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

项目摘要/摘要 免疫疗法正在迅速成为癌症的主流治疗方法，然而，只有不到30%的 患者从这种方法中受益。因此，迫切需要开发新的免疫治疗药物。 适用于对目前可用的免疫疗法无效的患者。我们的目标是找出这样的小说 通过研究驱动不同基因产生和维持的分子机制 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 对从中国分离的纯化的CD8+CTL的100多个转录本进行了最大规模的无偏见调查 肿瘤的治疗-早期肺癌患者的天真和挑衅地表明TRM是关键 调节强大的抗肿瘤免疫反应的参与者(自然免疫学2017)。我们透露，TRM 在具有高密度肿瘤浸润性淋巴细胞(TIL)的肿瘤中选择性地浓缩并显示 增强的细胞毒性和增殖，意味着更好的抗肿瘤活性。我们还展示了一个更高的 肿瘤中TRM细胞的密度预示着更好的生存结果。鉴于只有一小部分肿瘤是 对于富含TRM的，它安装了有效的抗肿瘤反应，理解关键信号是至关重要的 推动肿瘤内TRM细胞的生成和维持。我们假设TRM的发展可能包括 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(肿瘤)的反应。在这里，我们建议直接分析转录组和表观基因组 利用基因组研究肿瘤浸润性TRM CTL以确定调控TRM发育的分子途径 单细胞测序、ATAC测序、组蛋白芯片测序等工具。我们还将在功能上 验证一个如此重要的候选通路，即CD39信号，在促进TRM方面的作用，考虑到 我们最近的研究表明，CD39在富含TRM的TIL上有不同程度的高表达。CD39已被认为与 免疫抑制和T记忆形成，因此CD39表达的后果是 不清楚。鉴于肿瘤浸润性TRM启动了临床上有益的抗肿瘤免疫反应，我们 推测TRM细胞表面CD39的高表达可能优先保护其免受ATP诱导的细胞损伤 死亡，并使肿瘤内的存留。我们将评估CD39在体内的功能作用 利用CD39去除检测肿瘤浸润性TRM的生成和维持 可移植和自体小鼠肿瘤模型和CD8+T细胞特异性CD39基因敲除小鼠。 此外，CD39下调或过表达的研究将在患者来源的肿瘤中进行- TRM细胞的体外侵袭及其对TRM活化、凋亡、增殖、细胞毒和细胞因子的影响 将对生产进行评估。总而言之，我们的研究将对分子信号提供重要的见解 促进强大的抗肿瘤TRM免疫反应，这可能使免疫治疗靶向 关键途径和疫苗设计将有助于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.