Molecular Cloning of a Novel Human CC Chemokine Secondary Lymphoid-Tissue Chemokine That Is a Potent Chemoattractant for Lymphocytes and Mapped to Chromosome 9p13*

By searching the Expressed Sequence Tag (EST) data base, we identified partial cDNA sequences potentially encoding a novel human CC chemokine. We determined the entire cDNA sequence which encodes a highly basic polypeptide of 134 amino acids total with a putative signal peptide of 23 amino acids. The predicted mature protein of 111 amino acids has the four canonical cysteine residues and shows 21–33% identity to other human CC chemokines, but has a unique carboxyl-terminal extension of about 30 amino acids which contains two extra cysteine residues. The mRNA was expressed strongly in tissues such as the lymph nodes, Appendix, and spleen. The recombinant protein, which was produced by the baculovirus system and purified to homogeneity, was a highly efficient chemoattractant for certain human T cell lines and a highly potent one for freshly isolated peripheral blood lymphocytes and cultured normal T cells expanded by phytohemagglutinin and interleukin 2. Unlike most other CC chemokines, however, this novel chemokine was not chemotactic for monocytes or neutrophils, suggesting that it is specific for lymphocytes. From these results, we designated this novel CC chemokine as SLC fromsecondary lymphoid-tissuechemokine. SLC fused with the secreted form of alkaline phosphatase (SLC-SEAP) was used to characterize the SLC receptor. Binding of SLC-SEAP to freshly isolated lymphocytes was blocked by SLC (IC50, 0.12 nm) but not by any other CC chemokine so far tested, suggesting that resting lymphocytes express a class of receptors highly specific for SLC. By using somatic cell hybrids, radiation hybrids, and selected yeast and bacterial artificial chromosome clones, we mapped the SLC gene (SCYA21) at chromosome 9p13 and between chromosomal markers, D9S1978(WI-8765) and AFM326vd1, where the gene for another novel CC chemokine termed ELC from EBI1-ligandchemokine (SCYA19) also exists. Collectively, SLC is a novel CC chemokine specific for lymphocytes and, together with ELC, constitutes a new group of chemokines localized at chromosome 9p13.

It is now known that structurally related polypeptides, collectively called chemokines, play important roles in inflammatory and immunological responses primarily by virtue of their ability to recruit selective subsets of leukocytes (1,2). Some chemokines may also be involved in constitutive migration and homing of lymphocytes (3,4). Furthermore, some chemokines have been shown to have other biological activities such as suppression of hematopoiesis (5-7), stimulation of angiogenesis (8), suppression of angiogenesis (9,10), suppression of apoptosis (11), and suppression of infection of human immunodeficiency viruses (12)(13)(14). The chemokines are grouped into two major subfamilies from the arrangement of the amino-terminal two of the four canonical cysteine residues. One amino acid separates the two cysteines in the CXC chemokines, whereas the two cysteines are adjacent in the CC chemokines. The CXC chemokine genes are clustered at chromosome 4q12-q21, whereas the CC chemokine genes at chromosome 17q11.2. Most CXC chemokines are primarily chemotactic for neutrophils, whereas most CC chemokines are chemotactic for monocytes. Furthermore, most CXC and CC chemokines share receptors with some other members of the respective subfamilies (1,2). Thus, the functional redundancy may be more or less the norm of the chemokine system. In this context, three novel CC chemokines, TARC, 1 LARC, and ELC, that we have recently identified, are quite unique among the known CC chemokines because they are all functionally specific for lymphocytes, each interacts with a class of receptors not shared by any other CC chemokines so far tested, and their genes are distinctly mapped at chromosomes 16q13, 2q33-q37, and 9p13, respectively (15)(16)(17)(18)(19)(20). Thus, these chemokines may constitute a new category of CC chemokines, each playing a specific role in the immune system. Furthermore, besides CXC and CC chemokines, new molecules related to the chemokine superfamily have been identified. Lymphotactin/SCM-1 is a cytokine which carries only the second and the fourth of the four canonical cysteine residues and seems to act specifically on lymphocytes (21,22). Fractalkine is a transmembrane molecule that carries a chemokine-like domain 1 The abbreviations and other trivial names used are: TARC, thymus and activation-regulated chemokine; LARC, liver and activation-regulated chemokine; ELC, EBI1-ligand chemokine; SCM-1, single C motif 1; EST, expressed sequence tags; SLC, secondary lymphoid-tissue chemokine; FCS, fetal calf serum; PBMC, peripheral blood mononuclear cells; PBL, peripheral blood lymphocytes; PHA, phytohemagglutinin; IL, interleukin; PT, pertussis toxin; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; RANTES, regulated upon activation, normal T cell-expressed and secreted; RACE, rapid amplification of cDNA end; PCR, polymerase chain reaction; SEAP, secreted form of alkaline phosphatase; YAC, yeast artificial chromosome; BAC, bacterial artificial chromosome; G-protein, heterotrimeric guanine nucleotide-binding regulatory protein; CCR, CC chemokine receptor; EBI1, Epstein-Barr virus-induced gene 1; kb, kilobase(s); bp, base pair(s); MES, 2-morpholinoethanesulfonic acid; FPLC, fast protein liquid chromatography; contig, group of overlapping clones; AP, alkaline phosphatase. with a distinct CX 3 C motif on top of an extended mucin-like stalk. A soluble form of fractalkine was shown to be efficiently chemotactic for monocytes and T cells (23).
