清脆的
GNAS复合轨迹
T细胞
生物
效应器
癌症研究
体内
离体
背景(考古学)
细胞
表型
细胞生物学
基因剔除小鼠
调节器
细胞毒性T细胞
功能(生物学)
计算生物学
小发夹RNA
基因敲除
基因组编辑
细胞疗法
细胞培养
信号转导
遗传筛选
免疫学
基因敲除
HEK 293细胞
CD8型
电池类型
基因敲除
细胞信号
移植
作者
Qi Liu,Peixin Amy Chen,Esha Urs,Shimin Zhang,Maya M. Arce,Charlotte H. Wang,Zhongmei Li,Jin Seo,Nupura Kale,Taylor N. LaFlam,Fanglue Peng,Eric Shifrut,Greg Allen,Justin Eyquem,Katherine C. Fuh,Stacie E. Dodgson,Jason G. Cyster,Alexander Marson,Julia Carnevale
出处
期刊:
[Cold Spring Harbor Laboratory]
日期:2025-09-24
被引量:2
标识
DOI:10.1101/2025.09.23.678127
摘要
Large-scale CRISPR screening in human T cells holds significant promise for identifying genetic modifications that can enhance cellular immunotherapy. However, many genetic regulators of T cell performance in solid tumors may not be readily revealed in vitro. In vivo screening in tumor-bearing mice offers greater physiological relevance, but has historically been limited by low intratumoral T cell recovery. Here, we developed a new model system that achieves significantly higher human T cell recovery from tumors, enabling genome-wide in vivo screens with small numbers of mice. Tumor-infiltrating T cells in this model exhibit hallmarks of dysfunction compared to matched splenic T cells, creating an ideal context for screening for genetic modifiers of T cell activity in the tumor microenvironment. Using this platform, we performed two genome-wide CRISPR knockout screens to identify genes regulating T cell intratumoral abundance and effector function (e.g., IFN-γ production). The intratumoral abundance screen uncovered the P2RY8-Gα13 GPCR signaling pathway as a negative regulator of human T cell infiltration into tumors. The effector function screen identified GNAS (Gαs), a central signaling mediator downstream of multiple GPCRs that sense different suppressive ligands, as a key regulator of T cell dysfunction in tumors. Targeted GNAS knockout rendered T cells resistant to multiple suppressive cues and significantly improved therapeutic performance across diverse solid tumor models. Moreover, combinatorial knockout of P2RY8 (trafficking) and GNAS (effector function) further enhanced overall tumor control, demonstrating that genetic modifications targeting distinct T cell phenotypes can be combined to improve therapeutic potency. This flexible and scalable in vivo screening platform can be adapted to diverse tumor models and pooled CRISPR libraries, enabling future discovery of genetic strategies that equip T cell therapies to overcome barriers imposed by solid tumors.
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