n e w s and vi e w s
High endothelial venules through a transcriptomics lens Steven D Rosen & Richard Daneman Transcriptional profiling of endothelial cells from diverse secondary lymphoid organs reveals distinctions that underlie their functional specification.
npg
© 2014 Nature America, Inc. All rights reserved.
H
igh endothelial venules (HEVs) are the portals of entry for blood-borne lymphocytes into secondary lymphoid organs such as lymph nodes and Peyer’s patches (PPs). Lee et al. now provide a genome-wide transcriptomics analysis of endothelial cells, including those isolated from HEVs1. Their study contributes what is expected to be an essential resource for future work on this critical microvasculature and provides many new insights into HEV biology and lymphocyte migration, including a previously unknown role for the B cell lectin CD22 (Siglec-2) in the homing of B cells to PPs. The field of lymphocyte homing and recirculation began with the monumental work of James Gowans, who first described the extensive migration of small lymphocytes from the blood into lymph nodes and PPs through specialized high-walled venules, now called ‘high endothelial venules’2. Noting that HEVs are infiltrated extensively, yet very selectively, by lymphocytes, Gowans posited that there must be a “special affinity” between lymphocytes and the endothelium of these vessels2. The past 50 years of research have seen tremendous progress in understanding the migration (‘homing’) of lymphocytes across HEVs and how this “special affinity” is achieved. Naive B lymphocytes and T lymphocytes and certain memory populations interact with HEVs through a multistep adhesion cascade3–6. Homing to different lymphoid organs involves distinct mechanisms, with those for peripheral lymph nodes (PLNs) being the best understood. Homing to PLNs begins with the tethering and rolling of lymphocytes on HEVs, mediated by L-selectin’s engaging the sulfated, sialylated and fucosylated determinant sialyl α2-3-galactose β1-4[fucose α1-3][SO3-6] N-acetylglucosamine (6-sulfosialyl Lewis X) that is presented on a set of HEV-expressed mucins (the peripheral lymph node addressin (PNAd) complex). Rolling lymphocytes encounter immobilized chemokines Steven D. Rosen is in the Department of Anatomy and Program in Immunology, University of California, San Francisco, California, USA. Richard Daneman is in the Department of Pharmacology and the Department of Neuroscience, University of California, San Diego, California, USA. e-mail: [email protected]
906
(such as CCL21) on the luminal aspect of HEVs and, through signaling by chemokine receptors (such as CCR7 for CCL21), undergo activation of the integrin LFA-1. The activated integrin engages intercellular adhesion molecules expressed on HEVs, which leads to the arrest of the lymphocytes. The cells migrate intralumenally on HEVs and subsequently transmigrate into the node. For homing to PPs, overlapping but distinct mechanisms are involved. L-selectin mediates the initial rolling step, but the nature of the carbohydraterecognition determinant on PP HEVs differs from that on PLN HEVs. In a further distinction, the velocity of rolling is reduced through the interaction of integrin α4β7 (expressed on lymphocytes) with its ligand MAdCAM-1 (on HEVs). Activation by chemokines (by an overlapping set, including CCL21) leads to integrin-mediated arrest of the lymphocytes through both interactions between LFA-1 and intercellular adhesion molecules and those between the integrin α4β7 and MAdCAM-1. It is now clear that the “special affinity” of lymphocytes for HEVs (as well as that of various leukocyte subpopulations for other vascular beds) is determined by the combinatorial use of adhesion molecules and chemo kines in a multistep process. Despite this new understanding, many questions remain. For example, are there additional adhesive and signaling mechanisms that confer specificity in homing? How are the intraluminal migration and transendothelial migration of lymphocytes regulated? What are the programs for the specification and maintenance of HEVs? The objective of the study by Lee et al. is