The data base of expressed sequence tags (EST) consists of partial single pass cDNA sequences from various tissues (24). Analysis of the EST data base is becoming a powerful approach to look for new members of gene families. Recently, we have identified a number of novel human CC chemokines by initially probing the EST data base with nucleotide and amino acid sequences of known CC chemokines (16,18,25). Here we report another novel human CC chemokine termed SLC from secondary lymphoid-tissue chemokine which was also initially identified in the EST data base. SLC is expressed strongly in lymphoid tissues such as lymph nodes, appendix, and spleen and is specifically chemotactic for lymphocytes. Freshly isolated peripheral blood lymphocytes possess a class of receptors binding SLC with high affinity and specificity. The SLC gene (SCYA21) is mapped to chromosome 9p13 where the gene for another novel CC chemokine ELC (SCYA19) also exists (18).

EXPERIMENTAL PROCEDURES
Cells-Human T cell lines, Hut78 and Hut102, and a human monocytic cell line, THP-1, were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS). 293/EBNA-1 cells were purchased from Invitrogen (San Diego, CA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS. Sf9 insect cells were maintained at 27°C in Grace insect medium (Life Technologies, Inc.) supplemented with 10% FCS. High Five TM cells were purchased from Invitrogen and maintained at 27°C in EX-CELL 400 medium (JRH Bioscience, Lenexa, KS). Peripheral blood mononuclear cells (PBMC) were isolated from EDTA-treated venous blood obtained from healthy adult donors by using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). Granulocytes were isolated from the pellet of Ficoll-Paque gradient by dextran sedimentation and hypotonic lysis of residual erythrocytes. Peripheral blood lymphocytes (PBL) (Ͼ97% lymphocytes) were negatively selected by MiniMACS (Milteny Biotec, Bergisch, Germany) after staining with fluorescein isothiocyanate-conjugated anti-CD14. In some experiments, T cells were expanded from PBMC by stimulation with phytohemagglutinin (PHA) (Life Technologies, Inc.) for 2 days and cultivation for another week with a supplement of 400 units/ml IL-2 (Shionogi, Osaka, Japan) (Ͼ99% CD3 ϩ T cells). In some experiments, cells were preincubated with 500 ng/ml pertussis toxin (PT) (Sigma) at 37°C for 90 min before assays.
EST Data Base Search-The EST data base (24), a division of Gen-Bank TM , was searched with nucleotide sequences and amino acid sequences of various CC chemokines as queries using the data base search and analysis service Search Launcher (27) available on the World Wide Web. The program used was Basic Local Alignment Search Tool (BLAST) (28).
Isolation and Characterization of cDNA Clones-By using primers based on an EST sequence (GenBank TM accession number W84422), cDNA clones were obtained by the method of rapid amplification of cDNA ends (RACE) (29). In brief, PCR was performed with 0.5 ng of human fetal lung cDNA commercially available for RACE-PCR (CLON-TECH, Palo Alto, CA) in 10 l of reaction mixture containing 0.2 mM each dNTP, 10 pmol each of primers (5Ј-RACE primer, 5Ј-CCTTCTTG-CATCTTGGGTTCAGGCTTC; 3Ј-RACE primer, 5Ј-GAAGCCTGAAC-CAAGATGCAAGAAGG; AP1 primer (CLONTECH) which is complementary to part of the cDNA adaptor ligated at both ends of the cDNA), 2.5 units of TAKARA LA Taq (Takara, Kyoto, Japan), 1 ϫ buffer supplied with the polymerase, and 0.55 g of TaqStart antibody (CLONTECH). The PCR conditions were 94°C for 1 min, 5 cycles of 94°C for 30 s and 72°C for 2 min, 5 cycles of 94°C for 30 s and 70°C for 2 min, and 25 cycles of 94°C for 30 s and 68°C for 2 min. The amplification products were cloned into pCR-II vector (Invitrogen) by T-A ligation and sequenced on both strands by using gene-specific primers and commercially available primers.
