The Dual Role of High Endothelial Venules in Cancer Progression versus Immunity
Authors: Stefan Milutinovic, Jun Abe, Andrew Godkin, Jens V. Stein, Awen Gallimore
Journal: Trends in cancer (2020)

High endothelial venules (HEVs) are specialised blood vessels, found in secondary lymph nodes, where they play an important role in initiating and regulating immunity.Recent studies indicate that HEVs can also provide an exit route for tumour cell dissemination and the establishment of metastases.The dual roles of HEVs imply that they can both limit and accelerate progression of cancer.It is important to address gaps in our understanding of HEV remodelling in order to uncover routes to promoting immune activation via HEV, whilst simultaneously limiting their ability to disseminate cancer cells. Secondary lymphoid organs (SLOs) are important initiators and regulators of immunity. To carry out this function, the blood vasculature must deliver oxygen and nutrients and recruit circulating lymphocytes into the SLO parenchyma, where they encounter cognate antigen. High endothelial venules (HEVs) are specialised postcapillary venules that specifically serve this function and are found in all SLOs except spleen. It is becoming clear that alterations to HEV network density and/or morphology can result in immune activation or, as recently implicated, in providing an exit route for tumour cell dissemination and metastases. In this review, the structural plasticity of HEVs, the regulatory pathways underpinning this plasticity, and the relevance of these pathways to cancer progression will be discussed. Secondary lymphoid organs (SLOs) are important initiators and regulators of immunity. To carry out this function, the blood vasculature must deliver oxygen and nutrients and recruit circulating lymphocytes into the SLO parenchyma, where they encounter cognate antigen. High endothelial venules (HEVs) are specialised postcapillary venules that specifically serve this function and are found in all SLOs except spleen. It is becoming clear that alterations to HEV network density and/or morphology can result in immune activation or, as recently implicated, in providing an exit route for tumour cell dissemination and metastases. In this review, the structural plasticity of HEVs, the regulatory pathways underpinning this plasticity, and the relevance of these pathways to cancer progression will be discussed. HEVs comprise high endothelial cells (HECs) that are readily distinguished from other blood endothelial cells by their characteristic plump, cuboidal morphology, first described in the late 19th century [1.Thomé R. Endothelien als phagocyten (aus den lymphdrüsen von Macacus cynomolgus).Arch. Mikrosk. Anat. 1898; 52: 820-842Crossref Scopus (24) Google Scholar]. Whilst they share common pan-endothelial cell (EC) markers such as CD31 and VE-cadherin [2.Pfeiffer F. et al.Distinct molecular composition of blood and lymphatic vascular endothelial cell junctions establishes specific functional barriers within the peripheral lymph node.Eur. J. Immunol. 2008; 38: 2142-2155Crossref PubMed Scopus (60) Google Scholar], HECs preferentially express genes that are important for the role of HEVs in lymphocyte recruitment and immunological defence [3.Lee M. et al.Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.Nat. Immunol. 2014; 15: 982-995Crossref PubMed Scopus (80) Google Scholar]. A key example is peripheral node addressin (PNAd), which acts as an adhesion molecule for CD62L (L-selectin)-expressing lymphocytes [3.Lee M. et al.Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.Nat. Immunol. 2014; 15: 982-995Crossref PubMed Scopus (80) Google Scholar], an interaction that mediates the tethering and rolling of lymphocytes along HEVs, marking the initial stages of the multistep adhesion cascade, and ultimately leading to lymphocyte extravasation into the lymph node (LN) parenchyma [4.Girard J-P. et al.HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes.Nat. Rev. Immunol. 2012; 12: 762-763Crossref PubMed Scopus (353) Google Scholar,5.Miyasaka M. Tanaka T. Lymphocyte trafficking across high endothelial venules: dogmas and enigmas.Nat. Rev. Immunol. 