to extend the knowledge of HEVs through a gene-expression analysis of purified high endothelial cells (HECs)1. It has long been established that there is important structural and functional heterogeneity in the vasculature, both in different segments of the vascular tree (arteries, capillaries and veins) and in the same segments in different organs throughout the body. This heterogeneity is important in allowing region-specific regulation of blood flow, permeability, clotting, leukocyte trafficking and other vascular functions. Several groups have used the purification of endothelial cells and micro array analysis to identify regional vascular
gene expression, including the identification of transcriptomic signatures of endothelial cells in the central nervous system that form the blood-brain barrier and liver endothelial cells that form discontinuous sinusoidal liver vessels7,8. Here, Lee et al. aim to identify the molecular signatures of HECs and capillary endothelial cells (CAP ECs) in different lymphoid organs. In particular, they address two questions: first, what makes HECs different from CAP ECs, and second, what are the differences between HECs in these organs? To accomplish this, Lee et al. isolate HECs and CAP ECs by flow cytometry from dissociated PLNs, mesenteric lymph nodes (MLNs) and PPs1. They first enrich the samples for blood vascular endothelial cells by positive selection with monoclonal antibody to the adhesion molecule CD31 (PECAM-1) and by negative selection to remove cells of hematolymphoid lineages, lymphatic ECs and stromal cells. They then sort capillary endothelial cells and HECs with the monoclonal antibody MECA-99 to recognize CAP ECs, with the monoclonal antibody MECA-79 to recognize the sulfated L-selectin ligands of PLN HEVs and with monoclonal antibody to MAdCAM-1 to stain PP HEVs and MLN HEVs. Since Lee et al. adhere to standards of the Immunological Genome Project, it is anticipated that their new data sets can be incorporated into that resource and thus be generally available to the community. Principal-component analysis of genes expre ssed differently by the various EC populations demonstrates that the largest variability is between CAP ECs and HECs. There are also tissue-specific differences among HECs (PLN versus MLN versus PP) (Fig. 1), consistent with known distinctions in homing mechanisms, as well as surprising differences among CAP ECs. The ana lysis provides lists of signature gene sets for HECs and CAP ECs (in which differences in expression hold for all three organs), as well as genes with different expression between HEC populations. Lee et al. highlight several examples of genes with higher expression in HECs than in CAP ECs1. Consistent with their trafficking functions, HECs have higher expression of genes encoding certain chemokines (Ccl21, Cxcl9 and Cxcl10) and molecules involved in
volume 15 number 10 october 2014 nature immunology
n e w s and vi e w s
Lymphatic system
PLN
Principal-component analysis
HEV
Afferent lymphatic vessel Capillary Artery
PLNs
Vein
Efferent lymphatic vessel
MLN PP HEC HEV
Afferent lymphatic vessel
PC2
PP CAP
Capillary
Kim Caesar/Nature Publishing Group
Artery
© 2014 Nature America, Inc. All rights reserved.
PLN HEC
PLN CAP
MLNs
npg
MLN HEC MLN CAP
PPs
Vein
Efferent lymphatic vessel
PC1
PP Follicle-associated epithelium HEV Capillary Artery
Vein
Figure 1 The transcriptomic resource. The secondary lymphoid organs (left) include PLNs, MLNs and PPs (details, middle). Lee et al. isolate HECs and CAP ECs from pooled inguinal, axillary and brachial lymph nodes for PLN samples, and isolate HECs and CAP ECs from MLNs and PPs. Capillaries deliver nutrients to the lymph tissues, whereas HEVs regulate the trafficking of lymphocytes from the blood into the lymphoid organs. Principal-component analysis (right) of gene expression in HECs and CAP ECs isolated from PLNs, MLNs and PPs shows that the biggest difference in gene-expression profiles (PC1) is between HECs and CAP ECs, whereas the next biggest difference (PC2) distinguishes the anatomical source (PLN, MLN or PP) of each subset of endothelial cells.
