Cytokine-Dependent Modification of IL-12p70 and IL-23 Balance in Dendritic Cells by Ligand Activation of Vα24 Invariant NKT Cells

Yasushi Uemura, Tian-Yi Liu, Yayoi Narita, Motoharu Suzuki, Ryusuke Nakatsuka, Tomoyuki Araki, Masahito Matsumoto, Leo Kei Iwai, Narumi Hirosawa, Yoshikazu Matsuoka, Mari Murakami, Takashi Kimura, Makoto Hase, Hirao Kohno, Yutaka Sasaki, Yasuko Ichihara, Osamu Ishihara, Hirosato Kikuchi, Yasushi Sakamoto, Shun-Chang Jiao, Satoru Senju and Yoshiaki Sonoda
J Immunol July 1, 2009, 183 (1) 201-208; DOI: https://doi.org/10.4049/jimmunol.0900873

Abstract

CD1d-restricted invariant NKT (iNKT) cells play crucial roles in various types of immune responses, including autoimmune diseases, infectious diseases and tumor surveillance. The mechanisms underlying their adjuvant functions are well understood. Nevertheless, although IL-4 and IL-10 production characterize iNKT cells able to prevent or ameliorate some autoimmune diseases and inflammatory conditions, the precise mechanisms by which iNKT cells exert immune regulatory function remain elusive. This study demonstrates that the activation of human iNKT cells by their specific ligand α-galactosylceramide enhances IL-12p70 while inhibiting the IL-23 production by monocyte-derived dendritic cells, and in turn down-regulating the IL-17 production by memory CD4+ Th cells. The ability of the iNKT cells to regulate the differential production of IL-12p70/IL-23 is mainly mediated by a remarkable hallmark of their function to produce both Th1 and Th2 cytokines. In particular, the down-regulation of IL-23 is markedly associated with a production of IL-4 and IL-10 from iNKT cells. Moreover, Th2 cytokines, such as IL-4 and IL-13 play a crucial role in defining the biased production of IL-12p70/IL-23 by enhancement of IL-12p70 in synergy with IFN-γ, whereas inhibition of the IFN-γ-promoted IL-23 production. Collectively, the results suggest that iNKT cells modify the IL-12p70/IL-23 balance to enhance the IL-12p70-induced cell-mediated immunity and suppress the IL-23-dependent inflammatory pathologies. These results may account for the long-appreciated contrasting beneficial and adverse consequence of ligand activation of iNKT cells.

Invariant NKT (iNKT)4 cells are a unique subset of T lymphocytes characterized by the expression of a semi-invariant TCR composed of a canonical invariant TCR α-chain (Vα14-Jα18 for mice and Vα24-Jα18 for humans) and a TCR β-chain using limited Vβ segments (Vβ8.2, Vβ7, and Vβ2 in mice and a Vβ11 in humans). The biased TCR usage appears to confer on iNKT cells the capacity to recognize a limited number of glycosphingolipids presented by CD1d on APCs (1).

α-Galactosylceramide (α-GalCer), a synthetic glycosphingolipid originally derived from a marine sponge, potently activates iNKT cells to rapidly produce large amounts of both Th1 and Th2 cytokines (2). The activation of iNKT cells by α-GalCer leads to downstream activation of other cell types in the immune system, such as CTL, NK cells, and dendritic cells (DCs) (3, 4, 5). Bystander activation of these cells is crucial to the protective antitumor and microbial immunity (6, 7, 8). In contrast, iNKT cells are also involved in the prevention of some autoimmune diseases (9, 10). These findings indicate that iNKT cells can exert both immune stimulatory and immune regulatory functions (11, 12, 13).

It is widely accepted that organ-specific autoimmune diseases are the result of dysregulated autoantigen-specific Th1 responses (14). Therefore, the beneficial effect of iNKT cells in autoimmunity is thought to be due to its inhibition of Th1 response. In particular, the release of IL-4 or IL-10 from iNKT cells is implicated in the amelioration of some autoimmune diseases and inflammatory conditions (9, 10, 15, 16). However, the precise mechanisms underlying the immune regulatory functions are not well understood. In addition, the simultaneous production of Th1 cytokines by α-GalCer-activated iNKT cells has made it difficult to explain the contradictory roles of iNKT cells (11, 12, 13).

The observation that mice deficient in IFN-γ have increased susceptibility rather than resistance to autoimmunity led to the hypothesis that iNKT cells play a role in regulating a T cell subset other than Th1 cells (17). Recently, IL-23-dependent pathogenic Th17 cells have shown to be implicated in a number of inflammatory processes and autoimmune diseases (18, 19, 20, 21, 22, 23). Although iNKT cells have been shown to play crucial roles in regulating autoimmunity, it remains unclear whether iNKT cells can regulate IL-23 and the downstream Th17 responses.

IL-12p70 and IL-23 are members of a small family of heterodimeric cytokines predominantly produced by DCs and macrophages (24). Both cytokines share a common p40 subunit that is covalently linked either to p35 subunit to form IL-12p70 or to a p19 subunit to form IL-23 (25). Whereas the IL-12p70 is involved in the induction and amplification of the Th1 response, IL-23 is critical for the survival and expansion of Th17 cells (26). Studies using IL-23p19- or IL-12p35-deficient models of autoimmunity (19, 20, 27), tumors (28), and inflammatory bowel disease (29) have identified IL-23 rather than IL-12, as the major factor responsible for lesions caused by chronic inflammation, acting through the induction of a Th17 response.

Because of its important role in shaping of the immune response, the cross-talk between iNKT cells and DCs has been a field of extensive study in recent years (30, 31, 32, 33, 34). The activation of iNKT cells with α-GalCer induces a rapid differentiation and maturation of DCs to produce IL-12p70, followed by enhancement of Th1 immune responses (33, 35, 36). In contrast, some studies have demonstrated a contribution of regulatory DCs acting downstream of iNKT cell activation (31, 32). Considering the ability of iNKT cells to either enhance or suppress the immune responses, it is conceivable that the activation of iNKT cells regulates the commitment toward an IL-12p70-induced Th1 response or an IL-23-facilitated Th17 response through an altered DC response. Therefore, evaluating the IL-23 production by DCs in conjunction with IL-12p70 may be important to elucidate the exact nature of their ability to regulate the immune response.

The present study investigated the role of human iNKT cells in the regulation of IL-12p70 and IL-23 production by DCs. The study further ascertained whether iNKT cells could regulate the IL-17 production by CD4+ Th cells via DCs.

