of September 13, 2015.
This information is current as
Murine T Cells
and Externalization of Phosphatidylserine by
-Dependent Shedding of CD62L
and ATP Released from Injured Cells
Haag and Friedrich Koch-Nolte
FriedrichKrebs, Peter Bannas, Björn Rissiek, Michel Seman,
Felix Scheuplein, Nicole Schwarz, Sahil Adriouch, Christian
2009; 182:2898-2908; ;
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The Journal of Immunology
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NAD?and ATP Released from Injured Cells Induce
P2X7-Dependent Shedding of CD62L and Externalization of
Phosphatidylserine by Murine T Cells1
Felix Scheuplein,2*†Nicole Schwarz,2* Sahil Adriouch,*‡Christian Krebs,* Peter Bannas,*
Bjo ¨rn Rissiek,* Michel Seman,‡Friedrich Haag,* and Friedrich Koch-Nolte3*
Extracellular NAD?and ATP trigger the shedding of CD62L and the externalization of phosphatidylserine on murine T cells.
These events depend on the P2X7ion channel. Although ATP acts as a soluble ligand to activate P2X7, gating of P2X7by NAD?
requires ecto-ADP-ribosyltransferase ART2.2-catalyzed transfer of the ADP-ribose moiety from NAD?onto Arg125 of P2X7.
Steady-state concentrations of NAD?and ATP in extracellular compartments are highly regulated and usually are well below the
threshold required for activating P2X7. The goal of this study was to identify possible endogenous sources of these nucleotides. We
show that lysis of erythrocytes releases sufficient levels of NAD?and ATP to induce activation of P2X7. Dilution of erythrocyte
lysates or incubation of lysates at 37°C revealed that signaling by ATP fades more rapidly than that by NAD?. We further show
that the routine preparation of primary lymph node and spleen cells induces the release of NAD?in sufficient concentrations for
ART2.2 to ADP-ribosylate P2X7, even at 4°C. Gating of P2X7occurs when T cells are returned to 37°C, rapidly inducing
CD62L-shedding and PS-externalization by a substantial fraction of the cells. The “spontaneous” activation of P2X7during
preparation of primary T cells could be prevented by i.v. injection of either the surrogate ART substrate etheno-NAD or ART2.2-
inhibitory single domain Abs 10 min before sacrificing mice. The Journal of Immunology, 2009, 182: 2898–2908.
play important roles also as signaling molecules in the extracellu-
lar environment (1, 2). In the context of the immune system of
higher organisms, it has been proposed that the release of NAD?
and ATP from lysed cells may alert cells of the immune system to
tissue damage (2–6). Indeed, lymphocytes and macrophages are
equipped with numerous sensors for extracellular NAD?and ATP,
including nucleotide-metabolizing ecto-enzymes and nucleotide
receptors (7, 8).
Mono-ADP-ribosyltransferases (ARTs)4transfer the ADP-ri-
bose moiety from NAD?to specific amino acids in target proteins
(9, 10). This is the mechanism by which several bacterial toxins,
like cholera- and pertussis-toxin, cause pathology after translocat-
ing into mammalian host cells (11). ART2.2 is a GPI-anchored,
raft-associated ecto-enzyme prominently expressed by murine T
AD?and ATP are universal currencies of energy me-
tabolism and are found in all cells in all kingdoms of
life. Mounting evidence indicates that these nucleotides
cells (12–14). ART2.2 catalyzes ADP-ribosylation of CD8, the
integrin LFA-1, the P2X7receptor, and several other target pro-
teins (12–15). T cell activation induces the metalloprotease-medi-
ated shedding of a soluble, enzymatically active isoform of
ART2.2 (16). ART2-deficient mice (17) exhibit reduced sensitiv-
ities to Con-A-induced hepatitis (18).
The type II transmembrane protein CD38 is a potent ecto-NAD-
glycohydrolase (ecto-NADase) (19, 20) expressed by lymphocytes,
endothelial cells, and several other cell types. CD38-deficient mice
show impaired humoral immune responses, neutrophil chemotaxis,
and dendritic cell (DC) trafficking (21–23). Cells from CD38-de-
ficient mice do not metabolize ecto-NAD?efficiently, and the re-
sulting higher levels of ecto-NAD?lead to a higher level of
ART-mediated cell surface protein ADP-ribosylation (24).
CD38-deficient mice show enhanced sensitivity to insulin-de-
pendent diabetes mellitus, which is dependent on the presence
of ART2.2 (25). Likely, this reflects the enhanced activity of
ART2.2 in these mice as a consequence of increased levels of
The cytolytic P2X7receptor is a homo-trimeric, ligand-gated,
nonselective ion channel that has sparked interest because of its
peculiar ability to induce the formation of a large nonselective
membrane pore (4, 26–28). High concentrations of extracellular
ATP (0.2–2 mM) are required to gate P2X7. Much lower con-
centrations of extracelluar NAD?(2–20 ?M) suffice to gate
P2X7on cells coexpressing ART2.2 (13). ART2.2-catalyzed
ADP-ribosylation at residue R125 presumably positions the
common nucleotide-diphosphate moiety into the ligand binding
site at the interface of adjacent subunits of the homotrimeric
receptor (29). Activation of P2X7on T cells by ATP or by NAD-
dependent ADP-ribosylation initiates a cascade of events, includ-
ing influx of calcium, the shedding of the L-selectin/CD62L hom-
ing receptor, and the externalization of phosphatidylserine (PS) on
the outer leaflet of the cell membrane (13, 29, 30). Chronic
*Institute of Immunology, University Hospital, Hamburg, Germany;†The Jackson
Laboratory, Bar Harbor, ME, 04609, and‡Inserm U905 and Faculte ´ de me ´dicine et de
pharmacie, Universite ´ de Rouen, Rouen, France
Received for publication May 28, 2008. Accepted for publication January 3, 2009.
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.
1This work was supported by Deutsche Forschungsgemeinschaft Grant No310/6 (to
F.K.-N. and F.H.) and by stipends from the Werner Otto Foundation (to C.K. and
P.B.) and from the Fondation pour la Recherche Medical (to S.A.).
2F.S. and N.S. contributed equally to this study.
