of September 16, 2015.This information is current as

Cells In VivoInducing ART2-Mediated Death of Naive TParticipates in T Cell Homeostasis by

Released during Inflammation+NAD

Friedrich Koch-Nolte, Friedrich Haag and Michel SemanSahil Adriouch, Sandra Hubert, Severine Pechberty,

http://www.jimmunol.org/content/179/1/186doi: 10.4049/jimmunol.179.1.186

2007; 179:186-194; ;J Immunol 

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, 23 of which you can access for free at: cites 39 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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NAD� Released during Inflammation Participates in T CellHomeostasis by Inducing ART2-Mediated Death of Naive TCells In Vivo1

Sahil Adriouch,*†‡ Sandra Hubert,*† Severine Pechberty,* Friedrich Koch-Nolte,†‡

Friedrich Haag,†‡ and Michel Seman2*†

Mono ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on mouse T lymphocytes, which catalyze the transfer ofADP-ribose groups from NAD� onto several target proteins. In vitro, ADP-ribosylation by ART2 activates the P2X7 ATPreceptor and is responsible for NAD�-induced T cell death (NICD). Yet, the origin of extracellular NAD� and the role ofNICD in vivo remain elusive. In a model of acute inflammation induced by polyacrylamide beads, we demonstrate releaseof NAD� into exudates during the early phase of the inflammatory response. This leads to T cell depletion in the draininglymph nodes from wild-type and, more severely, from mice lacking the CD38 NAD� glycohydrolase, whereas no effect isobserved in ART2-deficient animals. Intravenous injection of NAD� used to exacerbate NICD in vivo results in fast anddramatic ART2- and P2X7-dependent depletion of CD4� and CD8� T lymphocytes, which can affect up to 80% of peripheralT cells in CD38�/� mice. This affects mainly naive T cells as most cells surviving in vivo NAD� treatment exhibit thephenotype of recently activated/memory cells. Consistently, treatment with NAD� abolishes primary Ab response to aT-dependent Ag in NICD-susceptible CD38�/� mice but has no effect on the secondary response when given several daysafter priming. Unexpectedly NAD� treatment improves the response in their wild-type BALB/c counterparts. We proposethat NAD� released during early inflammation facilitates the expansion of primed T cells, through ART2-driven death ofresting cells, thus contributing to the dynamic regulation of T cell homeostasis. The Journal of Immunology, 2007, 179:186 –194.

M ammalian mono-ADP-ribosyltransferases (ART)3

constitute a family of ectoenzymes structurally relatedto bacterial toxins catalyzing the transfer of the ADP-

ribose group from NAD� onto amino acid residues of target pro-teins (1–3). ART1–ART4 paralogs are GPI-anchored in the outerleaflet of the plasma membrane and are thus responsible for uniqueposttranslational modifications of proteins in the extracellularcompartment. Like phosphorylation mediated by protein kinases,ART-mediated modification of proteins by ADP-ribosylationleads to structural changes that can either inhibit or stimulatetarget protein functions. ART activities in vitro can be split intothose relevant to trans-ADP-ribosylation of free soluble targetssuch as cytokines and growth factors or proteins produced by

neighboring cells and those relevant to cis-ADP-ribosylation ofproteins present on the surface of the ART-expressing cells (3).Although ADP-ribosylation of proteins can easily be shown invitro (2, 4 – 6), its reality and role in vivo are still poorly doc-umented (7, 8).

In the mouse, two of the six ART paralogs, namely, ART2.1and ART2.2, are expressed on the surface of most mature pe-ripheral T lymphocytes (1, 9). It was first shown that a GPI-anchored ART is expressed on the surface of mouse cytotoxicT cell lines and that incubation of cells with NAD� leads to theinhibition of both T cell proliferation and cytotoxic activity (6).This inhibition was associated with ADP-ribosylation of severalmembrane proteins such as LFA-1, CD8, CD27, CD43, CD44,or CD45 (10 –12). Recently, the association of ART2 with lipidrafts has been shown to focus ART2 on specific targets (13). Itwas postulated that ADP-ribosylation of coreceptors inhibitsTCR-signaling by altering receptor association as well as cellcontacts and T cell trafficking (10, 12). We subsequentlyshowed that incubation with NAD� leads to the rapid inductionof ART-dependent T cell death in vitro detectable at NAD con-centrations as low as 1 �M, a phenomenon that we proposed tocall NAD�-induced T cell death (NICD) for NAD�-inducedcell death (4, 14). We also discovered that NICD results fromthe activation of the P2X7 ATP receptor, which is another tar-get of murine ART2 (14).

P2X7 belongs to the P2X family of ATP-gated ion channelsexpressed on different cell types in the immune system includingT lymphocytes (15, 16). P2X7 activation triggers calcium flux,shedding of CD62L, phosphatidylserine (PS) exposure, opening ofa large nonselective membrane pore and ultimately cell death byapoptosis (17–19). Incubation of mouse T lymphocytes with

*University Denis Diderot-Paris 7, EA1556, Paris, France; †Institut National de laSante et de la Recherche Medicale Unite 519, Faculty of Medicine and Pharmacy,Rouen, France; ‡Institute of Immunology, University Medical Center, Hamburg-Ep-pendorf, Hamburg, Germany