Materials and Methods

Abs and reagents

α-GalCer (KRN7000) was purchased from Funakoshi. Recombinant human (rh) IL-4 and rhGM-CSF were from Primmune. The rhIL-2, rhIL-10, rhIL-13, rhIL-23, and rhIFN-γ were from R&D Systems. Anti-invariant NKT cell (6B11, a mAb specific for the invariant Vα24JαQ CDR3 loop), anti-CD3 (HIT3a), anti-CD4 (RPA-T4), anti-CD28 (CD28.2), anti-CD80 (L307.4), anti-CD83 (HB15e), anti-CD86 (FUN-1) mAb, isotype control, and 7-aminoactinomycin D were from BD Pharmingen. Anti-Vα24 (C15) and anti-CD8β (2ST8.5H7) mAbs were from Beckman Coulter. Anti-IL-4, anti-IL-10, anti-IL-12 (24910), anti-IL-13, anti-IL-23, and anti-IFN-γ Abs for neutralizing, anti-CD40L mAb for blocking, and control IgG were from R&D Systems. Anti-CD45RA, anti-CD45RO and anti-CD14 microbeads were from Miltenyi Biotec. Anti-CD1d (55.3.1) mAb was provided by Dr. S. A. Porcelli (Albert Einstein College of Medicine, Bronx, NY).

Human Vα24 iNKT cells

The derivation of human Vα24 iNKT (iNKT) cells has been previously described (33). Briefly, PBMCs from the peripheral blood of healthy volunteers were cultured in RPMI 1640 medium (Sigma-Aldrich) containing 5% heat-inactivated human serum and α-GalCer (10 ng/ml). At day 15, Vα24+6B11+ cells were sorted with FACSAria automated cell sorter (BD Biosciences). The sorted cells were restimulated in the presence of rhIL-2 (10 U/ml) with irradiated (4500 cGy) autologous PBMCs prepulsed for 5 h with α-GalCer (100 ng/ml). After expansion for 9 days, CD4+CD8β− and CD4−CD8β− subsets, respectively, were sorted to >99% purity.

Generation of human monocyte-derived DCs and NKT-DC cultures

The induction of human monocyte-derived-DCs was done as described (37). Briefly, CD14+ monocytes were isolated from PBMCs by positive magnetic sorting using CD14 microbeads and were cultured at 1.0 × 106 cells/ml in the presence of rhGM-CSF and rhIL-4 (50 ng/ml each). On day 6, nonadherent DCs were harvested and served as immature DCs. Further differentiation into mature DCs was induced by treatment with live Escherichia coli (100 bacteria per DC) or attenuated Mycobacterium tuberculosis strain H37Ra (Difco) at the concentration of 100 μg/ml. iNKT cells were cultured with DCs at a NKT cell to DC ratio of 1:10 in the presence or absence of α-GalCer.

Isolation and activation of naive and memory CD4+ Th cells

CD4+ Th cells were isolated from PBMCs with a CD4+ T cell isolation kit II (Miltenyi Biotec). CD4+CD45RO− naive Th cells were further isolated by negative selection from the CD4 Th cell fraction with CD45RO microbeads. CD4+CD45RA− memory Th cells were isolated with CD45RA microbeads. The naive or the memory Th cells (1.0 × 105) were stimulated with autologous DCs (3.0 × 104) in the presence of 100 pg/ml staphylococcal enterotoxin B (SEB; Sigma-Aldrich).

Flow cytometry

Flow cytometry was performed with a FACSCalibur flow cytometer (BD Biosciences) and the data were processed using the CellQuest software program (BD Biosciences).

Measurement of cytokines

The levels of cytokines in the culture supernatants were evaluated with ELISA (human IL-12/23p40, IL-12p70, and IL-17; R&D Systems and human IL-23; eBioscience).

Real-time quantitative RT-PCR

Total RNA was extracted with the RNeasy Mini kit (Qiagen). The Omniscript RT kit (Qiagen) was used for cDNA synthesis. Genomic DNA was digested and removed using an RNase-Free DNase kit (Qiagen). Transcripts were quantified by real-time quantitative PCR on an ABI PRISM 7500 sequence detector (PerkinElmer Applied Biosystems) with TaqMan Gene expression Assays (Applied Biosystems) and reagents according to the instructions. The following probes were used, as identified by Applied Biosystems assay identification number: IL-12/23p40; Hs01011519_m1, IL-12p35; Hs01073449_g1, and IL-23p19; Hs00900829_g1. For relative quantification, the gene expression in each sample was normalized by comparison with the GAPDH mRNA expression using the ddCT method as described (38).

Statistics

Statistical analyses were performed using Student’s t test. The values were considered to be statistically significant at a value of p < 0.05.

Results

Human DN and CD4+ Vα24 iNKT cells

To search for the role of human iNKT cells in the regulation of IL-12p70 and IL-23 production by DCs, the CD4−CD8β− (DN) iNKT cells and the CD4+CD8β− (CD4+) iNKT cells of healthy individuals were separated from peripheral blood. They expressed TCR Vα24 and Vβ11, specifically recognized α-GalCer in a CD1d-restricted manner (33). The CD4+ and DN iNKT cells produced both Th1 and Th2 cytokines on stimulation, although the DN iNKT cells revealed a relative Th1 bias (33, 39, 40). In contrast to the mouse NKT cells, which have been recently reported to produce IL-17 and IL-21 (41, 42), the human iNKT cells stimulated with anti-CD3/anti-CD28 mAbs did not produce them (see supplemental Fig. S1 and data not shown).5

IL-12p70 and IL-23 production by bacteria-activated monocyte-derived DCs

It is conceivable that certain conditions in which DCs produce both IL-12p70 and IL-23 are feasible for identifying whether human iNKT cells may regulate the IL-12p70/IL-23 balance. Preliminary studies showed that immature monocyte-derived DCs cultured with iNKT cells in the presence of α-GalCer produced IL-12p70 but not IL-23 (data not shown). To determine the conditions permissive for the production of both IL-12p70 and IL-23 by monocyte-derived DCs, DCs were stimulated for 24 h with live E. coli, attenuated M. tuberculosis (strain H37Ra), or different classes of the TLR ligands (e.g., peptidoglycan, a TLR2 ligand; Pam3CSK4, a TLR1/2 ligand; LPS, a TLR4 ligand; and R848, a TLR8 ligand) and the production of IL-12p70 and IL-23 was evaluated. Stimulation with the live E. coli or the attenuated M. tuberculosis (strain H37Ra) induced both IL-12p70 and IL-23 production in DCs (see supplemental Fig. S2).5 However, the DCs stimulated with a TLR ligand that induced high levels of CD83 expression, were unable to produce these cytokines (see supplemental Fig. S3 and data not shown).5

Activation of iNKT cells by α-GalCer enhances phenotypic maturation of bacteria-activated DCs

Having identified the live E. coli and the attenuated M. tuberculosis (strain H37Ra) as the potent inducers of both IL-12p70 and IL-23 production in monocyte-derived DCs enabled the investigation of the regulation of these cytokines by iNKT cells. Monocyte-derived DCs were stimulated with H37Ra (H37Ra-DC) for 24 h in the presence of iNKT cells, together with or without α-GalCer (Fig. 1⇓). When iNKT cells were activated by α-GalCer, DC maturation induced by H37Ra was further enhanced compared with that given vehicle control as evidenced by the up-regulation of the CD80, CD83, and CD86. Similar results were also obtained when E. coli was used for DC stimulation (data not shown).

