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J. Biol. Chem., Vol. 275, Issue 26, 20157-20167, June 30, 2000

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

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

Received for publication, November 24, 1999, and in revised form, April 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It has been an established concept that immunologically naive T cells can be activated most efficiently or even exclusively by special subsets of antigen-presenting cells, termed dendritic 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 pathogens, and tumor-associated antigens (2-5). Members of the DC family have been identified in many organs, including (a) spleen (splenic DC), thymus (thymic DC), lymph nodes (interdigitating cells), and tonsils (tonsil DC); (b) epidermis (Langerhans cells) and mucosal surfaces of the oral cavity, intestinal tract, and respiratory tract; (c) dermis (dermal DC) and other connective tissues (interstitial DC), d) peripheral blood (blood DC); and (e) afferent lymphatics (veiled cells) (6).

DC are characterized morphologically by the extension of long, lamellar dendrites. Upon activation with proinflammatory stimuli, DC acquire surface expression of relatively large amounts of major histocompatibility complex class I and class II 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 allow DC to establish intimate, antigen-specific interaction with T cells. DC are also capable of incorporating exogenous antigens efficiently by phagocytosis, endocytosis, and pinocytosis (7), and surface expression of IgG and IgE receptors and carbohydrate receptors appears to contribute to this function (8-11). DC are highly mobile leukocytes, migrating across different tissues via blood or lymphatic vessels (12). This mobility is mediated in part by the expression of homing receptors (e.g. CD44, CD62P ligand, and cutaneous lymphocyte-associated antigen) and chemokine receptors (e.g. CXC chemokine receptor-1, -2, and -3 and CC chemokine receptor-1 through -7) (6, 13-16). Finally, DC elaborate a wide variety of cytokines (interleukin (IL)-1, IL-6, IL-12, and tumor necrosis factor-) and chemokines (IL-8, macrophage inflammatory protein-1 and -1, DC-chemokine-1, and thymus-expressed chemokine) (17-21), thereby regulating the magnitude and direction of T cell activation.

The ultimate goal in our laboratories has been to understand the biology of DC at a molecular level. As an initial step, we established a stable, long term DC line from the epidermis of newborn BALB/c mice. This DC line, termed "XS52," retains important 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 profiles (19, 22-31). Taking the advantage of having relatively large numbers of a pure DC population (with cell doubling time of 18-24 h), we chose to employ the subtractive cloning strategy for the identification of genes that are expressed preferentially by a DC line. Here we report the results of this molecular approach, focusing on the characterization of one of the novel genes identified by the subtraction.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals-- Female BALB/c mice (6-10 weeks old) and New Zealand White rabbits (4-20 weeks old) were housed in the pathogen-free facility of the Animal Resource Center at the University of Texas Southwestern Medical Center. All of the experiments were conducted according to the guidelines of the National Institutes of Health.

Cell Lines-- XS52 cells are a long term DC line established from the epidermis of BALB/c mice (22). This line was maintained and expanded in complete RPMI 1640 supplemented with mouse recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) (1 ng/ml) and NS47 fibroblast culture supernatant (10% v/v) as a source of CSF-1 (22, 23). The phenotypic and functional features of this DC line have been described elsewhere (19, 22-31). The J774 cells are a long term macrophage line established from BALB/c mice; this line was purchased from the American Tissue Type Collection (ATCC, Manassas, VA) and maintained 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 T cell line (34, 35), Raw macrophage line (ATCC), HDK-1 CD4⁺ Th1 clone and D10 CD4⁺ Th2 line (kindly provided by Dr. Nancy Street, University of Texas Southwestern Medical Center), 5C5 and 2G9 B cell hybridoma clones (provided by Dr. Mansour Mohamadzadeh, University of Texas Southwestern Medical Center), and COS-1 line (ATCC).

Cell Isolation-- Epidermal cells were isolated from abdominal skin of BALB/c mice using two sequential trypsin treatments and then enriched for Langerhans cells by centrifugation over Histopaque (1.083; Sigma) as before (30, 36). In some experiments, the epidermal cells harvested from the medium/Histopaque interface (interface epidermal cells) were depleted of Langerhans cells by anti-Ia mAb plus complement treatment as before (30). Splenic DC were isolated as described previously (36). Briefly, spleen cell suspensions were prepared by mechanical dissociation, followed by collagenase treatment (1% collagenase, 1 h, 37 °C). After lysis of erythrocytes, splenic cells were subjected to gradient centrifugation with Percoll (Amersham Pharmacia Biotech); cells collected from the interface between 1.035 and 1.075 g/ml Percoll were then incubated on tissue culture plates. After a 90-min incubation, nonadherent cells were removed by extensive pipetting, and the adherent cells were cultured overnight. Cells released during the second culture period were harvested and used as splenic DC preparations.

Identification of Dectin-1 cDNA Clone by the Subtractive Cloning Strategy-- A subtractive cDNA library was constructed using the methods reported by Rubenstein et al. (37). Briefly, poly(A)⁺ RNAs isolated from the XS52 DC line were reverse-transcribed into cDNAs, ligated unidirectionally to the  ZapII phages (Stratagene, La Jolla, CA), and then converted into a single-stranded phagemid library in which cDNAs were synthesized as an antisense strand (pBluescript II SK(), Strategene). This cDNA library (1.2 µg) was hybridized with 50 µg of biotinylated poly(A)⁺ RNA isolated from the J774 macrophage line. Unhybridized cDNAs were purified by the streptavidin-phenol extraction method and converted into a double-stranded form by Taq DNA polymerase. Subsequently, the resulting "DC-specific" cDNA library was screened sequentially by differential colony hybridization, slot-blotting, and Northern blotting.