Received for publication December 20, 2006. Accepted for publication April14, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Ligue Nationale Contre le Cancer, theAssociation pour la Recherche sur le Cancer, and the Ministere de la Recherche (toM.S.); the Fondation pour la Recherche Medicale (to S.A.), and the DeutscheForschungsgemeinschaft (to F.K.-N. and F.H.).2 Address correspondence and reprint requests to Prof. Michel Seman, Institut Na-tional de la Sante et de la Recherche Medicale, U519, Rouen, F-76000, France. E-mailaddress: michel.seman@univ-rouen.fr3 Abbreviations used in this paper: ART, ADP-ribosyltransferase; PI, propidiumiodide; NADase, NAD� glycohydrolase; ADH, alcohol dehydrogenase; PES,phenazyne ethosulfate; PS, phosphatidylserine; NICD, NAD�-induced T cell death.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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micromolar NAD� concentrations induces all these effects,whereas P2X7 activation by ATP requires millimolar concentra-tions (14, 17). NICD depends on ART2 and P2X7 as assessed bythe resistance observed with T cells from ART2-deficient or P2X7-deficient mice (14, 20, 21). Moreover, C57BL/6 T cells, whichharbor a P451L natural mutation in the C-terminal intracytoplas-mic domain of P2X7 that severely impairs the response to ATP(22), are relatively resistant also to NICD in vitro, further support-ing the conclusion that P2X7 is required for NAD�-induced T cellapoptosis.

The origin of the ecto-NAD� ART-substrate is still poorlyunderstood. The intracellular NAD� concentration is in therange of 1 mM, whereas the plasma concentration is in therange of 0.1 �M, below the Km of ARTs (5, 23, 24). It isspeculated that high amounts of ecto-NAD� can be releasedduring tissue injury as a consequence of cell lysis. However,nucleotides such as ATP or NAD� also seem to be released bynonlytic processes under various physiological conditions in-cluding hypoxia, inflammation, and mechanical or chemical ac-tivation (25–27). Connexin 43 hemichannels, for instance, me-diate transmembrane NAD� fluxes in intact cells (25). Hence,high local NAD� concentrations may be reached under variousphysiological and pathophysiological situations which wouldpermit ADP-ribosylation of membrane proteins on ART-ex-pressing neighboring cells. The fate of this NAD� is controlledby ecto-NADases. As recently shown, the ecto-NADase CD38expressed on B lymphocytes, competes with ARTs for theNAD� substrate, controls its availability and therefore the levelof ART2-catalyzed ADP-ribosylation on the surface of mouse Tlymphocytes (28). This regulation is likely to occur in vivo insecondary lymphoid tissues where T and B lymphocytes are inclose contact.

The aim of the present study was to evaluate NAD� releaseduring inflammation and to explore its consequence on T cell pop-ulations in vivo. We used a model of inflammation induced bypolyacrylamide beads to investigate NAD� release in exudatesduring the course of an acute inflammatory response. We thenexplored the effect of NAD� on the different T cell subsets inlymph nodes drained by the inflammatory site or after systemicinjection of NAD�. Our results show that high amounts of NAD�

are released during acute inflammation. They also demonstrate thatNAD� can induce strong T cell depletion in vivo and that cellssurviving NAD� treatment are enriched in activated/memory Tlymphocytes. We finally show that NAD� treatment in vivo affectsAb production to T dependent Ags providing evidence that NICDparticipates in T cell homeostasis in the course of the immuneresponse.

Materials and MethodsMice

BALB/c mice were obtained from the Centre d’Elevage Janvier (Le GenestSaint Isle, France). ART2-deficient mice were generated by standard ho-mologous recombination procedures and backcrossed for 12 generationsonto the BALB/c background as described elsewhere (20). CD38-deficientmice were gifts from Dr. F. Lund (Trudeau Institute, New York, NY) (29).All mice were maintained under standard conditions in the animal quarterof the Institute Jacques Monod (University Paris 7, Paris, France). Exper-iments were performed according to European regulations.

Reagents and Abs

ATP, NAD�, propidium iodide (PI), Con A, and Neurospora crassaNAD� glycohydrolase (NADase) were obtained from Sigma-Aldrich. PE-and FITC-conjugated anti-B220, anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD44, anti-CD69, anti-CD62L, anti-CD11b, anti-Gr1, and An-nexin VFITC were from BD Pharmingen. ART2.2-specific Nika 102 mAb(9) was purified from hybridoma supernatant by affinity chromatography

on protein G-Sepharose (Pharmacia) and conjugated to FITC (Sigma-Aldrich). P2X7-specific Hano44 mAb was obtained by genetic immuniza-tion of rats and fusion of spleen cells with Sp2/0 myeloma cells as de-scribed previously (9, 30). Abs were purified from hybridoma supernatantsand conjugated to Alexa Fluor 488 (Molecular Probes) according to themanufacturer’s instructions. SRBC were purchased from Eurobio.

Assay for phosphatidylserine exposure and cell death

Single-cell suspensions from lymph nodes were prepared and processed byflow cytometry on a FACSCalibur instrument using the CellQuest-Pro pro-gram (BD Biosciences) as described elsewhere (4, 9). B cells were depletedby magnetic cell separation using anti-B220 Abs (BD Pharmingen) con-jugated to Adembeads prepared according to the manufacturer’s instruc-tions (Ademtech). Purity of T cells was always �95% as verified by FACSanalysis using PE-conjugated anti-B220 and FITC-conjugated anti-CD3.Following treatment with NAD� or ATP for 30 min at 37°C, cells werewashed in RPMI 1640 medium (Invitrogen Life Technologies) and wereresuspended in annexin V binding buffer (BD Pharmingen). They were stainedfor 20 min on ice with FITC-conjugated annexin V (1 �g/ml) and propidiumiodide (10 �g/ml).

