CD1d-Restricted and TCR-Mediated Activation of Vα14 NKT Cells by Glycosylceramides

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Science  28 Nov 1997:
Vol. 278, Issue 5343, pp. 1626-1629
DOI: 10.1126/science.278.5343.1626


Natural killer T (NKT) lymphocytes express an invariant T cell antigen receptor (TCR) encoded by the Vα14 and Jα281 gene segments. A glycosylceramide-containing α-anomeric sugar with a longer fatty acyl chain (C26) and sphingosine base (C18) was identified as a ligand for this TCR. Glycosylceramide-mediated proliferative responses of Vα14 NKT cells were abrogated by treatment with chloroquine–concanamycin A or by monoclonal antibodies against CD1d/Vβ8, CD40/CD40L, or B7/CTLA-4/CD28, but not by interference with the function of a transporter-associated protein. Thus, this lymphocyte shares distinct recognition systems with either T or NK cells.

An unusual lineage of lymphocytes, Vα14 NKT cells, are characterized by their development before thymus formation (1), their expression of an invariant TCR encoded by the Vα14 and Jα281 gene segments (2,3) mainly associated with Vβ8.2 (4), and by the coexpression of the NK1.1 receptor, a marker of NK cells (5). The invariant Vα14 TCR is essential for the development and function of Vα14 NKT cells (6-8). Contrary to the general rule that the interaction of the TCR with the major histocompatibility complex (MHC) molecules leads to the development of T cells, Vα14 NKT cells are selected by CD1d, a nonclassical class Ib molecule (9); mutant mice deficient in CD1d lack Vα14 NKT cells (10). However, a ligand for invariant Vα14 TCR has not yet been identified. Here, we attempt to define a ligand with which to specifically activate Vα14 NKT cells and characterize their activation mechanisms.

Because another CD1 molecule, human CD1b, presents glycolipids (11, 12), and because the development and selection of Vα14 NKT cells are independent of transporter-associated protein (TAP) essential for peptide presentation on MHC (13), we studied glycolipids as candidate Vα14 TCR ligands. Synthetic glycolipids (14) were used to avoid effects of minor contaminants in biological samples. Moreover, we generated Vα14 NKT mice expressing the invariant Vα14 and Vβ8.2 transgenes in a recombination activating gene (RAG)–deficient background (RAG−/−Vα14tg Vβ8.2tg) that only have Vα14 NKT cells, but no T, B, or NK cells (15). Spleen cells from Vα14 NKT mice were cocultured with fractionated dendritic cells (DCs) from NK-only (RAG−/−) mice (no T, B, or Vα14 NKT cells) pulsed with various glycosylceramides, and their proliferative responses were measured.

Vα14 NKT cells incorporated [3H]thymidine ([3H]TdR) after stimulation with α-galactosylceramide (α-GalCer), whereas activation with ceramide itself or β-galactosylceramide (β-GalCer) resulted in no proliferative responses (Fig. 1, A and C). Because α-glucosylceramide (α-GlcCer) as well as α-GalCer stimulated Vα14 NKT cells readily, the α-anomeric conformation of sugar moiety is essential. Indeed, in diglycosylated ceramides (Fig. 1, B and C), the α-anomeric configuration of the inner sugar is important. Galα1-6Galα1-1′Cer, Galα1-6Glcα1-1′Cer, Galα1-2Galα1-1′Cer, or Galβ1-3Galα1-1′Cer, whose inner sugar is either α-glucose or α-galactose despite their α- or β-anomer of the outer sugar moiety, could stimulate Vα14 NKT cells, whereas Galβ1-4Glcβ1-1′Cer with the β-anomer inner sugar could not.

Figure 1

Proliferative responses of Vα14 NKT cells by glycosylceramides. Fractionated DCs prepared from RAG / mice as described by Crowley et al. (30) were pulsed with ceramide [100 ng/ml in 0.1% dimethyl sulfoxide (DMSO)–RPMI], glycosylceramides (100 ng/ml in 0.1% DMSO-RPMI), or control vehicle (0.1% DMSO-RPMI). Spleen cells (2 × 105) from Vα14 NKT mice (15) were cocultured with pulsed DCs. Three days later, 0.5 μCi of [3H]TdR was added for 12 hours and [3H]TdR uptake was measured. (A) Stimulation with monoglycosylated ceramides. (B) Stimulation with diglycosylated ceramides. (C) Schematic representation of glycosylceramides. The results are expressed as mean counts per minute of three cultures ± SD.

