Identification of a Novel, Dendritic Cell-associated Molecule,Dectin-1, by Subtractive cDNA Cloning*

Received for publication, November 24, 1999, and in revised form, April 20, 2000Published, JBC Papers in Press, April 12, 2000, DOI 10.1074/jbc.M909512199

Kiyoshi Ariizumi, Guo-Liang Shen, Sojin Shikano, Shan Xu, Robert Ritter III,Tadashi Kumamoto, Dale Edelbaum, Akimichi Morita, Paul R. Bergstresser,and Akira Takashima‡

From the Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9069

Dendritic cells (DC) are special subsets of antigen pre-senting cells characterized by their potent capacity toactivate immunologically naive T cells. By subtractingthe mRNAs expressed by the mouse epidermus-derivedDC line XS52 with the mRNAs expressed by the J774macrophage line, we identified five novel genes thatwere expressed selectively by this DC line. One of thesegenes encoded a type II membrane-integrated polypep-tide of 244 amino acids containing a putative carbohy-drate recognition domain motif at the COOH-terminalend. This molecule, termed “dectin-1,” was expressedabundantly at both mRNA and protein levels by theXS52 DC line, but not by non-DC lines (including theJ774 macrophage line). Dectin-1 mRNA was detectedpredominantly in spleen and thymus (by Northern blot-ting) and in skin-resident DC, i.e. Langerhans cells (byreverse transcription-polymerase chain reaction). Affin-ity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated fromthe XS52 DC line and from the dectin-1 cDNA-trans-fected COS-1 cells. His-tagged recombinant proteinscontaining the extracellular domains of dectin-1 showedmarked and specific binding to the surface of T cells andpromoted their proliferation in the presence of anti-CD3monoclonal antibody at suboptimal concentrations.These in vitro results suggest that dectin-1 on DC maybind to as yet undefined ligand(s) on T cells, therebydelivering T cell co-stimulatory signals. Not only dothese results document the efficacy of subtractive cDNAcloning for the identification of unique genes expressedby DC, they also provide a framework for studying thephysiological function of dectin-1.

It has been an established concept that immunologicallynaive T cells can be activated most efficiently or even exclu-sively by special subsets of antigen-presenting cells, termeddendritic cells (DC)1 (1). DC play central roles in the induction

of cellular immune reactions against a wide variety of antigens,including chemical haptens, foreign proteins, infectious patho-gens, and tumor-associated antigens (2–5). Members of the DCfamily have been identified in many organs, including (a)spleen (splenic DC), thymus (thymic DC), lymph nodes (inter-digitating cells), and tonsils (tonsil DC); (b) epidermis (Lange-rhans cells) and mucosal surfaces of the oral cavity, intestinaltract, and respiratory tract; (c) dermis (dermal DC) and otherconnective tissues (interstitial DC), d) peripheral blood (bloodDC); and (e) afferent lymphatics (veiled cells) (6).

DC are characterized morphologically by the extension oflong, lamellar dendrites. Upon activation with proinflamma-tory stimuli, DC acquire surface expression of relatively largeamounts of major histocompatibility complex class I and classII molecules as well as co-stimulatory molecules (e.g. CD40,CD80, and CD86) and adhesion molecules (e.g. CD11a, CD11c,CD54, CD58, and CD102) (1, 6). These surface molecules allowDC to establish intimate, antigen-specific interaction with Tcells. DC are also capable of incorporating exogenous antigensefficiently by phagocytosis, endocytosis, and pinocytosis (7),and surface expression of IgG and IgE receptors and carbohy-drate receptors appears to contribute to this function (8–11).DC are highly mobile leukocytes, migrating across differenttissues via blood or lymphatic vessels (12). This mobility ismediated in part by the expression of homing receptors (e.g.CD44, CD62P ligand, and cutaneous lymphocyte-associatedantigen) and chemokine receptors (e.g. CXC chemokine recep-tor-1, -2, and -3 and CC chemokine receptor-1 through -7) (6,13–16). Finally, DC elaborate a wide variety of cytokines (in-terleukin (IL)-1b, IL-6, IL-12, and tumor necrosis factor-a) andchemokines (IL-8, macrophage inflammatory protein-1a and-1g, DC-chemokine-1, and thymus-expressed chemokine) (17–21), thereby regulating the magnitude and direction of T cellactivation.

The ultimate goal in our laboratories has been to understandthe biology of DC at a molecular level. As an initial step, weestablished a stable, long term DC line from the epidermis ofnewborn BALB/c mice. This DC line, termed “XS52,” retainsimportant features of resident DC in the epidermis of skin (i.e.Langerhans cells), including their surface phenotype, antigen-presenting capacity, and cytokine and cytokine receptor pro-files (19, 22–31). Taking the advantage of having relativelylarge numbers of a pure DC population (with cell doubling time

* This work was supported by National Institutes of Health GrantsRO1-AR44189, RO1-AR35068, RO1-AR43777, and RO1-AI43262; agrant from Taisho Pharmaceutical Co., Ltd., Ohmiya, Japan; and anaward from the Centre de Recherches et Investigations Epidermiqueset Sensorielles (to A. T.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s)AF262985.

‡ To whom correspondence should be addressed: Dept. of Dermatol-ogy, University of Texas Southwestern Medical Center, 5323 HarryHines Blvd., Dallas, TX 75235-9069. Tel.: 214-648-3419; Fax: 214-648-3472; E-mail: atakas@mednet.swmed.edu.

1 The abbreviations used are: DC, dendritic cells; aa, amino acid(s);

CRD, carbohydrate recognition domain; DCIR, DC immunoreceptor;DHFR; dihydrofolate reductase, CSF, colony-stimulating factor; GM-CSF, granulocyte/macrophage colony-stimulating factor; IL, interleu-kin; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, im-munoreceptor tyrosine-based inhibitory motif; Ab, antibody; mAb,monoclonal Ab; nt, nucleotide(s); FITC, fluorescein isothiocyanate;PCR, polymerase chain reaction.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 26, Issue of June 30, pp. 20157–20167, 2000© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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of 18–24 h), we chose to employ the subtractive cloning strat-egy for the identification of genes that are expressed preferen-tially by a DC line. Here we report the results of this molecularapproach, focusing on the characterization of one of the novelgenes identified by the subtraction.

EXPERIMENTAL PROCEDURES

Animals—Female BALB/c mice (6–10 weeks old) and New ZealandWhite rabbits (4–20 weeks old) were housed in the pathogen-free facil-ity of the Animal Resource Center at the University of Texas South-western Medical Center. All of the experiments were conducted accord-ing to the guidelines of the National Institutes of Health.

Cell Lines—XS52 cells are a long term DC line established from theepidermis of BALB/c mice (22). This line was maintained and expandedin complete RPMI 1640 supplemented with mouse recombinant gran-ulocyte/macrophage colony-stimulating factor (GM-CSF) (1 ng/ml) andNS47 fibroblast culture supernatant (10% v/v) as a source of CSF-1 (22,23). The phenotypic and functional features of this DC line have beendescribed elsewhere (19, 22–31). The J774 cells are a long term macro-phage line established from BALB/c mice; this line was purchased fromthe American Tissue Type Collection (ATCC, Manassas, VA) and main-tained in complete RPMI 1640 in the absence of added growth factors.We also used the Pam 212 keratinocyte line (32), NS fibroblast lines (22,23, 33), 7-17 dendritic epidermal gd T cell line (34, 35), Raw macrophageline (ATCC), HDK-1 CD41 Th1 clone and D10 CD41 Th2 line (kindlyprovided by Dr. Nancy Street, University of Texas Southwestern Med-ical Center), 5C5 and 2G9 B cell hybridoma clones (provided by Dr.Mansour Mohamadzadeh, University of Texas Southwestern MedicalCenter), and COS-1 line (ATCC).

