Figure 8. Analysis of the functional characteristics of MDSC. Panel A: MDSC were isolated from IL-2 and IL-2+resveratrol treatment (labeled as IL-MDSC and R-MDSC respectively). LAK cells from IL-2 treated mice were isolated and incubated with MDSC for 2 h, and then used as effectors in the cytotoxicity assay against EC cell targets. Panel B: 3H-thymidine incorporation assay to measure the proliferation of the LAK cells in the panel A. Panel C: 16106 MDSC were intravenously transferred to naive mice. VLS was then induced in the recipient mice. Panel D & E: Total RNA was extracted from the MDSC with the respective treatment. Expression of Arg1 (D) and AhR (E) were detected by QPCR. The relative expression was normalized to the endogenous 18S. doi:10.1371/journal.pone.0035650.g008 To further evaluate whether these MDSCs play a suppressive role in the development of VLS in vivo, we transferred these MDSCs intravenously into na¨ıve mice and induced VLS in the recipient mice on the same day of the cell transfer. The results showed that R-MDSC caused marked inhibition of VLS; again, IL-MDSC did not inhibit VLS at all (Fig. 8C). Correspondently, we found that resveratrol induced the expression of Arg1 in MDSC but IL-2 did not. Interestingly, IL-2 and resveratrol co- treatment induced the most expression of Arg1 (Fig. 8D). This could be because resveratrol is more effective in activated cells than unactivated cells [30]. We also found that R-MDSC and IL- MDSC have different expression level of AhR, which is the main receptor for resveratrol-derived signaling [23]. We found that resveratrol increased the expression of AhR in MDSC but IL-2 did not. Similar to the expression of Arg 1, IL-2 and resveratrol co- treatment induced the most expression of AhR (Fig. 8E). All together, our results indicated that resveratrol can elicit or Resveratrol in IL-2 Therapy against Melanoma enhance the suppressive functionality of MDSC that could contribute to the suppression of VLS. Resveratrol induced expression of FoxO1 in tumor cells Thus far, we demonstrated that resveratrol can ameliorate IL-2 toxicity while promoting the effectiveness of IL-2 in metastatic melanoma therapy. We showed that resveratrol-treated MDSC could contribute to the suppression of the cytolytic activity of LAK cells on ECs. However, MDSC are generally recognized for their role in helping evasion of tumor by suppressing the host immunity in situ of the tumor. Our results raised the possibility that resveratrol possesses the specific characteristic that can overcome the suppressive effect of MDSC to the tumor. To explore the possible mechanisms, we studied whether resveratrol played a role in the expression of the tumor suppressor genes by analysis of FoxO1 gene expression. To this end, we resected the primary melanoma in the skin and examined the transcripts of FoxO1 by QPCR. The results showed that either resveratrol or IL-2 treatment can induce the expression of FoxO1 in the tumor in vivo. IL-2 and resveratrol co-treatment induced the highest expression of FoxO1 (Fig. 9A). Correspondently, the growth of the tumor was significantly inhibited by the treatment which was demonstrated by the decreased tumor weight while IL-2 and resveratrol co-treatment can exert the maximal inhibitory effects on the tumor growth (Fig. 9B). We also cultured B16F10 cells with IL-2 and/or different dose of resveratrol. We found that resveratrol could induce the expression of FoxO1 in a dose-dependant manner. However, IL-2 alone did not induce the expression of FoxO1. This concentration of IL-2 is effective in LAK cell development [26]. With IL-2 and resveratrol co-treatment, the expression levels of FoxO1 were not different from the resveratrol treatment alone (Fig. 9C), which suggest that IL-2 does not directly affect the expression of FoxO1 in B16F10 tumor cells. However, in the in vivo experiment, HDIL-2 administration did induce the expression of FoxO1 in the primary tumors (Fig. 9A). These results indicate that the release of other factors from the HDIL-2-induced inflammation could trigger the expression of FoxO1 in the tumor, and such factors work together with resveratrol co-treatment to further enhance the expression of FoxO1. Resveratrol makes melanoma more susceptible to the cytotoxicity of LAK cells by increasing the sensitivity of the tumor cells to the cytotoxicity It has been shown that resveratrol can suppress melanoma growth by inducing the cell-cycle interruption and apoptosis in the tumor cells; resveratrol also can enhance the chemical cytotoxicity to the tumor by the chemotherapeutic agents [24,25]. In this study, we studied whether resveratrol could affect the LAK cytotoxicity to the tumor. To this end, LAK cells were used as the effectors and generated in vitro from splenocytes of normal mice as described in the Materials and Methods. B16F10 cells were used as the targets. We pre-incubated the targets with resveratrol at 25 mM for 2 h at 37uC before the 4 h cytotoxicity assay. The results showed that the pre-treatment of resveratrol significantly increased the number of the killed targets by LAK cells (Fig. 9D). The above results suggested that resveratrol can directly induce the expression of the tumor suppressor gene FoxO1, thereby leading to the suppression of the tumor growth and cell death by triggering autophagy [31,32]. IL-2 can facilitate such effects by the unknown factors from the HDIL-2-induced inflammation. Mean- while, resveratrol directly enhances the susceptibility of B16F10 tumor cells to the LAK cytotoxicity. Together, these mechanisms PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35650