修饰的安卡拉痘苗病毒(MVA)是基因工程改造的非复制型病毒载体。MVA的瘤内施用诱导环GMP-AMP合酶介导的I型干扰素(IFN)应答和高水平的转基因的产生,所述转基因被工程化到病毒基因组中,例如肿瘤抗原以构建癌症疫苗。尽管I型IFN对于建立CD8介导的抗肿瘤反应至关重要,这种细胞因子家族也可能产生免疫抑制机制。
进行体外测定以评价辛伐他汀和阿托伐他汀对I型IFN信号传导和抗原呈递的活性。IFNα/β受体1的表面水平,牛血清白蛋白-荧光素5(6)-异硫氰酸盐的内吞作用,信号转导和转录激活因子(STAT)磷酸化,在鼠成纤维细胞系L929中评估IFN刺激的基因和实时PCR。在表达卵清蛋白的B16黑素瘤(OVA)和Lewis肺癌(LLC)-OVA肿瘤模型中,进行体内实验以表征辛伐他汀对MVA诱导的先天免疫应答和对基于MVA的抗肿瘤疫苗的抗肿瘤作用。RNAseq分析,消耗单克隆抗体,和流式细胞术用于评估MVA介导的免疫应答。
在这项工作中,由于他汀类药物能够降低IFN-α/β受体1的表面表达水平和减少网格蛋白介导的内吞作用,我们将常用的他汀类药物鉴定为有效的IFNα药物抑制剂.辛伐他汀和阿托伐他汀有效地消除了8小时对IFNα的转录组反应,并增加了体内呈递与主要组织相容性复合物(MHC)I类结合的OVA衍生肽的树突状细胞的数量。辛伐他汀的腹膜内或肌内给药减少了肿瘤周围给药MVA介导的炎症反应,并增强了编码肿瘤相关抗原的MVA的抗肿瘤活性。协同抗肿瘤作用关键取决于CD8+细胞,而它们通过CD4+淋巴细胞的消耗而显著改善,调节性T细胞,或NK细胞。MVA-OVA单独或联合辛伐他汀增强的B细胞,CD4+淋巴细胞,CD8+淋巴细胞,和肿瘤引流淋巴结中的肿瘤特异性CD8+。然而,只有治疗组合增加了肿瘤微环境和脾脏中这些淋巴细胞群的数量。
总之,辛伐他汀对IFNα功能的阻断显着增强了淋巴细胞浸润和MVA的抗肿瘤活性,促使一个可行的药物再利用。
Modified vaccinia virus Ankara (MVA) are genetically engineered non-replicating viral vectors. Intratumoral administration of MVA induces a cyclic GMP-AMP synthase-mediated type I interferon (IFN) response and the production of high levels of the transgenes engineered into the viral genome such as tumor antigens to construct cancer vaccines. Although type I IFNs are essential for establishing CD8-mediated antitumor responses, this cytokine family may also give rise to immunosuppressive mechanisms.
In vitro assays were performed to evaluate the activity of simvastatin and atorvastatin on type I IFN signaling and on antigen presentation. Surface levels of IFN α/β receptor 1, endocytosis of bovine serum albumin-fluorescein 5 (6)-isothiocyanate, signal transducer and activator of transcription (STAT) phosphorylation, and real-time PCR of IFN-stimulated genes were assessed in the murine fibroblast cell line L929. In vivo experiments were performed to characterize the effect of simvastatin on the MVA-induced innate immune response and on the antitumor effect of MVA-based antitumor vaccines in B16 melanoma expressing ovalbumin (OVA) and Lewis lung carcinoma (LLC)-OVA tumor models. RNAseq analysis, depleting monoclonal antibodies, and flow cytometry were used to evaluate the MVA-mediated immune response.
In this work, we identified commonly prescribed statins as potent IFNα pharmacological inhibitors due to their ability to reduce surface expression levels of IFN-α/β receptor 1 and to reduce clathrin-mediated endocytosis. Simvastatin and atorvastatin efficiently abrogated for 8 hours the transcriptomic response to IFNα and enhanced the number of dendritic cells presenting an OVA-derived peptide bound to major histocompatibility complex (MHC) class I. In vivo, intraperitoneal or intramuscular administration of simvastatin reduced the inflammatory response mediated by peritumoral administration of MVA and enhanced the antitumor activity of MVA encoding tumor-associated antigens. The synergistic antitumor effects critically depend on CD8+ cells, whereas they were markedly improved by depletion of CD4+ lymphocytes, T regulatory cells, or NK cells. Either MVA-OVA alone or combined with simvastatin augmented B cells, CD4+ lymphocytes, CD8+ lymphocytes, and tumor-specific CD8+ in the tumor-draining lymph nodes. However, only the treatment combination increased the numbers of these lymphocyte populations in the tumor microenvironment and in the spleen.
In conclusion, blockade of IFNα functions by simvastatin markedly enhances lymphocyte infiltration and the antitumor activity of MVA, prompting a feasible drug repurposing.