CD83 Is a Sialic Acid-Binding Ig-Like Lectin (Siglec) Adhesion Receptor that Binds and a Subset of Activated CD8+ T Cells This information is current as of September 25, 2021. Nathalie Scholler, Martha Hayden-Ledbetter, Karl-Erik Hellström, Ingegerd Hellström and Jeffrey A. Ledbetter J Immunol 2001; 166:3865-3872; ; doi: 10.4049/jimmunol.166.6.3865 http://www.jimmunol.org/content/166/6/3865 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CD83 Is a Sialic Acid-Binding Ig-Like Lectin (Siglec) Adhesion Receptor that Binds Monocytes and a Subset of Activated CD8؉ T Cells1

Nathalie Scholler,2* Martha Hayden-Ledbetter,† Karl-Erik Hellstro¨m,* Ingegerd Hellstro¨m,* and Jeffrey A. Ledbetter†

To help determine CD83 function, a cDNA encoding a soluble containing the CD83 extracellular domain was fused with a mutated human IgG1 constant region (CD83Ig) and expressed by stable transfection of Chinese hamster ovary cells. Purified .CD83Ig bound to peripheral blood monocytes and a subset of activated CD3؉CD8؉ lymphocytes but did not bind to FcR Monocytes that had adhered to plastic lost their ability to bind to CD83Ig after 90 min of in vitro incubation. CD83Ig bound to

two of five T cell lines tested, HPB-ALL and Jurkat. The binding to HPB-ALL cells significantly increased when they were grown Downloaded from at a low pH (pH 6.5), whereas binding to Jurkat cells increased after was induced with anti-Fas mAb. and monocytic lines did not bind CD83Ig and neither did CD56؉ NK cells or granulocytes. Full-length CD83 expressed by a transfected carcinoma line mediated CD83-dependent adhesion to HPB-ALL cells. CD83Ig immunoprecipitated and immunoblotted a 72-kDa protein from HPB-ALL cells. Binding of CD83Ig to HPB-ALL cells was eliminated by neuraminidase treatment of the cells. We conclude that CD83 is an adhesion receptor with a counterreceptor expressed on monocytes and a subset of activated or stressed

T lymphocytes, and that interaction between CD83 and its counterreceptor is dependent upon the state of glycosylation of a 72-kDa http://www.jimmunol.org/ counterreceptor by sialic acid residues. In view of the selectivity of the expression of CD83 and its ligand, we postulate that the interaction between the two plays an important role in the induction and regulation of immune responses. The Journal of Immunology, 2001, 166: 3865–3872.

he marker CD83 is highly restricted to mature dendritic that includes B-G, butyrophilin, MOG, BT, BT2, B7c, B7-1, and cells (DC),3 including Langerhans cells and interdigitat- B7-2 (8–11). However, its function is not known. We now present T ing reticulum cells in the T cells zones of lymphoid or- data suggesting that CD83 mediates adhesion of DC to circulating gans (1, 2). It was independently discovered by two different monocytes and to a fraction of activated T cells or stressed T cells teams, in 1992 by Zhou et al. (2) and in 1993 by Kozlow et al. (3). by a specific binding of CD83 to a 72-kDa counterreceptor (li- by guest on September 25, 2021 Mouse CD83 was recently cloned and is up-regulated during DC gand). We further show that CD83Ig binding to its ligand is elim- maturation (4). CD83 transcripts are also detectable in mouse and inated by neuraminidase, an enzyme specific for the most common human brain mRNA by Northern hybridization, although it is not sialic acid, N-acetylneuraminic acid. Thus, CD83Ig binds to a car- known what cells within the brain express CD83 (3, 5). bohydrate epitope that depends on sialic acid residues. This clas- Isolation of cDNA encoding CD83 revealed that it is a 45-kDa sifies CD83 as a sialic acid-binding Ig-like lectin (Siglec; Ref. 12). type 1 membrane glycoprotein member of the Ig superfamily (2). Our data further suggest that the formation of the carbohydrate It is composed of a single extracellular V-type Ig-like domain, a epitope recognized by CD83 is influenced by cell growth condi- transmembrane region, and a 40-aa short cytoplasmic domain. The tions and can be rapidly altered by cellular stress and early tran- CD83 structure is similar to that of several other members of the sition to apoptosis. Ig superfamily. CD83 shows highly restricted cellular expression, it shares 23% overall identity with myelin protein Po, the most abundant glycoprotein in the peripheral myelin of mammals (6, 7), Materials and Methods and has significant homologies with the B7 ancestral family CD83Ig fusion protein construction A population highly enriched for DC was isolated from 200 ml of human peripheral blood by discontinuous Nycodenz gradient centrifugation, as Laboratories of *Tumor Immunology and †Immunobiology, Pacific Northwest Re- search Institute, Seattle, WA 98122 described elsewhere (13). Nycodenz was purchased as Nycoprep (13% (w/v) Nycodenz, 0.58% (w/v) NaCl, 5 mM Tris-HCl, pH 7.2, density ϭ Received for publication September 26, 2000. Accepted for publication January 8, 1.068 Ϯ 0.001, 335 Ϯ 5 mOsm/kg) from Nycomed Pharma (Oslo, Nor- 2001. way). At the end of the purification procedure, RNA was directly extracted The costs of publication of this article were defrayed in part by the payment of page from DC by TRIzol (Life Technologies, Grand Island, NY) and reverse charges. This article must therefore be hereby marked advertisement in accordance transcripted (Superscript II; Life Technologies). cDNA from DC was am- with 18 U.S.C. Section 1734 solely to indicate this fact. plified with PCR primers containing a 5Ј HindIII site: gaataagctt atg tcg cgc 1 This work was supported by Pacific Northwest Research Institute and by National ggc ctc cag ctt ctg ctc c and a 3Ј BglII site in the antisense primer: gag cca Institutes of Health Grants CA90143 (to J.A.L.), CA79490 (to K.E.H.) and CA85780 gca gca gga gaagatctt ccg ctc tgt att tc. The PCR product (457 bp) was (to I.H.). cloned into pCDNA1 human IgG1 (a gift from Robert Peach, Bristol Myers 2 Address correspondence and reprint requests to Dr. Nathalie Scholler, Laboratory of Squibb Pharmaceutical Institute, Princeton, NJ). DNA from recombinant Tumor Immunology, Pacific Northwest Research Institute, 720 Broadway, Seattle, colonies was amplified by Qiagen plasmid maxi (Qiagen, Valencia, WA 98122. E-mail address: [email protected] CA), sequenced, and transfected into COS7 cells. After 3 days, the pres- 3 Abbreviations used in this paper: DC, ; Siglec, sialic acid-binding ence of soluble protein in cell supernatant was checked by Western blot Ig-like lectin; PVDF, polyvinylidene difluoride. analysis and the fusion protein was purified by protein A-Sepharose 4B