3Address correspondence and reprint requests to Dr. Friedrich Koch-Nolte, Institute
of Immunology, University Hospital, Hamburg, Germany. E-mail address: nolte@
4Abbreviations used in this paper: ART, ADP-ribosyltransferase; etheno-NAD?,
1,N6-Ethenonicotinamide adenine dinucleotide; DC, dendritic cell; PS, phosphatidyl-
serine; WT, wild type; PI, propidium iodide; sdAb, single domain Ab.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
by guest on September 13, 2015
activation of P2X7induces apoptosis and cell lysis (13). The nat-
ural allelic P451L polymorphism in the C-terminal tail of murine
P2X7distinguishes common strains of laboratory mice (31). Wild-
type (WT) P451 is expressed by BALB/c and most other strains of
mice, whereas the 451L variant is expressed by C57BL/6 and DBA
mice (31). The 451L variant displays normal membrane currents,
but impaired pore formation (31, 32). Naive BALB/c T cells are
very sensitive to NAD?and ATP, whereas C57BL/6 T cells are
less sensitive (13).
The plasma membrane of living cells is impermeable to NAD?
and ATP, but they can be released from cells by lytic and nonlytic
mechanisms (33, 34). Intracellular levels of NAD?and ATP are in
the upper micromolar and lower millimolar range, whereas serum
concentrations are two to three orders of magnitude lower and are
kept low by ecto-nucleotidases. The duration of extracellular sig-
naling via NAD?is controlled by the CD38 family ecto-NAD-
glycohydrolases/ADP-ribosylcyclases that hydrolyze NAD?to
ADP-ribose and the CD203 family of phosphodiesterases that
hydrolyze NAD?to nicotinamide mononucleotide and AMP (20,
35, 36). Similarly, the concentration of ATP and the duration of
signaling via ATP in the extracelluar compartment are controlled
by the CD39 family of ecto-nucleotidases and the CD203 family of
phosphodiesterases which hydrolyze ATP to ADP and/or AMP
The principle aim of this study was to determine whether en-
dogenous sources of NAD?and/or ATP can activate P2X7. To
address this question, we used two model systems, hemolysis and
mechanical manipulation of cells as during the routine preparation
of primary cells from lymph nodes and spleen. Our results show
that both scenarios, indeed, result in the release of endogenous
nucleotides in sufficient concentration to activate P2X7, with ATP-
mediated effects dissipating more rapidly than those mediated by
Materials and Methods
Chemicals and Abs
ADP-ribose, ATP, NAD?, etheno-NAD?, and KN-62 (1-(N,O-bis(5-
isoquinolinesulfonyl)-N-methyl-L-tyrosyl)-4-phenylpiperazine) were obtained
from Sigma-Aldrich. PE- and FITC-conjugated mAbs and Annexin-V
were purchased from BD Pharmingen, including anti-CD3? (145-
2C11), anti-CD4 (RM4–5), anti-CD8 (53–5.8), anti-CD38 (90), and
anti-CD62L (MEL-14).32P-NAD?was obtained from Amersham Bio-
sciences. mAbs Nika102 (anti-ART2.2) pAb K1G (anti-P2X7) and sin-
gle domain Abs s?16a and l-17 (anti-ART2.2) were prepared as de-
scribed previously (12, 40, 41).
Animals and cells
BALB/c mice were obtained from The Jackson Laboratory or Charles
River Laboratories. CD38-deficient mice (21) and ART2-deficient mice
(17) were backcrossed onto the BALB/c background for 12 generations.
The ART2?/?lines are deficient in both ART2.1 and ART2.2 (17). P2X7-
deficient mice were from the twelfth backcross generation to C57BL/6
mice (3). Where indicated, mice received i.v. injections of PBS, single
domain Abs in PBS, or etheno-NAD?in PBS (200 ?l into the tail vein).
Mice were sacrificed by exposure to O2/CO2. All animal experiments were
performed in accord with the German animal-protection law. Single-cell
suspensions were prepared from lymph nodes and spleens in cold (4°C)
RPMI 1640 medium by gentle dissection and passage through Nitex mem-
brane (125-?m mesh, Tetko). Alternatively, single cell suspensions were
prepared by two other techniques: 1) from spleen by gentle perfusion
with 10 ml ice cold medium using a 1.0-gauge needle and 10 ml syringe
or 2) from lymph nodes and spleen by gentle pipetting following pre-
incubation in medium containing 10 mg/ml collagenase (Life Technol-
ogies) for 60 min at 4°C. B cells were depleted using magnetic cell
separation with Dynabead-immobilized goat anti-mouse IgG (Dynal
Biotech) at 4°C as described (13). Purity of T cells was always ?95%
as verified by FACS analyses using PE-conjugated anti-B220 and FITC-
Preparation of erythrocyte lysates
Blood was collected from mice by retroorbital puncture into ice cold hep-
arinized Eppendorf tubes. Erythrocytes were pelleted by centrifugation at
4°C, washed once in ice cold PBS, and were resuspended in 1.5 volumes
of ice cold PBS. Cells were ruptured on ice by ultrasonication in an MK2
ultrasonic disintegration machine (MSE Scientific Instruments) by two 10-
second sonifications at a power setting of 12 micron. Cellular debris was
removed by centrifugation (15,000 ? g) for 5 min at 4°C. Clarified lysates
were diluted in ice cold PBS. Aliquots of lysates (50 ?l) were added to cell
suspensions in RPMI 1640 medium (50 ?l, 4 ? 105cells). NAD?con-
centrations in cell lysates were determined by a sensitive cycling assay
(42). ATP concentrations were measured with a luciferase-based assay
system according to the manufacturer’s instructions (Sigma-Aldrich). In
some cases, lysates were incubated at 37°C for the indicated times before
addition to T cells.
Assay for phosphatidylserine exposure and propidium iodide
Following treatment with exogenous NAD?, ATP, or erythrocyte lysates
for the indicated times at 4 or 37°C, cells were washed in RPMI 1640
medium adjusted to 2 mM CaCl2, and were stained for 20 min on ice with
FITC-conjugated Annexin-V (1 ?g/ml) (BD Biosciences) and PI (10 ?g/
ml) before flow cytometry. In some cases, KN-62 was added during or after
treatment with NAD?and ATP. Relatively high concentrations (10 ?M)
were used because KN-62 exhibits low potency at mouse P2X7receptors
(43, 44). For temperature response analyses, cells were incubated in the
absence or presence of ATP for 30 min at the indicated temperature in a
PCR machine. For pulse chase analyses, cells were treated with NAD?or
ATP for 30 min at 4 or 37°C. Cells were then washed and incubated further
for 60 min at 4 or 37°C before staining with Annexin-V/PI as above.