Because α-GalCer and α-GlcCer, which differ only in the configuration of the 4-hydroxyl group on the carbohydrate, showed no functional differences, the 4-hydroxyl configuration of the sugar seems not to be important. However, α-mannosyl ceramide (α-ManCer), which showed no stimulatory activity, has the 2-hydroxyl group with an axial configuration that differs from that with an equatorial bond on α-GalCer or α-GlcCer, suggesting the importance of the configuration of the 2-hydroxyl group on the sugar moiety, probably for the TCR contact site of this glycolipid (Fig. 1, A and C).

A mutant derivative lacking the 3- and 4-hydroxyl groups on the phytosphingosine of α-GalCer(3,4-deoxy α-GalCer) was not stimulatory, indicating that the 3,4-hydroxyl groups of the phytosphingosine are also important (Fig. 1, A and C). Although the 3-hydroxyl group of the sphingosine plays a crucial role in the sphingolipid-mediated fusion of Semliki Forest virus (16), we could not determine whether the 3- and 4-hydroxyl groups were important for the TCR contact sites or for the stabilization of glycolipid conformation.

CD1d is essential for this ligand presentation and recognition by the invariant Vα14 TCR, because proliferative responses of Vα14 NKT cells were abrogated by monoclonal antibody (mAb) against CD1d, Vβ8, B7, CTLA-4, CD28, CD40, or CD40L but not against H-2Kb or I-Ab (Fig.2, A and B), indicating that α-GalCer–mediated stimulation of Vα14 NKT cells is CD1d-restricted and TCR/costimulatory molecule–dependent. NKT cell hybridomas have been reported to have CD1d autoreactivity (9). This discrepancy may be explained by the different TCR expression of hybridomas made by the fusion of thymocytes lacking Vα14 TCR expression on the surface (3). β2-Microglobulin (β2M)−/− DCs did not stimulate Vα14 NKT cells, whereas TAP / DCs could (Fig. 2C), supporting nonpeptide ligand presentation by class Ib molecule. On the basis of an analogy with lipoglycan presentation (12), it is conceivable that chloroquine (Chl) or concanamycin A (CMA), which prevents acidification or transportation to late endosomes (17), could inhibit α-GalCer presentation by DCs. Treatment of DCs with these drugs before pulse with α-GalCer inhibited proliferation of Vα14 NKT cells, whereas treatment after pulse with α-GalCer failed. This finding suggests a requirement for endosomal function in α-GalCer presentation (Fig. 2D).

Figure 2

Mode of recognition and activation of Vα14 NKT cells in the induction phase. Cells were prepared as described in Fig. 1 except for the materials indicated below. (A) CD1d-dependent recognition. DCs (2 × 104) preincubated with anti-FcγR (50 μg/ml) (2.4G2) were reacted with anti-CD1d (1B1; Pharmingen) or its control antibody (rat immunoglobulin G2b,κ ). (B) Blocking of Vα14 NKT cell activation. Monoclonal antibodies (50 μg/ml; Pharmingen) against CD1d (1B1), Vβ8 (MR5-2), B7-1(1G10), B7-2 (PO3.1), CD28 (37.51), CTLA-4 (UC10-4F10-11), CD40 (HM40-3), CD40L (MR1), H-2Kb (AF6-88.5), and I-Ab (AF6-120.1), or control mAb, were used. The data were expressed as percent inhibition of the experimental counts per minute over the control counts per minute (81,336 ± 4050 counts per minute). (C) Requirement of MHC class I–like molecules but not TAP for stimulation of Vα14 NKT cells. α-GalCer–pulsed (closed symbols) or vehicle-pulsed (open symbols) DCs prepared from β2M / , TAP / , or RAG / mice were used (30). (D) Effects of Chl or CMA on α-GalCer presentation by DCs. Chl (100 μM; Sigma) or CMA (10 nM; Wako Pure Chemical Industries) was added to the culture of DCs (2 × 104) 1 hour before (Chl or CMA→α-GalCer) or 2 hours after (α-GalCer→ Chl or CMA) the beginning of the 4-hour pulse with α-GalCer. α-GalCer– or vehicle-pulsed DCs without treatment were used as positive (Pos contr) or negative (Neg contr) controls, respectively. The results are expressed as mean counts per minute of three cultures ± SD.

