Costimulatory Pathways Functions of NKT Cells by CD28 and CD40 Differential Regulation of Th1 And
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Differential Regulation of Th1 and Th2 Functions of NKT Cells by CD28 and CD40 Costimulatory Pathways1 Yoshihiro Hayakawa,* Kazuyoshi Takeda,2†‡ Hideo Yagita,†‡ Luc Van Kaer,§ Ikuo Saiki,* and Ko Okumura†‡ V␣14 NKT cells produce large amounts of IFN-␥ and IL-4 upon recognition of their specific ligand ␣-galactosylceramide (␣- GalCer) by their invariant TCR. We show here that NKT cells constitutively express CD28, and that blockade of CD28-CD80/ CD86 interactions by anti-CD80 and anti-CD86 mAbs inhibits the ␣-GalCer-induced IFN-␥ and IL-4 production by splenic V␣14 NKT cells. On the other, the blockade of CD40-CD154 interactions by anti-CD154 mAb inhibited ␣-GalCer-induced IFN-␥ production, but not IL-4 production. Consistent with these findings, CD28-deficient mice showed impaired IFN-␥ and IL-4 production in response to ␣-GalCer stimulation in vitro and in vivo, whereas production of IFN-␥ but not IL-4 was impaired in CD40-deficient mice. Moreover, ␣-GalCer-induced Th1-type responses, represented by enhanced cytotoxic activity of splenic or hepatic mononuclear cells and antimetastatic effect, were impaired in both CD28-deficient mice and CD40-deficient mice. In contrast, ␣-GalCer-induced Th2-type responses, represented by serum IgE and IgG1 elevation, were impaired in the absence of the CD28 costimulatory pathway but not in the absence of the CD40 costimulatory pathway. These results indicate that CD28- CD80/CD86 and CD40-CD154 costimulatory pathways differentially contribute to the regulation of Th1 and Th2 functions of V␣14 NKT cells in vivo. The Journal of Immunology, 2001, 166: 6012–6018. atural killer T cells, which include heterogeneous pop- production of IFN-␥ by ␣-GalCer-activated V␣14 NKT cells, ulations, represent a novel lymphoid lineage distinct which requires IL-12 production by DC (15). On the other hand, N from conventional T cells, B cells, or NK cells (1, 2). V␣14 NKT cells also produce large amounts of IL-4 in the primary The TCR␣ expressed on the majority of NKT cells consists of a response and have been considered to play a role for the develop- single invariant V␣14-J␣281 chain paired preferentially with ment of Th2 responses (9, 10, 16–19). Since IL-4 and IFN-␥ have V8.2, V2, or V7, and recognizes glycolipid Ags or particular opposite effects on Th1/Th2 development, the role for V␣14 NKT hydrophobic peptides presented by the MHC class Ib molecule cells in the regulation of immune responses remains controversial CD1d (1–3). Although the physiological Ags for NKT cells still (9–11). In the present study, we examined the involvement of remain unclear, ␣-galactosylceramide (␣-GalCer),3 a glycolipid CD28- and CD40-mediated costimulatory pathways in IL-4 and derived from a marine sponge, has been identified to act as a spe- IFN-␥ production by ␣-GalCer-stimulated V␣14 NKT cells in by guest on September 30, 2021. Copyright 2001 Pageant Media Ltd. cific ligand for V␣14 NKT cells (4–6). It has been reported that vitro and in vivo. We found differential contributions of these co- ␣-GalCer selectively stimulates V␣14 NKT cells to rapidly pro- stimulatory pathways to IL-4 and IFN-␥ production by V␣14 NKT duce large amounts of IFN-␥ and IL-4 and to exhibit cytotoxic and cells. Selective manipulation of V␣14 NKT cell functions by antitumor activities (7, 8). Moreover, ␣-GalCer-induced V␣14 ␣-GalCer and the blockade of costimulatory pathways, which can NKT cell activation secondarily resulted in the induction and mod- potentially modulate systemic immune responses, is discussed. ulation of innate (NK cell) and adaptive (T cell and B cell) immune responses (9–13). The presentation of ␣-GalCer by CD1d ex- Materials and Methods pressed on certain APC, especially dendritic cells (DC), efficiently Mice induced V␣14 NKT cell activation (3, 4, 7, 14). It has been re- http://classic.jimmunol.org ported that CD40-CD154 interactions are critically involved in the Male C57BL/6 (B6) wild-type mice were purchased from Clear Japan (To- kyo, Japan). B6 CD28-deficient (CD28Ϫ/Ϫ) mice were originally pur- chased from The Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facility. B6 CD1-deficient (CD1Ϫ/Ϫ) mice were generated as *Department of Pathogenic Biochemistry, Research Institute of Natural Medicine, previously described (20). B6 CD40-deficient (CD40Ϫ/Ϫ) mice were Toyama Medical and Pharmaceutical University, Toyama, Japan; †Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; ‡Core Research kindly provided by H. Kikutani (Osaka University, Osaka, Japan) (21). All for Evolutional Science and Technology of Japan Science and Technology Corpora- mice were maintained under specific pathogen-free conditions and used at § Downloaded from tion, Tokyo, Japan; and Department of Microbiology and Immunology, Howard 6–7 wk of age. Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232 Reagents Received for publication December 4, 2000. Accepted for publication March 7, 2001. ␣-GalCer [(2S,3S,4R)-1-o-(␣-D-galactopyranosyl)-2-(N-hexacosanoylamino)- The costs of publication of this article were defrayed in part by the payment of page 1,3,4-octadecanetiol] was provided by Y. Koezuka and K. Motoki (Kirin charges. This article must therefore be hereby marked advertisement in accordance Brewery, Gumma, Japan) and was prepared as described previously (4, 8). with 18 U.S.C. Section 1734 solely to indicate this fact. Purified mAbs (no azide/low endotoxin grade) against mouse CD86 (PO. 3), 1 This work was supported by a Grant-in Aid for Scientific Research from the Min- CD154 (MR1), and IL-12 (C17.8) and control hamster IgG (A19-4) were istry of Education, Science, and Culture, Japan. purchased from PharMingen (San Diego, CA). Control rat IgG was purchased 2 Address correspondence and reprint requests to Dr. Kazuyoshi Takeda, Department from Sigma (St. Louis, MO). The hybridoma-producing anti-mouse CD154 of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, mAb (MR1) was obtained from American Type Culture Collection (Manassas, Tokyo 113-8421, Japan. E-mail address: [email protected] VA) (22). The hybridomas producing anti-mouse CD80 mAb (RM80) and 3 Abbreviations used in this paper: ␣-GalCer, ␣-galactosylceramide; MNC, mononu- anti-mouse CD86 mAb (PO.3) were established in our laboratory (23). The clear cells; DC, dendritic cells. mAbs were prepared from these hybridoma as described previously (23). Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 6013 Flow cytometric analysis Cytotoxic assay Surface phenotype of the cells was characterized by three-color flow cy- Cytolytic activity was assessed against NK-susceptible YAC-1 target cells tometry as previously described (24). Briefly, 1 ϫ 106 cells were first and NK- and Fas ligand-resistant B16-BL6 cells by a standard 51Cr release preincubated with anti-CD16/32 (2.4G2) mAb to avoid the nonspecific assay as previously described (26, 27). Both target cells were cultured in binding of Abs to Fc␥R. Then the cells were incubated with a saturating RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, and 25 mM amount of biotinylated isotype-matched control mAbs (Ha4/8, A19-3, NaHCO3. As effector cells, hepatic and splenic MNC were isolated from G235-2356, or R3-34), anti-CD28 (37.51), anti-CD152/CTLA-4 (UC10- the mice 24 h after i.p. injection of 2 g/200 lof␣-GalCer or 200 lof 4F10-11), anti-CD137/4-1BB (1AH2), anti-CD134/OX40 (OX86), anti- the vehicle (0.5% polysorbate 20). Target cells (106) were labeled with 100 51 CD27 (LG.3A10), anti-CD30 (mCD30.1), and anti-CD154/CD40L (MR1) Ci/ml Na2 CrO4 for 60 min at 37°C in RPMI 1640 medium containing mAb before incubation with FITC-conjugated anti-NK1.1 (PK136) mAb, 10% FCS. Labeled target cells (104/well) were incubated in a total volume Cy-Chrome-conjugated anti-CD3⑀ mAb (145-2C111), and PE-conjugated of 200 l with effector cells in 10% FCS-RPMI 1640 in 96-well U-bottom streptavidin. All staining reagents were obtained from PharMingen. After plates. The plates were centrifuged before incubation, and after4hthe washing with PBS, the stained cells were analyzed on a FACSCalibur supernatant was harvested and counted in a gamma counter. Specific lysis (Becton Dickinson, San Jose, CA). was calculated as previously described (26, 27). In vitro stimulation with ␣-GalCer Experimental lung metastasis Splenic mononuclear cells (MNC, 5 ϫ 105) were cultured with 100 ng/ml Log-phase cell cultures of B16-BL6 were harvested with 1 mM EDTA in ␣-GalCer or vehicle (0.1% DMSO) as a control in RPMI 1640 medium PBS, washed three times with serum-free RPMI 1640, and resuspended to 4 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 25 appropriate concentrations in PBS. B16-BL6 cells (5 ϫ 10 /100 l) were ␣ mM NaHCO3 in humidified 5% CO2 at 37°C in 96-well U-bottom plates injected i.v. into syngeneic B6 mice, and then -GalCer (2 g/200 l) or 200 (Costar, Cambridge, MA). In the blocking experiments, anti-CD80 l of vehicle was i.p. administered on days 0, 4, and 8. On day 14, the number (RM80), anti-CD86 (PO.3), anti-CD154 (MR1), anti-IL-12 (C17.8), and of tumor colonies in the lung was counted under a dissecting microscope. isotype-matched control mAbs were added at 10 g/ml each in the culture. After incubation for 72 h, the cell-free culture supernatants were harvested Statistical analysis to detect cytokine levels by ELISA. Data were analyzed using a two-tailed Student t test. All p values Ͻ 0.05 ELISA were considered as significant.