In colony hybridization, colonies were transferred onto nylon membranes and hybridized differentially with total cDNA probes prepared from XS52 DC-derived poly(A)⁺ RNAs and from J774 macrophage-derived poly(A)⁺ RNAs. In slot blotting, the cDNA inserts were polymerase chain reaction-amplified, slot-blotted onto nylon membranes, and hybridized with total cDNA probes from XS52 DC and from J774 macrophages. In both screening steps, we isolated only those clones that were hybridized strongly with XS52 DC-derived total cDNA probes but 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 µg/lane) isolated from XS52 DC or J774 macrophages by using RNA-STAT60 (Tel-TestB; Friendswood, TX) were size-fractionated on a vertical agarose gel, transferred onto a nylon membrane, and hybridized with the above ³²P-labeled probes. Once again, we selected those cDNA clones that showed strong hybridization only with the XS52 DC-derived probes. Finally, 50 cDNA clones selected by Northern blotting were sequenced and subjected to homology search using the GenBank^TM and EMBL data bases.

Tissue and Cell Distributions of Dectin-1 mRNA Expression-- Total RNAs were isolated from several different lines or from different mouse organs. In some experiments, XS52 DC and J774 macrophages were cultured for 48 h in the presence or absence of 10 ng/ml of either GM-CSF or CSF-1 before RNA isolation. Northern blotting was carried out as described above using ³²P-labeled dectin-1 cDNA probe (clone 1C11-5) or the glyceraldehyde-3-phosphate dehydrogenase control probe (38). Reverse transcription-polymerase chain reaction was carried out as described previously (30, 38). Briefly, total RNA isolated from the interface epidermal cells was reverse-transcribed into cDNA and then PCR-amplified with the following primer sets: 5'-AGGCCCTATGAAGAACTACAGACA-3' (nt 1384-1407) and 5'-TGGCCAGGACAGCATAAGGAA-3' (nt 1830-1811) for dectin-1; 5'-TACAGGCTCCGAGATGAACAACAA-3' (nt 450-473) and 5'-TGGGGAAGGCATTAGAAACAGTC-3' (nt 899-921) for IL-1, or 5'-GTGGGCCGCTCTAGGCACCAA-3' (nt 25-45) and 5'-CTCTTTGATGTCACGCACGATTTC-3' (nt 541-564) for -actin. The PCR products were separated on agarose gel, transferred onto a membrane, and hybridized with ³²P-labeled dectin-1, IL-1, or -actin cDNA probe.

Preparation of His-Dectin-1 Fusion Proteins-- His-dectin-1 fusion protein consisting of, from the N terminus, hexahistidine and the extracellular domain of dectin-1 was produced in Escherichia coli as follows. The DNA fragment encoding an extracellular domain (aa 73-244) of dectin-1 was PCR-amplified, and BamHI and SmaI sites were then attached to the resulting DNA fragment at the 5'- and 3'-end, respectively. Using BamHI and SmaI sites, the fragment was ligated to immediately downstream of the hexahistidine sequence in pQE-30 vector (Qiagen, Chatsworth, CA). This recombinant vector was introduced into E. coli; His-dectin-1 protein was extracted from isopropyl-1-thio--D-galactopyranoside-treated E. coli in 8 M urea, 100 mM sodium phosphate, 10 mM Tris/HCl (pH 8.0) buffer and purified using Ni²⁺-nitrilotriacetic acid resin (Qiagen). Approximately 5-8 mg of proteins were recovered 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, pH 7.4), relatively small fractions (4-20%) of the His-dectin-1 proteins were recovered in a soluble form, and this fraction was used in the functional studies (see below). As a control protein with a His tag, His-dihydrofolate reductase (DHFR) was produced in E. coli transformed 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 Biochemicals) with pSecTag vector (Invitrogen, Carlsbad, CA) that contained the DNA insert encoding, from the N terminus, the Ig leader sequence, hexahistidine sequence, TEV site, and extracellular domain (aa 73-244) of dectin-1. Culture supernatants collected at 48-60 h after transfection were purified using the Ni²⁺-nitrilotriacetic acid resin.

Immunoblotting and Flow Cytometric Analyses-- A synthetic 21-aa peptide containing a cysteine residue at the N terminus of the 20-aa sequence GRNPEEKDNFLSRNKENHKP corresponding to aa 75-94 of dectin-1 was used to immunize rabbits. After three rounds of standard immunization, serum was collected and subjected to affinity purification of peptide-specific Ab using the same 21-mer peptide. Briefly, high pressure liquid chromatography-purified peptide was conjugated to the thiol 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-conjugated thiol coupling gel, and peptide-specific antibodies were then eluted, after extensive washing with the phosphate buffer, with 100 mM glycine-HCl (pH 2.5). After neutralization, the fractions showing significant A₂₈₀ values were dialyzed against buffer B (10 mM NaH₂PO₄, 20 mM NaCl, pH 7.0).