2004; 4: 360-370Crossref PubMed Scopus (327) Google Scholar]. The L-selectin/PNAd interaction is crucial for enabling naïve and central memory T cells as well as naïve B cells to home to LNs [4.Girard J-P. et al.HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes.Nat. Rev. Immunol. 2012; 12: 762-763Crossref PubMed Scopus (353) Google Scholar]. Ectopic HEVs have also been found at sites of chronic inflammatory diseases [6.Aloisi F. Pujol-Borrell R. Lymphoid neogenesis in chronic inflammatory diseases.Nat. Rev. Immunol. 2006; 6: 205-217Crossref PubMed Scopus (681) Google Scholar], infection [7.Neyt K. et al.Tertiary lymphoid organs in infection and autoimmunity.Trends Immunol. 2012; 33: 297-305Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar], and, as described in detail in this review, in cancer [8.Martinet L. et al.Human solid tumors contain high endothelial venules: association with T- and B-lymphocyte infiltration and favorable prognosis in breast cancer.Cancer Res. 2011; 71: 5678-5687Crossref PubMed Scopus (267) Google Scholar]. In each of these settings, HEVs have been implicated in driving immune reactions, possibly by enabling homing of naïve immune cells to the diseased site [6.Aloisi F. Pujol-Borrell R. Lymphoid neogenesis in chronic inflammatory diseases.Nat. Rev. Immunol. 2006; 6: 205-217Crossref PubMed Scopus (681) Google Scholar, 7.Neyt K. et al.Tertiary lymphoid organs in infection and autoimmunity.Trends Immunol. 2012; 33: 297-305Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 8.Martinet L. et al.Human solid tumors contain high endothelial venules: association with T- and B-lymphocyte infiltration and favorable prognosis in breast cancer.Cancer Res. 2011; 71: 5678-5687Crossref PubMed Scopus (267) Google Scholar]. Since the development, structure, and function of HEVs have been described in detail in previous reviews, a summary of the key features of HEV is described in Box 1. The functional and structural plasticity of HEVs in response to LN stimulation is also well documented. Alcian blue dye infusion, together with angiography, has been used to show extensive LN expansion and HEV remodelling in response to antigen stimulation [9.Herman P.G. et al.Blood microcirculation in the lymph node during the primary immune response.J. Exp. Med. 1972; 136: 697-714Crossref PubMed Scopus (73) Google Scholar,10.Anderson N.D. et al.Microvascular changes in lymph nodes draining skin allografts.Am. J. Pathol. 1975; 81: 131-160PubMed Google Scholar]. This was characterised by an increase in HEV length and branching patterns [9.Herman P.G. et al.Blood microcirculation in the lymph node during the primary immune response.J. Exp. Med. 1972; 136: 697-714Crossref PubMed Scopus (73) Google Scholar,10.Anderson N.D. et al.Microvascular changes in lymph nodes draining skin allografts.Am. J. Pathol. 1975; 81: 131-160PubMed Google Scholar] and accompanied by increased blood flow and lymphocyte trafficking [11.Hay J.B. Hobbs B.B. The flow of blood to lymph nodes and its relation to lymphocyte traffic and the immune response.J. Exp. Med. 1977; 145: 31-44Crossref PubMed Scopus (121) Google Scholar]. Whilst these early studies revealed the contribution of EC proliferation in HEV plasticity, it has become clear that the co-operative activities of vascular, stromal [notably fibroblastic reticular cells (FRCs)], and immune cells are required for HEV remodelling and development of an immune response (Figure 1).Box 1HEV Structure and FunctionIn LNs, HEVs are localised to the cortical–paracortical junction and paracortex (where the T cell zone is located) and form part of the venular tree, a distinct hierarchy of venules that branch out from the largest collecting vein (order I) to the smallest postcapillary venules (order V) (Figure I) [74.von Andrian U.H. Intravital microscopy of the peripheral lymph node microcirculation in mice.Microcirculation. 