chemokine transcellular transport (Darc) and chemokine scavenging (Ackr2). CXCL9 and CXCL10 have previously been linked to the recruitment of activated T cells and monocytes across HEVs during inflammation4,5. Notably, HECs have higher expression of genes encoding molecules involved in the synthesis of sphingosine 1-phosphate (Sphk1 and Asah2). Sphingosine 1-phosphate from HEVs may be relevant to integrin-mediated arrest of lymphocytes or may exert autocrine activities on the endothelial cells. HECs have higher expression of Ch25h and Cyp27a1, which encode enzymes involved in the synthesis of 25-hydroxycholesterol and 27-hydrocholesterol, precursors of B cell chemoattractants. Lee et al. note that the gene encoding the 7-hydroxylase CYP7B1, which is responsible for generating active ligands, is expressed in stromal cells, whereas the gene encoding the degradative hydroxysteroid dehydrogenase HSD3B7 is expressed in HECs1; this suggests the existence of a HEV–stromal cell gradient of the ligands. Such a gradient could be pertinent to the transient retention of B cells around HEVs preceding their entry into follicles6. Genes involved in innate defense also
have higher expression in HECs, including those encoding complement components, a pattern receptor for Gram-negative bacteria, an antimicrobial protein and inhibitors of neutrophil proteases. These factors may offer protection to HEVs against various insults that might otherwise compromise this critical portal. The genes with higher expression in CAP ECs also suggest some interesting physiological possibilities. CAP ECs have higher expression of genes encoding molecules involved in angiogenesis, including components of the VEGF, PDGF, Notch, TGF-β and Wnt signaling pathways. The 'enrichment' for these pathways in capillaries may reflect the need to expand the microvasculature when the lymphoid organ is activated. In line with more general capillary endothelial function is the ‘enrichment’ for genes encoding molecules involved in water transport (Aqp7 and Aqp11), pH control (Car4 and Car7) and lipid transport (Cd36). There are also differences in CAP ECs from different lymphoid organs, indicative of distinct, as-yet-undefined functions of the capillary bed in each organ.
nature immunology volume 15 number 10 october 2014
A particular strength of this study is its sophisticated analysis of the glycobiological implications of the HEC transcriptomes. Applying several gene-ontology approaches, Lee et al. document that the gene signature of HECs shows considerable enrichment for glycosylation-related transcripts relative to their abundance in CAP ECs, which is not surprising given the critical function of L-selectin in homing1. N-acetyllactosamine galactosyl β1-4 N-acetylglucosamine (LacNAc;) serves as framework for the elaboration of 6-sulfosialyl Lewis X. Notably, genes encoding many of the enzymes involved in the synthesis of LacNAc show higher expression in the transcriptomes of HECs than in those of CAP ECs. Chst4 and Fut7, which encode the key enzymes that provide the 6-sulfation and α1-3 fucos ylation, respectively, of N-acetylglucosamine (GlcNAc) in LacNAc, are among the top 100 HEC signature genes with the greatest difference in expression in HECs versus CAP ECs. Although it is expressed in PP HECs, Chst4 has much higher expression (~17-fold) in PLN HECs, a finding consistent with the much smaller amount of 6-sulfo-sialyl Lewis X on PP HEVs and the dispensability of this enzyme for the homing of lymphocytes to PPs9,10. The lower avidity of L-selectin ligands in PP HEVs, which underlies the higher rolling velocity of lymphocytes in this vascular bed, may be due to the reduced abundance of 6-sulfation on sialyl Lewis X. Searching for additional mucin-like ligands for L-selectin, the authors spot upregulation of Parm1 in PLN HECs relative to its expression in CAP ECs. They