The structural and functional relation between the lengths of fatty acyl chain and sphingosine base and activity of α-GalCer was examined (Fig. 3, A and B). The most effective lengths of fatty acyl chain and sphingosine base were C26 and C18, respectively, whereas the short fatty acyl or short sphingosine base lost their activity, indicating the hydrophobic interaction of α-GalCer with CD1d. The α-GalCer with fatty acyl (C26) and sphingosine base (C18) is estimated to be about 34 Å long, with the fatty acyl chain, the sphingosine base, and the sugar moiety being 28, 17, and 8 Å long, respectively (18). Recent studies on the crystal structure of CD1d molecule indicate that the binding groove has two large hydrophobic pockets about 30 Å long and 10 to 15 Å wide (19). Therefore, the findings indicate that the α-GalCer with fatty acyl (C26) and sphingosine base (C18) may be suitable for binding to these two pockets of CD1d, possibly through hydrophobic interactions.

Figure 3

Effects of lengths of fatty acyl chain and sphingosine base of α-GalCer on activation of Vα14 NKT cells. α-GalCers with different lengths of fatty acyl chains as indicated by X (A) and those of sphingosine base as indicated by Y (B) were used. The results are expressed as mean counts per minute of three cultures ± SD.

To investigate the selectivity of Vα14 NKT cell activation with α-GalCer, we cultured spleen cells with α-GalCer from wild-type littermates and Vα14 NKT-deficient, Vα14 NKT, and NK-only mice whose FACS (fluorescent-activated cell sorting) profiles are shown in Fig.4A (top). Proliferative responses were observed in Vα14 NKT and wild-type mice, but not in the mice without Vα14 NKT cells (NK-only mice or Vα14 NKT-deficient mice) (Fig. 4A). In addition, Vα14 NKT mice produced large amounts of interleukin-4 (IL-4) and interferon γ (Fig. 4B) and also killed the YAC-1 cells upon stimulation with α-GalCer, whereas Vα14 NKT-deficient or NK-only mice did not (Fig. 4C). Thus, α-GalCer selectively activates Vα14 NKT cells in vivo but not other lymphocytes. Vα14 NKT cells directly kill target tumor cells by an NK-like mechanisms and inhibit tumor growth and metastasis in vivo after activation with α-GalCer (20) or IL-12 (8), confirming the previous data on the protection of tumor metastasis by the treatment of tumor-bearing mice with α-GalCer (21).

Figure 4

Selective stimulation of Vα14 NKT cells by α-GalCer. (A) FACS profiles and in vitro proliferative responses. α-GalCer–pulsed DCs were cocultured with spleen cells from wild-type (Jα281+/+) mice, Vα14 NKT-deficient (Jα281 / ) mice, NK-only (RAG-1 / ) mice, or Vα14 NKT (RAG / Vα14tg Vβ8.2tg) mice as described in Fig. 1. Profiles by FACS analysis (31) are also shown. (B) Production of IL-4 and IFN-γ by α-GalCer activation. Spleen cells (2 × 105) were cultured at 37°C for 48 hours with α-GalCer (100 ng/ml), immobilized anti-CD3ɛ (10 μg/ml) (2C11; Pharmingen), or control vehicle. Cytokine production in the supernatants was assayed by an enzyme-linked immunosorbent assay (ELISA) kit (Endogen). (C) α-GalCer–mediated cytotoxicity in vitro. Mice were injected intraperitoneally with either α-GalCer (100 μg/kg in 0.025% Polysolvate 20), or vehicle–phosphate-buffered saline (PBS) (0.025% Polysolvate 20 in PBS). Twenty-four hours later, spleen cells were assayed for 4 hours on 51Cr-labeled YAC-1 (H-2k/d) cells (1 × 104) as described (8). The results are expressed as mean counts per minute of three cultures ± SD.

Monogalactosylceramide is the smallest size glycosphingolipid, but β-GalCer has been detected mainly in mammals (22). Most mammalian normal tissues have ceramides composed of sphingosine with the 4,5-trans carbon-carbon double bond in the sphingosine backbone, whereas α-GalCer has phytosphingosine without this carbon double bond (23). Although α-GalCer has been isolated from marine sponges and has been hardly detected in normal mammalian tissues (24), a form of α-anomeric monoglycolipid or glycolipid with phytosphingosine has been detected in certain bacteria (25) and in some conditions of mammalian tissues, such as fetus (26), cancer cells (27), kidney or intestine (28), or in some cultured cells (29). An α-glycosylceramide or a natural ligand similar to this glycolipid may thus exist in restricted mammalian tissues or be expressed on cells after activation or during malignancy.

  • * To whom correspondence should be addressed. E-mail: taniguti{at}


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