XS52 DC were homogenized in 10 mM HEPES (pH 7.3) with 5-10 strokes with a 27-gauge needle on a 1-ml syringe and centrifuged for 10 min at 1,000 × g, and the resulting supernatants ("crude lysates") were fractionated into cytosolic and membrane fractions by centrifugation for 40 min at 100,000 × g. In some experiments, whole cell extracts were prepared from several different cell lines in 0.3% Triton X-100 in PBS, followed by centrifugation for 10 min at 1,000 × g. The full-length cDNA for dectin-1 was excised from clone 1C11-5 by digestion with EcoRI and XbaI restriction enzymes and inserted into a mammalian expression vector pZeoSV₂(+) (Invitrogen). COS-1 cells were transfected with the resulting vector or an empty vector alone using FuGene 6, and membrane fractions were prepared at 72 h after transfection. These samples were separated by 10-20% or 4-20% SDS-PAGE, transferred onto polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA), and then blotted with 0.72 µg/ml of affinity-purified rabbit anti-dectin-1 or control rabbit IgG. After extensive wash, the membrane was blotted with horseradish peroxidase-conjugated anti-rabbit IgG (Zymed Laboratories Inc., San Francisco, CA) and then developed with the ECL system (Amersham Pharmacia Biotech).

Different cell lines were fixed with 2% paraformaldehyde in PBS and then incubated with 1 µg/ml of anti-dectin-1 or control rabbit IgG, followed by incubation with FITC-conjugated anti-rabbit IgG. Splenic DC preparations were subjected to double staining with anti-dectin-1 followed by phosphatidylethanolamine-conjugated secondary Ab and with FITC-conjugated anti-Ia or anti-CD11c mAb (Pharmingen, San Diego, CA). These samples were analyzed by FACScan (Becton-Dickinson, Mountain View, CA) as before (22).

His-Dectin-1 Binding Assays-- Binding properties of His-dectin-1 were examined in binding buffer (Hank's balanced salt solution containing 1.3 mM CaCl₂, 3% fetal calf serum, and 10 mM HEPES) by using four different protocols. First, soluble fractions of bacterially produced His-dectin-1 proteins were labeled with biotin (Pierce) and tested for the binding to selected cell lines (2 × 10⁶ cells/ml). Following a 30-min incubation on ice with 5 µg/ml of biotinylated His-dectin-1, the cells were washed and incubated with FITC-conjugated streptavidin at 1:100 dilution (Jackson Immunoresearch Laboratories, West Grove, PA). Samples were then analyzed by FACScan. In some experiments, the activated D10 T cells were cultured for 16 h in the presence of 4 µg/ml tunicamycin (Sigma) before analyses, or they were incubated with 0.25% trypsin in PBS for 20 min at 37 °C. For the N-glycosidase treatment, the cells were first fixed with 0.4% paraformaldehyde in PBS for 3 h at 4 °C, washed with PBS, and then incubated with 600 milliunits/ml neuraminidase (Roche Molecular Biochemicals) for 3 h at 37 °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 Molecular Biochemicals) in the same buffer for 16 h at 37 °C. To determine the extent of deglycosylation achieved by the above treatment with tunicamycin or N-glycosidase, the same T cell samples were tested for their ability to bind biotinylated phytohemagglutinin and biotinylated wheat germ agglutinin (both purchased from Sigma).

Second, soluble fractions of bacterially produced His-dectin-1 proteins (3 µg in 100 µl of 100 mM phosphate buffer, pH 6.5) were labeled with 100 µCi of ¹²⁵I (ICN, Costa Mesa, CA) in the presence of an IODO-BEAD (Pierce). After a 10-min incubation at room temperature, the reaction was stopped by the removal of the bead and the addition of 100 µl of 3% bovine serum albumin. The ¹²⁵I-labeled His-dectin-1 (0.7 µg/ml, specific activity: 5 × 10⁶ cpm/µg) was then incubated with the activated D10 T cells (1 × 10⁷ cells/ml). After a 90-min incubation on ice, the cell suspensions were overlaid on the top of 700 µl of 100% fetal calf serum and centrifuged at 600 × g for 1 min to remove the unbound probes. The cellular pellets were washed three more times in PBS and then counted for radioactivities. To test the specificity, the incubation was carried out in the presence of "cold" His-dectin-1 (76 µg/ml) or anti-dectin-1 (50 µg/ml); His-DHFR or control IgG at the same concentrations were added to serve as controls.

Third, His-dectin-1 proteins produced by COS-1 cells were biotinylated, incubated at 5 µg/ml with the activated D10 T cells (2 × 10⁶ cells/ml), and then analyzed by FACScan as described above. To test the specificity, nonlabeled, bacterially produced His-dectin-1 or His-DHFR (160 µg/ml) was added to the incubation. Finally, ¹²⁵I-labeled, bacterially produced His-dectin-1 proteins were incubated for 120 min on ice with agarose beads that were conjugated with mannose, fucose, lactose, GluNAc, or GalNAc (all purchased from Sigma). Specific binding was then examined by counting the radioactivities that were eluted by the addition of the corresponding carbohydrates.