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 3866 CD83 IS A SIALIC ACID-BINDING Ig-LIKE LECTIN (SIGLEC) ADHESION RECEPTOR affinity chromatography (Zymed, South San Francisco, CA). Stable trans- DMEM medium. In some experiments, incubations were carried in DMEM fectants were generated in Chinese hamster ovaries cells by using CD83Ig medium that had been supplemented with 2-fold serial dilutions of sucrose cDNA cloned into pD18 (14). (from 0.6 to 0.1 M). CD83 retrovirus construction and generation of transfected cell Labeling of beads line A total of 50 ␮g of material to be labeled (CD83Ig, anti-CD83 mAb, and CD83 cDNA was cloned into pLNCX vector (15). DNA from recombinant a mixture of anti-CD28 mAb and anti-CD3 mAb) were conjugated to mag- colonies was amplified by Qiagen plasmid maxi kit and transfected into netic beads (Tosylativated Dynabeads M-450; Dynal, Lake Success, NY) ecotropic packaging cells (PE501) by using a calcium phosphate method according to a published protocol (19). Beads conjugated with CD83Ig or (16). PE501 viral supernatant was used to infect PG13 cells, a primate- anti-CD83 mAb were used to test the specificity of CD83Ig fusion protein. specific packaging line. PG13 supernatant was harvested, filtered, and used Anti-CD28/anti-CD3 mAb-conjugated beads were used to stimulate T to infect 1C, a colon carcinoma line derived in our laboratory. Recombi- cells. nant colonies were selected by G418 (Life Technologies). T cell stimulation with anti-CD28/anti-CD3 mAb-coated beads Media for cell culture and flow cytometry PBMC were incubated 5 days with conjugated or nonconjugated beads (control) in RPMI medium at 37°C. After that time, the beads were mag- Cells were cultured with a standard medium (referred to as RPMI medium), netically removed and the cells resuspended in RPMI medium supple- which consisted of RPMI 1640 (Life Technologies) supplemented with mented with 10 IU/ml of rIL-2 (Roche Molecular Biochemicals, Indianap- glutamine (1%; Life Technologies), penicillin/streptomycin (1%; Life olis, IN) and cultivated for 2 wk. Technologies), and 10% FCS (Atlanta Biological, Norcross, GA). All la- beling for flow cytometry was conducted at 4°C in a medium that consisted Cell lysate preparation of DMEM (Life Technologies) supplemented with 5% FCS without azide 7 8 (referred to as DMEM medium). In some experiments, this medium was HPB-ALL (5 ϫ 10 –10 ) cells were washed three times in ice-cold PBS, Downloaded from supplemented with 0.6 M sucrose (Sigma-Aldrich, St. Louis, MO). resuspended in 4 ml of lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail tablets, Purification of PBL and of monocytes complete, mini; Roche Molecular Biochemicals), and incubated on ice for 30 min. Lysates were centrifuged for 30 min at 12,000 ϫ g at 4°C, and the ϫ 7 PBMCs (5–10 10 ) were isolated from 50–100 ml fresh blood from supernatants were harvested. In some experiments, HPB-ALL cells were healthy donors by sedimentation in Ficoll-Paque Plus (Amersham Phar- treated by neuraminidase before lysis, as described in the flow cytometry macia Biotech, Uppsala, Sweden) and washed twice in RPMI medium. For analysis section. experiments involving T cell activation, the PBMCs were resuspended in http://www.jimmunol.org/ RPMI medium and stimulated with Abs or with Ab-conjugated beads as Immunoprecipitation described below. Supernatants of cell lysates were incubated for 1 h with 50 ␮l of strepta- Induction of cellular stress and apoptosis vidin-Sepharose 4B conjugate (Zymed) at 4°C. Streptavidin-Sepharose was removed by centrifugation, and 1 ml of lysate was incubated overnight Induction of cellular stress of HPB-ALL cells was conducted in four dif- at 4°C with 50 ␮g of biotinylated CD83Ig. Immunoprecipitated ferent ways: 1) by incubating the cells in a T75 culture flask with an airtight were separated by3hofincubation with streptavidin-Sepharose followed lid for 2–4 days in RPMI medium without HEPES buffer; 2) by suspending by six washes with 1 ml of lysis buffer and one wash with PBS. Strepta- ϫ 6 2–5 10 cells in 1 ml of RPMI medium, seeding them into six-well plates, vidin-Sepharose was then resuspended in 2ϫ SDS sample buffer (Novex, and exposing them for 4 min to UV irradiation by an antimicrobial UV San Diego, CA) with 1% 2-ME and boiled for 10 min, after which 20 ␮l lamp placed 50 cm above the cells inside a biosafety cabinet; 3) by incu- of the supernatant was loaded on a 4–12% gradient gel (Novex). Subse- by guest on September 25, 2021 bating the cells in culture medium at different pH (6–7.4) or in culture quently, the migration gel was blotted and probed as described in the fol- medium supplemented with 25 mM HEPES buffer (Life Technologies); or lowing section. 4) by exposing the cells to oxidative stress. This was accomplished by adding to the medium 2-fold serial dilutions of hydrogen peroxide (10 mM, Western blotting analysis 5 mM, 2.5 mM, 1.25 mM, and 0.625 mM) and incubating 2–5 ϫ106 cells for 10 min at 37°C. After two washes, the cells were incubated in RPMI Supernatants of cell lysates, streptavidin-Sepharose-purified samples, and CD83Ig were eluted in SDS-PAGE sample buffer containing 2-ME and medium at 37°C until labeling1hto7days later. ␮ Jurkat cell apoptosis was induced by anti-CD95 (Fas) mAb from Beck- then boiled. CD83Ig (10 g) was also reduced with 10 mM DL-DTT (Sig- man Coulter (Palantine, IL). Petri dishes were coated with 1 ␮g/ml anti- ma) for 30 min at 37°C and free sulfhydryl residues were alkylated with 25 murine IgM mAb from Beckman Coulter in bicarbonate buffer (Sigma) for mM of iodoacetamide, pH 8 (Sigma), for1hat37°C. Samples were run on 2 h at 37°C. Jurkat cells were washed three times with PBS and incubated a tris-glycine 4–12% gradient gel (Novex). The amount of proteins in cell for 1 h with 4-fold serial dilutions anti-Fas mAb. After two washes with lysates was quantified with micro bicinchoninic acid protein reagent kit culture medium, anti-Fas-coated Jurkat cells were incubated in the anti- (Pierce) according to the manufacturer’s instructions. After migration pro- murine IgM-coated petri dish in culture medium at 37°C overnight. teins were transferred to polyvinylidene difluoride (PVDF) membranes (Novex), they first were probed with biotinylated CD83Ig 0.5 ␮g/ml, Flow cytometry analysis washed four times (WesternBreeze; Invitrogen, Carlsbad, CA), and then probed with 1:5000 streptavidin-HRP (BD PharMingen). The signal was Monoclonal Abs recognizing the following Ags were used: CD83 detected by ECL (Amersham Pharmacia Biotech) according to the manu- (HB15A), IgG1 and IgG2a isotype controls from Immunotech, and CD11b facturer’s protocol and quantified by using OptiQuant version 03.00 (Pack- (17), CD4, CD8, and CD3 from BD PharMingen (Lexington, KY). To ard Instruments, Meriden, CT). detect apoptosis, we used an annexin V kit (Beckman Coulter) according to the manufacturer’s instructions. CD83Ig, CD80Ig (14), and CD40Ig (18) Adhesion assays fusion proteins were biotinylated with EZ-Link N-hydroxy-succinimi-bi- Wild-type 1C cultured human carcinoma cells and CD83-transfected cells otin (Pierce, Rockford, IL) kit according to the manufacturer’s procedure. obtained from these (1C/CD83) were seeded at 5 ϫ 105 cells/well into a Insomeexperimentscells,theHPB-ALLlinewereincubatedwithneuramini- 48-well plate (Costar, Cambridge, MA) and incubated in culture medium at dase (Sigma) for 15 min at 37°C (1 U/5 ϫ 106cells) or with neuraminidase 37°C for 24 h to allow their adhesion. After 24 h, the plates were washed together with 2.5 mg/ml of a sialidase inhibitor (2,3-dehydro-2-deoxy-N- one time with fresh medium to remove nonadherent cells. Nonstressed or acetylneuraminic acid, Sigma). Cells were washed twice in DMEM me- HPB-ALL cells stressed by growth at low oxygen level were labeled with dium and labeled at 4°C for 45 min with 1 ␮g/ml biotinylated CD83Ig 50 ␮Ci of 51Cr (Amersham Pharmacia Biotech) for 45 min at 37°C then followed by two washes in DMEM medium and labeled for 15 min at 4°C ϫ 6 ␮ ␮ washed twice and resuspended in RPMI medium at 2.5 10 cells/ml in with 3 l/100 l of PE streptavidin (BD PharMingen). ␮ ␤ ␮ the presence of 10 g/ml anti- 2 integrin mAb 60.3 (20) or with 10 g/ml 51 Cold competition experiments anti-CD83 mAb. Next, 2-fold serial dilutions of Cr-labeled HPB-ALL cells were distributed and incubated for 1–4 h at 37°C with 1C or 1C/CD83 Cells or beads were washed twice and incubated for 15–30 min at 4°C with cells. Finally, the cells were washed once with PBS and once according to 50 ␮g/ml unlabeled CD83Ig or with 20 ␮g/ml anti-CD83 mAb in DMEM a procedure derived from gravity flow wash (21). According to this pro- medium. The cells then were labeled with PE-conjugated anti-CD83 mAb cedure, the plates were immersed in PBS in a large container and sus- or with 1 ␮g/ml biotin-conjugated CD83Ig as described previously in pended upside down above the bottom of the container for 15 min to allow The Journal of Immunology 3867 nonadherent cells to detach. They then were turned slowly right side up, ever, after reduction with DTT and alkylation with iodoacetamide, were removed from the container, and the PBS in each well was removed CD83Ig migrated as a single band of an ϳ98-kDa monomer (Fig. by aspiration. The adherent cells remaining after this procedure were lysed 1D, lane 2). by 100 ␮l of PBS plus 0.2% Triton X-100 (Fisher Scientific, Fairlawn, NJ). Lysates (40 ␮l) were transferred into LumaPlate-96 plates (Packard Instru- ments) and counted with a Top-Count NXT (Packard Instruments). CD83Ig binds to circulating monocytes and to a subset of activated T lymphocytes Results According to flow cytometry analysis of fresh PBMC, biotinylated Construction of CD83Ig and verification of its activity CD83Ig was found to bind to Ͻ1% of CD3ϩ cells and to ϳ4% of Ϫ We constructed a CD83Ig fusion protein as described in Materials CD3 cells in the lymphocyte-scatter gate (gate 1), whereas bio- and Methods (Fig. 1A). It was engineered without an Ig hinge tinylated CD40Ig used as control did not bind (Fig. 2, B and C). In region between the coding sequence for CD83 extracytoplasmic the larger cell-scatter gate (gate 2), biotinylated CD83Ig bound to Ͼ lowϩ domain (432 bp) and the CH2 and CH3 domains and contains two 75% of cells that expressed CD11b (Fig. 2D), CD4 (data mutations, one at 231 bp, transforming valine to proline, and the not shown), and CD14 (Fig. 2E), i.e., cells with the distinctive other at 531 bp, transforming a proline to a serine. These structural characteristics of circulating monocytes. When CD83Ig labeling was performed in the presence of anti-CD83 mAb (HB15A), the modifications eliminated the binding to FcR. CD83Ig did not bind ϩ to cells expressing Fc␥RI (U937), Fc␥RII (normal B cells and B binding of CD83Ig to the CD14 cells consistently increased up to cell leukemia lines, Raji, Ramos, Bjab), or Fc␥RIII (blood CD16 90% (Fig. 2F), whereas an isotype control mAb did not increase plus NK cells; data not shown). To check the specificity and proper the binding of CD83Ig to these cells. The binding of CD83Ig to folding of the CD83Ig fusion protein, experiments were performed monocytes was specific, because biotinylated CD40Ig did not bind Downloaded from that showed that PE-labeled anti-CD83 mAb bound to CD83Ig- at all (data not shown). This suggests that the HB15A mAb binding conjugated beads and that a PE-labeled isotype control mAb did epitope is distinct from the active binding site of CD83 ligand, not (Fig. 1B). The binding of PE-labeled anti-CD83 mAb to the consistent with the lack of function described for this anti-CD83 CD83Ig-conjugated beads was partially blocked by preincubation mAb. The CD83Ig binding to monocytes decreased after 90 min of with an unlabeled anti-CD83 mAb (20 ␮g/ml) for 15 min at 4°C culture (Fig. 3, A and B). This decrease was less in the presence of

