Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.
Methods Mol Biol. 2022;2463:269-288. doi: 10.1007/978-1-0716-2160-8_19.
Natural killer (NK) cells are an important component of the cancer immune surveillance system. They are regulated by germline-encoded receptors that activate and inhibit their effector function, such as secretion of cytokines and direct lysis of tumor cells and virus-infected cells. Without the need to be primed by prior exposure to tumor antigen, NK cells can detect ligands expressed on tumor cells and selectively kill these cells. NK cells are under strict control by inhibitory receptors that bind to HLA class I on target cells and block early activation signals, thus preventing lysis of target cells. The sensitivity to lysis by NK cells is therefore determined to a large extent by the expression of HLA class I molecules on tumor cells. In addition to receptor-ligand interactions that occur at NK-target cell synapses, many other factors determine the sensitivity of tumor cells to lysis by NK. Intrinsic properties of tumor cells, such as their metabolism and signaling networks establish a threshold above which they will succumb to the death pathways triggered by NK cell attack. Here we provide a protocol for a genome-wide CRISPR screen in tumor cells to identify factors that regulate their sensitivity to primary human NK cells. Tumor cells first transduced for expression of Cas9 are then transduced with a guide RNA (gRNA) library and co-cultured with NK cells. Deep sequencing of the library generated from the genome of tumor cells that survived the selection by NK cells and analysis of the distribution of guide RNAs is performed to identify genes that promote either sensitivity or resistance to NK-mediated killing. The contribution of individual genes to tumor sensitivity can be validated by knockouts using individual gRNAs. The techniques and workflow described here could be applied to primary tumors from cancer patients and reveal tumor-specific points of vulnerability that could be exploited for cancer immunotherapy, such as checkpoint blockade or expression of chimeric antigen receptors specifically designed to activate NK cell cytotoxicity.
自然杀伤 (NK) 细胞是癌症免疫监视系统的重要组成部分。它们受胚系编码的受体调控，这些受体激活并抑制其效应功能，如细胞因子的分泌以及肿瘤细胞和病毒感染细胞的直接溶解。NK 细胞无需预先暴露于肿瘤抗原即可检测到肿瘤细胞上表达的配体，并选择性地杀死这些细胞。NK 细胞受到抑制性受体的严格控制，这些受体与靶细胞上的 HLA 类 I 结合并阻断早期激活信号，从而防止靶细胞的溶解。因此，NK 细胞的裂解敏感性在很大程度上取决于肿瘤细胞上 HLA 类 I 分子的表达。除了在 NK-靶细胞突触处发生的受体-配体相互作用外，许多其他因素决定了肿瘤细胞对 NK 溶解的敏感性。肿瘤细胞的固有特性，如它们的代谢和信号网络，建立了一个阈值，超过该阈值，它们将屈服于 NK 细胞攻击触发的死亡途径。在这里，我们提供了一种在肿瘤细胞中进行全基因组 CRISPR 筛选的方案，以鉴定调节其对原代人 NK 细胞敏感性的因素。首先转导表达 Cas9 的肿瘤细胞，然后转导带有指导 RNA (gRNA) 文库，并与 NK 细胞共培养。对 NK 细胞选择后存活下来的肿瘤细胞基因组文库进行深度测序，并分析指导 RNA 的分布，以鉴定促进 NK 介导杀伤敏感性或抗性的基因。可以使用单个 gRNA 通过敲除来验证单个基因对肿瘤敏感性的贡献。此处描述的技术和工作流程可应用于来自癌症患者的原发性肿瘤，并揭示肿瘤特异性的脆弱点，这些脆弱点可用于癌症免疫治疗，如检查点阻断或专门设计用于激活 NK 细胞细胞毒性的嵌合抗原受体的表达。
