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High Sensitivity of CD4+CD25+ Regulatory T
Cells to Extracellular Metabolites
Nicotinamide Adenine Dinucleotide and ATP:
A Role for P2X 7 Receptors
Fred Aswad, Hiroki Kawamura and Gunther Dennert
J Immunol 2005; 175:3075-3083; ;
doi: 10.4049/jimmunol.175.5.3075
http://www.jimmunol.org/content/175/5/3075
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Copyright © 2005 by The American Association of
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References
The Journal of Immunology
High Sensitivity of CD4ⴙCD25ⴙ Regulatory T Cells to
Extracellular Metabolites Nicotinamide Adenine Dinucleotide
and ATP: A Role for P2X7 Receptors1
Fred Aswad,2 Hiroki Kawamura,2 and Gunther Dennert3
A
fundamental question in immunology is how nonresponsiveness to self-Ags is maintained while enabling
an efficient response to infections. Tolerance is established by a central mechanism in the thymus and is maintained in
the periphery by regulatory T (Treg)4 cells, which control autoreactive T cells. The importance of peripheral tolerance is well documented by experiments in which elimination of Treg cells results
in severe autoimmune pathology (1, 2). Given the pivotal role of
Treg cells, the question of how these cells are regulated remains to
be elucidated. It is well documented that immune responses to
microbial infections often trigger autoimmunity (3, 4), which
raises the question of why Treg cells fail to protect against breakage of tolerance in the course of an infection. An explanation could
be that the function of Treg cells is curtailed in favor of a more
potent response to the infectious pathogen. It is postulated that
there are mechanisms that inactivate Treg cells at sites of an infection. A common feature of infections is tissue inflammation and
necrosis, associated with the release of cellular components into
the extracellular space. Some of these components could provide a
danger signal that modulates Treg cell functions. We tested this by
examining the effects of two common cell metabolites that are
released from necrotic cells and undergo rapid degradation outside
cells.
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
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.
Materials and Methods
Mouse strains and injections
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 ␮l 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
Received for publication March 25, 2005. Accepted for publication June 24, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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:
[email protected]
4
Abbreviations used in this paper: Treg cell, regulatory T cell; Bz-ATP, benzoylbenzoyl-ATP; Foxp3, forkhead/winged helix transcription factor gene.
Copyright © 2005 by The American Association of Immunologists, Inc.
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 1⫻ 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.
0022-1767/05/$02.00
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Although regulatory lymphocytes play an important role in the immune system, the regulation of their functions is poorly
understood and remains to be elucidated. In this study we demonstrate that micromolar concentrations of the common cell
metabolite NAD induce death in murine forkhead/winged helix transcription factor gene-expressing CD4ⴙCD25ⴙ regulatory T
cells with high efficiency and within minutes. Similar, but less dramatic, effects are demonstrable with ATP and its nonhydrolysable derivative, benzoylbenzoyl-ATP. Other T cell subsets are more resistant, with CD8 cells being the least sensitive and CD4
cells expressing intermediate sensitivity. The higher sensitivity of CD4ⴙCD25ⴙ cells is demonstrable in vivo. Injection of NAD or
benzoylbenzoyl-ATP causes preferential induction of a cell death signal in CD4ⴙCD25ⴙ cells. Transmission of the death signal
requires functional P2X7 receptors, pointing to a role for these receptors in regulation and homeostasis of CD4ⴙCD25ⴙ regulatory
T cells. Consistent with this, P2X7R gene-deleted mice possess increased levels of forkhead/winged helix transcription factor
gene-expressing CD4ⴙCD25ⴙ cells. The Journal of Immunology, 2005, 175: 3075–3083.
3076
To assay the function of CD4⫹CD25⫹ T cells, purified CD4⫹CD25⫺ T
cells (5 ⫻ 104/well) were cultured with 10 ␮g/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 ⫻ 105
cells/well) was irradiated with 3000 rad. [3H]thymidine (Amersham Biosciences; 0.5 ␮Ci/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 (ⱕ5 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 ␮M 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
FIGURE 1. CD4⫹CD25⫹ cells express ART-2 enzyme activity. B6
spleen cells were incubated with 300 ␮M etheno-NAD for 30 min at 37°C.