Inflammation

Inflammation was induced as previously described (31). Briefly, animalswere shaved and 800 �l of a sterile 67% suspension (53 mg dry weight/ml)of Biogel P100 (Bio-Rad) in PBS was injected s.c. into the bottom of theback. At given times, pouches were incised on sacrificed animals. TheBiogel mass was collected under a dissecting microscope with a spatulaand resuspended in 0.5 ml of PBS/20 U/ml heparin. Tubes were weighedbefore and after addition of the beads to determine the Biogel mass recov-ered. After vigorous shaking to extract cells and exudate components fromthe gel, beads were allowed to sediment and supernatants were collected.Cells were then recovered by centrifugation and cell-free exudates wereimmediately used for NAD� measurement or frozen and kept at �20°C.Total cells in exudates were counted on a hemocytometer. Cell subpopu-lations were differentially evaluated by flow cytometry after staining withPE-conjugated anti-Gr-1 and FITC-conjugated anti-CD11b.

NAD measurements

NAD� concentrations were determined by a cyclic enzymatic assay asdescribed previously (23). The first step of the cycle is an enzymatic re-action catalyzed by the NAD�-dependent alcohol dehydrogenase (ADH).The next steps are nonenzymatic reactions. NADH2 produced during theenzymatic step is oxidized by phenazyne ethosulfate (PES) which, oncereduced, is itself oxidized by thiazolyl blue (MTT). Oxidized NAD� andPES are used in the next cycle. Absorbance of the reduced form of MTTis measured at 570 nm in a two-chamber spectrophotometer. A “premix”solution containing 3.2 ml of sodium-1.5 M (pH 7.8) bicine (N,N-bis(2-hydroxyethyl)glycine) buffer, 2.4 ml of 10 M ethanol, 2.0 ml of 10 mMMTT, 1.0 ml of 0.2 M EDTA, 0.4 ml of 100 mg/ml BSA, and 1.0 ml ofH2O was prepared. Measurements were performed by mixing in the dark0.3 ml of premix solution with 0.1 ml of 40 mM PES, 0.1 ml of 1 mg/mlADH in sodium-bicine buffer (0.1 M; pH 8.8), and 0.1 ml of a standardNAD� solution or sample. OD was followed over a period of 3–5 min.NAD� concentration was deduced from standard curves. Determination ofthe initial speed of the cyclic reaction, for each known concentration ofNAD� allowed us draw a standard curve: V � f[C], which then permitsaccurate calculation of NAD� concentration in samples. We routinely usedinitiation by NAD� for inflammatory exudates (colorless in our model) butinitiation by ADH for plasma. All reagents were purchased fromSigma-Aldrich.

Immunization and Ab titration

Mice were immunized i.p. with 0.2 ml of a 10% sheep erythrocyte sus-pension (Eurobio) in PBS (32). Serum samples were collected at appro-priate times and hemagglutination titers were determined by serial dilutionsof 50 �l of serum in PBS with 25 �l of a 0.4% SRBC suspension.

Data representation and statistical analysis

Results are expressed as the mean � SD. Significance was assessed byStudent’s t test with p at least inferior to 0.05 indicating statisticalsignificance.

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ResultsNAD� is released during acute inflammation

To evaluate whether NAD� is released during inflammation and todetermine its concentration in inflammatory exudates, we took ad-vantage of the experimental model of acute inflammation inducedby s.c. injection of Biogel polyacrylamide beads (31, 33). In thismodel, surgical recovery of the beads and their suspension in adefined volume of PBS gives access to the number of cells and tothe real concentration of NAD� trapped per unit of gel mass re-covered. This method, which is not traumatic for cells, allows thedetermination of free NAD� concentration in inflammatory exu-dates. Wild-type BALB/c mice were injected with Biogel andsacrificed at various time points to follow the kinetics of NAD�

concentration and cellular infiltration during the course of the in-flammatory reaction (Fig. 1B). Cells recovered in the gel werecounted and NAD� concentration was determined by an enzy-matic assay. In this model, maximal cell infiltration is achievedafter 24 h and up to 48 h and primarily correspond to neutrophilsas assessed by Gr-1/CD11b staining (Fig. 1A), which are thenprogressively replaced by macrophages (31). Remarkably, freeNAD� was detected in exudates. The concentration peaked at 12 hafter induction of inflammation, a time when the cellular responsewas still building up (Fig. 1B). This suggests that NAD� is ac-tively released from cells as an early response to inflammatorystimuli as opposed to being passively set free as a result of celllysis or neutrophil apoptosis.

The influence of CD38 and ART2 in this process was exploredby performing similar experiments in CD38 and ART2-deficientBALB/c mice. Twenty-four hours after treatment with Biogel, the

number of neutrophils was lower in exudates from CD38�/� micecompared with wild type (Fig. 2A). This is consistent with theestablished role of CD38 in neutrophil chemotaxis and homing inresponse to inflammatory stimuli (34). At peak, NAD� concentra-tion reached �10 �M in exudates from CD38�/� BALB/c micebut �4 �M in their wild-type or ART2�/� counterparts (Fig. 2B).This indicated that the CD38 NADase participates in the clearanceof NAD� at the inflammatory site. Yet, NAD� release presentedthe same kinetics as in wild-type animals with a peak at 12 hpreceding massive neutrophil influx in the pouch (Fig. 2C). NAD�

release in exudates had no effect on the basal plasma level, whichremained unchanged throughout the inflammatory reaction (Fig.2B and data not shown). Higher NAD� concentrations were foundin plasma of untreated (data not shown) and Biogel-treatedCD38�/� mice compared with wild-type animals. This may eitherreflect elevated steady-state NAD� levels in these animals or haveresulted from NAD� released by hemolysis occurring duringblood collection and preparation.

