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An in-vivo RNAi approach to identify and evaluate suppressors of anti-tumor T cell immunity

Final Report Summary - ANTITUMOR IMMUNITY (An in-vivo RNAi approach to identify and evaluate suppressors of anti-tumor T cell immunity)

Recent years and decades have witnessed an extension of our current view of the immune system from a strict involvement in fighting invading pathogens to more pleiotropic functions. Multiple lines of evidence solidified the long-standing hypothesis of an additional and prominent role of immune cells in safeguarding us from cancer development to a well-accepted paradigm. Immunocompromised patients and mouse models engineered to lack major components of the immune system are prone to develop malignancies. Among many involved cell types, CD8+ T cells stand out as critical mediators of anti-tumor immunity, and various strategies ranging from vaccination to adoptive T cell therapy have been tried to exploit these effects therapeutically – so far, however, with limited clinical success. Cancer cells can employ a variety of mechanisms (e.g. reduced antigen expression, secretion of immunosuppressive factors, the recruitment of accessory suppressive cell types and the expression of ligands for negative immune-modulatory feedback receptors such as PD-1) to evade immune surveillance, and this evasion is thought to be a key event in tumor progression. Importantly, functionally suppressed CD8+ T cells often remain within the tumor microenvironment, suggesting that strategies aimed at re-establishing their anti-tumor activity may provide a promising therapeutic avenue. Indeed, blocking antibodies against CTLA-4 and PD1 have been approved for the treatment of skin cancer, lung cancer as well as bladder cancer and show potent activity in many other cancer types. These impressive results have triggered a lot of excitement about therapeutic targeting of pathways involved in suppression of tumor-associated CD8+ T cells, which will require a better understanding of key mediators and mechanisms involved in this process.
Here, we aimed at systematically identifying and functionally exploring new key players in the suppression of T cell anti-tumor immunity using an innovative approach by combining well-established genetically engineered mouse models (GEMMs) of human cancer, T cell receptor (TCR) / cognate antigen transgenic systems, and state-of-the-art in vivo RNAi technologies. To systematically probe the biology of dysfunctional anti-tumor T cell responses and explore candidate targets for the development of immune-modulatory therapies we established scalable experimental in vivo systems for screening and evaluating genes involved in the suppression of anti-tumor T cell responses in a model of acute myeloid leukemia (AML) and in a model of pancreatic ductal adenocarcinoma (PDAC). In brief, we generated T cell focused candidate shRNA libraries and probed them in multiplexed in vivo RNAi screens to systematically identify key genes involved in the suppression of anti-tumor T cell responses. We employed next generation sequencing to assess changes in the abundance of individual genetic elements (shRNA expression cassettes) in the context of an anti-tumor T cell response, and – after setting up a novel bioinformatic analysis pipeline – could identify both, already established as well as new modulators of T cell function. Furthermore, the dual setup of our approach allowed us to pinpoint common, as well as tumor type specific mediators of T cell dysfunction. We are currently following up on the function of these genes to evaluate their suitability as drug targets.
In sum, we have established a robust screening platform that allows multiplexed interrogation of gene function in the context of anti-tumor T cell immunity. With this we could identify genes that are involved in the functional suppression of tumor reactive CD8+ T cells in vivo, which may be exploited as novel drug targets.