Samples were stained for CD25, CD4, CD8, and etheno-ADP-ribose-specific 1G4 Ab and analyzed by FACS. To generate the histograms, FACS
data were gated for CD4⫹CD25⫺, CD4⫹CD25⫹, or CD8⫹ cells. 䡺, Samples not incubated with etheno-NAD; f, samples treated with ethenoNAD. This experiment was repeated at least three times with similar
results.
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 ␮M 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.
Statistical analysis
Results are expressed as the mean ⫾ SD. Statistical significance of differences between experimental groups was calculated by Student’s t test.
Results
CD4⫹CD25⫹ Treg cells express ART-2 activity and rapidly
undergo cell death when incubated with NAD
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 ADPribosyltransferase 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
FIGURE 2. CD4⫹CD25⫹ Treg cells disappear from spleen cell cultures
incubated with NAD. A and B, B6 spleen cells were incubated with 500
␮M NAD for the indicated periods of time; stained for CD25, CD4, and
CD8; and analyzed by FACS. Scattergrams of untreated controls and the 30
min end point are shown. Numbers in the scattergrams represent percentages specified by the gates. The plot on the right shows all data, of which
only the controls and respective end points are shown on the left. C, B6
spleen cells were incubated with the indicated concentrations of NAD for
30 min and analyzed as described above (A and B). D, B6 spleen cells were
incubated with or without 100 ␮M NAD for 15 min. CD4⫹ cells were
isolated by magnetic bead adsorption. In the isolated cell population,
FOXP3 mRNA was assayed by RT-PCR as described in Materials and
Methods. All experiments were repeated at least three times.
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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: PerCPconjugated 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 ⫻ 106
cells/ml in RPMI 1640) were incubated with 300 ␮M etheno-NAD (SigmaAldrich) for 30 min at 37°C, followed by incubation with etheno-ADPribose-specific Ab 1G4, provided by Dr. R. Santella (Mailman School of
Public Health of Columbia University, New York, NY) (9) and FITCconjugated 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).
SENSITIVITY OF CD4⫹CD25⫹ Treg CELLS TO NAD AND ATP
The Journal of Immunology
Purinergic receptor P2X7 is required for NAD-induced death in
CD4⫹CD25⫹ cells
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 ␮M. Fig. 4 shows 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).
To provide additional evidence for the involvement of P2X7Rs,
spleen cells from P2X7R⫺/⫺ mice (5) were incubated with NAD
and assayed for annexin V staining. Fig. 5 shows that although B6
CD4⫹CD25⫹ T cells underwent a significant increase in annexin
V staining under normal culture conditions, cells from P2X7R⫺/⫺
mice showed no increase, even at 500 ␮M NAD. Conventional
CD4 and CD8 cells from P2X7R⫺/⫺ mice were also resistant to
induction of annexin V staining by NAD. These results demonstrate that functional P2X7Rs are required for induction of rapid
cell death in CD4⫹CD25⫹ cells. Moreover, the finding that death
of CD4⫹CD25⫹ cells lacking functional P2X7Rs and cultured under normal culture conditions was very low, suggested that components in the medium or released by cells, one of which is most
likely NAD, induce death via signaling through P2X7Rs.
ATP and Bz-ATP induce rapid death in CD4⫹CD25⫹ cells
FIGURE 3. NAD induces rapid and preferential annexin V staining in
CD4⫹CD25⫹ cells. A and B, B6 spleen cells were incubated with 500 ␮M
NAD for the indicated periods of time and then stained for CD25, CD4,
CD8, and annexin V and analyzed by FACS. To generate the histograms,
FACS data were gated for CD4⫹CD25⫺, CD4⫹CD25⫹, or CD8⫹ cells as
shown in Fig. 2. Histograms of untreated controls and the 30 min end point
are shown, and numbers in histograms indicate the percentage of annexin
V-staining cells. The plot in B shows all data, of which only the controls
and respective end points are shown in A. C, B6 spleen cells were incubated with the indicated concentrations of NAD for 30 min, then stained for
CD25, CD4, CD8, and annexin V and analyzed as described in A and B.