verify expression of the encoded mucin in PLN HEVs and document that it reacts with the monoclonal antibody MECA-79, which supports the proposal that it is another member of the PNAd complex. As for adhesive ligands on PP HEVs, it is very satisfying to see that Madcam1 ranks as the gene with the greatest difference in expression by PP HECs relative to its expression in PLN HECs. The most exciting biological finding of the study is triggered by the observation that St6gal1 has higher expression in PP HECs than in PLN HECs or CAP ECs. This gene encodes an α2-6 sialyltransferase that acts on LacNAc to produce sialyl α2-6-galactosyl β1-4 N-acetylglucosamine. This structure is a known recognition determinant for CD22, a B cell Siglec previously linked to the accumulation of B cells in bone marrow11. Indeed, PP HECs are stained by a chimera of CD22 and immunoglobulin Fc, whereas PLN HECs stain minimally with this chimera. Short-term migration studies of wild-type mice given Cd22–/– donor lymphocytes or St6gal1–/– recipients given wild-type lymphocytes point to the involvement of CD22 in the homing of B cells to PPs. Such a 907
n e w s and vi e w s Of additional interest will be the investigation of HEVs in tertiary lymphoid organs (TLOs). These organized collections of lymphoid cells, which have HEV-like vessels, arise in settings of chronic inflammation, infection, autoimmunity and cancer6,14,15. Increasing evidence indicates that TLOs initiate adaptive immune responses to local antigens. The presence of TLOs can result in either deleterious consequences (chronic inflammation or graft rejection) or beneficial consequences (cancer or infection)6,14,15. As HEV-like vessels are critical to the genesis and maintenance of TLOs14, fuller understanding of these vessels gained from the Lee et al. resource1 and follow-up studies may open up new opportunities for therapeutic intervention in a wide range of diseases. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
1. Lee, et al. Nat. Immunol. 15, 982–995 (2014). 2. Gowans, J.L. & Knight, E.J. Proc. R. Soc. Lond. B Biol. Sci. 159, 257–282 (1964). 3. Butcher, E.C. & Picker, L.J. Science 272, 60–66 (1996). 4. von Andrian, U.H. & Mempel, T.R. Nat. Rev. Immunol. 3, 867–878 (2003). 5. Miyasaka, M. & Tanaka, T. Nat. Rev. Immunol. 4, 360–370 (2004). 6. Girard, J.P. et al. Nat. Rev. Immunol. 12, 762–773 (2012). 7. Seaman, S. et al. Cancer Cell 11, 539–554 (2007). 8. Daneman, R. et al. PLoS ONE 5, e13741 (2010). 9. Kawashima, H. et al. Nat. Immunol. 6, 1096–1104 (2005). 10. Uchimura, K. et al. Nat. Immunol. 6, 1105–1113 (2005). 11. Nitschke, L. et al. J. Exp. Med. 189, 1513–1518 (1999). 12. Stevens, S.K. et al. J. Immunol. 128, 844–851 (1982). 13. Kimura, N. et al. J. Biol. Chem. 282, 32200–32207 (2007). 14. Ruddle, N.H. & Akirav, E.M. J. Immunol. 183, 2205–2212 (2009). 15. Pitzalis, C. et al. Nat. Rev. Immunol. 14, 447–462 (2014).
npg
© 2014 Nature America, Inc. All rights reserved.
mechanism may help explain why B cells have a homing ‘preference’ for PPs rather than PLNs, relative to that of T cells12. The more complete recognition determinant for CD22 on HEVs may be a GlcNAc-6-sulfated version of sialyl α2-6-galactosyl β1-4 N-acetylglucosamine13, a critical point that remains to be explored with sulfotransferase-null mice (in particular Chst2–/– mice)9,10. Of course, this transcriptomic data set is just the start. It undoubtedly provides a fantastic resource, but much hard work still remains to be done to achieve full understanding of how the molecules encoded by HEC-expressed genes of the HEC signature regulate the development, structure and function of HEVs in the various lymphoid organs. In the future, combining cellpurification techniques with RNA sequencing, proteomics, glycomics, metabolomics and other ‘-omics’ approaches will provide even broader understanding of this microvasculature.
