Department of Medical Microbiology and Infection Control, Amsterdam UMC Locatie VUmc, Amsterdam, The Netherlands.
Amsterdam institute for Infection and Immunity, Infectious Diseases, Amsterdam, Netherlands.
J Immunother Cancer. 2023 Apr;11(4). doi: 10.1136/jitc-2022-005839.
Interferon (IFN)-β induction via activation of the stimulator of interferon genes (STING) pathway has shown promising results in tumor models. STING is activated by cyclic dinucleotides such as cyclic GMP-AMP dinucleotides with phosphodiester linkages 2'-5' and 3'-5' (cGAMPs), that are produced by cyclic GMP-AMP synthetase (cGAS). However, delivery of STING pathway agonists to the tumor site is a challenge. Bacterial vaccine strains have the ability to specifically colonize hypoxic tumor tissues and could therefore be modified to overcome this challenge. Combining high STING-mediated IFN-β levels with the immunostimulatory properties of could have potential to overcome the immune suppressive tumor microenvironment.
We have engineered to produce cGAMP by expression of cGAS. The ability of cGAMP to induce IFN-β and its IFN-stimulating genes was addressed in infection assays of THP-I macrophages and human primary dendritic cells (DCs). Expression of catalytically inactive cGAS is used as a control. DC maturation and cytotoxic T-cell cytokine and cytotoxicity assays were conducted to assess the potential antitumor response in vitro. Finally, by making use of different type III secretion (T3S) mutants, the mode of cGAMP transport was elucidated.
Expression of cGAS in results in a 87-fold stronger IFN-β response in THP-I macrophages. This effect was mediated by cGAMP production and is STING dependent. Interestingly, the needle-like structure of the T3S system was necessary for IFN-β induction in epithelial cells. DC activation included upregulation of maturation markers and induction of type I IFN response. Coculture of challenged DCs with cytotoxic T cells revealed an improved cGAMP-mediated IFN-γ response. In addition, coculture of cytotoxic T cells with challenged DCs led to improved immune-mediated tumor B-cell killing.
can be engineered to produce cGAMPs that activate the STING pathway in vitro. Furthermore, they enhanced the cytotoxic T-cell response by improving IFN-γ release and tumor cell killing. Thus, the immune response triggered by can be enhanced by ectopic cGAS expression. These data show the potential of -cGAS in vitro and provides rationale for further research in vivo.
通过激活干扰素基因刺激物（STING）途径诱导干扰素（IFN）-β已在肿瘤模型中显示出良好的效果。STING 可被环二核苷酸激活，例如具有磷酸二酯键 2'-5'和 3'-5'（cGAMPs）的环鸟苷酸-腺苷酸二核苷酸（cGAMPs），由环鸟苷酸-腺苷酸合成酶（cGAS）产生。然而，将 STING 途径激动剂递送到肿瘤部位是一个挑战。细菌疫苗株具有特异性定殖缺氧肿瘤组织的能力，因此可以对其进行修饰以克服这一挑战。结合高 STING 介导的 IFN-β水平和 的免疫刺激特性可能有潜力克服免疫抑制性肿瘤微环境。
我们通过表达 cGAS 使 产生 cGAMP。通过 THP-I 巨噬细胞和人原代树突状细胞（DC）的感染实验来解决 cGAMP 诱导 IFN-β及其 IFN 刺激基因的能力。使用无催化活性的 cGAS 表达作为对照。进行 DC 成熟和细胞毒性 T 细胞细胞因子和细胞毒性测定以评估体外抗肿瘤反应。最后，通过利用不同的 III 型分泌（T3S）突变体，阐明了 cGAMP 转运的模式。
在 中表达 cGAS 可使 THP-I 巨噬细胞中的 IFN-β 反应增强 87 倍。这种作用是通过 cGAMP 的产生介导的，并且是 STING 依赖性的。有趣的是，T3S 系统的针状结构对于上皮细胞中的 IFN-β诱导是必需的。DC 激活包括成熟标志物的上调和 I 型 IFN 反应的诱导。用挑战的 DC 与细胞毒性 T 细胞共培养显示出改善的 cGAMP 介导的 IFN-γ 反应。此外，用挑战的 DC 与细胞毒性 T 细胞共培养导致免疫介导的肿瘤 B 细胞杀伤得到改善。
可以被工程化为体外产生 cGAMPs，激活 STING 途径。此外，它们通过提高 IFN-γ 的释放和肿瘤细胞的杀伤来增强细胞毒性 T 细胞的反应。因此，通过异位表达 cGAS 可以增强 引发的免疫反应。这些数据显示了 的体外潜力，并为体内进一步研究提供了依据。