Cell Isolation—Epidermal cells were isolated from abdominal skin ofBALB/c mice using two sequential trypsin treatments and then en-riched for Langerhans cells by centrifugation over Histopaque (1.083;Sigma) as before (30, 36). In some experiments, the epidermal cellsharvested from the medium/Histopaque interface (interface epidermalcells) were depleted of Langerhans cells by anti-Ia mAb plus comple-ment treatment as before (30). Splenic DC were isolated as describedpreviously (36). Briefly, spleen cell suspensions were prepared by me-chanical dissociation, followed by collagenase treatment (1% collagen-ase, 1 h, 37 °C). After lysis of erythrocytes, splenic cells were subjectedto gradient centrifugation with Percoll (Amersham Pharmacia Biotech);cells collected from the interface between 1.035 and 1.075 g/ml Percollwere then incubated on tissue culture plates. After a 90-min incubation,nonadherent cells were removed by extensive pipetting, and the adher-ent cells were cultured overnight. Cells released during the secondculture period were harvested and used as splenic DC preparations.

Identification of Dectin-1 cDNA Clone by the Subtractive CloningStrategy—A subtractive cDNA library was constructed using the meth-ods reported by Rubenstein et al. (37). Briefly, poly(A)1 RNAs isolatedfrom the XS52 DC line were reverse-transcribed into cDNAs, ligatedunidirectionally to the l ZapII phages (Stratagene, La Jolla, CA), andthen converted into a single-stranded phagemid library in which cDNAswere synthesized as an antisense strand (pBluescript II SK(2), Strate-gene). This cDNA library (1.2 mg) was hybridized with 50 mg of bioti-nylated poly(A)1 RNA isolated from the J774 macrophage line. Unhy-bridized cDNAs were purified by the streptavidin-phenol extractionmethod and converted into a double-stranded form by Taq DNA polym-erase. Subsequently, the resulting “DC-specific” cDNA library wasscreened sequentially by differential colony hybridization, slot-blotting,and Northern blotting.

In colony hybridization, colonies were transferred onto nylon mem-branes and hybridized differentially with total cDNA probes preparedfrom XS52 DC-derived poly(A)1 RNAs and from J774 macrophage-derived poly(A)1 RNAs. In slot blotting, the cDNA inserts were polym-erase chain reaction-amplified, slot-blotted onto nylon membranes, andhybridized with total cDNA probes from XS52 DC and from J774macrophages. In both screening steps, we isolated only those clonesthat were hybridized strongly with XS52 DC-derived total cDNA probesbut not detectable with the J774 macrophage-derived cDNA probes.

Northern blotting was performed as described previously (38).Briefly, cDNA inserts were excised by enzymatic digestion and 32P-labeled. Total RNAs (10 mg/lane) isolated from XS52 DC or J774 macro-phages by using RNA-STAT60 (Tel-TestB; Friendswood, TX) were size-fractionated on a vertical agarose gel, transferred onto a nylonmembrane, and hybridized with the above 32P-labeled probes. Onceagain, we selected those cDNA clones that showed strong hybridizationonly with the XS52 DC-derived probes. Finally, 50 cDNA clones selectedby Northern blotting were sequenced and subjected to homology search

using the GenBankTM and EMBL data bases.Tissue and Cell Distributions of Dectin-1 mRNA Expression—Total

RNAs were isolated from several different lines or from different mouseorgans. In some experiments, XS52 DC and J774 macrophages werecultured for 48 h in the presence or absence of 10 ng/ml of eitherGM-CSF or CSF-1 before RNA isolation. Northern blotting was carriedout as described above using 32P-labeled dectin-1 cDNA probe (clone1C11-5) or the glyceraldehyde-3-phosphate dehydrogenase controlprobe (38). Reverse transcription-polymerase chain reaction was car-ried out as described previously (30, 38). Briefly, total RNA isolatedfrom the interface epidermal cells was reverse-transcribed into cDNAand then PCR-amplified with the following primer sets: 59-AGGCCCT-ATGAAGAACTACAGACA-39 (nt 1384–1407) and 59-TGGCCAGGACA-GCATAAGGAA-39 (nt 1830–1811) for dectin-1; 59-TACAGGCTCCGA-GATGAACAACAA-39 (nt 450–473) and 59-TGGGGAAGGCATTAGAA-ACAGTC-39 (nt 899–921) for IL-1b, or 59-GTGGGCCGCTCTAGGCAC-CAA-39 (nt 25–45) and 59-CTCTTTGATGTCACGCACGATTTC-39 (nt541–564) for b-actin. The PCR products were separated on agarose gel,transferred onto a membrane, and hybridized with 32P-labeled dectin-1,IL-1b, or b-actin cDNA probe.

Preparation of His-Dectin-1 Fusion Proteins—His-dectin-1 fusionprotein consisting of, from the N terminus, hexahistidine and the ex-tracellular domain of dectin-1 was produced in Escherichia coli asfollows. The DNA fragment encoding an extracellular domain (aa 73–244) of dectin-1 was PCR-amplified, and BamHI and SmaI sites werethen attached to the resulting DNA fragment at the 59- and 39-end,respectively. Using BamHI and SmaI sites, the fragment was ligated toimmediately downstream of the hexahistidine sequence in pQE-30 vec-tor (Qiagen, Chatsworth, CA). This recombinant vector was introducedinto E. coli; His-dectin-1 protein was extracted from isopropyl-1-thio-b-D-galactopyranoside-treated E. coli in 8 M urea, 100 mM sodium phos-phate, 10 mM Tris/HCl (pH 8.0) buffer and purified using Ni21-nitrilo-triacetic acid resin (Qiagen). Approximately 5–8 mg of proteins wererecovered from 1.5 liters of E. coli culture by elution at pH 5.9 and 4.9.After extensive dialysis against phosphate-buffered saline (PBS, pH7.4), relatively small fractions (4–20%) of the His-dectin-1 proteinswere recovered in a soluble form, and this fraction was used in thefunctional studies (see below). As a control protein with a His tag,His-dihydrofolate reductase (DHFR) was produced in E. coli trans-formed with the commercially available His-DHFR plasmid pQE-16(Qiagen) and purified as described above.

COS-1 cells were transfected using FuGene 6 (Roche Molecular Bio-chemicals) with pSecTag vector (Invitrogen, Carlsbad, CA) that con-tained the DNA insert encoding, from the N terminus, the Igk leadersequence, hexahistidine sequence, TEV site, and extracellular domain(aa 73–244) of dectin-1. Culture supernatants collected at 48–60 h aftertransfection were purified using the Ni21-nitrilotriacetic acid resin.

Immunoblotting and Flow Cytometric Analyses—A synthetic 21-aapeptide containing a cysteine residue at the N terminus of the 20-aasequence GRNPEEKDNFLSRNKENHKP corresponding to aa 75–94 ofdectin-1 was used to immunize rabbits. After three rounds of standardimmunization, serum was collected and subjected to affinity purifica-tion of peptide-specific Ab using the same 21-mer peptide. Briefly, highpressure liquid chromatography-purified peptide was conjugated to thethiol coupling gel in buffer A (50 mM Tris-HCl, 5 mM EDTA, pH 8.5).Following washing and equilibration with a 50 mM phosphate buffer(pH 6.5), 40 ml of serum was applied to the peptide antigen-conjugatedthiol coupling gel, and peptide-specific antibodies were then eluted,after extensive washing with the phosphate buffer, with 100 mM gly-cine-HCl (pH 2.5). After neutralization, the fractions showing signifi-cant A280 values were dialyzed against buffer B (10 mM NaH2PO4, 20mM NaCl, pH 7.0).

XS52 DC were homogenized in 10 mM HEPES (pH 7.3) with 5–10strokes with a 27-gauge needle on a 1-ml syringe and centrifuged for 10min at 1,000 3 g, and the resulting supernatants (“crude lysates”) werefractionated into cytosolic and membrane fractions by centrifugation for40 min at 100,000 3 g. In some experiments, whole cell extracts wereprepared from several different cell lines in 0.3% Triton X-100 in PBS,followed by centrifugation for 10 min at 1,000 3 g. The full-length cDNAfor dectin-1 was excised from clone 1C11-5 by digestion with EcoRI andXbaI restriction enzymes and inserted into a mammalian expressionvector pZeoSV2(1) (Invitrogen). COS-1 cells were transfected with theresulting vector or an empty vector alone using FuGene 6, and mem-brane fractions were prepared at 72 h after transfection. These sampleswere separated by 10–20% or 4–20% SDS-PAGE, transferred ontopolyvinylidene fluoride membrane (Millipore Corp., Bedford, MA), andthen blotted with 0.72 mg/ml of affinity-purified rabbit anti-dectin-1 orcontrol rabbit IgG. After extensive wash, the membrane was blotted

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with horseradish peroxidase-conjugated anti-rabbit IgG (Zymed Labo-ratories Inc., San Francisco, CA) and then developed with the ECLsystem (Amersham Pharmacia Biotech).

