Expression Cloning of the STRL33/BONZO/TYMSTR Ligand Reveals Elements of CC, CXC, and CX3C This information is current as of September 24, 2021. Alyson Wilbanks, Susan Carr Zondlo, Kristine Murphy, Simona Mak, Dulce Soler, Patricia Langdon, David P. Andrew, Lijun Wu and Michael Briskin J Immunol 2001; 166:5145-5154; ; doi: 10.4049/jimmunol.166.8.5145 Downloaded from http://www.jimmunol.org/content/166/8/5145

References This article cites 43 articles, 23 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/166/8/5145.full#ref-list-1

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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. Expression Cloning of the STRL33/BONZO/TYMSTR Ligand Reveals Elements of CC, CXC, and CX3C Chemokines

Alyson Wilbanks,1 Susan Carr Zondlo,1 Kristine Murphy,1 Simona Mak, Dulce Soler, Patricia Langdon, David P. Andrew,2 Lijun Wu, and Michael Briskin3

STRL33/BONZO/TYMSTR is an orphan and HIV/SIV coreceptor receptor that is expressed on activated T lympho- cytes. We describe an expression cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an ␣ (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting expression of this receptor Downloaded from in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4؉ T ,lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19؉ B cells and CD14؉ /macrophages and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact. The Journal of Immunology, 2001, 166: 5145–5154. http://www.jimmunol.org/

he chemokines comprise an ever growing family of small sets it is likely that overlapping gradients of different chemo- secreted involved in a number of inflammatory kines and combinations of receptor ligand interactions might T and immunological processes including lymphocyte hom- determine directionality of leukocyte trafficking from the cir- ing, suppression and stimulation of , suppression culation into tissues (9, 11). of HIV infection, and enhancement of CTL responses (1–3). All Recently, several GPCRs have been cloned with no known nat- chemokines to date (with one exception noted) exhibit a con- ural ligands and are thus referred to as “orphan GPCRs” (12). One served structure that is predominated by conserved disulfide orphan receptor referred to as STRL33/BONZO/TYMSTR (re- by guest on September 24, 2021 bonds, Cys1-Cys3 and Cys2-Cys4 (1, 3). To date there are four ferred to herein as Bonzo), simultaneously identified in three subfamilies, based on the arrangement of N-terminal cysteine groups, was shown to be expressed on activated T cells, and can residues including the CC, CXC, and one member each of the serve as an HIV/SIV coreceptor. Subsequent studies have shown C and the CX3C whereby, in each case, X represents any res- Bonzo to be a receptor more frequently expressed on memory T idue other than cysteine (1, 3). The selective binding of the cells with a preferential expression on CD8-positive T cells (but is chemokine subfamily members to G -coupled receptors expressed on CD4-positive cells as well) (13–15). As no known (GPCRs) results in the subclassification of chemokine receptors chemokines have been shown to interact with Bonzo, it has re- 4 as CCR1–11, CXC chemokine receptor (CXCR) 1–5, XCR1, mained an orphan receptor, while sequence comparisons strongly and CX3CR1 (1, 2, 4–8). The selectivity of these receptor li- suggest that it is a CCR. Here, we use a novel expression cloning gand interactions will, in part, contribute to the specificity by strategy to isolate a cDNA that upon transfection into 293 cell which leukocyte subsets might preferentially localize to differ- recipients mediates a robust chemotactic response to Bonzo recep- ent extravascular sites (4, 9, 10). However, in many instances, tor transfectants. This cDNA encoded a novel chemokine, which as multiple receptors appear to be expressed on individual sub- we term CXCL16. Although it is, in the strictest definition, a member of the CXC family, CXCL16 is distantly related to all known chemo- Millennium Pharmaceuticals, Cambridge, MA 02139 kines and might be phylogenetically closer to certain ␤ chemo- Received for publication November 27, 2000. Accepted for publication February kines than ␣ chemokines. Additionally, it has a novel feature 5, 2001. that is shared with the only known CX3C chemokine, neuro- The costs of publication of this article were defrayed in part by the payment of page tactin/fractalkine, in that sequence predicts that it is membrane charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. bound and suspended by a heavily glycosylated mucin stalk (16, 1 A.W., S.C.Z., and K.M. contributed equally to this work. 17). We show that Bonzo/CXCL16 interactions define a unique 2 Current address: CuraGen Corporation, 322 East Main Street, Branford, CT 06405. receptor ligand pair as CXCL16 fails to bind all known che- mokine receptors. We also show in this study that this ligand 3 Address correspondence and reprint requests to Dr. Michael J. Briskin, Millennium Pharmaceuticals, Sidney Street, Cambridge, MA 02139. E-mail address: functionally interacts with activated lymphocytes and describe [email protected] novel features of expression on leukocyte subsets. These data, 4 Abbreviations used in this paper: CXCR, CXC chemokine receptor; GPCR, G along with the structural features of both Bonzo and CXCL16, protein-coupled receptor; BAB, binding assay buffer; MDC, macrophage-derived chemokine; MIP, macrophage-inflammatory protein; SDF, stromal cell-derived indicate that this receptor ligand pair is unique among all mem- factor; SLC, secondary lymphoid chemokine; EF1, elongation factor-1. bers of this family known to date.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 5146 EXPRESSION CLONING OF THE STRL33/TYMSTR/BONZO LIGAND

