Understanding high endothelial venules: Lessons for cancer immunology

Abstract

High endothelial venules (HEVs) are blood vessels especially adapted for lymphocyte trafficking which are normally found in secondary lymphoid organs such as lymph nodes (LN) and Peyer's patches. It has long been known that HEVs develop in non-lymphoid organs during chronic inflammation driven by autoimmunity, infection or allografts. More recently, HEVs have been observed in solid, vascularized tumors and their presence correlated with reduced tumor size and improved patient outcome. It is proposed that newly formed HEV promote antitumor immunity by recruiting naive lymphocytes into the tumor, thus allowing the local generation of cancerous tissue-destroying lymphocytes. Understanding how HEVs develop and function are therefore important to unravel their role in human cancers. In LN, HEVs develop during embryonic and early post-natal life and are actively maintained by the LN microenvironment. Systemic blockade of lymphotoxin-β receptor leads to HEV de-differentiation, but the LN components that induce HEV differentiation have remained elusive. Recent elegant studies using gene-targeted mice have demonstrated clearly that triggering the lymphotoxin-β receptor in endothelial cells (EC) induces the differentiation of HEV and that CD11c⁺ dendritic cells play a crucial role in this process. It will be important to determine whether lymphotoxin-β receptor-dependent signaling in EC drives the development of HEV during tumorigenesis and which cells have HEV-inducer properties. This may reveal therapeutic approaches to promote HEV neogenesis and determine the impact of newly formed HEV on tumor immunity.

Keywords: EC, endothelial cells; FRC, fibroblast reticular cells; HEC, high endothelial cells; HEV, high endothelial venules; LN, lymph nodes; LPA, lysophosphatidic acid; LT, lymphotoxin; LT-βR, lymphotoxin-β receptor; MAdCAM, mucosal cell adhesion molecule; PNAd, peripheral node addressin; SIP, sphingosine-1-phosphate; T cell homing; TLO, tertiary lymphoid organ; VE-cadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor; dendritic cells; high endothelial venules; lymphotoxin-β receptor; tumor immunotherapy.

Figure 1.

The role of high endothelial venules in T lymphocyte dependent immunity. HEV recruit naive and central memory T lymphocytes from the bloodstream into lymph nodes where they scan antigen loaded dendritic cells that have migrated from infected, damaged or cancerous tissues (1). Following activation by antigen, activated T lymphocytes exit the lymph node via lymphatics and re-enter the bloodstream (2). Activated T lymphocytes are recruited to sites of inflammation by cytokine-activated blood vessels (which are not HEVs) to clear infected or cancerous tissue (3).

Figure 2.

The migration of immune cells in and out of lymph nodes via high endothelial venules and lymphatics. The main artery into the node arborizes into a capillary bed in the outer cortex that leads directly into the post-capillary venular network where HEVs are located. HEVs increase in size as they traverse the paracortex or T cell area of the node and merge with flat-walled venules in the medulla. HEV are ensheathed by fibroblast reticular cells (FRC) that are continuous with the FRC-coated conduits that form the supporting internal scaffold on which lymphocytes and antigen presenting cells crawl during immunosurveillance (insert). Under homeostatic conditions HEV are major portals for entry of naive (T_N), central memory (T_CM) T and B cells as well as precursors of conventional dendritic cells (pre-DCs), natural killer (NK) cells and plasmacytoid dendritic cells (pDCs). Effector T cells (T_E), NK cells, pDCs, neutrophils (PMN) and monocytes can be recruited by HEV in activated LN. Lymphatic vessels form a separate vascular system. Afferent lymphatics drain the surrounding area and deliver tissue-derived dendritic cells (DCs) to the FRC network and <70 kDa solutes to the basal lamina of HEV via the conduit system. Recirculating and activated lymphocytes leave via efferent lymphatics to re-enter the bloodstream.

Figure 3.

