Fas/Fas Ligand Interactions Promote Activation-Induced Cell Death of NK T Lymphocytes

This information is current as Maria C. Leite-de-Moraes, André Herbelin, Christine of September 23, 2021. Gouarin, Yasuhiko Koezuka, Elke Schneider and Michel Dy J Immunol 2000; 165:4367-4371; ; doi: 10.4049/jimmunol.165.8.4367 http://www.jimmunol.org/content/165/8/4367 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Fas/Fas Ligand Interactions Promote Activation-Induced Cell Death of NK T Lymphocytes1

Maria C. Leite-de-Moraes,2* Andre´Herbelin,† Christine Gouarin,† Yasuhiko Koezuka,‡ Elke Schneider,* and Michel Dy*

NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demon- strate herein that in addition to their IFN-␥ production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared Downloaded from the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, ␣-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism. The Journal of Immunology, 2000, 165: 4367–4371. http://www.jimmunol.org/ atural killer T lymphocytes are characterized by NK1.1 forin- or Fas-dependent mechanisms and have been reported to expression and usage of an invariant TCR encoded by prevent tumor metastasis (10–12). Our own studies and those of N V␣14-J␣281 gene segments preferentially associated others have provided evidence for a tight association between the with a highly skewed V␤ repertory, represented mainly by V␤8.2 development of autoimmune diseases, such as lupus erythematosus (1, 2). They express memory/activation cell markers, such as or diabetes, and the loss or dysfunction of NKT cells (13, 14). CD44 and CD69, and are positively selected by the nonpolymor- Activated NKT cells may also become dangerous to the organism, phic MHC class I-like molecule CD1d (1–4). It has also been as suggested by their implication in liver injury after Salmonella established that they specifically recognize ␣-galactosylceramide infection and in Con A-induced hepatitis (15, 16). It is therefore (␣-GalCer)3 or parasite glycosylphosphatidyl inositols presented conceivable that their life span is strictly controlled, leading to by guest on September 23, 2021 by CD1d molecules (3–5). their elimination as soon as they have fulfilled their regulatory An interesting feature of NKT cells consists of their ability to functions. In the present study we investigated the fate of NKT express both Th1 and Th2 cytokine profiles according to their cells after their stimulation with IL-12 plus IL-18 or their specific mode of activation and the cytokines present in their microenvi- ligand ␣-GalCer. We found that both TCR-dependent and -inde- ronment (6–10). Indeed, upon TCR cross-linking, they constitute pendent stimuli promoted activation and cell death, whose mech- mainly a source of IL-4 (6, 7), whereas a TCR-independent stim- anisms we further analyzed. ulation with IL-12 plus IL-18 promotes IFN-␥ production and cytotoxicity (10), thus enabling them to participate in either type of Materials and Methods immune response. Activated NKT cells kill their targets by per- Animals and reagents Wild-type, Fas-deficient C57BL/6-lpr/lpr and Fas ligand (FasL)-deficient *Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8603, Uni- C57BL/6-gld/gld mice were bred in our own facilities and used at the age of versite´Rene´Descartes, Paris V, Hoˆpital Necker, Paris, France; †Institut National de 6–8 wk, before the onset of lymphadenopathy in the mutant strain (17). In la Sante´ et de la Recherche Me´dicale, Unite´ 25, and Association Claude Bernard, some experiments 7-mo-old mice were used. RPMI 1640 (Life Technologies, ‡ Hoˆpital Necker, Paris, France; and Pharmaceutical Research Laboratory, Kirin Grand Island, NY) supplemented with 10% heat-inactivated FCS (TechGen, Brewery Co., Ltd., Gunma, Japan Les Ulis, France), 100 IU/ml penicillin, 100 ␮g/ml streptomycin, 10 mM Received for publication March 28, 2000. Accepted for publication July 28, 2000. HEPES buffer (all from Life Technologies), and 5 ϫ 10Ϫ5 M 2-ME was used ␥ The costs of publication of this article were defrayed in part by