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Relevance for T Cell-Mediated HepatitisProduction by Invariant NKT Cells: The Pro-Th1 Cytokine IL-12 Enhances IL-4

Schneider and André HerbelinDamotte, Michel Samson, Pierre Gourdy, Michel Dy, ElkeAumeunier, Jordan Denizeau, Emilie Philadelphe, Diane Ren Zhu, Séverine Diem, Luiza M. Araujo, Aude

http://www.jimmunol.org/content/178/9/5435doi: 10.4049/jimmunol.178.9.5435

2007; 178:5435-5442; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Pro-Th1 Cytokine IL-12 Enhances IL-4 Production byInvariant NKT Cells: Relevance for T Cell-Mediated Hepatitis1

Ren Zhu,* Severine Diem,* Luiza M. Araujo,* Aude Aumeunier,* Jordan Denizeau,*Emilie Philadelphe,* Diane Damotte,† Michel Samson,§ Pierre Gourdy,‡ Michel Dy,*Elke Schneider,* and Andre Herbelin2*

IL-12 is essential for invariant NKT (iNKT) cells because it can maintain a functionally active population and promote a cytokineprofile that is assumed to be mainly of the pro-Th1 type. We used the murine concanavalin A (Con A)-induced hepatitis model,in which iNKT cells, IL-12, IL-4, and IFN-� are equally requisite, to reevaluate this issue. We demonstrate that IL-12 interactsdirectly with iNKT cells, contributes to their recruitment to the liver, and enhances their IL-4 production, which is essential fordisease onset. IL-12-deficient mice were less susceptible to experimental hepatitis and their iNKT cells produced less IL-4 thantheir wild-type counterpart. A normal response could be restored by IL-12 injection, revealing its importance as endogenousmediator. In accordance with this observation, we found that iNKT cells expressed the IL-12R constitutively, in contrast toconventional T cells. Furthermore, the physiological relevance of our data is supported by the lower susceptibility to diseaseinduction of NOD mice, known for their inherent functional and numerical abnormalities of iNKT cells associated with decreasediNKT cell-derived IL-4 production and low IL-12 secretion. Taken together, our findings provide the first evidence that IL-12 canenhance the immune response through increased IL-4 production by iNKT cells, underscoring once more the functional plasticityof this subset. The Journal of Immunology, 2007, 178: 5435–5442.

I t is generally acknowledged that IL-12 plays an importantrole during antibacterial and antitumor responses because ofits capacity to promote a pro-Th1 cytokine profile (1). Its

participation in the regulation of a distinctive subpopulation ofmature T cells, namely invariant NKT (iNKT)3 cells, identified bytheir capacity to recognize glycolipids presented by the MHC classI-like CD1d molecule, has been reported more recently (2, 3).iNKT cells promptly produce a large spectrum of pro-Th1 as wellas pro-Th2 cytokines upon stimulation with their specific Ag�-galactosylceramide (�-GalCer) (2, 4). IL-12 not only inducesand locally recruits functionally active cells of this subset but alsoinfluences their cytokine pattern (5, 6). It is believed that this oc-

curs mainly through induction of a pro-Th1 phenotype, as reportedin models of antitumor response (7).

Little is known about the cross-talk between IL-12 and iNKTcells in vivo, prompting us to address this issue in a murine modelof Con A-induced hepatitis (8), in which both IL-12 and iNKTcells participate (9–11). This experimental disease engenders a Tcell activation-dependent acute liver-specific injury that closelyresembles human autoimmune hepatitis (8, 12). It is characterizedby a marked increase of plasma alanine transaminase (ALT) levelswithin 8–24 h after injection, as well as simultaneous hepatic in-filtration by immune cells, including T cells, followed by apoptosisand necrosis of hepatocytes.

It has been established that IL-4, IFN-�, and IL-12 are eachessential for the development of Con A-induced hepatitis (9, 13–15). As to the cellular origin of these cytokines, the requirement ofiNKT cells has been evidenced, based on studies with geneticallyengineered mice (10, 11). Indeed, the typical features of this subsetare consistent with a potential involvement in Con A-mediatedhepatitis, considering the relative abundance of NKT cells in theliver (16, 17) and their capacity to generate both IL-4 and IFN-�upon TCR engagement (18).

