Tumour immune evasion presents a significant challenge to the effectiveness of cancer immunotherapies. Recent advances in high-throughput screening techniques have uncovered that loss of antigen presentation and cytokine signalling pathways are central mechanisms by which tumours evade T cell immunity. To uncover additional vulnerabilities in tumour cells beyond the well-recognized antigen presentation pathway, we conducted a genome-wide CRISPR/Cas9 screen to identify genes that mediate resistance to chimeric-antigen receptor (CAR)-T cells, which function independently of classical antigen presentation. Our study revealed that loss of core-binding factor subunit beta (CBFβ) enhances tumour cell resistance to T cell killing, mediated through T cell-derived TNF. Mechanistically, RNA-sequencing and elemental analyses revealed that deletion of CBFβ disrupts numerous pathways including those involved in zinc homoeostasis. Moreover, we demonstrated that modulation of cellular zinc, achieved by supplementation or chelation, significantly altered tumour cell susceptibility to TNF by regulating the levels of inhibitor of apoptosis proteins. Consistent with this, treatment of tumour cells with a membrane-permeable zinc chelator had no impact on tumour cell viability alone, but significantly increased tumour cell lysis by CD8+ T cells in a TNF-dependent but perforin-independent manner. These results underscore the crucial role of intracellular zinc in regulating tumour cell susceptibility to T cell-mediated killing, revealing a novel vulnerability in tumour cells that might be exploited for the development of future cancer immunotherapeutics.

肿瘤免疫逃避对癌症免疫疗法的有效性提出了重大挑战。高通量筛选技术的最新进展发现，抗原呈递和细胞因子信号传导途径的丧失是肿瘤逃避 T 细胞免疫的核心机制。为了发现肿瘤细胞中除了公认的抗原呈递途径之外的其他脆弱性，我们进行了全基因组 CRISPR/Cas9 筛选，以鉴定介导嵌合抗原受体 (CAR)-T 细胞抗性的基因，该细胞的功能独立于经典抗原推介会。我们的研究表明，核心结合因子亚基β (CBFβ) 的缺失会增强肿瘤细胞对 T 细胞杀伤的抵抗力，这种杀伤作用是通过 T 细胞衍生的 TNF 介导的。从机制上讲，RNA测序和元素分析表明，CBFβ的缺失会破坏许多途径，包括参与锌稳态的途径。此外，我们证明，通过补充或螯合实现的细胞锌调节可通过调节凋亡蛋白抑制剂的水平显着改变肿瘤细胞对TNF的敏感性。与此一致的是，用膜渗透性锌螯合剂处理肿瘤细胞仅对肿瘤细胞活力没有影响，但显着增加 CD8+ T 细胞以 TNF 依赖性但不依赖穿孔素的方式裂解肿瘤细胞。这些结果强调了细胞内锌在调节肿瘤细胞对 T 细胞介导的杀伤敏感性中的关键作用，揭示了肿瘤细胞的一种新的脆弱性，可用于开发未来的癌症免疫疗法。