High Endothelial Venules Endothelial Cells Research Articles

Prediction Modeling of CAR-T Cell Therapy for Diffuse Large B-Cell Lymphoma Using Artificial Neural Networks on Tumor Vascular Phenotype

Chimeric antigen receptor T (CAR-T) cell therapy targeting CD19 only results in complete remission in approximately 30-40% of relapsed/refractory disease patients with diffuse large B-cell lymphoma (DLBCL). Failure may result from lack of CAR-T entry into the tumor. Endogenous T cell homing is initiated by binding to post-capillary venular endothelial cells (ECs). Naïve and central memory T cells use L-selectin binding to peripheral node addressin (PNad) on high endothelial venules (HEVs) of the lymph node while circulating effector memory T cells, lacking L-selectin, use other molecules to bind cytokine-induced endothelial proteins E-selectin, ICAM-1 and VCAM-1 on flat venules of inflamed tissues. Both of these venular EC types are present in inflamed lymph nodes. We hypothesized that the expression patterns of adhesion molecules on venular ECs in pre-treatment tumor biopsies could predict CAR-T efficacy. To test this, we applied cyclic immunofluorescence to 18 archived pre-treatment DLBCL patient FFPE biopsies (8 responders and 10 non-responders, defined by relapse at 12 months). We detected venular ECs using a combination of anti-CD31 and UEA lectin on larger diameter vessels and assessed expression for PNad, E-selectin, ICAM-1, VCAM-1. We detected endogenous CD8+ T cells and evaluated their expression of L-selectin, alpha 4 integrin and PSGL-1. See table 1. DAPI nuclear staining was included in each cycle. Images of each cycle were combined in ImageJ and imported into QuPath for analysis. Using Warpy, affine transformations were applied to DAPI staining to create overlays between imaging cycles. After alignment, representative images of several samples were randomly chosen for image training using a combination of Artificial Neural Networks (ANNs) and classifiers to perform identification and mask tissue regions of vessels and T-cells within the tissue. Positive and negative cells for each marker of interest (EC PNad, ICAM-1, E-Selectin, VCAM-1, and T cell alpha 4 integrin, L-Selectin, and PSGL-1) were used to create classification algorithms to determine marker expression for ECs and T-cells within the tissue. Results were analyzed by unpaired t-test and one-way ANOVA conducted in Graph-Pad Prism 9 software. Preliminary results from quantitative analysis of pre-treatment biopsies show a significant increase in CD31+/PNAD- venular ECs compared to non-responders that were double positive for ICAM1 and VCAM-1 (p=0.0273). This analysis also revealed a significant increase in CD31+/PNAD+ HEV ECs from responders that were triple positive for E-Selectin, ICAM-1, and VCAM-1 (p=0.0242). See figure 1. Our initial semi-quantitative analysis showed a trend toward elevated numbers of tissue-infiltrating CD3 + CD8 + T cells in the pre-treatment biopsies of responders. T cells expressing PSGL-1, a glycoprotein that may carry carbohydrate moieties recognized by E-Selectin and linked to T-cell exhaustion, was increased in the non-responder cohort. These data suggest an increased extent and activation state of intra-tumoral venular ECs and increased endogenous T-cell infiltrates, which may predict the outcome of CAR-T therapy. Analysis of T-cells in the pre-treatment biopsies leveraging machine learning algorithms is in progress and both complete quantitative findings and prognostic value of this approach will be presented. While our findings require further validation thru additional biopsy analysis, they provide emerging evidence for a rationale involving vascular readiness in DLBCL responder differences to CART therapy.

Stepwise transmigration of T- and B cells through a perivascular channel in high endothelial venules.

High endothelial venules (HEVs) effectively recruit circulating lymphocytes from the blood to lymph nodes. HEVs have endothelial cells (ECs) and perivascular sheaths consisting of fibroblastic reticular cells (FRCs). Yet, post-luminal lymphocyte migration steps are not well elucidated. Herein, we performed intravital imaging to investigate post-luminal T- and B-cell migration in popliteal lymph node, consisting of trans-EC migration, crawling in the perivascular channel (a narrow space between ECs and FRCs) and trans-FRC migration. The post-luminal migration of T cells occurred in a PNAd-dependent manner. Remarkably, we found hot spots for the trans-EC and trans-FRC migration of T- and B cells. Interestingly, T- and B cells preferentially shared trans-FRC migration hot spots but not trans-EC migration hot spots. Furthermore, the trans-FRC T-cell migration was confined to fewer sites than trans-EC T-cell migration, and trans-FRC migration of T- and B cells preferentially occurred at FRCs covered by CD11c+ dendritic cells in HEVs. These results suggest that HEV ECs and FRCs with perivascular DCs delicately regulate T- and B-cell entry into peripheral lymph nodes.