908
volume 15 number 10 october 2014 nature immunology
High endothelial venules through a transcriptomics lens Steven D Rosen & Richard Daneman Transcriptional profiling of endothelial cells from diverse secondary lymphoid organs reveals distinctions that underlie their functional specification.
npg
© 2014 Nature America, Inc. All rights reserved.
H
igh endothelial venules (HEVs) are the portals of entry for blood-borne lymphocytes into secondary lymphoid organs such as lymph nodes and Peyer’s patches (PPs). Lee et al. now provide a genome-wide transcriptomics analysis of endothelial cells, including those isolated from HEVs1. Their study contributes what is expected to be an essential resource for future work on this critical microvasculature and provides many new insights into HEV biology and lymphocyte migration, including a previously unknown role for the B cell lectin CD22 (Siglec-2) in the homing of B cells to PPs. The field of lymphocyte homing and recirculation began with the monumental work of James Gowans, who first described the extensive migration of small lymphocytes from the blood into lymph nodes and PPs through specialized high-walled venules, now called ‘high endothelial venules’2. Noting that HEVs are infiltrated extensively, yet very selectively, by lymphocytes, Gowans posited that there must be a “special affinity” between lymphocytes and the endothelium of these vessels2. The past 50 years of research have seen tremendous progress in understanding the migration (‘homing’) of lymphocytes across HEVs and how this “special affinity” is achieved. Naive B lymphocytes and T lymphocytes and certain memory populations interact with HEVs through a multistep adhesion cascade3–6. Homing to different lymphoid organs involves distinct mechanisms, with those for peripheral lymph nodes (PLNs) being the best understood. Homing to PLNs begins with the tethering and rolling of lymphocytes on HEVs, mediated by L-selectin’s engaging the sulfated, sialylated and fucosylated determinant sialyl α2-3-galactose β1-4[fucose α1-3][SO3-6] N-acetylglucosamine (6-sulfosialyl Lewis X) that is presented on a set of HEV-expressed mucins (the peripheral lymph node addressin (PNAd) complex). Rolling lymphocytes encounter immobilized chemokines Steven D. Rosen is in the Department of Anatomy and Program in Immunology, University of California, San Francisco, California, USA. Richard Daneman is in the Department of Pharmacology and the Department of Neuroscience, University of California, San Diego, California, USA. e-mail: [email protected]
906
(such as CCL21) on the luminal aspect of HEVs and, through signaling by chemokine receptors (such as CCR7 for CCL21), undergo activation of the integrin LFA-1. The activated integrin engages intercellular adhesion molecules expressed on HEVs, which leads to the arrest of the lymphocytes. The cells migrate intralumenally on HEVs and subsequently transmigrate into the node. For homing to PPs, overlapping but distinct mechanisms are involved. L-selectin mediates the initial rolling step, but the nature of the carbohydraterecognition determinant on PP HEVs differs from that on PLN HEVs. In a further distinction, the velocity of rolling is reduced through the interaction of integrin α4β7 (expressed on lymphocytes) with its ligand MAdCAM-1 (on HEVs). Activation by chemokines (by an overlapping set, including CCL21) leads to integrin-mediated arrest of the lymphocytes through both interactions between LFA-1 and intercellular adhesion molecules and those between the integrin α4β7 and MAdCAM-1. It is now clear that the “special affinity” of lymphocytes for HEVs (as well as that of various leukocyte subpopulations for other vascular beds) is determined by the combinatorial use of adhesion molecules and chemo kines in a multistep process. Despite this new understanding, many questions remain. For example, are there additional adhesive and signaling mechanisms that confer specificity in homing? How are the intraluminal migration and transendothelial migration of lymphocytes regulated? What are the programs for the specification and maintenance of HEVs? The objective of the