FIGURE 1.
FIGURE 1.

iNKT cells enhance phenotypic maturation of bacteria-activated DCs. a, Surface CD80, CD83, and CD86 expression on H37Ra-DCs cultured for 24 h in the presence of indicated DN iNKT cells or CD4+ iNKT cells with or without α-GalCer (100 ng/ml). b, H37Ra-DCs alone. c, Immature DCs (medium control). Filled histograms represent staining of DC surface molecules. Open histograms represent the isotype control. Viable DC gate was set on the basis of 7-aminoactinomycin D (7-AAD)- and Vα24-negative cells. The mean fluorescence intensity (MFI) of each histogram is shown for each panel (upper right corner). Results are representative of three separate experiments.

Activation of iNKT cells by α-GalCer is unique in its ability to enhance IL-12p70 while inhibiting IL-23 production by DCs

Next, the effect of iNKT cells on the cytokine production in DCs was examined. E. coli- or H37Ra-DCs were cultured with iNKT cells for 24 h in the presence of graded doses of α-GalCer and the production of IL-12/23p40, IL-12p70, and IL-23 was evaluated (Fig. 2⇓). The IL-12/23p40 production in E. coli- and H37Ra-DCs was slightly inhibited by the α-GalCer activation of iNKT cells (Fig. 2⇓, a and b). In contrast, the titration of α-GalCer showed dose-dependent increase of IL-12p70 (Fig. 2⇓, c and d) and decrease of IL-23 production (Fig. 2⇓, e and f) both in the E. coli- and H37Ra-DCs. An analysis using iNKT cells from other healthy donors showed a consistent pattern of IL-12p70 and IL-23 production by these DCs, irrespective of donor (see supplemental Fig. S4).5

FIGURE 2.
FIGURE 2.

iNKT cells markedly enhance IL-12p70, whereas they inhibit IL-23 production in bacteria-activated DCs. E. coli-DCs (left column) or H37Ra-DCs (right column) were cultured with the indicated iNKT cells for 24 h at increased concentrations of α-GalCer. IL-12/23p40 (a and b), IL-12p70 (c and d), and IL-23 (e and f) levels in the culture supernatants were evaluated with ELISA. Data represent mean ± SD of triplicate cultures. Results are representative of three separate experiments.

CD40 ligation by iNKT cells enhances IL-12p70 and IL-23 production in DCs

CD40L expressed by human iNKT cells stimulates immature monocyte-derived DCs to produce IL-12p70 through CD40 ligation and the IL-12p70 production was further enhanced by IFN-γ from iNKT cells (33). A blocking experiment was performed to investigate the influence of CD40L on IL-12p70 and IL-23 production in bacteria-activated DCs (Fig. 3⇓). Immature DCs (medium control), E. coli-DCs, or H37Ra-DCs were cultured with CD4+ iNKT cells together with α-GalCer for 24 h in the presence or absence of either anti-CD40L or anti-CD1d mAb. The IL-12p70 production was suppressed by the anti-CD40L mAb in all of the DCs tested (Fig. 3⇓, a–c). The IL-23 production was also suppressed by the anti-CD40L mAb in E. coli- or H37Ra-DCs (Fig. 3⇓, e and f). However, the influences of these mAbs on IL-23 production could not be detected in immature DCs (Fig. 3⇓d). Similar results were also obtained in the experiment in which DN iNKT cells were used (data not shown). These findings indicate that CD40 ligation by iNKT cells exert promoting effect on both the IL-12p70 and IL-23 production. Interestingly, the blocking mAb to CD1d suppressed the IL-12p70 production, whereas promoted the IL-23 production in both E. coli- and H37Ra-DCs, thus indicating that other factor(s) expressed by activated iNKT cells might play a crucial role in the enhancement of IL-12p70 and the suppression of IL-23.

FIGURE 3.
FIGURE 3.

CD40L on iNKT cells enhances bacteria-induced IL-12p70 and IL-23 production. Medium-DCs (a and d), E. coli-DCs (b and e), or H37Ra-DCs (c and f) were cultured with CD4+ iNKT cells for 24 h in the presence of α-GalCer (100 ng/ml), in the additional presence of a control IgG, an anti-CD1d, or an anti-CD40L mAb (10 μg/ml). IL-12p70 (a–c) and IL-23 (d–f) levels in the culture supernatants were evaluated with ELISA. Data represent mean ± SD of triplicate cultures. Results are representative of three separate experiments. *, p < 0.05 and **, p < 0.005.

iNKT-derived soluble factors regulate the biased cytokine production in DCs

Having established that iNKT cells produce large amount of cytokines with immune regulatory properties, the possible role of iNKT-derived soluble factors in the regulation of IL-12 and IL-23 was examined. DCs were stimulated with E. coli or H37Ra for 24 h in the presence or absence of a supernatant from CD3/28 prestimulated iNKT cells (33), and the production of IL-12/23p40, IL-12p70, and IL-23 was evaluated (Fig. 4⇓). The supernatants from iNKT cells inhibited the IL-12/IL-23p40 and IL-23 production (Fig. 4⇓, a, b, e, and f), whereas they enhanced the IL-12p70 production (Fig. 4⇓, c and d).

FIGURE 4.
FIGURE 4.

iNKT-derived soluble factor enhances IL-12p70, whereas it inhibits IL-23 production. E. coli-DCs (left column) or H37Ra-DCs (right) were cultured for 24 h in the presence of 25% cell-free supernatants that were from CD3/28 prestimulated iNKT cells. IL-12/23p40 (a and b), IL-12p70 (c and d), and IL-23 (e and f) levels in the culture supernatants were evaluated with ELISA. Medium control served as the reference for other culture conditions. Data represent mean ± SD of triplicate cultures. Results are representative of three separate experiments. *, p < 0.05 and **, p < 0.005.

IL-12p70 is a heterodimeric cytokine consisting of a p40 subunit paired with a p35 subunit. The p40 subunit can pair with a p19 subunit to form IL-23 (25). Therefore, the effect of the supernatants on the expression of the corresponding mRNA was analyzed (Fig. 5⇓). Consistent with the protein data, the supernatants from iNKT cells enhanced the expression of IL-12p35 mRNA (Fig. 5⇓b), and inhibited the expression of IL-12/23p40 and IL-23p19 mRNA (Fig. 5⇓, a and c). These findings suggest that iNKT cells regulate the IL-12p70/IL-23 production by enhancing the IL-12p35 and inhibiting the IL-23p19 and IL-12/23p40 expression, and the differential IL-12p35/IL-23p19 expression is mainly mediated by their soluble factors.

FIGURE 5.
FIGURE 5.

Expression of IL-12/23p40, IL-12p35, and IL-23p19 mRNA. H37Ra-DCs were cultured for 24 h in the presence or absence of 25% cell-free supernatants that were from CD3/28 prestimulated iNKT cells. IL-12/23p40 (a), IL-12p35 (b), and IL-23p19 (c) mRNA levels at indicated time points after stimulation were quantitatively measured by real-time RT-PCR. A medium control served as the reference for other culture conditions. The amount of mRNA is shown by arbitrary units relative to the amount of the GAPDH mRNA. Data represent mean ± SD of triplicate cultures. Results are representative of three separate experiments.