Inflammation induces NICD in vivo

In vitro, micromolar concentrations of NAD� are sufficient to in-duce rapid apoptosis of mouse T cells which can be detected byannexin V/PI staining (4, 14). We, therefore, next tested whetherNAD� present in inflammatory exudates could induce cell death invitro. As shown in Fig. 3A, exudates collected after 12 h fromCD38�/� BALB/c mice induced significant apoptosis of purifiedlymph node T cells. The effect was limited due to the dilution ofexudates that was necessary to extract the NAD� from the Biogel,leading to a working concentration in the range of 1.5 �M. Thenotion that NAD� in inflammatory exudates caused PS flashing issupported by the finding that PS flashing was inhibited by pretreat-ment of exudates with exogenous NADase. Moreover, inflammatoryexudates did not induce any detectable annexin V/PI staining ofART2-deficient T cells (data not shown).

These results prompted us to evaluate the effect of Biogel-in-duced inflammation on T cells in lymph nodes from wild-type,CD38�/�, and ART2�/� BALB/c mice. One day after injection ofBiogel beads s.c., in the bottom of the back, draining (inguinal andpara-aortic) and nondraining (axillary) nodes were separately col-lected. Cells were counted and analyzed by flow cytometry. Asignificant diminution of �20% in the number of CD3� T cellswas observed in the draining nodes of wild-type-treated mice butnot in the nondraining axillary nodes of the same animals (Fig.3B). A more severe reduction of �50% was observed in the drain-ing nodes from CD38�/� mice but no effect of inflammation wasobserved in ART2�/� mice treated under the same conditions(Fig. 3B). Together, these results demonstrated that NAD� re-leased during inflammation can reach the draining nodes and can

FIGURE 1. NAD� release during acute inflammation. Inflammatorypouches were induced in BALB/c mice with 0.8 ml of a 50% Biogel beadsuspension s.c. Cells and exudates were collected at the times indicated. A,Phenotype of inflammatory cells collected after 24 h and stained withCD11b/Gr-1 Abs. B, Kinetics of cell infiltration (E) and NAD� release(�) in inflammatory exudates (n � 6 mice/point). Error bars, SD.

FIGURE 2. Comparison of inflammatory response and NAD� release in wild-type, CD38�/�, and ART2�/� BALB/c mice. Inflammation was inducedas described in Fig. 1. A, Total cell number in inflammatory exudates 24 h after treatment with Biogel were determined in wild-type (WT; n � 11), CD38�/�

(n � 11), and in ART2�/� mice (n � 7). B, NAD� concentration in plasma and exudates at 12 h (n � 6 for each group). C, Kinetics of cell infiltrationand NAD� release in exudates from CD38�/� mice (n � 6). Error bars, SD.

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induce T cell depletion in vivo. Consistent with previous reports(28), CD38 evidently partially protects T cells from the ART2-dependent deleterious effect of NAD�.

In CD38�/� mice, T cell depletion in the draining nodes wasaccompanied by a relative increase in the fraction of CD62Llow

T cells (Fig. 4). This could reflect the resistance of CD62Llow Tcells to NAD�-induced death and/or shedding of CD62L bysurviving cells as a consequence of P2X7 activation (14). When10 mg of NAD� was mixed with Biogel, a more pronounced Tcell depletion and CD62L shedding was observed in draining

nodes but also in nondraining ones (Fig. 4). This confirmed thatNAD� released at local inflamed sites can reach draining lymphnodes (see Fig. 4, Biogel, middle panels) and even nondraininglymph nodes through lymphatic and blood circulations providedthat the NAD� amount is high enough (see Fig. 4, Biogel �NAD, right panels).

NAD� injection induces rapid T cell depletion in vivo

To further explore the effect of NAD� on T cell populations invivo, BALB/c mice were injected i.v. with the maximal tolerateddose of either ATP (6 mg) or NAD� (10 mg) in 0.2 ml of PBS.After 24 h, spleen and lymph nodes were recovered and the size ofthe T cell population was evaluated by CD3/B220 staining in flowcytometry. A significant reduction in the percentage of CD3� cellswas observed in lymphoid organs from NAD�-treated mice,whereas ATP treatment did not have any significant effects (Fig.5A). The differential effects of ATP and NAD� were consistentwith the higher concentration of ATP compared with NAD� re-quired to induce T cell death by P2X7 activation in vitro (14) andwith the short half-life of ATP in body fluids due to widely dis-tributed ecto-ATPases (35, 36). Reduction of the percentage ofCD3� cells was accompanied by a 40% drop in the absolute T cellcount in secondary lymphoid tissues (Fig. 5B). As expected, T celldepletion was more dramatic in CD38�/� mice compared withwild-type BALB/c mice, particularly in lymph nodes (Fig. 5C),whereas NAD� treatment had no effect on T cells in theirART2�/� counterparts (Fig. 5D). Evidently, NAD�-induced Tcell depletion in vivo is ART2 dependent, in accord with previousobservations in vitro (14, 20). Results in CD38�/� mice are inagreement with our previous report demonstrating the antagonisticeffect of the CD38 NADase on ART2-mediated T cell death (28).In CD38�/� BALB/c mice, T cell depletion was clearly detectableas early as 3 h after NAD� injection and reached its maximum at�70% after 1 day (Fig. 5E). NAD� treatment had little if anyeffects on B cell counts in spleen and lymph nodes. The effect onT lymphocytes was long-lasting and mice had not fully recoverednormal T cell numbers, 1 wk after NAD� treatment. These obser-vations strongly suggested that NAD� induces rapid T cell deathin vivo as already described in vitro (4).

