Mechanisms underlying tumor-infiltrating tissue resident memory T cell generation and maintenance

项目总结/摘要 免疫疗法正在迅速成为癌症的主流治疗方法，尽管如此， 患者受益于这种方法。因此，迫切需要开发新型免疫治疗剂 对于目前可用的免疫疗法没有反应的患者。我们的目标是找出这种新的 通过研究驱动一种独特的细胞生成和维持的分子机制， 一类肿瘤浸润性细胞毒性T淋巴细胞（CTL）-组织驻留记忆细胞（TRM）。我们最近 进行了最大的无偏调查，超过100个转录组从纯化的CD 8 + CTL分离， 肿瘤初治的早期肺癌患者，并明确表明，TRM是关键 参与介导强大的抗肿瘤免疫反应（Nature Immunology 2017）。我们发现TRM 选择性地富集在具有高密度肿瘤浸润淋巴细胞（TIL）的肿瘤中， 增强的细胞毒性和增殖，意味着更好的抗肿瘤活性。我们还发现， 肿瘤中TRM细胞的密度预测更好的存活结果。考虑到只有一部分肿瘤 富含TRM，它可以产生有效的抗肿瘤反应，因此了解关键信号至关重要， 驱动TRM细胞在肿瘤内的生成和维持。我们假设TRM的发展可能涉及 几个分子开关（如转录因子），调节迁移，组织保留，存活和 对局部环境的反应（肿瘤）。在这里，我们建议直接分析转录组和表观基因组的 肿瘤浸润性TRM CTL，以通过利用基因组 工具，如单细胞测序，ATAC测序和组蛋白ChIP-Seq。我们还将在功能上 验证一个这样重要的候选途径的作用，即，CD 39信号传导，在促进TRM，鉴于 我们最近的研究显示了在富含TRM的TIL上CD 39的差异性高表达。CD 39与 免疫抑制以及T记忆形成，因此CD 39表达的后果是 不清楚考虑到肿瘤浸润性TRM产生临床有益的抗肿瘤免疫应答，我们 假设TRM细胞上CD 39较高表达可优先保护它们免受ATP诱导的细胞 死亡并使其在肿瘤中持久存在。我们将评估CD 39在体内的功能作用， 通过测试利用CD 39消除的效果来产生和维持肿瘤浸润性TRM 可移植和自体小鼠肿瘤模型和CD 8 + T细胞特异性CD 39敲除小鼠。 此外，将在患者源性肿瘤中进行CD 39敲低或过表达研究。 体外浸润TRM细胞及其对TRM活化、凋亡、增殖、细胞毒性和细胞因子的影响 将对生产进行评估。总之，我们的研究将提供重要的见解的分子信号 其促进了强有力抗肿瘤TRM免疫应答，这可以使免疫靶向 关键途径和疫苗的设计，这将有助于TRM的产生。

Project Summary/Abstract Immunotherapy is rapidly becoming a mainstream treatment of cancers, nonetheless, less than 30% of patients benefit from this approach. Thus, there is an urgent need to develop novel immunotherapeutic agents for patients who do not respond to currently available immunotherapies. Our goal is to identify such novel targets by investigating the molecular mechanisms that drive the generation and maintenance of a distinct class of tumor-infiltrating cytotoxic T lymphocytes (CTLs)—tissue-resident memory cells (TRM). We recently performed the largest unbiased survey of over 100 transcriptomes from purified CD8+ CTLs isolated from tumors of treatment-naïve patients with early-stage lung cancer and provocatively showed that TRM were key players in mediating robust anti-tumor immune responses (Nature Immunology 2017). We revealed that TRM was selectively enriched in tumors with a high density of tumor-infiltrating lymphocytes (TILs) and displayed enhanced cytotoxicity and proliferation, implying better anti-tumor activity. We also showed that a higher density of TRM cells in tumors predicted better survival outcomes. Given that only a subset of tumors is enriched for TRM, which mounts an effective anti-tumor response, it is crucial to understand the key signals that drive TRM cell generation and maintenance within tumors. We hypothesize that TRM development likely involves several molecular switches (like transcription factors) that regulate migration, tissue retention, survival and response to local milieu (tumor). Here, we propose to directly analyze the transcriptome and epigenome of tumor-infiltrating TRM CTLs to define the molecular pathways governing TRM development by utilizing genomic tools such as single-cell sequencing, ATAC-sequencing and histone ChIP-Seq. We will also functionally validate the role of one such important candidate pathway, i.e., CD39 signaling, in promoting TRM, given that our recent studies show differential high expression of CD39 on TRM-rich TILs. CD39 has been associated with immunesuppression as well as T memory formation, hence the consequences of CD39 expression are unclear. Given that tumor-infiltrating TRM mount clinically beneficial anti-tumor immune responses, we hypothesize that higher expression of CD39 on TRM cells may preferentially protect them from ATP-induced cell death and enable persistence within tumors. We will evaluate the functional role of CD39 in vivo in the generation and maintenance of tumor-infiltrating TRM by testing effects of CD39 abrogation utilizing transplantable and autochthonous mouse tumor models and CD8+ T cell-specific CD39-knockout mice. Furthermore, CD39 knock-down or overexpression studies will be performed in patient-derived tumor- infiltrating TRM cells in vitro and the effects on TRM activation, apoptosis, proliferation, cytotoxicity and cytokine production will be evaluated. In summary, our studies will provide important insights into the molecular signals that promote robust anti-tumor TRM immune response, which may enable immunotherapeutic targeting of critical pathways and design of vaccines that will aid TRM generation.