Materials and Methods For the chemotaxis assays in the expression-cloning screen (and with mAbs, isolation of primary cells, and cell lines macrophage supernatants), exponentially growing Bonzo transfectants were resuspended at a density of 1 ϫ 107/ml in an assay buffer that con- Medium for transfectants was standard DMEM with 10% FCS (Life Tech- sisted of DMEM supplemented with 10% bovine calf serum. The cell sus- nologies, Gaithersburg, MD). L1-2 cells, a murine B cell lymphoma, were pension (100 ␮l) was placed in the upper chamber of a 24-well chemotaxis obtained from E. Butcher at Stanford University (Stanford, CA). Receptor plate (Costar; Corning Glass, Corning, NY), and 0.5 ml of the supernatant transfectants expressing Bonzo were generated as previously described (18, from each transfected well was placed in the lower chamber. The plates 19). Chemokine receptor transfectants encoding CXCR5, CCR1–9, and were then incubated for 6–24 h at 37°C. Numbers of migrating cells were CX3CR1 were maintained in RPMI 1649 medium (Life Technologies) quantitated on a FACScan (Becton Dickinson) using the acquisition phase with 0.88 g/L gentamicin (G418), 10% HyClone serum, 10 nM HEPES, at 30 s. cDNA pools that, upon transfection, mediated chemotaxis above 1% penicillin/streptomycin, 1% L-glutamine, 1 mM sodium pyruvate, and background were subsequently enriched as previously described (23). 55 nM 2-ME. Chemotaxis with purified CXCL16 (described below) and L1-2 receptor Chronically activated Th1, Tr1, and Th2 lymphocytes were prepared as transfectants was identical with the set-up for expression cloning with the previously described (20, 21) with the addition of generation of Tr1 sub- exception that defined concentrations of chemokines were used and the sets. For Tr1 lymphocytes IL-10 was used at 10 ng/ml. After initial acti- buffer was changed to 50% RPMI, 50% M199 medium, and 0.5% BSA. ϩ vation in the presence of anti-CD28 and anti-CD3 mAbs Th1, Tr1, and Th2 Chemotaxis with in vitro-derived effector cells and isolated CD4 tonsil lymphocytes were restimulated for 5 days with specific cytokines, but with lymphocytes was performed using 3-␮m pore diameter gelatin-coated the addition of anti-CD95 ligand (1 ␮g/ml) to prevent apoptosis. Activated transwell inserts followed by growth of 2 ϫ 105 ECV304 cells as previ- Th1, Tr1, and Th2 lymphocytes were maintained in this way for a maxi- ously described (20). An aliquot of 200 ␮l of cell suspension (input of 8 ϫ mum of three cycles. 105 cells) was added to each insert. After 2 h, the inserts were removed and Tonsils were obtained from Massachusetts Eye and Ear (Cambridge, the number of cells that had migrated through the ECV304 monolayer to MA). Tissue was macerated with surgical scissors, mixed with DMEM the lower well was counted for 30 s on a Becton Dickinson FACScan with (Life Technologies) and passed through a cell strainer (Becton Dickinson, the gates set to acquire the cells of interest. Using this technique 100% Downloaded from Franklin Lakes, NJ). Cells were washed three times with PBS before stain- migration would be 25,000 cells for Th1/Th2 cells, where this number ing. CD4ϩ T cells were isolated by positive magnetic selection using CD4 represents the cells in the lower well counted on the FACScan for 1 min. microbeads (Miltenyi Biotec, Auburn, CA) and the manufacturer’s In all cases the data points were the result of duplicate wells, with the mean instructions. value shown and the error bars representing the sample SD. PBMC were isolated by density gradient centrifugation using Lym- phoprep (Nycomed, Oslo, Norway). were seeded into T75 Sequencing and sequence analysis flasks, allowed to adhere, and cultured for 10 days in RPM1 1640 supple- Sequencing of the entire cDNA insert was accomplished in conjunction http://www.jimmunol.org/ mented with 2.5 mM HEPES, 20 ␮g/ml gentamicin, 2 mM L-glutamine, with Seqwrite (Houston, TX) and the Tufts University sequencing core 1% penicillin/streptomycin, 2% nonessential amino acids, and 1 mM so- facility. Overlapping primers (originally using an SP6 primer for the 3Ј end dium pyruvate (all obtained from Life Technologies). On the 10th day of and a primer from the EF1 promoter for the 5Ј sequence) made to both culture, cells were incubated with either 50 ng/ml LPS (Sigma, St. Louis, strands as sequence information was gathered, resulting in complete un- MO) or 10 ng/ml TNF-␣ (R&D Systems, Minneapolis, MN) for 4 and 24 h. ambiguous sequence of both strands of the insert. Sequence comparison Supernatants were drawn off the cells and used in chemotaxis experiments with known chemokines was performed with the Lasergene system with Bonzo/L1.2 transfectants and GusB (CCR11)/L1.2 transfectants as a (DNAstar, Madison, WI), using the Clustal method with a gap penalty of negative control. 10 and a gap length penalty of 10. Pairwise alignment parameters were: MAbs reactive with Bonzo were generated by immunizing mice with ktuple ϭ 2, gap penalty-5, window ϭ 4, and diagonals saved ϭ 4. L1.2 cells expressing high levels of transfected Bonzo, as previously de- scribed (19). These mAbs were screened to ensure selectivity on numerous Construction and purification of recombinant CXCL16 His- by guest on September 24, 2021 L1.2 transfectants expressing chemokine receptors (CCR1–CCR8, CXCR1–CXCR5, GPR5, V28, and GPR9-6) or orphan GPCRs (Bob, tagged protein LyGPR, AF, APJ, and RDC). Chemokines were obtained from R&D Sys- Fusion proteins consisting of regions of CXCL16 fused to a C-terminal 5ϫ tems, PeproTech (Rocky Hill, NJ), or were synthesized using an automated histidine (His) were made in the pEF1/V5-His vector from Invitrogen solid-phase peptide synthesizer using previously described methods (22). (Carlsbad, CA) by fusion with a PCR-generated fragment containing the MAbs to CXCL16 were generated by immunization of BALB/C mice with entire extracellular domain of CXCL16. A 5Ј primer starting at the initi- the synthetic chemokine and screening for reactivity by ELISA. ation methionine with a BamHI site and a 3Ј primer ending at Val155 with an XbaI site were used in the PCR. Primer sequences are available upon FACS staining request. PCR inserts were purified from agarose gels along with vector DNA digested with BamHI and XbaI and recombinants isolated by stan- Before the addition of primary Ab, PBMCs or tonsil cells were incubated dard procedures. Maxiprep (Qiagen, Chatsworth, CA) DNA was used in in PBS with 5% True Clot human serum (Scantibodies, Santee, CA) and transient transfections of 10-cm plates using Lipofectamine (Life Technol- mouse IgG (Sigma) to prevent nonspecific staining. Cells were incubated ogies). Three days after transfection, supernatant was collected and cen- for 30 min with specific mAbs or isotype controls (Sigma). FITC- or Cy5- trifuged, and the clarified supernatant was then run over 5 ml wheat germ goat anti-mouse IgG Abs (Jackson ImmunoResearch, West Grove, PA) agglutinin conjugated to agarose in a column (Vector Laboratories, Bur- were used as the secondary Abs at 1/200 dilutions. Abs against all surface lingame, CA). PBS was used as the wash buffer and eluted with 100 mM Ags including CD4, CD14, and CD19 directly conjugated to PE were ob- acetic acid (pH 2.8). The 10-ml elution was brought to pH neutrality with tained from PharMingen (San Diego, CA). Cell staining was analyzed on 5 ml 1 M Tris base (pH 10.5) and run over an nickel-nitrilotriacetic acid- a FACScan (Becton Dickinson) using the CellQuest program. agarose column (Qiagen) washed with 50 mM NaH2PO4 (pH 8), 300 mM NaCl, and 20 mM imidazole. Elution was performed with 50 mM ϫ cDNA library expression cloning NaH2PO4 (pH 8), 300 mM NaCl, and 250 mM imidazole in 5 1-ml aliquots. Samples were dialyzed using a 3-ml 10,000 m.w. cutoff Slide-a- An expression library was made from spleen mRNA purchased from Clon- lyzer (Pierce, Rockford, IL) into PBS overnight. OD280 readings were tech Laboratories (Palo Alto, CA). cDNA was prepared as previously de- taken to determine the protein concentration. scribed (23) using synthesis reagents from Life Technologies with the ex- A Glycoprotein Deglycosylation Kit (Calbiochem, San Diego, CA) was ception that an ECOR1 adapter (Pharmacia, Piscataway, NJ) was ligated to ␮ Ј used to deglycosylate 2 g of CXCL16 protein. The standard denaturing the 5 end to facilitate directional cloning into the elongation factor-1 protocol was followed, and 150 ng each of untreated and deglycosylase- (EF1)-based vector pcDEF3. cDNA was partitioned into 96 pools of 1000 treated protein was run on a 4–20% Tris-glycine gel (Novex, San Diego, colonies each, and plasmid DNAs were prepared as previously described CA). Protein was transferred onto a nitrocellulose membrane (Novex) and (23). hybridized with a CXCL16 mAb (SD7). The NEN (Boston, MA) Renais- Transfections were performed into 293T cells as described previously sance system was used for detection. (23) with the exception that 60,000 cells/well were plated in collagen- coated plates (Becton Dickinson). Approximately 18 h after transfection, Receptor binding assays medium was changed to 0.5 ml/well of standard DMEM/10% FCS. Forty- eight hours after changing medium, supernatant was harvested, cell debris Exponentially growing L1.2 Bonzo transfectants were counted on the day was removed by microcentrifugation, and the medium was then used in of the assay and resuspended in binding assay buffer (BAB; 10 mM chemotaxis assays. HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/0.05% sodium azide at The Journal of Immunology 5147

