Figure 3

S1P and FTY720 suppress NKT cell cytokine production through S1P₁. (a) Sorted NK1.1⁺TCR-β⁺ NKT cells (2 × 10⁵ per well) (upper panel) were incubated with S1P (1 μg/ml) or FTY720p (1 μg/ml) in the presence or absence of anti-CD3 mAb (0.5 μg/ml) for 48 h in 12-well plates and CD4⁺ T cells (1 × 10⁶ per well) (lower panel) were incubated in the presence or absence of anti-CD3 mAb(1 μg/ml)+anti-CD28(1 μg/ml) mAbs. The amount of IL-4, IFN-γ, IL-10, or IL-13 in the culture supernatant was measured by ELISA. (NS, not significant, ^*P<0.05 and ^**P<0.01, anti-CD3 vs anti-CD3+S1P or anti-CD3+FTY720p, anti-CD3+anti-CD28 mAbs vs anti-CD3+anti-CD28 mAbs+S1P or anti-CD3+anti-CD28 mAbs+FTY720p). (b) B6 and CD1d^−/− mice were intraperitoneally injected with S1P (20 μg in 300 μl PBS per mouse) or FTY720, or with α-GalCer (1 μg in 300 μl PBS)+S1P or FTY720, and IL-4, IFN-γ, IL-10, and IL-13 were measured at various time points by ELISA. The data shown are the means±s.d. for three mice in each group. Similar results were obtained from three independent experiments. (^*P<0.05 and ^**P<0.01, B6 mice +α-GalCer vs B6 mice+α-GalCer+S1P or B6 mice+α-GalCer+FTY720). (c) Liver mononuclear cells from the B6 mice were incubated with α-GalCer (220 ng), S1P (1 μg), PTX (50 ng), or FTY720p (1 μg), or with PTX, PTX+S1P, or FTY720p in the presence of α-GalCer. The production of IL-4 or IFN-γ was then measured in the culture supernatant by ELISA. The results shown are representative of four independent experiments (^*P<0.05 and ^**P<0.01). (d) Liver mononuclear cells from the B6 mice were incubated with α-GalCer (220 ng/ml) or with α-GalCer+S1P or FTY720p for 24 h, and the intracellular expression of T-bet and GATA-3 in gated NK1.1⁺TCR-β⁺ NKT or NK1.1⁻TCR-β⁺ T cells was evaluated. (^*P<0.05 and ^**P<0.01 α-GalCer vs α-GalCer +S1P or α-GalCer+FTY720). (e) The expression levels of T-bet and GATA-3 was estimated in B6 mouse splenocytes and liver mononuclear cells incubated with S1P (1 μg/ml) or FTY720p (1 μg/ml) in the presence of α-GalCer (200 ng/ml) for 24 h using real-time PCR. (^**P<0.01 α-GalCer vs α-GalCer+S1P or α-GalCer +FTY720). (Panels a–e) The data shown are the means±s.d. for three mice in each group, and representative of three independent experiments.