␤ http://www.jimmunol.org/ (Fig. 1B). Conversely, CD83Ig bound to anti-CD83 mAb-conju- the anti-CD18 ( 2 integrin) Ab 60.3 (Fig. 3, C and D), which gated beads whereas CD40Ig did not bind (Fig. 1C). The binding blocks the adhesion of monocytes to plastic (22–24); because ad- of CD83Ig to beads conjugated with anti-CD83 mAb was com- hesion induces activation, this suggests that expression of the CD83 ligand on monocyte correlates with a resting stage. In pletely blocked by preincubation with an unlabeled anti-CD83 ϩ mAb (20 ␮g/ml) for 15 min at 4°C (Fig. 1C, picture 2). 2-ME contrast, the binding of CD83Ig to CD3 T lymphocytes increased Ͻ incompletely reduced CD83Ig, which migrated as a mixture of a from 1% for resting cells (Fig. 2C) to 6% for cells activated by 60-kDa monomer and a 120-kDa dimer (Fig. 1D, lane 1). How- 2 wk of culture with anti-CD3/anti-CD28-conjugated beads (Fig. 4B). CD3ϩCD8ϩ T lymphocytes bound to CD83Ig (Fig. 4C), whereas CD3ϩCD4ϩ T lymphocytes did not (Fig. 4D). A total of 90% of the cells binding to CD83Ig were costained with annexin by guest on September 25, 2021 V (Fig. 4E) as compared with Ͻ6% of the cells binding to CD80Ig, used as control (Fig. 4F). Altogether, these data indicate that CD83Ig binds to a ligand the expression of which is regulated by cell activation and apoptosis. No binding of CD83Ig was found on