the payment of page as the culture medium. Murine IL-2, IL-4, IL-12, IL-18, and IFN- were pur- charges. This article must therefore be hereby marked advertisement in accordance chased from R&D Systems (Abingdon, U.K.). Human rIL-7 (sp. act., 8.8 ϫ with 18 U.S.C. Section 1734 solely to indicate this fact. 106 U/mg) was provided by Sanofi (Labege, France). Anti-IL-4 mAbs (11B11 ␥ 1 This work was supported by institutional funds from the Centre National de la and BVD6-24G2.3), anti-IFN- mAbs (AN18 and R46A2), and anti-CD3 Recherche Scientifique, Universite´Rene´Descartes-Paris V, the Association pour la mAb (145-2C11) were purified in our laboratory. The BVD6-24G2.3 clone Recherche sur le Cancer (ARC 9742), and the Ligue Nationale Contre le Cancer (Axe was obtained from DNAX (Palo Alto, CA). mAbs against CD8 (53.67), CD24 Immunologie, 1999). M.C.L.M. was supported by a grant from the Ligue Nationale (J11d), B220 (RA3-6B2), and Mac1 (M1/70) used for cell depletion were Contre le Cancer. purified in our laboratory. CD4-PE (YTS 191.1), PE- or FITC-conjugated CD8 ␣␤ 2 Address correspondence and reprint requests to Dr. Maria C. Leite-de-Moraes, Cen- (YTS 169.4), CD3-FITC (500-A2), TCR -FITC (H57-597), and streptavi- tre National de la Recherche Scientifique, Unité Mixte de Recherche 8603, Hoˆpital din-PE (SAV-PE) were purchased from Caltag (Le Perray en Yvelines, Necker, 161 rue de Se`vres, 75743 Paris Cedex 15, France. E-mail address: France). Biotinylated anti-NK1.1 (PK136) or anti-CD69 (H1.2F3), anti- [email protected] CD122-FITC (TM-␤1), anti-TCR␣␤-APC (H57-597), annexin V-FITC or 3 Abbreviations used in this paper: ␣-GalCer, ␣-galactosylceramide; FasL, Fas ligand; -PE, anti-Fas-PE (Jo2), SAV-Cy-Chrome, and blocking NA/LE anti-FasL SAV, streptavidin; PI, propidium iodide; CFSE, 5-(and 6-)-carboxyfluorescein diac- (clone Kay-10) were obtained from PharMingen (San Diego, CA), and anti-rat etate, succinimidyl ester. Ig-coated magnetic beads were purchased from Dynal (Compiegne, France).

The irreversible, large spectrum caspase inhibitor benzyl-oxy-carbonyl-Val- 1B). NKT cell activation was accompanied by increased prolifer- Ala-Asp (zVAD)-fmk, was purchased from Bachem (Voisins-le-Bretonneux, ation assessed by the fluorescent dye CFSE, which is a means of France). quantifying cell divisions by flow cytometry (19). Fig. 2A shows In vivo treatment, purification of NKT lymphocytes, stimulation, that 36% of the NKT population had divided after 3 days of culture and apoptosis assay in IL-12 plus IL-18. Yet, the number of cells effectively recovered Wild-type and C57BL/6-lpr/lpr mice received a single i.v. injection of 2 at this time point was surprisingly low, amounting merely to about ␮gof␣-GalCer (Kirin Brewery Co., Gunma, Japan) (18) dissolved in PBS 20% of the cells initially plated. This result could only be ex- containing 0.025% polysolvate 20 or vehicle alone and were sacrificed 2 or plained by the disappearance of NKT cells once they had been ϩ 18 h later. Freshly isolated splenocytes were enriched for CD4 and activated by IL-12 plus IL-18. CD4ϪCD8Ϫ T cells by immunomagnetic depletion of CD8ϩ, Mac1ϩ and ϩ Ϫ Ϫ B220 cells. Enriched CD8 CD24 NKT thymocytes were obtained as Activation-induced cell death of NKT lymphocytes in response to previously described (14). At least 90% of TCR␣␤ϩ splenocytes or thymocytes were obtained after depletion. For in vitro experiments, the en- IL-12 plus IL-18 riched lymphocyte population was further labeled with anti-TCR␣␤ and To test our hypothesis, we analyzed the viability of proliferating ␣␤ϩ ϩ anti-NK1.1 mAbs, and TCR NK1.1 NKT lymphocytes were sorted NKT lymphocytes gated from the population that had completed on a FACS Vantage cell sorter (Becton Dickinson, Mountain View, CA). Purity was Ͼ99% upon reanalysis. division. Using the apoptosis assay, based on the binding of an- Sorted lymphocytes were then stimulated at a concentration of 5 ϫ nexin V that occurs early in programmed cell death after the ex- 105/ml with IL-12 (10 ng/ml) plus IL-18 (100 ng/ml) in the presence or the