We have set up the experimental hepatitis model in NOD micebecause of their partially deficient iNKT cell population (19, 20)associated with aberrant IL-12 production (21, 22). We investi-gated how these anomalies together affected the cytokine patterngenerated during progression of the disease, as well as its severity.From our results, we conclude that the intrinsic capacity of iNKTcells to promptly produce IL-4 is directly influenced by IL-12,endowing them with a critical role in the onset of autoimmunehepatitis.

Materials and MethodsMice

Seven- to 8-wk-old wild-type and mutant (CD1d�/�, J�18�/�) NOD miceand wild-type and mutant (IL-12p40�/�) C57BL/6 mice were bred and

*Centre National de la Recherche Scientifique, Unite Mixte de Recherche 8147, Uni-versite Paris V, Hopital Necker, and †Hopital Europeen Georges Pompidou, Serviced’Anatomie et Cytologie Pathologiques, Paris, France; ‡Institut National de la Santeet de la Recherche Medicale, Unite 589, Institut L. Bugnard, Centre Hospitalier etUniversitaire Rangueil, Toulouse, France; and §Institut National de la Sante et de laRecherche Medicale, Unite 620, Faculte de Medecine-Pharmacie, Universite deRennes 1, Rennes, France

Received for publication September 7, 2006. Accepted for publication February2, 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 funding from the Centre National de la RechercheScientifique, University Rene Descartes-Paris V, the Chancellerie des Universitesde Paris (legs Poix), and from Institut National de la Sante et de la Recherche Medi-cale, Programme National de Recherche sur le Diabete 2004, and by fellowships fromthe Association pour la Recherche sur le Cancer (to R.Z.), the Academie de Medecine(to R.Z.), the Fondation pour la Recherche Medicale (to L.M.A.) and the AssociationFrancaise des Diabetiques (to A.A.).2 Address correspondence and reprint requests to Dr. Andre Herbelin, Centre Nationalde la Recherche Scientifique, Unite Mixte de Recherche 8147, Hopital Necker, 161rue de Sevres, 75783 Paris Cedex 15, France. E-mail address: herbelin@necker.fr3 Abbreviations used in this paper: iNKT, invariant NKT; ALT, alanine transam-inase; MNC, mononuclear cell; �-GalCer, �-galactosylceramide.

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

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maintained in our animal facility under specific pathogen-free conditions.The NOD mice used in this study were not diabetic. C57BL/6 IL-12p40�/�

mice from The Jackson Laboratory were provided by F. Bayard (InstitutNational de la Sante et de la Recherche Medicale Unite 589, Toulouse,France). Animal experiments were performed according to the institutionalcommittee of France.

Reagents

Con A and recombinant murine IL-12 were purchased from Sigma-Aldrichand R&D Systems, respectively. �-GalCer was provided by the Pharma-ceutical Research Laboratory of Kirin Brewery. Fluorochrome-conjugatedanti-TCR-� (clone H57-597), anti-CD5 (55-7.3), anti-CD19 (clone 1D3),anti-NK1.1 (clone PK136), anti-IFN-� (clone XMG1-2), anti-IL-4 (clone11B11), PE-conjugated annexin V, and corresponding isotype controlswere from BD Pharmingen. The Fc�R blocking mAb (clone 24G2.3) wasfrom DNAX. PE-conjugated anti-IL-12R�1 (IgG2a, clone 114) and itscorresponding mouse isotype control (clone H106.771) were from BDPharmingen and Immunotech, respectively. Allophycocyanin-conjugatedtetramers were prepared in our laboratory from the murine CD1d/�2-mi-croglobulin expression vector constructed by Sidobre and Kronenberg (23),loaded or not with �-GalCer.

Cell preparation and flow cytometry analysis

After perfusion with PBS, livers were removed and gently pressed througha 70-�m cell strainer. Parenchymal cells (pellet) were separated frommononuclear cells (MNC) by centrifugation at 50 � g for 5 min (24). Aftera single washing, MNC were separated using a 35% Percoll solution(Amersham Biosciences), and RBC were lysed in an ammonium chlo-ride buffer. For cell sorting, liver MNC were stained with FITC-labeledanti-CD19, PE-labeled anti-NK1.1, and allophycocyanin-labeled anti-CD5. Then, CD19�NK1.1�CD5� cells (iNKT) were sorted using aFACSVantage sorter (BD Biosciences). Purity was �98% after reanal-ysis. Membrane labeling as well as intracellular cytokine staining wereperformed as previously described (25). At least 1000 events gatedamong the population of interest were analyzed on a FACSCalibur cy-tometer using CellQuest software (BD Biosciences). The proportion of�-GalCer-unloaded CD1d tetramer-positive cells in gated TCR-�� Tcells was always below 0.5%.