Reciprocal control of IL-12p70 and IL-23 production is mainly mediated by iNKT-derived IL-4

The contribution of the iNKT-derived Th cytokines was examined to define the mechanism regulating the IL-12p70 and IL-23 production. DCs were stimulated with H37Ra for 24 h in the presence of a supernatant from CD3/28 prestimulated iNKT cells together with a neutralizing Ab to IL-4, IL-10, IL-13, or IFN-γ, and the production of IL-12p70 and IL-23 was evaluated (Fig. 6⇓). The neutralization of IFN-γ exerted an inhibitory effect on both IL-12p70 and IL-23 production, whereas the neutralization of IL-10 exerted a promoting effect on them. Importantly, the neutralization of IL-4 exerted an inhibitory effect on IL-12p70 production while it exerted a promoting effect on IL-23 production. The neutralization of IL-13 exerted an inhibitory effect on IL-12p70 production (Fig. 6⇓, a and b). However, no significant differences were observed in IL-23 production (Fig. 6⇓, c and d). Similar results were also obtained when E. coli were used for DC stimulation (data not shown). These findings suggest that IL-4 derived from iNKT cells is a major regulator that defines the enhanced IL-12p70 and inhibited IL-23 production by DCs.

FIGURE 6.
FIGURE 6.

IL-4 derived from iNKT cells enhances IL-12p70, whereas it inhibits IL-23 production. H37Ra-DCs were cultured for 24 h in the presence of 25% cell-free supernatants that were from CD3/28 prestimulated DN iNKT cells (left) and CD4 iNKT cells (right), in the additional presence of a neutralizing Ab to IL-4, IL-10, IL-13, or IFN-γ (10 μg/ml). IL-12p70 (a and b) and IL-23 (c and d) levels in the culture supernatants were evaluated with ELISA. The IgG control served as the reference for other culture conditions. N.S., Not significant. Data represent mean ± SD of triplicate cultures. Results are representative of three separate experiments.

IL-4 and IL-13 enhance IL-12p35 in synergy with IFN-γ while they inhibit IFN-γ-promoted IL-23p19 expression

DCs were stimulated with H37Ra in the presence of rhIL-4, rhIL-10, rhIL-13, rhIFN-γ, or combinations of these cytokines, and the expression of IL-12/23p40, IL-12p35, and IL-23p19 mRNA was evaluated to confirm the direct effect of these cytokines (Fig. 7⇓). The rhIFN-γ consistently enhanced the IL-12/23p40, IL-12p35, and IL-23p19 expression, whereas the rhIL-10 consistently decreased them (Fig. 7⇓, a–c). In contrast, the rhIL-4 or the rhIL-13 exerted a promoting effect on IL-12p35 expression (Fig. 7⇓b), whereas they exerted an inhibitory effect on IL-12/23p40 and IL-23p19 expression (Fig. 7⇓, a and c). Although the IL-12p35 expression was further amplified by rhIL-4 or rhIL-13 in synergy with IFN-γ (Fig. 7⇓e), the IFN-γ-promoted IL-12/23p40 and IL-23p19 expression was inhibited by the additional presence of either rhIL-4 or rhIL-13 (Fig. 7⇓, d and f). Notably, the simultaneous addition of IFN-γ, IL-4, and IL-13 induced a 11-fold increase in IL-12p35 expression in comparison to the addition of IFN-γ alone (Fig. 7⇓e), whereas the IL-12/23p40 and IL-23p19 expression was still below the control levels of H37Ra alone (Fig. 7⇓, d and f).

FIGURE 7.
FIGURE 7.

IL-4 and IL-13 enhances IL-12p35 in synergy with IFN-γ, whereas it inhibits IFN-γ-promoted IL-23p19 expression. H37Ra-DCs were cultured for 24 h in the presence of rhIL-4, rhIL-10, rhIL-13, rhIFN-γ (10 ng/ml each), or a combination of these cytokines. IL-12/23p40 (a and d), IL-12p35 (b and e), and IL-23p19 (c and f) mRNA levels were quantitatively measured by real-time quantitative RT-PCR. A medium control served as the reference for the other culture conditions. The amount of mRNA is shown by arbitrary units relative to the amount of the GAPDH mRNA. Data represent mean ± SD of triplicate cultures. Results are representative of three separate experiments.

iNKT cells reduce the capacity to produce IL-17 in memory Th cells via a down-regulation of DC-derived IL-23

The production of IL-17 in human Th cells is promoted by IL-23 in memory T cells but not in naive T cells (43, 44). Therefore, having demonstrated that iNKT cells are a potent regulator of IL-23 reduction, it is conceivable that activation of iNKT cells may inhibit the IL-17 production in memory cells via altered DC responses. Indeed, the naive T cells stimulated with SEB in the presence of immature DCs (medium control) or H37Ra-DCs never produced IL-17, whereas the memory T cells produced IL-17 (Fig. 8⇓a). Consistent with the IL-23 producing capacity (see supplemental Fig. S2),5 the robust IL-17 production was induced following treatment with H37Ra-DCs (Fig. 8⇓a). The addition of a neutralizing anti-IL-12p70 Ab in T cell cocultures with H37Ra-DCs enhanced IL-17 production by memory T cells, whereas an anti-IL-23 Ab inhibited the production of IL-17 (Fig. 8⇓b). To determine the role of iNKT cells in the regulation of Th17 response, naive or memory T cells were stimulated with SEB in the presence of H37Ra-DCs, together with iNKT cells in the presence or absence of α-GalCer (Fig. 8⇓c). The IL-17 production was markedly suppressed in memory T cells by α-GalCer activation of iNKT cells. In the additional presence of exogenous rhIL-23, the IL-17 production was observed to recover (Fig. 8⇓d). These findings suggest that iNKT cells have a potential to suppress the Th17 response via a down-regulation of IL-23 production by DCs.

FIGURE 8.
FIGURE 8.

iNKT cells reduce the capacity to produce IL-17 in memory Th cells via a down-regulation of DC-derived IL-23. a, Autologous naive or memory Th cells were cultured with medium-DCs or H37Ra-DCs at a T cell to DC ratio of 5:1 for 72 h in the presence or absence of SEB (100 pg/ml). b, Th cells were cultured as described in a in the additional presence of an anti-IL-12p70, an anti-IL-23, or control IgG (10 μg/ml). c, Th cells were cultured as described in a in the presence of iNKT cells, together with or without of α-GalCer. d, Memory Th cells were cultured as in a in the presence of iNKT cells together with α-GalCer. The rhIL-23 was added at indicated concentrations. IL-17 levels in the culture supernatants were evaluated with ELISA. Data represent mean ± SD of triplicate cultures. Results are representative of three separate experiments. **, p < 0.005.

Discussion

This study demonstrated that human iNKT cells regulate DC function to enhance the IL-12p70 and inhibit the IL-23 production, and thereby down-regulating the IL-17 production by memory CD4+ Th cells. The ability of the iNKT cells to regulate the differential production of IL-12p70/IL-23 is mainly mediated by their concordant cytokines. The modification of IL-12p70/IL-23 balance by iNKT cells may thus be one of the mechanisms that regulate the immune responses in various diseases.