Cells resistant to NICD exhibit the phenotype ofactivated/memory T cells

The effect of treatment with NAD� in vivo on different T cellsubsets was followed by examining cell phenotypes in lymphnodes 24 h after the injection of NAD� into CD38�/� BALB/cmice. The results indicate, first, that CD4� cells appear to be moresensitive to NICD than their CD8� counterparts (Fig. 6, A and B).

FIGURE 3. NAD� released in exudates induces T cell apoptosis invitro and T cell depletion in vivo. A, Induction of apoptosis in BALB/c Tcells by 12-h inflammatory exudates from CD38�/� BALB/c mice. Puri-fied T cells were incubated with exudates or NAD� solutions in PBS for 30min at 37°C and then stained with Annexin VFITC/PI. Right panel, 5 U ofNADase was added to T cells just before addition of the exudate. No gatewas applied from the scatters plots to analyze living and shrinking/dyingcells. The numbers indicated in the lower right quadrant represent thepercentage of apoptotic cells defined as annexin V�/PI�. Results are rep-resentative of six individual mice from three independent experiments. B,T cell number in draining (para-aortic and inguinal) and nondraining (ax-illary) lymph nodes in control or Biogel-treated wild-type (wt; n � 12),CD38�/� (n � 12), and ART2�/� (n � 6) BALB/c mice. Lymph nodeswere collected 24 h after induction of inflammation and cells were counted.T cell numbers were deduced from the percentage of CD3� cells estimatedafter CD3/B220 staining in flow cytometry. Vertical bars, SD and p valuesare from Student’s t test.

FIGURE 4. Analysis of T cells in draining and nondraining lymph nodes in CD38�/� BALB/c mice 24 h after induction of inflammation. Inflammationwas induced with 0.8 ml of a 50% Biogel bead suspension s.c. or Biogel containing 10 mg of NAD�. T cells from draining (para-aortic and inguinal) andnondraining (axillary) nodes from CD38�/� BALB/c mice were collected after 24 h and stained with anti-CD3-FITC/anti-B220-PE or anti-CD3-FITC/anti-CD62L-PE mAbs. Panels are representative of six mice from two independent experiments. Right panels, cells were gated on the CD3� population.

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Indeed, 78 � 11% of CD4� cells and 52 � 21% of CD8� cellswere affected in the lymph nodes of these animals 24 h after theinjection of NAD� ( p � 0.003, n � 10). This could be accountedfor by a difference in ART2 and/or P2X7 density on the plasmamembrane. In accord with a previous report, we show that CD8�

T cells express a higher level of ART2.2 on their surface thanCD4� cells as assessed by staining with ART2.2-specific Nika102mAb (Ref. 9) and Fig. 6C). Yet, CD8� T cells display a lowerP2X7 receptor density than CD4� cells as assessed by P2X7-spe-cific Hano44 mAb (Fig. 6C). This suggests that susceptibility toNICD is more influenced by the density of P2X7 than of ART2 onthe cell surface.

Second, cells surviving NAD� treatment in vivo show strikingphenotypic differences to cells of sham-treated mice. Indeed, in-creased percentages of CD44high and CD69high along with a de-creased percentage of CD62Lhigh T lymphocytes were observedamong cells resistant to NICD in vivo (Fig. 6D). These markers

characterize the pool of recently activated/memory T cells. Anal-ysis of cell numbers in vivo reveals that NAD� treatment has nodirect effect on the size of the CD44high T cell population and onlya slight effect on the size of the CD69high population (Fig. 6E). Yet,both populations seem to expand 10 days after treatment, a time atwhich the peripheral T cell pool is being replenished. Our resultstherefore indicate that NAD� treatment has a strong effect on na-ive T cells and that activated/memory T lymphocytes are resistantto NICD in vivo.

Analysis of ART2.2 expression after NAD� treatment, usingNika102 mAb, reveals that a significant fraction of the CD4� cellsand most of the CD8� cells resistant to NAD� in vivo do notexpress ART2.2 on their surface (Fig. 7A). Since activated T cellsshed ART2 from their surface (37), this along with their CD44,CD62L, and CD69 phenotypes supports the conclusion that endo-genously activated or memory cells are less sensitive to the dele-terious effect of NAD�. Moreover, CD4� and CD8� cells resistant

FIGURE 5. Injection of free NAD� induces T celldepletion in vivo. A, BALB/c mice were injected with0.2 ml of PBS or 0.2 ml of PBS containing 6 mg of ATPor 10 mg of NAD�. CD3/B220 staining was performedon spleen and lymph nodes (13 nodes collected) 24 hafter injection. Profiles are representative of at least 12mice. B, T cell numbers in spleen and lymph nodes fromBALB/c mice 24 h after treatment with NAD�. Themean value indicated by the horizontal bars correspondto 60 � 9 � 106 and 41 � 10 � 106 cells in the spleenof PBS- vs NAD-treated mice (n � 9, respectively, andto 24 � 5 � 106 and 15 � 4 � 106 cells in the lymphnodes of PBS- vs NAD-treated mice (n � 12), respec-tively. Statistical analyses were performed using Stu-dent’s t test. C, CD3/B220 staining in CD38�/�

