Maturation Results in Pronounced Changes in Expression Affecting Recognition by and This information is current as of September 23, 2021. Marieke Bax, Juan J. García-Vallejo, Jihye Jang-Lee, Simon J. North, Tim J. Gilmartin, Gilberto Hernández, Paul R. Crocker, Hakon Leffler, Steven R. Head, Stuart M. Haslam, Anne Dell and Yvette van Kooyk

J Immunol 2007; 179:8216-8224; ; Downloaded from doi: 10.4049/jimmunol.179.12.8216 http://www.jimmunol.org/content/179/12/8216 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2008/03/07/179.12.8216.DC1 Material References This article cites 70 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/179/12/8216.full#ref-list-1

Why The JI? Submit online. by guest on September 23, 2021

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Dendritic Cell Maturation Results in Pronounced Changes in Glycan Expression Affecting Recognition by Siglecs and Galectins1

Marieke Bax,2* Juan J. Garcı´a-Vallejo,2* Jihye Jang-Lee,† Simon J. North,† Tim J. Gilmartin,‡ Gilberto Herna´ndez,‡ Paul R. Crocker,§ Hakon Leffler,¶ Steven R. Head,‡ Stuart M. Haslam,† Anne Dell,† and Yvette van Kooyk3*

Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic reprogramming. Glycosylation is the most common form of posttranslational mod- ification of and has been implicated in multiple aspects of the . To investigate the involvement of Downloaded from glycosylation in the changes that occur during DC maturation, we have studied the differences in the glycan profile of iDC and mDC as well as their glycosylation machinery. For information relating to glycan biosynthesis, gene expression profiles of human -derived iDC and mDC were compared using a gene microarray and quantitative real-time PCR. This gene expression profiling showed a profound maturation-induced up-regulation of the glycosyltransferases involved in the expression of LacNAc, core 1 and sialylated structures and a down-regulation of genes involved in the synthesis of core 2 O-. Glycosylation changes during DC maturation were corroborated by mass spectrometric analysis of N- and O-glycans and by flow cytometry using plant http://www.jimmunol.org/ and glycan-specific Abs. Interestingly, the binding of the LacNAc-specific lectins -3 and -8 increased during mat- uration and up-regulation of expression by mDC correlated with an increased binding of -1, -2, and -7. The Journal of Immunology, 2007, 179: 8216–8224.

endritic cells (DC)4 are the most potent APC in the im- signaling cascade leading to the migration of DC to the neighbor- mune system. They reside as immature DC (iDC) in the ing lymph nodes. In these lymph nodes, the DC arrive as mature D peripheral tissues, where they sense for pathogens (1). DC (mDC), ready to interact with a naive lymphocyte carrying the

Pathogen recognition often results in the activation of iDC via appropriate TCR. mDC are characterized by the expression of high by guest on September 23, 2021 TLR present on their membrane or in intracellular compartments levels of MHC class II, costimulatory molecules, chemokines, and (2). The interaction of TLRs with their ligands elicits a complex , in contrast to iDC, which show low expression of these molecules and high levels of Ag-uptake receptors. Thus, DC suffer