2.5 ϫ 106/ml) at a density of 2.5 ϫ 106/ml. Purified CXCL16 was labeled with 125I using a sodium iodide method (Amersham, Arlington Heights, IL) and diluted in BAB to 4 nM. Cold CXCL16 (in PBS at 5.3 ␮M) was diluted for competition in BAB to 0.4, 2, 4, 20, 40, and 400 nM. Reactions con- sisted, in triplicate (for a final volume of 100 ␮l) of 50 ␮l of cells (1.25 ϫ 105 total), 25 ␮l 125I-CXCL16 (final concentration ϭ 1 nM), and 25 ␮l cold CXCL16 serially diluted from 100 to 0 nM. The specific activity of the labeled ligand was 7.8 ϫ 1010 nmol/cpm, total counts bound were 813.67, nonspecific counts were 222.33 resulting in 591.34 specific counts binding. For calculation of total 125I input, 50 ␮l cells was added to 25 ␮l 125I- CXCL16 and 25 ␮l BAB. All tubes were incubated for1hatroom tem- perature. Cells were spun down at 3500 rpm and washed five times in BAB ϩ 0.5 M NaCl. After the final wash, the cell pellet was resuspended in 100 ␮l wash buffer, and the 125I counts were calculated by a Cobra II Auto- Gamma scintillation counter (Packard, Meriden, CT) along with the total input sample. Binding data were calculated using a program written in Excel (L. Wu, unpublished observation). Calcium flux assays Bonzo/L1.2 transfectants or parental cells were washed once in PBS and resuspended in load buffer (HBSS, 20 mM HEPES, 2.5 mM probenecid, 0.1% BSA, and 1% FBS). Fluo-3 (Molecular Probes, Eugene, OR) was dissolved in 50% DMSO/50% pluronic acid and added to the cells at a final Downloaded from concentration of 4 ␮M. Cells were incubated for1hat37°C. Then, cells were washed twice in load buffer and plated into 96-well assay plates at 300,000 cells per well. The plate was spun for 5 min at 1200 rpm to pellet cells on the bottom of the well. Chemokine (50 ␮l) was added to a separate 96-well plate at varying concentrations to achieve final concentrations as ϩ indicated in the figure. Ca2 mobilization was then measured on a 96-well FIGURE 1. Sequence of phylogenetic analysis of CXCL16. A, Pre-

FLIPR System (Molecular Devices, Sunnyvale, CA). dicted signal peptide and transmembrane regions are indicated by a bold http://www.jimmunol.org/ Northern and Southern blots underline. Past the signal peptide and before the line demarcating the mu- cin domain is the predicted chemokine domain. All cysteines are indicated Human multiple tissue mRNA blots (Human I, II) from in bold. B, Dendrogram showing phylogenetic relationship of CXCL16 Clontech Laboratories were used for Northern blot analysis. A 400-bp with selected ␣ chemokines as well as MIP-1␣ and MIP-1␤. These se- Ј cDNA fragment representing the chemokine domain from the 5 EcoRI site quence data are available from GenBank under accession number to a EcoRV site was used as the probe template and primed with random AF337812. hexamers to produce an [␣-32P]dCTP-labeled probe. Hybridization was performed according to the manufacturer’s instructions with four addi- tional high stringency washes at 65°C in 0.1ϫ SSC and 0.1% SDS, and

then exposed to Kodak (Rochester, NY) XAR film with an intensifying by guest on September 24, 2021 screen. Blots were stripped and reprobed with a ␤-actin probe, the template of which was provided by the blot manufacturer. of six cysteines, with an N-terminal CYC motif consistent with For Southern blots, human genomic DNA (15 ␮g/reaction; Clontech members of the ␣ chemokine family (1, 3). The predicted chemo- Laboratories) was digested by BamHI, EcoRI, and HindIII and run on a kine domain is followed by a region that is largely composed of 1.4% agarose gel. DNA was transferred to a Protran nitrocellulose mem- S/T/G/P residues (37/84 aa or 44%), which is the hallmark of a brane (Schleicher & Schuell, Keene, NH) and cross-linked with a Strata- mucin domain (23), followed by a hydrophobic membrane span- linker. The probe template was the same as for the Northern blots. The blot was washed three times for 30 min at 60°C in 1ϫ SSC and 0.1% SDS, and ning a region of 25 residues and a short 28-residue cytoplasmic was exposed to film for 2 days at room temperature. tail, containing two additional cysteines in each of these regions. This overall structure is similar to the one CX3C chemokine, frac- Results talkine/neurotactin (16, 17), while a C-terminal mucin-like region Expression cloning reveals a novel chemokine has also been observed for JE (murine monocyte chemoattractant L1.2 Bonzo transfectants were tested in chemotaxis assays against protein-1) (24). Although this protein must be shed or proteolyti- a panel of all known chemokine receptor ligands (along with or- cally cleaved for chemotactic activity, there are no known dibasic phan chemokines), and no response was noted in any physiological cleavage sites before the transmembrane region. dose range (data not shown). As this data suggested that Bonzo Alignment of this new sequence (using only the chemokine do- likely binds an unknown ligand, we subsequently devised a strat- main) with a number of other ␣ chemokines shows it is distantly egy whereby we could transfect pools of cDNAs, harvest super- related to all CXC chemokines with the best homology with stro- natants, and ask whether we could detect a chemotactic response. mal cell-derived factor (SDF)-1 at 18.8% (Fig. 1B). Additional As Bonzo had previously been shown to be highly expressed in searches with different regions of the protein sequence reveal no spleen (13, 14), an expression library was constructed from spleen homology to any proteins in the database. Although the predicted mRNA. Transient transfection of 96 pools (representing ϳ1000 protein has characteristics of both CXC and CX3C chemokines, independent clones/pool) revealed one pool with a response above similarities to CC chemokines exist as well, including 1) homology background. Subsequent rounds of enrichment increased the ac- (closer than to any CXC chemokines) to macrophage-inflamma- tivity, and eventual deconvolution resulted in a single cDNA clone tory protein (MIP)-1␤ at 22%; 2) similarity to Eskine/CTAK and that, upon transfection, mediated a robust chemotactic response to Teck in having 27 residues between Cys 2 and 3, while this loop Bonzo receptor transfectants (data not shown). is generally no longer than 24 residues in all other chemokines (25); and 3) a relationship to secondary lymphoid chemokine Sequence analysis reveals a novel chemokine (SLC)/6-C-Kine in having six cysteine residues in its chemokine The isolated cDNA revealed a large open reading frame of 254 aa domain (26, 27). Due to the CYC motif, we provisionally refer to with an N-terminal hydrophobic predicted signal peptide of 27 aa this protein as a novel CXC chemokine termed CXCL16 (28) and (Fig. 1A). The signal peptide is followed by 90 residues with a total we propose to rename STRL33/BONZO/TYMSTR as CXCR6. 5148 EXPRESSION CLONING OF THE STRL33/TYMSTR/BONZO LIGAND Downloaded from http://www.jimmunol.org/

FIGURE 2. Analysis of CXCL16 RNA expression and genomic orga- nization. A, Membranes with poly(A)ϩ RNA from the indicated human tissues were hybridized with a chemokine domain CXCL16 probe (upper panels). ␤-Actin loading controls are shown in the panels below. The lower bands seen in muscle tissue lanes correspond to another ␤-actin isoform (Clontech Laboratories technical support, unpublished observations). B, by guest on September 24, 2021 Southern blot using the same probe, hybridized to genomic DNA digested with restriction enzymes as indicated.