FIGURE 1. Construction and verification of CD83Ig. A, CD83Ig was constructed by fusing the CD83 extracytoplasmic domain with a hinge- truncated human IgG1. B, CD83Ig-conjugated beads were labeled with a FIGURE 2. CD83Ig binds to circulating monocytes. PBMC from PE-labeled isotype control mAb (picture 1), a PE-labeled anti-CD83 mAb healthy donors were purified by Ficoll and stained immediately after pu- after a preincubation with an unlabeled anti-CD83 mAb (picture 2), or a rification. Cells were gated according to their forward and side angle PE-conjugated anti-CD83 mAb in DMEM medium (picture 3). C, Anti- scatter proprieties (A). In B and C, lymphocytes (gate 1) were labeled with CD83 mAb-conjugated beads were labeled with biotinylated CD40Ig plus FITC-conjugated anti-CD3 mAb and (B) with biotinylated CD83Ig plus PE PE streptavidin (picture 1), or with biotinylated CD83Ig plus PE strepta- streptavidin; or (C) with biotinylated CD40Ig plus PE streptavidin. In D–F, vidin after a preincubation with an unlabeled anti-CD83 mAb (picture 2), monocytes from a different donor (gate 2) were labeled with biotinylated or with biotinylated CD83Ig plus PE streptavidin in DMEM medium (pic- CD83Ig plus PE streptavidin and (D) FITC-conjugated CD11b. In E and F, ture 3). D, After treatment by 2-ME, CD83Ig migrated as a 60-kDa mono- monocytes were labeled with FITC-conjugated anti-CD14 mAb immedi- mer and a 120-kDa homodimerized band. E, After treatment by DTT and ately after purification (E) or after 30 min of incubation at 4°C with 20 iodoacetamide, CD83Ig migrated as a 98-kDa monomer band. ␮g/ml anti-CD83 mAb (F). 3868 CD83 IS A SIALIC ACID-BINDING Ig-LIKE LECTIN (SIGLEC) ADHESION RECEPTOR Downloaded from FIGURE 3. Adhesion down-regulates CD83 ligand expression on monocytes. Monocytes were labeled with CD83Ig plus PE streptavidin and FITC anti-CD11b (A) immediately after Ficoll preparation; (B) after 90 min in culture at 37°C; (C) after 24 h in culture at 37°C in medium; or (D) after 24 h in culture at 37°C in medium supplemented with the anti-CD18 mAb 60.3. http://www.jimmunol.org/

CD56-positive NK cells, nor to granulocytes or erythrocytes (data not shown).