*Department of Molecular Cell Biology and Immunology, Vrije Universiteit Univer- a dramatic change in phenotype and functionality upon maturation. sity Medical Center, Amsterdam, The Netherlands; †Division of Molecular Bio- This change is mediated by the modulation of a wide array of sciences, Imperial College, London, United Kingdom; ‡DNA Microarray Core Fa- molecules and ensures the development of a potent and specific cility, The Scripps Research Institute, La Jolla, CA 92037; §Wellcome Trust Biocentre, University of Dundee, Dundee, United Kingdom; and ¶Section Microbi- immune response. As a result, mDC have a limited Ag uptake and ology, Immunology, and Glycobiology, Department of Laboratory Medicine, Lund processing capacity, whereas Ag presentation and costimu- University, Lund, Sweden lation are promoted (3). Received for publication March 29, 2007. Accepted for publication October 5, 2007. To limit Ag uptake, C-type receptor (CLR) expression on The costs of publication of this article were defrayed in part by the payment of page the cell surface of DC is down-regulated during maturation. CLRs charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. constitute an important family of pattern recognition receptors ex- 1 This work was supported primarily by a Netherlands Organization of Scientific pressed on DC (4) and are well-characterized as Ag-uptake recep- Research Pioneer grant (to Y.v.K.) and in part by National Institute of General Med- tors for glycosylated structures (5) found on pathogens. Except for ical Sciences–The Consortium for Functional Glycomics GM62116. M.B. was sup- the recognition of pathogens, CLR have been implicated in several ported by a Vrije Universiteit Medical Center Institute for and Immunology PhD student grant, P.R.C. was supported by the Wellcome Trust, and H. L. was other functions, such as cell migration (6) and intercellular com- supported by the Swedish Research Council. A.D. is a Biotechnology and Biological munication (7). One of the best studied CLRs is the DC-specific Sciences Research Council Professorial Fellow. ICAM-3-grabbing nonintegrin, also known as DC-SIGN (8). Be- 2 M.B. and J.J.G.-V. contributed equally to this article. sides a pattern recognition receptor for HIV-1 (9), CMV (10), 3 Address correspondence and reprint requests to Dr. Y. van Kooyk, Department of Schistosoma mansoni (11), and other pathogens (5, 12), DC-SIGN Molecular Cell Biology and Immunology, Vrije Universiteit University Medical Cen- ter, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail address: has been shown to also recognize glycan epitopes on endogenous [email protected] ligands, such as ICAM-2 on endothelial cells (6), ICAM-3 on T 4 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; mDC, cells (8), or Mac1 on (13). Interaction of DC-SIGN mature DC; CLR, C-type lectin receptor; DC-SIGN, DC-specific ICAM-3-grabbing with its ligands regulates DC precursor migration into peripheral nonintegrin; MS, mass spectrometry; CRD, carbohydrate recognition domain; Ct, cycle threshold; Siglec, sialic acid-binding Ig superfamily lectin; MAA, Maackia tissues, stabilizes the DC-T cell contact surface in the immuno- amurensis agglutinin; SNA, Sambucus nigra agglutinin; RCA, Ricinus communis logical synapse (14), and allows neutrophils to induce DC matu- agglutinin. ration, respectively. Another CLR involved in intercellular com- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 munication is the -type C-type lectin, which www.jimmunol.org The Journal of Immunology 8217 binds to specific CD45 glycoforms on T cells (7) to down-regulate (Promega) following the manufacturer’s guidelines. Cells (0.5 ϫ 106) were effector T cell functions. Other members of the lectin superfamily washed twice with ice-cold PBS, pelleted, and lysed in 500 ␮l of lysis buffer. Lysates were incubated with biotin-labeled oligo(dT) for 5 min at that play an important role in the immune system are siglecs (15) 20 37°C and then 50 ␮l of the mix was transferred to streptavidin-coated tubes and galectins (16). The sialic acid-binding Ig superfamily lectins and incubated for 5 min at 37°C. After washing three times with 250 ␮lof ␮ ϫ (siglecs) are a class of Ig superfamily proteins which show binding washing buffer, 30 l of the reverse transcription mix (5 mM MgCl2,1 activity to specific glycan structures containing sialic acid (17). reverse transcription buffer, 1 mM dNTP, 0.4 U of recombinant RNasin Many siglecs have molecular features of inhibitory receptors, in- RNase inhibitor, 0.4 U of reverse transcriptase, 0.5 ␮g of random hexamers in nuclease-free water) were added to the tubes and incubated for 10 min cluding conserved tyrosine-based motifs. This is the case of si- at room temperature followed by 45 min at 42°C. To inactivate AMV glec-2 (CD22), involved in the control of the BCR signaling (18), reverse transcriptase and separate mRNA from the streptavidin-biotin com- and the siglec-3 (CD33) related siglecs, known to relay inhibitory plex, samples were heated at 99°C for 5 min, transferred to microcentrifuge signals or to inhibit activatory pathways when cross-linked with tubes and incubated in ice for 5 min, diluted 1/2 in nuclease-free water, and Ϫ activating receptors (19–22). Galectins have a binding specificity stored at 20°C until analysis. toward ␤-galactose-containing glycoconjugates (23). To date, 15 Real-time PCR members have been characterized and although all galectins re- ␤ Oligonucleotides were designed by using the computer software Primer quire -galactosides for binding, several structural and functional Express 2.0 (Applied Biosystems), synthesized by Invitrogen Life Tech- differences have been described. In the immune system, galectins nologies, and are published elsewhere. PCR were performed with the have been shown to operate at different levels in innate and adap- SYBR Green method in an ABI 7900HT sequence detection system (Ap- tive immune responses by modulating cell survival and cell acti- plied Biosystems). The reactions were set on a 96-well-plate by mixing 4 ␮l of the two times concentrated SYBR Green Master Mix (Applied Bio- vation or by influencing the Th1/Th2 balance (24). Downloaded from systems) with 2 ␮l of a oligonucleotide solution containing 5 nM/␮lof Classically, CLRs, siglecs, and galectins on immune cells have both oligonucleotides and 2 ␮l of a cDNA solution corresponding to 1/100 been studied considering that regulation of their function is of the cDNA synthesis product. The thermal profile for all the reactions achieved by the modulated expression of the receptor. However, was 2 min at 50°C, followed by 10 min at 95°C and then 40 cycles of 15 s their ligands may also be regulated, adding another layer of com- at 95°C and 1 min at 60°C. The housekeeping gene GAPDH was used as endogenous reference (29). To calculate the relative abundance of the plexity to the system. Importantly, whereas information about the genes, the formula 100 ϫ 2(Ct GAPDH-Ct glycosyltransferase) was used, where Ct expression of these receptors is widely available, only recently has is the cycle threshold. In this formula, the Ct value is defined as the number http://www.jimmunol.org/ the regulation of the glycosylation in cells of the immune system of PCR cycles at which the SYBR-green fluorescent signal exceeds the received more attention. Recent studies have shown that activation threshold of 0.2 relative units (26). by cytokines results in profound changes in the glycan profile of T Lectin staining cells (25) and on endothelial cells (26). In the case of DC, matu- ration has been shown to be accompanied by a decrease in ␣2,6- iDC and mDC were incubated with FITC- or biotin-labeled lectins (10 ␮g/ml) for 45 min at room temperature in TSM (20 mM Tris (pH 7.4), 150 sialylation (27), however, a more extensive biochemical and func- mM NaCl, 1 mM CaCl2, and 2 mM MgCl2) supplemented with 0.5% tional characterization of this important immune cell is still albumin from bovine serum (Fluka Biochemika). After washing the cells lacking. with TSM, FITC-labeled streptavidin was added to the cells incubated with In this study, we have investigated the changes in the glycosyl- biotin-labeled lectins for 30 min at room temperature. After another wash- by guest on September 23, 2021 ing step, cells were analyzed by immunofluorescence by collecting data for ation of DC that are associated with maturation with regards to the 104 cells per histogram (FACSCalibur; BD Biosciences). Corresponding glycosylation machinery. We evaluated changes in glycosylation- negative controls were performed using the FITC-labeled streptavidin related gene expression by quantitative real-time RT-PCR and a alone. glycosylation-related gene expression microarray. Mass spectro- MS analyses of glycans metric (MS) data were used as well as lectin binding to DC to confirm the data of the RT-PCR and microarray. In this study, we Core C of the Consortium for Functional Glycomics performed experi- show that maturation of DC results in large changes in the expres- ments to profile the N-glycans of iDC and mDC. The procedure was per- formed as previously described (25). N-glycans were released from ex- sion of glycosylation-related genes, involving fucosyltransferases, tracted of cell preparations by peptide N-glycosidase galactosyltransferases, and sialyltransferases. This results in a high (PNGase F) digestion and O-glycans were chemically released by reduc- expression of LacNAc structures, sialylated glycans, and Lewis tive elimination from the glycopeptides remaining after the release of N- structures. We further demonstrated that increased expression of glycans. Released glycans were permethylated using the sodium hydroxide these glycans result in binding epitopes for siglecs and galectins. procedure, and purified on a Sep-Pak C 18 cartridge, as previously de- scribed (30). Derivatized glycan samples were dissolved in methanol/water 8:2 (v/v) and mixed in a 1:1 ratio with 10 mg/ml 2,5-dihydroxybenzoic acid Materials and Methods in 80:20 (v/v) methanol/water. A total of 0.5- to 1-␮l aliquots were spotted Culture of DC from peripheral blood onto a target plate and dried under vacuum. MS spectra were obtained using a Voyager DE STR MALDI-TOF (Applied Biosystems) mass spec- were isolated from buffy coats of healthy blood donors (San- trometer in the reflectron mode with delayed extraction. Peaks observed in quin) through Ficoll gradient centrifugation and positive selection of the MS spectra were selected for further MS/MS. MS/MS data were ac- CD14ϩ cells using MACS sorting (Miltenyi Biotec). Isolated monocytes quired using a 4800 MALDI TOF/TOF (Applied Biosystems) mass spec- were cultured in RPMI 1640 (PAA Laboratories) supplemented with 10% trometer. The potential difference between the source acceleration voltage FCS (BioWhittaker), 10,000 U/ml penicillin (BioWhittaker), 10,000 U/ml and the collision cell was set to 1 kV and argon was used as collision gas. streptomycin (BioWhittaker), and 10,000 U/ml glutamine (Sigma-Aldrich) The 4700 Calibration Standard kit, calmix (Applied Biosystems), was used in the presence of IL-4 (500 U/ml; Schering-Plough) and GM-CSF (800 as the external calibrant for the MS mode and [Glu1]fibrinopeptide B hu- U/ml; Schering-Plough) for 7 days (28). Maturation was induced 6 days man (Sigma-Aldrich) was used as an external calibrant for the after isolation by incubation of iDC with 10 ng/ml LPS (from MS/MS mode. typhosa; Sigma-Aldrich) for 24 h. DC maturation was assessed by flow cytometric determination of the maturation markers MHC class II, CD80, Microarray analysis of glycosyltransferases using GLYCOv3 CD83, and CD86. RNA from iDC and mDC of three different donors was extracted using RNA isolation and cDNA synthesis TRIzol reagent according to the manufacturer’s protocol (Invitrogen Life Technologies). Total RNA was treated with DNase (Ambion), and purified mRNA from iDC and mDC was specifically isolated by capture of using the RNeasy kit (Qiagen). The RNA was amplified and biotin labeled poly(Aϩ) RNA in streptavidin-coated tubes using a mRNA Capture kit with the Bioarray High Yield RNA transcript labeling kit (Enzo Life Sci- (Roche). cDNA was synthesized using a Reverse Transcription System kit ences). Hybridization and scanning of the glycogene-chip v3, designed for 8218 GLYCOSYLATION CHANGES IN DC MATURATION

FIGURE 1. Expression levels of glycosylation-related genes in iDC. Glycosylation-related genes were ranked according to their expression levels by glycogene microarray (average Ϯ SD of the signal intensity obtained for iDC from three independent donors). Genes were clustered according to their

