Causes and consequences of oncolytic virus infection of non-tumoral cells in cancer immunotherapy

项目摘要 免疫疗法改变了我们看待和治疗癌症的方式。虽然有些患者会经历持久的 然而，大多数减免者不会作出反应。一些已知的反应障碍是缺乏免疫系统， 肿瘤微环境中的其他细胞类型抑制了浸润或浸润。这些患者可能 发现诱导从头免疫应答的免疫疗法，如溶瘤病毒，比 一种刺激现有免疫群体的方法溶瘤病毒选择性地在肿瘤中复制并溶解肿瘤 细胞，刺激针对病毒和肿瘤抗原的免疫反应，同时使健康组织不受伤害。 目前有一种溶瘤剂被批准用于临床。尽管最近的批准和快速 随着临床试验对溶瘤药物的评估不断扩大，人们对这些治疗对肿瘤的影响知之甚少。 常驻免疫细胞许多这些试验目前正在评估溶瘤牛痘病毒，因为它是一种理想的 免疫疗法的候选人。牛痘病毒完全在细胞质中复制，防止掺入宿主DNA， 具有易于工程化的双链DNA基因组，并刺激强大的免疫反应。在使用基因 具有病毒生长因子和胸苷激酶两者缺失的溶瘤痘苗病毒（vvDD）的工程菌株 为了增加其肿瘤选择性，我们已经证明，单剂量的这种疗法可以显着重塑肿瘤细胞， 肿瘤浸润。正如预期的那样，观察到CD 8 + T细胞浸润的主要增加，然而，我们还观察到， 令人惊讶的表型变化治疗后7天，观察到调节性CD 4 + T细胞（Treg）的损失。 使用表达GFP的病毒株（vvDD-GFP），我们惊奇地发现，治疗后一天， 选择性感染。由于这些细胞在肿瘤中经历缺氧，我们假设缺氧 导致这些亚群的感染，最终导致死亡。由此产生的微环境， 耗尽免疫调节细胞，更支持免疫浸润和抗肿瘤活性。我们将测试 通过（1）确定溶瘤病毒感染是否导致Treg的死亡并确定Treg的表达， Treg丢失对治疗效果的贡献和（2）剖析HIF 1 α对vvDD能力的贡献 来感染泰瑟姆这些研究的结论将为我们提供更好的了解机制 溶瘤性牛痘有了这些信息，设计更有效的溶瘤病毒也将成为可能 以确定最有可能对治疗作出反应的患者群体。

PROJECT SUMMARY Immunotherapy has changed the way we think about and treat cancer. While some patients experience durable remissions, the majority however, will not respond. Some known barriers to response are a lack of immune infiltrate or infiltrate that is suppressed by other cell types in the tumor microenvironment. These patients may find immunotherapies that induce a de novo immune response, such as oncolytic viruses, more successful than one that stimulates the existing immune populations. An oncolytic virus selectively replicates in and lyses tumor cells, stimulating an immune response against viral and tumor antigen, while leaving healthy tissue unharmed. Currently there is one oncolytic approved for use in the clinic. Despite this recent approval and the rapid expansion of clinical trials evaluating oncolytics, little is known about the effects of these treatments on tumor resident immune cells. Many of these trials are currently evaluating oncolytic vaccinia virus as it is an ideal candidate for immunotherapy. Vaccinia replicates entirely in the cytosol preventing incorporation into host DNA, has a dsDNA genome that is easily engineered, and stimulates robust immune responses. Using a genetically engineered strain of oncolytic vaccinia virus (vvDD) with deletions of both viral growth factor and thymidine kinase to increase its tumor selectivity, we have shown that a single dose of this therapy can dramatically remodel the tumor infiltrate. Major increases in CD8+ T cell infiltration are observed as expected, however we also observed surprising phenotypic changes. Seven days post-treatment a loss of regulatory CD4+ T cells (Treg) was seen. Using a GFP expressing strain of the virus (vvDD-GFP) we found that surprisingly, one day post-treatment Tregs were selectively infected. As these cells are experiencing hypoxia in the tumor, we hypothesize that hypoxia leads to infection of these subsets which culminates in their death. The resulting microenvironment, now depleted of immune regulatory cells, is more supportive of immune infiltration and anti-tumor activity. We will test this hypothesis by (1) determining if oncolytic viral infection leads to the death of Treg and determine the contribution of Treg loss to therapeutic efficacy and (2) dissecting the contribution of HIF1α to the ability of vvDD to infect Tregs. The conclusions from these studies will provide us with a better understanding of the mechanism of oncolytic vaccinia. With this information it will be possible to design more efficacious oncolytic viruses as well as determine the patient populations most likely to respond to therapy.