BALB/c 24 h after injection of 10 mg of NAD�. Resultsare representative of 12 individual mice. D, CD3/B220staining in ART2�/� BALB/c 24 h after injection of 10of mg NAD�. Results are representative of five indi-vidual mice. E, CD38�/� BALB/c mice were treatedwith 10 mg of NAD� i.v., and total cell counts weredetermined in spleen and lymph nodes at various times.T and B cell numbers were deduced from the percent-ages measured after CD3/B220 staining. Each point rep-resents the mean of six individual mice.

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to NICD in vivo are also poorly stained with Hano44 mAb com-pared with cells from untreated mice (Fig. 7A), consistent with thenotion that sensitivity to NICD is greater for cells expressing highlevels of P2X7. Correlatively, we show that activation of murine Tcells by Con A (Fig. 7B) or by anti-CD3 Abs (data not shown)leads to a down modulation of both P2X7 and ART2.2 on theplasma membrane.

Extracellular NAD� influences the immune response to SRBC

The influence of NAD� on the immune response was tested inwild-type, ART2�/�, and CD38�/� BALB/c mice after immuni-zation with SRBC. This Ag was selected for several reasons. First,SRBC Ab response is T dependent and quickly elicits detectableprimary and secondary serum Ab titers. Second, response to SRBCdoes not require the use of adjuvants, which are inflammatory anddelay Ag delivery. In a first series of experiments, mice weretreated with 10 mg of NAD� i.v. at the time of immunization with2 � 108 SRBC in saline i.p. Mice were bled after 6 days and

primary agglutination titers were determined. A much lower Abtiter was observed in CD38�/� mice treated with NAD� vs un-treated mice, whereas NAD� treatment did not show any detect-able effect on Ab induction in ART2�/� mice (Fig. 8A). This isconsistent with the dramatic T cell depletion induced by NAD� inCD38�/� animals and the resistance of T cells from ART2�/�

mice to NICD in vivo. Conceivably, elimination of most naive Tcells by NAD� in CD38�/� mice affects the recruitment of Ag-specific cells.

A second group of mice was treated with NAD� on day 4 afterpriming, a time when the response was still building up. They werethen boosted with SRBC on day 14 and the secondary responsewas tested on day 20. Agglutination titers were compared withthose in mice treated with NAD� on day 0 and to untreated mice.Results in Fig. 8B show that treatment with NAD� on day 4 afterpriming did not block the secondary SRBC response in CD38�/�

mice or even slightly increased it in comparison to untreatedCD38�/� mice. Notice that the secondary response in mice treatedwith NAD� on day 0 was equivalent to the primary one in un-treated animals. These results further support the notion that NICDaffects naive T cells, whereas activated T cells escape the delete-rious effect of NAD�.

Remarkably, different results where obtained in wild-typeBALB/c mice which are less sensitive to NICD than theirCD38�/� counterparts. Indeed, treatment with NAD� in thesemice led to a significant improvement of both primary and sec-ondary responses compared with controls (Fig. 8). This suggeststhat NAD�-induced partial T cell depletion of naive T cells, whichlikely corresponds to physiological situations in vivo, may facili-tate the expansion of Ag-primed T cells. Note that NAD� treat-ment at day 14, i.e., immediately before the secondary antigenicchallenge, had no effect on the secondary response in wild-type orin CD38�/� BALB/c mice (Fig. 8B). This result is consistent withthe idea that recently primed T cells responsible for the secondaryresponse are refractory to NICD.

FIGURE 6. T cell phenotype in CD38�/� BALB/c mice after treatmentwith NAD�. CD38�/� BALB/c mice were treated with 10 mg of NAD�

i.v. or 0.2 ml of PBS. A, Six peripheral lymph nodes were collected atdifferent times and total cells were counted. Cell numbers were calculatedafter CD3-FITC/B220-PE and CD8-PE/CD4-FITC staining. Each point isthe mean of three individual mice. B, CD4:CD8 ratio among B220-de-pleted lymph node cells 24 h after treatment with NAD� (representative ofseven mice). C, Comparison of P2X7 and ART2.2 expression on CD4�-and CD8-purified T cells in untreated mice after staining with P2X7-spe-cific mAb Hano44 or ART2.2-specific mAb Nika102. The mean fluores-cence intensity (MFI) values were 33 vs 50 for P2X7 expression on CD8�

vs CD4� cells, respectively, and 48 vs 21 for ART2.2 expression on CD8�

vs CD4� cells, respectively. The MFI values of the isotype controls werein the range of 5–7. These results are representative of at least four mice.D, Phenotypes of CD4� lymph node T cells were determined before or24 h after NAD� injection. Cells were gated on the CD4� population afterstaining with CD4-allophycocyanin/CD62L-FITC or CD4-allophycocya-nin/CD44-CyChrome/CD69-PE. Panels are representative of six individualmice from three independent experiments. E, Cell numbers were calculatedas in A from the percentages observed after CD4/CD44/CD69 staining.Each point is the mean of three individual mice.

