4DN Interrogation of T Cell Exhaustion in Cancer

项目摘要/摘要 增强T细胞识别和杀伤肿瘤细胞能力的免疫疗法已经发生了变革 在治疗人类癌症方面，但免疫疗法并不是对所有患者或癌症都有效，因此 需要研究持久的T细胞对癌症的反应的分子基础。一个关键的障碍 肿瘤浸润性T细胞的持续激活是T细胞耗尽的发展，这导致了 抑制性表面受体表达稳定，对肿瘤抗原反应差，细胞增殖和 T细胞在体内的持久性。然而，到目前为止，对基因调控机制的研究一直很困难。 控制人类T细胞衰竭的发展，因为缺乏敏感的基因组工具来研究 来自患者的初级免疫细胞。我们最近开发了一套高通量的表观基因组技术 这使得能够测量三维(3D)基因组构象和单细胞染色质 人类肿瘤原代T细胞的可及性。在拟议的研究中，我们的目标是利用这些方法 确定构成人类发育基础的4D核糖体(4dN)组织和可及性的变化 T细胞耗竭。在目标1中，我们将定义在人类T细胞耗竭过程中发生的3D基因组相互作用 晚期皮肤癌患者。精疲力竭相关的基因组构象将在 几种癌症类型以确定一致的耗竭情况，这些发现将与 染色质可及性和基因表达数据，以确定3D变化的转录效应。在目标2中，我们 将使用一种新的嵌合体来确定耗竭状态下调节性3D相互作用的动力学和可逆性 抗原受体(CAR)T细胞模型。在目标3中，我们将使用CRISPR/Cas9基因组干扰这些相互作用 在原代T细胞中进行编辑，结合单细胞表观基因组读出，设计出改进的、持久的、下一步- 世代免疫疗法。如果成功，这些发现将对未来的设计产生直接影响 免疫治疗策略，这将对癌症患者的临床护理产生重大影响。最后，我们 将通过自由分发协议和数据并发布客户 软件工具，我们将利用这些研究作为4DN网络中的协作启动点。我们期待着 这些结果将导致对T细胞耗竭的分子调控的新见解，并作为一种 萨特帕西博士建立独立实验室的有效研究计划 免疫学和基因组科学。

PROJECT ABSTRACT/SUMMARY Immunotherapies that enhance the ability of T cells to recognize and kill tumor cells have been transformational in the treatment of human cancer, but immunotherapy is not effective in all patients or cancers, and therefore studies interrogating the molecular basis for durable T cell responses to cancer are needed. A critical barrier for the sustained activation of tumor-infiltrating T cells is the development of T cell ‘exhaustion,’ which leads to the stable expression of inhibitory surface receptors, poor response to tumor antigens, and low cell proliferation and persistence of T cells in vivo. However, to date, it has been difficult to study the gene regulatory mechanisms that control the development of T cell exhaustion in humans, due to a lack of sensitive genomic tools to study primary immune cells from patients. We recently developed a suite of high-throughput epigenomic technologies that enable the measurement of three-dimensional (3D) genome conformation and single-cell chromatin accessibility in primary T cells from human tumors. In the proposed research, we aim to utilize these methods to identify changes in 4D nucleome (4DN) organization and accessibility that underlie the development of human T cell exhaustion. In Aim 1, we will define 3D genome interactions that occur in human T cell exhaustion in patients with advanced skin cancer. Exhaustion-associated genome conformation will be compared across several cancer types to identify a consensus exhaustion profile, and these findings will be integrated with chromatin accessibility and gene expression data to identify transcriptional effects of 3D changes. In Aim 2, we will determine the dynamics and reversibility of regulatory 3D interactions in exhaustion using a novel chimeric antigen-receptor (CAR)-T cell model. In Aim 3, we will perturb these interactions using CRISPR/Cas9 genome editing in primary T cells, coupled with single-cell epigenomic read-outs, to engineer improved, durable, next- generation immunotherapies. If successful, these findings will have a direct impact on the future design of immunotherapy strategies, which will have a significant impact on the clinical care of cancer patients. Finally, we will facilitate the dissemination of these findings by freely distributing protocols and data and releasing custom software tools, and we will use these studies as a collaborative launch point in the 4DN network. We anticipate that these results will lead to novel insights into the molecular regulation of T cell exhaustion and serve as an effective research program for Dr. Satpathy to establish his independent laboratory at the interface of immunology and genome science.