T Cell Co-stimulation Assay-- ELISA plates were coated with graded concentrations of anti-CD3 mAb (Pharmingen) together with bacterially produced His-dectin-1 (10 µg/ml). In some experiments, enzyme-linked immunosorbent assay plates coated with anti-CD3 mAb (0.3 µg/ml) and His-dectin-1 (10 µg/ml) were preincubated with graded concentrations of anti-dectin-1 or control IgG. Splenic T cells purified from 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 as before (22).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of Novel Genes Expressed by XS52 DC Line by Using the Subtractive Cloning Strategy-- Based on the hypothesis that one or more specific genes are expressed by DC, but not by other antigen-presenting cells (including macrophages), we hybridized the XS52 DC-derived cDNA library with excess amounts of biotinylated mRNAs isolated from the J774 macrophage line and isolated only the unhybridized cDNA clones. About 99% of the starting cDNA clones were eliminated by this procedure, estimated by counting the colony-forming units before and after subtraction (Table I). Subsequently, we constructed the "DC-specific" cDNA library from the unhybridized clones and tested this library by three rounds of screening. Of the 12,000 independent colonies analyzed first by colony hybridization, 226 colonies showed strong hybridization with the total cDNA probes from XS52 DC, but not with the probes from J774 macrophages. In other words, the overwhelming majority of the colonies failed to show any detectable hybridization with the DC probes (about 40-50% of the colonies), or they showed comparable levels of hybridization with the DC probes and the macrophage probes. We next tested these 226 clones in slot blotting and selected 140 clones that showed preferential hybridization with the DC probes. Finally, these 140 clones were examined for their expression levels in XS52 DC versus J774 macrophages by Northern blotting; we were able to confirm DC-specific expression for 50 of these clones (Table I).

Through partial sequencing and cross-hybridization, we found that the above 50 clones contained 11 distinct genes. A homology search of their nucleotide sequences revealed six genes that encoded currently recognized polypeptides, including C10 (a -chemokine) (39), IL-1 (40), cathepsin C (a cysteine 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 mouse equivalent of rat Rab-2 (a Ras-related protein) (44). The remaining five genes were judged to be distinct from any nucleotides currently registered in the GenBank^TM or EMBL data bank. We focused our subsequent effort on one of these novel genes.

Structural Features of Dectin-1 Polypeptide-- Clone 1C11-5 contained 735 nucleotides (nt) in its open reading frame, and it showed DC-specific hybridization in colony hybridization, slot blotting, and Northern blotting. As shown in Fig. 1A, the deduced amino acid sequence of this clone revealed a polypeptide of 244 aa with type II configuration, consisting of a cytoplasmic domain (aa 1-44), a putative transmembrane domain (aa 45-68), and extracellular domains (aa 69-244). The overall amino acid sequence of this polypeptide showed significant homology with several polypeptides, including (a) endothelial receptor (LOX-1) for oxidated low density lipoprotein (26.6% similarity by Clustral method analyzed with Lasergene Program; DNA Star, Madison, WI) (45), (b) a natural killer cell receptor CD94 (21.8%) (46, 47), (c) another receptor CD69 encoded in the natural killer gene complex (17.8%) (48), (d) asialoglycoprotein receptors, i.e. hepatic lectin-1 (18.9%) and hepatic lectin-2 (15.6%) (49, 50), and (e) low affinity Fc receptor CD23 (15.6%) (51). All of these molecules contain carbohydrate recognition domain (CRD) motifs and, thus, belong structurally to the family of C-type (Ca²⁺-dependent) lectins. Spiess has identified 13 invariant amino acid residues (including six cysteines playing a critical role in forming disulfide bridge frameworks) that are relatively conserved among CRD sequences in different C-type lectins (52). We identified a putative CRD motif at the COOH-terminal region (aa 119-244) of the 1C11-5 polypeptide sequence, and this motif contained 11 out of the 13 invariant residues, including all six cysteine residues (as indicated with asterisks in Fig. 1A). Moreover, this CRD motif showed relatively high degrees of homology (22.0-38.4% identities) with the CRD sequences found in other C-type lectins (Fig. 1B). Thus, we concluded that clone 1C11-5 encoded a unique polypeptide that belonged structurally to the C-type lectin family, and this polypeptide was designated as "DC-associated C-type lectin-1" or "dectin-1." It is also important to note that a putative immunoreceptor tyrosine-based activation motif (ITAM) (YXXL) (53, 54) was found in the cytoplasmic domain of dectin-1 (indicated with an underline in Fig. 1A).

View larger version (67K): [in this window] [in a new window]   Fig. 1.   Deduced amino acid sequence of dectin-1 and its homology with members of the C-type lectin family. A, deduced amino acid sequence of dectin-1 is shown, segmented into a cytoplasmic domain, a transmembrane domain, and extracellular domains containing a putative CRD motif at the COOH-terminal end. Asterisks and triangles indicate the invariant residues of C-type lectins and putative N-glycosylation sites, respectively. B, a putative CRD sequence of dectin-1 was aligned with the CRD sequences in other C-type lectins in mice (m), rats (r), and humans (h).

Cell and Tissue Distributions of Dectin-1 mRNA-- Northern blotting showed that dectin-1 mRNA (about 3.2 kilobases in size) was expressed at relatively high levels by XS52 DC, whereas it was detected at only negligible levels in J774 macrophages (Fig. 2A). Most importantly, dectin-1 mRNA was totally undetectable in other tested cell lines, including an additional macrophage line (Raw), a T cell line (7-17), two T cell lines (HDK-1 and D10), a B cell hybridoma clone (5C5), a keratinocyte line (Pam 212), and a fibroblast line (NS01). Because the XS52 DC line, but not other cell lines, had been expanded in the presence of added GM-CSF and CSF-1 (22, 23), we considered that dectin-1 mRNA expression might be simply induced by either of these growth factors. As shown in Fig. 2B, expression patterns of dectin-1 mRNA remained unchanged in both XS52 cells and J774 cells, regardless of the presence or absence of GM-CSF or CSF-1 in culture medium, thus excluding the possibility that dectin-1 expression was regulated by the added growth factors.