Single-Cell Analysis Reveals Heterogeneity of High Endothelial Venules and Different Regulation of Genes Controlling Lymphocyte Entry to Lymph Nodes

High-endothelial venules (HEVs) are specialized blood vessels allowing recirculation of naive lymphocytes through lymphoid organs. Here, using full-length, single-cell RNA sequencing, RNA fluorescence in situ hybridization (FISH), flow cytometry, and immunohistofluorescence, we reveal the heterogeneity of HEVs in adult mouse peripheral lymph nodes (PLNs) under conditions of homeostasis, antigenic stimulation, and after inhibition of lymphotoxin-β receptor (LTβR) signaling. We demonstrate that HEV endothelial cells are in an activated state during homeostasis, and we identify the genes characteristic of the differentiated HEV phenotype. We show that LTβR signaling regulates many HEV genes and pathways in resting PLNs and that immune stimulation induces a global and temporary inflammatory phenotype in HEVs without compromising their ability to recruit naive lymphocytes. Most importantly, we uncover differences in the regulation of genes controlling lymphocyte trafficking, Glycam1, Fut7, Gcnt1, Chst4, B3gnt3, and Ccl21a, that have implications for HEV function and regulation in health and disease.

Lysophosphatidic acid receptors LPA4 and LPA6 differentially promote lymphocyte transmigration across high endothelial venules in lymph nodes.

Naive lymphocytes continuously migrate from the blood into lymph nodes (LNs) via high endothelial venules (HEVs). To extravasate from the HEVs, lymphocytes undergo multiple adhesion steps, including tethering, rolling, firm adhesion and transmigration. We previously showed that autotaxin (ATX), an enzyme that generates lysophosphatidic acid (LPA), is highly expressed in HEVs, and that the ATX/LPA axis plays an important role in the lymphocyte transmigration across HEVs. However, the detailed mechanism underlying this axis's involvement in lymphocyte transmigration has remained ill-defined. Here, we show that two LPA receptors, LPA4 and LPA6, are selectively expressed on HEV endothelial cells (ECs) and that LPA4 plays a major role in the lymphocyte transmigration across HEVs in mice. In the absence of LPA4 expression, lymphocytes accumulated heavily within the HEV EC layer, compared to wild-type (WT) mice. This accumulation was also observed in the absence of LPA6 expression, but it was less pronounced. Adoptive transfer experiments using WT lymphocytes revealed that the LPA4 deficiency in ECs specifically compromised the lymphocyte transmigration process, whereas the effect of LPA6 deficiency was not significant. These results indicate that the signals evoked in HEV ECs via the LPA4 and LPA6 differentially regulate lymphocyte extravasation from HEVs in the peripheral LNs.

Constitutive Lymphocyte Transmigration across the Basal Lamina of High Endothelial Venules Is Regulated by the Autotaxin/Lysophosphatidic Acid Axis

Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC's effect was abrogated by ATX inhibition, whereas LPA's effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte-HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state.

Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules

While patrolling the body in search of foreign antigens, naive lymphocytes continuously circulate from the blood, through the lymph nodes, into the lymphatic vessels and back to the blood. This process, called lymphocyte recirculation, provides the body with effective immune surveillance for foreign invaders and for alterations to the body's own cells. However, the mechanisms that regulate lymphocyte recirculation during homeostasis remain incompletely characterized. Here we show that dendritic cells (DCs), which are well known for their role in antigen presentation to T lymphocytes, control the entry of naive lymphocytes to lymph nodes by modulating the phenotype of high endothelial venules (HEVs), which are blood vessels specialized in lymphocyte recruitment. We found that in vivo depletion of CD11c(+) DCs in adult mice over a 1-week period induces a reduction in the size and cellularity of the peripheral and mucosal lymph nodes. In the absence of DCs, the mature adult HEV phenotype reverts to an immature neonatal phenotype, and HEV-mediated lymphocyte recruitment to lymph nodes is inhibited. Co-culture experiments showed that the effect of DCs on HEV endothelial cells is direct and requires lymphotoxin-β-receptor-dependent signalling. DCs express lymphotoxin, and DC-derived lymphotoxin is important for lymphocyte homing to lymph nodes in vivo. Together, our results reveal a previously unsuspected role for DCs in the regulation of lymphocyte recirculation during immune surveillance.