Expression of CXCL16 RNA and Southern blot analysis To examine the tissue distribution of CXCL16, Northern blots were probed with a fragment encompassing the chemokine domain (Fig. 2A). A band of ϳ2.4 kb was seen with the highest expression in lung, liver, fetal liver, spleen, and peripheral blood leukocytes. FIGURE 3. CXCL16 is expressed on the surface of human monocytes Lower levels of expression were also observed in kidney, pan- and B cells and on tonsillar B cells A, PBMC from several donors were stained with an anti-CXCL16 mAb (SD7). Expression in one representative creas, lymph nodes, and placenta. Reprobing these blots with a ϩ ϩ donor was observed in both monocytes (CD14 ) and B cells (CD19 ). probe corresponding to the full length CXCL16 cDNA resulted in IgG1 isotype controls are in the bottom panels. B, Percentage of CXCL16ϩ extreme multiple banding, most likely due to repeat sequences cells varied by donor. C, Expression of CXCL16 on CD19 subsets of found in the 3Ј-most end of the cDNA and parts of the mucin freshly isolated tonsil lymphocytes. sequences as well (data not shown). Additionally, prior exposure of the blot before more stringent washes revealed the presence of other sized RNA species in certain tissues, indicating the possibil- As Northern blot analysis showed high expression in peripheral ity that related are expressed in some of the tissues examined blood leukocytes, we asked whether we could detect cell surface (data not shown). expression of CXCL16 on any leukocyte subsets. Staining was Southern blots containing human genomic DNA digested with observed on subsets of CD19ϩ and CD14ϩ peripheral blood leu- EcoRI, BamHI, or HindIII were also probed with the CXCL16 kocytes, indicating that APCs including B cells and monocyte/ chemokine domain probe (Fig. 2B). At moderate stringency, only macrophages express this chemokine as a cell surface ligand (Fig. one (BamHI and HindIII) or two (EcoRI) bands were observed, 3A). Although there was considerable variation in the percentage consistent CXCL16 existing as a single copy . of each cell type expressing CXCL16, this general staining pattern was observed in all donors examined (Fig. 3B). These observations CXCL16 is expressed on the surface of leukocyte subsets and raise the possibility that cell-cell contacts between activated T cell functional CXCL16 is shed from macrophages subsets known to express CXCR6 and APCs expressing CXCL16 Although sequence analysis suggests that CXCL16 can be pre- might result in subset-specific immune responses. In addition to sented as a cell surface molecule, we could not detect surface peripheral blood we also examined expression in tonsils and also expression of CXCL16 on our 293 transfectants (data not shown). found that subsets of CD19ϩ B cells stained specifically with anti The Journal of Immunology 5149

greatly increased on all three subsets, indicating that CXCR6 ex- pression might mark effector cells in settings of chronic inflam- mation (Fig. 5A). Upon examination of chemokine responsiveness, we observed that all three subsets subjected to multiple rounds of cytokine stimulation respond to RANTES, a ligand for both CCR5 and CCR1 (which marks all subsets), whereas only Th2 cells mi- grate in response to the CCR4 ligand macrophage-derived chemo- kine (MDC) (Fig. 5B). All three subsets migrate with similar ef- ficiency to CXCL16, showing that CXCR6 is functional on effector T cell subsets. We next asked whether activated cells from a chronically in- flamed tissue also express CXCR6 and respond to CXCL16. Stain- ing of tonsil-derived lymphocytes demonstrates that a subpopula- tion of CD4ϩ T cells expresses CXCR6 (Fig. 6A). Additionally, CXCL16 also mediates chemotaxis of the isolated CD4-positive cells. The observed chemotaxis is selectively blocked by anti- CXCR6 mAb 7F3, which indicates that this is a unique receptor FIGURE 4. Chemotaxis induced by CXCL16 secreted from human ligand interaction (Fig. 6B). macrophages. Supernatants taken from 4- and 24-h cultures of monocyte- Downloaded from derived macrophages induce chemotaxis of CXCR6/L1-2 transfectants. Levels of secreted CXCL16, as measured by chemotaxis, increased after 24-h stimulation with LPS and TNF-␣, whereas control GusB (CCR11) transfectants fail to chemotax. http://www.jimmunol.org/ CXCL16 mAbs (Fig. 3C), suggesting that in settings of chronic inflammation CXCL16 might participate in cell-cell interactions as well. We also asked whether functional chemokine could be shed from the surface of the leukocyte subsets expressing surface CXCL16. Cultured macrophages were propagated by adherence to plastic, and CXCL16 expression was still observed after several days in culture (data not shown). After replacement of medium, supernatants were collected in the presence or absence of inflam- by guest on September 24, 2021 matory mediators and examined for chemotactic activity. No in- crease of cell surface staining was observed after 4- or 24-h incu- bation with LPS or TNF-␣ (data not shown). After 4 h, minimal chemotactic activity was seen above background, with no discern- able difference between supernatants from unstimulated and stim- ulated cells. However, after 24 h, significant increase in activity was seen that was moderately increased by LPS and increased by ϳ2-fold in the presence of TNF-␣, whereas control orphan GPCR (GusB/CCR11) transfectants fail to chemotax (Fig. 4). These data indicate that functional chemokine was shed into the medium, sug- gesting that either increases in gene expression or processing might contribute to observed increase in biological activity.