Different cell lines were fixed with 2% paraformaldehyde in PBS andthen incubated with 1 mg/ml of anti-dectin-1 or control rabbit IgG,followed by incubation with FITC-conjugated anti-rabbit IgG. SplenicDC preparations were subjected to double staining with anti-dectin-1followed by phosphatidylethanolamine-conjugated secondary Ab andwith FITC-conjugated anti-Ia or anti-CD11c mAb (Pharmingen, SanDiego, CA). These samples were analyzed by FACScan (Becton-Dickin-son, Mountain View, CA) as before (22).

His-Dectin-1 Binding Assays—Binding properties of His-dectin-1were examined in binding buffer (Hank’s balanced salt solution con-taining 1.3 mM CaCl2, 3% fetal calf serum, and 10 mM HEPES) by usingfour different protocols. First, soluble fractions of bacterially producedHis-dectin-1 proteins were labeled with biotin (Pierce) and tested forthe binding to selected cell lines (2 3 106 cells/ml). Following a 30-minincubation on ice with 5 mg/ml of biotinylated His-dectin-1, the cellswere washed and incubated with FITC-conjugated streptavidin at 1:100dilution (Jackson Immunoresearch Laboratories, West Grove, PA).Samples were then analyzed by FACScan. In some experiments, theactivated D10 T cells were cultured for 16 h in the presence of 4 mg/mltunicamycin (Sigma) before analyses, or they were incubated with0.25% trypsin in PBS for 20 min at 37 °C. For the N-glycosidase treat-ment, the cells were first fixed with 0.4% paraformaldehyde in PBS for3 h at 4 °C, washed with PBS, and then incubated with 600 milli-units/ml neuraminidase (Roche Molecular Biochemicals) for 3 h at37 °C, followed by the second 16-h incubation at room temperature.Subsequently, the samples were washed with 50 mM phosphate buffer(pH 8.0) and treated with 10 units/ml N-glycosidase (Roche MolecularBiochemicals) in the same buffer for 16 h at 37 °C. To determine theextent of deglycosylation achieved by the above treatment with tunica-mycin or N-glycosidase, the same T cell samples were tested for theirability to bind biotinylated phytohemagglutinin and biotinylated wheatgerm agglutinin (both purchased from Sigma).

Second, soluble fractions of bacterially produced His-dectin-1 pro-teins (3 mg in 100 ml of 100 mM phosphate buffer, pH 6.5) were labeledwith 100 mCi of 125I (ICN, Costa Mesa, CA) in the presence of anIODO-BEAD (Pierce). After a 10-min incubation at room temperature,the reaction was stopped by the removal of the bead and the addition of100 ml of 3% bovine serum albumin. The 125I-labeled His-dectin-1 (0.7mg/ml, specific activity: 5 3 106 cpm/mg) was then incubated with theactivated D10 T cells (1 3 107 cells/ml). After a 90-min incubation onice, the cell suspensions were overlaid on the top of 700 ml of 100% fetalcalf serum and centrifuged at 600 3 g for 1 min to remove the unboundprobes. The cellular pellets were washed three more times in PBS andthen counted for radioactivities. To test the specificity, the incubationwas carried out in the presence of “cold” His-dectin-1 (76 mg/ml) oranti-dectin-1 (50 mg/ml); His-DHFR or control IgG at the same concen-trations were added to serve as controls.

Third, His-dectin-1 proteins produced by COS-1 cells were biotiny-lated, incubated at 5 mg/ml with the activated D10 T cells (2 3 106

cells/ml), and then analyzed by FACScan as described above. To test thespecificity, nonlabeled, bacterially produced His-dectin-1 or His-DHFR(160 mg/ml) was added to the incubation. Finally, 125I-labeled, bacteri-ally produced His-dectin-1 proteins were incubated for 120 min on icewith agarose beads that were conjugated with mannose, fucose, lactose,GluNAc, or GalNAc (all purchased from Sigma). Specific binding wasthen examined by counting the radioactivities that were eluted by theaddition of the corresponding carbohydrates.

T Cell Co-stimulation Assay—ELISA plates were coated with gradedconcentrations of anti-CD3e mAb (Pharmingen) together with bacteri-ally produced His-dectin-1 (10 mg/ml). In some experiments, enzyme-

linked immunosorbent assay plates coated with anti-CD3 mAb (0.3mg/ml) and His-dectin-1 (10 mg/ml) were preincubated with gradedconcentrations of anti-dectin-1 or control IgG. Splenic T cells purifiedfrom normal BALB/c mice were cultured in these wells (105 cells/well)for 4 days, pulsed with [3H]thymidine for 16 h, and then harvested asbefore (22).

RESULTS

Identification of Novel Genes Expressed by XS52 DC Line byUsing the Subtractive Cloning Strategy—Based on the hypoth-esis that one or more specific genes are expressed by DC, butnot by other antigen-presenting cells (including macrophages),we hybridized the XS52 DC-derived cDNA library with excessamounts of biotinylated mRNAs isolated from the J774 macro-phage line and isolated only the unhybridized cDNA clones.About 99% of the starting cDNA clones were eliminated by thisprocedure, estimated by counting the colony-forming units be-fore and after subtraction (Table I). Subsequently, we con-structed the “DC-specific” cDNA library from the unhybridizedclones and tested this library by three rounds of screening. Ofthe 12,000 independent colonies analyzed first by colony hy-bridization, 226 colonies showed strong hybridization with thetotal cDNA probes from XS52 DC, but not with the probes fromJ774 macrophages. In other words, the overwhelming majorityof the colonies failed to show any detectable hybridization withthe DC probes (about 40–50% of the colonies), or they showedcomparable levels of hybridization with the DC probes and themacrophage probes. We next tested these 226 clones in slotblotting and selected 140 clones that showed preferential hy-bridization with the DC probes. Finally, these 140 clones wereexamined for their expression levels in XS52 DC versus J774macrophages by Northern blotting; we were able to confirmDC-specific expression for 50 of these clones (Table I).

Through partial sequencing and cross-hybridization, wefound that the above 50 clones contained 11 distinct genes. Ahomology search of their nucleotide sequences revealed sixgenes that encoded currently recognized polypeptides, includ-ing C10 (a b-chemokine) (39), IL-1b (40), cathepsin C (a cys-teine protease) (41), spermidine/spermine N1-acetyltransferase(42), and A1 (a hemopoietic cell-specific early response gene)(43). We have also identified a gene that encodes a mouseequivalent of rat Rab-2 (a Ras-related protein) (44). The re-maining five genes were judged to be distinct from any nucle-otides currently registered in the GenBankTM or EMBL databank. We focused our subsequent effort on one of these novelgenes.

Structural Features of Dectin-1 Polypeptide—Clone 1C11-5contained 735 nucleotides (nt) in its open reading frame, and itshowed DC-specific hybridization in colony hybridization, slotblotting, and Northern blotting. As shown in Fig. 1A, the de-duced amino acid sequence of this clone revealed a polypeptideof 244 aa with type II configuration, consisting of a cytoplasmicdomain (aa 1–44), a putative transmembrane domain (aa 45–68), and extracellular domains (aa 69–244). The overall aminoacid sequence of this polypeptide showed significant homology

TABLE ISummary of isolation of dendritic cell-specific genes

Genes expressed selectively by the XS52 DC line were enriched by subtractive hybridization of the XS52 DC cDNA library (1.5 3 107) withbiotinylated mRNA prepared from the J774 macrophage line. The 12,000 clones in the resulting subtractive cDNA library (1.6 3 105) weresequentially screened by colony hybridization (first), slot blot hybridization (second), and Northern blotting (third).

