Defining High Endothelial Venules and Tertiary Lymphoid Structures in Cancer

High endothelial venules (HEVs) are structurally distinct blood vessels that develop during embryonic and neonatal life in all secondary lymphoid organs except the spleen. HEVs are critical for initiating and maintaining immune responses because they extract naive and memory lymphocytes from the bloodstream, regardless of antigen receptor specificity, and deliver them to antigen-presenting cells inside lymph nodes under homeostatic conditions. HEVs also develop postnatally in nonlymphoid organs during chronic inflammation driven by autoimmunity, infection, allografts, and cancer. Extranodal HEVs are usually surrounded by dense lymphocytic infiltrates organized into lymph-node like, T- and B-cell-rich areas called tertiary lymphoid structures (TLS). HEV neogenesis is thought to facilitate the generation of tissue-destroying lymphocytes inside chronically inflamed tissues and cancers. We are studying the mechanisms underpinning HEV neogenesis in solid cancers and the role of homeostatic T-cell trafficking in controlling cancer immunity. In this chapter we describe methods for identifying HEV in tissue sections of cancerous tissues in humans and mice using immunohistochemical staining for the HEV-specific marker peripheral lymph node addressin (PNAd). L-selectin binding to PNAd is a necessary first step in homeostatic lymphocyte trafficking which is the defining function of HEV. We also describe methods to measure L-selectin-dependent homing of lymphocytes from the bloodstream into lymphoid tissues and tumors in preclinical cancer models