study by Lee et al. is to extend the knowledge of HEVs through a gene-expression analysis of purified high endothelial cells (HECs)1. It has long been established that there is important structural and functional heterogeneity in the vasculature, both in different segments of the vascular tree (arteries, capillaries and veins) and in the same segments in different organs throughout the body. This heterogeneity is important in allowing region-specific regulation of blood flow, permeability, clotting, leukocyte trafficking and other vascular functions. Several groups have used the purification of endothelial cells and micro array analysis to identify regional vascular
gene expression, including the identification of transcriptomic signatures of endothelial cells in the central nervous system that form the blood-brain barrier and liver endothelial cells that form discontinuous sinusoidal liver vessels7,8. Here, Lee et al. aim to identify the molecular signatures of HECs and capillary endothelial cells (CAP ECs) in different lymphoid organs. In particular, they address two questions: first, what makes HECs different from CAP ECs, and second, what are the differences between HECs in these organs? To accomplish this, Lee et al. isolate HECs and CAP ECs by flow cytometry from dissociated PLNs, mesenteric lymph nodes (MLNs) and PPs1. They first enrich the samples for blood vascular endothelial cells by positive selection with monoclonal antibody to the adhesion molecule CD31 (PECAM-1) and by negative selection to remove cells of hematolymphoid lineages, lymphatic ECs and stromal cells. They then sort capillary endothelial cells and HECs with the monoclonal antibody MECA-99 to recognize CAP ECs, with the monoclonal antibody MECA-79 to recognize the sulfated L-selectin ligands of PLN HEVs and with monoclonal antibody to MAdCAM-1 to stain PP HEVs and MLN HEVs. Since Lee et al. adhere to standards of the Immunological Genome Project, it is anticipated that their new data sets can be incorporated into that resource and thus be generally available to the community. Principal-component analysis of genes expre ssed differently by the various EC populations demonstrates that the largest variability is between CAP ECs and HECs. There are also tissue-specific differences among HECs (PLN versus MLN versus PP) (Fig. 1), consistent with known distinctions in homing mechanisms, as well as surprising differences among CAP ECs. The ana lysis provides lists of signature gene sets for HECs and CAP ECs (in which differences in expression hold for all three organs), as well as genes with different expression between HEC populations. Lee et al. highlight several examples of genes with higher expression in HECs than in CAP ECs1. Consistent with their trafficking functions, HECs have higher expression of genes encoding certain chemokines (Ccl21, Cxcl9 and Cxcl10) and molecules involved in
volume 15 number 10 october 2014 nature immunology
n e w s and vi e w s
Lymphatic system
PLN
Principal-component analysis
HEV
Afferent lymphatic vessel Capillary Artery
PLNs
Vein
Efferent lymphatic vessel
MLN PP HEC HEV
Afferent lymphatic vessel
PC2
PP CAP
Capillary
Kim Caesar/Nature Publishing Group
Artery
© 2014 Nature America, Inc. All rights reserved.
PLN HEC
PLN CAP
MLNs
npg
MLN HEC MLN CAP
PPs
Vein
Efferent lymphatic vessel
PC1
PP Follicle-associated epithelium HEV Capillary Artery
Vein
Figure 1 The transcriptomic resource. The secondary lymphoid organs (left) include PLNs, MLNs and PPs (details, middle). Lee et al. isolate HECs and CAP ECs from pooled inguinal, axillary and brachial lymph nodes for PLN samples, and isolate HECs and CAP ECs from MLNs and PPs. Capillaries deliver nutrients to the lymph tissues, whereas HEVs regulate the trafficking of lymphocytes from the blood into the lymphoid organs. Principal-component analysis (right) of gene expression in HECs and CAP ECs isolated from PLNs, MLNs and PPs shows that the biggest difference in gene-expression profiles (PC1) is between HECs and CAP ECs, whereas the next biggest difference (PC2) distinguishes the anatomical source (PLN, MLN or PP) of each subset of endothelial cells.