Culture of liver MNC

Liver MNC were suspended in RPMI 1640 medium supplemented with10% heat-inactivated FCS, antibiotics, and 0.05 mM 2-ME. MNC (1.5 �105/well) were seeded into 96-well flat-bottom culture plates and incubateddirectly with or without �-GalCer (100 ng/ml) for 48 h. In some experi-ments, total liver MNC (1.2 � 105/well) or purified liver iNKT cells (2.5 �104/well) were stimulated with IL-12 (25 ng/ml) or medium for 24 h fol-lowed by additional culture with �-GalCer (100 ng/ml) or Con A (5 �g/ml)for 24 h. Culture supernatants were collected for cytokine determination.

Hepatitis induction

Con A was dissolved in sterile PBS and injected i.v. into mice at a dose of20 mg/kg in a final volume of 200 �l. Animals were sacrificed after 12 h,when blood and livers were recovered. In some experiments, groups ofmice were injected i.p. with IL-12 (250 ng/mouse), or PBS alone, 1 hbefore Con A injection. Anti-IL-4 mAb (clone 11B11, 0.5 mg/mouse) wasinjected i.p. 24 h before Con A.

Transaminase measurement and cytokine assay

Plasma from individual mice was recovered 12 h after Con A injectionwhen ALT activities were measured as units per liter by an automatedphotometric assay.

IL-4 and IFN-� in sera and supernatants were quantified using standardsandwich ELISA, as previously described (26). IL-12 (p40) was measuredusing ELISA kits from BD Biosciences. Note that the monoclonal captureAb used in the IL-12 immunoassay is not allele-specific and recognizesIL-12 variants represented in both C57BL/6 and NOD mouse strains (27).The sensitivity limits of the assays were 20 pg/ml.

Histological examination

Paraffin-embedded liver sections were stained with H&E and examined bya pathologist (D. Damotte, Hopital Europeen Georges Pompidou, ParisFrance) under light microscopy in a blinded assay.

Real-time PCR

Total RNA was extracted from 7 � 105 mouse spleen or liver iNKT cellsusing the SV Total RNA isolation kit (Promega) and �550 ng of total RNA

was extracted and subjected to a reverse transcription reaction using highcapacity cDNA archive kit (Applied Biosystems). A total 16.5 ng of cDNAequivalent was used as a template. The mRNA levels of IL-12R�1, IL-12R�2, and 18 S were determined with the 7000 sequence detection systemABI Prism Sequence Detector (Applied Biosystems), using the double-strand-specific SYBR Green (Applied Biosystems) dye system. The primersequences were: mouse IL-12R�1 (forward) 5�-TGAGTGCTCCTGGCAGTATG-3� and (reverse) 5�-TATGGTTCGGAGGGACAAAG-3�; mouseIL-12R�2 (forward) 5�-AGTCTCACATTACTGC-3� and (reverse) 5�-TCAGGTTGTGCTGTCGAGTC-3�; and mouse 18 S (forward) 5�-CGCCCTAGAGGTGAAATTC-3� and (reverse) 5�-TTGGCAAATGCTTTCGCTC-3�.

PCR was performed as follows: initial DNA denaturation for 10 min at95°C followed by 40 cycles at 95°C for 15 s, an annealing step, and ex-tension at 60°C during 1 min. The expression level of each gene wasnormalized and expressed as the ratio of 18 S mRNA as an internal stan-dard. Migration on a 2% agarose gel of the PCR product established thatit was unique and had the correct base pair size. The accuracy of theamplification was controlled by cDNA sequencing.

Statistical analysis

Data are expressed as mean � SEM. Nonparametric unpaired comparisonswere performed using the Mann-Whitney U or the Student t test. Values ofp � 0.05 were considered significant.