ternalization of phosphatidylserine, (20), we found that nearly 40% absence of IL-7 (40 ng/ml). In some experiments, zVAD-fmk or blocking of divided NKT cells were about to die (Fig. 2A). anti-FasL mAb was added at a concentration of 50 ␮Mor10␮g/ml, re- Downloaded from spectively. After 1, 2, and 3 days of incubation, cells were washed in PBS In accordance with these findings, an important percentage of and stained with annexin V-FITC and propidium iodide (PI) according to dead NKT lymphocytes was detected after 3 days of stimulation the manufacturer’s instructions. with IL-12 plus IL-18, similar to that for cells cultured in medium In another series of experiments, NKT lymphocytes were incubated alone (Fig. 2B). In contrast, only about 10% of cells died in the ␮ with 1 M 5-(and 6-)-carboxyfluorescein diacetate, succinimidyl ester presence of IL-7 (Fig. 2B). This survival effect might be mediated (CFSE; Molecular Probes, Leiden, The Netherlands) at 37°C for 5 min. Labeled cells were washed and then stimulated with IL-12 (10 ng/ml) plus at least in part through up-regulation of the anti-apoptotic molecule

IL-18 (100 ng/ml). After different periods of incubation, they were washed Bcl-2 (21, 22). Yet, even though IL-7 saved NKT cells from spon- http://www.jimmunol.org/ in PBS, stained with annexin V and PI, and analyzed for apoptotic cells. taneous cell death, it did not prevent apoptosis induced by IL-18 Supernatants were harvested in all experiments and stored at Ϫ80°C until plus IL-12 (Fig. 2B). This was also true when IL-2 or IL-4 was IL-4 and IFN-␥ assays as previously described (7, 10). used instead of IL-7, since neither factor could restore NKT cell Flow cytometric analysis survival (data not shown). Cells were stained in PBS containing 2% FCS and 0.01 M sodium azide and were incubated for 30 min with appropriate dilutions of various mAbs Implication of Fas/FasL interactions in NKT cell apoptosis coupled to biotin, PE, APC, or fluorescein. For biotinylated mAbs, induced by IL-12 plus IL-18 SAV-PE or SAV-Cy-Chrome was used as a second-step reagent. At least 4 We have previously demonstrated that the cytotoxic functions ac- 10 live lymphoid cells were acquired in each run and analyzed on a FAC- by guest on September 23, 2021 Scalibur (Becton Dickinson) cytometer using CellQuest software. quired by NKT cells stimulated by IL-18 plus IL-12 involve the Detection of FasL mRNA by RT-PCR Crude RNA was extracted from sorted TCR␣␤ϩNK1.1ϩ NKT lymphocytes after 18 h of stimulation with IL-18 and IL-12 using TRIzol reagent (Life Technologies, Cergy-Pontoise, France), according to the manufacturer’s instructions. The semiquantitative RT-PCR technique used was based on the comparison between FasL mRNA levels and those of the ␤ transcripts encoding the ubiquitous housekeeping gene 2-microglobulin as described previously (7). The following primers (synthesized by Bio- probe, Montreuil, France) were used: FasL 5Ј, CTA CCA CCF CCA TCA Ј ␤ CAA CC; FasL 3 , CAA CCT CTT CTC CTC CAT TA; 2-microglobulin Ј ␤ Ј 5 , TGA CCG GCT TGT ATG CTA TC; and 2-microglobulin 3 , CAG TGT GAG CCA GGA TAT AG. Statistics Data were expressed as the mean Ϯ SD, and differences between means were evaluated using Student’s t test. Results and Discussion Activation of NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro We have recently demonstrated that in the absence of TCR engagement, NKT cells produce IFN-␥ and become cytotoxic upon stimulation with IL-12 plus IL-18, while either factor alone has no significant effect (10). In the present study we addressed the ques- FIGURE 1. IL-18 plus IL-12 activate NKT lymphocytes. Sorted ␣␤ϩ ϩ tion of whether the acquisition of these functional capacities was TCR NK1.1 thymocytes from wild-type (C57BL/6) mice were cultured (2.5 ϫ 105 cells/ml) with IL-18 (100 ng/ml) plus IL-12 (10 ng/ml) or accompanied by other features of cellular activation. As shown in medium. A, Twenty-four hours later, CD69 expression on stimulated (solid Fig. 1A, we found that a 24-h incubation of FACS-sorted, NKT line) and control cells (broken