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

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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.