These experiments were repeated at least three times with similar results.
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 ␮M 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 ␮M
ATP. Effects on conventional CD4 and CD8 cells were concordant, but much less pronounced.
To determine the kinetics of death induction, cells were incubated with 500 ␮M ATP for various times. Fig. 6B shows that at
2 min there was a significant decrease in CD4⫹CD25⫹ cells as
well as an increase in the percentage of annexin V-staining cells,
effects that were even more pronounced at 15 min. Concordant, but
less dramatic, effects were seen with conventional CD4 and CD8
cells. To confirm that ATP-induced death of CD4⫹CD25⫹ cells
was mediated by P2X7Rs, effects in the presence of KN-62 and in
spleen cells from P2X7R⫺/⫺ mice were assayed. At the high concentration of 500 ␮M ATP, there was no increase in annexin V
staining of B6 cells in the presence of KN-62, or in cells from
P2X7R⫺/⫺ mice (Figs. 4 and 5). These results show that
CD4⫹CD25⫹ cells are highly sensitive to ATP-induced death, and
this response requires functional P2X7Rs.
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
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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 ␮M NAD induced a
substantial decrease in CD4⫹CD25⫹ cells. In contrast, there was
almost no effect on CD4⫹CD25⫺ and CD8⫹CD25⫺ cells.
These results suggest that NAD contact causes preferential elimination of Treg cells in spleen cell cultures. However, although
CD4 and CD25 are markers for Treg cells, the expression of Foxp3
is a more definitive marker of regulatory cells (14, 15). Therefore,
spleen cells were incubated for 15 min with 100 ␮M NAD, and
Foxp3 mRNA expression in CD4 cells was assayed by RT-PCR.
Fig. 2D shows that the Foxp3 signal disappeared after incubation
with NAD. This effect was higher than expected, because only
⬃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 ␮M 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 ␮M 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.
3077
3078
SENSITIVITY OF CD4⫹CD25⫹ Treg CELLS TO NAD AND ATP
of ATP induces effects in CD4⫹CD25⫹ cells similar to those of
ATP and that these effects are mediated by P2X7Rs.
CD4⫹CD25⫹ cells die upon engagement of P2X7R
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 con-
tact. 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
FIGURE 5. CD4⫹CD25⫹
cells
from
P2X7R⫺/⫺ mice are resistant to NAD- and ATPinduced cell death. Spleen cells from B6 or
P2X7R⫺/⫺ mice were incubated with 500 ␮M
NAD or ATP for 30 min, then stained for CD25,
CD4, CD8, and annexin V and analyzed by FACS.
To generate the histograms, FACS data were gated
for CD4⫹CD25⫺, CD4⫹CD25⫹, or CD8⫹ cells.
This experiment was repeated at least three times
with similar results.
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FIGURE 4. KN-62 inhibits NAD- and
ATP-induced cell death in CD4⫹CD25⫹
cells. B6 spleen cells were incubated with
500 ␮M NAD or ATP for 30 min, then
stained for CD25, CD4, CD8, and annexin V
and analyzed by FACS. To generate the histograms, FACS data were gated for
CD4⫹CD25⫺, CD4⫹CD25⫹, or CD8⫹ cells.
Where indicated, cells were preincubated for
10 min with 20 ␮M KN-62 before addition
of NAD or ATP, and incubation in the presence of KN-62 was continued for 30 min.
This experiment was repeated at least three
times with similar results.
The Journal of Immunology
3079
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 ␮M 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.