Activated T lymphocyte subsets express CXCR6 and functionally respond to CXCL16 CXCR6 was originally cloned from activated T lymphocytes, and recent reports have demonstrated the expression of CXCR6 on activated CD4ϩ and CD8ϩ T lymphocyte subsets along with NK cells (13, 14, 29–31). To look at expression and function of Bonzo in activated T cells, we generated T helper subsets by polarization in the presence of specific Th1, Th2, and Tr1 cytokines in condi- tions of repeated rounds of cytokine stimulation to mimic settings FIGURE 5. Expression and function of CXCR6 on effector cell subsets. A, Th1, Th2, and Tr1 effector cells were generated from umbilical blood of chronic inflammation (21). Upon initial expansion in the pres- ϩ ence of Th1, Th2, or Tr1 cytokines, we observed expression of CD4 lymphocytes and stained after 1 cycle (primary stimulation) of cy- tokine stimulation with anti CCR7 mAb 7H12, anti CCR4 mAb 1G1, and CCR7 on all subsets, whereas CCR4 (as previously demonstrated) anti-CXCR6 mAb 7F3. B, After two rounds of cytokine stimulation (sec- is only expressed on Th2 cells, and CXCL16 was low or undetec- ondary stimulation) mAb staining was repeated. C, After repeated rounds ted on all subsets (Fig. 5A). Upon multiple rounds of cytokine of stimulation, only Th2 cells migrate to MDC, whereas all three subsets stimulation, levels of CCR7 were reduced in all subsets, suggest- show similar response to both RANTES and CXCL16. Chemokine con- ing a transition to a memory phenotype, whereas CCR4 expression centrations were 100 nM for CXCL16 and 100 ng/ml of MDC and was greatly increased on Th2 cells. CXCR6 expression was also RANTES. 5150 EXPRESSION CLONING OF THE STRL33/TYMSTR/BONZO LIGAND

FIGURE 6. Expression and function of CXCR6 on tonsil CD4-positive lymphocytes A, CD4ϩ T cells were isolated from fresh ton- sils and stained with anti-CXCR6 mAb 7F3 or an isotype control. B, Tonsil-derived CD4ϩ T cells chemotax to the indicated concentrations of CXCL16. Anti-CXCR6 mAb 7F3 reduced migration to background levels. CXCR6 L1.2

transfectants were simultaneously run in the Downloaded from same assay as a control. http://www.jimmunol.org/

Recombinant CXCL16 is heavily glycosylated might bind to these (and other nonchemokine-binding GPCRs),

To further examine chemokine binding and receptor specificity, a these data clearly illustrate that, for known chemokine receptors, by guest on September 24, 2021 recombinant form of CXCL16 was constructed into a fusion pro- only CXCR6 functionally responds to CXCL16. We next asked tein, which encompassed the entire predicted extracellular domain whether CXCL16 could specifically signal a rise in intracellular fused to a C-terminal polyhistidine sequence. Although CXCL16 calcium in the CXCR6-transfected cell line. CXCL16 mediates a has only one predicted N-linked glycosylation site (NETT at re- dose-dependent rise in intracellular calcium that is similar to that ␣ sides 168–171), we do predict a heavily glycosylated mucin se- seen for SDF-1 , the ligand for CXCR4, which is expressed on quence that is likely to be rich in O-linked glycans. The expression construct encoding recombinant CXCL16 was transfected into 293T cells and purified over successive wheat germ agglutinin and nickel-nitrilotriacetic acid columns. Western blotting with a poly- clonal Ab to the chemokine domain detects a prominent species of ϳ Mr of 40 kDa, which is twice the size of the predicted protein backbone of Mr 19 kDa. Treatment of the recombinant protein with a mixture of deglycosidases including N-glycosidase F, endo-␣- N-acetylgalactosaminidase, ␣2–3,6,8,9-neuraminidase, B1,4-ga- lactosidase, and ␤-N-acetylglucosaminidase resulted in a signifi- cant increase in mobility and a shift in Mr from 40 to 23 kDa (Fig. 7). This verifies the prediction that CXCL16, similar to fractalkine, is highly glycosylated (16).

Only CXCR6 functionally responds to CXCL16 Purified CXCL16 was tested in a chemotaxis assay and exhibited a robust response to CXCR6-L1-2 transfectants, showing a typical bell-shaped response with peak activity ranging from 10 to 50 nM (Fig. 8A). CXCL16 was then tested in a chemotaxis assay against FIGURE 7. Analysis of purified CXCL16 protein. Purified protein (150 a panel of all known chemokine receptors including CCR1-9, ng) was resolved on a 4–20% Tris-glycine gel, transferred to nitrocellulose, CXCR1-5, and CX3CR1. Although known ligands for these re- and probed with a 1/5 dilution of a supernatant containing an anti-CXCL16 ceptors exhibited a robust response in this assay (data not shown), mAb. Mobility is decreased to a size roughly corresponding to the protein recombinant CXCL16 failed to mediate a response to any receptor backbone after incubation in the presence of a glycosidase mixture including other than CXCR6 at all concentrations tested (Fig. 8B). Although N-glycosidase F, endo-␣-N-acetylgalactosaminidase, ␣2–3,6,8,9-neuromini- this experiment does not exclude the possibility that CXCL16 dase, ␤1,4-galactosidase, and ␤-N-acetylglucosaminidase. The Journal of Immunology 5151

L1-2 cells (Fig. 8C). As a control, parental L1-2 cells only re- sponded to SDF-1␣, showing that CXCR6 can specifically signal in response to CXCL16. Radiolabeled CXCL16 was used as a probe to directly examine binding to the CXCR6-transfected L1.2 cells. Increasing concen- trations of unlabeled CXCL16 competitively inhibited 125I-labeled

CXCL16 binding, with an IC50 of 1 nM (Fig. 9A), whereas labeled CXCL16 could not be inhibited by nonspecific chemokines (data not shown). Additionally, CXCL16 binding was not specific for control chemokine receptor transfectants including CCR6 and CCR7 (data not shown). Scatchard analysis demonstrates that

binding is high affinity with an average KD of 1 nM and with ϳ4000 binding sites/transfectant, indicating that CXCL16 is a high affinity, selective ligand for CXCR6 (Fig. 9B). These data indicate that although CXCL16 might have a function in direct cell-cell interactions as has been postulated for fractalkine, it is clearly ca- pable of functioning in a manner similar to more classical chemo- kines. Soluble CXCL16 might be rapidly shed from the cell sur-

face and participate in recruitment of lymphocyte subpopulation to Downloaded from sites of immune and inflammatory responses as well. It is possible that mechanisms of shedding in a particular microenvironment may represent another level of control of CXCL16-mediated phys- iological responses.