FIGURE 1. Partial iNKT cell deficiency and decreased IL-12 produc-tion in NOD mice. A, MNC were isolated from the liver of naive femaleNOD and C57BL/6 mice, labeled with fluorochrome-conjugated anti-TCR� and CD1d/�-GalCer tetramer, and analyzed for the incidence ofiNKT cells per organ. Data are mean � SEM from four separate experi-ments. B, MNC were recovered from NOD and C57BL/6 livers and stim-ulated for 48 h with �-GalCer, followed by ELISA of IL-4 and IFN-� inculture supernatants. Data are mean � SEM from four mice in each group.C, Five hours after Con A injection, IL-12 levels were measured in theserum of NOD and C57BL/6 mice using ELISA. Data are mean � SEM of7–11 mice in each group.

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ResultsPartial iNKT cell deficiency, together with decreased IL-4 andIL-12 production, renders NOD mice less sensitive toCon A-induced hepatitis

We used the Con A-induced hepatitis model, which depends oniNKT cells, IL-4, IFN-�, and IL-12 for disease onset (9–11, 13–15), to investigate the mode of action of iNKT cells in vivo. Spe-cifically, we regarded the relationship of iNKT cells with endog-enous IL-12 and its impact on their cytokine profile and diseaseprogression. Furthermore, we addressed the physiological rele-vance of our findings in the NOD mouse, which is characterized byan inherent partial iNKT cell deficiency. We have reported beforethat iNKT cells are numerically and functionally impaired inspleen and thymus of NOD mice relative to C57BL/6 controls (19,28). As shown in Fig. 1, this subset was also diminished in the liver(Fig. 1A), leading to reduced IL-4 and IFN-� production by hepaticMNC in response to the iNKT cell-specific ligand �-GalCer (Fig.1B). Considering the importance of endogenous IL-12 for themaintenance of a functional iNKT pool and its critical contributionto experimental hepatitis, we compared its concentrations in theserum of NOD and C57BL/6 mice injected with Con A.

We found a significant decrease in the NOD strain (Fig. 1C),prompting us to examine how these deficiencies in the liver ofNOD mice affected the severity of hepatic injury induced by ConA injection at an optimal dose of 20 mg/kg. Hepatitis was evalu-ated by measuring the activity of plasma ALT, 12 h posttreatmentwhen the enzymatic activity was maximal (data not shown). ALTlevels were about 3-fold lower in NOD than in control C57BL/6mice (Fig. 2A). The important hepatic damage that developedwithin 24 h was also much attenuated in this strain, as revealed byH&E staining of liver sections (data not shown). The persistingmoderate liver injury disappeared in J�18�/� NOD mice, whichare totally devoid of iNKT cells, proving that these cells are in-dispensable for disease progression.

Analyzing the kinetics of cytokine production, we found thatIL-4 was promptly and transiently secreted into the serum of NODor C57BL/6 mice. IL-4 secretion reached maximal levels 2 h afterCon A injection and became undetectable within 5 h, whereasIFN-� levels increased only slightly at 2 h and peaked 12 h post-treatment (Fig. 2B). As shown in Fig. 2, B and C, the decreasedsusceptibility to hepatic injury in NOD mice coincided with a dras-tic reduction of maximal circulating IL-4 levels measured 2 h afterinjection, relative to C57BL/6 controls. By contrast, IFN-� pro-duction was not impaired at this time point, but even significantlyincreased (Fig. 2D) in NOD vs C57BL/6 mice 12 h after Con Ainjection (Fig. 2, B and E).

Knowing that IL-12 contributes to the development of Con A-induced hepatitis (9) and is aberrantly produced in NOD mice (21,22), we examined whether exogenous IL-12 could restore normalcytokine production and responsiveness to Con A in this strain. Asillustrated in Fig. 2A, this was indeed the case because a singleinjection 1 h before induction of hepatitis was sufficient to elicit a4-fold increase in ALT plasma levels in wild-type NOD mice to-gether with improved early IL-4 and IFN-� production (Fig. 2, Cand D). It is important to note that the treatment with IL-12 en-hanced neither the severity of hepatitis nor IL-4 production inNOD mice lacking iNKT cells, indicating that at this early time

