Elucidating the Role of Tregs in Cancer Metastasis and T Cell Dysfunction

项目摘要 胰腺导管腺癌(PDA)是导致癌症相关死亡的第四大原因，5年生存率低达10%。致命性归因于早期转移、较晚发现和治疗耐药。细胞毒性CD8T细胞是免疫监视的主要介体，对肿瘤的根除至关重要。然而，在PDA和其他癌症中，CD8T细胞逐渐失去其细胞毒功能，因此表现为精疲力竭。免疫检查点阻断旨在通过阻断抑制信号来纠正T细胞耗竭，并已彻底改变了许多恶性肿瘤的治疗方法。然而，PDA在很大程度上是抵抗的。因此，这项资助旨在了解导致PDA中CD8 T细胞耗尽的机制，以及PDA如何在免疫治疗后逃避激活的免疫反应。此外，这笔赠款还将研究PDA转移的癌细胞内在和外在机制，以开发合理的治疗方法。我的实验室从免疫耐药肿瘤中获得了新的癌细胞系，称为逃逸变异体，以研究免疫逃逸的机制。我发现，将逃逸变异细胞原位移植到免疫活性小鼠体内会产生多器官转移疾病，这在免疫疗法NA·1VE细胞系中是看不到的。因此，逃逸变异型肿瘤可以模拟PDA患者的转移性疾病。批量RNA测序发现了一个单一基因DDR2，与免疫治疗的原始细胞相比，该基因在多个逃逸变异系中显著上调。DDR2编码一种受体酪氨酸激酶，与其他恶性肿瘤的转移有关，并与PDA患者生存率降低有关。对逃逸变异型肿瘤微环境的研究证实，瘤内调节性T细胞(Tregs)积聚。我发现Treg耗竭显著减少了肿瘤负担和转移，并减少了CD8T细胞耗竭的发展。我将测试一个中心假设，即免疫疗法选择促进肿瘤内Tregs的转移性癌细胞，从而导致CD8T细胞功能障碍。在第一个目标中，我将调查免疫治疗逃逸是如何驱动Treg聚集和转移的。首先，我将测试Treg蓄积是否通过肿瘤内常规的CD4T细胞分化而发生。接下来，我将测试强制表达DDR2是否会导致转移，而切除DDR2是否会阻止转移。在第二个目标中，我将确定对防止PDA转移至关重要的CD8T细胞亚群。首先，我将确定CD8T细胞是否是减少肿瘤负担和消除Treg缺失所见的转移所必需的。接下来，我将使用单细胞RNA测序来鉴定富含Treg耗竭的抗转移CD8T细胞的转录特征。建议的研究策略的完成将揭示PDA的转移和CD8T细胞功能障碍的机制，并确定一个治疗靶点(S)，希望降低PDA的致死率。

PROJECT ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths and has a dismal 5-year survival rate of 10%. Lethality is attributed to early metastasis, late detection, and therapeutic resistance. Cytotoxic CD8 T cells are the principal mediators of immune surveillance and critical for cancer eradication. However, in PDA and other cancers, CD8 T cells progressively lose their cytotoxic function, and are thus characterized as exhausted. Immune checkpoint blockade aims to correct T cell exhaustion by blocking inhibitory signaling and has revolutionized the treatment of many malignancies. However, PDA is largely resistant. As such, this grant seeks to understand the mechanisms driving CD8 T cell exhaustion in PDA and how PDA evades an activated immune response after immunotherapy treatment. Additionally, this grant will study cancer cell-intrinsic and -extrinsic mechanisms of PDA metastasis, to develop rational therapies. My lab derived new cancer cell lines from immunotherapy resistant tumors, termed escape variants, to study mechanisms of immune evasion. I found that orthotopic implantation of escape variant cells into immunocompetent mice yields multi-organ metastatic disease, which is not seen with the immunotherapy na·1ve cell line. Thus, escape variant tumors can model metastatic disease of PDA patients. Bulk RNA sequencing identified a single gene, Ddr2, that was significantly upregulated across multiple escape variant lines compared to immunotherapy na"ive cells. Ddr2 encodes a receptor tyrosine kinase, is linked to metastasis in other malignancies, and is correlated with reduced patient survival in PDA. Investigation into the tumor microenvironment of escape variant tumors identified an accumulation of intratumoral regulatory T cells (Tregs). I found that Treg depletion significantly reduced tumor burden and metastasis and reduced the development of CD8 T cell exhaustion. I will test the central hypothesis that immunotherapy selects for metastatic cancer cells that promote intratumoral Tregs that cause CD8 T cell dysfunction. In the first Aim, I will investigate how immunotherapy escape drives Treg accumulation and metastasis. First, I will test if Treg accumulation occurs through conventional CD4 T cell differentiation within the tumor. Next, I will test if forced expression of Ddr2 drives metastasis and ablation of Ddr2 prevents metastasis. In the second Aim, I will identify the CD8 T cell subset critical for preventing PDA metastasis. First, I will determine if CD8 T cells are required for the reduction in tumor burden and abrogation of metastasis seen with Treg depletion. Next, I will use single cell RNA sequencing to identify the transcriptional signature of anti-metastatic CD8 T cells enriched with Treg depletion. Completion of the proposed research strategy will reveal mechanisms of metastasis and CD8 T cell dysfunction in PDA and identify a therapeutic target(s) in the hope of reducing PDA lethality.