Northern Blot Analysis-The 0.5-kb BamHI-XbaI fragment of the SLC cDNA containing the entire coding region was labeled with 32 P using Prime-It II kit (Stratagene, La Jolla, CA) at a specific activity of 3.1 ϫ 10 9 cpm/g. The probe hybridized with a single band in human genomic DNA digested with EcoRI, HindIII, or BamHI at high stringency conditions. Northern blot filters containing human poly(A) ϩ RNA from various tissues (2 g/lane) were purchased from CLONTECH. Filters were hybridized with the 32 P-labeled probe at 42°C for 16 h in a solution consisting of 50% formamide, 5 ϫ SSPE, 10 ϫ Denhardt's solution, 2% SDS, and 100 g/ml denatured salmon sperm DNA, washed twice at 42°C for 30 min in 0.1 ϫ SSC and 0.1% SDS, and exposed to x-ray films (Eastman Kodak, New Haven, CT) at Ϫ80°C with an intensifying screen for 8, 24, 48, and 96 h.
Preparation of Recombinant SLC Protein-Recombinant SLC was produced by using Bac-to-Bac baculovirus expression system (Life Technologies, Inc.) following the protocol recommended by the manufacturer. Briefly, the BamHI-XbaI fragment of the SLC cDNA containing the entire coding region was subcloned into BamHI-XbaI sites of pFastBac 1 baculovirus transfer vector in the downstream of the polyhedrin promoter. The resulting recombinant plasmid termed pFastBac-NCC8.1 was transfected into Escherichia coli DH10Bac containing shuttle vector "bacmid" with a mini-attTn7 target site and the helper plasmid. The mini-Tn7 element on the pFastBac 1 plasmid transposes to the mini-attTn7 target site on the bacmid in the presence of transposition proteins provided by the helper plasmid. Insertion of the mini-Tn7 into the mini-attTn7 target site disrupts the coding sequence for the LacZ ␣ peptide, making colonies containing recombinant bacmids white in the presence of 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside. The resulting recombinant bacmid was transfected into Sf9 cells using Cellfectin reagent (Life Technologies, Inc.), and the recombinant viruses were obtained. High Five TM cells (Invitrogen) were infected with the recombinant viruses at multiplicity of infection of 10 -20. The culture supernatants collected 2 days after infection were mixed with 0.1 volume of 500 mM MES, pH 6.5, and applied to 1-ml cation-exchange Hitrap-S column (Pharmacia) equilibrated with 50 mM MES, pH 6.5, and 100 mM NaCl. The column on the FPLC system (Pharmacia) was eluted with 45 ml of a linear gradient of 0.1-1.0 M NaCl in 50 mM MES at a rate of 1 ml/min. The fractions containing recombinant SLC were pooled and injected into a reverse-phase high performance liquid chromatography column (4.6 ϫ 250 mm Cosmocil 5C4-AR-300, Cosmo Bio, Tokyo, Japan) equilibrated with 0.05% trifluoroacetic acid. The column was eluted with 60 ml of a linear gradient of 0 -60% acetonitrile in 0.05% trifluoroacetic acid at a flow rate of 1 ml/min. Fractions containing recombinant SLC were pooled and lyophilized. Protein concentrations were determined by BCA kit (Pierce). Endotoxin levels, which were determined by the Limulus amoebocyte lysate assay (QCL-1000, BioWhittaker, Walkersville, MD), were always Ͻ4 pg/g of recombinant SLC. The amino-terminal sequence analysis was done on a protein sequencer (Shimazu, Tokyo, Japan).