Microanatomy of lymphocyte-endothelial interactions at the high endothelial venules of lymph nodes.

Lymphocyte trafficking into lymph nodes and Peyer's patches is mediated primarily by specifically differentiated venules, called high endothelial venules (HEVs), located in the tissue parenchyma. HEVs have a unique morphology and phenotype, which enables them to interact with circulating lymphocytes efficiently. That is, the HEV endothelial cells have a tall and plump appearance, and constitutively express multiple adhesion molecules and chemokines on their surface. These molecules can interact with cognate receptors on circulating lymphocytes, thereby mediating the stepwise and sequential lymphocyte adhesion and transendothelial migration (TEM) at the HEV endothelial luminal surface. This review summarizes the fine morphological aspects of the unique HEV endothelial cells, with special reference to the spatial distribution of the adhesion molecules and chemokines that regulate lymphocyte migration.

Involvement of the Lysophosphatidic Acid-Generating Enzyme Autotaxin in Lymphocyte-Endothelial Cell Interactions

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.

Plasmacytoid dendritic cells employ multiple cell adhesion molecules sequentially to interact with high endothelial venule cells - molecular basis of their trafficking to lymph nodes

Plasmacytoid dendritic cells (pDCs) are natural type I IFN-producing cells found in lymphoid tissues, where they support both innate and adaptive immune responses. They emigrate from the blood to lymph nodes, apparently through high endothelial venules (HEVs), but little is known about the mechanism. We have investigated the molecular mechanisms of pDC migration using freshly isolated DCs and HEV cells. We found that pDCs bound avidly to HEV cells and then transmigrated underneath them. Two observations suggested that these binding and migration steps are differentially regulated. First, treatment of pDCs with pertussis toxin blocked transmigration but not binding. Second, pDCs were able to bind but not to transmigrate under non-HEV endothelial cells, although the binding was observed to both HEV and non-HEV endothelial cells. Antibody inhibition studies indicated that the binding process was mediated by alphaL and alpha4 integrins on pDCs and by intercellular adhesion molecule (ICAM)-1, ICAM-2 and vascular cell adhesion molecule-1 on HEVs. The transmigration process was also mediated by alphaL and alpha4 integrins on pDCs, with junctional adhesion molecule-A on HEV cells apparently serving as an additional ligand for alphaL integrin. These data show for the first time that pDCs employ multiple adhesion molecules sequentially in the processes of adhesion to and transmigration through HEVs.

免疫細胞は自分の行き先をどのようにして見つけるのか？

Lymphocytes are mobile and travel from blood to lymph and then return to blood, which ensures a coordinated expression of immunological functions. In lymph nodes (LN) and Peyer's patches, the port of entry for naive T and B cells is the high endothelial venules (HEVs). The endothelial cells of HEVs are characterized by the luminal presentation of various adhesion molecules and chemokines. Some of the endothelial adhesion molecules are responsible for the tissue-specific trafficking of lymphocytes by giving lymphocytes positional cues, and hence are called vascular addressins. The peripheral LN addressins (PNAd) include several sialomucins and the mucosal LN addressin is MAdCAM-1. The complementary lymphocyte homing receptors for these addressins are L-selectin for PNAd, and α4β7 integrin for MAdCAM-1. While L-selectin and their counter-receptors are primarily involved in lymphocyte rolling along the endothelial surface, LFA-1 and its counter-receptor ICAMs are thought to be involved in firm adhesion of lymphocytes. During the course of rolling, lymphocytes are rapidly and transiently activated by chemokines expressed in HEV, which leads to rapid activation of LFA-1-mediated lymphocyte adhesion to HEV. How the subsequent transmigration of lymphocytes is controlled remains poorly characterized. Here We briefly review the molecular mechanisms controlling chemokine-driven lymphocyte trafficking across HEVs.