1996; 3: 287-300Crossref PubMed Scopus (166) Google Scholar]. Intravital microscopy studies revealed that the major sites of lymphocyte recruitment occur in venule branches that are of the order III–V, which constitutes all HEVs [74.von Andrian U.H. Intravital microscopy of the peripheral lymph node microcirculation in mice.Microcirculation. 1996; 3: 287-300Crossref PubMed Scopus (166) Google Scholar] (Figure I). HECs have also been shown to exhibit tissue-specific specialisation [3.Lee M. et al.Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.Nat. Immunol. 2014; 15: 982-995Crossref PubMed Scopus (80) Google Scholar]. Whole-genome expression profiling of HECs isolated from gut-associated lymphoid tissue (GALT) showed preferential expression of the enzyme β-galactoside α-2,6-sialyltransferase 1 (ST6Gal I), whilst HECs isolated from peripheral LNs had lower expression levels of this enzyme [3.Lee M. et al.Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.Nat. Immunol. 2014; 15: 982-995Crossref PubMed Scopus (80) Google Scholar]. ST6Gal I was subsequently identified as a vascular addressin for the targeting of B cells to GALT [3.Lee M. et al.Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.Nat. Immunol. 2014; 15: 982-995Crossref PubMed Scopus (80) Google Scholar]. Focal sites of recruitment are found within HEVs. Single-cell RNA sequencing has revealed that HECs under homeostatic conditions display heterogenous expression of several genes, including CCL21, within the same SLO [75.Veerman K. et al.Single-cell analysis reveals heterogeneity of high endothelial venules and different regulation of genes controlling lymphocyte entry to lymph nodes.Cell Rep. 2019; 26: 3116-3131Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. This may reflect specialised functions of individual HECs and the establishment of preferential sites for immune cell transmigration (termed 'exit ramps') in different regions of the SLO (e.g., paracortex versus medulla) [76.Bajenoff M. et al.Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes.Immunity. 2006; 25: 989-1001Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar]. Furthermore, important differences in the regulation of genes controlling lymphocyte trafficking were also revealed by single-cell RNA sequencing [75.Veerman K. et al.Single-cell analysis reveals heterogeneity of high endothelial venules and different regulation of genes controlling lymphocyte entry to lymph nodes.Cell Rep. 2019; 26: 3116-3131Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. Several genes involved in the synthesis of the 6-sulfo sLeX epitope, which decorate PNAd, including the fucosyltransferase FucT-VII (Fut7) and core 2 branching enzyme Core2 GlcNAcT (Gcnt1), were found to require higher levels of LTβR-dependent signalling for expression than the sulfotransferase GlcNAc6ST-2 (Chst4) [75.Veerman K. et al.Single-cell analysis reveals heterogeneity of high endothelial venules and different regulation of genes controlling lymphocyte entry to lymph nodes.Cell Rep. 2019; 26: 3116-3131Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. Such differential sensitivity of HEV genes to LTβR-dependent signalling may in part explain the cellular and spatial heterogeneity of peripheral LN HEVs [75.Veerman K. et al.Single-cell analysis reveals heterogeneity of high endothelial venules and different regulation of genes controlling lymphocyte entry to lymph nodes.Cell Rep. 2019; 26: 3116-3131Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar].Figure 1Vascular-Stromal Elements Involved in Lymph Node (LN) High Endothelial Venule (HEV) Remodelling.Show full captionThe LN feeding arteriole branches into capillaries, which in turn branch into postcapillary HEVs (thickened blue segment). Cross-section of vascular-stromal elements found in the T zone are depicted. HEVs are suspended in a reticular network characterised by collagen-rich fibrils ensheathed by reticular cells, termed fibroblastic reticular cells (FRCs), which form a network supporting immune cell migration and survival. In addition to FRCs, T cells, B cells, and dendritic cells (DCs) are involved in mediating HEV remodelling in response to ovalbumin in complete Freund's adjuvant (OVA/CFA) immunisation. The initiation phase is driven by CD11cMed DCs (purple), whilst the expansion phase is driven by T and B cells. CD11cHigh DCs (yellow) are important in the resolution of this response. FRCs are also in close proximity to lymphatic vessels and may have a role in the regulation of this compartment as well. Figure adapted from [20.Dasoveanu D.C. et al.Regulation of lymph node vascular-stromal compartment by dendritic cells.Trends Immunol. 