line) was analyzed. B, The percentage of cells with IL-12 plus IL-18 resulted in a significant up-regulation blasts among live TCR␣␤ϩNK1.1ϩ cells stimulated with IL-18 plus IL-12 of the activation marker CD69. This effect coincided with another (E) or medium (‚) was determined daily for 3 days, according to forward manifestation of cell activation, namely an increase in the propor- light scatter characteristics. Similar results were observed in three separate tion of blast cells, as judged by light scatter characteristics (Fig. experiments. The Journal of Immunology 4369 Downloaded from http://www.jimmunol.org/ FIGURE 2. IL-18 plus IL-12 mediate activation-induced cell death of ϩ ϩ NKT lymphocytes. A, Sorted TCR␣␤ NK1.1 cells from the thymus of FIGURE 3. IL-18 plus IL-12 up-regulate both FasL and Fas expression. wild-type mice were labeled with CFSE and stimulated (2.5 ϫ 105 cells/ A, RNA was extracted from sorted TCR␣␤ϩNK1.1ϩ thymocytes from ml) with (solid line) or without (broken line) IL-18 plus IL-12. Three days wild-type mice after an 18-h incubation with medium or IL-18 (100 ng/ml) later, cells were analyzed for CFSE fluorescence. The percentage of viable plus IL-12 (10 ng/ml). RT-PCR was then performed, and 2-fold diluted ␤ cells (PI negative) that had divided at least once in response to IL-18 plus RNA from these cells was analyzed for FasL mRNA expression. 2-Mi- ␤ IL-12, based on CFSE staining, is represented in the first histogram. These croglobulin ( 2-m) mRNA expression served as an internal control. B,In cells were gated and analyzed for annexin V labeling, as shown in the parallel, Fas expression by TCR␣␤ϩNK1.1ϩ cells stimulated with IL-18 ϩ ϩ second histogram. B, Sorted TCR␣␤ NK1.1 lymphocytes from wild- plus IL-12 (open histogram) or cultured in medium (closed histogram) was type mice were cultured in medium alone or together with IL-7 (40 ng/ml), analyzed by flow cytometry. Similar results were obtained in at least two by guest on September 23, 2021 IL-18 (100 ng/ml) plus IL-12 (10 ng/ml), or all three cytokines together. experiments. The percentage of dead cells was determined 3 days later by PI staining. Similar results were observed in three separate experiments.

Fas pathway (10). It could therefore be argued that this mechanism was also responsible for the death of the effector cells themselves. Consistent with this view, we observed that IL-12 plus IL-18 induced FasL transcription (Fig. 3A) and up-regulated the surface expression of Fas (Fig. 3B), which is spontaneously displayed by NKT cells. Considering the importance of caspases in the signal transduc- tion pathway leading to apoptosis (23), we evaluated the effect of the irreversible inhibitor zVAD-fmk, which blocks most of these proteases, on the viability of NKT cells treated with IL-12 plus IL-18. Fig. 4 shows that the inhibition of the caspase cascade did indeed strikingly reduce cell death. To test whether NKT cell apoptosis was Fas dependent, we stimulated NKT cells with IL-12 plus IL-18 in the presence of blocking anti-FasL mAb. Fig. 4 clearly shows that anti-FasL mAb inhibited NKT cell apoptosis. Taken together, our findings support the implication of the Fas death pathway in the apoptosis of activated NKT cells. FIGURE 4. IL-18 plus IL-12-induced NKT cell apoptosis is Fas depen- ϩ ϩ To confirm this view, we analyzed the effect of IL-12 plus IL-18 dent and is blocked by zVAD-fmk. Sorted TCR␣␤ NK1.1 thymocytes ϫ on NKT lymphocytes from Fas-deficient, C57BL/6-lpr/lpr mice. from wild-type (C57BL/6) or C57BL/6-lpr/lpr mice were cultured (2.5 105 cells/ml) with IL-18 (100 ng/ml) plus IL-12 (10 ng/ml) in the presence We found that Fas deficiency markedly diminished apoptosis of or the absence of zVAD-fmk (50 ␮M) or anti-FasL mAb (10 ␮g/ml). IL-7 NKT cells exposed to IL-12 plus IL-18 (Fig. 4). Similar results (40 ng/ml) was added at the onset of all cultures. Three days later, cells were obtained with FasL-deficient C57BL/6-gld/gld mice (data not were harvested, dead cells were determined by PI staining, and their per- shown), proving the involvement of both Fas and FasL in this centage was calculated in relation to cells cultured in IL-7, which were activation-induced cell death. Increased survival of Fas-deficient considered 100% viable. Data represent the mean Ϯ SD from three sepa- .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .NKT cells could not be explained by a poor responsiveness to rate experiments 4370 ACTIVATION AND APOPTOSIS OF NK T CELLS