expression level in absent (not detected in microarray), low expressed (below P25), highly expressed (above P75), or intermediate (rest), and according to their function in Term (involved in the terminal modification of glycans), GAG (glycosaminoglycan biosynthesis), or HKG (necessary for the synthesisof Downloaded from core N-glycans, and the initiation of O-glycans and ). The proportion of Term, GAG, and HKG genes in each gene expression cluster and the list of Term genes in the cluster of gene expression low, intermediate, and high is provided. Bars corresponding to Term genes in the high expression cluster are displayed as empty bars and the expression levels for each gene are provided as the average Ϯ SD of the signal intensity obtained for iDC from three independent donors. http://www.jimmunol.org/ the Consortium for Functional Glycemics, were performed according to lated healthy donors. Raw data files for each of the experiments per- Affymetrix’s recommended protocols. The expression data obtained with formed are available at the Consortium for Functional Glycomics ϳ this chip array contain the expression of 2000 human and mouse tran- website (www.functionalglycomics.org/fg). scripts relevant to glycosylation. A complete description for the GLYCov3 array is available at www.functionalglycomics.org/static/consortium/recources/ The transcript levels of 147 of the 258 glycosylation-related resourcecoree.shtml. genes measured were detected as present, according to the mi- croarray internal controls (34) (Fig. 1, supplementary table Ia).5 Flow cytometry The transcripts marked as present were ranked and clustered ac- Siglec-Fc chimeras were used as tissue culture supernatants derived from cording to their relative expression level in high (first quartile), low ␮ stably transfected Chinese hamster ovary cells at 10 g/ml and were in- (third quartile), and moderately expressed (rest), as shown in Fig. by guest on September 23, 2021 cubated for 45 min on room temperature with FITC-labeled anti-human-Fc 1. To facilitate analysis, the transcripts were subdivided according Abs 1:200 (Jackson ImmunoResearch) in TSM supplemented with 0.5% albumin. After incubation, flow cytometric analysis using a FACSCalibur to their function in HKG (involved in the synthesis of core struc- flow cytometer and CellQuest software (BD Biosciences) was performed. tures, sugar donors, or transporters), Term (glycosylation related- As a negative control, cells were treated with 10 ␮lofVibrio cholerae genes implicated in the terminal modification of glycans), and neuraminidase (Boehringer) in 100 ␮l of PBS for 60 min at 37°C. GAG (involved in the synthesis of glycosaminoglycans). As ex- Galectins-3, -4, and -8 were produced as recombinant proteins in Esch- erichia coli and FITC labeled as previously described (31, 32). DC were pected, HKG transcripts showed the highest expression levels, incubated in HBBS (Invitrogen Life Technologies) medium containing 0.5 constituting the majority (80%) of the first quartile, while GAG M 30 min at room temperature to remove membrane-bound en- and Term were widely distributed over the first quartile and the dogenous galectins. Subsequently, cells were incubated with the FITC- moderately expressed gene cluster. Expression of genes involved labeled galectins in HBBS (Invitrogen Life Technologies) supplemented in glycosaminoglycan biosynthesis was markedly reduced as com- with 1% BSA and 2 mM 2-ME. Cells were washed with TSM and immu- nofluorescence analyzed using a FACSCalibur flow cytometer and pared with terminal glycosyltransferases, with only two genes stand- CellQuest software (BD Biosciences). ing up, NDST2 and CSGlcAT, involved in the synthesis of heparan and chondroitin sulfate. Interestingly, six transcripts encoding for en- Results zymes involved in terminal modifications of glycans were found in Microarray analysis of glycosylation-related genes in iDC this cluster, namely: ST6Gal-1, MGAT4B, ␤3GnT-5, ␤4GalT-1, and iDC are located in the peripheral tissues, where they establish mul- ST3Gal V. The high transcript levels found for these genes predicts an tiple interactions with surrounding cells, many of which are ex- abundance of triantennary N-glycans (MGAT4B), type 2 chains ␤ ␣ pected to be carbohydrate mediated. Although much is known ( 4GalT-1), 2,6-sialylated structures (ST6Gal-1), and glycolipids of ␤ about the lectins expressed by iDC, their glycans and glycosylation the lacto- ( 3GnT-5), and ganglioseries (ST3Gal-V). The range of machinery have been poorly described. The glycosylation machin- expression levels for moderately expressed transcripts (42–206, coef- ϭ ery consists of a set of enzymes, transporters, regulatory mole- ficient of variation 5.5%) was narrower compared with that of ϭ cules, cofactors, sugar donors, and other molecules present mainly highly expressed transcripts (206–1075, coefficient of variation in the endoplasmic reticulum and the Golgi apparatus that are im- 10.2%), indicating that the regulation of the glycosylation machinery plicated in the biosynthesis of glycans. The expression level of the may be more sensitive to subtle changes in the levels of moderately different components of the glycosylation machinery has been re- expressed genes. The transcripts found in the intermediate cluster ␣ ported to correlate with the array of glycans expressed by a certain cell (Fig. 1) suggest the presence of 3-fucosylated and 6-O-sulfated ␣ (26, 33) and, therefore, can be used to establish a prediction of the structures, as well as 2,3-sialylated and polysialylated glycans. The glycan profile. To account for interindividual variability, microarray relative expression levels of glycosylation-related genes involved in analysis was performed on RNA extracted from monocyte-derived iDC generated according to standard methods (28) from three unre- 5 The online version of this article contains supplemental material. The Journal of Immunology 8219

Table I. Assignments of molecular ions (͓M ϩ Na͔ϩ) observed in MALDI-TOF spectra of permethylated N-glycans of iDC and mDCa

iDC N-Glycans mDC N-Glycans

Signal Signal (m/z) Molecular assignments (m/z) Molecular assignments

1580.4 Hex5HexNAc2 1580.7 Hex5HexNAc2 1784.5 Hex HexNAc 1785.0 Hex HexNAc High 6 2 6 2 1988.7 Hex HexNAc 1989.2 Hex HexNAc High and man 7 2 7 2 hybrid type 2192.9 Hex8HexNAc2 2193.4 Hex8HexNAc2 2397.0 Hex9HexNAc2 2397.7 Hex9HexNAc2

Hybrid 2029.7 Hex6HexNAc3 2030.3 Hex6HexNAc3

2070.8 Hex5HexNAc4 2071.3 Hex5HexNAc4 NS 2245.9 FucHex5HexNAc4 2245.5 FucHex5HexNAc4

TFuc 2592.2 Fuc3Hex5HexNAc4 2593.9 Fuc3Hex5HexNAc4 2432.0 NeuAcHex HexNAc 2432.7 NeuAcHex HexNAc Biantennary 5 4 5 4 2793.3 NeuAc2Hex5HexNAc4 2793.3 NeuAc2Hex5HexNAc4 2606.2 NeuAcFucHex HexNAc 2606.9 NeuAcFucHex HexNAc S 5 4 5 4 2967.4 NeuAc2FucHex5HexNAc4 2967.4 NeuAc2FucHex5HexNAc4

TFuc 2780.3 NeuAcFuc2Hex5HexNAc4 2780.3 NeuAcFuc2Hex5HexNAc4 Downloaded from 3141.7 NeuAc2Fuc2Hex5HexNAc4

2520.1 Hex6HexNAc5 2520.8 Hex6HexNAc5 2694.3 FucHex HexNAc 2694.3 FucHex HexNAc NS 6 5 6 5 TFuc 2868.6 Fuc2Hex6HexNAc5 2868.2 Fuc2Hex6HexNAc5 3043.4 Fuc3Hex6HexNAc5

2881.4 NeuAcHex6HexNAc5 2881.4 NeuAcHex6HexNAc5 3242.6 NeuAc Hex HexNAc 3242.6 NeuAc Hex HexNAc http://www.jimmunol.org/ Triantennary 2 6 5 2 6 5 3603.9 NeuAc3Hex6HexNAc5 3605.0 NeuAc3Hex6HexNAc5 3055.5 NeuAcFucHex6HexNAc5 3056.4 NeuAcFucHex6HexNAc5 S 3416.8 NeuAc2FucHex6HexNAc5 3417.8 NeuAc2FucHex6HexNAc5 3301.7 NeuAcFucHex6HexNAc6 3301.3 NeuAcFucHex6HexNAc6

3476.9 NeuAcFuc2Hex6HexNAc6 TFuc 3229.6 NeuAcFuc2Hex6HexNAc5 3230.6 NeuAcFuc2Hex6HexNAc5 3404.5 NeuAcFuc2Hex6HexNAc5

3143.6 FucHex7HexNAc6 3144.5 FucHex7HexNAc6 3591.7 FucHex8HexNAc7 3592.0 FucHex8HexNAc7 by guest on September 23, 2021 4041.5 FucHex9HexNAc8 5215.7 Hex12HexNAc11 5216.7 Hex12HexNAc11 4490.9 FucHex HexNAc NS 10 9 4942.4 FucHex11HexNAc10 5389.7 FucHex12HexNAc11