FIGURE 7. ART2 and P2X7 expression after NAD� treatment in vivo.A, Lymph node-purified T cells from control or NAD�-treated CD38�/�

BALB/c mice were stained with PE-conjugated anti-CD4 or anti-CD8 andcounterstained with FITC-conjugated mAb Nika102 mAb for ART2.2 ex-pression and Alexa Fluor 488-conjugated mAb Hano44 for P2X7 expres-sion 24 h after treatment with NAD� or PBS. The MFI values in PBS vsNAD treatment were, respectively, 50 vs 35 (P2X7 expression on CD4�);33 vs 18 (P2X7 expression on CD8�); 28 vs 23 (ART2.2 expression onCD4�); and 46 vs 23 (ART2.2 expression on CD8�). The MFI values ofthe isotype controls were in the range of 5–7. Panels are representative ofthree individual mice. B, Lymph node-purified T cells from wild-typeBALB/c mice were cultured for 24 h with Con A (2 �g/ml) in RPMI1640/10% FCS and stained for expression of P2X7 and ART2.

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DiscussionThe discovery of mono-ART in vertebrates has opened a new fieldin cell communication by introducing new ectoenzymes catalyzingposttranslational modifications of proteins using NAD� in the ex-tracellular compartment (3). ART1 and ART2 are presently thetwo best-characterized paralogs (1), but their role in vivo remainselusive. In mice, ART2 is expressed on most peripheral T cells andremarkably induces P2X7 activation by ADP-ribosylation leadingto cell death in vitro (4, 14). Yet, the origin of endogenous extra-cellular NAD� is still a matter of debate, in which both lytic andnonlytic processes can be envisioned (25–27). Experiments re-ported herein provide the first evidence that a significant amount ofNAD� is present in exudates during the early phase of the inflam-matory response. Inflammation induced by polyacrylamide beadsis Ag free and thus reflects tissue reaction to a foreign bioincom-patible material. NAD� release precedes massive neutrophil influxinto the inflammatory site. Hence, NAD� release may not be con-ditioned by massive infiltration of neutrophils. Oxidative stressinduced by activated neutrophils, as well as hypoxia resulting fromthe pressure of the Biogel mass under the skin, may contribute tothe liberation of NAD� by neighboring cells through a nonlyticprocess. Importantly, the high local NAD� concentration in exu-dates, which can reach �10 �M, has no effect on the plasmaNAD� level. Local NAD� release at an inflammatory site is thusunlikely to have a global effect on T cell populations in the wholebody. Yet, NAD� concentrations in the range of 3 �M were foundin the plasma of CD38�/� mice vs �0.3 �M in their wild-typecounterparts. Whether such a high concentration reflects the phys-iological level in these mice or results from NAD� released in theabsence of the major NAD�-hydrolase CD38 during blood prep-

aration remains questionable. Micromolar NAD� concentrationsare sufficient to induce T cell death in vitro (4) and a NAD� con-centration in exudates is sufficient to induce detectable NICD invitro. Yet, nonmanipulated CD38�/� mice have T cell numbersand subsets similar to those in wild-type animals. It seems thuslikely that the high NAD� plasma level in CD38�/� animals cor-responds to an experimental artifact. More importantly, NAD�

released at a local inflammatory site induces a significant T celldepletion in the draining but not in the nondraining nodes fromwild-type or highly sensitive CD38�/� BALB/c mice developinginflammation to Biogel. This depletion is accompanied by CD62Lshedding on a significant fraction of remaining T cells, an earlyevent associated with P2X7 activation (18). This, in combinationwith T cell resistance in ART2�/� mice, despite NAD� release atthe inflammatory site, provides the first evidence that NICD canoccur under physiopathological situations in vivo.

Consistently, injection of NAD�, the ART2 substrate, into nor-mal mice expressing both ART2 and P2X7 induces massive T celldepletion detectable within a few hours following injection. Insupport of our findings, accumulation of apoptotic T cells in theliver has been described in the C57BL/6 mouse strain within 12 hafter NAD� injection (7), although these mice harbor a partiallydeficient P2X7 receptor (22). In our experiments, the depletion isaggravated particularly in CD38�/� mice where it affects �80% ofthe T cell population in peripheral lymphoid tissues. No effect oftreatment on the thymus was observed (data not shown), in agree-ment with the absence of ART2 on thymocytes (38). The highsensitivity of CD38�/� vs wild-type BALB/c mice to the delete-rious effect of NAD� is consistent with our previous observationindicating that the CD38 NADase, mainly expressed on B lym-phocytes, controls the level of ART2-mediated ADP-ribosylationof T cell surface proteins and, therefore, T cell apoptosis in un-fractionated peripheral lymphoid populations (28). The resistanceof T cells from ART2�/� BALB/c mice clearly establishes that Tcell depletion after NAD� treatment results from ART2- andtherefore P2X7-dependent NICD.