Because iNKT cells produce a variety of cytokines and express membrane-bound factors, the effects on DCs should be dependent on the balance of positive and negative factors expressed by iNKT cells. IL-4 and IL-10 characterize iNKT cells able to prevent or ameliorate some autoimmune diseases and inflammatory conditions (45). However, the precise mechanism and where they act have remained elusive. The current results indicate that the point of action of IL-4 and IL-10 is DCs and these cytokines effectively down-regulate IL-23 production (Figs. 6⇑ and 9⇓).

FIGURE 9.
FIGURE 9.

The hypothesized mechanisms underlying the induction of beneficial Th1 responses and suppression of harmful Th17 responses by α-GalCer activated NKT cells. IL-4, IL-13, and IFN-γ from iNKT cells exert promoting effect on IL-12p70 production by DCs. In contrast, IL-4, IL-13, and IL-10 from iNKT cells exert inhibitory effect on IL-23. Th2 cytokines such as IL-4 and IL-13 from α-GalCer-activated iNKT cells define the enhancement of IL-12p70 and the inhibition of IL-23. The dominant production of IL-12p70 and the reduced production of IL-23 promote the intensive cell-mediated immunity via activation of NK cells, CTLs, and Th1 cells while suppressing the Th17-dependent inflammatory conditions.

In the iNKT-DC coculture experiment, iNKT cells activated by α-GalCer consistently enhanced the IL-12p70 production by bacteria-activated DCs while inhibiting the IL-23 production. CD40 ligation by CD40L expressed on activated iNKT cells induces IL-12p70 production by immature monocyte-derived DCs (33). In addition, stimulation with single TLRs induces IL-23 production by monocyte-derived DCs, only in the additional presence of CD40 ligation by T cells (46). Consistent with these findings, CD40 ligation by iNKT cells exerted a promoting effect on both IL-12p70 and IL-23 production by bacteria-activated DCs (Fig. 3⇑), indicating a possibility that a factor other than the CD40 ligation played a decisive role in the down-regulation of IL-23.

Data from the ELISA and quantitative gene expression analyses clearly showed the crucial roles of iNKT cell-derived soluble factor for the differential production of IL-12p70/IL-23 (Figs. 5⇑ and 7⇑). These data provide evidence that 1) soluble factor from iNKT cells consistently enhances the expression of IL-12p35, whereas it suppresses both IL-23p19 and the common IL-12/23p40 subunit; 2) IFN-γ-from iNKT cells enhances IL-12p35, IL-12p40, and IL-23p19, whereas IL-10 suppresses all of them; 3) IL-4 and IL-13 enhance IL-12p35, whereas they suppress IL-23p19 and IL-12/23p40; 4) IL-4 and IL-13 enhance IL-12p35 expression in synergy with IFN-γ, whereas they suppress IL-23p19 expression even in the presence of IFN-γ; and 5) the pattern of IL-12p70 and IL-23 induction was reflected in the expression of IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40.

Despite efficient inhibition of IL-12p35 by IL-10, the supernatants containing IL-10 from iNKT cells even enhanced the IL-12p35 expression (Fig. 5⇑b). It is conceivable that the combined effect of positive signals for IL-12p35 expression induced by IFN-γ, IL-4, and IL-13 may be dominant over a negative signal induced by IL-10 (Fig. 7⇑e). In contrast, the observation that the supernatants from iNKT cells preferentially decreased the IL-23p19 expression (Fig. 5⇑c), thus indicating that the combined effect of IL-4, IL-10, and IL-13, which act as negative signals for IL-23p19 expression, may exceed a positive signal induced by IFN-γ (Fig. 7⇑f). These observations suggest that IL-4 and IL-13 from iNKT cells play a crucial role in defining the biased production of IL-12p70/IL-23. It is also of particular interest that both the enhancement of IL-12p35 and the suppression of IL-23p19 concurrently occurred, thus suggesting a mechanism that explains the two beneficial effects of the α-GalCer-activated iNKT cells in the enhancement of IL-12p70-induced cell-mediated immunity and the suppression of IL-23-dependent inflammatory pathologies (Fig. 9⇑).

The iNKT cells in humans are composed of two major distinct subsets, DN and CD4+ iNKT cells. The CD4+ and DN iNKT cells stimulated with anti-CD3/28 mAbs produced both Th1 and Th2 cytokines, although the CD4+ iNKT cells produced a larger amount of Th2 cytokines (see supplemental Fig. S5).5 The supernatant from CD4+ iNKT cells more effectively inhibited the IL-23 and less efficiently enhanced IL-12p70 than that from DN iNKT cells (Fig. 4⇑). The efficiency of IL-23 suppression was clearly associated with their capacity to produce Th2 cytokines. Therefore, CD4+ iNKT cells may play a more important role in the attenuation of IL-23-dependent pathologies. In addition, the balance between these subpopulations may influence the IL-12p70/IL-23 balance through the bystander mechanism.

The potential anti-inflammatory benefit of iNKT cells in multiple sclerosis is suggested by a higher IL-4 to IFN-γ ratio among CD4+ iNKT cells during the subsequent remission (47). Further support for the role of Th2 cytokines in protection of autoimmune encephalomyelitis derives from studies conducted using treatment with OCH ((2s,3s,4r)-1-O-(α-D-galactopyranosyl)-N-tetracosanoyl-2-amino-1,3,4-nonanetriol), an analog of α-GalCer with a truncated sphingosine chain, which promotes the production of IL-4 by iNKT cells (16). Therefore, OCH may be an excellent therapeutic option for diseases resolved by down-regulation of IL-23.

It is now clear that IL-23 does not act on naive CD4+ Th cells to induce their differentiation, but instead acts on already differentiated Th17 cells (43, 44). The current study showed that the activation of iNKT cells by α-GalCer suppressed the IL-17 production by memory CD4+ Th cells, and it was responsible for a down-regulation of IL-23 production by DCs (Fig. 8⇑). There is evidence that the presence of IL-12p70 reveals antagonistic effects for IL-23/IL-17 axis (48). Accordingly, a predominant IL-12p70 production induced by iNKT-DC interaction may also play a role in the inhibition of IL-17 production (Fig. 9⇑). The current findings might be of relevance for some microbial infections in which the TLR-dependent maturation of tissue-resident DCs results in the up-regulation of the endogenous glycolipids that can be directly recognized by iNKT cells (49, 50). It is conceivable that the activation of iNKT cells during infections might similarly regulate the IL-12/23 balance in DCs, thereby down-regulating the Th17 responses and limiting the subsequent tissue damage. Recently, TGF-β in combination with IL-1β, IL-6, or IL-21 was identified as a critical factor in human naive Th differentiation (51, 52). More recently, it has become clear that mouse iNKT cells play an important role in impeding the commitment of naive CD4+ T cells to the Th17 linage. The mechanism used by iNKT cells to inhibit Th17 commitment requires IL-4, IL-10, and IFN-γ (53). The role of human iNKT cells in DC-mediated Th17 cell differentiation, however, still remains to be elucidated.

