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MDSC suppressive activity was determined by admixing MDSCs with CFSE-labeled HA-specific CD8+ (clone 4) or CD4+ (6.5) T cells pulsed with their relevant peptide in the presence or absence of sildenafil (Fig. 7, A and B). Although the addition of tumor-derived MDSCs significantly impaired antigen-specific T cell proliferation as demonstrated by the low percentage of CFSElow clonotypic T cells, sildenafil almost completely restored both CD4+ and CD8+ responsiveness of these antigen-specific T cells. The absence of sildenafil-mediated enhancement in T cell function in the groups lacking CD11b+ cells underscores the targeted role of sildenafil on the MDSC population. Because in a Th1 cell�prone environment MDSC suppression is only NOS2 dependent (34), we examined the role of PDE5 in MDSCs in a C57BL/6 background where NOS2�/� mice are also available. CD11b+ MDSCs were isolated from either C57BL/6-NOS2+/+ or B16GM-bearing C57BL/6-NOS2�/� B16GM melanoma-bearing mice. A suppression assay was performed by stimulating OVA-specific CD4+ T cells with the relevant peptide in the presence or absence of MDSCs obtained from either NOS2+/+ or NOS2�/� tumor-bearing mice (Fig. 7 C). Although the addition of C57BL/6-NOS2+/+ MDSCs induced considerable T cell suppression, no suppression was observed with MDSCs from NOS2�/� mice. Furthermore, although PDE5 inhibition reversed MDSC suppression in NOS2+/+ mice, sildenafil failed to augment T cell responsiveness in the NOS2�/�-derived MDSC suppression assay. These results confirm the role of NOS2 in MDSC-mediated T cell suppression (Fig. 7 C) and underscore the ability of PDE5 inhibition to reverse the two major suppressive pathways in MDSCs (ARG1 and NOS2). Safety concerns and adverse effects have been studied carefully. The most common adverse effects are headaches and upper GI distress. These are not usually severe and are self-limited when the drug is stopped. 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