Targeting Lymph Node Dependent Immune Tolerance in Cancer

项目摘要 背景：恶性黑色素瘤和胰腺导管腺癌（PDAC）通常扩散到淋巴 淋巴结（LN）在远处组织中生长之前。虽然淋巴结转移通常归因于被动的 淋巴结转移的机制及其在肿瘤中的作用 进展仍然知之甚少。LN是适应性免疫反应的教育中心， 大多数潜在的肿瘤反应性淋巴细胞。最近，我们发现，在定植淋巴结，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， 在整个宿主中循环，导致全身耐受性，促进远处部位的转移性播种。 假设和目的：我们假设通过靶向诱导LN中的免疫耐受， 我们既能防止远处转移又能诱导肿瘤消退。我们将使用多种尖端技术 方法来确定LN耐受诱导的细胞和分子机制，并开发方法 这些目标将在以下目标中实现： 具体目的：目的1：确定淋巴结转移诱导肿瘤特异性免疫应答的机制。 免疫耐受性通过它们的免疫抑制淋巴细胞群的活化而增强。目标2：确定 表观遗传调控在LN转移和耐受诱导中的作用。目标3：调查 靶向LN中的肿瘤免疫耐受的免疫治疗方法。 研究设计和方法：使用高含量的多重显微镜，质谱仪，光转换- 基于谱系追踪，TCR测序和抗原呈递的遗传小鼠模型，我们将剖析 我们假设LN中的细胞相互作用是耐受诱导和传播的原因， 验证人体组织和数据集中的发现。LN转移瘤细胞表现出保守和稳定的 转录谱指示表观遗传重编程。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转移性转录特征，和 使用T-ATAC-seq来阐明耐受性T细胞内的表观遗传景观的变化， 肿瘤抗原我们将评估我们的新型免疫刺激抗体缀合物的能力，靶向 特别是针对LN，重编程转移性LN中的APC，使用HDAC抑制剂重编程恶性和非恶性淋巴瘤， 和淋巴细胞群远离肿瘤免疫耐受，并联合收割机这些策略，以引起强大的 在黑素瘤和PDAC的小鼠模型中的抗肿瘤组合疗法。 预期成果和影响：在成功完成这项工作后，我们将确定 LN转移诱导全身耐受的机制，并评估了新的免疫抑制剂， 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.