CRISPR Screening in T Cells Identifies Potential Immunotherapy Targets

CD8⁺ T cells are the mainstay of anticancer immunity and have become the focus of new approaches for cancer immunotherapy. To identify previously unknown negative regulators of T cell–mediated antitumor responses, Dong, Wang, Chow, Ye, and colleagues performed genome-scale CRISPR screens in CD8⁺ T cells and tested their tumor infiltration and degranulation. For the in vivo T-cell infiltration screen, naïve OT-1 T cells specific to ovalbumin peptide were transduced with a mouse genome-scale single-guide RNA (sgRNA) library and adoptively transferred to mice bearing orthotopic or subcutaneous breast cancer tumors expressing ovalbumin. A second screen for factors affecting degranulation ability was performed by pulsing breast cancer cells with ovalbumin peptide and co-culturing with naïve OT-1 T cells transduced with the sgRNA library. The three top genes identified from both infiltration and degranulation screens were Dhx37, Lyn, and Odc1. sgRNAs targeting these genes were individually tested, confirming Dhx37 as a negative regulator of T cells. Single-cell RNA sequencing of sgDhx37-transduced adaptively transferred OT-1 tumor infiltrating lymphocytes (TIL) demonstrated significant changes in the transcriptome. Infection of primary murine CD8⁺ T cells with adeno-associated virus expressing sgDhx37 led to a significant increase of degranulation and enhanced efficacy of adoptive T-cell therapy. Transcriptome profiling of Dhx37-mutant CD8⁺ T cells revealed an enrichment of differentially expressed genes associated with T cell activation and NFκB signaling. Immunostaining of human T cells showed that DHX37 localized in the nucleus and bound to the NFκB binding protein PDCD11 and the NFκB transcription factor p65. DHX37 expression was higher in activated T cells and exhausted cells. Expression of DHX37 mRNA was also detected in a fraction of peripheral blood T cells, tissue resident T cells, and TILs. Collectively, these results demonstrate that DHX37 suppresses T-cell activation and effector function, has a role in modulating NFκB signaling, and may serve as a potential target to enhance the efficacy of immune-cell therapies.

Dong MB, Wang G, Chow RD, Ye L, Zhu L, Dai X, et al. Systematic immunotherapy target discovery using genome-scale in vivo CRISPR screens in CD8 T cells. Cell 2019;178:1189–204.E23.

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