3316.7 Fuc2Hex7HexNAc6 3316.4 Fuc2Hex7HexNAc6 TFuc 3490.1 Fuc3Hex7HexNAc6 3489.6 Fuc3Hex7HexNAc6 4215.1 Fuc2Hex9HexNAc8

3330.7 NeuAcHex7HexNAc6 3331.7 NeuAcHex7HexNAc6 3504.8 NeuAcFucHex7HexNAc6 3505.9 NeuAcFucHex7HexNAc6 3692.0 NeuAc2Hex7HexNAc6 3693.1 NeuAc2Hex7HexNAc6 3781.0 NeuAcHex8HexNAc7 3783.2 NeuAcHex8HexNAc7 3866.1 NeuAc2FucHex7HexNAc6 3867.3 NeuAc2FucHex7HexNAc6 3954.2 NeuAcFucHex8HexNAc7 3956.4 NeuAcFucHex8HexNAc7 4053.2 NeuAc3Hex7HexNAc6 4054.5 NeuAc3Hex7HexNAc6 Tetra-antennary 4141.3 NeuAc2Hex8HexNAc7 4142.6 NeuAc2Hex8HexNAc7 4228.4 NeuAc3FucHex7HexNAc6 4228.6 NeuAc3FucHex7HexNAc6 4315.3 NeuAc2FucHex8HexNAc7 4316.7 NeuAc2FucHex8HexNAc7 4405.6 NeuAcFucHex9HexNAc8 4405.9 NeuAcFucHex9HexNAc8 4415.4 NeuAc4Hex7HexNAc6 4504.7 NeuAc Hex HexNAc 4503.9 NeuAc Hex HexNAc S 3 8 7 3 8 7 4590.2 NeuAc2Hex9HexNAc8 4590.2 NeuAc2Hex9HexNAc8 4678.1 NeuAc3FucHex8HexNAc7 4678.1 NeuAc3FucHex8HexNAc7 4765.7 NeuAc2FucHex9HexNAc8 4767.2 NeuAc2FucHex9HexNAc8 4952.4 NeuAc3FucHex9HexNAc8 5040.6 NeuAc2Hex10HexNAc9 5040.4 NeuAc2Hex10HexNAc9 5129.0 NeuAc3FucHex9HexNAc8 5128.6 NeuAc3FucHex9HexNAc8 5575.1 NeuAc3FucHex10HexNAc9

3680.0 NeuAcFuc2Hex7HexNAc6 3680.1 NeuAcFuc2Hex7HexNAc6 3853.8 NeuAcFuc2Hex7HexNAc6 TFuc 4129.5 NeuAcFuc2Hex8HexNAc7 4302.2 NeuAcFuc3Hex8HexNAc7 4852.2 NeuAc3Fuc2Hex8HexNAc7 5302.8 NeuAc3Fuc2Hex9HexNAc8 Polylactosamine-elongated glycans are depicted in bold. NS, Unsialylated; S, sialylated. High man, High mannose-type N-glycans. TFuc, Glycans decorated with Fucose in one or more of the antennae. 8220 GLYCOSYLATION CHANGES IN DC MATURATION the terminal modification of glycans was further confirmed by quan- B), increase in ␣3-fucosylation (FUT4), increase in ␣2,3 sialyla- titative real-time PCR using a set of primers (26) to assay for 75 tion (ST3Gal-4/6) and ␣2,8-sialylation (ST8Sia-4), a decrease in transcripts, including Gal-, GlcNAc-, Fuc-, sialyl-, and sulfotrans- ␣2,6-sialylation (ST6Gal-1), and an increase in GlcNAc 6-O sul- ferases (data not shown). fation (GST-5). Sialylated core 1 O-glycans are expected to in- crease their abundance with respect to core 2 O-glycans due to the Analysis of N- and O-glycans on iDC concomitant up-regulation of core 1 ␤3GalT and ST3Gal-1/2 and Two pools of iDC from at least five unrelated donors were sub- the down-regulation of C2GnT-1 (Fig. 2B). jected to glycan profiling by MALDI-TOF MS analysis. Portions Several other models have been used in the literature to induce of representative MALDI-TOF MS profiles of iDC with the most DC maturation, based on the targeting of different TLR (36). To likely structures based on glycan compositions, MS/MS fragmen- examine whether the modulation of glycosylation-related genes tation patterns and gas chromatography-MS linkage analysis, are observed was purely dependent on TLR4 signaling or was due to shown in Tables I and II. The MALDI-TOF MS spectra of N- a more general maturation-dependent effect, the expression of a set glycans showed high mannose (m/z 1580.4, 1784.5, 1988.7, of 75 glycosylation-related genes was assessed in poly I:C (TLR3) 2192.9, and 2397.0) and complex type glycans, the latter compris- or flagellin (TLR5) treated DC. Whereas LPS induced the stron- ing bi-, tri-, and tetra-antennary structures which are mono-, di-, gest modulatory response in the expression of glycosylation-re- tri-, and tetrasialylated. Some of the complex N-glycans also carry lated transcripts, maturation induced by TL3 or TLR5 had com- fucose (compatible with (s)LeX) and/or poly-N-acetyllactosamine parable effects (data not shown). (m/z 2432.0–5215.7, Table I), as would be predicted from the gene expression data. The O-glycan profile (Table II) demonstrated that Effects of maturation on the glycan profile of DC Downloaded from the most abundant glycan species is sialylated core 1 (m/z 895.5). Two pools of mDC from at least five unrelated donors were sub- Rigorous MS/MS analyses indicated that the sialic acid can be jected to glycan profiling by MALDI-TOF MS analysis and com- present either attached to the Gal or GalNAc residue of the core 1 pared with the profile obtained from iDC (Tables I and II). The structure (data not shown). Disialylated core 1 structures (m/z N-glycan profile of mDC comprised of high mannose (m/z 1580.7, 1256.8) as well as unsialylated and sialylated core 2 glycans are 1785.0, 1989.2, 2193.4, and 2397.7) and complex bi-, tri- and tet- also present (m/z 983.6, 1344.8, and 1706.0). ra-antennary glycans, which were sialylated, fucosylated, and con- http://www.jimmunol.org/ tained poly-N-acetyllactosamine structures (m/z 2070.8 to 5575.1). Analysis of the glycosylation-related gene expression profile Studies have demonstrated that relative quantitation based on sig- during DC maturation nal intensities of permethylated glycans analyzed by MALDI-TOF Upon Ag capture, DC undergo a maturation process characterized MS is a reliable method, especially when comparing signals over a by the up-regulation of molecules involved in migration, Ag pre- small mass range within the same spectrum (37). The mDC N-glycan sentation, and costimulation (3). Activation of iDC with the TLR4 profile exhibited an increase in antennal fucosylation relative to the ligand LPS has been classically used as a model for the study of iDC. This is exemplified by comparing the relative intensities of fu- DC maturation (35). To investigate whether the maturation process cose-containing glycan signals, for example, the ratio of m/z 2606.9 to by guest on September 23, 2021 also affects the glycosylation of DC, we assessed the expression of 2780.3, which correspond to a mono- (NeuAcFucHex5HexNAc4) and glycosylation-related genes in DC incubated in the presence or difucosylated (NeuAcFuc2Hex5HexNAc4) biantennary structure, re- absence of LPS (10 ng/ml, 24 h). spectively (Table I), was altered from 7:1 in iDC to 2:1 in mDC. Also, An initial screening by a glycogene-oriented microarray showed the ratio of signals at m/z 3056.4 (monofucosylated triantennary N- that the expression levels of the majority of HKG transcripts were glycan) to 3230.6 (difucosylated triantennary N-glycans) was altered not significantly affected by maturation, while a large group of from 10:1 in iDC to 3:1 in mDC. This observation was further sup- GAG transcripts was down-regulated and many Term transcripts ported by an increase of 3,4-linked GlcNAc in mDC compared with were up-regulated (Fig. 2A), indicating that GAG biosynthesis iDC in the gas chromatography-MS linkage analyses on partially might be turned down during maturation while dramatic changes methylated alditol acetates (data not shown). Therefore, an increase in might occur on the terminal modifications of N- and O-glycans. the expression of FUT4 is consistent with the increase in ␣3-fucosy- This was confirmed by gene expression profiling by quantitative lation observed from these data. In addition, an increase in N-glycan real-time PCR performed on a set of glycosylation-related genes structures with poly-N-acetyllactosamine chains is also observed (Ta- selected from the list of up- and down-regulated Term transcripts ble I). The abundance of signals above m/z 3956.4, which from com- in the glycogene microarray. Based on the modulation of the dif- positions must contain at least one LacNAc repeat, has increased in ferent Term transcripts (Fig. 2B), the maturation process is pre- the mDC compared with iDC. For example, there is a relative increase dicted to be accompanied by an increase in poly-N-acetyllac- in the flanking signals (e.g., m/z 4678.1, NeuAc3FucHex8HexNAc7 ␤ ␤ tosamine chains (up-regulation of 3GnT-2 and 4GalT-4), and 4767.2, NeuAc2FucHex9HexNAc8) of m/z 4591.0, decrease in N-glycan branching (down-regulation of MGAT-4A/ NeuAc2Hex8HexNAc7.