FIGURE 2. IL-12 restores the susceptibility of NOD mice to iNKTcell-dependent hepatitis. NOD wild-type or iNKT cell-deficient micewere injected 1 h before Con A administration with IL-12 or PBS. A,ALT levels were determined in plasma 12 h after Con A injection. Dataare mean � SEM of 3–5 mice in each group. B, Kinetics of Con A-in-duced IL-4 and IFN-� secretion in serum of NOD and C57BL/6 wild-type mice were determined by ELISA and shown as the mean � SEMof 2–14 mice in each group. C–E, Serum levels of IL-4 and IFN-� wereassayed 2 h (C and D) or 12 h (E) after Con A injection. Data aremean � SEM of 3–5 mice in each group. Male C57BL/6 wild-type mice

were used for comparison. Of note, the susceptibility of female NODmice to Con A-induced hepatitis was also significantly increased byexogenous IL-12, but the ensuing acute liver injury led to rapid deathwithin 12 h.

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point they constitute the unique source of IL-4. Furthermore, itsneutralization by pretreatment with anti-IL-4 Abs prevented thedevelopment of Con A-induced hepatitis in NOD mice, (with adecrease in the ALT levels from 5103 � 1757 UI/L (n 3) to1621 � 904 UI/L (n 6); p � 0.05), as reported previously inC57BL/6 mice (Refs. 9–11, 13–15 and data not shown). By con-trast, the lack of iNKT cells had no evident affect on IFN-� pro-duction, which was similar in wild-type and iNKT-deficient NODmice 2 and 12 h after Con A injection (Fig. 2, D and E). Likewise,iNKT cells were not the major source of IFN-� in response toexogenous IL-12 because its concentration was not significantlydecreased 2 and 12 h posttreatment in the serum of iNKT cell-deficient mice, which implies that IFN-� is mainly generated bycells other than iNKT cells in our experimental set up.

iNKT cells constitutively express the IL-12R�

To compare the time course of IL-4 and IL-12 production, wemeasured both cytokines in the serum of C57BL/6 mice shortlyafter Con A injection, before maximal levels were attained. Asillustrated in Fig. 3A, the serum of naive mice contained alreadydetectable levels of IL-12, thus preceding IL-4 that was detectedhalf an hour later. IL-12 increased gradually about 4-fold between0 and 1.5 h posttreatment, paralleled by a more rapid increase ofserum IL-4 levels (Fig. 3A).

Knowing that endogenous IL-12 production occurred alreadyin untreated mice, we addressed the question whether iNKTcells expressed the corresponding receptor. To this end, IL-12R�1 subunit expression was analyzed by flow cytometry in

FIGURE 3. Constitutive expression of IL-12R by iNKTcells. A, IL-4 and IL-12 concentrations were measured byELISA in the serum of wild-type female C57BL/6 mice beforeand shortly after Con A injection (30, 60, and 90 min). Data aremean � SEM of 3–10 mice per group. Backgrounds levels inserum of IL-12- and IL-4-deficient mice, which served as neg-ative controls, were 30 � 2 pg/ml (n 2 mice) and 0 pg/ml(n 2 mice), respectively. B, Hepatic and splenic MNC wereisolated from naive female C57BL/6 and NOD mice andstained with anti-IL-12R�1 mAb or irrelevant isotype-matchedmAb as negative control (dotted line histogram). iNKT andconventional T cells were gated as TCR�� CD1d/�-GalCertetramer-positive and TCR�� CD1d/�-GalCer tetramer-nega-tive populations, respectively. Percentages shown are mean �SEM of iNKT cells expressing IL-12R�1 (n 5–6 mice pergroup). C, Expression of IL-12R in purified spleen and liveriNKT cells. Total RNA was extracted from purified spleen andliver iNKT (CD19�NK1.1�CD5�) cells from wild-typeC57BL/6 mice and subjected to a reverse transcription reaction(see Materials and Methods for details). IL-12R�1 and IL-12R�2 mRNA levels are expressed as mean � SEM from du-plicate samples of an index, with 18 S as an internal reference.