NAD and Bz-ATP induce a death signal in CD4⫹CD25⫹ cells
of normal, but not P2X7R⫺/⫺, mice in vivo
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
FIGURE 7. Bz-ATP
induces
more efficient cell death than ATP in
CD4⫹CD25⫹ cells and requires
functional P2X7Rs. A, B6 spleen
cells were incubated with the indicated concentrations of Bz-ATP for
120 min, then stained for CD25,
CD4, CD8, and annexin V and analyzed by FACS. To generate histograms, FACS data were gated for
CD4⫹CD25⫺, CD4⫹CD25⫹, or
CD8⫹ cells. Scattergrams and histograms of untreated controls and the
highest Bz-ATP concentrations are
shown. The plots show all data from
scattergrams and histograms, of
which only the controls and respective end points are shown on the left.
B, Spleen cells from P2X7R⫺/⫺ mice
were incubated with 300 ␮M BzATP for 120 min and processed and
analyzed as described in A. These experiments were repeated twice with
similar results.
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FIGURE 6. CD4⫹CD25⫹
cells
are more sensitive to ATP-induced
cell death than conventional CD4⫹
and CD8⫹ cells. A, B6 spleen cells
were incubated with the indicated
concentrations of ATP for 120 min,
then stained for CD25, CD4, CD8,
and annexin V and analyzed by
FACS. To generate the histograms,
FACS data were gated for
CD4⫹CD25⫺, CD4⫹CD25⫹, or
CD8⫹ cells. Scattergrams and histograms of untreated controls and the
highest ATP concentrations are
shown. The plots show all data from
scattergrams and histograms, of
which only the controls and respective end points are shown on the left.
This experiment was repeated at least
three times with similar results. B, B6
spleen cells were incubated with 500
␮M ATP for the times indicated, then
stained for CD25, CD4, CD8, and annexin V and analyzed by FACS as
described in A. This experiment was
repeated at least three times with similar results.
3080
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
FIGURE 9. Effects of NAD and Bz-ATP on
CD4⫹CD25⫹ cells in B6 and P2X7R⫺/⫺ mice. A,
Groups of three B6 and P2X7R⫺/⫺ mice received 10 mg
of NAD in 300 ␮l of PBS i.v. once or additional injections 30 and 60 min later. Spleen cells were harvested
2 h later; stained for CD25, CD4, CD8, and annexin V;
and analyzed by FACS. To generate histograms, FACS
data were gated for CD4⫹CD25⫹ or CD4⫹CD25⫺ cells.
B, Groups of three B6 and P2X7R⫺/⫺ mice received one
injection of 1 mg of Bz-ATP/mouse. Spleen cells were
harvested 2 h later and processed as described in A.
These experiments were repeated twice with similar
results.
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.
Although these results show that NAD can exert effects on
CD4⫹CD25⫹ cells in vivo and by action on P2X7Rs, they raise the
question of why such high doses are required. A likely reason is
that effects are limited by rapid, CD38-mediated, NAD hydrolysis
(21). We therefore tested whether injection of a lower dose of a
nonhydrolysable P2X7 ligand, i.e., Bz-ATP, has effects on
CD4⫹CD25⫹ cells. Fig. 9B shows that this is indeed the case.
Injection of 1 mg of Bz-ATP induced annexin V staining in
CD4⫹CD25⫹ cells of B6, but not P2X7R⫺/⫺, mice. Therefore, the
action of NAD is most likely local and in the direct vicinity of
lysing cells.
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
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FIGURE 8. CD4⫹CD25⫹ cells rapidly lyse upon engagement of the
P2X7R. A, CD4⫹CD25⫹ and CD4⫹CD25⫺ cells were isolated, and purity
was analyzed by FACS. B, The isolated CD4⫹CD25⫹ and CD4⫹CD25⫺
cells were treated with 500 ␮M ATP for 15 min, and cell viability was
determined by trypan blue exclusion. ⴱ, p ⬍ 0.02; ⴱⴱ, p ⬍ 0.01 (control vs
ATP-treated cells). Error bars indicate SD values from triplicate samples.
This experiment was repeated three times with similar results.
