Differential Regulation of Th1 and Th2 Functions of NKT Cells by CD28 and CD40 Costimulatory Pathways

Yoshihiro Hayakawa, Kazuyoshi Takeda, Hideo Yagita, Luc Van Kaer, Ikuo Saiki and Ko Okumura
J Immunol May 15, 2001, 166 (10) 6012-6018; DOI: https://doi.org/10.4049/jimmunol.166.10.6012

Abstract

Vα14 NKT cells produce large amounts of IFN-γ and IL-4 upon recognition of their specific ligand α-galactosylceramide (α-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 α-GalCer-induced IFN-γ and IL-4 production by splenic Vα14 NKT cells. On the other, the blockade of CD40-CD154 interactions by anti-CD154 mAb inhibited α-GalCer-induced IFN-γ production, but not IL-4 production. Consistent with these findings, CD28-deficient mice showed impaired IFN-γ and IL-4 production in response to α-GalCer stimulation in vitro and in vivo, whereas production of IFN-γ but not IL-4 was impaired in CD40-deficient mice. Moreover, α-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, α-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 Vα14 NKT cells in vivo.

Natural killer T cells, which include heterogeneous populations, represent a novel lymphoid lineage distinct from conventional T cells, B cells, or NK cells (1, 2). The TCRαβ expressed on the majority of NKT cells consists of a single invariant Vα14-Jα281 chain paired preferentially with Vβ8.2, Vβ2, or Vβ7, and recognizes glycolipid Ags or particular hydrophobic peptides presented by the MHC class Ib molecule CD1d (1, 2, 3). Although the physiological Ags for NKT cells still remain unclear, α-galactosylceramide (α-GalCer),3 a glycolipid derived from a marine sponge, has been identified to act as a specific ligand for Vα14 NKT cells (4, 5, 6). It has been reported that α-GalCer selectively stimulates Vα14 NKT cells to rapidly produce large amounts of IFN-γ and IL-4 and to exhibit cytotoxic and antitumor activities (7, 8). Moreover, α-GalCer-induced Vα14 NKT cell activation secondarily resulted in the induction and modulation of innate (NK cell) and adaptive (T cell and B cell) immune responses (9, 10, 11, 12, 13). The presentation of α-GalCer by CD1d expressed on certain APC, especially dendritic cells (DC), efficiently induced Vα14 NKT cell activation (3, 4, 7, 14). It has been reported that CD40-CD154 interactions are critically involved in the production of IFN-γ by α-GalCer-activated Vα14 NKT cells, which requires IL-12 production by DC (15). On the other hand, Vα14 NKT cells also produce large amounts of IL-4 in the primary response and have been considered to play a role for the development of Th2 responses (9, 10, 16, 17, 18, 19). Since IL-4 and IFN-γ have opposite effects on Th1/Th2 development, the role for Vα14 NKT cells in the regulation of immune responses remains controversial (9, 10, 11). In the present study, we examined the involvement of CD28- and CD40-mediated costimulatory pathways in IL-4 and IFN-γ production by α-GalCer-stimulated Vα14 NKT cells in vitro and in vivo. We found differential contributions of these costimulatory pathways to IL-4 and IFN-γ production by Vα14 NKT cells. Selective manipulation of Vα14 NKT cell functions by α-GalCer and the blockade of costimulatory pathways, which can potentially modulate systemic immune responses, is discussed.

Materials and Methods

Mice

Male C57BL/6 (B6) wild-type mice were purchased from Clear Japan (Tokyo, Japan). B6 CD28-deficient (CD28−/−) mice were originally purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facility. B6 CD1-deficient (CD1−/−) mice were generated as previously described (20). B6 CD40-deficient (CD40−/−) mice were kindly provided by H. Kikutani (Osaka University, Osaka, Japan) (21). All mice were maintained under specific pathogen-free conditions and used at 6–7 wk of age.

Reagents

α-GalCer [(2S,3S,4R)-1-o-(α-d-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetiol] was provided by Y. Koezuka and K. Motoki (Kirin Brewery, Gumma, Japan) and was prepared as described previously (4, 8). Purified mAbs (no azide/low endotoxin grade) against mouse CD86 (PO. 3), CD154 (MR1), and IL-12 (C17.8) and control hamster IgG (A19-4) were purchased from PharMingen (San Diego, CA). Control rat IgG was purchased from Sigma (St. Louis, MO). The hybridoma-producing anti-mouse CD154 mAb (MR1) was obtained from American Type Culture Collection (Manassas, VA) (22). The hybridomas producing anti-mouse CD80 mAb (RM80) and anti-mouse CD86 mAb (PO.3) were established in our laboratory (23). The mAbs were prepared from these hybridoma as described previously (23).

