Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-γ, GM-CSF, or IL-4, and on DCs by IFN-γ, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-γ, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.
The natural killer T (NKT) cell ligand α-galactosylceramide (α-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12–mediated antitumor activities. Because of these similarities between the activities of α-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by α-GalCer. We first established, using purified subsets of various lymphocyte populations, that α-GalCer selectively activates NKT cells for production of interferon (IFN)-γ. Production of IFN-γ by NKT cells in response to α-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, α-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1−/− or Vα14−/− mice. This effect of α-GalCer required the production of IFN-γ by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of α-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-γ production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by α-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.
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