RESUMO
CD8 T cells play essential roles in anti-tumor immune responses. Here, we performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings and identified regulators of tumor infiltration and degranulation. 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 triple-negative breast cancer in vivo. Immunological characterization in mouse and human CD8 T cells revealed that DHX37 suppresses effector functions, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating NF-κB. These data demonstrate high-throughput in vivo genetic screens for immunotherapy target discovery and establishes DHX37 as a functional regulator of CD8 T cells.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , RNA Helicases/genética , Animais , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citocinas/genética , Citocinas/metabolismo , Feminino , Humanos , Memória Imunológica , Imunoterapia , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , RNA Helicases/deficiência , RNA Guia de Cinetoplastídeos/metabolismo , TranscriptomaRESUMO
Hoxb8 cells are immortalized myeloid progenitors that maintain their multipotent potential and can be differentiated into neutrophils. Genetic modification of Hoxb8 cells can be used as a model system for the functional analysis of regulators of neutrophil maturation and effector functions, such as transcription factors. Here we describe the generation of transcription factor (TF) knockout Hoxb8 cell lines in vitro with the lentivirus (lenti)CRISPR-Cas 9 technique. After their differentiation into neutrophils, the study of their maturation profile, morphology, and effector functions, including NETosis, phagocytosis, and ROS production, is described.
Assuntos
Proteínas de Homeodomínio , Neutrófilos , Neutrófilos/metabolismo , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Diferenciação Celular/genéticaRESUMO
The advent of genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening has advanced the understanding of molecular systems within cells. Here, we demonstrate the utility of sequentially performed CRISPR knockout screens that use an existing library to explore a biological question across the human genome, and then the remaining cells are used to examine each gene candidate against one common gene of interest. We call this approach "Many vs One" CRISPR screening, made possible by a modified 7SK promoter in place of the U6 promoter to drive expression of a single guide RNA. Inserting this novel 7SK promoter into the ubiquitously used lentiCRISPRv2 backbone is crucial, because it overcomes the need for a substantial increase in CRISPR library coverage during screening, sample processing, and next generation sequencing. This new 7SK vector equals the original lentiCRISPRv2 in lentiviral titer, knockout efficiency, and ease of use.