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. Author manuscript; available in PMC: 2020 Apr 27.
Published in final edited form as: Clin Cancer Res. 2018 Jan 23;24(8):1891–1904. doi: 10.1158/1078-0432.CCR-17-0691

Figure 2: iNOS inhibition restores FcR mediated NK cell functions and signal transduction in the presence of MDSC.

Figure 2:

Female Balb/c mice were inoculated with 1x105 4T1 tumor cells or were left un-injected (no tumor-control). Following the establishment of tumors, mice were treated daily with (a) intraperitoneal injections of IgG (250 μg), anti-TGF-β antibody (200 μg), or anti-IL-10 antibody (250 μg) for four consecutive days prior to NK cell isolation for the ADCC assay. The mice were sacrificed 24 hrs after the last treatment. Each group consists of pooled samples from spleens of 4-5 mice. Values represent mean ± SD from one experiment. (b) Mice were treated with PBS (vehicle), arginase inhibitor nor-NOHA (20 mg/kg) i.p or the IDO inhibitor 3-methyltryptophan (MT, 400 mg/kg) via oral gavage prior to NK cell isolation for the ADCC assay. (c) Mice were given intraperitoneal injections of PBS (vehicle) or iNOS inhibitor, L-NIL (20 mg/kg) for one week. NK cells isolated from the spleen were employed in a standard ADCC assay using trastuzumab-coated CT26-HER2 positive tumor cells as targets. (d) NK cells and MDSC from the peripheral blood of melanoma patients were co-cultured overnight at a 1:1 ratio with or without the nitric oxide inhibitor L-NIL (2.5 mM). ADCC function of NK cells displayed as the mean percent lysis of cetuximab-coated HT-29 tumor cells at the 10:1 effector to target ratio. Means ± SE from four independent experiments are shown, p<0.05. Significance was determined using a linear mixed model. Treatment of NK cells with L-NIL alone did not enhance ADCC activity (not shown). (e) Autologous NK cells and MDSC isolated from peripheral blood of melanoma patients were co-cultured in 96 well plates coated with human IgG or media with or without the iNOS inhibitor L-NIL (2.5 mM). Supernatants were harvested after 48 hrs and analyzed for levels of IFNγ by ELISA. Values represent mean ± SE from three independent experiments, p<0.05. Significance was determined using a linear mixed model. (f) p-Erk expression in NK cells co-cultured overnight with MDSC in the presence or absence of L-NIL (2.5 mM). p-Erk levels are expressed as the average fold change ± SE from three independent experiments, p<0.05. Significance was determined using a linear mixed model.