2016; 37: 764-777Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar,71.Fletcher A.L. et al.Lymph node fibroblastic reticular cells in health and disease.Nat. Rev. Immunol. 2015; 15: 350-361Crossref PubMed Scopus (159) Google Scholar].View Large Image Figure ViewerDownload Hi-res image Download (PPT) In LNs, HEVs are localised to the cortical–paracortical junction and paracortex (where the T cell zone is located) and form part of the venular tree, a distinct hierarchy of venules that branch out from the largest collecting vein (order I) to the smallest postcapillary venules (order V) (Figure I) [74.von Andrian U.H. Intravital microscopy of the peripheral lymph node microcirculation in mice.Microcirculation. 1996; 3: 287-300Crossref PubMed Scopus (166) Google Scholar]. Intravital microscopy studies revealed that the major sites of lymphocyte recruitment occur in venule branches that are of the order III–V, which constitutes all HEVs [74.von Andrian U.H. Intravital microscopy of the peripheral lymph node microcirculation in mice.Microcirculation. 1996; 3: 287-300Crossref PubMed Scopus (166) Google Scholar] (Figure I). HECs have also been shown to exhibit tissue-specific specialisation [3.Lee M. et al.Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.Nat. Immunol. 2014; 15: 982-995Crossref PubMed Scopus (80) Google Scholar]. Whole-genome expression profiling of HECs isolated from gut-associated lymphoid tissue (GALT) showed preferential expression of the enzyme β-galactoside α-2,6-sialyltransferase 1 (ST6Gal I), whilst HECs isolated from peripheral LNs had lower expression levels of this enzyme [3.Lee M. et al.Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.Nat. Immunol. 2014; 15: 982-995Crossref PubMed Scopus (80) Google Scholar]. ST6Gal I was subsequently identified as a vascular addressin for the targeting of B cells to GALT [3.Lee M. et al.Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.Nat. Immunol. 2014; 15: 982-995Crossref PubMed Scopus (80) Google Scholar]. Focal sites of recruitment are found within HEVs. Single-cell RNA sequencing has revealed that HECs under homeostatic conditions display heterogenous expression of several genes, including CCL21, within the same SLO [75.Veerman K. et al.Single-cell analysis reveals heterogeneity of high endothelial venules and different regulation of genes controlling lymphocyte entry to lymph nodes.Cell Rep. 2019; 26: 3116-3131Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. This may reflect specialised functions of individual HECs and the establishment of preferential sites for immune cell transmigration (termed 'exit ramps') in different regions of the SLO (e.g., paracortex versus medulla) [76.Bajenoff M. et al.Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes.Immunity. 2006; 25: 989-1001Abstract Full Text Full Text PDF PubMed Scopus (704) Google Scholar]. Furthermore, important differences in the regulation of genes controlling lymphocyte trafficking were also revealed by single-cell RNA sequencing [75.Veerman K. et al.Single-cell analysis reveals heterogeneity of high endothelial venules and different regulation of genes controlling lymphocyte entry to lymph nodes.Cell Rep. 2019; 26: 3116-3131Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. Several genes involved in the synthesis of the 6-sulfo sLeX epitope, which decorate PNAd, including the fucosyltransferase FucT-VII (Fut7) and core 2 branching enzyme Core2 GlcNAcT (Gcnt1), were found to require higher levels of LTβR-dependent signalling for expression than the sulfotransferase GlcNAc6ST-2 (Chst4) [75.Veerman K. et al.Single-cell analysis reveals heterogeneity of high endothelial