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Having demonstrated that PDE5 inhibition can impair MDSC suppressive mechanisms in BALB/c and C57BL/6 tumor-bearing mice, we next sought to determine whether similar results could be obtained in humans. Head and neck cancers are known to be highly immunosuppressive. Their high levels of GM-CSF production are likely the major mediator of immune suppression observed in these patients and are probably responsible for the intratumoral infiltration by MDSCs (36). In fact, peripheral blood lymphocytes (PBLs) from these patients are functionally impaired in their ability to be activated and to proliferate upon stimulation (37). Similar results were also seen in prostate cancer (38) and in nonsmall cell cancer (35). Although this anergic state in solid tumors may be attributable to the ARG1- and/or NOS-dependent suppressive activity of MDSCs, MDSC-mediated immunosuppression has not been previously reported in hematological malignancies. PBMCs from MM patients were stimulated with anti-CD3/CD28 antibody-coated beads in the presence of N-(omega)-hydroxy-nor-L-arginine (NorNOHA; an ARG1-specific inhibitor), NG-monomethyl-L-arginine (L-NMMA; an NOS2 inhibitor), both inhibitors, or neither. As shown in Fig. 8 A (top), T cell expansion was considerably enhanced in the presence of both NorNOHA and L-NMMA, whereas the single inhibitors failed to increase T cell proliferation over the baseline. Interestingly, sildenafil yielded results equivalent to the combination of NorNOHA and L-NMMA. These results suggest involvement of both ARG1 and NOS2 in MDSC-mediated immunosuppression in myeloma and confirm the in vitro results demonstrating the ability of PDE5 inhibitors to affect both pathways. Recent data from our lab identified human MDSCs as ARG+, CD14+ cells (unpublished data). We therefore examined T cell expansion in CD14+-depleted PBMCs under the same conditions (Fig. 8 A, bottom). Although CD14+ depletion alone increased CD3+ T cell expansion fourfold, pharmacologic inhibitors failed to further enhance proliferation, suggesting that CD14+ cells are the mediators of ARG1- and NOS2-mediated immunosuppression in MM. As seen with purified mouse T cells (Fig. 5 B), sildenafil also failed to enhance the CD3+ T cell proliferation of CD14-depleted PBMCs from cancer patients.
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