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. 2017 Feb 13;8:129. doi: 10.3389/fimmu.2017.00129

Figure 5.

Figure 5

Myeloid-derived suppressor cells (MDSCs) inhibited IFN-γ production of NKT cells via membrane-bounded transforming growth factor β (TGF-β) in a cell contact-dependent manner. (A) Membrane-bound TGF-β is the main factor for suppression of NKT cell IFN-γ secretion. NKT cells (CD3+NK1.1+) were isolated from liver tissues of wild-type (WT) mice, co-cultured with MDSCs sorted from livers of tumor-bearing (TB) mice at the ratio of 1:1 under various conditions, including either transwell (0.4 µm) separation or in the presence of 10 ng/ml anti-TGF-β1 mAb or 0.5 ng/ml rmTGF-β1 for 6 h, and then cells were then stimulated with PMA (25 ng/ml) plus ionomycin (1 µg/ml). Eighteen hours later, supernatants were harvested for determination of IFN-γ levels using ELISA kit. Results (mean ± SEM) of triplicated wells from one representative experiment are shown. (B) Expression levels of arginase 1 (Arg-1) and inducible nitric oxide synthase (iNOS) were not changed in liver tissues of TB mice. Livers were isolated from either WT or TB mice (day 15 posttumor inoculation), and mRNA levels of Arg-1 and iNOS were determined via quantitative real-time PCR (n = 3). Results from one representative experiment are shown. (C) Reactive oxygen species (ROS) expression was increased in the liver of TB mice. Livers were isolated from either WT or TB mice 15 days posttumor inoculation. ROS levels were determined via ROS assay kit as described in Section “Materials and Methods” (n = 3). Results from one representative experiment are shown. (D) Arg-1, iNOS, and ROS inhibitors failed to restore IFN production of NKT cells in vitro. NKT cells were co-cultured with MDSCs isolated from the liver of TB mice at the ratio of 1:1 as described above in the absence or presence of various inhibitors, including N-hydroxy-nor-arginine (1 mM), catalase (1,000 U/ml), or l-NIL (0.5 µM) for 6 h. Then cells were stimulated with PMA plus ionomycin, and IFN-γ levels at the supernatant were determined using ELISA kit. Results from triplicated wells (mean ± SEM) are shown.