ROLES AND MECHANISMS OF TERTIARY LYMPHOID STRUCTURES IN ANTI-TUMOR IMMUNITY

PROJECT SUMMARY Tertiary lymphoid structures (TLS) are T and B lymphocytes in aggregation with antigen presenting cells, fibroblasts, and high endothelial venules observed in some chronically inflamed tissues. TLS formation in tumors of cancer patients is associated with improved adaptive immune function and increased overall survival. In surgically resected human pancreatic ductal adenocarcinoma (PDAC), a significant proportion of patients form spontaneous TLS in their primary tumors. Other groups have demonstrated similar findings, but no evidence currently exists showing TLS directly influence T cell immunity. In this application we propose to investigate if TLS enhance T cell immunity in the context of PDAC tumorigenesis and the mechanism of how this occurs. Our preliminary data in TLS+ mouse and human PDAC tumors shows TCF1+ stem-like T cells (T-stem) are enriched specifically within TLS relative to the surrounding tumor and stroma. We posit that TLS in PDAC tumors support the development of stem-like T cell phenotypes (T-stem), dependent on interleukin-21 signaling. These unique T cell subsets have previously been shown to sustain cellular immunity during chronic infection and cancer, providing continual repletion of memory, effector, and terminally differentiated cells following antigenic recall or immune checkpoint blockade treatment. We model this biology using a panel of mouse PDAC cell lines derived from transgenic mouse tumors that conditionally express mutant KRas and p53, the most common driver mutations in PDAC, and encode for a model MHCI-restricted antigen to track tumor antigen-specific responses. These cancer cell lines, despite sharing a similar genetic and mutational background, induce tumors with unique phenotypes and heterogenous tumor microenvironments. We will deploy a novel method to induce TLS in these mouse tumors through lymphotoxin beta receptor (LTBR) agonism, whereby some of our mouse tumor lines are permissive for TLS formation and others are resistant. We will test if the TLS-mediated generation of T-stem is responsible for improved tumor control in CD8 conditional TCF1-knockout mice. We will determine if their enrichment in TLS is due to IL-21 signals derived directly from TLS that promote their formation, independent of lymphatic involvement, utilizing a combination of cell lineage tracing and genetically modified mouse models. We will complement these studies by investigating tumor samples from PDAC patients for phenotypic pathways unique to specific regions of these tumors. We will achieve this by performing spatial transcriptomics on human PDAC tumor sections analyzed by an innovative algorithm that transforms spatial variable genes into functional tissue modules to determine region-specific immune phenotypes localized to the TLS compared to non-TLS regions and tumors without TLS. This approach identifies the mechanism of how TLS supports T cell function in authentic mouse models of TLS+ PDAC and translates these findings to human cancer patients so novel immunotherapy options designed to elicit and activate TLS can be offered to patients with a treatment resistant malignancy.