Department of Cancer Immunology and Immune Modulation, Boehringer Ingelheim, Biberach an der Riss, Germany.
Department of Drug Discovery Sciences, Boehringer Ingelheim, Biberach an der Riss, Germany.
Elife. 2023 Sep 21;12:e84314. doi: 10.7554/eLife.84314.
Cytotoxic CD8 +T lymphocytes (CTLs) are key players of adaptive anti-tumor immunity based on their ability to specifically recognize and destroy tumor cells. Many cancer immunotherapies rely on unleashing CTL function. However, tumors can evade killing through strategies which are not yet fully elucidated. To provide deeper insight into tumor evasion mechanisms in an antigen-dependent manner, we established a human co-culture system composed of tumor and primary immune cells. Using this system, we systematically investigated intrinsic regulators of tumor resistance by conducting a complementary CRISPR screen approach. By harnessing CRISPR activation (CRISPRa) and CRISPR knockout (KO) technology in parallel, we investigated gene gain-of-function as well as loss-of-function across genes with annotated function in a colon carcinoma cell line. CRISPRa and CRISPR KO screens uncovered 187 and 704 hits, respectively, with 60 gene hits overlapping between both. These data confirmed the role of interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and autophagy pathways and uncovered novel genes implicated in tumor resistance to killing. Notably, we discovered that encoding the integrin-linked kinase-associated serine/threonine phosphatase 2 C, a gene previously unknown to play a role in antigen specific CTL-mediated killing, mediate tumor resistance independently from regulating antigen presentation, IFN-γ or TNF-α responsiveness. Moreover, our work describes the contrasting role of soluble and membrane-bound ICAM-1 in regulating tumor cell killing. The deficiency of membrane-bound ICAM-1 (mICAM-1) or the overexpression of soluble ICAM-1 (sICAM-1) induced resistance to CTL killing, whereas PD-L1 overexpression had no impact. These results highlight the essential role of ICAM-1 at the immunological synapse between tumor and CTL and the antagonist function of sICAM-1.
细胞毒性 CD8+T 淋巴细胞（CTL）是适应性抗肿瘤免疫的关键参与者，因为它们能够特异性识别和破坏肿瘤细胞。许多癌症免疫疗法依赖于释放 CTL 功能。然而，肿瘤可以通过尚未完全阐明的策略来逃避杀伤。为了更深入地了解抗原依赖性肿瘤逃逸机制，我们建立了一个由肿瘤和原代免疫细胞组成的人共培养系统。使用该系统，我们通过进行互补的 CRISPR 筛选方法，系统地研究了肿瘤抵抗的内在调节剂。通过并行利用 CRISPR 激活（CRISPRa）和 CRISPR 敲除（KO）技术，我们研究了在一个结肠癌细胞系中具有注释功能的基因的基因获得功能和丧失功能。CRISPRa 和 CRISPR KO 筛选分别发现了 187 和 704 个靶点，其中 60 个基因靶点在两者之间重叠。这些数据证实了干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）和自噬途径的作用，并揭示了新的与肿瘤抵抗杀伤相关的基因。值得注意的是，我们发现编码整合素连接激酶相关丝氨酸/苏氨酸磷酸酶 2C 的基因，该基因以前未知在抗原特异性 CTL 介导的杀伤中发挥作用，独立于调节抗原呈递、IFN-γ或 TNF-α反应性来介导肿瘤抵抗。此外，我们的工作描述了可溶性和膜结合的 ICAM-1 调节肿瘤细胞杀伤的对比作用。膜结合 ICAM-1（mICAM-1）的缺乏或可溶性 ICAM-1（sICAM-1）的过表达诱导了对 CTL 杀伤的抵抗，而 PD-L1 的过表达没有影响。这些结果强调了 ICAM-1 在肿瘤和 CTL 之间免疫突触中的重要作用以及 sICAM-1 的拮抗剂功能。