Molecular Characterization of NF-HEV, a Nuclear Factor Preferentially Expressed in Human High Endothelial Venules

Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers's patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.

Molecular and functional characterization of SLC26A11, a sodium‐independent sulfate transporter from high endothelial venules

Lymphocyte emigration from the blood into most secondary lymphoid organs and chronically inflamed tissues occurs at the level of high endothelial venules (HEV). A unique characteristic of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. We have previously shown that sulfate uptake into HEVEC is mediated by two distinct functional classes of sulfate transporters: Na+-coupled transporters and sulfate/anion exchangers. Here, we report the molecular characterization from human HEVEC of SLC26A11, a novel member of the SLC26 sulfate/anion exchanger family. Functional expression studies in COS-7 and Sf9 insect cells revealed that SLC26A11 is targeted to the cell membrane and exhibits Na+-independent sulfate transport activity, sensitive to the anion exchanger inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Northern blot analysis showed the highest SLC26A11 transcript levels in placenta, kidney, and brain. The SLC26A11 gene mapped to human chromosome 17q25, very close to the hereditary hearing loss diseases loci DFNA20, DFNA26, and USH1G. RT-PCR analysis of SLC26 sulfate transporters in human HEVEC revealed coexpression of SLC26A11 with SLC26A2/DTDST and lack of SLC26A1/SAT1, SLC26A3/DRA, and SLC26A8/TAT1. Together, our results indicate that SLC26A11 is a novel Na+-independent sulfate transporter that may cooperate with SLC26A2 to mediate DIDS-sensitive sulfate uptake into HEVEC.

Expression of Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) During Small-Bowel Graft Rejection in Rats

Background: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a critical endothelial adhesion molecule for lymphocyte trafficking to gut-associated lymphoid tissues (GALT) under physiological conditions. It is expressed on special postcapillary venules, that is the high endothelial venules (HEV) in GALT. In this study, we investigated changes in MAdCAM-1 expression during small-bowel graft rejection. Methods: Orthotopic small-bowel transplantation (SBT) with portacaval drainage was performed from brown Norway (BN) rats to Lewis (LEW) rats. Isografted (LEW → LEW) and untransplanted animals served as controls. Animals were killed on days 3, 4, 5, 6 and 7 after SBT. Cryostat sections (6 μm thick) were prepared from normal small-bowel tissues and small-bowel grafts, including Peyer patches (PPs). Indirect immunoperoxidase staining was performed using monoclonal antibodies (mAbs) against rat MAdCAM-1 (OST12). Results: In the PPs of controls, MAdCAM-1 antibodies specifically stained the endothelial cells of HEV, which were predominantly located in the interfollicular areas. In the allografts on day 4 after SBT, the MAdCAM-1 expression was weaker on the HEV in the PPs than in the controls. In the lamina propria of controls, a faint expression of MAdCAM-1 on vessels was observed. On day 4 after allogeneic SBT, the MAdCAM-1 was more strongly expressed on the endothelial cells of the vessels at the base of the villi. As rejection developed, the MAdCAM-1 expression on the vessels progressed toward the villus tip. Conclusions: The change of MAdCAM-1 expression may be involved in the development of small-bowel graft rejection. The vessels at the base of villi, which is associated with lymphocyte recruitment, may become a site of intense immune reactivity in the early phase of small-bowel allograft rejection.

Heterogeneity of Endothelial Cells: The Specialized Phenotype of Human High Endothelial Venules Characterized by Suppression Subtractive Hybridization