Immunoprecipitation and Western blot analyses
T cells (1 ? 107/ml) were incubated for 20 min at 4 or 37°C in the presence
of radiolabeled32P-NAD?(1 ?M, 5?Ci/ml) in RPMI 1640 medium con-
taining 1 mM ADP-ribose. Cells were then washed in cold PBS to remove
free NAD?. Cells were lysed in 250 ?l PBS, 1% Triton X-100, 1 mM
AEBSF at 4°C for 30 min. Cell lysates were precleared by centrifugation
(14,000 ? g at 4°C for 30 min) followed by incubation with 20 ?l Protein-
G-Sepharose (Pharmacia Biotech) for 60 min at 4°C. Immunoprecipitation
was performed in parallel with K1G anti-P2X7immune serum (1 ?l) (40),
LFA-1 specific mAb M17/4 (1 ?g), or CD8-specific mAb 53–6.7 (1 ?g)
each immobilized on 20 ?l Protein-G-Sepharose. Proteins were size frac-
tionated by SDS-PAGE on precast Nupage (10%) gels (Invitrogen) and
blotted onto nitrocellulose membranes. Radioactivity was detected by au-
toradiography by exposing the membrane to Kodak X-omat Films at
?80°C for 48 h.
Exposure of murine T cells to exogenous NAD?or ATP causes
shedding of CD62L and externalization of phosphatidylserine
Treatment of purified murine T cells with exogenous NAD?or
ATP at 37°C induced, within minutes, the externalization of phos-
phatidylserine on the outer leaflet of the plasma membrane (Fig.
1A, panels 2 and 4) and the shedding of L-selectin/CD62L (Fig.
1B, panels 2 and 4). T cells from P2X7-deficient mice (3) were
completely resistant to the effects of NAD?and ATP (Fig. 1, C
and D), indicating that these effects are dependent on P2X7. Con-
sistently, WT cells incubated with NAD?or ATP in the presence
of KN-62, a specific inhibitor of P2X7(45), neither shed CD62L
nor exposed phosphatidylserine (Fig. 1, A and B, panels 3 and 5).
In the case of T cells obtained from C57BL/6 mice that express the
451L P2X7variant (31), much higher concentrations of NAD?and
ATP were required to induce PS exposure and CD62L shedding
than by T cells from BALB/c mice that express WT P2X7(Fig. 1C,
panels 2–5 vs Fig. 1A, panels 2 and 4). NAD-mediated but not
ATP-mediated activation of P2X7requires functional ART2.
Hence, T cells from ART2-deficient mice (17) were sensitive to
direct activation of P2X7with the soluble ligand ATP (Fig. 1, A
and B, panel 9), but were resistant to NAD-induced activation of
P2X7(Fig. 1, A and B, panel 7), which requires ART2-catalyzed
ADP-ribosylation of P2X7(29).
2899The Journal of Immunology
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NAD?and ATP released from lysed erythrocytes activate P2X7
on murine T cells
The membrane of living cells is impermeable to NAD?or ATP.
Cells lysed during tissue injury or in the course of necrotic cell
death present a potential source of extracellular nucleotides. To
test whether nucleotides released from lysed cells can activate
P2X7, we exposed purified T cells to crude cell lysates generated
by ultrasonication of mouse erythrocytes (Fig. 2). We chose eryth-
rocytes for these analyses because the lysis of erythrocytes occurs
under various pathological conditions in vivo (46–48) and because
erythrocyte lysis is commonly used to deplete these cells during
the preparation of primary lymphocytes (49).
The results shown in Fig. 2 demonstrate that T cells, indeed,
respond in a dose-dependent manner to erythrocyte lysates with
externalization of PS and shedding of CD62L (Fig. 2, A and B,
panels 1 and 3). As in case of exogenously added nucleotides (Fig.
1), these responses were suppressed by the P2X7antagonist KN-62
(Fig. 2, A and B, panels 2 and 4). Note the lower intensity of
Annexin-V staining in cells treated with concentrated vs dilute
lysates (Fig. 2A, panels 1 and 3). This likely reflects the presence
induce P2X7-dependent externalization of
phosphatidylserine and shedding of CD62L by
T cells. A and B, Purified lymph node T cells
from BALB/c WT, and ART2?/?mice were
incubated without (panels 1 and 6) or with 25
?M NAD?or 250 ?M ATP for 30 min at
37°C. Parallel incubations were performed in
the presence of the P2X7antagonist KN-62
(10 ?M) (panels 3, 5, 8, and 10). Cells
were washed and stained with Annexin-
VFITCand PI (A) or with anti-CD62LPE
and anti-CD3FITC(B) before FACS analy-
sis. C and D, Purified lymph node T cells
from C57BL/6 WT, and P2X7
were incubated without (panels 1 and 6) or
with 25 ?M, 250 ?M NAD?250 ?M
ATP, or 2.5 mM ATP for 30 min at 37°C.
Cells were stained as in A and B before
FACS analysis. Numbers indicate the per-
centage of cells in each quadrant (in B and
D those in the upper and lower right quad-
rants). Results are representative of four
Exogenous NAD?and ATP
2900 ACTIVATION OF P2X7BY ENDOGENOUS NUCLEOTIDES
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of endogenous annexins released from lysed erythrocytes that
compete with the fluorochrome-conjugated Annexin-V for binding
to externalized PS on T cells. Note further that ART2-deficient
cells responded only to concentrated lysates (Fig. 2, A and B, pan-
els 5 and 7) whereas WT T cells responded to both concentrated
and diluted lysates (Fig. 2, A and B, panels 1 and 3). These findings
indicate that diluted lysates contain NAD?but not ATP in suffi-
cient concentration to activate P2X7, whereas concentrated lysates
also contain sufficient ATP.
To better assess the relative contributions of NAD?and ATP
from erythrocyte lysates on L-selectin shedding and PS exposure
by T cells, we performed comparative dose response analyses with
erythrocyte lysates and exogenous NAD?, ATP, or mixtures of
NAD?and ATP. The results confirm the complete resistance of
ART2-deficient T cells to NAD?(Fig. 3A), whereas WT T cells
exhibit a very high sensitivity to NAD?(EC502.6 ?M). In con-
trast, both cell types respond with a similar dose dependency to
ATP (EC50120 ?M) (Fig. 3B). The dose responses of WT and
ART2-deficient T cells to mixtures of NAD?and ATP (e.g., at a
ratio of 1:10; Fig. 3C) were similar to those obtained with eryth-
rocyte lysates (Fig. 3D).