FIGURE 6. Fas/FasL interactions are implicated in the apoptosis of NKT cells following ␣-GalCer treatment. Wild-type (C57BL/6) or C57BL/ 6-lpr/lpr mice received a single injection of ␣-GalCer (2 ␮g/mouse) and were killed 18 h later. The percentage of TCR␣␤intNK1.1ϩ cells among enriched splenocytes from both strains treated with vehicle or ␣-GalCer is depicted. Data represent the mean Ϯ SD from three different experiments. .p Ͻ 0.05 ,ء Downloaded from

we observed that NKT cells disappeared from both the spleen and the thymus, as shown in Fig. 5A. The loss of this population occurred after its functional activation in terms of IL-4 and IFN-␥ production, which was already detected 2 h postinjection, when the

frequency of NKT cells in the two organs was not yet diminished http://www.jimmunol.org/ (data not shown). The disappearance of NKT cells after ␣-GalCer injection could be the consequence of NK1.1 down-modulation rather than depletion. This conclusion was not consistent with the fact that the number of TCR␣␤intCD122ϩ cells, which have been characterized as NKT lymphocytes (1, 2), diminished similarly after in vivo treatment with ␣-GalCer (0.8% TCR␣␤intCD122ϩ cells among TCR␣␤ϩ splenocytes of mice having received ␣-GalCer vs 3.2% in vehicle controls). The proof that NKT cells were actually un- by guest on September 23, 2021 dergoing apoptosis after injection of ␣-GalCer was provided by their binding of annexin V, which is an early event of programmed FIGURE 5. In vivo treatment with ␣-GalCer induces NKT cell deple- cell death, while a much lower proportion of NKT cells was lation. Wild-type mice were injected once with ␣-GalCer (2 ␮g/mouse) and beled in vehicle controls (Fig. 5B). ␣␤int ϩ were killed 18 h later. A, The percentage of TCR NK1.1 cells among Interestingly, in vivo treatment with ␣-GalCer not only affects ␣␤ϩ TCR -enriched lymphocytes, obtained as described in Materials and peripheral NKT cells, but also depletes this population from the Methods, is represented in each quadrant. B, Annexin V staining was an- ϩ Ϫ thymus, which might have important implications for its steady alyzed among gated TCR␣␤intNK1.1 PI splenocytes from wild-type mice injected with vehicle or ␣-GalCer. Data represent a typical experi- state and selection. A previous in vivo study has shown that ad- ment of three. ministration of anti-CD3 mAb, which activates both T and NKT cells, results in the disappearance of the latter population from the IL-12 plus IL-18, since they were similarly activated in terms of spleen, but not from the thymus (25). The lack of effect in this IFN-␥ production (up to 700 ng/2.5 ϫ 105 NKT cells), up-regu- organ is probably due to the inability of anti-CD3 mAb to stimulation of CD69 expression (ϳ2-fold after 18 h of stimulation), and late thymic NKT cells (6), conversely to ␣-GalCer, which can blast transformation (data not shown). Despite the drastically re- activate this thymic subset in vivo (our unpublished observations). duced cell death among NKT lymphocytes lacking functional Fas We investigated the involvement of the Fas pathway in NKT receptors, a significant percentage remained apoptotic and was res- cell depletion in vivo, again taking advantage of the Fas deficiency cued by the caspase inhibitor zVAD. These findings suggest that a in C57BL/6-lpr/lpr mice. The absence of functional Fas did not second caspase-dependent, but Fas-independent, pathway was in- prevent ␣-GalCer-induced activation in terms of CD69 up-regula- volved in the death of NKT cells stimulated with IL-12 plus IL-18. tion and IL-4 production in response to in vivo treatment (data not shown). Yet, as shown