Flow cytometric analysis

Surface phenotype of the cells was characterized by three-color flow cytometry as previously described (24). Briefly, 1 × 106 cells were first preincubated with anti-CD16/32 (2.4G2) mAb to avoid the nonspecific binding of Abs to FcγR. Then the cells were incubated with a saturating amount of biotinylated isotype-matched control mAbs (Ha4/8, A19-3, G235-2356, or R3-34), anti-CD28 (37.51), anti-CD152/CTLA-4 (UC10-4F10-11), anti-CD137/4-1BB (1AH2), anti-CD134/OX40 (OX86), anti-CD27 (LG.3A10), anti-CD30 (mCD30.1), and anti-CD154/CD40L (MR1) mAb before incubation with FITC-conjugated anti-NK1.1 (PK136) mAb, Cy-Chrome-conjugated anti-CD3ε mAb (145-2C111), and PE-conjugated streptavidin. All staining reagents were obtained from PharMingen. After washing with PBS, the stained cells were analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA).

In vitro stimulation with α-GalCer

Splenic mononuclear cells (MNC, 5 × 105) were cultured with 100 ng/ml α-GalCer or vehicle (0.1% DMSO) as a control in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM l-glutamine, and 25 mM NaHCO3 in humidified 5% CO2 at 37°C in 96-well U-bottom plates (Costar, Cambridge, MA). In the blocking experiments, anti-CD80 (RM80), anti-CD86 (PO.3), anti-CD154 (MR1), anti-IL-12 (C17.8), and isotype-matched control mAbs were added at 10 μg/ml each in the culture. After incubation for 72 h, the cell-free culture supernatants were harvested to detect cytokine levels by ELISA.

ELISA

IFN-γ and IL-4 levels in the culture supernatants and serum were evaluated using specific ELISA kits (Endogen, Boston, MA) according to the manufacturer’s instructions. For serum IgG1- or IgG2a-specific ELISA, microtiter plates (Immulon 2HB, 96-well; Dynex Technologies, Chantilly, VA) were coated with monoclonal anti-IgG1 or anti-IgG2a (PharMingen) at 10 μg/ml in PBS overnight at 4°C. The plates were blocked with PBS containing 1% BSA for 1 h and washed extensively with 0.05% Tween 20 in PBS. Serial dilutions of serum samples were incubated for 2 h at 37°C. The plates were then washed with 0.05% Tween 20 in PBS and overlaid with biotin-conjugated isotype-specific mAbs, including anti-mouse IgG1 (Serotec, Oxford, U.K.) and IgG2a (PharMingen), washed, and then developed with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and o-phenylendiamine (Wako Pure Chemical, Osaka, Japan). After termination of the reaction with 2 N H2SO4, OD at 490/595 nm was measured on a microplate reader (Bio-Rad, Hercules, CA). Concentrations were calculated on the basis of standard curves of Ab isotypes (PharMingen) run in parallel ELISA. Total serum IgE was quantitated by IgE-specific sandwich ELISA as previously described (25).

Cytotoxic assay

Cytolytic activity was assessed against NK-susceptible YAC-1 target cells and NK- and Fas ligand-resistant B16-BL6 cells by a standard 51Cr release assay as previously described (26, 27). Both target cells were cultured in RPMI 1640 medium containing 10% FCS, 2 mM l-glutamine, and 25 mM NaHCO3. As effector cells, hepatic and splenic MNC were isolated from the mice 24 h after i.p. injection of 2 μg/200 μl of α-GalCer or 200 μl of the vehicle (0.5% polysorbate 20). Target cells (106) were labeled with 100 μCi/ml Na251CrO4 for 60 min at 37°C in RPMI 1640 medium containing 10% FCS. Labeled target cells (104/well) were incubated in a total volume of 200 μl with effector cells in 10% FCS-RPMI 1640 in 96-well U-bottom plates. The plates were centrifuged before incubation, and after 4 h the supernatant was harvested and counted in a gamma counter. Specific lysis was calculated as previously described (26, 27).