venules and different regulation of genes controlling lymphocyte entry to lymph nodes.Cell Rep. 2019; 26: 3116-3131Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. Such differential sensitivity of HEV genes to LTβR-dependent signalling may in part explain the cellular and spatial heterogeneity of peripheral LN HEVs [75.Veerman K. et al.Single-cell analysis reveals heterogeneity of high endothelial venules and different regulation of genes controlling lymphocyte entry to lymph nodes.Cell Rep. 2019; 26: 3116-3131Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. The LN feeding arteriole branches into capillaries, which in turn branch into postcapillary HEVs (thickened blue segment). Cross-section of vascular-stromal elements found in the T zone are depicted. HEVs are suspended in a reticular network characterised by collagen-rich fibrils ensheathed by reticular cells, termed fibroblastic reticular cells (FRCs), which form a network supporting immune cell migration and survival. In addition to FRCs, T cells, B cells, and dendritic cells (DCs) are involved in mediating HEV remodelling in response to ovalbumin in complete Freund's adjuvant (OVA/CFA) immunisation. The initiation phase is driven by CD11cMed DCs (purple), whilst the expansion phase is driven by T and B cells. CD11cHigh DCs (yellow) are important in the resolution of this response. FRCs are also in close proximity to lymphatic vessels and may have a role in the regulation of this compartment as well. Figure adapted from [20.Dasoveanu D.C. et al.Regulation of lymph node vascular-stromal compartment by dendritic cells.Trends Immunol. 2016; 37: 764-777Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar,71.Fletcher A.L. et al.Lymph node fibroblastic reticular cells in health and disease.Nat. Rev. Immunol. 2015; 15: 350-361Crossref PubMed Scopus (159) Google Scholar]. HEV remodelling in response to immunisation with antigen [ovalbumin in complete Freund's adjuvant (OVA/CFA)] has been extensively studied and is a well-regulated process that occurs in three distinct phases [12.Chyou S. et al.Fibroblast-type reticular stromal cells regulate the lymph node vasculature.J. Immunol. 2008; 181: 3887-3896Crossref PubMed Scopus (101) Google Scholar, 13.Kumar V. et al.Optical projection tomography reveals dynamics of HEV growth after immunization with protein plus CFA and features shared with HEVs in acute autoinflammatory lymphadenopathy.Front. Immunol. 2012; 3: 282Crossref PubMed Scopus (35) Google Scholar, 14.Webster B. et al.Regulation of lymph node vascular growth by dendritic cells.J. Exp. Med. 2006; 203: 1903-1913Crossref PubMed Scopus (146) Google Scholar, 15.Tzeng T.-C. et al.CD11c(hi) dendritic cells regulate the re-establishment of vascular quiescence and stabilization after immune stimulation of lymph nodes.J. Immunol. 2010; 184: 4247-4257Crossref PubMed Scopus (31) Google Scholar, 16.Chyou S. et al.Coordinated regulation of lymph node vascular-stromal growth first by CD11c+ cells and then by T and B cells.J. Immunol. 2011; 187: 5558-5567Crossref PubMed Scopus (85) Google Scholar, 17.Benahmed F. et al.Multiple CD11c+ cells collaboratively express IL-1beta to modulate stromal vascular endothelial growth factor and lymph node vascular-stromal growth.J. Immunol. 2014; 192: 4153-4163Crossref PubMed Scopus (28) Google Scholar]. The initiation phase, which is T and B cell independent [16.Chyou S. et al.Coordinated regulation of lymph node vascular-stromal growth first by CD11c+ cells and then by T and B cells.J. Immunol. 2011; 187: 5558-5567Crossref PubMed Scopus (85) Google Scholar], lasts for 2 days and is driven by CD11c+ dendritic cells (DC) [14.Webster B. et al.Regulation of lymph node vascular growth by dendritic cells.J. Exp. Med. 2006; 203: 1903-1913Crossref PubMed Scopus (146) Google Scholar]. Upregulation of vascular endothelial growth factor (VEGF) by FRCs is in part stimulated by the release of IL-1β from CD11c+ DCs and monocytes [17.Benahmed F. et al.Multiple CD11c+ cells collaboratively express IL-1beta to modulate stromal vascular endothelial growth factor and lymph node vascular-stromal growth.J. Immunol. 