Resveratrol in IL-2 Therapy against Melanoma Figure 7. Expansion of MDSC by IL-2 and/or resveratrol treatment. Splenocytes and the infiltrating cells were stained with FITC-conjugated anti-CD11b and PE-conjugated anti-Gr-1 mAbs. The stained cells were analyzed by flow cytometry. The representative dot plots from spleen (A), lung (B), and liver (C) and the statistical analysis of the percentage mean 6SEM of MDSC (D) were shown. doi:10.1371/journal.pone.0035650.g007 resveratrol can prevent VLS by protecting ECs from ultrastruc- tural damage and apoptosis. Resveratrol induced expansion and suppressive functionality of myeloid-derived suppressor cells (MDSC) To explore the underneath mechanisms that contribute to the suppressive function of resveratrol on VLS, we studied the contribution of the suppressor cells in the inhibition of VLS by resveratrol. To this end, we focused on studies of Treg and MDSC. Our previous study showed that IL-2 treatment could induce expansion of CD4+FoxP3+ Treg; these Treg can suppress the cytolytic killing of ECs by LAK cells in vitro [12]. In the current study, we measured the percentage of Treg in spleen, lung and liver. On day 12, the splenocytes and the infiltrating-mononuclear cells from lung and liver were prepared for staining of CD4 and FoxP3. The results showed that IL-2 caused an expansion of Treg in spleen, lung and liver. However, resveratrol treatment did not induce their expansion; instead, it inhibited Treg expansion to some extent (Fig. 6). Simultaneously, we stained cells for Gr-1 and CD11b expression. The results showed that either resveratrol or IL-2 could induce the expansion of MDSC. Moreover, combina- tion of IL-2+resveratrol caused a further induction of MDSC (Fig. 7). These results suggested the possibility that MDSC rather than Treg probably contributed to the suppression of VLS in resveratrol treatment. To address this further, we examined the suppressive ability of MDSC on LAK cytotoxicity and proliferation. To this end, LAK cells from IL-2-treated mice were isolated, and then incubated with MDSC at 10:1 ratio (LAK: MDSC) for 2 h at 37uC. For the cytotoxicity assay, the mixture of the culture was added to 51Cr- labeled EC target (TME cell line) at 50:1 ratio (E:T) followed by 4 h 51Cr-release assay. For the proliferation assay, the mixture of the culture was supplemented with 3H-thymidine and continued culture for 16 h and then the 3H-thymidine incorporation was measured. To our surprise, the results showed that MDSC from IL-2+resveratrol group (named as R-MDSC) significantly inhib- ited LAK cytotoxicity and proliferation; however, MDSC from IL- 2 group (named as IL-MDSC) did not show any suppressive effects on either LAK cytotoxicity or proliferation (Fig. 8A & 8B). PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35650