Experimental lung metastasis

Log-phase cell cultures of B16-BL6 were harvested with 1 mM EDTA in PBS, washed three times with serum-free RPMI 1640, and resuspended to appropriate concentrations in PBS. B16-BL6 cells (5 × 104/100 μl) were injected i.v. into syngeneic B6 mice, and then α-GalCer (2 μg/200 μl) or 200 μl of vehicle was i.p. administered on days 0, 4, and 8. On day 14, the number of tumor colonies in the lung was counted under a dissecting microscope.

Statistical analysis

Data were analyzed using a two-tailed Student t test. All p values < 0.05 were considered as significant.

Results

Constitutive expression of CD28 on NKT cells

To examine the expression of costimulatory receptors on NKT cells, freshly isolated hepatic MNC from B6 mice were subjected to three-color staining with biotin-conjugated mAb against CD28, CD152 (CTLA-4), CD27, CD30, CD134 (OX40), or CD137 (4-1BB), followed by FITC-conjugated anti-NK1.1 mAb, Cy-Chrome-conjugated anti-CD3 mAb, and PE-conjugated streptavidin. Then the expression of each receptor was analyzed on electronically gated NK1.1+ CD3+ (NKT), NK1.1− CD3+ (T), or NK1.1+ CD3− (NK) cells as represented in Fig. 1⇓A. The vast majority of NKT cells exhibited high levels of CD28 expression, which was equivalent to that on conventional T cells (Fig. 1⇓B). None of the other molecules were expressed by the cell types that were examined. CD152 was not detected in NKT cells even by the intracellular staining. Although most NK cells and T cells expressed CD27, NKT cells did not express CD27. Similar results were obtained for splenic NKT cells or hepatic and splenic NKT cells isolated at 3 h after α-GalCer injection (data not shown). These results indicated a unique expression profile of costimulatory receptors on NKT cells, which was apparently distinct from conventional T cells and NK cells.

FIGURE 1.
FIGURE 1.

Constitutive expression of CD28 but not other costimulatory receptors on NKT cells. Freshly isolated hepatic MNC were stained with biotinylated mAbs against the indicated molecules or with control IgG, followed by PE-conjugated streptavidin, FITC-conjugated anti-NK1.1 mAb, and Cy-Chrome-conjugated anti-CD3 mAb. Expression of the respective molecules was analyzed by flow cytometry on electronically gated CD3+ NK1.1+ NKT, CD3+ NK1.1− T, or CD3− NK1.1+ NK cells (boxed in A). B, Solid lines indicate the staining with respective mAb, and dotted lines indicate the staining with isotype-matched control IgG.

Involvement of CD28-CD80/CD86 and CD154-CD40 costimulatory pathways in IFN-γ and IL-4 production by α-GalCer-stimulated NKT cells

We next investigated whether Vα14 NKT cells require CD28-mediated costimulation for IFN-γ and IL-4 production in response to their specific ligand, α-GalCer. As shown in Fig. 2⇓A, high levels of IFN-γ and IL-4 were detected in the supernatant of splenic MNC when cultured with α-GalCer for 72 h. These cytokines were not detected when splenic MNC from CD1-deficient mice were cultured with α-GalCer, indicating the dependence on CD1-restricted NKT cells as previously reported (4, 12) (Fig. 2⇓A). The blockade of CD28-mediated costimulation by anti-CD80 and anti-CD86 mAbs resulted in a marked but partial inhibition of IFN-γ production and an almost complete inhibition (>95% in three experiments) of IL-4 production (Fig. 2⇓A). Since it has been reported that IL-12 produced by DC plays a critical role in the α-GalCer-induced IFN-γ production (15) and that CD40-CD154 interactions are required for the α-GalCer-induced IL-12 production (28), we also investigated the effect of blocking mAbs against CD154 and IL-12. Rapid induction of CD154 on α-GalCer-activated NKT cells was confirmed in vitro (Fig. 2⇓B) as previously reported (28). Anti-CD154 mAb alone or anti-IL-12 mAb alone inhibited IFN-γ production and conversely increased IL-4 production by 2- to 2.5-fold (Fig. 2⇓A). The combination of these two mAbs did not result in further inhibition of IFN-γ production, suggesting that the contribution of CD154 to IFN-γ production was mediated by IL-12 as previously reported (15, 28). When combined with anti-CD80/CD86 mAbs, anti-IL-12 further inhibited IFN-γ production, whereas anti-CD154 mAb did not. This suggested that the CD80/CD86-independent IFN-γ production, at least in part, was IL-12 dependent but CD154 independent. Notably, anti-CD80/CD86 mAbs completely inhibited IL-4 production even in the presence of anti-IL-12 or anti-CD154 mAb. Taken together, these results indicated that the α-GalCer-induced IFN-γ production by Vα14 NKT cells was mostly dependent on both CD28-CD80/CD86 and CD154/CD40 costimulatory pathways, whereas IL-4 production was absolutely dependent on the CD28-CD80/CD86 pathway but instead suppressed by the CD154/CD40 pathway.