Discussion http://www.jimmunol.org/ We report the use of an expression cloning strategy to isolate a novel chemokine that mediates chemotaxis of receptor transfec- tants expressing the orphan GPCR, STRL33/Bonzo/TYMSTR (13, 14, 31), This chemokine contains an N-terminal CXC motif and is now named, according to recent systematic nomenclature (28), CXCL16; we also renamed STRL33/Bonzo/TYMSTR as CXCR6. This receptor ligand pair appears unique as no other receptors tested functionally respond to this new ligand. Although we have not ruled out the fact that CXCL16 might directly bind to other by guest on September 24, 2021 chemokine receptors (and other GPCRs as well), our data suggest that this interaction represents one of the few examples of exclu- sivity between a single ligand and chemokine receptor. It should also be noted that a murine cDNA encoding CXCL16 was also identified as a Bonzo ligand, and the human ortholog was also reported with essentially the same sequence as the one reported in this manuscript, with the exception of a longer signal peptide (32). This longer sequence is likely incorrect, as 1) we functionally cloned a shorter version (which is efficiently secreted) lacking this extended sequence; 2) this extended sequence does not align with the murine sequence; and 3) it fails to show a hydrophobic core commonly seen in observed signal peptides. CXCR6 has close homology to CCR7 and CCR9, which is of interest as SLC and Teck, which share some structural similarity to CXCL16, bind to these receptors, respectively (4, 8, 20, 33). Ad- ditionally, CXCR6 has been localized to 3, which clusters with other CC receptors as well. CXCL16 has been iden- tified on a first pass sequence of a genomic fragment from chro- mosome 17, meaning that CXCL16 would localize with CC, and FIGURE 8. Selective chemotaxis and calcium flux of L1.2 Bonzo trans- not CXC, chemokines. This genomic fragment (AC015913) is fectants in response to CXCL16. CXCL16 mediates selective chemotaxis to 100% similar to the CXCL16 cDNA over bp 77–220 (residues L1.2 CXCR6 (A and B) transfectants. A, Dose response of L1.2 Bonzo trans- 27–75). Based on the intron-exon structure of several other CC and fectants to CXCL16 in a chemotaxis assay. B, Results of chemotaxis assay CXC chemokines, this region corresponds to the predicted second using 10 nM CXCL16 in chemotaxis buffer and the following L1.2-transfected exon of the chemokine region of CXCL16. Therefore, although we cell lines: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, and Bonzo. Chemo- taxis buffer was used to measure background chemotaxis. Also tested (data not use the definition of the CYC motif in the N terminus to call shown): CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, and CXCL16 a CXC chemokine, one might want to consider the pos- CCR9. All assays were also performed at 100 nM with similar results (data not sibility that this is a completely novel class of chemokine that does shown). C, CXCL16 mediates specific calcium flux in only CXCR6/L1.2 not fall into any single system of classification. transfectants. SDF-1␣ mediates calcium flux in both CXCR6 and control L1.2 Although CXCR6 is phylogenetically related to chemokine re- cells, indicating a response to endogenously expressed CXCR4. ceptors, it is structurally diverse in that it lacks cysteine residues in 5152 EXPRESSION CLONING OF THE STRL33/TYMSTR/BONZO LIGAND

125 FIGURE 9. Binding of CXCL16 to the CXCR6 chemokine receptor in L1.2-transfected cells. A, I-CXCL16 is specific for L1.2 CXCR6 transfectants. Downloaded from Panel shows binding activity of L1.2 CXCR6 transfectants with 1 nM 125I-CXCL16 with or without increasing concentrations of cold competitor. The calculated value of 50% inhibition is 1 nM CXCL16. B, Scatchard analysis of binding data. Results shown are the average of two experiments. The calculated Kd is 1 nM.

