Tumor and Immune Cell Dynamics during Immunotherapy and Cancer Progression

项目总结 旨在重新激活肿瘤特异性免疫的免疫疗法已经改变了癌症的治疗 细胞反应，特别是针对T细胞上抑制性受体的检查点阻断疗法。虽然 这些疗法取得了持久的临床疗效，许多患者无反应或短期复发。 有效的免疫治疗策略的基本原理设计需要对动力学有详细的了解 肿瘤和免疫系统之间的关系。我的目标是使用组合来解码这些动态交互 体内模型、基因组技术和对患者数据的大规模分析，以获得对 肿瘤进展和对免疫治疗反应的生物学过程。在F99阶段，我的目标是 识别对检查点封锁有反应的免疫细胞的来源。一个悬而未决的问题是， T细胞对检查点封锁的反应依赖于先前存在的肿瘤浸润性淋巴细胞或 关于新T细胞的募集。到目前为止，在我的论文工作中，我使用了单细胞RNA和T细胞受体 PD-1阻断前后部位匹配的肿瘤活检组织克隆T细胞的(TCR)测序 免疫治疗的反应动力学。我发现，对PD-1阻断做出反应的克隆扩张的T细胞 富含以前在同一肿瘤中从未观察到的新克隆，这是我们的一种现象 术语克隆替换。然而，新T细胞的来源以及其他免疫细胞群体的作用 目前尚不清楚这一过程中的具体情况。我假设新的T细胞来自次级淋巴器官和 克隆性替代需要克服抗原提呈细胞启动T细胞的缺陷 在PD-1封锁之后。我将使用流式细胞术、单细胞测序和批量TCR的组合 对体内同基因小鼠肿瘤模型进行测序以确定对PD-1和 CTLA-4封锁。此外，我将使用遗传小鼠模型来确定PD-L1在抗原上表达的作用 呈现细胞，特别是常规的1型树突状细胞，对PD-1的阻断效率。在K00阶段， 目的是阐明染色体外DNA扩增、先天免疫信号和染色体外DNA扩增之间的关系。 检查站封锁的效果。导致癌基因扩增的拷贝数变化频繁发生 在环状染色体外DNA(EcDNA)上，由于缺乏 着丝粒，并通过cGAS-STING途径与细胞质DNA感知的丧失有关。此外， 通过cGAS刺痛的先天免疫信号已被证明可以提高检查点封锁的效果。 我认为，胞质DNA感知的丧失是允许染色体外DNA产生的，这是 促进肿瘤进展不仅通过癌基因扩增，而且还通过受损的先天免疫 信号、限制免疫监视和检查点封锁的效果。共同努力，这项工作将提供 对免疫和肿瘤动力学的新见解，这些动力学是癌症进展和反应的基础 免疫疗法。

PROJECT SUMMARY Treatment of cancer has been transformed by immunotherapies that aim to reactivate tumor-specific immune cell responses, in particular checkpoint blockade therapies that target inhibitory receptors on T cells. Although these therapeutics have achieved durable clinical responses, many patients do not respond or shortly relapse. Rationale design of effective immunotherapy strategies requires a detailed understanding of the dynamics between tumors and the immune system. My goal is to decode these dynamic interactions using a combination of in vivo models, genomic technologies, and large-scale analyses of patient data to gain novel insights into the biological processes that underlie cancer progression and response to immunotherapy. In the F99 phase, I aim to identify to origin of immune cells that respond to checkpoint blockade. An outstanding question is whether the T cell response to checkpoint blockade relies on reinvigoration of pre-existing tumor-infiltrating lymphocytes or on recruitment of novel T cells. In my dissertation work so far, I have used single cell RNA and T cell receptor (TCR) sequencing of site-matched tumor biopsies before and after PD-1 blockade to profile clonal T cell dynamics in response to immunotherapy. I found that T cells that clonally expand in response to PD-1 blockade are enriched for novel clones that have not been previously observed in the same tumor, a phenomenon we term clonal replacement. However, the source of novel T cells as well as the role of other immune cell populations in this process remain unclear. I hypothesize that novel T cells are derived from secondary lymphoid organs and that overcoming deficiencies in T cell priming by antigen presenting cells is required for clonal replacement following PD-1 blockade. I will use a combination of flow cytometry, single cell sequencing, and bulk TCR sequencing of in vivo syngeneic mouse tumor models to determine the origin of T cells that respond to PD-1 and CTLA-4 blockade. Further, I will use genetic mouse models to determine the role of PD-L1 expression on antigen presenting cells, in particular conventional type 1 dendritic cells, on PD-1 blockade efficiency. In the K00 phase, I aim to elucidate the relationship between extrachromosomal DNA amplification, innate immune signaling, and the efficacy of checkpoint blockade. Copy number alterations leading to oncogene amplification frequently occurs on circular extrachromosomal DNA (ecDNA), which are commonly found in the cytoplasm due to lack to centromeres and associate with loss of cytosolic DNA sensing through the cGAS-STING pathway. Further, innate immune signaling through cGAS-STING has been shown to improve the efficacy of checkpoint blockade. I propose that loss of cytosolic DNA sensing is permissive for extrachromosomal DNA production, which promotes tumor progression not only through oncogene amplification but also through impaired innate immune signaling, limiting immune surveillance and the efficacy of checkpoint blockade. Together, this work will provide novel insights into immune and tumor dynamics that underlie cancer progression and response to immunotherapy.