FIGURE 2.
FIGURE 2.

A, Distinct effects of anti-CD80/CD86 and anti-CD154 mAbs on IFN-γ and IL-4 production by α-GalCer-stimulated splenic MNC. Freshly isolated splenic MNC (5 × 105 cells) from naive B6 mice (wild-type) or CD1-deficient mice (CD1 −/−) were cultured with α-GalCer (100 ng/ml) or vehicle (0.1% DMSO) in 96-well U-bottom plates. Anti-CD80 and anti-CD86 mAbs, anti-CD154 mAb, anti-IL-12 mAb, and/or control IgG were added at 10 μg/ml each into the culture. After 72 h, culture supernatants were harvested and the levels of IFN-γ and IL-4 were determined by ELISA. Data are represented as mean ± SD of triplicate wells. Similar results were obtained in three independent experiments. ∗, p < 0.01. B, Rapid induction of CD154 on α-GalCer-stimulated NKT cells. Expression of CD154 was analyzed by flow cytometry on electronically gated CD3+ NK1.1+ NKT cells in splenic MNC at 3 h after stimulation with α-GalCer (100 ng/ml) or vehicle (0.1% DMSO). Solid lines indicate the staining with anti-CD154 mAb, and dotted lines indicate the staining with isotype-matched control IgG.

α-GalCer-induced IFN-γ production is impaired in both CD28- and CD40-deficient mice, but IL-4 production is impaired in CD28-deficient mice only

To confirm the differential contribution of CD28- and CD40-mediated costimulatory pathways to IFN-γ and IL-4 production by Vα14 NKT cells, we next investigated the α-GalCer-induced IFN-γ and IL-4 production by using splenic MNC from CD28- or CD40-deficient mice. As represented in Fig. 3⇓, splenic MNC from CD28-deficient mice showed greatly impaired production of both IFN-γ and IL-4 as compared with those from wild-type mice. Splenic MNC from CD40-deficient mice showed similarly impaired IFN-γ production but intact IL-4 production, which was comparable to wild-type mice (Fig. 3⇓). We also examined IFN-γ and IL-4 production in vivo by administrating α-GalCer into CD28- or CD40-deficient mice (Fig. 4⇓). In preliminary experiments, we found that serum IFN-γ levels peaked at 16 h and serum IL-4 peaked at 3 h after i.p. injection of α-GalCer to wild-type mice (data not shown). The serum IFN-γ elevation at 16 h after α-GalCer administration was significantly impaired in both CD28- or CD40-deficient mice (Fig. 4⇓). The serum IL-4 elevation at 3 h after α-GalCer administration was abrogated in CD28-deficient mice but rather enhanced in CD40-deficient mice (Fig. 4⇓). No apparent shift in the kinetics of serum IFN-γ and IL-4 levels was observed in these mutant mice (data not shown). These results indicated that CD28-mediated costimulation was required for both IFN-γ and IL-4 production by Vα14 NKT cells, whereas CD40-mediated costimulation was only required for IFN-γ production both in vitro and in vivo.

FIGURE 3.
FIGURE 3.

α-GalCer-induced IFN-γ and IL-4 production by splenic MNC from CD28−/− or CD40−/− mice. Splenic MNC (5 × 105 cells) freshly isolated from wild-type, CD28−/−, or CD40−/− mice were stimulated with α-GalCer (100 ng/ml) in 96-well U-bottom plates. After 72 h, culture supernatants were harvested and the levels of IFN-γ and IL-4 were determined by ELISA. Data are represented as the mean ± SD of triplicate wells. Similar results were obtained in three independent experiments. ∗, p < 0.01.