ϩ The CD83 ligand is expressed on two of five tested T FIGURE 4. CD83 ligand expression is up-regulated on CD8 -activated lymphocyte lines and its expression is influenced by cellular lymphocytes. PBL were activated 5 days with anti-CD3/antiCD28- conjugated beads and, after removal of beads, were incubated for 2 wk in stress medium supplement with 10 IU/ml of IL-2. Lymphocytes then were la- The binding of CD83Ig to B, T, myeloid, and monocyte cell lines beled with PE streptavidin and FITC isotype control mAb (A). In B–E, was examined. CD83Ig did not bind to any of four B cell lines lymphocytes were labeled with biotinylated CD83Ig plus PE streptavidin by guest on September 25, 2021 (Nalm6, Reh, Bjab, DHL10), or to two myeloid cell lines (Thp1, and (B) FITC anti-CD3 mAb; (C) FITC anti-CD8 mAb; (D) FITC anti- HL60) or to the monocyte cell line U937 (data not shown). Sim- CD4 mAb; or (E) FITC annexin V. As control, lymphocytes were labeled ilarly, no binding was observed to Molt4, CEM, Hut78, or Jurkat with biotinylated CD80Ig plus PE streptavidin and FITC annexin V (F). cells in exponential growth. However, CD83Ig did bind to HPB- ALL cells (25) in light-scatter gates 1 and 2 (Fig. 5, A and B). binding to cells undergoing apoptosis. Jurkat cells were incubated Interestingly, we found that CD83Ig binding increased after 24 h with serial dilutions of anti-Fas Ab, as described in Materials and of incubation in a flask with a tightly closed lid, and this increase Methods. Although the binding of biotinylated CD86Ig, which was was greater for cells in gate 2 than for cells in gate 1 (Fig. 5, C–F). used as a control, did not vary (data not shown), apoptotic Jurkat This suggested that expression of the CD83 ligand might be reg- cells expressed the CD83 ligand after 12 h of treatment with anti- ulated by cell growth conditions and/or by cellular stress and led us Fas Ab coincidentally with the binding of annexin V. The binding to study the effect of cellular stress, including apoptotic events, on of CD83Ig to Jurkat cells was proportional to the concentration of CD83Ig binding. The increase of CD83Ig binding on HPB-ALL anti-Fas mAb (Fig. 5, G–I). cells in gate 2 was not induced by UV irradiation, by hydrogen Unlabeled CD83Ig blocks biotinylated CD83Ig binding in peroxide, or by incubation in a tight-close lid flask in the presence hypertonic medium but not in DMEM medium of HEPES (data not shown). This suggested that an additional condition was required to increase the binding of CD83Ig. Addi- We were unable to block the binding of biotin-conjugated CD83Ig tional experiments demonstrated that the CD83Ig binding increase to HPB-ALL cells with unconjugated CD83Ig and therefore hy- in gate 2 was correlated with the pH of the culture medium. When pothesized that there could be a rapid, receptor-mediated internal- cells were incubated in a tight-close lid flask at pH 7, 10% of ization of the fusion protein, as described in some other systems HPB-ALL cells bound to CD83Ig in gate 2 (Fig. 5E), whereas at (27). Thus, we tested CD83 ligand endocytosis in the presence of pH 6.5 the binding increased up to 44% (Fig. 5F). In contrast, in a high sucrose hypertonic medium known to block internalization gate 1 the binding of CD83Ig to HPB-ALL cells did not signifi- by preventing clathrin-coated pit formation (28). Fig. 6 shows that cantly vary with the pH (Fig. 5, C and D). Finally, 5–6% of it was possible to block the binding of biotin-conjugated CD83Ig CD83Ig-bearing cells in gate 1 (Fig. 5, C and D) and none of the with unlabeled CD83Ig in the presence of 0.6 M sucrose and that CD83Ig-bearing cells in gate 2 (Fig. 5, E and F) were labeled by the blocking was proportional to the sucrose concentration. annexin V. Thus, CD83Ig binding to HPB-ALL cells depends on the pH of the cell medium and is not dependent on apoptosis, CD83Ig immunoprecipitates and immunoblots a 72-kDa protein because the cells that bound CD83Ig did not bind annexin V. from HPB-ALL cell lysates Because HPB-ALL cells are not sensitive to apoptosis mediated Lysates of HPB-ALL cells were immunoprecipitated with biotin- by anti-Fas mAb (26), we chose Jurkat cells for studying CD83Ig conjugated CD83Ig and collected on streptavidin-Sepharose beads. The Journal of Immunology 3869

FIGURE 5. Cellular stress up-regulated CD83 ligand expression on T lymphocyte cell lines. A, HPB-ALL cells were analyzed in two light-scatter gates. B, HPB- ALL cells from gate 2 were stained with PE streptavidin as a negative control (black area) or with biotinylated CD83Ig plus PE streptavidin (lines). The signal was brighter when cells were cultivated with a tightly closed lid (straight line) than in standard culture conditions (dotted line). In C–F, HPB-ALL cells were labeled with biotinylated CD83Ig plus PE streptavidin and FITC an- nexin V. HPB-ALL cells were cultivated at pH 7 and gated on gate 1 (C)orgate2(D). HPB-ALL cells were cultivated at pH 6.5 and gated on gate 1 (E) or gate 2 (F). In G–I, cells from the Jurkat line were stained with biotinylated CD83Ig plus PE streptavidin after being cultured in the presence of (G)10␮g/ml isotype control Downloaded from mAb; (H)1␮g/ml anti-Fas mAb; or (I)5␮g/ml anti-Fas mAb. http://www.jimmunol.org/

After washing, the beads were eluted with SDS sample buffer and 2-fold serial dilutions). Biotinylated CD83Ig was detected in the separated on tris-glycine 4–12% gradient gels (Fig. 7). After trans- control lane (lane 2). Thus, the ligand epitope detected by CD83Ig fer, filters were blotted with biotinylated CD83Ig. Fig. 7 shows that is not destroyed by boiling in SDS. a 72-kDa molecule binds to CD83Ig in HPB-ALL cell lysates, which were immunoprecipitated with biotinylated CD83Ig (lane The CD83 ligand binding site contains sialic acid 1) or directly blotted without immunoprecipitation (lanes 3–6, Experiments were performed to test whether the epitope detected

by CD83Ig on the 72-kDa protein was part of the protein itself or by guest on September 25, 2021 was a carbohydrate attached to the protein. We found that a treat- ment of HPB-ALL cells with neuraminidase diminished the CD83Ig binding (Fig. 8B), although treatment with sialidase usu- ally enhances cell-cell interactions (29, 30) by removing nega- tively charged sialic acids. Inhibition of neuraminidase by a siali- dase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, prevented the reduction of CD83Ig binding to HPB-ALL (Fig. 8C) as well as to fresh monocytes (data not shown). Lysates were pre- pared from HPB-ALL cells before or after treatment by neuramin- idase, and their protein concentrations were quantified. Even