No. of selected clones/No. of tested clones

Recovery ateach step Method

%

Subtraction 1.6 3 105/1.5 3 107 1.1 XS52 DC mRNA minus J774 mRNAFirst screening 226/12,000 1.9 Colony hybridizationSecond screening 140/226 62 Slot blot hybridizationThird screening 50/140 36 Northern blotting

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with several polypeptides, including (a) endothelial receptor(LOX-1) for oxidated low density lipoprotein (26.6% similarityby Clustral method analyzed with Lasergene Program; DNAStar, Madison, WI) (45), (b) a natural killer cell receptor CD94(21.8%) (46, 47), (c) another receptor CD69 encoded in thenatural killer gene complex (17.8%) (48), (d) asialoglycoproteinreceptors, i.e. hepatic lectin-1 (18.9%) and hepatic lectin-2(15.6%) (49, 50), and (e) low affinity Fce receptor CD23 (15.6%)(51). All of these molecules contain carbohydrate recognitiondomain (CRD) motifs and, thus, belong structurally to thefamily of C-type (Ca21-dependent) lectins. Spiess has identified13 invariant amino acid residues (including six cysteines play-ing a critical role in forming disulfide bridge frameworks) thatare relatively conserved among CRD sequences in differentC-type lectins (52). We identified a putative CRD motif at theCOOH-terminal region (aa 119–244) of the 1C11-5 polypeptidesequence, and this motif contained 11 out of the 13 invariantresidues, including all six cysteine residues (as indicated withasterisks in Fig. 1A). Moreover, this CRD motif showed rela-tively high degrees of homology (22.0–38.4% identities) withthe CRD sequences found in other C-type lectins (Fig. 1B).Thus, we concluded that clone 1C11-5 encoded a uniquepolypeptide that belonged structurally to the C-type lectin fam-ily, and this polypeptide was designated as “DC-associatedC-type lectin-1” or “dectin-1.” It is also important to note that aputative immunoreceptor tyrosine-based activation motif(ITAM) (YXXL) (53, 54) was found in the cytoplasmic domain ofdectin-1 (indicated with an underline in Fig. 1A).

Cell and Tissue Distributions of Dectin-1 mRNA—Northernblotting showed that dectin-1 mRNA (about 3.2 kilobases insize) was expressed at relatively high levels by XS52 DC,whereas it was detected at only negligible levels in J774 macro-phages (Fig. 2A). Most importantly, dectin-1 mRNA was totallyundetectable in other tested cell lines, including an additionalmacrophage line (Raw), a gd T cell line (7-17), two ab T celllines (HDK-1 and D10), a B cell hybridoma clone (5C5), akeratinocyte line (Pam 212), and a fibroblast line (NS01). Be-cause the XS52 DC line, but not other cell lines, had beenexpanded in the presence of added GM-CSF and CSF-1 (22, 23),we considered that dectin-1 mRNA expression might be simplyinduced by either of these growth factors. As shown in Fig. 2B,

expression patterns of dectin-1 mRNA remained unchanged inboth XS52 cells and J774 cells, regardless of the presence orabsence of GM-CSF or CSF-1 in culture medium, thus exclud-ing the possibility that dectin-1 expression was regulated bythe added growth factors.

As noted in Fig. 2C, abundant expression of dectin-1 mRNAwas detected in spleen and thymus, the tissues known to con-tain relatively large numbers of DC (1, 6). Unexpectedly,Northern blotting failed to reveal dectin-1 mRNA expression inskin, the tissue from which the XS52 DC line had been estab-lished (22). This did not mean, however, that dectin-1 mRNAwas absent from this tissue, because a strong PCR signal wasdetected in epidermal cells freshly isolated from BALB/c mice(Fig. 2D). With respect to the source of dectin-1 mRNA expres-sion, depletion of the Ia1 epidermal cell population (i.e. Lange-rhans cells) abrogated almost completely dectin-1 mRNA, aswell as IL-1b mRNA, which is known to be expressed exclu-sively by Langerhans cells in murine epidermis (18, 55, 56).This corroborates our observations that dectin-1 mRNA wasdetected by Northern blotting in the XS52 Langerhans cell-likeline, but not in cell lines derived from other epidermal cellpopulations, i.e. the Pam 212 keratinocyte line and the 7-17epidermal gd T cell line (Fig. 2A). These results suggest thatdectin-1 mRNA is expressed constitutively and preferentiallyby Langerhans cells in the epidermis.

Identification of Dectin-1 Protein—To study dectin-1 proteinexpression, we produced rabbit Ab against a synthetic peptidecorresponding to aa 75–94 of dectin-1 and purified them byaffinity chromatography. The resulting anti-dectin-1 Ab recog-nized in immunoblotting a bacterially produced recombinantfusion protein of about 27 kDa consisting of a hexahistidine tagand an extracellular domain sequence (aa 73–244) of dectin-1(Fig. 3). The same Ab also immunolabeled a major band of 43kDa in crude lysates of XS52 DC. By contrast, control rabbitIgG failed to label any specific bands. When XS52 cell lysateswere fractionated into a membrane fraction and a cytosolicfraction, the 43-kDa immunoreactivity was detected primarilyin the membrane fraction (Fig. 4), in accordance with the pre-dicted molecular structure. The above molecular size of dec-tin-1 protein detected in immunoblotting was considerablylarger than the size predicted from the full-length amino acid

FIG. 1. Deduced amino acid se-quence of dectin-1 and its homologywith members of the C-type lectinfamily. A, deduced amino acid sequenceof dectin-1 is shown, segmented into acytoplasmic domain, a transmembranedomain, and extracellular domains con-taining a putative CRD motif at theCOOH-terminal end. Asterisks and trian-gles indicate the invariant residues of C-type lectins and putative N-glycosylationsites, respectively. B, a putative CRD se-quence of dectin-1 was aligned with theCRD sequences in other C-type lectins inmice (m), rats (r), and humans (h).

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sequence (28 kDa). This discordance most likely resulted fromglycosylation, because N-glycosidase treatment of XS52 DClysates reduced substantially the molecular size of dectin-1immunoreactivity (data not shown) and because dectin-1 con-tains two putative N-glycosylation sites at aa 185 and 233 (asindicated with triangles in Fig. 1A). Thus, dectin-1 is expressedby XS52 DC as a 43-kDa membrane-associated glycoprotein.

To determine the extent to which dectin-1 protein expressionoccurred in a DC-specific manner, we tested whole cell extractsprepared from several different cell lines by immunoblotting.As shown in Fig. 5, the 43-kDa immunoreactivity was, again,

detected in XS52 DC extracts, whereas it was totally undetect-able in extracts from other tested cell lines, including J774macrophage, HDK-1 ab T cell, 7-17 epidermal gd T cell, 2G9 Bcell hybridoma, Pam 212 keratinocyte, and NS47 fibroblastlines. Dectin-1 protein with the same molecular weight wasalso detected in a second DC line (XS106) derived from A/J mice(data not shown). These results, together with our observationwith Northern blotting, document that dectin-1 mRNA andprotein are expressed selectively by DC lines.

As shown in Fig. 6, a 43-kDa immunoreactivity was detectedin the membrane fraction prepared from COS-1 cells that hadbeen transfected with dectin-1 cDNA (clone 1C11-5), and thisband closely corresponded in molecular size to the naturaldectin-1 protein produced by XS52 DC. No immunoreactivity

FIG. 2. Cell- and tissue-specific ex-pression of dectin-1. Total RNAs wereisolated from XS52 DC, J774 and Rawmacrophages, 7-17 dendritic epidermal gdT cells, HDK-1 Th1 cells, D10 Th2 cells,5C5 B cell hybridoma, Pam 212 keratino-cytes, and NS01 fibroblasts (A) or fromthe indicated tissues in adult BALB/cmice (C). B, XS52 cells and J774 cellswere cultured for 48 h in the presence orabsence of GM-CSF (10 ng/ml) or CSF-1(10 ng/ml) before isolation of RNA. TheRNA samples were then examined byNorthern blotting for dectin-1 or glyceral-dehyde-3-phosphate dehydrogenase(GAPDH). D, interface epidermal cellsisolated from BALB/c mice were exam-ined for dectin-1 mRNA expression by re-verse transcriptase-PCR. Some sampleswere treated with anti-Ia mAb plus com-plement to deplete Langerhans cells; theextent of depletion was assessed by meas-uring IL-1b mRNA, which is known to beexpressed exclusively by Langerhans cellswithin murine epidermal cells.