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Cell- and tissue-specific expression of dectin-1. Total RNAs were isolated from XS52 DC, J774 and Raw macrophages, 7-17 dendritic epidermal T cells, HDK-1 Th1 cells, D10 Th2 cells, 5C5 B cell hybridoma, Pam 212 keratinocytes, and NS01 fibroblasts (A) or from the indicated tissues in adult BALB/c mice (C). B, XS52 cells and J774 cells were cultured for 48 h in the presence or absence of GM-CSF (10 ng/ml) or CSF-1 (10 ng/ml) before isolation of RNA. The RNA samples were then examined by Northern blotting for dectin-1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). D, interface epidermal cells isolated from BALB/c mice were examined for dectin-1 mRNA expression by reverse transcriptase-PCR. Some samples were treated with anti-Ia mAb plus complement to deplete Langerhans cells; the extent of depletion was assessed by measuring IL-1 mRNA, which is known to be expressed exclusively by Langerhans cells within murine epidermal cells.

As noted in Fig. 2C, abundant expression of dectin-1 mRNA was detected in spleen and thymus, the tissues known to contain relatively large numbers of DC (1, 6). Unexpectedly, Northern blotting failed to reveal dectin-1 mRNA expression in skin, the tissue from which the XS52 DC line had been established (22). This did not mean, however, that dectin-1 mRNA was absent from this tissue, because a strong PCR signal was detected in epidermal cells freshly isolated from BALB/c mice (Fig. 2D). With respect to the source of dectin-1 mRNA expression, depletion of the Ia⁺ epidermal cell population (i.e. Langerhans cells) abrogated almost completely dectin-1 mRNA, as well as IL-1 mRNA, which is known to be expressed exclusively by Langerhans cells in murine epidermis (18, 55, 56). This corroborates our observations that dectin-1 mRNA was detected by Northern blotting in the XS52 Langerhans cell-like line, but not in cell lines derived from other epidermal cell populations, i.e. the Pam 212 keratinocyte line and the 7-17 epidermal T cell line (Fig. 2A). These results suggest that dectin-1 mRNA is expressed constitutively and preferentially by Langerhans cells in the epidermis.

Identification of Dectin-1 Protein-- To study dectin-1 protein expression, we produced rabbit Ab against a synthetic peptide corresponding to aa 75-94 of dectin-1 and purified them by affinity chromatography. The resulting anti-dectin-1 Ab recognized in immunoblotting a bacterially produced recombinant fusion protein of about 27 kDa consisting of a hexahistidine tag and an extracellular domain sequence (aa 73-244) of dectin-1 (Fig. 3). The same Ab also immunolabeled a major band of 43 kDa in crude lysates of XS52 DC. By contrast, control rabbit IgG failed to label any specific bands. When XS52 cell lysates were fractionated into a membrane fraction and a cytosolic fraction, the 43-kDa immunoreactivity was detected primarily in the membrane fraction (Fig. 4), in accordance with the predicted molecular structure. The above molecular size of dectin-1 protein detected in immunoblotting was considerably larger than the size predicted from the full-length amino acid sequence (28 kDa). This discordance most likely resulted from glycosylation, because N-glycosidase treatment of XS52 DC lysates reduced substantially the molecular size of dectin-1 immunoreactivity (data not shown) and because dectin-1 contains two putative N-glycosylation sites at aa 185 and 233 (as indicated with triangles in Fig. 1A). Thus, dectin-1 is expressed by XS52 DC as a 43-kDa membrane-associated glycoprotein.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Identification of dectin-1 protein. Whole cell extracts prepared 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) were examined for the immunoreactivity to anti-His-dectin-1 Ab (top panel) or control IgG (middle panel). The same samples were also stained with Coomassie Blue (bottom panel).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Membrane association of dectin-1 protein. Crude lysates prepared mechanically from XS52 DC were centrifuged at 100,000 × g for 40 min, and the pellet (membrane fraction) and the supernatant (cytosolic fraction) were examined by immunoblotting with anti-dectin-1 or control IgG. The data shown are representative of three independent experiments.

To determine the extent to which dectin-1 protein expression occurred in a DC-specific manner, we tested whole cell extracts prepared 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 undetectable in extracts from other tested cell lines, including J774 macrophage, HDK-1 T cell, 7-17 epidermal T cell, 2G9 B cell hybridoma, Pam 212 keratinocyte, and NS47 fibroblast lines. Dectin-1 protein with the same molecular weight was also detected in a second DC line (XS106) derived from A/J mice (data not shown). These results, together with our observation with Northern blotting, document that dectin-1 mRNA and protein are expressed selectively by DC lines.

View larger version (58K): [in this window] [in a new window]   Fig. 5.   Cell type specificity of dectin-1 protein expression. Whole cell extracts were prepared from the indicated cell lines in the cell lysis buffer containing Triton X-100. These samples were then examined by immunoblotting for dectin-1 immunoreactivity (top) and by Coomassie Blue staining for protein profiles (bottom). The data shown are representative of two independent experiments.

