Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated Valpha14 NKT cells

Abstract

We have recently identified alpha-galactosylceramide (alpha-GalCer) as a specific ligand for an invariant Valpha14/Vbeta8.2 T cell receptor exclusively expressed on the majority of Valpha14 NKT cells, a novel subset of lymphocytes. Here, we report that alpha-GalCer selectively activates Valpha14 NKT cells resulting in prevention of tumor metastasis. The effector mechanisms of the ligand-activated Valpha14 NKT cells seem to be mediated by natural killer (NK)-like nonspecific cytotoxicity. Indeed, the cytotoxic index obtained by alpha-GalCer-activated Valpha14 NKT cells was reduced by the addition of cold target tumor cells or by treatment with concanamycin A, which inhibits activation and secretion of perforin, but not by mAbs against molecules involved in the NKT cell recognition and conventional cytotoxicity, such as CD1d, Vbeta8, NK1. 1, Ly49C, Fas, or Fas ligand. These results suggest that the ligand-activated Valpha14 NKT cells kill tumor cells directly through a CD1d/Valpha14 T cell receptor-independent, NK-like mechanism.

Figure 1

Inhibition of liver metastasis of B16 melanoma cells by in vivo treatment with α-GalCer. (A) Melanoma antigens in metastasized livers measured by RIA using ¹²⁵I-labeled M2590 mAb. B16 melanoma cells (3 × 10⁶ per mouse) were inoculated in the spleen of wild-type (Jα281^+/+), Vα14 NKT-deficient (Jα281^−/−), and Vα14 NKT (RAG^−/−/Vα14tg Vβ8.2tg) mice, followed by intraperitoneal injection with either α-GalCer (100 μg/kg) or vehicle on days 1, 5, and 9. The mice were sacrificed and examined on day 14. Each group consisted of three mice. The data are expressed as a mean cpm value of triplicate samples with standard errors. (B) Photographic views of metastasized livers from mice treated with α-GalCer or vehicle.

Figure 2

Cytotoxicity of spleen cells from Vα14 NKT mice after in vivo treatment with α-GalCer. Vα14 NKT mice were injected intraperitoneally with either α-GalCer (100 μg/kg) or vehicle, and 24 hr later spleen cells were used as effector cells for cytotoxic assay on B16 melanoma cells at indicated effector/target ratios.

Figure 3

Effector mechanism of α-GalCer-activated Vα14 NKT cells. (A) α-GalCer-activated Vα14 NKT cells were incubated with ⁵¹Cr-labeled B16 melanoma cells in the presence of various numbers of unlabeled tumor cells (B16 or EL-4) as cold targets. Cytotoxic assays were performed at an E/T ratio of 25:1. (B) Effect of CMA treatment on α-GalCer-activated Vα14 NKT cells. α-GalCer-activated Vα14 NKT cells were treated with indicated doses of CMA for 2 hr, and their cytotoxic activity was assessed on ⁵¹Cr-labeled B16 melanoma at an E/T ratio of 50:1.

Figure 4

Molecular requirements in the effector phase of cytotoxic function of α-GalCer-activated Vα14 NKT cells. (A) Effects of various mAbs on Vα14 NKT cell-mediated cytotoxicity. Fifty micrograms per ml of the indicated mAbs were added to the cytotoxic assays. (B) Cytotoxic activity of Vα14 NKT cells on α-GalCer-pulsed class I-deficient RMA-S cells and their CD1d-transfectants. RMA-S and RMA-S-CD1.1 cells were pulsed with α-GalCer and used as target cells. The data are expressed as a mean value of triplicate cultures with standard deviations.

Figure 5

Cytotoxicity of NK and Vα14 NKT cells against FBL-3 erythroleukemia cells. (A) Fluorescence-activated cell sorter profiles of FBL-3. Cells were stained with anti-H-2K^b (AF6–88.5) or H-2D^b (KH95) antibodies. Isotype-matched antibody was used as a negative control. (B) Cytotoxic activity. Freshly isolated spleen cells from Vα14 NKT mice (•) or RAG^−/− (NK) mice (○) were assayed for their cytotoxicity by the 4-hr ⁵¹Cr-release assay on FBL-3 tumor cells expressing MHC class I molecules on the cell surface.