Table II. Assignments of molecular ions (͓M ϩ Na͔ϩ) observed in MALDI-TOF spectra of permethylated O-glycans of iDC and mDC

iDC O-Glycans mDC O-Glycans

Signal (m/z) Molecular assignments Signal (m/z) Molecular assignments

895.5 NeuAcHexHexNAc-itol 895.5 NeuAcHexHexNAc-itol

983.6 Hex2HexNAc2-itol 1256.7 NeuAc2HexHexNAc-itol 1256.8 NeuAc2HexHexNAc-itol 1344.7 NeuAcHex2HexNAc2-itol 1344.8 NeuAcHex2HexNAc2-itol 1616.8 NeuAc3HexHexNAc-itol 1705.9 NeuAc2Hex2HexNAc2-itol 1706.0 NeuAc2Hex2HexNAc2-itol The Journal of Immunology 8221 Downloaded from

FIGURE 2. Changes in the expression of glycosylation-related genes during DC maturation. The number of transcripts that exhibited in the microarray at least a 2-fold up- or down-regulated, or remained stable for the Term, GAG, and HKG groups, is shown in A. From the Term group, a set of transcripts that showed significant changes was selected and their expression levels assayed in iDC and mDC from five different donors by real-time PCR (B).

The 2- to 3-fold decrease in the expression of MGAT4A/B LacNAc and poly-N-acetyllactosamine (Fig. 4A) have been http://www.jimmunol.org/ could not be correlated with a decrease in N-glycan branching, reported as ligands for several galectins (39). To test whether because the majority of the complex-type N-glycans species ob- the up-regulation of these structures on mDC would lead to an served in iDC and mDC were of the tetra-antennary type (Table I). increased interaction with galectin, binding of galectin 3, 4, and This apparent paradox could be explained by the high basal levels 8 was tested by flow cytometry. Only galectins 3 and 8 showed observed for MGAT4A/B, among the highest for the group of binding to iDC, which could be blocked by lactose (Fig. 4B) branching GlcNAc-transferases (supplementary table Ia). Also, the and increased during maturation (Fig. 4B). The increase in ga- enzymes they encode for have been recently shown to have the lectin binding correlated with an increased binding of the ␤-gal- highest kinetic efficiency for the triantennary N-glycan product of actoside-specific lectin Ricinus communis agglutinin (RCA) the GnT-II and GnT-V reactions, which indicates that the preced- (Fig. 4C). by guest on September 23, 2021 ing branch formations on the Man␣1-6 arm by other GnTs pro- mote the actions of both GnT-IV enzymes (38). Discussion In the O-glycan spectrum (Table II), the sialic acid on the signal We have investigated the array of glycans expressed on iDC, its at m/z 895.5 is exclusively attached to the Gal residue of the core regulation during DC maturation, and the functional consequences 1 O-glycan structure (data not shown). This result is consistent for the binding of several carbohydrate recognition molecules of with the increased expression of ST3Gal-1 and ST3Gal-2 and a importance in the immune system. The data showed the presence decrease in ST6Gal-1 expression in mDC compared with iDC. of abundant amounts of mono-, di-, and trisialylated tri- and tetra- antennary N-glycans, with some glycan species decorated with Functional aspects of DC glycosylation during maturation Lewis-type fucose or elongated with poly-N-acetyllactosamine, Both the glycosylation-related gene expression profile and the gly- while the dominant O-glycan structure was the sialyl-T Ag. Mat- can profile demonstrated an increase in poly-N-acetyllactosamine- uration resulted in the modulation of the expression levels of sev- elongated glycans (see Fig. 4A) and ␣2,3/␣2,8 sialylated structures eral glycosylation-related genes, mainly affecting the synthesis of (Fig. 3A), suggesting that binding of galactose- and sialic acid- i-type elongated poly-N-acetyllactosamine chains, and the terminal specific lectins, such as galectins and siglecs, might be affected capping of glycans by sialic acid and fucose, which correlated with during maturation. The binding of (siglec-1), CD22 the changes observed in the glycan profiles. These glycan changes (siglec-2), siglecs-3, -5, -7, -9, and -10 and galectins-3, -4, and -8 are expected to play an important role in the biology of the DC, to iDC and mDC was tested by flow cytometry. Only sialoadhesin, because their up-regulation correlated with an increased binding of CD22 and siglec-7 bound with high affinity to iDC and the binding their receptors, the carbohydrate recognition molecules galectin-3 could be prevented by neuraminidase treatment (Fig. 3B). As ex- and -8, as well as sialoadhesin, CD22, and -7. pected, the binding of the ␣2,3-specific siglec sialoadhesin in- Galectins are animal lectins recognizing ␤-galactose that com- creased with maturation (Fig. 3B), consistent with the staining ob- prise a family of 15 members, all containing conserved carbohy- tained with the plant lectin Maackia amurensis agglutinin (MAA), drate-recognition domains (CRD) of ϳ130 aa responsible for car- although to a lower extent (Fig. 3C). The presence and maturation- bohydrate binding (40, 41). Two types of galectins exist, those dependent increase of polysialic acid could be confirmed by using with one CRD (galectin-1, -2, -3, -5, -7, -10, -11, -13, -14, and the specific Ab 735 (Fig. 3D), and correlates nicely with an in- -15), or with two homologous CRDs separated by a linker (galec- creased binding of the ␣2,8-specific siglec-7. Surprisingly, the tin-4, -6, -8, -9, and -12). Galectin-3 is unique in that it contains a binding of CD22 also increased with maturation (Fig. 3B), corre- long N-terminal region rich in Pro and Gly that is involved in lating with an increased number of ␣2,6-linked sialic acids on oligomerization (42). Although all galectins bind ␤-galactose, their mDC, as shown by the plant lectin Sambucus nigra agglutinin fine specificity varies (31, 39, 41). Galectin-1 prefers terminal Lac- (SNA) (Fig. 3C). NAc in its core disaccharide binding site, although ␣2,3-sialylation 8222 GLYCOSYLATION CHANGES IN DC MATURATION

FIGURE 4. Binding of galectins to DC. The glycan structures that serve as ligands for galectin-3 and -8 are depicted in A. FACS analysis of iDC (thin line) and mDC (thick line) stained with FITC-labeled galectin-3 or -8. Galectin binding could be inhibited by preincubating galectins with lactose (dotted line). Results are representative of up to seven experiments and differences in the median fluorescence intensity were statistically signifi- cant (Mann-Whitney, p Ͻ 0.05) (B). The galactose-specific plant lectin RCA-II showed an increased binding to mDC (C).