There is much evidence supporting an association between autoimmune diseases and a previous infection (54, 55). The importance of IL-23-IL-17 axis in several microbial infections is underlined by previous studies (23, 56). In the current study, the DCs stimulated with attenuated M. tuberculosis H37Ra that have been used for the induction of autoimmune encephalomyelitis were analyzed (10, 17, 57). In this case, the presence of supernatant from iNKT cells completely suppressed the IL-23p19 expression at early time points after H37Ra stimulation but the expression was elevated at later time points (Fig. 5⇑c). Thereby, repeated administration of α-GalCer to autoimmune susceptible individuals with specific infections may exert preventative effects on the onset of autoimmune diseases. This possibility is partially supported by the observation that the protective effect of iNKT cells in autoimmune encephalomyelitis is needed for repeated administration of α-GalCer (58).

IL-12 and IL-23 production are differentially regulated by various TLR ligands expressed by microbes (48). In addition, IL-23 production is either enhanced or inhibited by IFN-γ depending on the TLR involved in the induction of IL-23 (59). Therefore, the activation of iNKT cells by α-GalCer may profoundly and differentially affect the IL-12p70/IL-23 balance depending on microbial derived signals to which DCs are exposed. However, this still remains an unresolved issue.

There is evidence suggesting the tumorigenic properties of IL-23 and IL-17 (28, 60, 61). The use of microbial adjuvants for cancer immunotherapy may be detrimental because their synergistic pairs may induce undesirable effects by their induction of IL-23 (62). The current finding in which iNKT cells enhanced IL-12p70 while inhibiting IL-23 might therefore be relevant for the use of the self-iNKT cell-adjuvant capable of inducing the intensive Th1 response and suppressing the tumorigenic milieu (Fig. 9⇑).

In conclusion, this study information regarding the ability of iNKT cells to down-regulate the IL-23 production by DCs. The findings also indicate a distinct regulatory role of the soluble factors of iNKT cells in the differential production of IL-12p70 and IL-23. Therefore, the mode of action of iNKT cells in vivo may be more complex than previously appreciated, thus encompassing the influence on DCs, NK cells, and T cells. Further elucidation of the iNKT cell biology and their immunoregulatory role will therefore be necessary to achieve the rational use of iNKT cells for human diseases.

Acknowledgments

We are grateful to Y. Nishimura (Kumamoto University) for helpful suggestions. We also thank S. Torigoe (Kansai Medical University) for valuable assistance in the preparation of the manuscript.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported in part by Grants-in-Aid 21710069, 21791572, 21791473, 21791958, 21602005, 21591251, 20591158, 20591190, 19890205, 19790637, and 18790689 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by research grants from the 21st Century Center of Excellence Program, Ministry of Education, Mitsubishi Pharma Research Foundation (05), Takeda Science Foundation (05), Ochiai Memorial Award (07, 08), Ichiro Kanehara Foundation (08), Kanzawa Medical Research Foundation (09), and Kowa Life Science Foundation (09).

  • ↵2 Y.U., T.-Y.L., Y.N., and M.S. contributed equally to this study as lead authors.

  • ↵3 Address correspondence and reprint requests to Dr. Yasushi Uemura, Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, Moriguchi, Osaka, 570-8506, Japan. E-mail address: uemuraya{at}takii.kmu.ac.jp

  • ↵4 Abbreviations used in this paper: iNKT, invariant NKT; α-GalCer, α-galactosylceramide; DN, double negative; DC, dendritic cell; rh, recombinant human; SEB, staphylococcal enterotoxin B.

  • ↵5 The online version of this article contains supplemental material.

  • Received March 17, 2009.
  • Accepted April 21, 2009.