2014; 192: 4153-4163Crossref PubMed Scopus (28) Google Scholar]. This initiation phase is marked by increased VEGF-driven EC proliferation, FRC proliferation, and a modest increase in HEV EC numbers [14.Webster B. et al.Regulation of lymph node vascular growth by dendritic cells.J. Exp. Med. 2006; 203: 1903-1913Crossref PubMed Scopus (146) Google Scholar,16.Chyou S. et al.Coordinated regulation of lymph node vascular-stromal growth first by CD11c+ cells and then by T and B cells.J. Immunol. 2011; 187: 5558-5567Crossref PubMed Scopus (85) Google Scholar]. FRCs, which are normally tightly wrapped around vessels, stretch and begin to detach to allow for the second phase, characterised by vascular expansion, to occur [15.Tzeng T.-C. et al.CD11c(hi) dendritic cells regulate the re-establishment of vascular quiescence and stabilization after immune stimulation of lymph nodes.J. Immunol. 2010; 184: 4247-4257Crossref PubMed Scopus (31) Google Scholar]. During this phase, detachment of FRCs is mediated by CLEC-2 expressing CD11c+ cells [18.Acton S.E. et al.Dendritic cells control fibroblastic reticular network tension and lymph node expansion.Nature. 2014; 514: 498-502Crossref PubMed Scopus (152) Google Scholar], whilst podoplanin (PDPN) expression by FRCs is critical in the maintenance of overall HEV integrity during LN expansion and lymphocyte homing through its interaction with CLEC-2-expressing platelets [19.Herzog B.H. et al.Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2.Nature. 2013; 502: 105-109Crossref PubMed Scopus (203) Google Scholar]. In addition to EC and FRC proliferation, the expansion phase marks extensive increases in total HEV length and branching patterns [13.Kumar V. et al.Optical projection tomography reveals dynamics of HEV growth after immunization with protein plus CFA and features shared with HEVs in acute autoinflammatory lymphadenopathy.Front. Immunol. 2012; 3: 282Crossref PubMed Scopus (35) Google Scholar] and lasts between days 2 and 5 [16.Chyou S. et al.Coordinated regulation of lymph node vascular-stromal growth first by CD11c+ cells and then by T and B cells.J. Immunol. 2011; 187: 5558-5567Crossref PubMed Scopus (85) Google Scholar]. This phase is strongly dependent on T and B cells [16.Chyou S. et al.Coordinated regulation of lymph node vascular-stromal growth first by CD11c+ cells and then by T and B cells.J. Immunol. 2011; 187: 5558-5567Crossref PubMed Scopus (85) Google Scholar], implying that HEV expansion reflects an effort to support the development of an adaptive immune response [20.Dasoveanu D.C. et al.Regulation of lymph node vascular-stromal compartment by dendritic cells.Trends Immunol. 2016; 37: 764-777Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar]. Recently, multicoloured fluorescent fate-mapping models, which enable labelling of adult LN ECs with specific colours to allow for subsequent progeny tracking at the single-cell level, have revealed that HECs act as local progenitors to create both capillaries and HEV neo-vessels during vascular expansion [21.Mondor I. et al.Clonal proliferation and stochastic pruning orchestrate lymph node vasculature remodeling.Immunity. 2016; 45: 877-888Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. In addition to structural changes, HEVs revert to an immature phenotype (MAdCAM-1highHEC-6STlow) during this phase, which is caused by impaired afferent lymphatic vessel function [22.Liao S. Ruddle N.H. Synchrony of high endothelial venules and lymphatic vessels revealed by immunization.J. Immunol. 2006; 177: 3369-3379Crossref PubMed Scopus (148) Google Scholar], resulting in a dilution of DC-secreted LTβR ligands [23.Moussion C. Girard J-P. Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules.Nature. 2011; 479: 542-546Crossref PubMed Scopus (195) Google Scholar]. The vascular-stromal quiescence phase can last for several weeks and is mediated by CD11chi DCs [15.Tzeng T.-C. et al.CD11c(hi) dendritic cells regulate the re-establishment of vascular quiescence and stabilization after immune stimulation of lymph nodes.J. Immunol. 