Systematic identification of immunotherapy targets using genome-scale in vivo CRISPR screens in CD8+ cytotoxic T cells [scRNA-Seq]

CD8+ cytotoxic T cells play essential roles in anti-tumor immune responses. Here, we performed in vivo screens in CD8+ T cells and identified regulators of tumor infiltration and killing, which are directly relevant to cancer immunotherapy. Unlike in vitro screens, the in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. The infiltration and degranulation screens converged on an RNA helicase Dhx37. Dhx37 knockout enhanced the efficacy of antigen-specific CD8+ T cells against cancer in vivo. Immunological characterization in mouse and human CD8+ T cells revealed that DHX37 suppresses effector function, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating the NF-kB pathway. These data demonstrated the power of high-throughput in vivo genetic screens for immunotherapy target discovery, and uncovered DHX37 as a functional regulator of CD8+ T cells. Overall design: Rag-/- were transplanted with E0771-mCherry-OVA cells. OT-1; Cas9 CD8+ T cells were harvested and transduced with either lenti-Vector or lenti-sgDhx37. Transduced T cells were then adoptively transferred into the tumor-bearing mice. Tumor-infiltrating lymphocytes were subsequently FACS isolated and subject to 10x Genomics single cell RNA-seq.