The EC50values obtained with exogenous NAD?or ATP cor-
respond well to our measurements of NAD?and ATP in erythro-
cyte lysates and those of previous studies (50–52). Experiments
performed with ART2-deficient T cells, sensitive only to ATP,
show that the effects obtained with the EC50of ATP (?120 ?M)
are reproduced with erythrocyte lysates diluted ?10-fold, consis-
tent with the interpolated ATP concentration in undiluted erythro-
cyte lysates of 1.0 mM (?400 ?M, n ? 13). Similarly, experi-
ments performed with WT T cells show that the effects obtained
with the EC50of NAD?(?2.6 ?M) are reproduced with erythro-
cyte lysates diluted 100-fold, consistent with the interpolated con-
centration of NAD?in undiluted erythrocyte lysates of 260 ?M
(?80 ?M, n ? 13).
In these experiments, we consistently noted a slightly higher
proportion of cells “spontaneously” exposing PS in preparations
obtained from WT mice vs ART2-deficient mice (i.e., compare
control values for WT and ART2-deficient cells in the absence of
exogenously added nucleotides or lysates in Fig. 3, A–D). The
hypothesis that this observation reflects the exposure of cells to
NAD?before or during cell preparation will be addressed further
The P2X7-inducing activities in erythrocyte lysates fade upon
incubation at 37°C
The fate and half-life of extracellular nucleotides is determined by
numerous nucleotide-metabolizing ecto-enzymes such as CD38
and CD39, many of which are expressed by erythrocytes (53, 54).
One should, therefore, expect that ultrasonication of erythrocytes
would expose the released NAD?and ATP to nucleotide-degrad-
ing enzymes. To determine whether nucleotide turnover affects the
P2X7-inducing potential of erythrocyte lysates, we incubated these
lysates for various times at 37°C and then assayed the capacity of
the lysates to induce PS exposure by T cells (Fig. 4). A substantial
reduction in the capacity of lysates to induce PS exposure was seen
already after 10 min of preincubation (Fig. 4A, panels 2 and 5 and
Fig. 4B) and all PS-inducing activity was lost within 60 min (Fig.
4A, panels 3 and 6 and Fig. 4B). Taken together, these results
indicate that erythrocyte lysates contain both, NAD?and ATP, in
sufficient concentrations to activate P2X7on T cells, and further
that both nucleotides are degraded with a half-life of less than 10
min upon incubation at 37°C. The results again pinpoint a clear
difference in the background proportion of freshly prepared T cells
from WT mice vs ART2-deficient mice, exposing PS on their sur-
face (Fig. 4B). The following experiments were designed to further
explore this phenomenon.
P2X7-dependent PS exposure and shedding of CD62L. Purified lymph
node T cells from BALB/c WT and ART2?/?mice were incubated with
fresh erythrocyte lysates in two different concentrations (diluted 1/7.5, 1/20
in PBS) for 30 min at 37°C. Parallel incubations were performed in the
presence of 10 ?M KN-62 (panels 2, 4, 6, and 8). Cells were washed and
stained with Annexin-VFITCand PI (A) or with anti-CD62LPEand anti-
CD3FITC(B) before FACS analysis. Numbers indicate the percentage of
cells in each quadrant (in B those in the upper and lower right quadrants).
Results are representative of four independent experiments.
NAD?and ATP released from lysed erythrocytes induce
lysate-induced PS exposure by T cells. Purified lymph node T cells from
BALB/c WT (f) and ART2?/?mice (E) were incubated with the indi-
cated concentrations of NAD?(A), ATP (B), a mixture of NAD?and ATP
(C), or fresh erythrocyte lysates (D) for 30 min at 37°C. Cells were then
washed and subjected to FACS analyses as in Fig. 1. Vital cells correspond
to Annexin-V-negative and PI-negative cells (i.e., cells in the lower left
quadrant of FACS plots as shown in Figs. 1 and 2). Results are represen-
tative of two independent experiments.
Comparative dose response analyses of NAD-, ATP-, and
2901The Journal of Immunology
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In vivo blockade of ART2.2 prevents the spontaneous PS
flashing and shedding of CD62L by freshly prepared lymph
The “spontaneous” externalization of PS and shedding of CD62L
by a fraction of primary WT T cells from freshly prepared lymph
nodes (Fig. 1 and 4B) might reflect their prior encounter with ex-
tracellular nucleotides released from endogenous sources. The
spontaneous PS exposure and shedding of CD62L evidently de-
pend on functional ART2 and P2X7, as these phenomena are not
observed in T cell preparations from ART2-deficient (Fig. 1, A and
B, panel 6) or P2X7-deficient (Fig. 1, C and D, panel 6) mice.
Spontaneous PS exposure and shedding of CD62L was specific for
T cells, consistent with the notion that the effect is dependent on
ART2.2, which is expressed by T cells, but not by B cells, NK
cells, or macrophages (12). The finding that ART2-deficient cells
do not spontaneously expose PS (Fig. 1A, panel 6) or shed CD62L
(Fig. 1B, panel 6) indicates that these effects are caused by endog-
enous NAD?rather than by endogenous ATP because ART2-de-
ficient T cells retain unabated sensitivity to exogenous ATP (Fig.
1A, panel 9). Consistently, T cells from CD38-deficient mice
which lack the major NAD-hydrolyzing ecto-enzyme (21, 24)
show markedly increased levels of spontaneous PS externalization
(Fig. 5A, panel 1) and CD62L shedding (Fig. 5A, panel 3).
In principle, the spontaneous PS exposure and CD62L shedding
could reflect exposure of cells to extracellular NAD?either before,
i.e., in vivo, or during cell preparation, i.e., after sacrificing of the
mouse. To distinguish between these possibilities, we sought con-
ditions that would prevent these reactions either by blocking
ART2.2 or by blocking the activation of P2X7. To this end, we first
used two distinct, recently described single domain Abs (sdAbs)
that block the enzymatic and cytotoxic activities of ART2.2 (41).