chemokine transcellular transport (Darc) and chemokine scavenging (Ackr2). CXCL9 and CXCL10 have previously been linked to the recruitment of activated T cells and monocytes across HEVs during inflammation4,5. Notably, HECs have higher expression of genes encoding molecules involved in the synthesis of sphingosine 1-phosphate (Sphk1 and Asah2). Sphingosine 1-phosphate from HEVs may be relevant to integrin-mediated arrest of lymphocytes or may exert autocrine activities on the endothelial cells. HECs have higher expression of Ch25h and Cyp27a1, which encode enzymes involved in the synthesis of 25-hydroxycholesterol and 27-hydrocholesterol, precursors of B cell chemoattractants. Lee et al. note that the gene encoding the 7-hydroxylase CYP7B1, which is responsible for generating active ligands, is expressed in stromal cells, whereas the gene encoding the degradative hydroxysteroid dehydrogenase HSD3B7 is expressed in HECs1; this suggests the existence of a HEV–stromal cell gradient of the ligands. Such a gradient could be pertinent to the transient retention of B cells around HEVs preceding their entry into follicles6. Genes involved in innate defense also
have higher expression in HECs, including those encoding complement components, a pattern receptor for Gram-negative bacteria, an antimicrobial protein and inhibitors of neutrophil proteases. These factors may offer protection to HEVs against various insults that might otherwise compromise this critical portal. The genes with higher expression in CAP ECs also suggest some interesting physiological possibilities. CAP ECs have higher expression of genes encoding molecules involved in angiogenesis, including components of the VEGF, PDGF, Notch, TGF-β and Wnt signaling pathways. The 'enrichment' for these pathways in capillaries may reflect the need to expand the microvasculature when the lymphoid organ is activated. In line with more general capillary endothelial function is the ‘enrichment’ for genes encoding molecules involved in water transport (Aqp7 and Aqp11), pH control (Car4 and Car7) and lipid transport (Cd36). There are also differences in CAP ECs from different lymphoid organs, indicative of distinct, as-yet-undefined functions of the capillary bed in each organ.
nature immunology volume 15 number 10 october 2014
A particular strength of this study is its sophisticated analysis of the glycobiological implications of the HEC transcriptomes. Applying several gene-ontology approaches, Lee et al. document that the gene signature of HECs shows considerable enrichment for glycosylation-related transcripts relative to their abundance in CAP ECs, which is not surprising given the critical function of L-selectin in homing1. N-acetyllactosamine galactosyl β1-4 N-acetylglucosamine (LacNAc;) serves as framework for the elaboration of 6-sulfosialyl Lewis X. Notably, genes encoding many of the enzymes involved in the synthesis of LacNAc show higher expression in the transcriptomes of HECs than in those of CAP ECs. Chst4 and Fut7, which encode the key enzymes that provide the 6-sulfation and α1-3 fucos ylation, respectively, of N-acetylglucosamine (GlcNAc) in LacNAc, are among the top 100 HEC signature genes with the greatest difference in expression in HECs versus CAP ECs. Although it is expressed in PP HECs, Chst4 has much higher expression (~17-fold) in PLN HECs, a finding consistent with the much smaller amount of 6-sulfo-sialyl Lewis X on PP HEVs and the dispensability of this enzyme for the homing of lymphocytes to PPs9,10. The lower avidity of L-selectin ligands in PP HEVs, which underlies the higher rolling velocity of lymphocytes in this vascular bed, may be due to the reduced abundance of 6-sulfation on sialyl Lewis X. Searching for additional mucin-like ligands for L-selectin, the authors spot upregulation of Parm1 in PLN HECs relative to its expression in CAP ECs. They verify expression of the