ated ligands has been associated with a multitude of functional effects that include the modulation of , cell activation,

chemoattraction, cell growth, or (51), while galectin-8 Downloaded from has been associated with adhesion to the and -related reorganization of the cytoskeleton (52, 53). The modulation of the expression of different sialyltransferases during maturation has also a clear repercussion on siglec binding. Siglecs are a family of type 1 membrane proteins with variable

numbers of Ig domains and one N-terminal V-set domain where http://www.jimmunol.org/ sialic acid binding is mediated via well-characterized molecular interactions (54). Siglecs can be divided into two subsets: the CD33-related siglecs (siglec-3, -5, -6, -7, -8, -9, -10, and -11) and a group comprising sialoadhesin, -associated (siglec-4) and CD22, which are more distantly related. Strikingly, FIGURE 3. Binding of siglecs to DC. The glycan structures that may all siglecs except siglec-4 are expressed by cells of the immune serve as ligands for siglec-1, -2, and -7 are depicted in A. FACS analysis system and most of them have two or more ITIMs. Although the of iDC (thin line) and mDC (thick line) stained with siglec-1, -2, and -7/Fc function of the -restricted CD22 has been analyzed in detail chimeras. Siglec binding could be inhibited by neuraminidase treatment of (18), the functions of the other siglecs remain poorly understood by guest on September 23, 2021 DC before staining (dotted line). Results are representative of up to seven (54). The binding of CD22, and specially sialoadhesin, and si- experiments and differences in the median fluorescence intensity were sta- glec-7 to iDC was very strong and could, in the three cases, be Ͻ tistically significant (Mann-Whitney, p 0.05) (B). FACS analysis of up-regulated during maturation. The preferred ligand of CD22 is ␣ ␣ 2,3- and 2,6-linked sialic acid with the plant lectins MAA and SNA, NeuAc␣2-6Gal␤1-4GlcNAc, but CD22 has also been shown to respectively, is shown in C. FACS analysis of polysialic acid (PSA) on iDC interact less strongly with other glycans capped with ␣2,6-linked (thin line) and mDC (thick line) with the mAb 735 (D). sialic acid (55). The binding does not differ greatly for several sialylated proteins suggesting that the presence and density of the on the Gal may be tolerated, with equal or slightly decreased af- carbohydrate, but not the backbone, determine binding finity (43). Galectin-3 prefers to bind Gal␤1-4GlcNAc and the (18). Interestingly, the increase in the binding of CD22, or the affinity may increase with extensions on the Gal by another Lac- ␣2,6-specific plant lectin SNA, did not correlate with the expres- NAc or by ␣-linked Gal(NAc). The N-terminal CRD of galectin-8 sion of the enzyme involved in the synthesis of its glycan, prefers ␣2,3-sialylated galactosides as found in GM3 and GD1a, ST6Gal-1, which decreased dramatically. This could be explained but in intact galectin-8 the two CRDs can cooperate to give high- by the maturation-triggered mobilization of molecules stored in affinity cell surface binding via LacNAc residues in N-linked gly- granules, a phenomenon that has been previously described on DC cans (31, 32). The CRDs of galectin-4 bind to the 3-O-sulfated (56, 57), and needs to be further investigated. The interaction be- glycolipids SM4, SM3, and SB1a, but also to some glycoproteins tween DC and B lymphocytes has been shown to result in multiple (44). iDC express relatively high levels of the transcripts for the effects, such as the inhibition of B cell apoptosis (58), blockage of enzymes ␤4GalT-1, -4, and iGnT, previously described to be in- the production of IgE (59), or Ag presentation (60). volved in the expression of poly-N-acetyllactosamine chains (45), In contrast to CD22, the macrophage siglec sialoadhesin prefers which are clearly detected by MS analyses of the N-glycans. This ␣2,3-linked sialic acid (61), as found on the sialomucins P- observation could explain the high binding of galectin-3 and -8 to glycoprotein ligand 1 and CD43 (62), both expressed on DC (63, iDC. The binding of galectin-3 and -8 increases during maturation, 64). As shown by MALDI-TOF mass spectrometry, the O-glycan correlating with an increase in the expression of poly-N-acetyllac- NeuAc␣2-3Gal␤1-3GalNAc (sialyl-T Ag) is highly abundant on tosamine elongated glycans possibly due to the up-regulation of both iDC and mDC and correlates with a high siglec-1 binding. ␤4GalT-4 and other enzymes potentially involved in the synthesis and DC coexist in the spleen, tonsils, and lymph of these structures, such as ␤4GalT-5 (46) and ␤3GnT-2 (47). nodes (65–67). Interaction of siglec-1 with its ligand on DC may Binding of other galectins to monocyte-derived DC has been serve two purposes, either to enhance intercellular contact or to shown previously to induce maturation, giving rise to mDC with a induce or modulate signaling on DC. strong proinflammatory capacity, as demonstrated for galectin-1 Siglec-7 is a CD33-related siglec containing intracellular inhib- (48, 49) and -9 (50). Interaction of galectin-3 with its cell-associ- itory motifs that is expressed on NK cells and to a lesser extent, The Journal of Immunology 8223

ϩ monocytes, macrophages, DC, and a subset of CD8 T cells (68). 14. van Gisbergen, K. P., L. C. Paessens, T. B. Geijtenbeek, and Y. van Kooyk. 2005. In vitro-binding studies have revealed that siglec-7 shows a Molecular mechanisms that set the stage for DC-T cell engagement. Immunol. Lett. 97: 199–208. marked preference for glycoconjugates bearing ␣(2,8)-linked di- 15. Crocker, P. R., and A. Varki. 2001. Siglecs in the immune system. Immunology sialic acid (GD3 and GT1b) and branched ␣(2,6)-linked sialic acid 103: 137–145. 16. Liu, F. T. 2005. Regulatory roles of galectins in the immune response. Int. Arch. (69). The function of siglec-7 on NK cells appear to be related with Immunol. 136: 385–400. a down-modulation of the NK killing activity (70), therefore trans 17. Varki, A., and T. Angata. 2006. Siglecs–the major subfamily of I-type lectins. interactions between NK cells and DC could serve this function. Glycobiology 16: 1R–27R. 18. Nitschke, L. 2005. The role of CD22 and other inhibitory co-receptors in B-cell Siglec-7 is also expressed on DC, and cis interactions could thus activation. Curr. Opin. Immunol. 17: 290–297. exist and be involved in regulating DC activation, as speculated for 19. Ulyanova, T., D. D. Shah, and M. L. Thomas. 2001. Molecular cloning of MIS, the cis interactions on NK cells. a myeloid inhibitory siglec, that binds protein-tyrosine phosphatases SHP-1 and SHP-2. J. Biol. Chem. 276: 14451–14458. In conclusion, our results provide a detailed description of the 20. Falco, M., R. Biassoni, C. Bottino, M. Vitale, S. Sivori, R. Augugliaro, glycans expressed on DC, their regulation during maturation, and L. Moretta, and A. Moretta. 1999. Identification and molecular cloning of p75/ some functional repercussions with regards to interactions in trans AIRM1, a novel member of the sialoadhesin family that functions as an inhibitory receptor in human natural killer cells. J. Exp. Med. 190: 793–802. with receptors expressed on other immune cells or in cis on DC. 21. Ulyanova, T., J. Blasioli, T. A. Woodford-Thomas, and M. L. Thomas. 1999. The DC appear to be rich in galactosylated and sialylated structures, sialoadhesin CD33 is a myeloid-specific inhibitory receptor. Eur. J. Immunol. 29: 3440–3449. which are up-regulated during maturation, resulting in an enhanced 22. Paul, S. P., L. S. Taylor, E. K. Stansbury, and D. W. McVicar. 2000. Myeloid binding of the lectins galectin-3 and -8, and sialoadhesin, CD22, specific human CD33 is an inhibitory receptor with differential ITIM function in and siglec-7. Further research is expected to shed more light on the recruiting the phosphatases SHP-1 and SHP-2. Blood 96: 483–490. 23. Barondes, S. H., V. Castronovo, D. N. Cooper, R. D. Cummings, K. Drickamer, Downloaded from functional consequences of these interactions. T. Feizi, M. A. Gitt, J. Hirabayashi, C. Hughes, K. Kasai, et al. 1994. Galectins: a family of animal ␤-galactoside-binding lectins. Cell 76: 597–598. 24. Rabinovich, G. A., and A. Gruppi. 2005. Galectins as immunoregulators during Acknowledgments infectious processes: from microbial invasion to the resolution of the disease. The mAb 735 was provided by Dr. M. Muhlenhoff (Medizinische Hoch- Parasite Immunol. 27: 103–114. schule Hannover, Hannover, Germany). Galectins-3 and -4 were produced 25. Comelli, E. M., M. Sutton-Smith, Q. Yan, M. Amado, M. Panico, T. Gilmartin, by B. Kahl-Knutson, and galectin-8 was produced by S. Carlsson. We also T. Whisenant, C. M. Lanigan, S. R. Head, D. Goldberg, et al. 2006. Activation of murine CD4ϩ and CD8ϩ T lymphocytes leads to dramatic remodeling of thank G. Kraal, W. van Dijk, and T. K. van den Berg for critical reading N-linked glycans. J. Immunol. 177: 2431–2440. http://www.jimmunol.org/ of the manuscript and helpful suggestions. The glycan analyses were per- 26. Garcia-Vallejo, J. J., W. van Dijk, B. van het Hof, I. van Die, M. A. Engelse, formed by the Analytical Glycotechnology Core of the Consortium for V. W. van Hinsbergh, and S. I. Gringhuis. 2006. Activation of human endothelial Functional Glycomics (GM62116). cells by tumor necrosis factor-␣ results in profound changes in the expression of glycosylation-related genes. J. Cell Physiol. 206: 203–210. 27. Jenner, J., G. Kerst, R. Handgretinger, and I. Muller. 2006. Increased ␣2,6-sia- Disclosures lylation of surface proteins on tolerogenic, immature dendritic cells and regula- The authors have no financial conflict of interest. tory T cells. Exp. Hematol. 34: 1212–1218. 28. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble by cultured human dendritic cells is maintained by granulocyte/macrophage col- References ony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor ␣. J. Exp. Med. 179: 1109–1118.