As illustrated in Fig. 5, a single i.v. injection of either sdAbs
s?16a or l-17 10 min before sacrificing effectively blocked both
PS externalization (Fig. 5A, panel 2 and Fig. 5B) and shedding of
CD62L (Fig. 5A, panel 4 and Fig. 5B), even in case of the highly
susceptible cells from CD38-deficient mice. As a complementary
approach, we used etheno-NAD?as ART-substrate (55). Etheno-
ADP-ribosylation does not activate P2X7and, in addition, pre-
vents activation of P2X7by subsequent ADP-ribosylation (13,
29). Indeed, as in case of the sdAbs, a single i.v. injection of
etheno-NAD?10 min before sacrificing effectively blocked
both PS externalization and shedding of CD62L (Fig. 5B) by
freshly prepared cells. These results strongly suggest that ex-
posure of cells to endogenous NAD?occurred during sacrific-
ing and/or cell preparation.
T cells prepared at 4°C do not externalize PS, but do so when
returned to 37°C
To further test the hypothesis that NAD?is released during cell
preparation, we next analyzed the effects of temperature during
and after cell preparation on spontaneous PS externalization and
CD62L shedding (Figs. 6 and 7). Cells that had been prepared and
kept at 4°C did not spontaneously externalize PS (Fig. 6A, panels
1 and 5). In contrast, cells prepared and kept at 37°C for 30 min
contained substantial numbers of spontaneously PS-exposing cells,
with higher proportions of such cells in preparations from CD38-
deficient vs WT mice (Fig. 6A, panels 2 and 6). Remarkably, cells
prepared at 4°C did expose PS when subsequently incubated at
37°C, with cells from CD38-deficient mice again showing stronger
responses than WT cells (Fig. 6A, panels 3 and 7). Further, cells
prepared at 37°C did not re-internalize PS when subsequently
their P2X7-inducing activity. Erythrocyte lysates were diluted 1/10 in PBS
(A) or as indicated (B) and preincubated for 0, 10, or 60 min at 37°C.
Purified T cells from BALB/c WT and ART2?/?mice were then incubated
with pretreated erythrocyte lysates for 30 min at 37°C. Cells were washed
and stained with Annexin-VFITCand PI. Results in B are presented as
percentage of viable (PS?/PI?) cells. Results are representative of three
Incubation of erythrocyte lysates at 37°C results in loss of
posure and shedding of CD62L by freshly prepared T cells. Ten minutes
before sacrificing, BALB/c WT, ART2?/?, and CD38?/?mice received
injections of PBS (control) or PBS containing 300 ?g ART2.2-specific
sdAb s plus 16a. Additional CD38?/?mice received injections of sdAb
l-17 or 2 mg etheno-NAD?. Lymph node cell suspensions were prepared
and incubated for 30 min at 37°C before staining with Annexin-VFITC,
anti-CD3APC, and PI or with anti-CD62LPEand anti-CD3FITCbefore FACS
analysis. A, FACS plots from CD38?/?mice after injections with PBS or
sdAb s?16a. B, Results are presented as percentage of vital (PS?/PI?) T
cells and as percentage of CD62L?T cells (gated on CD3?cells). Results
are representative of three independent experiments.
Blocking ART2.2 with sdAbs prevents spontaneous PS-ex-
2902ACTIVATION OF P2X7BY ENDOGENOUS NUCLEOTIDES
by guest on September 13, 2015
returned to 4°C (Fig. 6A, panels 4 and 8). These results imply that
the spontaneous activation of P2X7is caused by NAD?released
from cells during preparation rather than by exposure of cells to
NAD?before preparation in vivo, because cells exposed to NAD?
before killing of the animal, i.e., at 37°C, should still have exhib-
ited externalized PS when subsequently prepared and kept at 4°C.
Similar results were obtained when exogenous NAD?(25 ?M)
was added to the buffer during cell preparation (Fig. 6B), i.e., cells
exposed to NAD?at 4°C externalized little if any PS (panels 1
and 5), while cells prepared at 37°C (panels 2 and 6) responded
vividly with PS externalization as did cells that were returned to
37°C for 30 min after preparation at 4°C (panels 3 and 7). Note
that addition of exogenous NAD?to WT cells resulted in a re-
sponse of similar magnitude as the spontaneous response of CD38-
deficient cells (to endogenous NAD?) (Fig. 6B, panel 2 vs Fig. 6A,
panel 6). Note further that the level of PS exposure by CD38KO
cells in response to endogenously released NAD?already was
near maximal, i.e., was enhanced only slightly by the addition of
exogenous NAD?(panel 6 in Fig. 6A vs panel 6 in Fig. 6B). This
indicates that P2X7is already ADP ribosylated to a large extent on
CD38KO cells during cell preparation.
Fig. 7 illustrates more detailed analyses of the temperature-de-
pendency of PS-externalization (Fig. 7, A and B) and CD62L-
shedding (Fig. 7, C and D) by T cells induced by exogenously
added ATP and NAD?. The results reveal that T cells do not
externalize PS or shed CD62L at 4°C, even when exposed to rel-
atively high concentrations of ATP (250 ?M) or NAD?(25 ?M).
Cells exposed to ATP did respond vividly at room temperature
(20°C) (Fig. 7, B and D), while cells exposed to NAD?showed
maximal shedding only at 37°C (Fig. 7, A and C). These results
indicate that the soluble ligand ATP is a better agonist for P2X7
than the covalently attached ADP-ribose. Consistent with this in-
terpretation, the temperature response curves were shifted even
further to the left when T cells were treated with the highly potent
P2X7agonist benzoyl-ATP (results not shown).
Note that the temperature response curves of CD38-deficient
cells in the absence of exogenous nucleotides resemble those of
WT cells exposed to exogenously added NAD?(Fig. 7, A and C).
Moreover, ART2-deficient cells, which respond normally to exog-
enous ATP, neither spontaneously expose PS or shed CD62L nor
do so in response to exogenous NAD?at any temperature. These
results substantiate the interpretation that NAD?rather than ATP
is the effective signaling molecule inducing spontaneous PS expo-
sure and CD62L shedding during T cell preparation.
ADP-ribosylation of cell surface proteins proceeds more
efficiently at 4°C than at 37°C
To assess the extent of cell surface protein ADP-ribosylation at
different temperatures, we used a previously described FACS-
based assay using the NAD?analog etheno-NAD?and the
when returned to 37°C. Purified lymph node T cells from BALB/c WT
(?/?) and CD38-deficient mice (?/?) were incubated for 30 min at 4 or
37°C in the absence (A) or presence (B) of exogenous NAD?(25 ?M).
Cells were washed and stained with Annexin-VFITCand PI for FACS anal-
yses either directly or following a further incubation for 60 min at 4 or
37°C as indicated. Numbers in A and B indicate the percentage of cells in
each quadrant. Results are representative of three independent experiments.