in Fig. 6, it rendered NKT cells insensitive Fas-dependent activation-induced cell death in vivo, after to activation-induced cell death, implicating Fas/FasL interactions treatment with the specific ligand of NKT lymphocytes, in the disappearance of ligand-activated NKT cells. ␣ -GalCer We analyzed whether NKT cells accumulate with age in Fas- The results obtained to date raised the question of whether acti- deficient mice. Seven-month-old C57BL/6-lpr/lpr mice presented vation-induced cell death of NKT lymphocytes occurred exclu- a slight increase in the percentage as well as the number of sively in response to TCR-independent stimulation or whether TCR␣␤ϩNK1.1ϩ splenocytes compared with controls (16.2 Ϯ TCR ligation induced the same effect. To address this issue, we 4.3 ϫ 105 vs 9.3 Ϯ 2.7 ϫ 105 cells for C57BL/6-lpr/lpr and wild- used the cognate Ag ␣-GalCer, whose function as a specific in- type mice, respectively). In vivo, this slight accumulation of NKT ducer of NKT cell activation has been established both in vitro and cells in old C57BL/6-lpr/lpr mice might be explained by the im- in vivo (10, 24). Within 18 h after a single injection of ␣-GalCer, plication of other death receptors (23) in the apoptosis of these The Journal of Immunology 4371 lymphocytes. It might also be argued that a marked accumulation wards IFN-␥ production by IL-12: influence of the microenvironment on the of NKT cells cannot be observed in Fas-deficient mice because functional capacities of NK T cells. Eur. J. Immunol. 28:1507. 10. Leite-de-Moraes, M. C., A. Hameg, A. Arnould, F. Machavoine, Y. Koezuka, they are kept in pathogen-free conditions where exogenous acti- E. Schneider, A. Herbelin, and M. Dy. 1999. A distinct IL-18-induced pathway vation of NKT cells is unlikely. to fully activate NK T lymphocytes independently from TCR engagement. J. Im- munol. 163:5871. In conclusion, our data provide evidence for a new Fas-depen- 11. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, dent mechanism controlling the life span of activated NKT cells in M. Kanno, and M. Taniguchi. 1997. Requirement for V␣14 NKT cells in IL-12- response to the cognate Ag ␣-GalCer as well as to TCR-indepen- mediated rejection of tumors. Science 278:1623. 12. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, H. Sato, E. Kondo, dent (IL-12 plus IL-18) stimulation. A strict surveillance of these M. Harada, H. Koseki, T. Nakayama, et al. 1998. Natural killer-like nonspecific autoreactive effector cells seems requisite considering their func- tumor cell lysis mediated by specific ligand-activated V␣14 NKT cells. Proc. tional capacities, which ensure a prompt riposte during the early Natl. Acad. Sci. USA 95:5690. 13. Mieza, M. A., T. Itoh, J. Q. Cui, Y. Makino, T. Kawano, K. Tsuchida, T. Koike, stages of the immune response but may become harmful thereafter, T. Shirai, H. Yagita, A. Matsuzawa, et al. 1996. Selective reduction of V␣14ϩ causing damage to the organism itself. NK T cells associated with disease development in autoimmune-prone mice. J. Immunol. 156:4035. 14. Gombert, J. M., A. Herbelin, E. Tancrede-Bohin, M. Dy, C. Carnaud, and Acknowledgments J. F. Bach. 1996. Early quantitative and functional deficiency of NK1ϩ-like thy- We are grateful to Corinne Garcia (Institut National de la Santeétdela mocytes in the NOD mouse. Eur. J. Immunol. 26:2989. 15. Ishigaki, M., H. Nishimura, Y. Naiki, K. Yoshioka, T. Kawano, Y. Tanaka, Recherche Me´dicale, Unite´373) for cell sorting. We thank Anne Arnould M. Taniguchi, S. Kakumu, and Y. Yoshikai. 1999. The roles of intrahepatic and François Machavoine (Centre National de la Recherche Scientifique, V␣14ϩNK1.1ϩ T cells for liver injury induced by Salmonella infection in mice. Unité Mixte de Recherche 8603) for helpful technical assistance. We are Hepatology 29:1799. 16. Kaneko, Y., M. Harada, T. Kawano, M. Yamashita, Y. Shibata, F. Gejyo, particularly indebted to Sanofi (Labege, France) for providing hrIL-7. Downloaded from T. Nakayama, and M. Taniguchi. 2000. 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