encoded mucin in PLN HEVs and document that it reacts with the monoclonal antibody MECA-79, which supports the proposal that it is another member of the PNAd complex. As for adhesive ligands on PP HEVs, it is very satisfying to see that Madcam1 ranks as the gene with the greatest difference in expression by PP HECs relative to its expression in PLN HECs. The most exciting biological finding of the study is triggered by the observation that St6gal1 has higher expression in PP HECs than in PLN HECs or CAP ECs. This gene encodes an α2-6 sialyltransferase that acts on LacNAc to produce sialyl α2-6-galactosyl β1-4 N-acetylglucosamine. This structure is a known recognition determinant for CD22, a B cell Siglec previously linked to the accumulation of B cells in bone marrow11. Indeed, PP HECs are stained by a chimera of CD22 and immunoglobulin Fc, whereas PLN HECs stain minimally with this chimera. Short-term migration studies of wild-type mice given Cd22–/– donor lymphocytes or St6gal1–/– recipients given wild-type lymphocytes point to the involvement of CD22 in the homing of B cells to PPs. Such a 907
n e w s and vi e w s Of additional interest will be the investigation of HEVs in tertiary lymphoid organs (TLOs). These organized collections of lymphoid cells, which have HEV-like vessels, arise in settings of chronic inflammation, infection, autoimmunity and cancer6,14,15. Increasing evidence indicates that TLOs initiate adaptive immune responses to local antigens. The presence of TLOs can result in either deleterious consequences (chronic inflammation or graft rejection) or beneficial consequences (cancer or infection)6,14,15. As HEV-like vessels are critical to the genesis and maintenance of TLOs14, fuller understanding of these vessels gained from the Lee et al. resource1 and follow-up studies may open up new opportunities for therapeutic intervention in a wide range of diseases. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
1. Lee, et al. Nat. Immunol. 15, 982–995 (2014). 2. Gowans, J.L. & Knight, E.J. Proc. R. Soc. Lond. B Biol. Sci. 159, 257–282 (1964). 3. Butcher, E.C. & Picker, L.J. Science 272, 60–66 (1996). 4. von Andrian, U.H. & Mempel, T.R. Nat. Rev. Immunol. 3, 867–878 (2003). 5. Miyasaka, M. & Tanaka, T. Nat. Rev. Immunol. 4, 360–370 (2004). 6. Girard, J.P. et al. Nat. Rev. Immunol. 12, 762–773 (2012). 7. Seaman, S. et al. Cancer Cell 11, 539–554 (2007). 8. Daneman, R. et al. PLoS ONE 5, e13741 (2010). 9. Kawashima, H. et al. Nat. Immunol. 6, 1096–1104 (2005). 10. Uchimura, K. et al. Nat. Immunol. 6, 1105–1113 (2005). 11. Nitschke, L. et al. J. Exp. Med. 189, 1513–1518 (1999). 12. Stevens, S.K. et al. J. Immunol. 128, 844–851 (1982). 13. Kimura, N. et al. J. Biol. Chem. 282, 32200–32207 (2007). 14. Ruddle, N.H. & Akirav, E.M. J. Immunol. 183, 2205–2212 (2009). 15. Pitzalis, C. et al. Nat. Rev. Immunol. 14, 447–462 (2014).
npg
© 2014 Nature America, Inc. All rights reserved.
mechanism may help explain why B cells have a homing ‘preference’ for PPs rather than PLNs, relative to that of T cells12. The more complete recognition determinant for CD22 on HEVs may be a GlcNAc-6-sulfated version of sialyl α2-6-galactosyl β1-4 N-acetylglucosamine13, a critical point that remains to be explored with sulfotransferase-null mice (in particular Chst2–/– mice)9,10. Of course, this transcriptomic data set is just the start. It undoubtedly provides a fantastic resource, but much hard work still remains to be done to achieve full understanding of how the molecules encoded by HEC-expressed genes of the HEC signature regulate the development, structure and function of HEVs in the various lymphoid organs. In the future, combining cellpurification techniques with RNA sequencing, proteomics, glycomics, metabolomics and other ‘-omics’ approaches will provide even broader understanding of this microvasculature.
908
volume 15 number 10 october 2014 nature immunology