T cells prepared at 4°C do not externalize PS but do so
NAD?induced PS externalization and CD62L shedding.
Purified lymph node T cells from BALB/c WT, ART2?/?,
and CD38?/?mice were incubated for 30 min at the in-
dicated temperatures in the absence or presence of exog-
D). Washed cells were stained with Annexin-VFITCand PI
(A and B), or with anti-CD62LPEand anti-CD3FITC(C and
D) before FACS analyses. Results are representative of
three independent experiments.
Temperature dependency of ATP and
2903The Journal of Immunology
by guest on September 13, 2015
etheno-adenosine-specific mAb 1G4 to detect etheno-ADP-ri-
bosylation of cell surface proteins (Fig. 8) (55). In accord with
previous reports, ART2-deficient cells did not show any detect-
able etheno-ADP-ribosylation of cell surface proteins (Fig. 8A).
Remarkably, on WT T cells, the extent of cell surface protein
ADP-ribosylation decreased with increasing temperatures (Fig.
8A), i.e., was much higher on cells incubated at 4°C than on
those incubated at 37°C (Fig. 8B, panels 1 and 2). Further,
subjecting cells that had been labeled at 4°C to a subsequent
incubation at 37°C resulted in a marked decrease in the level of
protein ADP-ribosylation (Fig. 8A, panel 3 vs panel 1). In con-
trast, subjecting cells that had been labeled at 37°C to a sub-
sequent incubation at 4°C resulted in little if any detectable
changes in the level of protein ADP-ribosylation (Fig. 8A, panel
4 vs panel 2). Similar results were obtained with CD38-defi-
cient T cells (Fig. 8A, panels 5–8). These results show that
ADP-ribosylation of cell surface proteins proceeds efficiently at
4°C and, further, suggest that labeling is reversed at 37°C, e.g.,
by enzymatic removal of the etheno-ADP-ribose group and/or
internalization or shedding of labeled proteins.
Differential radiolabeling of P2X7, LFA-1, and CD8 in cells
prepared from WT vs CD38-deficient mice
ART2.2 is known to ADP-ribosylate several distinct T cell surface
proteins (14, 29, 56). ADP-ribosylation sites on cell surface pro-
teins already occupied during cell preparation would not be avail-
able to subsequent ADP-ribosylation, e.g., upon addition of exog-
enously added NAD?. To determine to which extent the exposure
of cells to NAD?released during cell preparation affects the ADP-
ribosylation of cell surface proteins to a subsequent exposure to
exogenous NAD?, we incubated freshly prepared T cells from WT
and CD38-deficient mice at 4 or 37°C with exogenously added
radioactive NAD?(1 ?M), followed by lysis of cells and immu-
noprecipitation of known ART2-target proteins with specific Abs
Incubation of cells with radiolabeled NAD?at 4°C leads to
covalent radiolabeling of numerous cell surface proteins in T cells
from both, WT and CD38KO mice (Fig. 9A, lanes 1 and 2). Cells
from ART2KO mice do not incorporate any radiolabel under these
conditions (data not shown). The results confirm the efficient ac-
tivity of ART2 at 4°C and indicate that numerous binding sites are
still available for ADP-ribosylation. The reduced radiolabeling of
P2X7in cells from CD38KO vs WT mice (Fig. 9A, lanes 5 and 6)
is consistent with the notion that most ADP-ribosylation sites on
P2X7are already occupied on cells from CD38KO mice as a result
of prior ADP-ribosylation by NAD?released during cell prepara-
tion. Incubation of cells with radiolabeled NAD?at 37°C results
in much lower radiolabeling of proteins than incubation with
NAD?at 4°C (Fig. 9B vs Fig. 9A), as in case of labeling with
etheno-NAD?(Fig. 8A), consistent with reversion of ADP-ribo-
sylation at 37°C. Note further that at 37°C, overall radiolabeling of
proteins is lower in WT than CD38KO cells (Fig. 9B, lanes 1 and
2), presumably due to CD38-mediated NAD?hydrolysis by WT
The results of this study demonstrate that P2X7on T cells can be
activated by endogenous sources of NAD?and ATP released from
lysed cells. We show that massive lysis of erythrocytes can result
in the release of sufficient quantities of these nucleotides to activate
P2X7. Our results further indicate that techniques routinely used in
immunology laboratories to prepare primary lymphocytes from
spleen and lymph nodes cause the release of NAD?in sufficient
quantity to gate P2X7and to phenotypically and functionally alter
a substantial fraction of cells.
In accord with previous studies (13, 57, 58), we show in this
study that exposure of T cells to exogenous ATP or NAD?triggers
the P2X7-dependent externalization of phosphatidylserine and
efficiently at 4°C than at 37°C. Purified lymph node T cells from BALB/c
WT, ART2?/?, and CD38?/?mice were incubated for 30 min at the in-
dicated temperatures with 25 ?M (A) or 5 ?M (B) etheno-NAD?. Cells
were washed and stained with anti-etheno-adenosine mAb 1G4Alexa488for
FACS analyses either directly or following a further incubation for 60 min
at 4 or 37°C as indicated. Results are representative of three independent
ADP-ribosylation of cell surface proteins proceeds more
sylated at 4 and 37°C. Purified lymph node T cells from BALB/c WT and
CD38?/?mice were incubated for 20 min at 4°C (A) or at 37°C (B) in the
presence of exogenous32P-NAD?(1 ?M). Cells were washed and lysed in
1% Triton X-100. Cell lysates (cl) were clarified by high speed centrifu-
gation and subjected to immunoprecipitation with Abs directed against
LFA-1, P2X7, or CD8. Radiolabeled proteins in immunoprecipitates were
detected by SDS-PAGE autoradiography. The results are representative of
three independent experiments.
