The finding that NKT cells recognize α-GalCer presented by DCs in a CD1d-dependent manner represents a novel recognition mechanism in the immune system (15
). NKT cells, which can produce both IFN-γ and IL-4 (16
), play an important role in immunoregulation and have been considered to play a central role as innate effector cells involved in both the protection and the onset of immune diseases (18
). The NKT cell ligand α-GalCer has a strong immunopotentiating effect in vivo, and this chemical mediates strong antitumor activity (3
). Therefore, it is important to dissect the mechanism by which α-GalCer activates NKT cells.
The previous finding (3
) that NKT-deficient mice did not respond to α-GalCer strongly suggested that NKT cells may be the primary target cells to α-GalCer. However, it still remained unclear whether only NKT cells responded to α-GalCer. To answer this question, we used highly purified splenic NK cells, NKT cells, CD4+
T cells, and CD8+
T cells and determined their responsiveness to α-GalCer in the presence of DCs. The data illustrated in Fig. clearly demonstrate that NKT cells are the only cells that respond to α-GalCer (3
). It is surprising that neither classical NK cells nor mainstream CD4+
T cells or CD8+
T cells revealed a significant response to α-GalCer even in the presence of DCs. Together with previous findings (3
), the present data indicate that α-GalCer selectively stimulates NKT cells in the presence of DCs.
Recently, the mechanisms of activation of naive CD4+
T cells through interaction with DCs have been examined (12
). Cell–cell adhesion between CD4+
T cells and DCs through CD40/CD40L and B7.1/CD28 resulted in the activation of both DCs and T cells, which triggered the production of IL-12 by DCs and IFN-γ by Th1 cells (12
). Such conditioned DCs were able to prime cytotoxic T cells (22
). This recognition system has resemblance to that discussed here. As shown in Fig. , IL-12 production by DCs appears to be essential for NKT cell activation by α-GalCer, because neutralization of endogenously produced IL-12 by anti–IL-12 mAb caused a strong inhibition of IFN-γ production by NKT cells. The important role of CD40/CD40L for the production of IFN-γ in the cocultures of DCs and NKT cells with α-GalCer is also apparent from these experiments (Fig. B). As demonstrated in Fig. C, DCs produce IL-12 only when they are cultured with α-GalCer in the presence of NKT cells, indicating that direct contact between α-GalCer–bound DCs and NKT cells may be essential for IL-12 production by DCs. This interaction may be required for the production of IFN-γ by IL-12–activated NKT cells, because mAbs directed against CD40/CD40L greatly inhibited IFN-γ production by NKT cells (Fig. ). These findings indicate that the interaction of NKT cells with DCs may be very similar to the interaction of helper T cells with DCs (22
). Since the interactions between DCs and NKT cells occur very quickly after administration of α-GalCer, NKT cells may be able to condition DCs very early in an immune response, and affect subsequent adaptive responses.
In this paper, we also demonstrate that α-GalCer upregulates IL-12R expression in vivo (Fig. ). IL-12R upregulation is blocked by mAbs against IL-12 or IFN-γ and is absent in CD1d−/− and NKT-deficient mice (Figs. and ). Moreover, activation of NKT cells in vitro and in vivo results in a strong induction of IL-12Rβ1 and IL-12Rβ2 on these cells (Fig. , C and D). Therefore, we speculate that the following series of events is induced upon culture of α-GalCer with DCs and NKT cells: (a) α-GalCer first binds to CD1d molecules on DCs; (b) NKT cells recognize α-GalCer–bound DCs via their TCRs and also interact with DCs via CD40/CD40L; (c) during this interaction, DCs produce IL-12; (d) the endogenously produced IL-12 stimulates IFN-γ production by NKT cells; and (e) IFN-γ produced by NKT cells upregulates IL-12R on NKT cells in an autocrine manner. The dramatic synergistic effect of suboptimal α-GalCer and exogenously administered IL-12 indicates that expression of IL-12Rβ1 and β2, detected by quantitative RT-PCR, is functionally upregulated in vivo. Moreover, since this synergistic effect of α-GalCer and IL-12 was not demonstrated in NKT-deficient mice, we conclude that in wild-type mice coadministration of α-GalCer and IL-12 leads to upregulation of IL-12R on CD1-dependent NKT cells.
Both α-GalCer and IL-12 have been demonstrated to exhibit potent antitumor activity in vivo. IL-12 has multiple effects on the immune system that are beneficial for the induction of antitumor immunity in vivo (28
). However, the unexpected severe side effects of IL-12 have made it difficult to use this cytokine in clinical trials (31
). We demonstrated that α-GalCer synergistically acts with small doses of IL-12 in vivo to activate NKT cells and to induce IFN-γ production (Fig. ). These findings suggest that coadministration of α-GalCer with IL-12 could be used as a new approach for tumor immunotherapy.
Recent studies have demonstrated that Th1 immunity regulated by IL-12 and IFN-γ plays a critical role in the induction of protective immunity against tumors and infectious agents (32
). Although NKT cells are involved in both Th1 and Th2 immunity through IFN-γ or IL-4 production, the immunomodulating protocol using α-GalCer and IL-12 preferentially induces NKT cells that produce large amounts of IFN-γ (34
). These NKT cells may facilitate the development of Th1-dominant cellular immunity essential for the induction of protective immunity against tumors and some infectious agents. Recently, it was demonstrated that α-GalCer can stimulate human NKT cells in a CD1d-dependent manner (35
), indicating that our proposed immunotherapy protocol using α-GalCer and IL-12 will be useful for the application to human immune diseases, including cancer.