Targeting Lymph Node Dependent Immune Tolerance in Cancer

项目概要 背景：恶性黑色素瘤和胰腺导管腺癌 (PDAC) 通常扩散至淋巴 在远处组织中生长之前的淋巴结（LN）。虽然淋巴结转移通常归因于被动 肿瘤淋巴管的引流、淋巴结转移的机制及其在肿瘤中的功能作用 进展仍知之甚少。 LN 是适应性免疫反应的教育中心和港口 大多数潜在的肿瘤反应性淋巴细胞。最近，我们发现在定植的淋巴结中，肿瘤 细胞通过与白细胞的相互作用诱导肿瘤特异性免疫耐受，随后 在整个宿主体内循环，产生全身耐受性，促进远处部位的转移播种。 假设和目标：我们假设通过针对 LN 中免疫耐受的诱导， 我们既可以预防远处转移，又可以诱导肿瘤消退。我们将运用多项尖端技术 鉴定 LN 耐受诱导的细胞和分子机制并开发方法的方法 为了打破它。这些目标将通过以下目标来实现： 具体目标：目标 1：确定淋巴结转移诱导肿瘤特异性的机制 通过激活免疫抑制淋巴细胞群来实现免疫耐受。目标 2：确定 表观遗传调控在淋巴结转移和耐受诱导中的作用。目标 3：调查 针对 LN 中肿瘤免疫耐受的免疫治疗方法。 研究设计和方法：使用高内涵多重显微镜、质谱流式细胞仪、光转换- 基于谱系追踪、TCR 测序和抗原呈递的基因小鼠模型，我们将剖析 我们假设 LN 中的细胞相互作用负责耐受诱导和传播，并且 验证人体组织和数据集中的发现。 LN转移性肿瘤细胞表现出保守且稳定 转录谱表明表观遗传重编程。我们将评估表观遗传改变的作用 使用亚硫酸氢盐测序和 ATAC-seq 赋予 pro-LN 转移转录特征，以及 使用 T-ATAC-seq 来阐明耐受 T 细胞内表观遗传景观的变化，这些变化可识别 肿瘤抗原。我们将评估我们的新型免疫刺激抗体缀合物的靶向能力 特别针对 LN，要重新编程转移性 LN 中的 APC，请使用 HDAC 抑制剂来重新编程恶性 和淋巴细胞群体远离肿瘤免疫耐受，并结合这些策略来引发强大的 黑色素瘤和 PDAC 小鼠模型的抗肿瘤联合疗法。 预期成果和影响：这项工作成功完成后，我们将确定 淋巴结转移诱导全身耐受的机制，并评估新型免疫治疗 克服这些机制的策略。

Project Summary Background: Malignant melanoma and pancreatic ductal adenocarcinoma (PDAC) typically spread to lymph nodes (LNs) prior to outgrowth in distant tissues. While metastasis to LNs is frequently attributed to passive drainage from tumor lymphatics, the mechanisms enabling LN metastasis and its functional role in tumor progression remain poorly understood. LNs are education hubs of the adaptive immune response and harbor the majority of potentially tumor-reactive lymphocytes. Recently, we discovered that in colonizing LNs, tumor cells induce tumor-specific immune tolerance through their interactions with leukocytes that subsequently circulate throughout the host, resulting in systemic tolerance that facilitates metastatic seeding of distant sites. Hypothesis and objective: We hypothesize that by targeting the induction of immune tolerance in LNs, we can both prevent distant metastasis and induce tumor regression. We will use multiple cutting-edge methods to identify the cellular and molecular mechanisms of LN tolerance induction and develop approaches for breaking it. These goals will be pursued in the following aims: Specific Aims: Aim 1: Identify the mechanisms by which lymph node metastases induce tumor-specific immune tolerance through their activation of immunosuppressive lymphocyte populations. Aim 2: Determine the role of epigenetic regulation in LN metastasis and tolerance induction. Aim 3: Investigate immunotherapeutic approaches targeting tumor immune tolerance in LNs. Study design and methods: Using high-content multiplexed microscopy, mass cytometry, photoconversion- based lineage tracing, TCR sequencing, and genetic mouse models of antigen presentation, we will dissect the cellular interactions in LNs that we hypothesize are responsible for tolerance induction and dissemination, and validate the findings in human tissues and datasets. LN metastatic tumor cells exhibit conserved and stable transcriptional profiles indicative of epigenetic reprogramming. We will assess the role of epigenetic alterations in conferring a pro-LN metastatic transcriptional signature using bisulfite sequencing and ATAC-seq, and employ T-ATAC-seq to elucidate changes in the epigenetic landscape within tolerized T cells that recognize tumor antigens. We will evaluate the ability of our novel immunostimulatory antibody conjugates, targeted specifically to LNs, to reprogram APCs in metastatic LNs, employ HDAC inhibitors to reprogram both malignant and lymphocyte populations away from tumor immune tolerance, and combine these strategies to elicit robust anti-tumor combination therapies in mouse models of melanoma and PDAC. Expected results and impact: Upon successful conclusion of this work, we will have identified the mechanisms by which LN metastasis induces systemic tolerance, and evaluated novel immunotherapeutic strategies to overcome these mechanisms.