As shown in Fig. 6, a 43-kDa immunoreactivity was detected in the membrane fraction prepared from COS-1 cells that had been transfected with dectin-1 cDNA (clone 1C11-5), and this band closely corresponded in molecular size to the natural dectin-1 protein produced by XS52 DC. No immunoreactivity was detected after transfection with the vector alone. An additional band of about 40 kDa was also detected only in the COS-1 membrane fraction; the molecular identity of this second band remains unclear at present. In flow cytometric analyses, anti-dectin-1 Ab recognized dectin-1 proteins on the surface of paraformaldehyde-fixed XS52 cells (Fig. 7A, left panel). A similar staining profile was also observed in the absence of fixation (data not shown). Consistent with our observations in immunoblotting, no significant staining was observed with anti-dectin-1 Ab for NS47 fibroblasts (Fig. 7A, right panel) or other cell lines, including macrophage, B cell, T cell, and keratinocyte lines (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Production of dectin-1 protein by introducing the dectin-1 cDNA clone. COS-1 cells were transfected with pZeoSV-Dec1KZ vector encoding the full-length dectin-1 polypeptide (derived from clone 1C11-5) or pZeoSV2(+) control vector. Membrane fractions prepared from these transfectants or from XS52 DC were examined by immunoblotting. The data shown are representative of three independent experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 7.   Surface expression of dectin-1 on DC. A, XS52 DC and NS47 fibroblasts were fixed with paraformaldehyde and incubated with anti-dectin-1 Ab (closed histograms) or control IgG (open histograms), followed by FITC-conjugated, anti-rabbit IgG. B, splenic DC preparations isolated 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-Ia mAb (left panel) or anti-CD11c mAb (right panel). Data shown are the histograms for dectin-1 staining within the Iahigh or CD11c+ 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 three independent experiments.

Because dectin-1 mRNA was expressed most abundantly in spleen, we next examined dectin-1 protein expression in this tissue. Splenic DC preparations isolated by our standard protocol contained routinely 40-70% DC, as assessed by morphology and surface expression of Ia (major histocompatibility complex class II) molecules and CD11c (36). As shown in Fig. 7B, dectin-1 expression was detected in some, but not all, cells in this preparation, and a majority of the cells expressing dectin-1 co-expressed Ia molecules at relatively high levels. On the other hand, when dectin-1 expression was analyzed in the gated population of Ia^high cells, it became clear that dectin-1 protein was not expressed by all Ia^high cells (Fig. 7C, left panel). The percentage of dectin-1⁺ cells in the Ia^high population varied from 25 to 32% in four independent experiments. Likewise, dectin-1 expression was detected in 20-42% of the CD11c⁺ population (Fig. 7C, right panel). These observations suggested that dectin-1 protein was expressed on the surface of some, but not all, splenic DC. It is to be noted that dectin-1 expression was also detectable in minor fractions of the Ia population as well as the CD11c population, with the implication that expression of dectin-1 does not occur exclusively within the DC populations in living animals. Considering the data together, it is reasonable to conclude that dectin-1 is a unique polypeptide that is characterized by the inclusion of a CRD motif and by its preferential expression by some DC populations in both lymphoid and nonlymphoid tissues.

Functional Potential of Dectin-1-- As an initial step to study the function, we tested whether His-dectin-1 fusion protein would bind to any cell types. As shown in Fig. 8A, His-dectin-1 proteins produced in E. coli migrated as a 27-kDa band, closely corresponding to the predicted molecular size of 21 kDa. Recombinant proteins produced and secreted by COS-1 cells, however, showed significantly higher molecular masses (37 and 39 kDa) than the predicted size (25 kDa), consistent with our hypothesis that dectin-1 is a heavily glycosylated polypeptide. On the other hand, it remains unclear whether the 37 and 39 bands differ each other only in the extent of glycosylation. Soluble fractions of the bacterially produced His-dectin-1 were labeled with biotin and tested for binding. Among several tested cell lines, only the D10 T cell line showed modest, but significant, binding (Fig. 8B). When tested after stimulation with concanavalin A, D10 T cells showed markedly elevated binding of His-dectin-1. Likewise, two additional T cell lines (HDK-1 and 7-17) also showed significant binding of His-dectin-1 only in the activated states. Binding of His-dectin-1 to the activated T cells was also confirmed by using FITC-conjugated mAb against the His tag (data not shown). ¹²⁵I-Labeled His-dectin-1 (produced in E. coli) bound significantly to the surfaces of activated D10 T cells. Importantly, this binding was competed with "cold" His-dectin-1, but not with His-DHFR, and it was inhibited by anti-dectin-1 Ab, but not by control Ab, thus documenting the specificity (Fig. 8C). Moreover, the glycosylated His-dectin-1 proteins (produced in COS-1 cells) also bound to the activated D10 T cells in a manner that was inhibitable by bacterially produced His-dectin-1 but not by His-DHFR (Fig. 8D). We noticed that the recombinant proteins produced in COS-1 cells were less efficient than those produced in E. coli in their T cell binding ability; mechanisms for this functional disparity remain to be determined. Nevertheless, these results indicate that extracellular domains of dectin-1 bind specifically to one or more putative molecules that are expressed on activated T cells.

View larger version (43K): [in this window] [in a new window]   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 using Ni2+-NTA resin and then examined by immunoblotting with anti-dectin-1 Ab. B, a soluble fraction of the bacterially produced His-dectin-1 was biotinylated and examined for the binding to the indicated cell lines. The three T cell lines were tested for His-dectin-1 binding before (resting) and after concanavalin A stimulation (activated). The binding of biotinylated His-dectin-1 (closed histograms) or biotinylated His-DHFR control (open histograms) was examined with FITC-conjugated streptavidin. Data shown are representative of three different experiments. C, a soluble fraction of 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 or His-DHFR (upper panels) or anti-dectin-1 or control IgG (lower panels). Data shown are representative of three independent experiments, showing the mean ± S.D. from triplicate samples. D, activated D10 T cells were incubated with biotinylated His-dectin-1 (produced in COS-1 cells) in the presence of no competitor (open histogram), nonlabeled bacterially produced His-dectin-1 (closed histogram), or His-DHFR (dotted line).

