A high‐throughput RNAi screen for detection of immune‐checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes

The success of T cell‐based cancer immunotherapy is limited by tumor's resistance against killing by cytotoxic T lymphocytes (CTLs). Tumor‐immune resistance is mediated by cell surface ligands that engage immune‐inhibitory receptors on T cells. These ligands represent potent targets for therapeutic inhibition. So far, only few immune‐suppressive ligands have been identified. We here describe a rapid high‐throughput siRNA‐based screening approach that allows a comprehensive identification of ligands on human cancer cells that inhibit CTL‐mediated tumor cell killing. We exemplarily demonstrate that CCR9, which is expressed in many cancers, exerts strong immune‐regulatory effects on T cell responses in multiple tumors. Unlike PDL1, which inhibits TCR signaling, CCR9 regulates STAT signaling in T cells, resulting in reduced T‐helper‐1 cytokine secretion and reduced cytotoxic capacity. Moreover, inhibition of CCR9 expression on tumor cells facilitated immunotherapy of human tumors by tumor‐specific T cells in vivo . Taken together, this method allows a rapid and comprehensive determination of immune‐modulatory genes in human tumors which, as an entity, represent the ‘immune modulatome’ of cancer. Synopsis A novel in vitro screening methodology involving high‐throughput RNAi‐based gene knockdown in short‐term cocultures identifies immune‐modulatory molecules that mediate tumor resistance to cytotoxic T cells. Breast cancer cells express on their surface multiple molecules that inhibit T cell‐mediated tumor cell killing CCR9 strongly inhibits the destruction of breast cancer, as well as melanoma and pancreatic cancer, by tumor‐specific cytotoxic T cells CCR9 directly inhibits T cell function in a cell–cell contact‐dependent manner CCR9 engagement by T cells modulates effector cytokine secretion through the regulation of STAT pathway activation CCR9 knockdown in melanoma cells results in efficient rejection of xenotransplanted melanoma tumors in vivo Graphical Abstract A novel in vitro screening methodology involving high‐throughput RNAi‐based gene knockdown in short‐term cocultures identifies immune‐modulatory molecules that mediate tumor resistance to cytotoxic T cells.