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Furthermore, numerous factors are present in tumor-bearing hosts that could impair the generation of an effective immune response such as the defective maturation of DCs (7), the accumulation of suppressive MDSCs (4), T cell anergy (27), and/or the accumulation of T reg cells (28). These populations were, therefore, examined separately. Flow cytometric analysis of sildenafil-treated or untreated mice failed to reveal important differences in DC maturation, CD4+Foxp3+ T reg cells, or CD11b+/Gr-1+ MDSC accumulation (unpublished). We evaluated the effect of in vitro PDE5 inhibition on purified CD11c+, CD11b+, CD4+, and CD8+ cells isolated from C26GM tumor-bearing mice. This enabled us to examine purified populations, thereby eliminating the potential for exogenous influences. Although sildenafil treatment increased the intracellular concentration of cGMP in all the populations examined (Fig. 5 A), it had no effect on the proliferation of purified CD4+ and CD8+ T cells stimulated with either ConA (Fig. 5 B) or anti-CD3/CD28 beads (Fig. S5, available at http://www.jem.org/cgi/content/full/jem.20061104/DC1), nor on CD11c maturation (Fig. 5 C) as determined by B7.2 or MHC class II up-regulation. It did, however, demonstrate the ability to reverse the suppressive phenotype of MDSCs. In light of recent data identifying IL-4R as a functional suppressive marker for MDSCs (29), we examined IL-4R expression on CD11b+ cells cultured in the presence or absence of sildenafil. Fig. 5 D shows that sildenafil significantly decreases IL-4R expression on MDSCs, suggesting a down-regulation of their suppressive pathways. 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The following cell lines were used: CT26, a carcinogen-induced, undifferentiated colon carcinoma obtained from BALB/c mice (51); TS/A, a mouse mammary adenocarcinoma derived from BALB/c mice (52); MCA203, a C57BL/6-derived fibrosarcoma (53); and B16-GM, a C57BL/6 melanoma cell line genetically modified to secrete GM-CSF (54). The 4T1-HA cell line was obtained by lentiviral transduction of 4T1 mammary carcinoma and was provided by K. Whartenby (Johns Hopkins University, Baltimore, MD). These cell lines were grown in DMEM or RPMI 1640 (Invitrogen) with 10% FBS (Invitrogen). The C26GM cell line derived from the C26 colon carcinoma was genetically modified to produce GM-CSF (8) and was grown in the presence of 800 µg/ml G418.