References

  1. ↵
    Godfrey, D. I., H. R. MacDonald, M. Kronenberg, M. J. Smyth, L. Van Kaer. 2004. NKT cells: what’s in a name?. Nat. Rev. Immunol. 4: 231-237.
    OpenUrlCrossRefPubMed
  2. ↵
    Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, et al 1997. CD1d-restricted and TCR-mediated activation of Vα14 NKT cells by glycosylceramides. Science 278: 1626-1629.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Carnaud, C., D. Lee, O. Donnars, S. H. Park, A. Beavis, Y. Koezuka, A. Bendelac. 1999. Cutting edge: Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J. Immunol. 163: 4647-4650.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Fujii, S., K. Shimizu, C. Smith, L. Bonifaz, R. M. Steinman. 2003. Activation of natural killer T cells by α-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein. J. Exp. Med. 198: 267-279.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Taniguchi, M., M. Harada, S. Kojo, T. Nakayama, H. Wakao. 2003. The regulatory role of Vα14 NKT cells in innate and acquired immune response. Annu. Rev. Immunol. 21: 483-513.
    OpenUrlCrossRefPubMed
  6. ↵
    Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, M. Taniguchi. 1997. Requirement for Vα14 NKT cells in IL-12-mediated rejection of tumors. Science 278: 1623-1626.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Smyth, M. J., N. Y. Crowe, D. G. Pellicci, K. Kyparissoudis, J. M. Kelly, K. Takeda, H. Yagita, D. I. Godfrey. 2002. Sequential production of interferon-γ by NK1.1+ T cells and natural killer cells is essential for the antimetastatic effect of α-galactosylceramide. Blood 99: 1259-1266.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Gonzalez-Aseguinolaza, G., L. Van Kaer, C. C. Bergmann, J. M. Wilson, J. Schmieg, M. Kronenberg, T. Nakayama, M. Taniguchi, Y. Koezuka, M. Tsuji. 2002. Natural killer T cell ligand α-galactosylceramide enhances protective immunity induced by malaria vaccines. J. Exp. Med. 195: 617-624.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Sharif, S., G. A. Arreaza, P. Zucker, Q. S. Mi, J. Sondhi, O. V. Naidenko, M. Kronenberg, Y. Koezuka, T. L. Delovitch, J. M. Gombert, et al 2001. Activation of natural killer T cells by α-galactosylceramide treatment prevents the onset and recurrence of autoimmune type 1 diabetes. Nat. Med. 7: 1057-1062.
    OpenUrlCrossRefPubMed
  10. ↵
    Jahng, A. W., I. Maricic, B. Pedersen, N. Burdin, O. Naidenko, M. Kronenberg, Y. Koezuka, V. Kumar. 2001. Activation of natural killer T cells potentiates or prevents experimental autoimmune encephalomyelitis. J. Exp. Med. 194: 1789-1799.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Wilson, S. B., T. L. Delovitch. 2003. Janus-like role of regulatory iNKT cells in autoimmune disease and tumour immunity. Nat. Rev. Immunol. 3: 211-222.
    OpenUrlCrossRefPubMed
  12. ↵
    Kronenberg, M.. 2005. Toward an understanding of NKT cell biology: progress and paradoxes. Annu. Rev. Immunol. 23: 877-900.
    OpenUrlCrossRefPubMed
  13. ↵
    Godfrey, D. I., M. Kronenberg. 2004. Going both ways: immune regulation via CD1d-dependent NKT cells. J. Clin. Invest. 114: 1379-1388.
    OpenUrlCrossRefPubMed
  14. ↵
    Charlton, B., K. J. Lafferty. 1995. The Th1/Th2 balance in autoimmunity. Curr. Opin. Immunol. 7: 793-798.
    OpenUrlCrossRefPubMed
  15. ↵
    Hong, S., M. T. Wilson, I. Serizawa, L. Wu, N. Singh, O. V. Naidenko, T. Miura, T. Haba, D. C. Scherer, J. Wei, et al 2001. The natural killer T-cell ligand α-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice. Nat. Med. 7: 1052-1056.
    OpenUrlCrossRefPubMed
  16. ↵
    Miyamoto, K., S. Miyake, T. Yamamura. 2001. A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells. Nature 413: 531-534.
    OpenUrlCrossRefPubMed
  17. ↵
    Ferber, I. A., S. Brocke, C. Taylor-Edwards, W. Ridgway, C. Dinisco, L. Steinman, D. Dalton, C. G. Fathman. 1996. Mice with a disrupted IFN-γ gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156: 5-7.
    OpenUrlAbstract
  18. ↵
    Becher, B., B. G. Durell, R. J. Noelle. 2002. Experimental autoimmune encephalitis and inflammation in the absence of interleukin-12. J. Clin. Invest. 110: 493-497.
    OpenUrlCrossRefPubMed
  19. ↵
    Cua, D. J., J. Sherlock, Y. Chen, C. A. Murphy, B. Joyce, B. Seymour, L. Lucian, W. To, S. Kwan, T. Churakova, et al 2003. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421: 744-748.
    OpenUrlCrossRefPubMed
  20. ↵
    Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham, J. D. Sedgwick, T. McClanahan, R. A. Kastelein, D. J. Cua. 2005. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201: 233-240.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Chen, Y., C. L. Langrish, B. McKenzie, B. Joyce-Shaikh, J. S. Stumhofer, T. McClanahan, W. Blumenschein, T. Churakovsa, J. Low, L. Presta, et al 2006. Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis. J. Clin. Invest. 116: 1317-1326.
    OpenUrlCrossRefPubMed
  22. ↵
    Kebir, H., K. Kreymborg, I. Ifergan, A. Dodelet-Devillers, R. Cayrol, M. Bernard, F. Giuliani, N. Arbour, B. Becher, A. Prat. 2007. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat. Med. 13: 1173-1175.
    OpenUrlCrossRefPubMed
  23. ↵
    Khader, S. A., G. K. Bell, J. E. Pearl, J. J. Fountain, J. Rangel-Moreno, G. E. Cilley, F. Shen, S. M. Eaton, S. L. Gaffen, S. L. Swain, et al 2007. IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nat. Immunol. 8: 369-377.
    OpenUrlCrossRefPubMed
  24. ↵
    Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, et al 2000. Novel p19 protein engages IL-12p40 to form a cytokine: IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13: 715-725.
    OpenUrlCrossRefPubMed
  25. ↵
    Kastelein, R. A., C. A. Hunter, D. J. Cua. 2007. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu. Rev. Immunol. 25: 221-242.
    OpenUrlCrossRefPubMed
  26. ↵
    Aggarwal, S., N. Ghilardi, M. H. Xie, F. J. de Sauvage, A. L. Gurney. 2003. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J. Biol. Chem. 278: 1910-1914.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Murphy, C. A., C. L. Langrish, Y. Chen, W. Blumenschein, T. McClanahan, R. A. Kastelein, J. D. Sedgwick, D. J. Cua. 2003. Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J. Exp. Med. 198: 1951-1957.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Langowski, J. L., X. Zhang, L. Wu, J. D. Mattson, T. Chen, K. Smith, B. Basham, T. McClanahan, R. A. Kastelein, M. Oft. 2006. IL-23 promotes tumour incidence and growth. Nature 442: 461-465.
    OpenUrlCrossRefPubMed
  29. ↵
    Hue, S., P. Ahern, S. Buonocore, M. C. Kullberg, D. J. Cua, B. S. McKenzie, F. Powrie, K. J. Maloy. 2006. Interleukin-23 drives innate and T cell-mediated intestinal inflammation. J. Exp. Med. 203: 2473-2483.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Fujii, S., K. Liu, C. Smith, A. J. Bonito, R. M. Steinman. 2004. The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation. J. Exp. Med. 199: 1607-1618.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Kojo, S., K. Seino, M. Harada, H. Watarai, H. Wakao, T. Uchida, T. Nakayama, M. Taniguchi. 2005. Induction of regulatory properties in dendritic cells by Vα14 NKT cells. J. Immunol. 175: 3648-3655.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Chen, Y. G., C. M. Choisy-Rossi, T. M. Holl, H. D. Chapman, G. S. Besra, S. A. Porcelli, D. J. Shaffer, D. Roopenian, S. B. Wilson, D. V. Serreze. 2005. Activated NKT cells inhibit autoimmune diabetes through tolerogenic recruitment of dendritic cells to pancreatic lymph nodes. J. Immunol. 174: 1196-1204.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Liu, T. Y., Y. Uemura, M. Suzuki, Y. Narita, S. Hirata, H. Ohyama, O. Ishihara, S. Matsushita. 2008. Distinct subsets of human invariant NKT cells differentially regulate T helper responses via dendritic cells. Eur. J. Immunol. 38: 1012-1023.
