ArrayExpress Uploaderarrayexpress_sid7525shRNA targeting Lacz1-sp1spleenshRNA targeting Lacz1-sp2spleenshRNA targeting Lacz1-sp3spleenshRNA targeting Ppp2r2d2-sp1spleenshRNA targeting Ppp2r2d2-sp2spleenshRNA targeting Ppp2r2d2-sp3spleenshRNA targeting Egr23-sp1spleenshRNA targeting Egr23-sp2spleenshRNA targeting Egr23-sp3spleenshRNA targeting Ptpn24-sp1spleenshRNA targeting Ptpn24-sp2spleenshRNA targeting Ptpn24-sp3spleenshRNA targeting Arhgap55-sp1spleenshRNA targeting Arhgap55-sp2spleenshRNA targeting Arhgap55-sp3spleenshRNA targeting Alk6-sp1spleenshRNA targeting Alk6-sp2spleenshRNA targeting Alk6-sp3spleenshRNA targeting Lacz1-tu1TumorshRNA targeting Lacz1-tu2TumorshRNA targeting Lacz1-tu3TumorshRNA targeting Ppp2r2d2-tu1TumorshRNA targeting Ppp2r2d2-tu2TumorshRNA targeting Ppp2r2d2-tu3TumorshRNA targeting Egr23-tu1TumorshRNA targeting Egr23-tu2TumorshRNA targeting Egr23-tu3TumorshRNA targeting Ptpn24-tu1TumorshRNA targeting Ptpn24-tu2TumorshRNA targeting Ptpn24-tu3TumorshRNA targeting Arhgap55-tu1TumorshRNA targeting Arhgap55-tu2TumorshRNA targeting Arhgap55-tu3TumorshRNA targeting Alk6-tu1TumorshRNA targeting Alk6-tu2TumorshRNA targeting Alk6-tu3Tumor06Recent work has shown that cytotoxic T cells play a central role in immune-mediated control of cancers1-3, and monoclonal antibodies that...24476824E-GEOD-53388/profile/8773/arrayexpressuploader336cellcentraldeepmelanomaspleenDec.12, 2014FalseE-GEOD-53388_A-AFFY-45immunotherapy-targets-in-tumorsImmunotherapy Targets in TumorsNov.12, 2014Recent work has shown that cytotoxic T cells play a central role in immune-mediated control of cancers1-3, and monoclonal antibodies that target inhibitory receptors on T cells can induce significant clinical benefit in patients despite advanced disease4-6. However, many of the regulatory pathways that result in loss of T cell function within immunosuppressive tumors remain unknown. Here we show that such regulatory mechanisms can be systematically discovered in vivo in the tumor microenvironment. We devised a pool shRNA screening approach aimed at identifying genes that block the function of tumor-infiltrating CD8 T cells. We postulated that shRNAs targeting key inhibitors would enable robust T cell infiltration and proliferation in tumors, despite multiple inhibitory signals. Candidate shRNAs were discovered by transfer of shRNA-transduced T cells into tumor-bearing mice, followed by deep sequencing to quantify the representation of all hairpins in tumors and lymphoid organs. A subset of shRNAs induced T cell accumulation in tumors but not the spleen, demonstrating feasibility of discovering shRNAs with differential action across tissues. One of the targets was Ppp2r2d, a regulatory subunit of the family of PP2A phosphatases7. Control shRNA-transduced T cells underwent apoptosis upon recognition of melanoma cells, while Ppp2r2d shRNA-transduced T cells accumulated in tumors due to enhanced proliferation and reduced apoptosis. Ppp2r2d shRNAexpressing T cells also significantly delayed tumor growth. This in vivo approach has wide applications to dissect complex immune functions in relevant tissue microenvironments. OT-I derived T cells were transduced with shRNA's and adoptively transferred to B16 tumor bearing mice. Following incubation the T cells were purified from either tumors or spleens. The shRNA's targeted either a control gene (LacZ) or one of 5 selected genes found to regulate the presence of T cells in tumors vs in spleenhttp://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-53388mousehttp://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-53388/samples/