FIGURE 4.
FIGURE 4.

Serum IFN-γ and IL-4 levels in CD28−/− or CD40−/− mice after α-GalCer treatment. Serum samples were obtained from wild-type, CD28−/−, or CD40−/− mice at 3 and 16 h after i.p. injection of vehicle (200 μl) or α-GalCer (2 μg/200 μl). IFN-γ and IL-4 levels in the serum were determined by ELISA. Data are represented as the mean ± SD of five mice in each group. Serum IFN-γ and IL-4 in the vehicle-injected mice were not detectable (data not shown). Similar results were obtained in two independent experiments. ∗, p < 0.01.

Impairment of α-GalCer-induced cytolytic activity and antimetastatic effect in CD28- and CD40-deficient mice

We next investigated the effect of CD28 or CD40 deficiency on α-GalCer-induced cytotoxic activity of splenic and hepatic MNC, which represents the Th1-like function of NKT cells, since we have found that α-GalCer-induced cytotoxicity and antimetastatic activity were dependent on the IFN-γ produced by α-GalCer-activated Vα14 NKT cells and IFN-γ-activated NK cells.4 As reported (8), i.p. injection of α-GalCer into wild-type mice induced substantial cytotoxic activities of splenic and hepatic MNC against both NK-susceptible YAC-1 and NK-resistant B16-BL6 target cells (Fig. 5⇓). These cytotoxic activities were diminished by NK cell depletion by anti-asialo GM1 Ab administration4 (data not shown). In contrast, such an α-GalCer-induced cytotoxic activity was not observed in splenic or hepatic MNC from CD28- or CD40-deficient mice (Fig. 5⇓). Then we examined the antimetastatic effect of α-GalCer in an experimental lung metastasis model of the B16-BL6 melanoma, which is mediated by Vα14 NKT cells (8). As previously reported (8), α-GalCer administration greatly reduced the lung metastasis of B16-BL6 melanoma cells in wild-type mice (Fig. 6⇓). In contrast, no significant antimetastatic effect of α-GalCer was observed in CD28-, CD40- (Fig. 6⇓), or IFN-γ-deficient mice (data not shown).4 These results indicated that both CD28- and CD40-mediated costimulatory pathways were required for the Th1-like functions of Vα14 NKT cells in vivo.

FIGURE 5.
FIGURE 5.

Impairment of α-GalCer-induced cytotoxicity in CD28−/− or CD40−/− mice. Wild-type, CD28−/−, or CD40−/− mice were i.p. injected with vehicle (200 μl) or α-GalCer (2 μg/200 μl). Hepatic and splenic MNC were prepared 24 h later, and the cytotoxicity against YAC-1 and B16-BL6 was tested by 51Cr release assay at the indicated E:T ratios. Data are represented as the mean ± SD of triplicate wells. Similar results were obtained in three independent experiments.

FIGURE 6.
FIGURE 6.

Impairment of α-GalCer-induced antimetastatic effect in CD28−/− or CD40−/− mice. B16-BL6 (5 × 104/100 μl) were i.v. injected into wild-type, CD28−/−, or CD40−/− mice. α-GalCer (2 μg/200 μl) or vehicle (200 μl) was i.p. injected three times on days 0, 4, and 8. On day 14, the number of tumor colonies in the lung was counted under a dissecting microscope. Data are represented as the mean ± SD of five mice in each group. Similar results were obtained in two independent experiments. ∗, p < 0.01.

Involvement of CD28-CD80/CD86 and CD154-CD40 costimulatory pathways in α-GalCer-induced Th2-like functions of NKT cells in vivo

It has been also reported that α-GalCer administration biases the subsequent immune responses toward Th2 type, as represented by enhanced IgE and IgG1 production, which is mediated by IL-4 secreted from Vα14 NKT cells (9, 10). We therefore investigated the contribution of CD28 and CD40 costimulatory pathways to the development of α-GalCer-induced Th2-type immune responses in vivo. As shown in Fig. 7⇓A, administration of α-GalCer significantly increased the serum IgE and IgG1 levels and conversely reduced the IgG2a level in wild-type mice as previously reported (10). In contrast, no significant elevation of serum IgE and IgG1 levels or reduction of the IgG2a level was observed in CD28-deficient mice, indicating that the Th2-like function of α-GalCer-stimulated Vα14 NKT cells required CD28-mediated costimulation. In CD40-deficient mice, administration of α-GalCer induced marginal but significant elevation of serum IgE and IgG1 levels, suggesting that CD40 was not essential for this response. The impaired elevation of serum IgE and IgG1 levels in CD40-deficient mice as compared with wild-type mice appeared to result from the defect in Ig class switching of CD40-deficient B cells in a later stage of the response. To minimize the effect of CD40 deficiency on the later stage, we administered anti-CD154 mAb only once before the administration of α-GalCer into wild-type mice. As shown in Fig. 7⇓B, anti-CD154 mAb significantly enhanced the elevation of serum IgE and IgG1 levels after α-GalCer administration. This suggested that the CD154-CD40 interaction played a suppressive role in the α-GalCer-induced and Vα14 NKT cell-mediated Th2-type response in vivo.