High endothelial venules (HEVs) are specialized postcapillary venules, found in lymphoid organs and chronically inflamed tissues, that support high levels of lymphocyte extravasation from the blood. Molecular characterization of HEV endothelial cells (HEVECs) has been hampered by difficulties in their purification and in vitro maintenance. To overcome these limitations, we developed a strategy combining the use of freshly purified HEVECs ( approximately 98% positive for the HEV-specific marker MECA-79) and the recently described polymerase chain reaction (PCR)-based cDNA subtraction cloning procedure called suppression subtractive hybridization (SSH). Subtracted probes prepared by SSH from small amounts of total RNA were used to screen a HEVEC cDNA library. This resulted in cloning of 22 cDNAs preferentially expressed in HEVECs, which encode the promiscuous chemokine receptor DARC, mitochondrial components, and matricellular proteins. The latter included hevin, thrombospondin-1, and mac25/IGFBP-rP1, which is a secreted growth factor-binding protein previously found to accumulate specifically in tumor blood vessels. Biochemical and histochemical analysis confirmed the identification of mac25 and DARC as novel markers of the HEVECs. Ultrastructural immunolocalization revealed a noticeable association of mac25 and MECA-79 antigens with microvillous processes near the endothelial cell junctions, suggesting a role for mac25 in the control of lymphocyte emigration. This study shows that PCR-based SSH is useful for cloning of differentially expressed genes in very small samples.

Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules.

High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na(+)-coupled anion transporters and exhibits 40-50% amino acid identity with the rat renal Na(+)/sulfate cotransporter, NaSi-1, as well as with the human and rat Na(+)/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na(+)-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription-PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na(+)-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV.

Sulfated Sialyl Lewis X, the Putative L-Selectin Ligand, Detected on Endothelial Cells of High Endothelial Venules by a Distinct Set of Anti-Sialyl Lewis X Antibodies

Endothelial cells of high endothelial venules (HEV) in human peripheral lymph nodes expressed a distinct type of sialyl Lewis X antigen, which was detected preferentially with a set of anti-sialyl Lewis X antibodies, 2F3, 2H5 and HECA-452 in immunohistochemistry, while another set of anti-sialyl Lewis X antibodies, FH-6 and CSLEX-1, failed to detect it. The adhesion of cells expressing L-selectin to HEV was inhibited by members of the former set of antibodies in Stamper-Woodruff assays performed on frozen sections of human peripheral lymph nodes. Transfection of a cultured endothelial cell line with a human α1→3 fucosyltransferase, Fuc-T VII, resulted in the expression of a distinct type of sialyl Lewis X antigen having the reactivity similar to that of HEV;i.e.,the antigen appearing on the transfectant clone was detectable only with the set of 2F3, 2H5 and HECA-452, but not with the set of FH-6 and CSLEX-1. Treatment of transfectant cells with sodium chlorate, a metabolic inhibitor of sulfation, resulted in reactivity to the members of the latter set of antibodies, suggesting that sulfation of sialyl Lewis X moiety was the cause of the discrepancy in the reactivity of the anti-sialyl Lewis X antibodies. When tested against various authentic sulfated sialyl Lewis X determinants, 6-sulfo sialyl Lewis X and 6,6′-bis-sulfo sialyl Lewis X were found to be reactive to the antibodies, 2F3, 2H5 and HECA-452, but not with antibodies FH-6 and CSLEX-1, suggesting that the distinct type of sialyl Lewis X determinant on the HEV endothelial cells and Fuc-T VII-transfected endothelial cell clone are mainly 6-sulfo and/or 6,6′-bis-sulfo sialyl Lewis X determinants.

High endothelial venules of the rat lymph node. A review and a question: is their activity antigen specific?

The high endothelial venules (HEVs) of the lymph nodes are sites for transvascular lymphocyte traffic. Due mostly to the wide scale of variations manifested by the HEVs and to frequently restricted conditions of observation, reports often differed on their morphological or functional features, which has led to opposing views on aspects of the functioning of HEVs. In the present review, we analyze previous reports and attempt to derive comprehensive proposals to reconcile variations in actual observations under diverse conditions. This analysis shows that the features typical of the HEV endothelial cells (HEV cells) are stimulated to emerge by antigens and the proper lymphocytes and mediators. The stimulation would implicate drained lymphocytes migrating in the perivascular channel, immediately cuffing an HEV's endothelium. A marked pleomorphism of HEV cells betrays the fact that they undergo individual stimulation and a somewhat heterogeneous activity. Other facts indicate that the subendothelial spaces of HEV cells are sites of interactions between drained lymphocytes, HEV cells, and recruited blood lymphocytes. Facts also reveal time- and site-related variations in the intensity of recruitment of blood lymphocytes by HEV cells and topographically related variations in the nature of the recruited cells. Analysis of some other observations, often ignored, lead to the conclusion that recruitment of lymphocytes by HEV cells for the sake of participating in local specific immune activities is antigen specific, despite the implication of homing receptors of lymphocytes.