An important question then concerned whether His-dectin-1 fusion proteins containing a CRD sequence would recognize carbohydrate moieties. ¹²⁵I-Labeled His-dectin-1 failed to show specific binding to any of the tested carbohydrate probes, i.e. agarose beads conjugated with mannose, fucose, lactose, GluNAc, or GalNAc (data not shown). Furthermore, pretreatment of T cells with tunicamycin, which diminished substantially the binding of a bona fide lectin, phytohemagglutinin, had only marginal effects on the binding of His-dectin-1 (Fig. 9A). Likewise, N-glycosidase treatment of paraformaldehyde-fixed T cells had no effects on the binding of His-dectin-1, whereas the same treatment markedly reduced the binding of wheat germ agglutinin (WGA) (Fig. 9B). By marked contrast, a brief exposure of activated T cells to trypsin abrogated their dectin-1 binding capacity almost completely (Fig. 9C). Thus, dectin-1 appears to bind to one or more trypsin-sensitive, tunicamycin/N-glycosidase-resistant ligands on T cells. We interpreted these results to suggest that although dectin-1 belongs structurally to the C-type lectin family by the inclusion of a CRD motif, it may not function as a conventional C-type lectin in the ligand specificity.

View larger version (28K): [in this window] [in a new window]   Fig. 9.   Characterization of the dectin-1-binding moiety on activated T cells. The activated D10 T cells were tested for their ability to bind His-dectin-1 and the indicated lectins following treatment with tunicamycin (A), N-glycosidase (B), or trypsin (C). A, the cells were cultured for 16 h in the presence (closed histogram) or absence (open histogram) of 4 µg/ml tunicamycin. B, the cells were fixed with paraformaldehyde and then subjected to the neuraminidase/N-glycosidase digestion (closed histogram) or the digestion with neuraminidase alone (open histogram). C, the cells were incubated with 0.25% trypsin (closed histogram) or PBS alone (open histogram) for 20 min at 37 °C. Data shown in the upper panels indicate the binding of bacterially produced His-dectin-1 (open and closed histograms) or His-DHFR control (dotted lines). The data in the lower panels indicate the binding of biotinylated phytohemagglutinin (PHA) or wheat germ agglutinin (WGA) (open and closed histograms, respectively) or His-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 three independent experiments.

To study the biological outcome of dectin-1 binding, splenic T cells were incubated on plates coated with His-dectin-1 (10 µg/ml). As shown in Fig. 10 (left panel), immobilized His-dectin-1 alone failed to induce significant T cell proliferation. However, His-dectin-1 promoted marked proliferation of T cells when anti-CD3 mAb was co-immobilized onto the same plates at suboptimal concentrations. For example, in the presence of 0.1-0.3 µg/ml of anti-CD3 mAb, His-dectin-1 caused more than 10-fold augmentation. On the other hand, His-dectin-1 had almost no effect on T cell proliferation that was triggered by higher concentrations of anti-CD3 mAb. In dose-response experiments, 3 µg/ml of His-dectin-1 was found to be sufficient for this biological activity (data not shown). Importantly, the co-stimulatory activity of His-dectin-1 was blocked almost completely with anti-dectin-1 Ab, but not with control IgG (Fig. 10, right panel). These results have revealed the potential of dectin-1 to deliver co-stimulatory signals to T cells.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Co-stimulatory potential of dectin-1. Enzyme-linked immunosorbent assay plates were coated with the indicated concentrations of anti-CD3 mAb together with His-dectin-1 (10 µg/ml) or buffer alone. Splenic T cells purified from 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 (left panel). Enzyme-linked immunosorbent assay plates coated with anti-CD3 mAb (0.3 µg/ml) and His-dectin-1 (10 µg/ml) were incubated with the indicated concentrations of anti-dectin-1 or control IgG. Splenic T cells were cultured in these wells and examined for [3H]thymidine uptake as described above. Data shown are representative of three independent experiments, showing the mean ± S.D. (n = 3) of [3H]thymidine uptake.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

One major technical barrier for studying the biology of DC at molecular levels had been the unavailability of pure DC preparations. This barrier has been overcome recently by the development of refined methods for isolating and culturing DC in large quantities (57-59) and by the establishment of stable DC lines (22, 60-65). By the PCR-based differential display between "freshly isolated" immature Langerhans cells versus "cultured" mature Langerhans cells, Ross et al. (66) have identified about 500 cDNA fragments whose expression was either up- or down-regulated during their maturation. By screening a cDNA library constructed from the cultured Langerhans cells with differential hybridization, they also identified that the expression of fascin, an actin-bundling protein, was markedly up-regulated during maturation (67). By searching the Human Genome Sciences nucleotide data bases with a consensus motif of C-type lectins, Bates et al. (68) identified a new C-type lectin, termed DC immunoreceptor (DCIR), which was expressed by short term human DC cultures generated from CD34⁺ progenitors as well as from CD14⁺ monocytes. Taking advantage of the XS52 DC line maintaining many features of skin-resident DC (19, 22-31), we employed the subtractive cDNA cloning strategy to identify the genes expressed predominantly by this DC line. We have identified 11 different genes (including five novel genes) that were expressed selectively by XS52 cells, documenting the efficacy of our approach.