(See previous page). Distinguishing properties of high endothelial venules. (A) High endothelial venules (HEV) are lined with plump high endothelial cells (HEC) which contrast with flat endothelial cells (EC) lining non-specialized post-capillary venules. HEC are supported by a thick basal lamina and perivascular sheath of fibroblast reticular cells (FRC). HEV are also characterized by the presence of lymphocytes (Ly) within the endothelial cell lining and basal lamina as shown by transmission electron micrography. (B) HEV in subcutaneous (peripheral) lymph nodes of mice such as axillary LN selectively express peripheral LN addressin (PNAd) and HEV in mucosal associated lymphoid organs such as Peyer's patches selectively express the mucosal addressin MAdCAM-1. However HEV in other mucosal associated lymphoid organs such as mesenteric LN co-express PNAd and MAdCAM-1. C57BL/6 mice were injected with anti-PNAd (MECA-79) or anti-MAdCAM-1 (MECA-89) antibody and vibratome sections processed for whole mount immunohistochemistry. Scale bar is 50 μM for LN and 100 μM for Peyer's patches.

Figure 4.

Lymphocyte transmigration across high endothelial venules is a multistage process. High endothelial cells express a molecular address that captures and arrests blood-borne lymphocytes on the inner, luminal surface (1). Arrested lymphocytes crawl over the endothelial lining before transmigrating across the wall of HEV. Transmigration can be separated into distinct stages according to the location of migrating lymphocytes. Lymphocytes first transmigrate the endothelial lining where they can accumulate in HEV pockets (2). Lymphocytes can be retained in the sub-endothelial space (3) before completing diapedesis by crossing the basal lamina and perivascular sheath to enter the LN parenchyma (4). Inhibition of ADAM/MMPs arrests lymphocytes within the endothelial lining (stage 2) and the endothelial lining is thickened due to accumulated lymphocytes as shown by transmission electron micrography.

Figure 5.

Tumor-infiltrating lymphocytes and tertiary lymphoid organs in colorectal cancer. The location and phenotype of CD3+ lymphocytes infiltrating the tumor tissue has been correlated with patient outcome (A). Lymphocytes are also found in tumor-induced tertiary lymphoid organs/lymphoid follicles in the peritumoral area (B). Tumor-infiltrating lymphocytes could be recruited directly from the bloodstream following their activation in draining LN or in peritumoral TLO and release into the circulation, as outlined in Figure. 1. Lymphocytes activated in peritumoural TLOs could bypass the bloodstream and migrate directly into the adjacent tumor tissue. Lymphocytes in cryostat sections of tumors were stained either for CD3 (A) or mismatch repair enzyme MLH1 (B).

Figure 6.

The development of high endothelial venules inside and outside of lymph nodes. Lymph node: HEV develop as an integral part of the blood vasculature during embryonic and early post-natal life. Mucosal addressin (MAdCAM-1) is expressed on blood vessel endothelial cells in the late embryo. Luminal expression of peripheral node addressin (PNAd) is induced on MAdCAM-1 expressing blood vessels early in post-natal life and MAdCAM-1 expression is either maintained or downregulated. Engagement of lymphotoxin-β receptor (LT-βR) on endothelial cells drives the development of PNAd expressing HEV. Dendritic cells (DCs) and lymphatics vessels are required to maintain fully differentiated PNAd⁺ HEV and the size of the HEV network is regulated by CCR7⁺ DCs. The stimuli that organize the surrounding basal lamina, perivascular sheath and connecting conduits are not known. Tumor: Tumor-derived factors, such as vascular endothelial growth factor, stimulate the growth of new blood vessels to nourish the growing tumor. In mice, tumor-derived ligands for LT-βR stimulate HEV neogenesis and in primary non-invasive breast cancer, dendritic cells (DC) are a candidate HEV-inducer cell since they are a major source of lymphotoxin-β. Whether tumor-derived HEV arise from pre-existing blood vessels during tumor angiogenesis or develop from circulating endothelial progenitor cells during tumor vasculogenesis remains to be determined.

Figure 7.

Manipulating tumor blood vessels to promote T lymphocyte homing in cancer immunotherapy. Left Tumor blood vessels are anergic to inflammatory cytokines that normally upregulate endothelial cell (EC) expression of homing-associated molecules for T lymphocytes. Tumor-derived factors such as endothelin-B and vascular endothelial growth factor also limit the expression of homing-associated molecules thereby restricting the recruitment of T lymphocytes. Right The recruitment of pericytes to immature tumor blood vessels leads to vessel normalization which is associated with increased immune cell infiltration and reduce tumor growth. Vessel normalization reverses EC anergy and upregulates expression of homing-associated molecules which recruit cancer-destroying T lymphocyte. Tumor-derived HEV may recruit naive and central memory lymphocytes and allow the generation of tissue-destroying lymphocytes within the tumor tissue. The development of HEV in tumours may occur independently of vessel normalization.