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  • br Materials and Methods br Results br


    Materials and Methods
    Discussion Even though T cell checkpoint inhibitors alone have achieved impressive clinical benefit in some cancers, their application as single agents has had limited efficacy (Hamid et al., 2013; Wolchok et al., 2013). The resistance to immunotherapy is in part mediated by the immunosuppressive microenvironment in the tumor tissue, and identification of such mechanisms is highly prudent in order to develop appropriate combination strategies. Tumor-infiltrating suppressive myeloid puromycin selection concentration have been demonstrated to be an important component of the inhibitory tumor microenvironment and include macrophages with the M2 phenotype and MDSCs. Depending on the tumor model; different myeloid populations may be dominant. Reports have shown that some tumor models such as prostate cancer and lung carcinoma models are dominated by MDSCs and these MDSCs heavily infiltrate tumors and systemic organs such as spleen, blood, and bone marrow (Srivastava et al., 2012; Xu et al., 2013). In contrast, other tumor models have few MDSCs infiltrating the tumor or accumulating in the spleen, and are primarily dominated by macrophages (Mok et al., 2014; Pyonteck et al., 2013). While studying the role of IDO in immune suppression, we found that tumor IDO expression induces recruitment of MDSCs to tumors in several mouse tumor models (Holmgaard et al., 2015). We thus established an IDO-expressing tumor model which allowed for studies of MDSC targeting in both MDSC-high and MDSC-low tumor types. Here we find high expression of CSF-1R on the dominant suppressive subset of MDSCs in B16-IDO tumors. Our data demonstrate that CSF-1R blockade with PLX647 decreases the quantity of MDSCs in B16-IDO tumors, leading to increased anti-tumor immunity. The blockade alone modestly enhances antitumor responses, promotes CTL infiltration, and slows tumor progression. However, the therapeutic effect of such inhibition is limited and does not induce tumor regression. Although CSF-1R blockade enhances the anti-tumor activity of myeloid cells and T cell responses, we found that its efficacy was blunted by high expression and upregulation of immune checkpoint molecules, which was consistent with the recent data from Zhu et al. (2014) in a pancreatic tumor model. Inhibition of CSF-1R, however, markedly puromycin selection concentration improved the efficacy of checkpoint- and IDO-based immunotherapy and led to regression of established tumors. The antitumor activity of a combined therapy with CSF-1R and T cell checkpoint blockade was mediated by inhibition of the myeloid cell-mediated immunosuppressive tumor microenvironment, resulting in increased tumor infiltration with activated T cells. These studies thus suggest that the resistance to immune checkpoint blockade could be alleviated by therapeutic strategies that reprogram dominant myeloid responses to allow for effective checkpoint therapy. To this end, our group has shown that pre-treatment levels of circulating MDSCs can predict therapeutic outcomes from CTLA-4 blockade in melanoma patients (Kitano et al., 2014). The findings above were not only restricted to the B16-IDO model, but were also noted in established CT26 colon tumors. While the IDO inhibitor therapy synergized with CSF-1R blockade in the B16-IDO tumor model, it was not as effective as the synergy between T cell checkpoint blockade and CSF-1R blockade. Tumor infiltration of MDSCs might be a result of high levels of IDO expression as recently published (Holmgaard et al., 2015), and thus, IDO inhibitors and CSF-1R blockade potentially target the same immune pathway, whereas the T cell checkpoint blockade and CSF-1R blockade therapies act on different immune components and pathways. Several preclinical studies have suggested that inhibition of CSF-1R signaling may alter the immunologic response of tumor-infiltrating MDSCs and/or tumor-infiltrating macrophages (DeNardo et al., 2011; Mitchem et al., 2013; Mok et al., 2014; Priceman et al., 2010; Pyonteck et al., 2013; Sluijter et al., 2014; Strachan et al., 2013; Xu et al., 2013; Zhu et al., 2014). Mok et al. (2014) targeted CSF-1R signaling using PLX3397 in a murine SM1 melanoma model. PLX3397 treatment depleted more than 80% of tumor-infiltrating macrophages, leading to an increased efficacy of adoptively transferred T cell based therapies. Other studies have shown that CSF-1R blockade therapy reduced the number of MDSCs as well as macrophages in tumor and systemic organs (Priceman et al., 2010; Xu et al., 2013). Using the selective inhibitor of CSF-1R, GW2580, Priceman et al. (2010) demonstrated that CSR-1R signaling regulated recruitment of both MDSCs and M2 macrophages to lung, melanoma, and prostate tumors.