Clone 1C11-5 encoded a novel, type II membrane-integrated polypeptide containing a single CRD motif at the COOH-terminal end. This polypeptide, termed dectin-1, was expressed on the surface of XS52 DC as a glycoprotein of about 43 kDa in size. Members of the C-type lectin family can be divided into two groups based on the molecular structures: (a) type I surface lectins with multiple CRDs on their NH₂-terminal ends and (b) type II surface lectins with a single CRD on their COOH termini. Importantly, DC have been shown to express both types of C-type lectins. Jiang et al. have cloned a novel, type I membrane-integrated C-type lectin, termed DEC-205, by using a DC-specific mAb (NLDC145) as a molecular probe (11). As observed for dectin-1, surface expression of DEC-205 was detected in some, but not all, splenic DC (69). Subsequently, Vremec and Shortman (70) found that DEC-205 is expressed preferentially by a specific subset of splenic DC called "lymphoid DC" as defined by their surface expression of CD8 homodimers. Sallusto et al. (10) reported that human DC express macrophage mannose receptor, a prototypic C-type lectin with the type I configuration. These two DC-associated C-type lectins, DEC-205 and macrophage mannose receptor, contain multiple CRD motifs (10 and 8, respectively) at their NH₂-terminal ends; thus, they belong to the type I surface lectin subfamily. Interestingly, both have been shown to mediate the uptake of glycosylated macromolecules by DC (10, 11).

DC also express CD23 (low affinity Fc receptor) (71) and CD69 (very early activation antigen) (72), both of which belong to the type II surface lectin subfamily. Although sharing the same molecular configuration, dectin-1 showed only limited degrees of sequence homology to CD23 (17.8% identity) or CD69 (15.6%), and the extent of homology was rather limited even within the highly conserved CRD motifs (22.0% with CD23 and 22.6% with CD69). More recently, Bates et al. (68) isolated a novel member (DCIR) by searching the nucleotide data bases with a motif (SCYWFSH) that is shared by hepatic lectin-1, hepatic lectin-2, and macrophage lectin. DCIR is expressed not only by DC but also by small fractions of other antigen presenting cells (monocytes, macrophages, and B cells), as we observed for dectin-1. Interestingly, DCIR contains, in the intracellular domain, a consensus immunoreceptor tyrosine-based inhibitory motif (ITIM) (54), suggesting its potential to deliver immunoregulatory signals (68). Dectin-1 showed relatively low sequence homology with DCIR (15.5% identity in overall sequence and 18.8% within the CRD region), and dectin-1 contained a putative ITAM motif, instead of the ITIM motif, in the cytoplasmic domain. ITAM motifs are also found in CD23 and macrophage lectin (51, 73), whereas an ITIM motif is present in CD72 (a B cell-associated C-type lectin) (74). It is, therefore, tempting to speculate that the ITAM-containing lectins and the ITIM-containing lectins may play counteracting roles in immunoregulation. Unfortunately, natural ligands recognized by those DC-associated C-type lectins (including dectin-1) remain mostly unknown, thus preventing us from directly testing this possibility. In sum, dectin-1 is the sixth member of the unique family of DC-associated C-type lectins. Very recently, we have identified the seventh member, termed "dectin-2," which resembles dectin-1 by virtue of the molecular configuration (a type II membrane-integrated polypeptide with a single CRD in the extracellular domain) and the expression profile (preferential expression by DC in both lymphoid and nonlymphoid tissues) (75).

With respect to the function of dectin-1, His-dectin-1 proteins bound to the surfaces of activated T cells, and immobilized His-dectin-1 promoted the proliferation of T cells only in the presence of anti-CD3 mAb at suboptimal concentrations. These observations suggest that dectin-1 proteins expressed on DC bind to putative ligand(s) on T cells and deliver co-stimulatory signals. On the other hand, His-dectin-1 (in a soluble form) and anti-dectin-1 Ab both failed to block DC-induced T cell activation in allogeneic mixed leukocyte reactions (data not shown), with the implication that the co-stimulatory property of dectin-1 may be replaceable by other molecules expressed on DC. His-dectin-1 did not bind to any of the conventional carbohydrate probes, and pretreatment of T cells with trypsin, but not with tunicamycin or N-glycosidase, abrogated their ability to bind His-dectin-1. These observations may imply that dectin-1 recognizes rather unique carbohydrate moieties or that dectin-1 must form homo- or heterodimers to exert high affinity binding, as has been reported for other type II lectins with single CRD motifs (76, 77). Alternatively, dectin-1 may recognize peptide or glycopeptide ligand(s). In fact, it is known that carbohydrates do not necessarily serve as natural ligands of all of the molecules that belong structurally to the C-type lectin family. For example, CD23, which has been shown to recognize Gal-Gal-NAc under certain experimental conditions (78), binds enzymatically de-glycosylated IgE and even recombinant (nonglycosylated) IgE produced in E. coli (79). Further studies are required to determine the physiological function of dectin-1 and to identify its ligand(s). Nevertheless, we believe that the present study has formed both technical and conceptual bases for such studies.

	ACKNOWLEDGEMENTS

We thank Drs. Nancy Street and Mansour Mohamadzadeh for providing T cell clones and B cell hybridoma clones, respectively. We are also grateful to Pat Adcock for secretarial assistance in the preparation of this manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants RO1-AR44189, RO1-AR35068, RO1-AR43777, and RO1-AI43262; a grant from Taisho Pharmaceutical Co., Ltd., Ohmiya, Japan; and an award from the Centre de Recherches et Investigations Epidermiques et Sensorielles (to A. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF262985.

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

Published, JBC Papers in Press, April 12, 2000, DOI 10.1074/jbc.M909512199

	ABBREVIATIONS

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, interleukin; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; Ab, antibody; mAb, monoclonal Ab; nt, nucleotide(s); FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES