceedingly short half-lives outside cells, due to an abundance of extracellular NAD- and ATP-degrading enzymes. Therefore, a rapid response of CD4+CD25+ cells to NAD and ATP compared with conventional T cells may cause a shift toward conventional T cells, enabling a more efficient immune response.
We demonstrate that CD4+CD25+ cells from P2X7R–/– mice exert normal suppression of T cell proliferation in vitro, which shows that P2X7Rs are not required for their function. The finding that P2X7R–/– mice have increased numbers of Foxp3-expressing CD4+CD25+ cells in the circulation and lymphoid organs points to the possibility that P2X7Rs play a role in Treg cell homeostasis. Low levels of Treg cells correlate with the development of autoimmunity, such as type I diabetes in NOD mice, and a role of P2X7Rs in this disease model has been suggested (45). Consistent with this, P2X7R–/– mice are relatively resistant to Ab-induced collagen arthritis (33).
In conclusion, our data show that NAD and ATP, at concentrations well below those inside cells, induce rapid and efficient death in CD4+CD25+ Treg cells via action on P2X7Rs. Conventional T cells are relatively resistant, resulting in preferential elimination of CD4+CD25+ cells. Our data prompt the hypothesis that metabolites NAD and ATP, released during cell necrosis, serve to limit the action of CD4+CD25+ Treg cells, thereby promoting increased responses of conventional T cells. This newly uncovered mechanism of cell regulation may lead to novel approaches to specifically eliminate Treg cells for therapeutic purposes.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by U.S. Public Health Service Grants AI40038 and AI43954. 
2 F.A. and H.K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Gunther Dennert, University of Southern California/Norris Comprehensive Cancer Center, P.O. Box 33800, 1441 Eastlake Avenue, M/S 73, Los Angeles, CA 90033-0800. E-mail address: dennert{at}usc.edu
4 Abbreviations used in this paper: Treg cell, regulatory T cell; Bz-ATP, benzoylbenzoyl-ATP; Foxp3, forkhead/winged helix transcription factor gene.
Received for publication March 25, 2005. Accepted for publication June 24, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.-1164. [Abstract]
induces Foxp3+ T-regulatory cells from CD4+ CD25– precursors. Am. J. Transplant. 4:1614.-1627. [Medline]
release from microglial cells through activation of ATP-primed P2X7 receptor channels. J. Neurosci. 22:3061.-3069. and IL-18 requires priming by lipopolysaccharide and is independent of caspase-1 cleavage. J. Biol. Chem. 276:3820.-3826. by microvesicle shedding. Immunity 5:825.-835.
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, Samples not incubated with etheno-NAD; 
, samples treated with etheno-NAD. This experiment was repeated at least three times with similar results.