Chemotaxis Assay-Migration of cells was assessed in a 48-well chemotaxis chamber (Neuroprobe, Cabin John, MD) as described previously (15). In brief, Hut78, Hut102, THP-1, PBMC, PBL, and cultured normal T cells expanded by PHA and IL-2 were suspended in RPMI 1640 containing 1% bovine serum albumin and 20 mM HEPES, pH 7.2, at 2 ϫ 10 6 /ml (Hut78, Hut102, THP-1, PBMC) or at 4 ϫ 10 6 /ml (PBL, cultured T cells). Granulocytes were suspended in Hank's balanced salt solution containing 1% bovine serum albumin at 1 ϫ 10 6 /ml. The lower wells were filled with 30 l of assay buffer without or with chemokines, while the upper wells were filled with 50 l of cell suspension. The lower and upper wells were separated by a polycarbonate filter (Neuroprobe) with 5-m pore size. Polyvinylpyrrolidone-free filters were used for assays with neutrophils, PBL, cultured T cells, and T-cell lines, whereas polyvinylpyrrolidone-treated filters were used for assays with monocytes (PBMC) and THP-1. In assays using PBL and cultured T cells, the surface of filters facing the lower wells was precoated with 5 g/ml fibronectin for 2 h at room temperature and extensively washed with distilled water. Similarly, in assays using T cell lines, the surface of filters facing the lower wells was precoated with 5 g/ml collagen type IV for 2 h at room temperature and extensively washed with distilled water. Assays were carried out at 37°C for 1 h with neutrophils, 2 h with PBMC (monocytes) and THP-1, or 4 h with PBL, cultured normal T cells, and established T cell lines (Hut78 and Hut102). Filters were washed, fixed, and stained with Diff-Quick (Harleco, Gibbstown, NJ). The number of migrated cells in five randomly selected high power (ϫ 400) fields was counted. All assays were done in triplicate.
Calcium Mobilization Assay-Cells were incubated at 37°C for 1 h with 2 M Fura-2/AM (Molecular Probe, Eugene, OR) at 1 ϫ 10 6 /ml in RPMI 1640 containing 1% FCS and 20 mM HEPES, pH 7.2, in the dark. Cells were washed three times and resuspended at 1.25 ϫ 10 6 /ml in phosphate-buffered saline containing 1% FCS, 50 mM CaCl 2 , and 50 mM MgCl 2 . Two ml of cell suspension was placed in a cuvette and set into a luminescence spectrometer (LS50B, Perkin-Elmer, Norwalk, CT) with constant stirring. Emission fluorescent intensity at the wavelength of 520 nm was measured upon excitation at 340 nm and 380 nm with a time resolution of 2.5 or 5 points/s. Data were presented by the ratio of F 340 divided by F 380 (R 340/380 ). After each measurement, R 340/380 at the presence and absence of excess calcium was determined by the sequential addition of 10 l of 10% Triton X-100 and 120 l of 0.5 M EGTA.
Ligand-binding Assay-This was carried out essentially as described previously (16). In brief, to produce a soluble fusion protein of SLC with the secreted form of placental alkaline phosphatase (SEAP) with the carboxyl-terminal histidine tag, the SLC cDNA was amplified by PCR using the 5Ј-SalI-SLC primer (5Ј-GCCGTCGACACAGACATGGCT-CAGTCACTGGCT-3Ј) and 3Ј-SLC-XbaI primer (5Ј-GCCTCTAGATG-GCCCTTTAGGGGTCTGTGACCG-3Ј), digested with SalI and XbaI, subcloned into SalI-XbaI sites of the pDREF-SEAP(His) 6 vector (16) to make pDREF-SLC-AP. 293/EBNA-1 cells were transfected with pDREF-SLC-AP using LipofectAMINE TM (Life Technologies, Inc.). After 96 h, the supernatants were centrifuged, filtered (0.45 m), and stored at 4°C with 20 mM HEPES, pH 7.4, and 0.02% sodium azide. Alkaline phosphatase (AP) activity was measured by using Great Es-cApe Detection Kit (CLONTECH) and expressed with relative light units/s. In binding assays, PBL (2 ϫ 10 5 ) were incubated with SLC-SEAP (4 ϫ 10 6 relative light units/s) in the absence or presence of increasing concentrations of SLC or 200 nM of various chemokines at 16°C for 1 h in 200 l of RPMI 1640 containing 1% bovine serum albumin, 20 mM HEPES, pH 7.2, and 0.02% sodium azide. After washing five times, cells were lysed in 50 l of 1% Triton X-100 in 10 mM Tris-HCl, pH 8.0. Lysates were heated at 65°C for 10 min to inactivate cellular phosphatases. After centrifugation, SEAP activity in 25 l of the lysate was determined by the chemiluminescent assay as described above. All assays were done in duplicate.