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spleen and liver MNC. As shown in Fig. 3B, IL-12R�1 wasdisplayed on iNKT but not on conventional T cells in spleenand liver of naive C57BL/6 wild-type mice. It was not signif-icantly up-regulated upon in vivo stimulation by Con A or�-GalCer (data not shown) and expressed more strongly byspleen rather than liver iNKT cells. The percentage of IL-12R�1� cells was higher in NOD than in C57BL/6 mice, bothin spleen and liver, excluding the possibility that impaired ex-pression of IL-12R�1 in NOD mice was responsible for IL-4deficiency (data not shown). IL-12R gene expression was fur-ther confirmed by real-time RT-PCR because both �1 and �2transcripts were consistently detected in purified spleen andliver iNKT cells with values of 23–25 threshold cycle (Fig. 3C).IL-12R�1 transcripts were more abundantly expressed in spleenthan in liver iNKT cells using 18 S as reference gene, similarlyto the corresponding protein.

IL-12 targets iNKT cells to increase their IL-4 production bothin vivo and in vitro

To prove that iNKT cells were directly responsible for IL-4 pro-duction and its enhancement by IL-12, we performed single-cellanalysis after intracellular staining of IL-4 in hepatic MNC fromNOD mice after injection of Con A, with or without exogenousIL-12. Liver iNKT cells, gated as TCR��CD1d/�-GalCer tet-ramer-positive cells produced IL-4 and IFN-� as soon as 90 minafter exposure to Con A, whether they were prepared from NODmice (Fig. 4A) or from C57BL/6 mice (data not shown), whereasconventional TCR��CD1d/�-GalCer tetramer-negative T cellswere virtually incapable of doing so at this time point. iNKT cellcounts were significantly lower in the liver of Con A-stimulatedNOD than C57BL/6 mice, as was the percentage of IL-4- or IFN-�-producing iNKT cells and the amount produced per cell. Pre-treatment with IL-12 increased both the percentage and the number

FIGURE 4. IL-12 enhances IL-4 productionby iNKT cells in vivo. Wild-type female NODmice were injected with IL-12 or PBS 1 h beforeCon A or PBS administration. At 90 min later,mice were sacrificed, and liver MNC in iNKTand conventional T cell gates, as described inFig. 3, were analyzed for IL-4 and IFN-� syn-thesis by intracytoplasmic staining. Irrelevantisotype-matched mAb were used as negativecontrols. A, Value in dot plots are percentages ofIL-4- or IFN-�-producing cells among totaliNKT and conventional T cells. B and C, Thepercentage (B) and number (C) of IL-4- or IFN-�-producing iNKT cells in the liver are ex-pressed as mean � SEM of 5–8 mice per group.

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of IL-4- and IFN-�-producing iNKT cells among liver MNC,suggesting that the cytokine contributed likewise to their he-patic recruitment (Fig. 4, B and C).

We further verified whether IL-12 could also induce IL-4 pro-duction by iNKT cells in vitro, by stimulating liver MNC fromC57BL/6, wild type, and iNKT cell-deficient NOD mice for 24 hwith Con A or �-GalCer after a 24-h preincubation with or without

IL-12. As shown in Fig. 5A, IL-4 was generated in response toeither stimulus and enhanced upon prior exposure to IL-12, pro-vided that iNKT cells were present in the liver. It was not detectedin supernatants of cells recovered from the iNKT cell-deficientNOD strain in response to the specific Ag �-GalCer and very lowafter stimulation with Con A, thus contrasting with IFN-� that wasdetected in response to Con A even in the absence of iNKT cells.The strict iNKT cell specificity of the effect of IL-12 on IL-4production was further supported by the use of electronicallysorted cells of this subset from C57BL/6 mice. Similarly to wholeliver MNC, both Con A and �-GalCer promoted substantial IL-4production that was about 3-fold enhanced by pretreatment with IL-12. Interestingly, purified iNKT cells cultured with exogenous IL-12alone secreted low but significant levels of both IFN-� and IL-4, re-vealing the autoreactivity of iNKT cells in response to IL-12 (Fig.5B). Altogether, our in vitro data demonstrate that IL-12 targets iNKTcells directly to enhance their IL-4 production.

IL-12 contributes to recruitment and activation of iNKT cellsduring Con A-induced hepatitis

We assessed the effect of IL-12 on the number of iNKT cells in theliver. As expected, a significant decrease occurred in mice havingreceived either IL-12 or Con A separately (Fig. 6A) (10, 29). Bycontrast, the iNKT cell compartment was partially restored whenIL-12 was administered before disease induction, suggesting thatthe cytokine increased cell survival or recruitment. In contrast, theconventional T cell compartment was not affected after IL-12 in-jection in the same conditions (data not shown).

