Enhancement of the synthetic ligand-mediated function of liver NK1.1Ag+ T cells in mice by interleukin-12 pretreatment

Abstract

We previously reported that mouse NK1.1 Ag+ T (NKT) cells activated by interleukin-12 (IL-12) act as anti-tumour/anti-metastatic effectors. However, IL-12 reportedly induces a rapid disappearance of liver NKT cells by activation-induced apoptosis. In the present study, however, we show that injection of IL-12 into mice merely down-regulates the NK1.1 expression of liver NKT cells and Vbeta8+ intermediate T-cell receptor cells and CD1d/alpha-galactosylceramide (alpha-GalCer)-tetramer reactive cells in the liver remained and did not decrease. Furthermore, when IL-12-pretreated (24 hr before) mice were injected with alpha-GalCer, not only serum interferon-gamma but also serum IL-4 concentrations increased several-fold in comparison to the control alpha-GalCer-injected mice. However, IL-12 pretreatment markedly up-regulated serum ALT levels and Fas-ligand expression on NKT cells after alpha-GalCer injection in middle-aged mice only. Consistently, the liver mononuclear cells (MNC) from IL-12-pretreated mice stimulated with alpha-GalCer in vitro produced much greater amounts of interferon-gamma and IL-4, and also showed a more potent cytotoxicity against tumour targets than those from mice pretreated with phosphate-buffered saline. Liver MNC from middle-aged mice, but not from young mice pretreated with IL-12, also showed increased cytotoxicity following in vitro alpha-GalCer stimulation against cultured hepatocytes. Furthermore, IL-12 treatment of middle-aged mice enhanced tumour necrosis factor receptor 1 mRNA expression in liver Vbeta8+ T cells, and in vitro experiments also revealed that IL-12 pretreatment of liver MNC from middle-aged mice enhanced their tumour necrosis factor-alpha production after alpha-GalCer stimulation. Synthetic ligand-mediated functions of NKT cells, including IL-4 production, are thus enhanced by IL-12 pretreatment.

Figure 1

NK1.1⁺T cells in the liver became undetectable after IL-12 injection into mice, whereas Vβ8⁺T cells with lower TCR and CD1d/α-GalCer tetramer binding Vβ8⁺T cells remain detectable. (a) NK1.1 expression of liver αβT cells. The middle-aged (25-week-old) mice were injected intraperitoneally with IL-12 and livers were obtained at the indicated times after IL-12 injections. Liver MNC were stained and analysed. Numbers of upper quadrants represent percentage of NK cells and NK1.1⁺T cells in the liver MNC. (b) Vβ8 expression of liver αβT cells. Numbers within left panels represent percentage ofVβ8T cells with intermediate TCR in whole liver MNC (indicated by squares). (c) CD1/α-GalCer tetramer binding Vβ8⁺T cells. Livers were obtained from middle-aged mice 24 hr after IL-12 injection and then the liver MNC were stained and analysed.

Figure 2

The serum IFN-γ, IL-4 and ALT levels after α-GalCer administration in young (6 weeks) and middle-aged (25 weeks) mice pretreated with IL-12 or PBS. Young and middle-aged mice pretreated with IL-12 or PBS 24 hr before were administered with α-GalCer intravenously and peripheral blood samples from five individual mice were collected at the indicated time points from the retro-orbital plexus. The data are expressed as the mean ± SE from five mice of each group.

Figure 3

(a) In vitroα-GalCer-stimulated IFN-γ and IL-4 production from liver MNC of mice pretreated with IL-12 or PBS. Liver MNC were obtained from young or middle-aged mice pretreated 24 hr before with IL-12 or PBS were stimulated with α-GalCer in vitro for 24 hr and culture supernatants were subjected to ELISA. (b) In vitroα-GalCer-stimulated IFN-γ and IL-4 production from liver MNC pretreated with IL-12 (25 ng/ml). Liver MNC were obtained from young or middle-aged mice and cultured with medium supplemented with IL-12 (in 5 μl PBS, final concentration; 25 ng/ml)or 5 μl PBS for 24 hr and thereafter were stimulated with α-GalCer in vitro for 24 hr and culture supernatants were subjected to ELISA. The data are expressed as the mean ± SE from four mice of each group.

Figure 4

Intracellular IFN-γ expression of liver MNC after α-GalCer administration. One hour after α-GalCer injection into middle-aged mice pretreated with IL-12 or PBS, liver MNC were isolated and stained with PE-antiαβ TCR antibody or FITC-anti-IFN-γ antibody as described in the Materials and Methods. Proportions (%) of IFN-γ-expressing cells in whole liver MNC are shown in left panels, while the mean fluorescence intensities (MFI) are shown in right panels (mean ± SE of three mice in each group). Similar results were obtained in two additional experiments.

Figure 5

The effect of IL-12 pretreatment in mice on the FasL expression on liver NKT cells after α-GalCer administration. One hour after α-GalCer administration, liver MNC were isolated and stained as described in the and analysed. Similar results were obtained in two additional experiments.

Figure 6

The effect of IL-12 pretreatment in middle-aged and young mice on the cytotoxicity of liver MNC against cultured hepatocytes stimulated with α-GalCer in vitro. The data are expressed as the mean ± SE from three independent experiments.

Figure 7

The serum TNF-α levels in mice after α-GalCer injection. Young and middle-aged mice pretreated with IL-12 or PBS 24 hr before were administered intravenously with α-GalCer and peripheral blood samples from mice were collected at the indicated time points from the retro-orbital plexus. The data are expressed as the mean ± SE from five mice of each group.

Figure 8

In vitro TNF-α production from liver MNC stimulated with α-GalCer. Liver MNC were isolated from livers of young and middle-aged mice and cultured with IL-12 (PBS as a control) for 24 hr and were further stimulated with α-GalCer and cultured for 24 hr. Culture supernatants at 2, 5 and 24 hr after α-GalCer stimulation were collected and were subjected to ELISA. The data are expressed as the mean ± SE from four mice of each group.

Figure 9

IL-12 receptor and TNF-α receptor expressions of liver Vβ8⁺T cells. (a) IL-12Rβ1 mRNA expression in Vβ8⁺liver MNC from young and middle-aged mice (upper panels) and mRNA expression levels relative to those of GAPDH (lower panels). (b) TNFR1 mRNA expression in Vβ8⁺ liver MNC from young and middle-aged mice pretreated with IL-12 or PBS (control) (upper panels) and the mRNA expression levels relative to those of GAPDH (lower panels). PCR bands of IL-12Rβ1 and TNFR1 were quantified based on the ratio of the signal intensity of the PCR products to those of the internal controls (GAPDH) as described in the Materials and Methods.