The dramatic effect of NAD� injection in vivo not only affectsthe number of T lymphocytes in peripheral organs from CD38�/�

BALB/c mice but also profoundly influences the distribution of Tcell subpopulations. The fraction of CD44high, CD62Llow andCD69high T cells is strongly enlarged, suggesting that cells resis-tant to NICD in vivo are activated/memory T cells. Three argu-ments support this conclusion. First, T cell activation leads to theshedding of ART2 by the TACE metalloproteinase and renderscells resistant to NICD (37, 38). Evidently, most of the CD4� andCD8� T cells present in lymph nodes from NAD�-treatedCD38�/� BALB/c mice do not express ART2.2 on their surface.Then, most of the CD4� and CD8� cells from NAD�-treated micehave a low P2X7 density on their surface compared with controls.We directly show that T cell activation induces a down-modulationof P2X7 expression and resistance to NICD. This is in agreementwith the low ability of CD44high activated/memory CD4� T cellsto open the P2X7 large pore permeable to ethidium bromide in thepresence of ATP (39). Finally, treatment with NAD� at the time ofpriming with SRBC inhibits the primary response in CD38�/�

BALB/c mice, but treatment on day 4 after priming has no effector even stimulates the subsequent response to antigenic challenge(Fig. 8). This demonstrates that primed T cells escape the delete-rious effect of NAD� administration. Altogether, these resultsdemonstrate for the first time that NAD� can modulate the pool ofnaive peripheral T cells in vivo, suggesting that endogenoussources of NAD� may exert a similar effect.

One important aspect of ART2 biology concerns its role in theregulation of the immune response. Previous experiments have

FIGURE 8. Effect of NAD� treatment on primary and secondary re-sponses to SRBC. A, To determine the effect of NAD� on the primaryresponse, CD38�/�, ART2�/�, and wild-type BALB/c mice were primedwith 0.2 ml of a 10% SRBC suspension in PBS i.p. One-half of themreceived 10 mg of NAD� in PBS or 0.2 ml of PBS i.v. at the same time asthe SRBC suspension. Agglutination titers were measured on day 6. Eachvalue represents the mean of six to eight individual mice from two inde-pendent experiments. B, To determine the effect of NAD� on the secondaryresponse, mice were primed with SRBC on day 0. For each line, a groupof six mice received 10 mg of NAD� i.v. on day 0, another group on day4, and the last group received NAD� on day 14. Controls received PBS onday 0. Mice were boosted with SRBC on day 14 and Ab titers were mea-sured on day 20. �, p � 0.05; ��, p � 0.01 compared to controls.

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shown that ART2 ADP-ribosylates several surface proteins includ-ing LFA-1, CD8, CD27, CD43, CD44, or CD45 (10–12). Thisclearly inhibits cell contact and T cell trafficking (12). ART2-me-diated ADP-ribosylation of membrane proteins would thus have aninhibitory effect on the expansion of the T cell response. Theseconclusions were drawn from experiments performed in C57BL/6mice, which express ART2 at a very high density on their T lym-phocytes (9) but have an impaired P2X7 receptor (22). Conversely,BALB/c T cells have a lower ART2 density but display a func-tional P2X7 receptor. Our present results indicate that in theBALB/c genetic background, CD4� cells, which are ART2low

P2X7high, are more prone to NICD in vivo than CD8� cells whichare ART2highP2X7low compared to the CD4�. This suggests thatsusceptibility to NICD is governed by P2X7 more than by ART2density.

Why should NAD� set free in the inflammatory phase of aninfection kill naive T cells during the early development of theadaptive immune response? This situation, as illustrated by ourexperiments in CD38�/� BALB/c mice treated with NAD� at thetime of immunization, could severely impair the response. Thesame experiments, however, show that the situation is different ina normal BALB/c mouse. Priming with NAD� does not impair theresponse to Ag but even leads to a weak but significant increase ofthe Ab titers. This is also verified during the secondary responsewhen mice are treated with NAD� 4 days after priming. In thesemice, naive T cells are protected from ART2-mediated P2X7activation and NICD by CD38 expressed on B lymphocytes.Hence, during the early phase of the immune response, whileactivated T cells down-modulate ART2 and P2X7 expressionon their surface, elimination of part of the naive T cells couldgive space for the expansion of the Ag-primed T cell populationand thus increase the response. Alternatively, CD4�Foxp3�

regulatory T cells may be highly sensitive to NICD, as recentlysuggested in C57BL/6 mice (21). Elimination of regulatory Tcells, which exert a negative effect, could contribute towardimproving the response. However, the extent to which the ef-fects of extracellular NAD� on the development of the immuneresponse can be attributed to ART-mediated NICD remainsquestionable. CD38 is not only a NADase but also an ADP-ribose cyclase. Cyclic ADP-ribose plays an important role inthe response of neutrophils and professional APCs to chemo-tactic signals (34, 40). Consequently, CD38�/� mice have adiminished Ab response to T-dependent Ags (29). The reducedresponse to SRBC of CD38�/� vs wild-type BALB/c mice (Fig.8A) is in fair agreement with this conclusion. Combined withNICD, a defect in Ag presentation may thus explain the dra-matic impairment of the primary SRBC response in CD38�/�

mice treated with a high dose of NAD�. Similarly, in CD38�/�

BALB/c mice, NAD� injection might increase cyclic ADP-ri-bose production and improve APC activation and trafficking,compensating the deleterious effect of NICD or even improvingthe response. The observation that NAD� administration doesnot enhance the primary response in ART2�/� mice, however,makes it likely that this effect is primarily due to ART2-mediated NICD.

Taken together, our results demonstrate that NAD� is releasedduring the early phase of inflammation and illustrate its role inimmune regulation in vivo. They also stress that ART2 and CD38are two important, but not necessarily opposed partners in thisregulation. Evidently, NICD is a phenomenon which can occur invivo. Yet further investigation is needed to define its main targetand its role in the regulation of T cell homeostasis and the controlof autoimmune diseases in normal mice.

DisclosuresThe authors have no financial conflict of interest.

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