SDS-PAGE autoradiography of target proteins ADP-ribo-
2904 ACTIVATION OF P2X7BY ENDOGENOUS NUCLEOTIDES
by guest on September 13, 2015
shedding of CD62L (Fig. 1). Using T cells from ART2-deficient
and WT mice as biological indicators, we can distinguish the ef-
fects of ATP on P2X7from those of NAD?on P2X7in case of
exogenously added nucleotides (Fig. 1) as well as in case of en-
dogenous nucleotides released from lysed cells (Fig. 2); ATP acts
as a soluble ligand that gates P2X7on both WT and ART2-defi-
cient cells, whereas NAD?gates P2X7via ART2.2-catalyzed
ADP-ribosylation of R125 on WT cells but not on ART2-deficient
cells (29). Even though much lower concentrations of NAD than
ATP suffice to activate P2X7, the ADP-ribosylgroup linked to
R125 seems to be a less potent agonist for P2X7than ATP; even
high concentrations of NAD induce a slower conversion of WT
cells from the Annexin-V positive/PI negative stage to the An-
nexin-V/PI double-positive stage than ATP (e.g., Fig. 1A, panels 2
and 4). Moreover, because ART2KO cells do not ADP-ribosylate
P2X7, activation of P2X7in these cells is entirely dependent on
ATP. Consistently, ART2KO cells respond more vividly than WT
cells to exogenous ATP (Fig. 1A, panels 4 and 9) as well as to
concentrated cell lysates (Fig. 2A, panels 1 and 5, Fig. 4, panels 1
and 4). Evidently, partial ADP-ribosylation of P2X7on WT cells
(in response to NAD released from cells) retards ATP-induced
conversion to the double-positive stage.
The results of dose response analyses permit an estimation of
the EC50values for nucleotide-induced activation of P2X7, i.e.,
120 ?M for ATP and 2.6 ?M for NAD?(Fig. 3). Our results
indicate that such concentrations are reached in the vicinity of
lysed cells. In physiological settings, the effects of extracellular
nucleotides released from lysed cells on T cells would depend on
both time and distance from the lysis event. Using dilution of
erythrocyte lysates to mimic increasing distance, we show that
ATP-mediated effects dominate only in the short range (i.e., at
high concentrations of lysates), whereas NAD-mediated effects are
effective also at longer ranges (i.e., at high dilutions of lysates)
(Fig. 2). Preincubating erythrocyte lysates at 37°C before T cell
exposure indicate that the effects of ATP dissipate faster than those
mediated by NAD?(Fig. 4). The relative duration of signaling by
these extracellular nucleotides is determined largely by nucleotide
degrading ecto-enzymes such as the transmembrane ecto-enzymes
CD38 and CD39 (21, 39, 59).
Massive lysis of erythrocytes is observed in a number of
pathological conditions, e.g., during malaria infection, geneti-
cally inherited hemolytic diseases, and adverse reactions to
blood transfusion (46–48). It is likely that the mechanisms de-
scribed in this study with mechanically disrupted erythrocytes
act also in these and other settings in vivo. Indeed, recent results
from three different mouse models of inflammation support the
notion that nucleotides released during tissue injury induce
P2X7activation on T cells in vivo (6, 18, 25). Firstly, injection
of Con A induces T cell-dependent hepatitis that is accompa-
nied by fulminant liver cell damage as evidenced by the release
of cytosolic enzymes into the circulation. Mice genetically de-
ficient in ART2 or P2X7develop a milder form of the disease,
correlating with decreased sensitivities of liver-resident iNK-T
cells in these mice to apoptosis induced by extracellular nucle-
otides (18). Secondly, genetic ablation of the major ecto-NAD-
glycohydrolase CD38 results in elevated tissue NAD levels and
enhanced levels of T cell surface ADP-ribosylation (24). Trans-
fer of the deficient CD38 allele into the autoimmune diabetes-
prone NOD/Lt background resulted in accelerated disease pro-
gression, correlating with an enhanced sensitivity of regulatory
T cells to ART2-dependent NAD-induced cell death in these
mice (25). Indeed, these changes were corrected when CD38
deficiency was combined with ART2 deficiency. Thirdly, local
inflammatory responses induced by s.c. injection of Biogel lead
to release of NAD into the inflammatory pouch, causing shed-
ding of CD62L by T cells in the draining but not in the nond-
raining lymph nodes (6).
The results obtained in this study with the second experimental
system, i.e., the mechanical manipulations during routine prepara-
tion of cells from lymphatic organs, are of special pertinence to
immunologists working with primary lymphocytes. Our results
show that routine cell preparation techniques can lead to the gating
of P2X7on a fraction of cells, and subsequently to the shedding of
CD62L and externalization of PS. Similar degrees of P2X7acti-
vation were observed whether cells were prepared from lymph
nodes or spleen and when cells were prepared by gentle passage
through nytex membranes, collagenase digestion, or perfusion
with medium (results not shown). This has important implications
for experiments designed to study lymphocyte functions both, in
vitro and after adoptive transfer in vivo. Our results show that cells
prepared and kept strictly at 4°C do not externalize PS or shed
CD62L (Fig. 6). However, when cells are prepared at 37°C or
when cells are returned to 37°C subsequent to a preparation at 4°C,
a substantial fraction of the cells do externalize PS and shed
CD62L. In the case of WT mice, a relatively small fraction of T
cells (5–10%) was affected (Fig. 6A, panels 6 and 7), whereas the
majority of T cells was affected in case of CD38-deficient mice
(Fig. 6A, panels 6 and 7), which lack the major NAD-hydrolizing
ecto-enzyme (21, 24). This finding, together with the observation
that ART2-deficient mice which lack the major T cell ecto-ADP-
ribosyltransferase (17) do not spontaneously shed CD62L or ex-
pose PS (Fig. 7) imply that NAD?but not ATP is released in
sufficient quantity to induce activation of P2X7during cell
Consistently, blocking the ADP-ribosylation of cell surface pro-
teins by i.v. injection of an ART2.2-inhibitory sdAb 10 min before
killing of the animal, completely prevented the subsequent shed-
ding of CD62L and externalization of PS by WT and CD38-defi-
cient cells (Fig. 5). Similar effects were achieved by injecting
etheno-NAD?before sacrifice, which results in the etheno-ADP-
ribosylation of P2X7, thereby blocking the subsequent activation
by ADP-ribosylation (Fig. 5). ART2.2-inhibitory sdAbs are not
expected to have any adverse side effects. Because sdAbs lack the
Fc domain, they cannot activate complement or Ab-dependent cy-
totoxicity. Moreover the small (15kd) sdAbs are rapidly eliminated
via the kidney, with a serum half life of ?5 min. Blockade of
ART2.2 by sdAbs is reversible and ART2.2 activity on lymph
node cells is largely restored 24 h after injection (41). However,
systemic administration of etheno-NAD?could have unwanted
side effects as this would provide other members of the ART-
family with substrate, leading to the etheno-ADP-ribosylation of
other cell surface proteins.