2010; 184: 4247-4257Crossref PubMed Scopus (31) Google Scholar]. This phase marks a reduction in HEV and FRC proliferation, reduction in HEV trafficking efficiency, and stabilisation of vessels by FRC reassembly around vessels [15.Tzeng T.-C. et al.CD11c(hi) dendritic cells regulate the re-establishment of vascular quiescence and stabilization after immune stimulation of lymph nodes.J. Immunol. 2010; 184: 4247-4257Crossref PubMed Scopus (31) Google Scholar]. HEVs also revert back to a mature phenotype during this period [22.Liao S. Ruddle N.H. Synchrony of high endothelial venules and lymphatic vessels revealed by immunization.J. Immunol. 2006; 177: 3369-3379Crossref PubMed Scopus (148) Google Scholar]. LN vascular expansion is then thought to eventually result in vascular regression following the resolution of the immune response [20.Dasoveanu D.C. et al.Regulation of lymph node vascular-stromal compartment by dendritic cells.Trends Immunol. 2016; 37: 764-777Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar]. Whilst both pre-existing and neo-vessels have been found to be pruned at the same pace during LN quiescence, the factors that induce such pruning remain to be identified [21.Mondor I. et al.Clonal proliferation and stochastic pruning orchestrate lymph node vasculature remodeling.Immunity. 2016; 45: 877-888Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. The development of mesoscopic imaging techniques (see Glossary), such as optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM), has allowed for the global analysis of HEV network remodelling in response to infection [24.Kumar V. et al.Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway.Blood. 2010; 115: 4725-4733Crossref PubMed Scopus (108) Google Scholar], immunisation, and autoimmune lymphadenopathy [13.Kumar V. et al.Optical projection tomography reveals dynamics of HEV growth after immunization with protein plus CFA and features shared with HEVs in acute autoinflammatory lymphadenopathy.Front. Immunol. 2012; 3: 282Crossref PubMed Scopus (35) Google Scholar]. Such studies have revealed key similarities and differences between different LN stimulation settings (Box 2). The total number of vessels and branching points, including each individual vessel length and diameter, can be extracted from LSFM/OPT-acquired datasets (see Figure I in Box 2). Since vessel elongation and a reduction in diameter may result from vessel stretching or an increase in number of HECs (reflecting expansion), flow cytometric analysis and enumeration of HEC populations represent an important complement to 3D imaging [13.Kumar V. et al.Optical projection tomography reveals dynamics of HEV growth after immunization with protein plus CFA and features shared with HEVs in acute autoinflammatory lymphadenopathy.Front. Immunol. 2012; 3: 282Crossref PubMed Scopus (35) Google Scholar,24.Kumar V. et al.Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway.Blood. 2010; 115: 4725-4733Crossref PubMed Scopus (108) Google Scholar]. Incorporation of these techniques into studies of LN remodelling should shed further light on the impact of alterations in HEV network/morphology on the course of antigen-specific immune responses in different settings.Box 23D Analysis of LN HEV RemodellingLymphocytic choriomeningitis virus (LCMV) is a widely used mouse model for examining immune responses during viral infection due to it being noncytolytic and capable of inducing a wide range of immune responses, depending upon which LCMV strain is used [78.Oldstone M.B.A. An odyssey to viral pathogenesis.Annu. Rev. Pathol. 2016; 11: 1-19Crossref PubMed Scopus (2) Google Scholar]. LCMV has a strong tropism for SLOs and its impact on HEV remodelling was assessed using OPT [24.Kumar V. et al.Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway.Blood. 2010; 115: 4725-4733Crossref PubMed Scopus (108) Google Scholar]. LCMV and OVA/CFA immunisation shared several features, including extent of HEV branching (arborisation) and expansion, which likely reflect shared regulatory mechanisms underpinning HEV remodelling [13.Kumar V. et al.O