Identification of novel tumor-associated immune modulators using a high-throughput RNAi screen

Immune surveillance by T cells is an important component of body’s defense against infection and disease, whereas the peripheral immune tolerance is important for defense against self-reactive T cells. The fine balance between the two is skewed in cancer, whereby the tumors exploit the immune tolerance mechanisms to escape recognition and elimination by the cytotoxic T lymphocytes (CTLs). One of the major routes of immune resistance of tumor cells is mediated by cell surface bound ligands that engage immune-inhibitory receptors on T cells. Targeting these immune-checkpoint ligands that inhibit immune rejection, e.g. via blocking antibodies, can restore immune surveillance and increase the efficacy of cancer immunotherapy. However, only a few such targets have been identified so far. The aim of this thesis was to establish a high-throughput screening assay that enables a rapid and comprehensive identification of cell surface genes with immune modulatory function in selected tumors. To this end, a tumor cell-T cell co-culture assay was established which combines siRNA-based gene knockdown with a luciferase-based assessment of T cell-mediated tumor cell killing. Applied to three independent parallel screens, this study uncovered a repertoire of novel and robust immune modulatory ligands on breast cancer cells that are also abundantly expressed by other cancer types. Amongst them, CCR9 was functionally validated as a strong inhibitor of T cell function and suppressor of T cell reactivity against breast cancer, malignant melanoma and pancreatic cancer. Knockdown of CCR9 resulted in increased tumor susceptibility towards immune lysis by antigen-experienced T cells along with the increased production of effector cytokines and cytolytic enzymes. CCR9-induced gene expression changes in the encountering T cells were consistent with an enhanced effector T cell phenotype. Mechanistically, CCR9 regulated T cell effector function through differential activation of the STAT signaling pathways, thereby representing a unique example of an alternative, TCR-independent route for effective immune suppression induced by a cell surface molecule. Additionally, the in vivo relevance of targeting CCR9 for adoptive cancer immunotherapy was explored in this study. Taken together, this study describes a rapid, high-throughput, siRNA-based screening approach that allows a comprehensive identification of immune-modulatory genes which, as an entity, represents the ‘immune modulatome’ of cancer. Screening additional tumor types for their immune modulatory signatures would help in uncovering additional targets for therapeutic inhibition