1. Steinman, R. M. 2003. The control of immunity and tolerance by dendritic cell. by guest on September 23, 2021 Pathol. Biol. 51: 59–60. 29. Garcia-Vallejo, J. J., B. van het Hof, J. Robben, J. A. van Wijk, I. van Die, 2. Iwasaki, A., and R. Medzhitov. 2004. Toll-like receptor control of the adaptive D. H. Joziasse, and W. van Dijk. 2004. Approach for defining endogenous ref- immune responses. Nat. Immunol. 5: 987–995. erence genes in gene expression experiments. Anal. Biochem. 329: 293–299. 3. Reis e Sousa, C. 2006. Dendritic cells in a mature age. Nat. Rev. Immunol. 6: 30. Sutton-Smith, M., H. R. Morris, P. K. Grewal, J. E. Hewitt, R. E. Bittner, 476–483. E. Goldin, R. Schiffmann, and A. Dell. 2002. MS screening strategies: investi- 4. Pyz, E., A. S. Marshall, S. Gordon, and G. D. Brown. 2006. C-type lectin-like gating the glycomes of knockout and myodystrophic mice and leukodystrophic receptors on myeloid cells. Ann. Med. 38: 242–251. human brains. Biochem. Soc. Symp. 69: 105–115. 5. van Kooyk, Y., A. Engering, A. N. Lekkerkerker, I. S. Ludwig, and 31. Carlsson, S., C. T. Oberg, M. C. Carlsson, A. Sundin, U. J. Nilsson, D. Smith, T. B. Geijtenbeek. 2004. Pathogens use carbohydrates to escape immunity in- R. D. Cummings, J. Almkvist, A. Karlsson, and H. Leffler. 2007. Affinity of duced by dendritic cells. Curr. Opin. Immunol. 16: 488–493. galectin-8 and its carbohydrate recognition domains for ligands in solution and at 6. Geijtenbeek, T. B., D. J. Krooshoop, D. A. Bleijs, S. J. van Vliet, the cell surface. Glycobiology 17: 663–676. G. C. van Duijnhoven, V. Grabovsky, R. Alon, C. G. Figdor, and Y. van Kooyk. 32. Patnaik, S. K., B. Potvin, S. Carlsson, D. Sturm, H. Leffler, and P. Stanley. 2006. 2000. DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking. Nat. Im- Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese munol. 1: 353–357. hamster ovary cells. Glycobiology 16: 305–317. 7. van Vliet, S. J., S. I. Gringhuis, T. B. Geijtenbeek, and Y. van Kooyk. 2006. 33. Comelli, E. M., S. R. Head, T. Gilmartin, T. Whisenant, S. M. Haslam, Regulation of effector T cells by antigen-presenting cells via interaction of the S. J. North, N. K. Wong, T. Kudo, H. Narimatsu, J. D. Esko, et al. 2006. A C-type lectin MGL with CD45. Nat. Immunol. 7: 1200–1208. focused microarray approach to functional glycomics: transcriptional regulation 8. Geijtenbeek, T. B., R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, of the glycome. Glycobiology 16: 117–131. G. J. Adema, Y. van Kooyk, and C. G. Figdor. 2000. Identification of DC-SIGN, 34. Lockhart, D. J., H. Dong, M. C. Byrne, M. T. Follettie, M. V. Gallo, M. S. Chee, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune M. Mittmann, C. Wang, M. Kobayashi, H. Horton, and E. L. Brown. 1996. responses. Cell 100: 575–585. Expression monitoring by hybridization to high-density oligonucleotide arrays. 9. Geijtenbeek, T. B., D. S. Kwon, R. Torensma, S. J. van Vliet, Nat. Biotechnol. 14: 1675–1680. G. C. van Duijnhoven, J. Middel, I. L. Cornelissen, H. S. Nottet, 35. Cella, M., F. Sallusto, and A. Lanzavecchia. 1997. Origin, maturation and antigen V. N. KewalRamani, D. R. Littman, et al. 2000. DC-SIGN, a dendritic cell- presenting function of dendritic cells. Curr. Opin. Immunol. 9: 10–16. specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100: 36. Reis e Sousa, C. 2004. Toll-like receptors and dendritic cells: for whom the bug 587–597. tolls. Semin. Immunol. 16: 27–34. 10. Halary, F., A. Amara, H. Lortat-Jacob, M. Messerle, T. Delaunay, C. Houles, 37. Wada, Y., P. Azadi, C. E. Costello, A. Dell, R. A. Dwek, H. Geyer, R. Geyer, F. Fieschi, F. Arenzana-Seisdedos, J. F. Moreau, and J. Dechanet-Merville. 2002. K. Kakehi, N. G. Karlsson, K. Kato, et al. 2007. Comparison of the methods for Human cytomegalovirus binding to DC-SIGN is required for dendritic cell in- profiling glycoprotein glycans–HUPO Human Disease Glycomics/Proteome Ini- fection and target cell trans-infection. Immunity 17: 653–664. tiative Multi-Institutional Study. Glycobiology 17: 411–422. 11. Meyer, S., E. van Liempt, A. Imberty, Y. van Kooyk, H. Geyer, R. Geyer, and 38. Oguri, S., A. Yoshida, M. T. Minowa, and M. Takeuchi. 2006. Kinetic properties I. van Die. 2005. DC-SIGN mediates binding of dendritic cells to authentic pseu- and substrate specificities of two recombinant human N-acetylglucosaminyltrans- do-Lewis Y glycolipids of Schistosoma mansoni cercariae, the first parasite-spe- ferase-IV isozymes. Glycoconj. J. 23: 473–480. cific ligand of DC-SIGN. J. Biol. Chem. 280: 37349–37359. 39. Hirabayashi, J., T. Hashidate, Y. Arata, N. Nishi, T. Nakamura, M. Hirashima, 12. Appelmelk, B. J., I. van Die, S. J. van Vliet, C. M. Vandenbroucke-Grauls, T. Urashima, T. Oka, M. Futai, W. E. Muller, et al. 2002. spec- T. B. Geijtenbeek, and Y. van Kooyk. 2003. Cutting edge: carbohydrate profiling ificity of galectins: a search by frontal affinity chromatography. Biochim. Biophys. identifies new pathogens that interact with dendritic cell-specific ICAM-3-grab- Acta 1572: 232–254. bing nonintegrin on dendritic cells. J. Immunol. 170: 1635–1639. 40. Houzelstein, D., I. R. Goncalves, A. J. Fadden, S. S. Sidhu, D. N. Cooper, 13. van Gisbergen, K. P., M. Sanchez-Hernandez, T. B. Geijtenbeek, and K. Drickamer, H. Leffler, and F. Poirier. 2004. Phylogenetic analysis of the ver- Y. van Kooyk. 2005. Neutrophils mediate immune modulation of dendritic cells tebrate galectin family. Mol. Biol. Evol. 21: 1177–1187. through glycosylation-dependent interactions between Mac-1 and DC-SIGN. 41. Leffler, H., S. Carlsson, M. Hedlund, Y. Qian, and F. Poirier. 2004. Introduction J. Exp. Med. 201: 1281–1292. to galectins. Glycoconj. J. 19: 433–440. 8224 GLYCOSYLATION CHANGES IN DC MATURATION