FIGURE 7.
FIGURE 7.

Involvement of CD28-CD80/CD86 and CD40-CD154 in α-GalCer-induced serum Ig responses. A, Serum Ig isotype levels in α-GalCer-treated wild-type, CD28−/−, or CD40−/− mice. Wild-type, CD28−/−, or CD40−/− mice were i.p. injected with vehicle (200 μl) or α-GalCer (2 μg/200 μl). B, Effect of anti-CD154 mAb on α-GalCer-induced serum Ig isotype levels. Wild-type mice were i.p. injected with 300 μg of anti-CD154 mAb or control IgG 3 h before the i.p. injection of vehicle (200 μl) or α-GalCer (2 μg/200 μl). In both experiments, mice were bled on day 7, and total IgE, IgG1, and IgG2a levels in the serum were measured by ELISA. Data are represented as mean ± SD of five mice in each group. Similar results were obtained in two independent experiments. ∗, p < 0.01.

Discussion

In this study, we demonstrated that murine NKT cells constitutively express CD28 and that CD28-mediated costimulation is required for production of both IFN-γ and IL-4 by Vα14 NKT cells in response to their specific ligand α-GalCer. Consequently, blockade of the CD28-mediated costimulation resulted in impairment of both Th1- and Th2-type responses (serum IFN-γ and IL-4 elevation, cytotoxicity induction, antimetastatic effect, and serum IgE/IgG1 elevation) induced by α-GalCer administration in vivo. In contrast, blockade of the CD40-CD154 interaction inhibited only the α-GalCer-induced Th1-type responses (serum IFN-γ elevation, cytotoxicity induction, and antimetastatic effect) but rather enhanced the Th2-type responses (serum IL-4 elevation and serum IgE/IgG1 elevation). These results indicate that Th1- and Th2-like functions of Vα14 NKT cells are differentially regulated by CD28- and CD40-mediated costimulatory pathways.

It has been well established that conventional T cells require a costimulatory signal, in addition to Ag-specific TCR-mediated signal, for their full activation (29). Such a costimulatory signal can be commonly transmitted by CD28 or some members of the TNF receptor superfamily, including CD27, CD30, CD134, and CD137, in conventional T cells (29, 30, 31, 32, 33, 34). It was also reported that CD161 transmitted a costimulatory signal into Vα24 NKT cells (35). We here showed that NKT cells also express CD28, which played a critical role in full activation of Vα14 NKT cell functions as manifested by IFN-γ and IL-4 production. It has been also reported that engagement of CD28 on NK cells promoted their proliferation, IFN-γ production, and cytolytic activity (36, 37). Therefore, CD28 appears to play important roles not only in adaptive immunity mediated by conventional T cells but also in innate immunity mediated by NK and NKT cells. It was notable that NKT cells did not express CD27, which has been implicated in activation of T cells and NK cells (38, 39) and T cell differentiation (40). This may be due to the unique ontogeny of NKT cells, which is distinct from conventional T cells (1, 2, 41).