Monitoring cell surface protein ADP-ribosylation using exog-
enously added etheno-NAD?(Fig. 8) or32P-NAD?(Fig. 9) con-
firmed that ART2.2-catalyzed ADP-ribosylation of cell surface
proteins proceeds efficiently at 4°C. In contrast, the gating of P2X7
requires elevated temperatures (Fig. 7). These findings imply that
unwanted activation of P2X7by ATP released from cells during
cell preparation can be prevented simply by keeping cells at 4°C
during preparation until the soluble ligand ATP is washed away.
However, ADP-ribosylation of P2X7in response to NAD?re-
leased from cells during cell preparation cannot be prevented by
keeping cells at 4°C because the covalently attached ADP-ribose
moiety cannot be removed by washing and thus will activate P2X7
when cells are returned to 37°C. To prevent Ab-induced modula-
tion of cell surface proteins, immunologists routinely perform
staining of lymphocytes for FACS-analyses at 4°C, whereas cells
2905The Journal of Immunology
by guest on September 13, 2015
are returned to 37°C for functional assays, e.g., in vitro TCR-
ligation and proliferation assays or in vivo migration studies. Un-
der such conditions, NAD-induced PS externalization and CD62L
shedding due to activation of P2X7by ADP-ribosylation could
Both the externalization of PS and the shedding of CD62L can
profoundly affect T cell functions. Externalization of PS is a com-
mon eat-me signal for macrophages, and such cells are equipped
with adapter proteins and cell surface receptors for binding PS-
exposing cells (60–62). Externalization of PS by T cells could
thus lead to enhanced binding to and/or phagocytic clearance by
macrophages. Moreover, PS exposure is associated with increased
cellular adhesion to endothelia and might promote the extravasa-
tion of T cells. CD62L is the major homing receptor for lymph
nodes, and cells lacking CD62L show impaired migration to pe-
ripheral lymph nodes (63–65). Metalloprotease-mediated shed-
ding of CD62L is triggered also upon activation of T cells by
engagement of the TCR or by mitogenic stimulation (66, 67). Our
results indicate that a substantial fraction of cells in primary lym-
phocyte preparations may exhibit a CD62L-negative phenotype as
a consequence of P2X7ADP-ribosylation during cell preparation
rather than as a sign of conventional T cell activation. Moreover,
it is possible that the constitutive externalization of PS described
for CD4?CD45RBlowcells is a consequence of exposure to NAD?
during cell preparation (68).
In this context it is important to note that common strains of
laboratory mice carry allelic variants of both P2X7and ART2
which affect the sensitivity to endogenously released nucleotides
(12, 31, 69). BALB/c mice express WT P2X7and both copies of
the duplicated ART2 locus. C57BL/6 mice carry the 451L allelic
variant of P2X7with impaired sensitivities to gating by ATP and
ADP-ribosylation (31, 32), as well as a defective ART2.1 allele,
while expressing the ART2.2 locus at much higher levels than
BALB/c mice (12, 69). Whether the reported high sensitivity of
naturally occurring regulatory T cells in C57BL/6 mice to activa-
tion of P2X7(70) is associated with NAD?released in vivo and/or
during cell preparation will be an important subject of future
The results of our experiments testing the temperature-depen-
dency of T cell surface ADP-ribosylation reactions indicate that
ADP-ribosylation of cell membrane proteins is reversible (Figs. 8
and 9), in accord with previous studies (56, 71). Enzymes capable
of reversing ADP-ribosylation include ADP-ribosylhydrolases
which can remove the entire ADP-ribose moiety (72, 73) and phos-
phodiesterases which remove only AMP, leaving ribose phosphate
attached to the target protein (74). To date ADP-ribosylhydrolases
have been described only as intracellular proteins (75), whereas
phosphodiesterase isoforms have been cloned and characterized
that function as membrane bound and secretory ecto-enzymes (76,
77). Because both labels used in this study (etheno-adenosine) and
(?-32P) would be removed by phosphodiesterases and ADP-ribo-
sylhydrolases, other tools will be required to determine the relative
contributions of these two enzyme families to reversion of protein
ADP-ribosylation. The differential labeling of P2X7vs LFA-1 and
CD8 at 4°C vs 37°C (Fig. 9) indicate that ADP-ribose moieties
buried in the ligand-binding pocket of P2X7may be better pro-
tected against de-ADP-ribosylating enzymes than ADP-ribose
moieties linked in a more exposed manner to other cell-membrane
That activation of P2X7by ADP-ribosylation can play a role in
physiological settings has been demonstrated previously by our
finding that NAD?released at inflammatory sites induces ART2-
dependent shedding of CD62L and T cell death in draining lymph
nodes (6). The results of the present study provide an additional
plausible scenario for the activation of P2X7in vivo, i.e., by
NAD?and ATP released during hemolysis. Malaria, for example,
is associated with periodic hemolysis and complex changes in T
cell function and apoptosis (47, 78). It will, therefore, be of interest
to determine whether and to what extent the genetic ablation or
pharmacological inhibition of ART2 and/or P2X7affects disease
progression in murine malaria models. Our results further demon-
strate that techniques routinely used in immunology laboratories to
prepare primary lymphocytes cause the release of sufficient quan-
tities of NAD?for ART2.2-catalyzed activation of P2X7. When
cells are returned to 37°C, this induces the externalization of PS
and shedding of CD62L, thereby likely altering T cell functions.
An efficient means to prevent the activation of P2X7during cell
preparation is an i.v. injection of ART2.2-blocking sdAbs shortly
We thank Dr. F. Lund, Saranac Lake, NY for providing CD38-deficient
mice, and Dr. C. Gable, Pfizer, for providing P2X7-deficient mice. This
work represents the partial fulfillment of the requirements for the graduate
thesis of FS. We thank Dunja Freese, Marion Nissen, Fabienne Seyfried,
and Dr. Kirsten Heiss, Hamburg, for assistance. FK-N, FH, and MS de-
signed and supervised the study. FS, and SA performed the experiments
shown in Figs. 1, 6 and 8B, NS those shown in Figs. 5, 7 and 8A, CK and
FS those shown in Figs. 2–4, and PB and FS those in Fig. 9, BR and NS
performed the NAD and ATP measurements. FK-N wrote the paper. We
thank Drs. H.-W. Mittru ¨cker and B. Fleischer, Hamburg, and Dr. O. Boyer,
Rouen, for critical reading of the manuscript.
The authors have no financial conflict of interest.
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