The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells

Abstract

The natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12-mediated antitumor activities. Because of these similarities between the activities of alpha-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by alpha-GalCer. We first established, using purified subsets of various lymphocyte populations, that alpha-GalCer selectively activates NKT cells for production of interferon (IFN)-gamma. Production of IFN-gamma by NKT cells in response to alpha-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, alpha-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1(-/-) or Valpha14(-/-) mice. This effect of alpha-GalCer required the production of IFN-gamma by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of alpha-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-gamma production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by alpha-GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer.

Figure 1

α-GalCer selectively activates NK1.1⁺TCR-α/β⁺ NKT cells. Spleen cells from C57BL/6 mice were separated into a variety of lymphoid cell subsets by cell sorting as described in Materials and Methods. Their responsiveness to α-GalCer in the presence of DCs was then determined by measuring IL-4 (▪) and IFN-γ () levels in the culture supernatants using ELISA. As a control, NK1.1⁺TCR-α/β⁺ NKT cells were cultured alone or with DCs in the absence of α-GalCer. The bars represent mean ± SE of triplicate samples.

Figure 2

Endogenously produced IL-12 and CD40/CD40L interaction during coculture of DCs and NKT cells is essential for NKT cell activation by α-GalCer. Purified NKT cells were cocultured with DCs in the presence of α-GalCer for 36 h. The IFN-γ levels in culture supernatants were then determined by ELISA. (A) The ability of anti–IL-12 mAb to block NKT cell activation by α-GalCer. Anti-CD8 mAb was used as control rat IgG Ab. (B) The ability of anti-CD40 mAb and anti-CD40L mAb to block NKT cell activation by α-GalCer. As a control, rat anti-CD8 IgG mAb was added to the culture. (C) IL-12 production by DCs cultured with α-GalCer and NKT cells. DCs (5 × 10⁵) were activated with 50 ng/ml of α-GalCer for 8 h in the presence or absence of NK1.1⁺TCR-α/β⁻ NK cells (10⁵) or NK1.1⁺TCR-α/β⁺ NKT cells (10⁵). The bars represent mean ± SE of triplicate samples.

Figure 3

Upregulation of IL-12R expression in the spleen upon in vivo administration of α-GalCer. C57BL/6 mice were injected intravenously with α-GalCer. Various times (0, 2, 4, and 6 h) after the treatment, spleen cells were prepared and their expression of IL-12Rβ1 (•) and IL-12Rβ2 (○) mRNA was measured by quantitative RT-PCR. The IL-12R mRNA levels are represented as an induction index, as described in Materials and Methods. The bars represent mean ± SE of triplicate samples.

Figure 4

Role of IL-12 and IFN-γ in the induction of IL-12R expression in the spleen. C57BL/6 mice were injected intravenously with α-GalCer. 4 h after the injection, spleen cells were prepared and their expression of IL-12Rβ1 () and IL-12Rβ2 (▪) mRNA was measured by quantitative RT-PCR. The IL-12R mRNA levels are presented as an induction index, as described in Materials and Methods. The blocking effect of anti–IL-12 mAb and anti–IFN-γ mAb was determined by injection of these mAbs (500 μg/mouse i.p.) into the mice 1 and 0 d before the treatment with α-GalCer. The bars represent mean ± SE of triplicate samples.

Figure 5

α-GalCer induces IL-12R expression on NKT cells. C57BL/6 wild-type mice, CD1d^−/− mice, and NKT-deficient mice were injected intravenously with vehicle () or α-GalCer (▪). 4 h after the injection, spleen cells were prepared and their expression of IL-12Rβ1 (A) and IL-12Rβ2 (B) mRNA was measured by quantitative RT-PCR. (C) Spleen cells from wild-type mice were cultured with DCs plus α-GalCer or vehicle for 8 h, NK1.1⁺TCR-α/β⁺ NKT cells were purified by cell sorting, and the expression of IL-12Rβ1 () and IL-12Rβ2 (▪) was examined by quantitative RT-PCR. (D) Wild-type mice were injected intravenously with α-GalCer or vehicle; after 6 h the spleens from these mice were isolated, and NK1.1⁺TCR-α/β⁺ NKT cells were purified by cell sorting, and expression of IL-12Rβ1 () and IL-12Rβ2 (▪) was examined by quantitative RT-PCR. The IL-12R mRNA levels are presented as an induction index, as described in Materials and Methods. The bars represent mean ± SE of triplicate samples.

Figure 6

Synergistic effect of α-GalCer and IL-12 in vivo. C57BL/6 mice were injected with a suboptimal dose of α-GalCer (200 ng/mouse i.v.) and 6 h later, mice were injected with a suboptimal dose of IL-12 (200 U/mouse i.p.). 1 d after the treatment with IL-12, the mice were killed and splenic natural killing activity (A) and serum IFN-γ levels (B) were determined as described in Materials and Methods. The bars represent mean ± SE of triplicate samples.