SENSITIVITY OF CD4⫹CD25⫹ Treg CELLS TO NAD AND ATP
The Journal of Immunology
3081
Discussion
Experiments are presented in this report showing that B6
CD4⫹CD25⫹ T cells express high sensitivity to the common cell
metabolites, NAD and ATP. A concentration of 1 ␮M NAD, i.e.,
1/1000 that inside cells and only ⬃10 times that in the extracellular
fluid (22–25), induces almost instantaneous death. Effects of ATP
are concordant, but not as impressive, because concentrations of
300 ␮M, 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
FIGURE 10. P2X7R⫺/⫺ mice possess more Foxp3-expressing CD4⫹CD25⫹
cells with normal regulatory function. A, Spleen cells, lymph node cells, and
PBL from P2X7R⫺/⫺ and wt B6 mice were first stained for CD25 and CD4,
then fixed and stained for Foxp3 and analyzed by FACS. The scattergrams
show data from CD4⫹ gated cells analyzed for the expression of CD25 and
Foxp3. Numbers in the scattergrams represent the relative percentage of each
respective cell population. These experiments were repeated twice with similar
results. B, CD4⫹CD25⫹ cells were isolated from normal B6 and P2X7R⫺/⫺
mice and cultured at the ratios indicated with B6 CD4⫹CD25⫺ cells as well as
an APC-containing population and anti-CD3 Ab. Cell proliferation was assayed by incorporation of [3H]TdR on day 3, as indicated. Error bars indicate
SDs from triplicate cultures.
Table I. Counts of CD4⫹ CD25⫹ or CD3⫹ cells in spleen, lymph nodes, or blood
Mice
WT B6
Compartment
b
Spleen
Lymph nodesc
Bloodd
Cell Type
⫹
⫹
CD4 CD25
CD3⫹
CD4⫹ CD25⫹
CD3⫹
CD4⫹ CD25⫹
CD3⫹
No. of mice ⫽ 3.
No. of cells per spleen.
No. of cells per lymph node of three lymph nodes extracted.
d
No. of cells per 1 ml of blood.
**, p ⬍ 0.01 (between B6 P2X7R⫺/⫺ and WT B6).
a
b
c
B6 P2X7R⫺/⫺a
a
No. of cells
% cells
No. of cells
% cells
1.27 ⫻ 10
2.25 ⫻ 107
3.05 ⫻ 104
5.20 ⫻ 105
1.88 ⫻ 103
3.09 ⫻ 105
1.59 ⫾ 0.10
28.13 ⫾ 3.62
3.11 ⫾ 0.27
52.03 ⫾ 6.17
0.14 ⫾ 0.04
23.07 ⫾ 4.38
2.42 ⫻ 10
2.76 ⫻ 107
4.69 ⫻ 104
6.20 ⫻ 105
2.80 ⫻ 104
5.32 ⫻ 105
3.03 ⫾ 0.12**
34.51 ⫾ 1.25
4.72 ⫾ 0.19**
61.13 ⫾ 3.18
2.09 ⫾ 0.23**
39.71 ⫾ 3.45
6
6
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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.
To examine whether CD4⫹CD25⫹ cells from P2X7R⫺/⫺ mice
are functional, the regulatory activity of these cells was assayed.
CD4⫹CD25⫹ cells were isolated by magnetic bead adsorption and
cell sorting from B6 and P2X7R⫺/⫺ mice. Cells of 95% homogeneity by FACS analysis were mixed at ratios between 1:1 and 1:8
with purified B6 CD4⫹CD25⫺ cells. APCs and anti-CD3 Ab were
added to induce proliferation of CD4⫹CD25⫺ cells (6). Fig. 10B
reveals that on a per cell basis, CD4⫹CD25⫹ cells from P2X7R⫺/⫺
mice were as effective in their suppressive activity as cells from B6
mice. Therefore, the absence of P2X7Rs appears to cause an increase in functional CD4⫹CD25⫹ Treg cells in vivo.
3082
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.
Acknowledgments
We thank Dr. C. Gabel for the generous gift of P2X7R⫺/⫺ mouse breeding
pairs, and Drs. William Stohl and Dixon Gray (University of Southern
California) for critical reading of this manuscript.
Disclosures
The authors have no financial conflict of interest.
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