It has been shown that IFN-γ production by Vα14 NKT cells in response to α-GalCer is predominantly mediated by IL-12 produced by DC and requires CD154-CD40 interaction (15). Our present observations that anti-IL-12 mAb or anti-CD154 mAb alone strongly inhibited the α-GalCer-induced IFN-γ production and that these mAbs did not exhibit an additive inhibitory effect (Fig. 2⇑A) are consistent with this notion. However, we also observed that the combination of anti-CD80/CD86 mAbs with anti-IL-12 mAb additively inhibited the IFN-γ production. This suggested that CD28-CD80/CD86 interactions regulate IFN-γ production by NKT cells in an IL-12-independent manner at least partly. This CD28-mediated IFN-γ production might be directly induced by transcriptional regulation of the IFN-γ gene by CD28-mediated signals as demonstrated in conventional T cells (42). Moreover, the CD28-mediated pathway and the CD40/IL-12-mediated pathway could interact mutually, since CD28-mediated costimulation stabilizes expression of CD154 on T cells (43, 44), and CD40-mediated activation up-regulates CD80/CD86 expression on DC (45). This explains why the IFN-γ production by α-GalCer-stimulated Vα14 NKT cells was mostly dependent on both CD28- and CD40-mediated costimulatory pathways.

In the present study, we observed that the induction of cytotoxic activity in liver or splenic MNC and the antimetastatic effect of α-GalCer were abolished in both CD28- and CD40-deficient mice (Figs. 5⇑ and 6⇑). This paralleled with the impairment of IFN-γ production in these mice (Fig. 4⇑). Both IL-12 and IL-4 have been implicated in cytolytic activation of Vα14 NKT cells (26, 27, 46). In our present observation, however, both the cytolytic activation and antimetastatic effect were abolished in CD40-deficient mice, which exhibited rather increased serum IL-4 levels upon α-GalCer administration. These results suggested that the α-GalCer-induced cytolytic activity and antimetastatic effect were associated with the production of IFN-γ, but not IL-4, by Vα14 NKT cells. Consistent with this notion, these responses were largely abolished in IFN-γ-deficient mice.4 On the other hand, we observed that the α-GalCer-induced serum IgE and IgG1 elevation was abolished in CD28-deficient mice but not in CD40-deficient mice (Fig. 7⇑A), which paralleled with the serum IL-4 elevation in these mice (Fig. 4⇑). Treatment with anti-CD154 mAb at α-GalCer administration rather augmented this response (Fig. 7⇑B). These results indicated that Th2-like function of Vα14 NKT cells was totally dependent on the CD28-mediated costimulation and rather suppressed by the CD40-mediated pathway, possibly due to a suppressive effect of IFN-γ on IgE/IgG1 production (47).

As represented in the present study, Vα14 NKT cells have been shown to produce both Th1-type (IFN-γ) and Th2-type (IL-4) cytokines upon stimulation with a specific ligand (α-GalCer) or anti-CD3 mAb (48, 49). Some recent studies have shown that α-GalCer treatment polarizes bystander immune responses toward a Th2 phenotype possibly through IL-4 production by Vα14 NKT cells (9, 10), whereas another study reported polarization toward a Th1 phenotype through IFN-γ production by Vα14 NKT cells (11). This discrepancy in the effects of α-GalCer might result from differences in dose, timing, and route of α-GalCer administration. Our present study suggests that such a Th1- or Th2-polarizing function of Vα14 NKT cells can be selectively modulated by the blockade of costimulatory pathways, as represented by polarization toward a Th2 phenotype by the blockade of CD154-CD40 interaction, at α-GalCer administration. Since NKT cells have been implicated in innate immunity against pathogens (50), antitumor responses (8, 51, 52), liver damage (53), and autoimmune diseases (54, 55, 56, 57), selective modulation of their functions with specific ligand and costimulatory blockade may be useful for prophylaxis and therapy of such diseases.

Acknowledgments

We thank Dr. Yasuhiko Koezuka and Kazuhiro Motoki (Pharmaceutical Research Laboratory, Kirin Brewery) for generously providing α-GalCer and Dr. Hisaya Akiba for technical assistance and helpful suggestions.

Footnotes

  • ↵1 This work was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.

  • ↵2 Address correspondence and reprint requests to Dr. Kazuyoshi Takeda, Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail address: ktakeda{at}med.juntendo.ac.jp

  • ↵3 Abbreviations used in this paper: α-GalCer, α-galactosylceramide; MNC, mononuclear cells; DC, dendritic cells.

  • 4 Y. Hayakawa, K. Takeda, H. Yagita, S. Kakuta, Y. Iwakura, L. V. Kaer, I. Saiki, and K. Okumura. Critical contribution of IFN-γ and NK cells, but not perforin-mediated cytotoxicity, to the antimetastatic activities of α-galactosylceramide. Submitted for publication.

  • Received December 4, 2000.
  • Accepted March 7, 2001.

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