Discovery of immune regulators in primary human T cells using genome-wide CRISPR screens.

The incredible success of cancer immunotherapies is still only limited to subsets of patients, which has motivated efforts to discover novel therapeutic targets to boost tumor killing by effector T cells. However, the translational potential of these discoveries may be limited by suboptimal correlation between cell line and animal models with human disease. Further, the majority of unbiased screening approaches are performed in cancer cells lines, rather than in the effector T cells themselves. We have developed a novel and flexible screening platform using sgRNA lentiviral infection with Cas9 protein electroporation (SLICE), to perform genome-wide CRISPR screens in primary human T cells at genome-wide scale. We have used this platform to survey the response of primary human CD8+ T cells to TCR stimulation. This screen revealed essential components of TCR signaling and inhibitors/agonists of T-cell proliferation. Targeted deletion of top candidate genes from the screen led to increased proliferation, increased expression of activation markers, and improved tumor killing in in vitro co-culture models. Coupling our screening platform with single-cell RNA-seq identified functional clustering of cell states across perturbations and showed that pooled screens in primary cells can now be analyzed at a single-cell level. In addition to finding ways to boost intrinsic T-cell activation, we also sought to adapt the SLICE screening platform to identify mediators of resistance to immunosuppressive forces in the tumor microenvironment. To model one such suppressive factor, we performed the screen in the presence of a potent adenosine agonist and found known and novel regulators of adenosine signaling in T cells. We are further integrating this platform with a two-cell co-culture system to attempt to find mechanisms of resistance to immunosuppressive cells in the tumor microenvironment. This novel platform provides the framework for unbiased, large-scale discovery of immune regulators as potential therapeutic targets in primary human T cells. This abstract is also being presented as Poster A90. Citation Format: Julia Carnevale, Eric Shifrut, Victoria Tobin, Theodore Roth, Jonathan Woo, Christina Bui, Jonathan Li, Morgan Diolaiti, Alan Ashworth, Alexander Marson. Discovery of immune regulators in primary human T cells using genome-wide CRISPR screens [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR08.