the N terminus and the third extracellular loop that are conserved tions might occur between APCs and CXCR6 expressed on acti- http://www.jimmunol.org/ in all other receptors to date (14). One might postulate that vated T cell subsets, thus raising the possibility that immune re- CXCR6, due to the loss of a critical disulfide bond, has a unique sponses may be facilitated by direct interactions mediated between membrane topology and therefore might bind to a structurally cell types expressing this receptor ligand pair. This expression pat- unique chemokine. Interestingly, a shorter synthetic version tern also raises the possibility that other CD14-derived cells, such CXCL16, truncated at S95 before the fifth and sixth cysteine res- as monocyte-derived dendritic cells, might express this ligand as idues, lacks the potency of the entire extracellular region and ap- well. Along these lines, we have observed expression of CXCL16 pears to be unstable at higher temperatures or pH (data not shown). in subsets (with a similar staining pattern to what we observe in In contrast, other chemokines that have been similarly truncated, macrophages) of monocyte-derived dendritic cells, cultured in the such as Teck and fractalkine, retain activity (D. Soler, unpublished presence of IL-4 and GM-CSF (data not shown). This, to some by guest on September 24, 2021 data), suggesting the possibility that the extra cysteine residues in extent, parallels observations showing expression of murine the chemokine domain are critical for overall structure and binding CXCL16 in spleen- and -derived dendritic cells and to this novel receptor. We have shown by HPLC analysis that indicates similarity in observations between these studies (32). synthetic truncated CXCL16 shows deficiencies in efficient folding Conversely, expression of murine CXCL16 in B cells was not (data not shown), which might require these residues as well. Al- reported in that study although this does not preclude the possi- though these extra cysteine residues might be critical for efficient bility that expression might be seen there as well, as it may have folding, it is also possible that, due to the unique features of yet to be examined. CXCR6, this receptor ligand interaction might be completely Expression of the other known membrane-bound chemokine, unique and require residues that have not been required for other CX3CL1 (fractalkine/neurotactin), has also been shown on den- chemokines studied thus far. dritic cells in epidermis and lymphoid tissues (34, 35). CX3CL1 is CXCL16 is the second example of a chemokine that has the also chemotactic for activated T cells and may play a role in re- sequence of a transmembrane protein and would be predicted to be cruitment and adhesion of T cells to secondary lymphoid tissues expressed on the cell surface. Interestingly, our attempts to show and directly interact with T cells in immune responses. Therefore, expression on the 293 cells (and Chinese hamster ovary cell trans- CXCL16 may represent a second member of the chemokine family fectants) failed to demonstrate cell surface expression of CXCL16 that can play a dual role in recruitment and retention of lympho- and likely explains our success in the identification of a soluble cyte subsets. Other chemokines, such as MDC, have been shown active form derived from a membrane-bound chemokine. This sug- to be expressed by dendritic cells and play a role in attracting gests that in certain settings, the chemokine can be efficiently shed activated T cell subsets (36). MDC and fractalkine both map to into the surrounding medium; accordingly, we did observe func- chromosome 16q13 (35). Although CXCL16 might share some tional activity from supernatants derived from cultured macro- functional relationship with these chemokines, it appears to reside phages. This activity was increased in the presence of LPS and on and, therefore, does not map to a chromosome TNF-␣, indicating that CXCL16 may be rapidly shed from the cell encoding either these chemokines or CXC chemokines as well, surface and participate in recruitment of lymphocyte subpopula- further illustrating the unique nature of this new ligand. tion to sites of both immune and inflammatory responses. It is We have observed CXCL16 expression on tonsil-derived possible that mechanisms of shedding in a particular microenvi- CD19ϩs and CXCR6 expression on tonsil CD4ϩs. This observa- ronment may represent a level of control of CXCL16-mediated tion raises the possibility that, within secondary lymphoid tissue, B physiological responses. cells expressing CXCL16 might be activated by a T cell-dependent Additionally, we observed cell surface expression on subsets of process. MDC and fractalkine have also been shown to be ex- peripheral blood leukocytes including CD19ϩ B cells and CD14ϩ pressed by activated B cells as well (36, 37), although expression monocytes. This expression pattern suggests that novel interac- of fractalkine was observed only at the RNA level. This is the first The Journal of Immunology 5153 report of surface expression of a chemokine on a B cell suggesting Acknowledgments that binding between this surface-encoded chemokine and its cog- We thank Nasim Kassam for technical assistance in generation of hybrid- nate receptor on T cells might contribute to T cell activation. These omas and Jose-Carlos Gutierrez-Ramos and Craig Gerard for critical read- B cell/T cell interactions may occur after CXCR6ϩ T cell/dendritic ing of this manuscript. We also thank Keith Robison for initial identifica- cell interactions have been initiated, and it is possible that tion of the identity of CXCL16 to SR-PSOX (see Note added in proof). CXCL16 may play a role in both of these steps. As the cytoplasmic tail of CXCL16 has a number of potential phosphorylation sites, it References 1. Baggiolini, M., B. Dewald, and B. Moser. 1997. Human chemokines: an update. will also be of interest to see whether interactions with CXCR6 Annu. Rev. Immunol. 15:675. result in signals transmitted through this chemokine, thus mediat- 2. Baggiolini, M. 1998. Chemokines and leukocyte traffic. Nature 392:565. ing signals in APCs in a novel manner. 3. Ward, S. G., K. Bacon, and J. Westwick. 1998. Chemokines and T lymphocytes: more than an attraction. Immunity 9:1. Although chemokines have been classified on the basis of the 4. Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, and structure surrounding the N-terminal cysteine residues, another L. T. Williams. 1998. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. classification has emerged as of late, subdividing chemokines into Natl. Acad. Sci. USA 95:258. either homeostatic (lymphoid) or inflammatory, based on their site 5. Imai, T., M. Baba, M. Nishimura, M. Kakizaki, S. Takagi, and O. Yoshie. 1997. of production and cell types that respond to them. Certain chemo- The T cell-directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4. J. Biol. Chem. 272:15036. kines are produced in lymphoid tissues such as BCA-1 (in stromal 6. Jarmin, D. I., M. Rits, D. Bota, N. P. Gerard, G. J. Graham, I. Clark-Lewis, and cells in B cell follicles) and SLC (in high endothelial venules and C. Gerard. 2000. Identification of the orphan G-protein-coupled receptor 2 as CCR10, a specific receptor for the chemokine ESkine. J. Immunol. 164:3460. by stromal cells in T cells) (26, 27, 38). Others are more known to 7. Yoshida, T., D. Izawa, T. Nakayama, K. Nakahara, M. Kakizaki, T. Imai, Downloaded from attract activated leukocytes and are induced by inflammatory stim- R. Suzuki, M. Miyasaka, and O. Yoshie. 1999. Molecular cloning of mXCR1, the murine SCM-1/lymphotactin receptor. FEBS Lett. 458:37. uli including IL-8, RANTES, monocyte chemoattractant protein-1, 8. Yoshida, R., T. Imai, K. Hieshima, J. Kusuda, M. Baba, M. Kitaura, MIP-1␣, and eotaxin (among others) (39–42), which are all M. Nishimura, M. Kakizaki, H. Nomiyama, and O. Yoshie. 1997. Molecular known to be more highly expressed at sites of inflammation. Al- cloning of a novel human CC chemokine EBI1-ligand chemokine that is a spe- cific functional ligand for EBI1, CCR7. J. Biol. Chem. 272:13803. though many chemokines fall into these categories, some do not 9. Campbell, J. J., and E. C. Butcher. 2000. Chemokines in tissue-specific and clearly fit either paradigm. MDC, in addition to having a role in microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 12:336. 10. Gunn, M. D., V. N. Ngo, K. M. Ansel, E. H. Ekland, J. G. Cyster, and http://www.jimmunol.org/ secondary lymphoid tissue, has also been shown to be produced in L. T. Williams. 1998. A B-cell-homing chemokine made in lymphoid follicles inflamed lungs (43, 44). CXL16 might prove to be a dual-function activates Burkitt’s lymphoma receptor-1. Nature 391:799. chemokine as well, as its expression is shown here to be in sec- 11. Foxman, E. F., J. J. Campbell, and E. C. Butcher. 1997. Multistep navigation and the combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139:1349. ondary lymphoid tissues and in addition in nonlymphoid tissues, 12. Heesen, M., M. A. Berman, A. Charest, D. Housman, C. Gerard, and M. E. Dorf. most notably in liver and lung tissues. Additionally, we show that 1998. Cloning and chromosomal mapping of an orphan chemokine receptor: mouse RDC1. Immunogenetics 47:364. activated T cell subsets including chronic Th1, Th2, Tr1, and 13. Deng, H. K., D. Unutmaz, V. N. KewalRamani, and D. R. Littman. 1997. Ex- ϩ CD4 T cells derived from tonsil functionally respond to this che- pression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 388:296. mokine. The observations of surface expression of CXCL16 on 14. Liao, F., G. Alkhatib, K. W. Peden, G. Sharma, E. A. Berger, and J. M. Farber. APCs suggest a potential homeostatic role in lymphoid tissue, 1997. STRL33, a novel chemokine receptor-like protein, functions as a fusion by guest on September 24, 2021 whereas the functional interaction with polarized effector cells sug- cofactor for both macrophage-tropic and T cell line-tropic HIV-1. J. Exp. Med. 185:2015. gests a role in chronic inflammation as well. 15. Loetscher, P., M. Seitz, M. Baggiolini, and B. Moser. 1996. Interleukin-2 regu- As the sequencing of the genome becomes complete, the con- lates CC chemokine receptor expression and chemotactic responsiveness in T lymphocytes. J. Exp. Med. 184:569. cept of “novel sequence identification” is rapidly becoming a thing 16. Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi, of the past. There is now a new challenge to assign function of D. R. Greaves, A. Zlotnik, and T. J. Schall. 1997. A new class of membrane- thousands of expressed sequenced clones where classification via bound chemokine with a CX3C motif. Nature 385:640. 17. Pan, Y., C. Lloyd, H. Zhou, S. Dolich, J. Deeds, J. A. Gonzalo, J. Vath, conventional means is not possible. CXCL16 was represented in M. Gosselin, J. Ma, B. Dussault, et al. 1997. Neurotactin, a membrane-anchored the database as an expressed sequence tag for 5 years, but due to chemokine upregulated in brain inflammation. [Published erratum appears in 1997 Nature 389:100.] Nature 387:611. lack of homology could not be identified by searching with related 18. Wu, L., N. Gerard, R. Wyatt, H. Choe, C. Parolin, N. Ruffing, A. Borsetti, sequences. Although newer searching algorithms that use second- A. A. Cardoso, E. Desjardin, W. Newman, et al. 1996. CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Na- ary structure and pattern predictions might solve some of this di- ture 384:179. lemma, the cloning of this chemokine raises the possibility that 19. Ponath, P. D., S. Qin, T. W. Post, J. Wang, L. Wu, N. P. Gerard, W. Newman, several other functionally relevant proteins of this class might also C. Gerard, and C. R. Mackay. 1996. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. J. Exp. Med. 183: be identified in this manner. The isolation of this structurally 2437. unique chemokine and demonstration of binding to a novel che- 20. Zabel, B. A., W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, E. C. Ebert, N. Kassam, S. Qin, M. Zovko, et al. 1999. Human G protein-coupled mokine receptor (the first to lack cysteine residues previously con- receptor GPR-9–6/CC chemokine receptor 9 is selectively expressed on intesti- sidered essential for function) breaks all known paradigms for nal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is re- known chemokine receptor/ligand interactions. Additionally, the quired for thymus-expressed chemokine-mediated chemotaxis. J. Exp. Med. 190: 1241. observation of both surface expression of CXCL16 on APCs and 21. Murphy, E., K. Shibuya, N. Hosken, P. Openshaw, V. Maino, K. Davis, shedding of active soluble material raises the possibility that this K. Murphy, and A. O’Garra. 1996. Reversibility of T helper 1 and 2 populations is lost after long-term stimulation. J. Exp. Med. 183:901. chemokine might play dual roles in inflammation and homeostasis. 22. Topham, P. S., V. Csizmadia, D. Soler, D. Hines, C. J. Gerard, D. J. Salant, and W. W. Hancock. 1999. Lack of chemokine receptor CCR1 enhances Th1 re- Note added in proof. While in revision it came to our attention sponses and glomerular injury during nephrotoxic nephritis. J. Clin. Invest. 104: 1549. that in an attempt to use expression cloning from monocytes to 23. Shyjan, A. M., M. Bertagnolli, C. J. Kenney, and M. J. Briskin. 1996. Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates struc- isolate novel receptors binding to OxLDL, a cDNA encoding a ␣ ␤ tural and functional similarities to the 4 7-integrin binding domains of murine receptor termed SR-PSOX (scavenger receptor that binds phos- MAdCAM-1, but extreme divergence of mucin-like sequences. J. Immunol. 156: phatidylserine and oxidized lipoprotein) was identified. This 2851. 24. Kawahara, R. S., and T. F. Deuel. 1989. Platelet-derived growth factor-inducible cDNA turns out to encode CXCL16, further illustrating the pos- gene JE is a member of a family of small inducible genes related to platelet factor sibility of additional functional roles of this protein. 4. J. Biol. Chem. 264:679. 5154 EXPRESSION CLONING OF THE STRL33/TYMSTR/BONZO LIGAND