    OpenUrlCrossRefPubMed
  34. ↵
    Baev, D. V., S. Caielli, F. Ronchi, M. Coccia, F. Facciotti, K. E. Nichols, M. Falcone. 2008. Impaired SLAM-SLAM homotypic interaction between invariant NKT cells and dendritic cells affects differentiation of IL-4/IL-10-secreting NKT2 cells in nonobese diabetic mice. J. Immunol. 181: 869-877.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Fujii, S., K. Shimizu, M. Kronenberg, R. M. Steinman. 2002. Prolonged IFN-γ-producing NKT response induced with α-galactosylceramide-loaded DCs. Nat. Immunol. 3: 867-874.
    OpenUrlCrossRefPubMed
  36. ↵
    Kitamura, H., K. Iwakabe, T. Yahata, S. Nishimura, A. Ohta, Y. Ohmi, M. Sato, K. Takeda, K. Okumura, L. Van Kaer, et al 1999. The natural killer T (NKT) cell ligand α-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J. Exp. Med. 189: 1121-1128.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Uemura, Y., T. Y. Liu, Y. Narita, M. Suzuki, S. Ohshima, S. Mizukami, Y. Ichihara, H. Kikuchi, S. Matsushita. 2007. Identification of functional type 1 ryanodine receptors in human dendritic cells. Biochem. Biophys. Res. Commun. 362: 510-515.
    OpenUrlCrossRefPubMed
  38. ↵
    Uemura, Y., M. Suzuki, T. Y. Liu, Y. Narita, S. Hirata, H. Ohyama, O. Ishihara, S. Matsushita. 2008. Role of human non-invariant NKT lymphocytes in the maintenance of type 2 T helper environment during pregnancy. Int. Immunol. 20: 405-412.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Lee, P. T., K. Benlagha, L. Teyton, A. Bendelac. 2002. Distinct functional lineages of human Vα24 natural killer T cells. J. Exp. Med. 195: 637-641.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Gumperz, J. E., S. Miyake, T. Yamamura, M. B. Brenner. 2002. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med. 195: 625-636.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Rachitskaya, A. V., A. M. Hansen, R. Horai, Z. Li, R. Villasmil, D. Luger, R. B. Nussenblatt, R. R. Caspi. 2008. Cutting edge: NKT cells constitutively express IL-23 receptor and RORγt and rapidly produce IL-17 upon receptor ligation in an IL-6-independent fashion. J. Immunol. 180: 5167-5171.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Coquet, J. M., K. Kyparissoudis, D. G. Pellicci, G. Besra, S. P. Berzins, M. J. Smyth, D. I. Godfrey. 2007. IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production. J. Immunol. 178: 2827-2834.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Bettelli, E., T. Korn, M. Oukka, V. K. Kuchroo. 2008. Induction and effector functions of TH17 cells. Nature 453: 1051-1057.
    OpenUrlCrossRefPubMed
  44. ↵
    van Beelen, A. J., Z. Zelinkova, E. W. Taanman-Kueter, F. J. Muller, D. W. Hommes, S. A. Zaat, M. L. Kapsenberg, E. C. de Jong. 2007. Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to promote interleukin-17 production in human memory T cells. Immunity 27: 660-669.
    OpenUrlCrossRefPubMed
  45. ↵
    Hammond, K. J., L. D. Poulton, L. J. Palmisano, P. A. Silveira, D. I. Godfrey, A. G. Baxter. 1998. α/β-T cell receptor (TCR)+CD4−CD8− (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10. J. Exp. Med. 187: 1047-1056.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Acosta-Rodriguez, E. V., G. Napolitani, A. Lanzavecchia, F. Sallusto. 2007. Interleukins 1β and 6 but not transforming growth factor-β are essential for the differentiation of interleukin 17-producing human T helper cells. Nat. Immunol. 8: 942-949.
    OpenUrlCrossRefPubMed
  47. ↵
    Araki, M., T. Kondo, J. E. Gumperz, M. B. Brenner, S. Miyake, T. Yamamura. 2003. Th2 bias of CD4+ NKT cells derived from multiple sclerosis in remission. Int. Immunol. 15: 279-288.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Roses, R. E., S. Xu, M. Xu, U. Koldovsky, G. Koski, B. J. Czerniecki. 2008. Differential production of IL-23 and IL-12 by myeloid-derived dendritic cells in response to TLR agonists. J. Immunol. 181: 5120-5127.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Brigl, M., L. Bry, S. C. Kent, J. E. Gumperz, M. B. Brenner. 2003. Mechanism of CD1d-restricted natural killer T cell activation during microbial infection. Nat. Immunol. 12: 1230-1237.
    OpenUrl
  50. ↵
    Salio, M., A. O. Speak, D. Shepherd, P. Polzella, P. A. Illarionov, N. Veerapen, G. S. Besra, F. M. Platt, V. Cerundolo. 2007. Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation. Proc. Natl. Acad. Sci. USA 104: 20490-20495.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    Manel, N., D. Unutmaz, D. R. Littman. 2008. The differentiation of human TH-17 cells requires transforming growth factor-β and induction of the nuclear receptor RORγt. Nat. Immunol. 9: 641-649.
    OpenUrlCrossRefPubMed
  52. ↵
    Yang, L., D. E. Anderson, C. Baecher-Allan, W. D. Hastings, E. Bettelli, M. Oukka, V. K. Kuchroo, D. A. Hafler. 2008. IL-21 and TGF-β are required for differentiation of human TH17 cells. Nature 454: 350-352.
    OpenUrlCrossRefPubMed
  53. ↵
    Mars, L. T., L. Araujo, P. Kerschen, S. Diem, E. Bourgeois, L. P. Van, N. Carrié, M. Dy, R. S. Liblau, A. Herbelin. 2009. Invariant NKT cells inhibit development of the Th17 lineage. Proc. Natl. Acad. Sci. USA 106: 6238-6243.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Uemura, Y., S. Senju, K. Maenaka, L. K. Iwai, S. Fujii, H. Tabata, H. Tsukamoto, S. Hirata, Y. Z. Chen, Y. Nishimura. 2003. Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs. J. Immunol. 170: 947-960.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Bach, J. F.. 2005. Infections and autoimmune diseases. J. Autoimmun. 25: 74-80.
    OpenUrlCrossRefPubMed
  56. ↵
    Happel, K. I., P. J. Dubin, M. Zheng, N. Ghilardi, C. Lockhart, L. J. Quinton, A. R. Odden, J. E. Shellito, G. J. Bagby, S. Nelson, J. K. Kolls. 2005. Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae. J. Exp. Med. 202: 761-769.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Hirata, S., S. Senju, H. Matsuyoshi, D. Fukuma, Y. Uemura, Y. Nishimura. 2005. Prevention of experimental autoimmune encephalomyelitis by transfer of embryonic stem cell-derived dendritic cells expressing myelin oligodendrocyte glycoprotein peptide along with TRAIL or programmed death-1 ligand. J. Immunol. 174: 1888-1897.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Singh, A. K., M. T. Wilson, S. Hong, D. Olivares-Villagómez, C. Du, A. K. Stanic, S. Joyce, S. Sriram, Y. Koezuka, L. Van Kaer. 2001. Natural killer T cell activation protects mice against experimental autoimmune encephalomyelitis. J. Exp. Med. 194: 1801-1811.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Gerosa, F., B. Baldani-Guerra, L. A. Lyakh, G. Batoni, S. Esin, R. T. Winkler-Pickett, M. R. Consolaro, M. De Marchi, D. Giachino, A. Robbiano, et al 2008. Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells. J. Exp. Med. 205: 1447-1461.
    OpenUrlAbstract/FREE Full Text
  60. ↵
    Numasaki, M., J. Fukushi, M. Ono, S. K. Narula, P. J. Zavodny, T. Kudo, P. D. Robbins, H. Tahara, M. T. Lotze. 2003. Interleukin-17 promotes angiogenesis and tumor growth. Blood 101: 2620-2627.
    OpenUrlAbstract/FREE Full Text
  61. ↵
    Kortylewski, M., H. Xin, M. Kujawski, H. Lee, Y. Liu, T. Harris, C. Drake, D. Pardoll, H. Yu. 2009. Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment. Cancer Cell. 15: 114-123.
    OpenUrlCrossRefPubMed
  62. ↵
    Napolitani, G., A. Rinaldi, F. Bertoni, F. Sallusto, A. Lanzavecchia. 2005. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat. Immunol. 6: 769-776.
    OpenUrlCrossRefPubMed
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