42. Hsu, D. K., R. Y. Yang, and F. T. Liu. 2006. Galectins in apoptosis. Methods 56. Zavasnik-Bergant, T., U. Repnik, A. Schweiger, R. Romih, M. Jeras, V. Turk, Enzymol. 417: 256–273. and J. Kos. 2005. Differentiation- and maturation-dependent content, localization, 43. Leppanen, A., S. Stowell, O. Blixt, and R. D. Cummings. 2005. Dimeric galec- and of cystatin C in human dendritic cells. J. Leukocyte Biol. 78: tin-1 binds with high affinity to ␣2,3-sialylated and non-sialylated terminal N- 122–134. acetyllactosamine units on surface-bound extended glycans. J. Biol. Chem. 280: 57. Klein, E., S. Koch, B. Borm, J. Neumann, V. Herzog, N. Koch, and T. Bieber. 5549–5562. 2005. CD83 localization in a recycling compartment of immature human mono- 44. Ideo, H., A. Seko, and K. Yamashita. 2005. Galectin-4 binds to sulfated glyco- cyte-derived dendritic cells. Int. Immunol. 17: 477–487. sphingolipids and carcinoembryonic antigen in patches on the cell surface of 58. Lindhout, E., A. Lakeman, and C. de Groot. 1995. Follicular dendritic cells in- human colon adenocarcinoma cells. J. Biol. Chem. 280: 4730–4737. hibit apoptosis in human B lymphocytes by a rapid and irreversible blockade of 45. Ujita, M., A. K. Misra, J. McAuliffe, O. Hindsgaul, and M. Fukuda. 2000. Poly- preexisting endonuclease. J. Exp. Med. 181: 1985–1995. N-acetyllactosamine extension in N-glycans and core 2- and core 4-branched 59. Obayashi, K., T. Doi, and S. Koyasu. 2007. Dendritic cells suppress IgE pro- O-glycans is differentially controlled by i-extension enzyme and different mem- duction in B cells. Int. Immunol. 19: 217–226. ␤ bers of the 1,4-galactosyltransferase gene family. J. Biol. Chem. 275: 60. Bergtold, A., D. D. Desai, A. Gavhane, and R. Clynes. 2005. Cell surface recy- 15868–15875. cling of internalized antigen permits dendritic cell priming of B cells. Immunity ␤ 46. Sato, T., and K. Furukawa. 2004. Transcriptional regulation of the human -1,4- 23: 503–514. galactosyltransferase V gene in cancer cells: essential role of transcription factor 61. Crocker, P. R., S. Kelm, C. Dubois, B. Martin, A. S. McWilliam, D. M. Shotton, Sp1. J. Biol. Chem. 279: 39574–39583. J. C. Paulson, and S. Gordon. 1991. Purification and properties of sialoadhesin, 47. Zhou, D., A. Dinter, G. R. Gutierrez, J. P. Kamerling, J. F. Vliegenthart, a sialic acid-binding receptor of murine tissue macrophages. EMBO J. 10: ␤ E. G. Berger, and T. Hennet. 1999. A -1,3-N-acetylglucosaminyltransferase 1661–1669. N ␤ with poly- -acetyllactosamine synthase activity is structurally related to -1,3- 62. van den Berg, T. K., D. Nath, H. J. Ziltener, D. Vestweber, M. Fukuda, I. van Die, Proc. Natl. Acad. Sci. USA galactosyltransferases. 96: 406–411. and P. R. Crocker. 2001. Cutting edge: CD43 functions as a T cell counterre- 48. Fulcher, J. A., S. T. Hashimi, E. L. Levroney, M. Pang, K. B. Gurney, ceptor for the macrophage adhesion receptor sialoadhesin (Siglec-1). J. Immunol. L. G. Baum, and B. Lee. 2006. Galectin-1-matured human monocyte-derived 166: 3637–3640. dendritic cells have enhanced migration through extracellular matrix. J. Immunol. 63. Laszik, Z., P. J. Jansen, R. D. Cummings, T. F. Tedder, R. P. McEver, and 177: 216–226.

K. L. Moore. 1996. P-selectin glycoprotein ligand-1 is broadly expressed in cells Downloaded from 49. Levroney, E. L., H. C. Aguilar, J. A. Fulcher, L. Kohatsu, K. E. Pace, M. Pang, of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells. K. B. Gurney, L. G. Baum, and B. Lee. 2005. Novel innate immune functions for Blood 88: 3010–3021. galectin-1: galectin-1 inhibits cell fusion by Nipah envelope glycoproteins and augments dendritic cell secretion of proinflammatory cytokines. J. Immunol. 64. Egner, W., B. D. Hock, and D. N. Hart. 1993. Dendritic cells have reduced cell Adv. Exp. Med. 175: 413–420. surface membrane glycoproteins including CD43 determinants. Biol. 50. Dai, S. Y., R. Nakagawa, A. Itoh, H. Murakami, Y. Kashio, H. Abe, S. Katoh, 329: 71–73. K. Kontani, M. Kihara, S. L. Zhang, et al. 2005. Galectin-9 induces maturation 65. Mebius, R. E., and G. Kraal. 2005. Structure and function of the spleen. Nat. Rev. of human monocyte-derived dendritic cells. J. Immunol. 175: 2974–2981. Immunol. 5: 606–616. 66. Willard-Mack, C. L. 2006. Normal structure, function, and histology of lymph 51. Dumic, J., S. Dabelic, and M. Flogel. 2006. Galectin-3: an open-ended story. http://www.jimmunol.org/ Biochim. Biophys. Acta 1760: 616–635. nodes. Toxicol. Pathol. 34: 409–424. 52. Levy, Y., S. Auslender, M. Eisenstein, R. R. Vidavski, D. Ronen, 67. Cesta, M. F. 2006. Normal structure, function, and histology of mucosa-associ- A. D. Bershadsky, and Y. Zick. 2006. It depends on the hinge: a structure- ated lymphoid tissue. Toxicol. Pathol. 34: 599–608. functional analysis of galectin-8, a tandem-repeat type lectin. Glycobiology 16: 68. Avril, T., H. Attrill, J. Zhang, A. Raper, and P. R. Crocker. 2006. Negative 463–476. regulation of leucocyte functions by CD33-related siglecs. Biochem. Soc. Trans. 53. Zick, Y., M. Eisenstein, R. A. Goren, Y. R. Hadari, Y. Levy, and D. Ronen. 2004. 34: 1024–1027. Role of galectin-8 as a modulator of cell adhesion and cell growth. Glycoconj. J. 69. Attrill, H., A. Imamura, R. S. Sharma, M. Kiso, P. R. Crocker, and 19: 517–526. D. M. van Aalten. 2006. Siglec-7 undergoes a major conformational change when 54. Crocker, P. R. 2005. Siglecs in innate immunity. Curr. Opin. Pharmacol. 5: complexed with the ␣(2,8)-disialylganglioside GT1b. J. Biol. Chem. 281: 431–437. 32774–32783. 55. Bakker, T. R., C. Piperi, E. A. Davies, and P. A. Merwe. 2002. Comparison of 70. Ikehara, Y., S. K. Ikehara, and J. C. Paulson. 2004. Negative regulation of T cell

CD22 binding to native CD45 and synthetic oligosaccharide. Eur. J. Immunol. receptor signaling by Siglec-7 (p70/AIRM) and Siglec-9. J. Biol. Chem. 279: by guest on September 23, 2021 32: 1924–1932. 43117–43125.