25. Baird, J. W., R. J. B. Nibbs, M. Komai-Koma, J. A. Connolly, K. Ottersbach, 36. Schaniel, C., E. Pardali, F. Sallusto, M. Speletas, C. Ruedl, T. Shimizu, T. Seidl, I. Clark-Lewis, F. L. Liew, and G. J. Graham. 1999. ESkine, a novel ␤-chemo- J. Andersson, F. Melchers, A. G. Rolink, and P. Sideras. 1998. Activated murine kine is differentially spliced to produce secretable and nuclear targeted isoforms. B lymphocytes and dendritic cells produce a novel CC chemokine which acts J. Biol. Chem. selectively on activated T cells. J. Exp. Med. 188:451. 26. Hedrick, J. A., and A. Zlotnik. 1997. Identification and characterization of a novel 37. Foussat, A., A. Coulomb-L’Hermine, J. Gosling, R. Krzysiek, ␤ chemokine containing six conserved cysteines. J. Immunol. 159:1589. I. Durand-Gasselin, T. Schall, A. Balian, Y. Richard, P. Galanaud, and D. Emilie. 27. Hromas, R., C. H. Kim, M. Klemsz, M. Krathwohl, K. Fife, S. Cooper, 2000. Fractalkine receptor expression by T lymphocyte subpopulations and in C. Schnizlein-Bick, and H. E. Broxmeyer. 1997. Isolation and characterization of vivo production of fractalkine in human. Eur. J. Immunol. 30:87. Exodus-2, a novel C-C chemokine with a unique 37-amino acid carboxyl-termi- 38. Nagira, M., T. Imai, K. Hieshima, J. Kusuda, M. Ridanpaa, S. Takagi, nal extension. J. Immunol. 159:2554. M. Nishimura, M. Kakizaki, H. Nomiyama, and O. Yoshie. 1997. Molecular 28. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and cloning of a novel human CC chemokine secondary lymphoid-tissue chemokine their role in immunity. Immunity 12:121. that is a potent chemoattractant for lymphocytes and mapped to chromosome 29. Sharron, M., S. Pohlmann, K. Price, E. Lolis, M. Tsang, F. Kirchhoff, 9p13. J. Biol. Chem. 272:19518. R. W. Doms, and B. Lee. 2000. Expression and coreceptor activity of STRL33/ 39. Bazzoni, F., M. A. Cassatella, F. Rossi, M. Ceska, B. Dewald, and M. Baggiolini. Bonzo on primary peripheral blood lymphocytes. Blood 96:41. 1991. Phagocytosing neutrophils produce and release high amounts of the neu- 30. Unutmaz, D., W. Xiang, M. J. Sunshine, J. Campbell, E. Butcher, and trophil-activating peptide 1/. J. Exp. Med. 173:771. D. R. Littman. 2000. The primate lentiviral receptor Bonzo/STRL33 is coordi- 40. Ponath, P. D., S. Qin, D. J. Ringler, I. Clark-Lewis, J. Wang, N. Kassam, nately regulated with CCR5 and its expression pattern is conserved between H. Smith, X. Shi, J.-A. Gonzalo, W. Newman, et al. 1996. Cloning of the human human and mouse. J. Immunol. 165:3284. eosinophil chemoattractant, eotaxin: expression, receptor binding and functional 31. Loetscher, M., A. Amara, E. Oberlin, N. Brass, D. Legler, P. Loetscher, M. properties provide a mechanism for the selective recruitment of eosinophils. D’Apuzzo, E. Meese, D. Rousset, J. L. Virelizier, et al. 1997. TYMSTR, a pu- J. Clin. Invest. 97:604. tative chemokine receptor selectively expressed in activated T cells, exhibits HIV-1 coreceptor function. Curr. Biol. 7:652. 41. Schall, T. J., K. Bacon, K. J. Toy, and D. V. Goeddel. 1990. Selective attraction 32. Matloubian, M., A. David, S. Engel, J. E. Ryan, and J. G. Cyster. 2000. A of monocytes and T lymphocytes of the memory phenotype by cytokine RAN- transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo. Nat. Im- TES. Nature 347:669. mun. 1:298. 42. Sherry, B., P. Tekamp-Olson, C. Gallegos, D. Bauer, G. Davatelis, S. D. Wolpe, Downloaded from 33. Zaballos, A., J. Gutierrez, R. Varona, C. Ardavin, and G. Marquez. 1999. Cutting F. Masiarz, D. Coit, and A. Cerami. 1988. Resolution of the two components of edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the macrophage inflammatory protein 1, and cloning and characterization of one of ␤ receptor for the chemokine TECK. J. Immunol. 162:5671. those components, macrophage inflammatory protein 1 . J. Exp. Med. 168:2251. 34. Papadopoulos, E. J., C. Sassetti, H. Saeki, N. Yamada, T. Kawamura, 43. Godiska, R., D. Chantry, C. J. Raport, S. Sozzani, P. Allavena, D. Leviten, D. J. Fitzhugh, M. A. Saraf, T. Schall, A. Blauvelt, S. D. Rosen, and S. T. Hwang. A. Mantovani, and P. W. Gray. 1997. Human macrophage-derived chemokine 1999. Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic up-regulated upon dendritic cell maturation. Eur. J. Immunol. 29:2551. cells, and natural killer cells. J. Exp. Med. 185:1595.

35. Kanazawa, N., T. Nakamura, K. Tashiro, M. Muramatsu, K. Morita, K. Yoneda, 44. Gonzalo, J. A., Y. Pan, C. M. Lloyd, G. Q. Jia, G. Yu, B. Dussault, C. A. Powers, http://www.jimmunol.org/ K. Inaba, S. Imamura, and T. Honjo. 1999. Fractalkine and macrophage-derived A. E. Proudfoot, A. J. Coyle, D. Gearing, and J. C. Gutierrez-Ramos. 1999. chemokine: T cell-attracting chemokines expressed in T cell area dendritic cells. Mouse monocyte-derived chemokine is involved in airway hyperreactivity and Eur. J. Immunol. 29:1925. lung inflammation. J. Immunol. 163:403. by guest on September 24, 2021