FIGURE 7. CD83Ig immunoprecipitates HPB-ALL lysates. A SDS tris- FIGURE 6. Unlabeled CD83Ig blocks biotinylated CD83Ig binding in glycine 4–12% gradient gel was loaded on lane 1 with HPB-ALL lysates hypertonic medium but not in DMEM medium. HPB-ALL cells were la- immunoprecipitated with 10 ␮g/ml of biotinylated CD83Ig and purified beled with PE streptavidin as a negative control (black) and with biotin- with streptavidin-Sepharose, on lane 2 with biotinylated CD83Ig alone ylated CD83Ig plus PE streptavidin as a positive control (white). Binding (200 ng), and on lanes 3–6 with 2-fold serial dilutions of HPB-ALL ly- was carried at 4°C in DMEM medium without azide (controls and dark sates. After migration, the gel was blotted on PVDF membrane, probed gray bar) or in the presence of unlabeled CD83Ig (20 ␮g/ml) and serial with biotinylated CD83Ig, and the signal was detected by streptavidin-HRP dilutions of sucrose. chemoluminescence. 3870 CD83 IS A SIALIC ACID-BINDING Ig-LIKE LECTIN (SIGLEC) ADHESION RECEPTOR

FIGURE 8. CD83 ligand binding site contains a sialic acid. A, HPB- ALL cells grow at pH 6.5 were stained with PE streptavidin as a negative control (dotted line) or with biotinylated CD83Ig plus PE streptavidin

(black area). B, HPB-ALL cells were stained with biotinylated CD83Ig Downloaded from plus PE streptavidin in flow cytometry medium (dotted line) or after a treatment with neuraminidase (1 U/ml) for 15 min at 37°C (black area). C, HPB-ALL cells were stained with biotinylated CD83Ig plus PE streptavi- din after a neuraminidase treatment (dotted line) or after neuraminidase treatment in presence of 2.5 mg/ml of a neuraminidase inhibitor for 15 min at 37°C (black area). D, A SDS tris-glycine 4–12% gradient gel was loaded

with 2-fold serial dilutions of HPB-ALL cell lysates, without (lanes 1 and http://www.jimmunol.org/ 2) and with neuraminidase treatment (lanes 3 and 4). Protein concentra- tions of cell lysates were determined by micro bicinchoninic acid to be 25 ␮g(lanes 1 and 3) and 50 ␮g(lanes 2 and 4). After migration, the gel was FIGURE 9. The binding between CD83 and CD83 ligand mediates ad- blotted on PVDF membrane and probed by biotinylated CD83Ig plus HRP- hesion. A, 1C wild-type cells (dotted line) or 1C/CD83 cells (black area) streptavidin. The signal was detected by chemoluminescence, scanned, and were labeled with PE anti-CD83 mAb. B, 51Cr-labeled HPB-ALL cells quantified by OptiQuant program. The difference of digital light units after either in exponential growth or stressed by growth at a low pH (pH 6.5), background subtraction was 37% between lanes 1 and 3 and 31% between were incubated 3 h with adherent 1C (wild type; white) or 1C/CD83 lanes 2 and 4. ␮ ␤ (black) in RPMI medium supplemented with 10 g/ml of anti- 2 integrin 51 mAb 60.3. C, Serial dilutions of Cr-labeled stressed HPB-ALL CD83 (5 by guest on September 25, 2021 ϫ105; 2.5 ϫ 105; 1.25 ϫ 105; 0.625 ϫ 105/well) were incubated for 3 h with adherent wild-type 1C cells (squares) or transfected 1C/CD83 cells though the protein concentration of the cell lysates was unchanged, (circles) in RPMI medium (open) or supplemented with 10 ␮g/ml anti- the CD83Ig reactivity with the 72-kDa protein was reduced by CD83 mAb (filled). 31–37% (Fig. 8D) with neuraminidase treatment of HPB-ALL cells, as measured by OptiQuant program (Packard, Meriden, CT). This indicates that the ability of the CD83 ligand to bind to CD83 Discussion is dependent on glycosylation by sialic acid residues. CD83 is selectively expressed by mature DC and has been used to define the maturation stage and purity of DC populations (1, 31). The binding between CD83 and the CD83 ligand mediates Its restricted expression suggests that CD83 may have a special- adhesion ized function during Ag presentation by DC, perhaps contributing We used cells from 1C colon cancer line transfected with CD83 to their ability to efficiently activate T cells. However, little data retrovirus (1C/CD83; Fig. 9A) to test their adhesion to HPB-ALL has been presented to support this hypothesis, and anti-CD83 (which express the CD83Ig ligand); wild-type 1C cells were used mAbs have not been reported to alter the ability of DC to activate as a control. HPB-ALL cells were incubated for 3 days at pH 7.4 T cells or to synthesize cytokines. We present here evidence that or at pH 6.5, harvested, and labeled with chromium 51. After two CD83 is an adhesion receptor, because a soluble CD83Ig fusion washes to remove unincorporated radioactivity, 51Cr-labeled cells protein binds to blood monocytes, two of five leukemia T cell were incubated for 1–4 h with adherent 1C or 1C/CD83 in RPMI lines, and a subset of activated CD8ϩ lymphocytes and that this medium (pH 7.4) supplemented with either 10 ␮g/ml of the anti- binding increased as a result of cellular stress. In addition, trans- integrin mAb 60.3 to avoid nonspecific adherence or with 10 fection of CD83 into a carcinoma line allowed them to bind to ␮g/ml of an anti-CD83 mAb. The strongest binding was observed HPB-ALL T cells. These results suggest that CD83 contributes to after3hofincubation (Fig. 9, B and C). After three washes with cell adhesion by facilitating DC interactions with monocytes and PBS, adherent cells were lysed with 100 ␮l of lysate buffer, and 40 subpopulations of activated and/or stressed CD8ϩ T cells. ␮l was harvested and counted. HPB-ALL cells showed a higher DC can be derived from monocytes by stimulation with IL4 and level of adhesion to 1C/CD83 than to 1C, both in medium (data not GM-CSF and/or Ag (32–34). Because the CD83 ligand disappears shown) and in the presence of 60.3 (Fig. 9B). The anti-CD83 mAb quickly during monocyte culture, adhesion between CD83 on DC blocked the adhesion of HPB-ALL cells incubated at pH 6.5 to and its ligand on monocytes may represent a regulatory mecha- 1C/CD83 (Fig. 9C). This adhesion pattern exactly followed the nism to control APC maturation. Conversely, mature DC may pattern of the CD83 ligand, indicating that the CD83-CD83 ligand stimulate the monocytes to release chemokines and/or cytokines to interaction mediates adhesion. amplify immune responses. The Journal of Immunology 3871

In cultures of lymphocytes, binding of CD83Ig was seen on 6% References ϩ of CD8 T cells after 2 wk of activation through their CD3 and 1. Zhou, L. J., and T. F. Tedder. 1995. Human blood dendritic cells selectively CD28 receptors. This binding increased 2- to 3-fold in the absence express CD83, a member of the immunoglobulin superfamily. J. Immunol. 154: 3821. of APC, and addition of mature DC but not CD83Ig to the T cell 2. Zhou, L. J., R. Schwarting, H. M. Smith, and T. F. Tedder. 1992. A novel cell- cultures down-regulated the expression of CD83 ligand (data not surface molecule expressed by human interdigitating reticulum cells, Langerhans shown). This suggests that CD83-specific interactions between cells, and activated lymphocytes is a new member of the Ig superfamily. J. Im- munol. 149:735. subpopulations of T cells and DC may be important when the T 3. Kozlow, E. J., G. L. Wilson, C. H. Fox, and J. H. Kehrl. 1993. Subtractive cDNA cells are at a specific stage of maturation. It is not yet known cloning of a novel member of the Ig gene superfamily expressed at high levels in whether or not the activated T cells that expresses the CD83 ligand activated B lymphocytes. Blood 81:454. 4. Berchtold, S., T. Jones, P. Muhl-Zurbes, D. Sheer, G. Schuler, and represent subpopulations of stressed cells; however, activated cells A. Steinkasserer. 1999. The human dendritic cell marker CD83 maps to chro- that bound CD83Ig were also annexin V positive. Neither is it mosome 6p23. Ann. Hum. Genet. 63:181. known whether interaction of the ligand-positive stressed cells 5. Twist, C. J., D. R. Beier, C. M. Disteche, S. Edelhoff, and T. F. Tedder. 1998. The mouse CD83 gene: structure, domain organization, and localization. with DC is a mechanism for removal or rescue of such cells. Immunogenetics 48:383. A recent publication by Cramer et al. (35) suggests that mouse 6. Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. CD83 ligand is expressed by B cells labeled with an anti-CD45R Role of myelin Po protein as a homophilic adhesion molecule. Nature 344:871. ϩ 7. Filbin, M. T., and G. I. Tennekoon. 1991. The role of complex carbohydrates in mAb (B220), because mouse CD83Ig bound to B220 splenocytes adhesion of the myelin protein, Po. Neuron 7:845. from normal BALB/c mice but not to splenocytes from ␮MT (B 8. Pham-Dinh, D., M. G. Mattei, J. L. Nussbaum, G. Roussel, P. Pontarotti, N. Roeckel, I. H. Mather, K. Artzt, K. F. Lindahl, and A. Dautigny. 1993. Myelin/ cell knockout) mice. In contrast, in human peripheral blood we did oligodendrocyte glycoprotein is a member of a subset of the immunoglobulin not detect any CD83Ig binding to B cells labeled with an anti- superfamily encoded within the major histocompatibility complex. Proc. Natl. Downloaded from CD19 mAb, while in mouse blood we could detect human CD83Ig Acad. Sci. USA 90:7990. 9. Vernet, C., J. Boretto, M. G. Mattei, M. Takahashi, L. J. Jack, I. H. Mather, binding to circulating monocytes (data not shown). In our study, S. Rouquier, and P. Pontarotti. 1993. Evolutionary study of multigenic families we demonstrated that cellular stress including cell growth at low mapping close to the human MHC class I region. J. Mol. Evol. 37:600. pH or progression into apoptosis after anti-Fas stimulation can 10. Gruen, J. R., S. R. Nalabolu, T. W. Chu, C. Bowlus, W. F. Fan, V. L. Goei, H. Wei, R. Sivakamasundari, Y. Liu, H. X. Xu, et al. 1996. A transcription map increases the binding of CD83Ig. It is possible that the CD83Ig of the major histocompatibility complex (MHC) class I region. Genomics 36:70.