FIG. 3. Identification of dectin-1 protein. Whole cell extracts pre-pared by Triton X-100 from J774 macrophages (lane 1) or from XS52 DC(lane 2) or His-dectin-1 fusion proteins produced in E. coli (lane 3) wereexamined for the immunoreactivity to anti-His-dectin-1 Ab (top panel)or control IgG (middle panel). The same samples were also stained withCoomassie Blue (bottom panel).

FIG. 4. Membrane association of dectin-1 protein. Crude lysatesprepared mechanically from XS52 DC were centrifuged at 100,000 3 gfor 40 min, and the pellet (membrane fraction) and the supernatant(cytosolic fraction) were examined by immunoblotting with anti-dec-tin-1 or control IgG. The data shown are representative of three inde-pendent experiments.

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was detected after transfection with the vector alone. An addi-tional band of about 40 kDa was also detected only in theCOS-1 membrane fraction; the molecular identity of this sec-ond band remains unclear at present. In flow cytometric anal-yses, anti-dectin-1 Ab recognized dectin-1 proteins on the sur-face of paraformaldehyde-fixed XS52 cells (Fig. 7A, left panel).A similar staining profile was also observed in the absence offixation (data not shown). Consistent with our observations inimmunoblotting, no significant staining was observed with an-ti-dectin-1 Ab for NS47 fibroblasts (Fig. 7A, right panel) orother cell lines, including macrophage, B cell, T cell, and kera-tinocyte lines (data not shown).

Because dectin-1 mRNA was expressed most abundantly in

spleen, we next examined dectin-1 protein expression in thistissue. Splenic DC preparations isolated by our standard pro-tocol contained routinely 40–70% DC, as assessed by morphol-ogy and surface expression of Ia (major histocompatibility com-plex class II) molecules and CD11c (36). As shown in Fig. 7B,dectin-1 expression was detected in some, but not all, cells inthis preparation, and a majority of the cells expressing dectin-1co-expressed Ia molecules at relatively high levels. On theother hand, when dectin-1 expression was analyzed in thegated population of Iahigh cells, it became clear that dectin-1protein was not expressed by all Iahigh cells (Fig. 7C, left panel).The percentage of dectin-11 cells in the Iahigh population variedfrom 25 to 32% in four independent experiments. Likewise,dectin-1 expression was detected in 20–42% of the CD11c1

population (Fig. 7C, right panel). These observations suggestedthat dectin-1 protein was expressed on the surface of some, butnot all, splenic DC. It is to be noted that dectin-1 expressionwas also detectable in minor fractions of the Ia2 population aswell as the CD11c2 population, with the implication that ex-pression of dectin-1 does not occur exclusively within the DCpopulations in living animals. Considering the data together, itis reasonable to conclude that dectin-1 is a unique polypeptidethat is characterized by the inclusion of a CRD motif and by itspreferential expression by some DC populations in both lymph-oid and nonlymphoid tissues.

Functional Potential of Dectin-1—As an initial step to studythe function, we tested whether His-dectin-1 fusion proteinwould bind to any cell types. As shown in Fig. 8A, His-dectin-1proteins produced in E. coli migrated as a 27-kDa band, closelycorresponding to the predicted molecular size of 21 kDa. Re-combinant proteins produced and secreted by COS-1 cells, how-ever, showed significantly higher molecular masses (37 and 39kDa) than the predicted size (25 kDa), consistent with ourhypothesis that dectin-1 is a heavily glycosylated polypeptide.On the other hand, it remains unclear whether the 37 and 39bands differ each other only in the extent of glycosylation.Soluble fractions of the bacterially produced His-dectin-1 werelabeled with biotin and tested for binding. Among severaltested cell lines, only the D10 T cell line showed modest, butsignificant, binding (Fig. 8B). When tested after stimulationwith concanavalin A, D10 T cells showed markedly elevatedbinding of His-dectin-1. Likewise, two additional T cell lines(HDK-1 and 7-17) also showed significant binding of His-dec-tin-1 only in the activated states. Binding of His-dectin-1 to theactivated T cells was also confirmed by using FITC-conjugatedmAb against the His tag (data not shown). 125I-Labeled His-dectin-1 (produced in E. coli) bound significantly to the surfacesof activated D10 T cells. Importantly, this binding was com-peted with “cold” His-dectin-1, but not with His-DHFR, and itwas inhibited by anti-dectin-1 Ab, but not by control Ab, thusdocumenting the specificity (Fig. 8C). Moreover, the glycosy-lated His-dectin-1 proteins (produced in COS-1 cells) alsobound to the activated D10 T cells in a manner that wasinhibitable by bacterially produced His-dectin-1 but not byHis-DHFR (Fig. 8D). We noticed that the recombinant proteinsproduced in COS-1 cells were less efficient than those producedin E. coli in their T cell binding ability; mechanisms for thisfunctional disparity remain to be determined. Nevertheless,these results indicate that extracellular domains of dectin-1bind specifically to one or more putative molecules that areexpressed on activated T cells.

An important question then concerned whether His-dectin-1fusion proteins containing a CRD sequence would recognizecarbohydrate moieties. 125I-Labeled His-dectin-1 failed to showspecific binding to any of the tested carbohydrate probes, i.e.agarose beads conjugated with mannose, fucose, lactose, Glu-

FIG. 5. Cell type specificity of dectin-1 protein expression.Whole cell extracts were prepared from the indicated cell lines in thecell lysis buffer containing Triton X-100. These samples were thenexamined by immunoblotting for dectin-1 immunoreactivity (top) andby Coomassie Blue staining for protein profiles (bottom). The datashown are representative of two independent experiments.

FIG. 6. Production of dectin-1 protein by introducing the dec-tin-1 cDNA clone. COS-1 cells were transfected with pZeoSV-Dec1KZvector encoding the full-length dectin-1 polypeptide (derived from clone1C11-5) or pZeoSV2(1) control vector. Membrane fractions preparedfrom these transfectants or from XS52 DC were examined by immuno-blotting. The data shown are representative of three independentexperiments.

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NAc, or GalNAc (data not shown). Furthermore, pretreatmentof T cells with tunicamycin, which diminished substantially thebinding of a bona fide lectin, phytohemagglutinin, had onlymarginal effects on the binding of His-dectin-1 (Fig. 9A). Like-wise, N-glycosidase treatment of paraformaldehyde-fixed Tcells had no effects on the binding of His-dectin-1, whereas thesame treatment markedly reduced the binding of wheat germagglutinin (WGA) (Fig. 9B). By marked contrast, a brief expo-

sure of activated T cells to trypsin abrogated their dectin-1binding capacity almost completely (Fig. 9C). Thus, dectin-1appears to bind to one or more trypsin-sensitive, tunicamycin/N-glycosidase-resistant ligands on T cells. We interpretedthese results to suggest that although dectin-1 belongs struc-turally to the C-type lectin family by the inclusion of a CRDmotif, it may not function as a conventional C-type lectin in theligand specificity.

FIG. 7. Surface expression of dectin-1 on DC. A, XS52 DC and NS47 fibroblasts were fixed with paraformaldehyde and incubated withanti-dectin-1 Ab (closed histograms) or control IgG (open histograms), followed by FITC-conjugated, anti-rabbit IgG. B, splenic DC preparationsisolated from adult BALB/c mice were double-stained with anti-Ia mAb (y-axis) and with anti-dectin-1 (x-axis in the right panel) or control IgG(x-axis in the left panel). C, splenic DC were double-stained with anti-dectin-1 Ab (closed histograms) or control IgG (open histograms) with anti-IamAb (left panel) or anti-CD11c mAb (right panel). Data shown are the histograms for dectin-1 staining within the Iahigh or CD11c1 population (i.e.splenic DC), with the percentages of dectin-1 positive cells indicated with numbers. All of the data shown are representative of at least threeindependent experiments.

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To study the biological outcome of dectin-1 binding, splenic Tcells were incubated on plates coated with His-dectin-1 (10mg/ml). As shown in Fig. 10 (left panel), immobilized His-dec-tin-1 alone failed to induce significant T cell proliferation.However, His-dectin-1 promoted marked proliferation of T cellswhen anti-CD3 mAb was co-immobilized onto the same platesat suboptimal concentrations. For example, in the presence of0.1–0.3 mg/ml of anti-CD3 mAb, His-dectin-1 caused more than10-fold augmentation. On the other hand, His-dectin-1 hadalmost no effect on T cell proliferation that was triggered byhigher concentrations of anti-CD3 mAb. In dose-response ex-periments, 3 mg/ml of His-dectin-1 was found to be sufficient forthis biological activity (data not shown). Importantly, the co-

stimulatory activity of His-dectin-1 was blocked almost com-pletely with anti-dectin-1 Ab, but not with control IgG (Fig. 10,right panel). These results have revealed the potential of dec-tin-1 to deliver co-stimulatory signals to T cells.

DISCUSSION

One major technical barrier for studying the biology of DC atmolecular levels had been the unavailability of pure DC prep-arations. This barrier has been overcome recently by the de-velopment of refined methods for isolating and culturing DC inlarge quantities (57–59) and by the establishment of stable DClines (22, 60–65). By the PCR-based differential display be-tween “freshly isolated” immature Langerhans cells versus

FIG. 8. Binding of His-dectin-1 to T cells. A, recombinant His-dectin-1 proteins produced in E. coli or in COS-1 cells were purified usingNi21-NTA resin and then examined by immunoblotting with anti-dectin-1 Ab. B, a soluble fraction of the bacterially produced His-dectin-1 wasbiotinylated and examined for the binding to the indicated cell lines. The three T cell lines were tested for His-dectin-1 binding before (resting) andafter concanavalin A stimulation (activated). The binding of biotinylated His-dectin-1 (closed histograms) or biotinylated His-DHFR control (openhistograms) was examined with FITC-conjugated streptavidin. Data shown are representative of three different experiments. C, a soluble fractionof the bacterially produced His-dectin-1 was labeled with 125I and incubated with activated D10 T cells in the presence of “cold” His-dectin-1 orHis-DHFR (upper panels) or anti-dectin-1 or control IgG (lower panels). Data shown are representative of three independent experiments, showingthe mean 6 S.D. from triplicate samples. D, activated D10 T cells were incubated with biotinylated His-dectin-1 (produced in COS-1 cells) in thepresence of no competitor (open histogram), nonlabeled bacterially produced His-dectin-1 (closed histogram), or His-DHFR (dotted line).

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“cultured” mature Langerhans cells, Ross et al. (66) have iden-tified about 500 cDNA fragments whose expression was eitherup- or down-regulated during their maturation. By screening acDNA library constructed from the cultured Langerhans cellswith differential hybridization, they also identified that theexpression of fascin, an actin-bundling protein, was markedlyup-regulated during maturation (67). By searching the Human

Genome Sciences nucleotide data bases with a consensus motifof C-type lectins, Bates et al. (68) identified a new C-type lectin,termed DC immunoreceptor (DCIR), which was expressed byshort term human DC cultures generated from CD341 progen-itors as well as from CD141 monocytes. Taking advantage ofthe XS52 DC line maintaining many features of skin-residentDC (19, 22–31), we employed the subtractive cDNA cloningstrategy to identify the genes expressed predominantly by thisDC line. We have identified 11 different genes (including fivenovel genes) that were expressed selectively by XS52 cells,documenting the efficacy of our approach.

Clone 1C11-5 encoded a novel, type II membrane-integratedpolypeptide containing a single CRD motif at the COOH-ter-minal end. This polypeptide, termed dectin-1, was expressed onthe surface of XS52 DC as a glycoprotein of about 43 kDa insize. Members of the C-type lectin family can be divided intotwo groups based on the molecular structures: (a) type I surfacelectins with multiple CRDs on their NH2-terminal ends and (b)type II surface lectins with a single CRD on their COOHtermini. Importantly, DC have been shown to express bothtypes of C-type lectins. Jiang et al. have cloned a novel, type Imembrane-integrated C-type lectin, termed DEC-205, by usinga DC-specific mAb (NLDC145) as a molecular probe (11). Asobserved for dectin-1, surface expression of DEC-205 was de-tected in some, but not all, splenic DC (69). Subsequently,Vremec and Shortman (70) found that DEC-205 is expressedpreferentially by a specific subset of splenic DC called “lymph-oid DC” as defined by their surface expression of CD8a ho-modimers. Sallusto et al. (10) reported that human DC expressmacrophage mannose receptor, a prototypic C-type lectin withthe type I configuration. These two DC-associated C-type lec-tins, DEC-205 and macrophage mannose receptor, contain mul-tiple CRD motifs (10 and 8, respectively) at their NH2-terminalends; thus, they belong to the type I surface lectin subfamily.

FIG. 9. Characterization of the dectin-1-binding moiety on activated T cells. The activated D10 T cells were tested for their ability to bindHis-dectin-1 and the indicated lectins following treatment with tunicamycin (A), N-glycosidase (B), or trypsin (C). A, the cells were cultured for 16 hin the presence (closed histogram) or absence (open histogram) of 4 mg/ml tunicamycin. B, the cells were fixed with paraformaldehyde and thensubjected to the neuraminidase/N-glycosidase digestion (closed histogram) or the digestion with neuraminidase alone (open histogram). C, the cellswere incubated with 0.25% trypsin (closed histogram) or PBS alone (open histogram) for 20 min at 37 °C. Data shown in the upper panels indicatethe binding of bacterially produced His-dectin-1 (open and closed histograms) or His-DHFR control (dotted lines). The data in the lower panelsindicate the binding of biotinylated phytohemagglutinin (PHA) or wheat germ agglutinin (WGA) (open and closed histograms, respectively) orHis-DHFR control (dotted lines). The binding of biotinylated wheat germ agglutinin was reduced significantly by neuraminidase treatment alone(data not shown) and diminished further by the subsequent N-glycosidase treatment. All of the results shown are representative of threeindependent experiments.

FIG. 10. Co-stimulatory potential of dectin-1. Enzyme-linked im-munosorbent assay plates were coated with the indicated concentra-tions of anti-CD3 mAb together with His-dectin-1 (10 mg/ml) or bufferalone. Splenic T cells purified from normal BALB/c mice were culturedin these wells (105 cells/well) for 4 days, pulsed with [3H]thymidine for16 h, and then harvested (left panel). Enzyme-linked immunosorbentassay plates coated with anti-CD3 mAb (0.3 mg/ml) and His-dectin-1 (10mg/ml) were incubated with the indicated concentrations of anti-dec-tin-1 or control IgG. Splenic T cells were cultured in these wells andexamined for [3H]thymidine uptake as described above. Data shown arerepresentative of three independent experiments, showing the mean 6S.D. (n 5 3) of [3H]thymidine uptake.

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Interestingly, both have been shown to mediate the uptake ofglycosylated macromolecules by DC (10, 11).

DC also express CD23 (low affinity Fce receptor) (71) andCD69 (very early activation antigen) (72), both of which belongto the type II surface lectin subfamily. Although sharing thesame molecular configuration, dectin-1 showed only limiteddegrees of sequence homology to CD23 (17.8% identity) orCD69 (15.6%), and the extent of homology was rather limitedeven within the highly conserved CRD motifs (22.0% withCD23 and 22.6% with CD69). More recently, Bates et al. (68)isolated a novel member (DCIR) by searching the nucleotidedata bases with a motif (SCYWFSH) that is shared by hepaticlectin-1, hepatic lectin-2, and macrophage lectin. DCIR is ex-pressed not only by DC but also by small fractions of otherantigen presenting cells (monocytes, macrophages, and B cells),as we observed for dectin-1. Interestingly, DCIR contains, inthe intracellular domain, a consensus immunoreceptor tyro-sine-based inhibitory motif (ITIM) (54), suggesting its potentialto deliver immunoregulatory signals (68). Dectin-1 showed rel-atively low sequence homology with DCIR (15.5% identity inoverall sequence and 18.8% within the CRD region), and dec-tin-1 contained a putative ITAM motif, instead of the ITIMmotif, in the cytoplasmic domain. ITAM motifs are also foundin CD23 and macrophage lectin (51, 73), whereas an ITIMmotif is present in CD72 (a B cell-associated C-type lectin) (74).It is, therefore, tempting to speculate that the ITAM-contain-ing lectins and the ITIM-containing lectins may play counter-acting roles in immunoregulation. Unfortunately, natural li-gands recognized by those DC-associated C-type lectins(including dectin-1) remain mostly unknown, thus preventingus from directly testing this possibility. In sum, dectin-1 is thesixth member of the unique family of DC-associated C-typelectins. Very recently, we have identified the seventh member,termed “dectin-2,” which resembles dectin-1 by virtue of themolecular configuration (a type II membrane-integratedpolypeptide with a single CRD in the extracellular domain) andthe expression profile (preferential expression by DC in bothlymphoid and nonlymphoid tissues) (75).

With respect to the function of dectin-1, His-dectin-1 proteinsbound to the surfaces of activated T cells, and immobilizedHis-dectin-1 promoted the proliferation of T cells only in thepresence of anti-CD3 mAb at suboptimal concentrations. Theseobservations suggest that dectin-1 proteins expressed on DCbind to putative ligand(s) on T cells and deliver co-stimulatorysignals. On the other hand, His-dectin-1 (in a soluble form) andanti-dectin-1 Ab both failed to block DC-induced T cell activa-tion in allogeneic mixed leukocyte reactions (data not shown),with the implication that the co-stimulatory property of dec-tin-1 may be replaceable by other molecules expressed on DC.His-dectin-1 did not bind to any of the conventional carbohy-drate probes, and pretreatment of T cells with trypsin, but notwith tunicamycin or N-glycosidase, abrogated their ability tobind His-dectin-1. These observations may imply that dectin-1recognizes rather unique carbohydrate moieties or that dec-tin-1 must form homo- or heterodimers to exert high affinitybinding, as has been reported for other type II lectins withsingle CRD motifs (76, 77). Alternatively, dectin-1 may recog-nize peptide or glycopeptide ligand(s). In fact, it is known thatcarbohydrates do not necessarily serve as natural ligands of allof the molecules that belong structurally to the C-type lectinfamily. For example, CD23, which has been shown to recognizeGal-Gal-NAc under certain experimental conditions (78), bindsenzymatically de-glycosylated IgE and even recombinant (non-glycosylated) IgE produced in E. coli (79). Further studies arerequired to determine the physiological function of dectin-1 andto identify its ligand(s). Nevertheless, we believe that the pres-

ent study has formed both technical and conceptual bases forsuch studies.

Acknowledgments—We thank Drs. Nancy Street and Mansour Mo-hamadzadeh for providing T cell clones and B cell hybridoma clones,respectively. We are also grateful to Pat Adcock for secretarial assist-ance in the preparation of this manuscript.

REFERENCES

1. Banchereau, J., and Steinman, R. M. (1998) Nature 392, 245–2522. Steinman, R. M. (1991) Annu. Rev. Immunol. 9, 271–2963. Bhardwaj, N. (1997) J. Exp. Med. 186, 795–7994. Johnson, L. L., and Sayles, P. C. (1997) J. Exp. Med. 186, 1799–18025. Shurin, M. R. (1996) Cancer Immunol. Immunother. 43, 158–1646. Hart, D. N. J. (1997) Blood 90, 3245–32877. Lanzavecchia, A. (1996) Curr. Opin. Immunol. 8, 348–3548. Fanger, N. A., Wardwell, K., Shen, L., Tedder, T. F., and Guyre, P. M. (1992)

J. Immunol. 157, 541–5489. Maurer, D., Fiebiger, E., Ebner, C., Reiniger, B., Fisher, G. F., Wichlas, S.,

Jouvin, M., Schmitt-Egenolf, M., Kraft, D., Kinet, J., and Stingl, G. (1996)J. Immunol. 157, 607–616

10. Sallusto, F., Cella, M., Danieli, C., and Lanzavecchia, A. (1995) J. Exp. Med.182, 389–400

11. Jiang, W., Swiggard, W. J., Heufler, C., Peng, M., Mirza, A., Steinman, R. M.,and Nussenzweig, M. C. (1995) Nature 375, 151–155

12. Sallusto, F., and Lanzavecchia, A. (1999) J. Exp. Med. 189, 611–61413. Weiss, J. M., Sleeman, J., Renkl, A. C., Dittmar, H., Termeer, C. C., Taxis, S.,

Howells, N., Schopf, E., Ponta, H., Herrlich, P., and Simon, J. C. (1997)J. Cell Biol. 137, 1137–1147

14. Koszik, F., Strunk, D., Simonitsch, I., Picker, L. J., Stingl, G., and Payer, E.(1994) J. Invest. Dermatol. 102, 773–780

15. Robert, C., Fuhlbrigge, R. C., Kieffer, J. D., Ayehunie, S., Hynes, R. O., Cheng,G., von Andrian, U. H., and Kupper, T. S. (1999) J. Exp. Med. 189, 627–635

16. Dieu, M.-C., Vanbervliet, B., Vicari, A., Bridon, J.-M., Oldham, E., Aıt-Yahia,S., Briere, F., Zlotnik, A., Lebecque, S., and Caux, C. (1998) J. Exp. Med.188, 373–386

17. Caux, C., Massacrier, C., Vanbervliet, B., Dubois, B., Van Kooten, C., Durand,I., and Banchereau, J. (1994) J. Exp. Med. 180, 1263–1272

18. Heufler, C., Topar, G., Koch, F., Trockenbacher, B., Kampgen, E., Romani, N.,and Schuler, G. (1992) J. Exp. Med. 176, 1221–1226

19. Mohamadzadeh, M., Poltorak, A. N., Bergstresser, P. R., Beutler, B., andTakashima, A. (1996) J. Immunol. 156, 3102–3106

20. Adema, G. J., Hartgers, F., Verstraten, R., de Vries, E., Marland, G., Menon,S., Foster, J., Xu, Y., Nooyen, P., McClanahan, T., Bacon, K. B., and Figdor,C. G. (1997) Nature 387, 713–717

21. Vicari, A. P., Figueroa, D. J., Hedrick, J. A., Foster, J. S., Singh, K. P., Menon,S., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Bacon, K. B., and Zlotnik,A. (1997) Immunity 7, 291–301

22. Xu, S., Ariizumi, K., Caceres-Dittmar, G., Edelbaum, D., Hashimoto, K.,Bergstresser, P. R., and Takashima, A. (1995) J. Immunol. 154, 2697–2705

23. Takashima, A., Edelbaum, D., Kitajima, T., Shadduck, R. K., Gilmore, G. L.,Xu, S., Taylor, R. S., Bergstresser, P. R., and Ariizumi, K. (1995)J. Immunol. 154, 5128–5135

24. Xu, S., Ariizumi, K., Edelbaum, D., Bergstresser, P. R., and Takashima, A.(1995) Eur. J. Immunol. 25, 1018–1024

25. Kitajima, T., Ariizumi, K., Mohamadzadeh, M., Edelbaum, D., Bergstresser,P. R., and Takashima, A. (1995) J. Immunol. 155, 3794–3800

26. Kitajima, T., Ariizumi, K., Bergstresser, P. R., and Takashima, A. (1995)J. Immunol. 155, 5190–5197

27. Kitajima, T., Ariizumi, K., Bergstresser, P. R., and Takashima, A. (1996)J. Clin. Invest. 98, 142–147

28. Kitajima, T., Ariizumi, K., Bergstresser, P. R., and Takashima, A. (1996)J. Immunol. 157, 3312–3316

29. Yokota, K., Ariizumi, K., Kitajima, T., Bergstresser, P. R., Street, N. E., andTakashima, A. (1996) J. Immunol. 157, 1529–1537

30. Ariizumi, K., Kitajima, T., Bergstresser, P. R., and Takashima, A. (1995) Eur.J. Immunol. 25, 2137–2141

31. Mohamadzadeh, M., Ariizumi, K., Sugamura, K., Bergstresser, P. R., andTakashima, A. (1996) Eur. J. Immunol. 26, 156–160

32. Yuspa, S. H., Hawley-Nelson, P., Koehler, B., and Stanley, J. R. (1980) CancerRes. 40, 4694–4703

33. Schuhmachers, G., Xu, S., Bergstresser, P. R., and Takashima, A. (1995)J. Invest. Dermatol. 105, 225–230

34. Kuziel, W. A., Takashima, A., Bonyhadi, M., Bergstresser, P. R., Allison, J. P.,Tigelaar, R. E., and Tucker, P. W. (1987) Nature 328, 263–266

35. Matsue, H., Bergstresser, P. R., and Takashima, A. (1993) J. Immunol. 151,6012–6019

36. Matsue, H., Edelbaum, D., Hartmann, A. C., Morita, A., Bergstresser, P. R.,Yagita, H., Okumura, K., and Takashima, A. (1999) J. Immunol. 162,5287–5298

37. Rubenstein, J. L. R., Brice, A. E., Ciaranello, R. D., Denney, D., Porteus,M. M. H., and Usdin, T. B. (1990) Nucleic Acids Res. 18, 4833–4842

38. Ariizumi, K., Meng, Y., Bergstresser, P. R., and Takashima, A. (1995)J. Immunol. 154, 6031–6039

39. Orlofsky, A., Berger, M. S., and Prystowsky, M. B. (1991) Cell Regul. 2,403–412

40. Gray, P. W., Glaister, D., Ellson, C., Goeddel, D. V., and Pennica, D. (1986)J. Immunol. 137, 3644–3648

41. Ishidoh, K., Muno. D., Sato, N., and Kominami, E. (1991) J. Biol. Chem. 266,16312–16317

42. Fogel-Petrovic, M., Kramer, D. L., Ganis, B., Casero, R. A., Jr., and Porter,C. W. (1993) Biochim. Biophys. Acta 1216, 255–264

Identification of Dectin-1 by Subtractive Cloning20166

by guest on May 24, 2020

http://ww

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nloaded from

43. Lin, E. Y., Orlofsky, A., Berger, M. S., and Prystowsky, M. B. (1993) J. Immu-nol. 151, 1979–1988

44. Touchot, N., Chardin, P., and Traitian, A. (1987) Proc. Natl. Acad. Sci. U. S. A.84, 8210–8214

45. Sawamura, T., Kume, N., Aoyama, T., Moriwaki, H., Hoshikawa, H., Aiba, Y.,Tanaka, T., Miwa, S., Katsura, Y., Kita, T., and Masaki, T. (1997) Nature386, 73–77

46. Dissen, E., Berg, S. F., Westgaard, I. H., and Fossum, S. (1997) Eur.J. Immunol. 27, 2080–2086

47. Lazetic, S., Chang, C., Houchins, J. P., Lanier, L. L., and Phillips, J. H. (1997)J. Immunol. 157, 4741–4745

48. Hamann, J., Fiebig, H., and Strauss, M. (1993) J. Immunol. 150, 4920–492749. Spiess, M., Schwartz, A. L., and Lodish, H. F. (1985) J. Biol. Chem. 260,

1979–198250. McPhaul, M., and Berg, P. (1987) Mol. Cell. Biol. 7, 1841–184751. Kondo, H., Ichikawa, Y., Nakamura, K., and Tsuchiya, S. (1994) Int. Arch.

Allergy Immunol. 105, 38–4852. Spiess, M. (1990) Biochemistry 29, 10009–1001853. Reth, M. (1989) Nature 338, 383–38454. Vivier, E., and Daeron, M. (1997) Immunol. Today 18, 286–29155. Matsue, H., Cruz, P. D. Jr., Bergstresser, P. R., and Takashima, A. (1992)

J. Invest. Dermatol. 99, 537–54156. Enk, A. H., and Katz, S. I. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1398–140257. Caux, C., Dezutter-Dambuyant, C., Schmitt, D., and Banchereau, J. (1992)

Nature 360, 258–26158. Sallusto, F., and Lanzavecchia, A. (1994) J. Exp. Med. 179, 1109–111859. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S.,

Muramatsu, S., and Steinman, R. M. (1992) J. Exp. Med. 176, 1693–170260. Paglia, P., Girolomoni, G., Robbiati, F., Granucci, F., and Ricciardi-Castagnoli,

P. (1993) J. Exp. Med. 178, 1893–190161. Granucci, F., Girolomoni, G., Lutz, M. B., Foti, M., Marconi, G., Gnocchi, P.,

Nolli, L., and Ricciardi-Castagnoli, P. (1994) Eur. J. Immunol. 24,2522–2526

62. Girolomoni, G., Lutz, M. B., Pastore, S., Abmann, C. U., Cavani, A., andRicciardi-Castagnoli, P. (1995) Eur. J. Immunol. 25, 2163–2169

63. Elbe, A., Schleischitz, S., Strunk, D., and Stingl, G. (1994) J. Immunol. 153,2878–2889

64. Timares, L., Takashima, A., and Johnston, S. A. (1998) Proc. Natl. Acad. Sci.U. S. A. 95, 13147–13152

65. Winzler, C., Rovere, P., Rescigno, M., Granucci, F., Penna, G., Adorini, L.,Zimmermann, V., Davoust, J., and Ricciardi-Castagnoli, P. (1997) J. Exp.Med. 185, 317–328

66. Ross, R., Endlich, A., Kumpf, K., Schwing, J., and Reske-Kunz, A. B. (1998)J. Invest. Dermatol. 110, 57–61

67. Ross, R., Ross, X.-L., Schwing, J., Langin, T., and Reske-Kunz, A. B. (1998)J. Immunol. 160, 3776–3782

68. Bates, E. E. M., Fournier, N., Garcia, E., Valladeau, J., Durand, I., Pin, J. J.,Zurawski, S. M., Patel, S., Abrams, J. S., Lebecque, S., Garrone, P., andSaeland, S. (1999) J. Immunol. 163, 1973–1983

69. Inaba, K., Swiggard, W. J., Inaba, M., Meltzer, J., Mirza, A., Sasagawa, T.,Nussenzweig, M. C., and Steinman, R. M. (1995) Cell Immunol. 163,148–156

70. Vremec, D., and Shortman, K. (1997) J. Immunol. 159, 565–57371. Bieber, T., Rieger, A., Neuchrist, C., Prinz, J. C., Rieber, E. P., Boltz-Nitulescu,

G., Scheiner, O., Kraft, D., Ring, J., and Stingl, G. (1989) J. Exp. Med. 170,309–314

72. Bieber, T., Rieger, A., Stingl, G., Sander, E., Wanek, P., and Strobel, I. (1992)J. Invest. Dermatol. 98, 771–776

73. Sato, M., Kawakami, K., Osawa, T., and Toyoshima, S. (1992) J. Biochem.(Tokyo) 111, 331–336

74. Ying, H., Nakayama, E., Robinson, W. H., and Parnes, J. R. (1995) J. Immunol.154, 2743–2752

75. Ariizumi, K., Shen, G.-L., Shikano, S., Ritter, R., III, Zukas, P., Edelbaum, D.,Morita, A., and Takashima, A. (2000) J. Biol. Chem. 275, 11957–11963

76. Rice, K. G., Weisz, O., Barthel, T., Lee, R. T., and Lee, Y. C. (1990) J. Biol.Chem. 265, 18429–18434

77. Loeb, J. A., and Drickamer, K. (1988) J. Biol. Chem. 263, 9752–976078. Kijimoto-Ochiai, S., Horimoto, E., and Uede, T. (1994) Immunol. Let. 40, 49–5379. Vercelli, D., Helm, B., Marsh, P., Padlan, E., Geha, R. S., and Gould, H. (1989)

Nature 338, 649–651

Identification of Dectin-1 by Subtractive Cloning 20167

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Kumamoto, Dale Edelbaum, Akimichi Morita, Paul R. Bergstresser and Akira TakashimaKiyoshi Ariizumi, Guo-Liang Shen, Sojin Shikano, Shan Xu, Robert Ritter III, Tadashi

Subtractive cDNA CloningIdentification of a Novel, Dendritic Cell-associated Molecule, Dectin-1, by

doi: 10.1074/jbc.M909512199 originally published online April 21, 20002000, 275:20157-20167.J. Biol. Chem. 

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