Regulation of peripheral node addressin biosynthesis and tertiary lymphoid structures in tumors

Tumor-associated tertiary lymphoid structures (TA-TLS) are ectopic lymphoid aggregates that have considerable morphological, cellular, and molecular similarity to secondary lymphoid organs, particularly lymph nodes. Tumor vessels expressing peripheral node addressin (PNAd) are hallmark features of TA-TLS, and previously work from the lab demonstrated that development of this vasculature in murine melanoma depends on tumor necrosis factor receptor (TNFR) signaling. However, the scaffolding proteins and biosynthetic enzymes driving the biosynthesis of PNAd in tumor endothelial cells (TEC) are unknown. Also, it is unknown which of these PNAd associated components are regulated by TNFR signaling. Chapter 1 of this thesis demonstrates that scaffolding proteins and biosynthetic enzymes driving PNAd biosynthesis in lymph node high endothelial venules are expressed in TECs displaying PNAd. On the tumor vasculature, PNAd is displayed at low levels and this was associated with reduced expression of some PNAd components in TECs, including GST2, which is an important sulfotransferase for PNAd synthesis in lymph node high endothelial venules. Also, loss of PNAd on TECs in the absence of TNFR signaling is associated with reduced expression of scaffolding proteins podocalyxin and nepmucin, and sulfotransferase GST1. Lastly, checkpoint immunotherapy augments both the fraction of TECs expressing PNAd and their surface level of this ligand.

TA-TLS are found in a variety of primary and metastatic human tumors, where they are usually associated with enhanced survival and a favorable response to cancer therapies. In mice, tumors growing in the peritoneal cavity, lungs, and liver, but not those growing subcutaneously, develop TA-TLS in juxtaposition to vessels expressing PNAd. However, the cellular and molecular mediators governing TA-TLS development were unknown. Chapter 2 of this thesis demonstrates that a discrete population of cancer-associated fibroblasts (CAF) that express high levels of B-cell chemokine CXCL13, and B-cell survival factors BAFF and APRIL orchestrate TA-TLS formation in murine melanoma. These lymphoid tissue organizer molecules in CAFs are induced by TNFR signaling mediated by an unknown cell that is neither an adaptive nor innate immune lymphoid cell. CAFs form reticular networks that are co-extensive with B- and T-cell aggregates of TA-TLS. Initial organization of CAFs into these structures is mediated by CD8 T-cells, while robust CAF accumulation and the expansion of TA-TLS-associated reticular networks depends on CXCL13-mediated recruitment of B-cells expressing lymphotoxin-alpha-1, beta-2. Some of these cellular and molecular elements are also evident in TA-TLS associated with human melanoma. Lastly, immunotherapy induces more and larger TA-TLS that are more often organized with discrete T- and B-cell zones, and TA-TLS presence, number, and size are correlated with reduced tumor size and overall response to checkpoint immunotherapy. This work provides a platform for the development of novel strategies that manipulate PNAd expression on tumor vasculature and the formation of TA-TLS as a form of cancer immunotherapy.