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28. Heuser, J. E., and R. G. Anderson. 1989. Hypertonic media inhibit receptor- 37. Nicoll, G., J. Ni, D. Liu, P. Klenerman, J. Munday, S. Dubock, M. G. Mattei, and mediated endocytosis by blocking clathrin-coated pit formation. J. Cell Biol. P. R. Crocker. 1999. Identification and characterization of a novel siglec, siglec-7, 108:389. expressed by human natural killer cells and monocytes. J. Biol. Chem. 274: 29. Varki, A. 1994. ligands. Proc. Natl. Acad. Sci. USA 91:7390. 34089. 30. Varki, A. 1996. “Unusual” modifications and variations of vertebrate oligosac- 38. Kelm, S., A. Pelz, R. Schauer, M. T. Filbin, S. Tang, M. E. de Bellard, charides: are we missing the flowers for the trees? Glycobiology 6:707. R. L. Schnaar, J. A. Mahoney, A. Hartnell, P. Bradfield, et al. 1994. Sialoadhesin, 31. Zhou, L. J., and T. F. Tedder. 1995. A distinct pattern of cytokine myelin-associated glycoprotein and CD22 define a new family of sialic acid- by human CD83ϩ blood dendritic cells. Blood 86:3295. dependent adhesion molecules of the immunoglobulin superfamily. Curr. Biol. 32. Morse, M. A., L. J. Zhou, T. F. Tedder, H. K. Lyerly, and C. Smith. 1997. 4:965. Generation of dendritic cells in vitro from peripheral blood mononuclear cells 39. Freeman, S. D., S. Kelm, E. K. Barber, and P. R. Crocker. 1995. Characterization with granulocyte-macrophage-colony-stimulating factor, interleukin-4, and tumor of CD33 as a new member of the sialoadhesin family of cellular interaction necrosis factor-␣ for use in cancer immunotherapy. Ann. Surg. 226:6. molecules. Blood 85:2005. 33. Romani, N., D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, 40. Cornish, A. L., S. Freeman, G. Forbes, J. Ni, M. Zhang, M. Cepeda, R. Gentz, D. Niederwieser, and G. Schuler. 1996. Generation of mature dendritic cells from M. Augustus, K. C. Carter, and P. R. Crocker. 1998. Characterization of siglec-5, human blood: an improved method with special regard to clinical applicability. a novel glycoprotein expressed on myeloid cells related to CD33. Blood 92:2123. J. Immunol. Methods 196:137. 41. Floyd, H., J. Ni, A. L. Cornish, Z. Zeng, D. Liu, K. C. Carter, J. Steel, and 34. Zhou, L. J., and T. F. Tedder. 1996. CD14ϩ blood monocytes can differentiate P. R. Crocker. 2000. Siglec-8: a novel eosinophil-specific member of the immu- into functionally mature CD83ϩ dendritic cells. Proc. Natl. Acad. Sci. USA 93: noglobulin superfamily. J. Biol. Chem. 275:861. 2588. 42. Foussias, G., G. M. Yousef, and E. P. Diamandis. 2000. Identification and mo- 35. Cramer, S. O., C. Trumpfheller, U. Mehlhoop, S. More, B. Fleischer, and lecular characterization of a novel member of the siglec family (SIGLEC9). A. von Bonin. 2000. Activation-induced expression of murine CD83 on T cells Genomics 67:171. and identification of a specific CD83 ligand on murine B cells. Int. Immunol. 43. Zhang, J. Q., G. Nicoll, C. Jones, and P. R. Crocker. 2000. Siglec-9, a novel sialic 12:1347. acid binding member of the immunoglobulin superfamily expressed broadly on 36. Brinkman-Van der Linden, E. C., E. R. Sjoberg, L. R. Juneja, P. R. Crocker, human blood leukocytes. J. Biol. Chem. 275:22121. N. Varki, and A. Varki. 2000. Loss of N-glycolylneuraminic acid in human evo- 44. Crocker, P. R., M. Vinson, S. Kelm, and K. Drickamer. 1999. Molecular analysis lution: implications for sialic acid recognition by siglecs. J. Biol. Chem. 275: of sialoside binding to sialoadhesin by NMR and site-directed mutagenesis. Bio- Downloaded from 8633. chem. J. 341:355. http://www.jimmunol.org/ by guest on September 25, 2021 1772 CORRECTIONS

Wang, Y., D. Feng, G. Liu, Q. Luo, Y. Xu, S. Lin, J. Fei, and L. Xu. 2008. ␥-Aminobutyric acid transpoter 1 negatively regulates T cell-mediated immune responses and ameliorates autoimmune inflammation in the CNS. J. Immunol. 181: 8226–8236.

In Fig. 4, the wrong labels were assigned to Fig. 4, B and C, during the final formatting of the figure. The data and conclusions in the manuscript are not affected. The correct Fig. 4 is shown below. The published legend is correct but is shown again for reference.

FIGURE 4. Cell proliferation and cytokine production. A, At the peak of the disease (days 18–22 p.i.), a proliferation assay was performed. Stimulation index (SI) was the ratio of MOG35–55-stimulated MNC proliferation/spontaneous MNC proliferation. Quantification of cytokine mRNA expression in (B) spleen and (C) spinal cord from WT mice or GAT-1Ϫ/Ϫ mice with EAE was accomplished using real-time PCR. Results are shown as means Ϯ SD. Data .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ء ;p Ͻ 0.05 ,ء .(are representative of three individual experiments (n ϭ 5 mice/group

Scholler, N., M. Hayden-Ledbetter, K.-E. Hellstro¨m, I. Hellstro¨m, and J. A. Ledbetter. 2001. CD83 is a sialic acid-binding Ig-like lectin (Siglec) adhesion receptor that binds monocytes and a subset of activated CD8ϩ T cells. J. Immunol. 166: 3865–3872.

The authors thank Ajit Varki for bringing to their attention that CD83 does not share any sequence homology with CD33 and therefore cannot be classified as a Siglec. The Journal of Immunology 1773

In the second paragraph of the Introduction, on page 3865, the authors wrote: “We now present data suggesting that CD83 mediates adhesion of DC to circulating monocytes and to a fraction of activated T cells or stressed T cells by a specific binding of CD83 to a 72-kDa counterreceptor (ligand). We further show that CD83Ig binding to its ligand is eliminated by neuraminidase, an enzyme specific for the most common sialic acid, N-acetylneuraminic acid. Thus, CD83Ig binds to a carbohydrate epitope that depends on sialic acid residues. This classifies CD83 as a sialic acid-binding Ig-like lectin.”

In the next-to-last paragraph of the Discussion, on page 3871, the authors wrote: “CD83 is structurally related to the B7 ancestral gene family, and its closest homology is 23% of identity with the myelin protein Po, which is an I-type lectin that recognizes a sulfated carbohydrate. Therefore, it is highly interesting that the CD83 ligand contains a sialic acid, classifying CD83 as a siglec i.e., it belongs to a subfamily of I-type lectins that can bind sialic acids and presently includes nine members.”

The authors correct the inaccurate classification of CD83 as a Siglec molecule, yet confirm their findings. CD83 has a single extracellular V-type Ig-like domain that binds to sialic acid residues. This classifies CD83 as an I-type lectin (1–3) but not as a Siglec, due to CD83 lack of homology with CD33.

Accordingly, the title of the article should have been: “CD83 is an I-type lectin adhesion receptor that binds monocytes and a subset of activated CD8ϩ T cells.”

Additions to the References are listed below.

Angata, T., and E. Brinkman-Van der Linden. 2002. I-type lectins. Biochim. Biophys. Acta. 1572: 294–316.

Crocker, P. R., and A. Varki. 2001. Siglecs, sialic acids and innate immunity. Trends Immunol. 22: 337–342.

King, M. W. 2006. Glycoproteins: roles in cellular homeostasis and disease. In Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2nd Ed. R. A. Meyers, ed. Wiley-VCH, Weinheim, Germany.