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The experiments described thus far demonstrate the ability of PDE5 inhibition to prime/augment antitumor immunity. Yet the mechanisms resulting in T cell activation are unknown. Because PDE5 is expressed in various cells of the immune system (including DCs, macrophages, and T cells [26]), PDE5 inhibitors could putatively target these various populations. 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failed to reveal important differences in DC maturation, CD4+Foxp3+ T reg cells, or CD11b+/Gr-1+ MDSC accumulation (unpublished).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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Figure 2. Sildenafil enhances antitumor CTL activity in vivo. (A) BALB/c mice were either challenged with CT26WT cells, vaccinated with 106 -irradiated CT26WT cells, or injected with PBS on day 0. Sildenafil was added to the drinking water where indicated. On day 12, all groups received 107 AH-1–pulsed CFSEhigh-labeled BALB/c splenocytes admixed with 107 HA-pulsed CFSElow-labeled BALB/c splenocytes. The mice were killed 40 h later. Single cell suspensions from spleens and tumor draining lymph nodes were analyzed by FACS. (B) BALB/c mice were either challenged with C26GM cells, vaccinated with 106 -irradiated C26GM cells, or injected with PBS on day 0. Sildenafil was added to the drinking water on day 0. After 5 d, the mice received CFSE-labeled target splenocytes as described in A. Analysis was performed as described in the Materials and methods.
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To extend this finding to other models, the experiment was repeated using the 4T1 mammary carcinoma genetically modified to express the influenza-derived HA as a model tumor antigen (Fig. 3 B). Mice were challenged on day 0 with 0.5 x 106 4T1-HA cells. On the next day they received 3 x 106 HA-specific naive CD8+ T cells. After adoptive transfer, the mice were either treated with sildenafil or left untreated. As with the C26GM experiments, no therapeutic benefit was observed with ACT alone, whereas sildenafil + ACT imparted the greatest antitumor benefit. Sildenafil treatment alone, instead, substantially delayed tumor progression in immunocompetent BALB/c mice but not in immune-deficient Rag-2–/– mice (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20061104/DC1). These data confirm the immunomodulatory properties of PDE5 inhibitors in augmenting the therapeutic efficacy of ACT and demonstrate a role of sildenafil in modifying the tumor microenvironment rendering it more susceptible to CTL-mediated cytotoxicity.
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