We tested these alternatives by evaluating the percentage of ap-optotic cells among liver MNC and found that it was increasedrather than diminished upon exposure to IL-12, as was their totalnumber (Fig. 6B). These data, along with those showing a signif-icant increase of cytokine-producing iNKT cells in response to

FIGURE 5. IL-12 enhances IL-4 production by iNKT cells in vitro. Atotal of 1.2 � 105 total liver MNC from wild-type or iNKT cell-deficientmice of the indicated strain (A) or 2.5 � 104 purified liver iNKT(CD19�NK1.1�CD5�) cells from wild-type C57BL/6 mice (B) were pre-incubated with IL-12 or medium for 24 h before culture with �-GalCer orCon A for an additional 24 h. IL-4 and IFN-� concentrations were mea-sured in supernatants using ELISA. Data in A are mean � SEM of four toeight mice per group and from three independent experiments, each in-cluding six to eight pooled livers (B).

FIGURE 6. IL-12 contributes to the recruitment of apoptotic iNKT cellsto the liver. Wild-type female NOD mice were injected with IL-12 or PBS1 h before Con A administration as compared with IL-12 or PBS alone.Liver MNC were stained, and analyzed in the iNKT cell gate, as describedin Fig. 1A. A, Total number of iNKT cells in the liver are expressed asmean � SEM. B, At 2 h after Con A injection, percentage and absolutenumber of apoptotic iNKT cells in the liver of NOD mice were comparedbetween the group pretreated with IL-12 and PBS 1 h before Con A admin-istration. Apoptotic cells were evaluated as Annexin V� 7-aminoactinomycinD� cells. Data are mean � SEM.

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IL-12 (Fig. 4), are consistent with enhanced activation and recruit-ment of iNKT cells rather than enhanced survival.

IL-12-deficient mice are less susceptible to Con A-inducedhepatitis

To ascertain the role of IL-12 in the susceptibility to Con A-in-duced hepatitis, we evaluated disease severity in C57BL/6 mice inwhich the p40 chain had been deleted. We observed that they wereless responsive to induction of hepatitis by Con A (Fig. 7A), har-bored less iNKT cells in their liver (Fig. 7B) and generated lowerIL-4 concentrations in serum (Fig. 7C). The number of iNKT cellswas not significantly diminished in the liver of untreated IL-12-deficient mice, as compared with controls (240,100 � 4746; n 5 vs 183,400 � 14,580; n 7). In response to exogenous IL-12,hepatic injury in deficient mice as well as IL-4 production regainedwild-type levels (Fig. 7, A and C). Importantly, disease exacerba-tion was prevented by pretreatment with anti-IL-4, proving thatIL-12 exerts its deleterious effect through induction of this pro-Th2cytokine.

DiscussionIt is generally acknowledged that IL-12 exerts its biological activ-ity mainly by promoting a Th1 immune response and that its det-

rimental effect on Con A-induced hepatitis occurs through thismechanism of action (9). Our results challenge this notion by pro-viding the first evidence that this cytokine can also enhance a Th2immune response by targeting iNKT for increased IL-4 produc-tion, in addition to that of IFN-� (30–32). In further support of thephysiological relevance of this mechanism of action, we found thatthe inherent functional and numerical abnormalities of iNKT cellsrendered NOD mice less susceptible to disease induction.

The use of IL-12p40-deficient mice in this experimental setuprevealed the importance of IL-12 as an endogenous modulator ofiNKT cell activity. Indeed, we found that these mice developed aless severe hepatitis and their iNKT cells produced less IL-4 thantheir wild-type counterparts. These data are consistent with thoseof Nicoletti et al. (9), who reported a prophylactic effect of anti-IL-12, whereas IL-12 aggravated the disease. Yet, in contrast toour study, genetic cytokine deficiency did not alter the severity ofthe disease, a discrepancy the researchers explained by compen-satory mechanisms in genetically engineered mice.

It is not clear yet how IL-12 increases IL-4 production by iNKTcells. Our in vitro data support a direct action of IL-12 on iNKTcells stimulated with Con A or �-GalCer, in accordance with thehigh IL-12R expression that we demonstrate herein, and their up-regulation upon stimulation with �-GalCer together with IL-12that has been previously reported by Kitamura et al. (30). More-over, IL-12 promotes increased recruitment of iNKT cells to theliver, thus providing another means of producing more IL-4 andaggravating the disease.