High endothelial venules of the rat lymph node. A review and a question: Is their activity antigen specific?

Background The high endothelial venules (HEVs) of the lymph nodes are sites for transvascular lymphocyte traffic. Due mostly to the wide scale of variations manifested by the HEVs and to frequently restricted conditions of observation, reports often differed on their morphological or functional features, which has led to opposing views on aspects of the functioning of HEVs. Methods In the present review, we analyze previous reports and attempt to derive comprehensive proposals to reconcile variations in actual observations under diverse conditions. Results This analysis shows that the features typical of the HEV endothelial cells (HEV cells) are stimulated to emerge by antigens and the proper lymphocytes and mediators. The stimulation would implicate drained lymphocytes migrating in the perivascular channel, immediately cuffing an HEV's endothelium. A marked pleomorphism of HEV cells betrays the fact that they undergo individual stimulation and a somewhat heterogeneous activity. Other facts indicate that the subendothelial spaces of HEV cells are sites of interactions between drained lymphocytes, HEV cells, and recruited blood lymphocytes. Facts also reveal time- and site-related variations in the intensity of recruitment of blood lymphocytes by HEV cells and topographically related variations in the nature of the recruited cells. Conclusions Analysis of some other observations, often ignored, lead to the conclusion that recruitment of lymphocytes by HEV cells for the sake of participating in local specific immune activities is antigen specific, despite the implication of homing receptors of lymphocytes. © 1996 Wiley-Liss, Inc.

Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC

High endothelial venules (HEV) in lymphoid tissues support high levels of lymphocyte extravasion from the blood. We purified high endothelial cells from human tonsils by immunomagnetic selection with MECA79 MAb to construct an HEV cDNA.library. Differential screening of this library using cDNA probes from HEV (plus) or flat-walled vessel (minus) endothelial cells allowed us to characterize a novel human cDNA expressed to high levels in HEV. The cDNA encodes a secreted acidic calcium-binding glycoprotein of 664 as residues, designated hevin, exhibiting 62% identity with the antiadhesive extracellular matrix protein SPARC, over a region of 232 as spanning more than four fifths of the SPARC coding sequence. The primary structure and sequence of hevin are similar to SPARC41ke proteins from rat and quail, called SC1 or GR1. Hevin could contribute to the Induction or maintenance of features of the HEV endothelium that facilitate lymphocyte migration.

Specific Expression of a Complex Sialyl Lewis X Antigen on High Endothelial Venules of Human Lymph Nodes: Possible Candidate for L-Selectin Ligand

L-selectin is a cell adhesion molecule that mediates homing of lymphocytes to the peripheral lymph nodes and is speculated to bind to the carbohydrate determinant which is specifically expressed by the endothelial cells of high endothelial venules (HEVs) in the peripheral lymph nodes. One of the murine monoclonal antibodies (2H5, IgM, κ), which was raised against sialyl Le x-active glycolipids which have long and complicated carbohydrate structures, was found to react strongly to the HEV endothelial cells of the human peripheral lymph nodes, while the typical anti-sialyl Le x antibodies SNH-3, FH-6 and CSLEX-1 failed to detect the antigen on HEV under the same condition. This new antibody was reactive to essentially all endothelial cells of HEV in the peripheral lymph nodes, and moderately to some endothelial cells in Payer′s patches and appendices, but never reacted with the endothelial cells of post capifiary venules in the spleen, thymus, or other organs. The 2H5 antibody detected three major glycoproteins of 90, 110 and 250 kDa, the most abundant molecular species being 110 kDa, in the stroma of human lymph nodes. In Stamper-Woodruff assays employing cryostat sections of human lymph nodes, the 2H5 antibody significantly inhibited the adhesion of peripheral lymphocytes to the endothelial cells of HEV. These results indicate that the carbohydrate antigens defined by the 2H5 antibody, most probably sialyl Le x determinants having complex carbohydrate structures, serve as the ligand for L-selectin on HEV endothelial cells.