Because it is well established that IL-4 and IFN-� are each es-sential for disease onset (13–15), Con A-induced iNKT cells couldequally well exert their deleterious functions through both of thesecytokines. However, it turned out that they are critical for the onsetof autoimmune hepatitis because of their unique, intrinsic capacityto produce IL-4. Indeed, in resistant Con A-treated NOD J�18�/�

mice IL-4 production was totally abrogated, whereas IFN-� levelswere not modified. Moreover, the early release of IL-4 in responseto Con A was exclusively due to iNKT cells in both NOD andC57BL/6 mice, as assessed by single-cell analysis after intracel-lular staining. This result is in agreement with the data reported byTaniguchi and colleagues (11) who proposed a major role foriNKT cell-derived IL-4 in disease progression through its capacityto up-regulate cell surface Fas ligand expression and Fas ligand-mediated cytotoxicity.

iNKT cells are remarkable for their uncommon ability to re-spond to self-Ags and their Th1/Th2 cytokine profile, which con-fers both immunostimulatory and immunosuppressive propertiesto this subset (31), depending on the stimulus encountered in themicroenvironment (32). IL-12, mainly produced by APC, is animportant constituent of this environment and has been commonlyassociated with the development of a Th1 immune response. Theexperimental hepatitis model reveals an unusual activity of IL-12,enabling iNKT cells to increase their IL-4 production and to exerta cytotoxic effect through this cytokine. Other research supportsthe view that iNKT cells stimulated with IL-12 can exert a cyto-toxic effect both via IFN-� and IL-4 production (33). It mighttherefore be postulated that endogenous IL-12 provides a generalmeans of amplifying the inherent activities of iNKT cells, by en-hancing not only IFN-� secretion (6), as previously reported inresponse to CD1d-presented self Ags but also IL-4 production.This hypothesis is strengthened by the constitutive surface expres-sion of IL-12R�1, which can be considered a distinctive marker ofiNKT because it is not shared by conventional T cells.

Our study provides new insights into the mechanisms throughwhich IL-12 and iNKT cells determine the onset of experimentalhepatitis. It would be interesting to investigate whether this kind of

FIGURE 7. IL-12-deficient mice are less susceptible to Con A-inducedhepatitis and their iNKT cells produce less IL-4. Wild-type and IL-12-p40�/� female C57BL/6 mice were injected with Con A. A, Plasma ALTactivities were determined 12 h after injection of Con A. They increasedwhen IL-12p40�/� mice received IL-12 before Con A, but returned tobaseline when IL-12p40�/� mice were injected with anti-IL-4 24 h beforeIL-12. B, At 2 h after Con A injection, the number of iNKT cells in the liverwas compared between C57BL/6 wild-type and IL-12p40�/� mice. Ofnote, iNKT cell counts do not differ significantly between naive IL-12-deficient and wild-type strain (data not shown). C, Serum levels of IL-4were measured by ELISA. The impaired production of IL-4 in IL-12p40�/� mice was restored after IL-12 injection 1 h before Con A. Dataare mean � SEM of 5–9 mice in each group.

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interaction takes place in other immune responses, such as anti-bacterial and antitumor responses.

We do not know yet how the conclusions drawn from the animalmodel apply to human disease. It has been reported that the num-ber of iNKT cells in PBMC varies considerably between healthysubjects, ranging from the limit of detection at 0.01% up to 1%(34). It should also be mentioned that in a recent study of childrensuffering from autoimmune hepatitis, IL-4 and IL-12 could be de-tected in PBMC (35). Whether increased levels of these two mol-ecules are associated with abnormally high numbers or activity ofiNKT in patients with T cell-mediated liver injuries remains to beestablished. If this were the case and increased iNKT cell fre-quency turned out to be a reliable indication of susceptibility toautoimmune hepatitis in humans, novel therapies based on deple-tion of iNKT cells could eventually be proposed.

AcknowledgmentsWe are grateful to Maria Leite-de-Moraes (Centre National de la Recher-che Scientifique, Unite Mixte de Recherche 8147) for helpful discussionsand critical comments and to Pauline Louche for technical assistance.

DisclosuresThe authors have no financial conflict of interest.

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