Chromosomal To determine the SLC gene locus, these DNA samples were analyzed by PCR using SLC primers (ϩ5Ј-GCCTTGCCACACTCTTTCTC and Ϫ5Ј-CAAGGAAGAG-GTGGGGTGTA). The PCR conditions were 35 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 1 min. PCR products were electrophoresed on 2% agarose gel. The SLC product was 218 bp in length. 2 Four yeast artificial chromosome (YAC) clones of a contig containing a chromosome marker AFM326vd1 were purchased from Research Genetics (Huntsville, AL), and their DNAs were prepared as described previously (25). Using these DNAs, PCR was carried out with the SLC primers and ELC primers (18). Furthermore, previously selected bacterial artificial chromosome (BAC) clones (Research Genetics) containing the DNA fragments of human chromosome 9p13 3 were examined by PCR using the SLC and ELC primers. Primer sequences for AFM326vd1 and the nearby markers were taken from the Whitehead Institute for Biomedical Research/MIT YAC data base.

RESULTS
Cloning of SLC cDNA-To find new members of the human CC chemokine family, we searched the EST data base (24) with amino acid sequences and nucleotide sequences of various CC chemokines as probes. We have found a number of EST sequences derived from a single species of cDNA encoding a novel CC chemokine (GenBank TM accession numbers W17274, W67885, W84375, W84422, T25128, W67812, AA027314, AA027315, AA149456, and AA151607) (Fig. 1). To determine the full-length cDNA, we carried out the 5Ј-and 3Ј-RACE using primers designed from W84422 and human fetal cDNA commercially available for RACE-PCR (CLONTECH) as templates. The 5Ј-RACE further extended 53 nucleotides beyond the 5Ј end of the overlapping ESTs. Sequence comparison of the RACE fragments with the ESTs showed no misincorporation of nucleotides during the RACE-PCR. The full-length cDNA is 852 bp with an open reading frame of 402 bp starting with the first methionine and encoding a polypeptide of 134 amino acid residues ( Fig. 2A). The nucleotide sequence flanking the first methionine codon conforms well to Kozak's rule (32). The 3Јnoncoding sequence contains a canonical polyadenylation signal (AATAAA) and a single copy of ATTTA that may be an mRNA destabilization signal (33).
The deduced polypeptide sequence contains a hydrophobic amino-terminal region characteristic of a signal peptide with a putative cleavage site between Gly-23 and Ser-24 ( Fig. 2A) (34). The calculated molecular weight and pI of the predicted mature protein are 12,237 and 10.72, respectively. The polypeptide contains no putative N-glycosylation site. As shown in Fig. 2B, the mature protein (indicated as SLC, see below) shows 21-33% identities with other CC chemokines and retains four properly placed cysteine residues as well as certain amino acid residues highly conserved among the CC chemokines such as Pro-41, Tyr-48, Phe-64, Trp-82, and Val-83. However, the polypeptide has a carboxyl-terminal extension of about 30 amino acids in comparison with other CC chemokines. This region, which is highly rich in basic amino acids and contains two cysteine residues, has no homologous sequences in the GenBank TM data base.
Expression in Human Tissues-We examined constitutive expression of this novel CC chemokine in various human tissues by Northern blot analysis (Fig. 3). Consistent with the size of the cDNA plus poly(A) tail, a single species of transcripts of about 0.9 kb was detected strongly in lymph nodes, intermediately in spleen, small intestine, thyroid gland, and trachea, and weakly in heart, pancreas, thymus, and colon. When the mRNA expression in various lymphoid tissues was examined further, the transcripts were present at high levels in lymph nodes and appendix, at intermediate levels in spleen, and at low levels in thymus. The expression was very low in bone marrow and fetal liver and virtually negative in peripheral blood leukocytes. From this pattern of tissue expression, we designated this novel CC chemokine as SLC from secondary lymphoid-tissue chemokine.
Production of Recombinant SLC Protein-To obtain the SLC protein, High Five TM insect cells (Invitrogen) were infected with a recombinant baculovirus encoding SLC under the control of the polyhedrin promoter. Recombinant SLC was purified from pooled culture supernatants by cation-exchange FPLC and reverse-phase high performance liquid chromatography. SLC was eluted from the reverse-phase column as a single major peak at the acetonitrile concentration of 28% (Fig. 4A). The purified SLC migrated as a single band of about 15 kDa on SDS-polyacrylamide gel electrophoresis (Fig. 4B). Amino acid sequence analysis demonstrated that the amino terminus of mature SLC started at Ser-24 as predicted (data not shown). The final yield of SLC was ϳ0.8 g/ml starting culture supernatants.
Chemotactic Activity of SLC-Previously, we showed that two human T cell lines, Hut78 and Hut102, responded well to a T-cell-directed CC chemokine TARC in the chemotaxis assay (15). Because SLC was expressed most abundantly in lymph nodes (Fig. 3), SLC might be chemotactic for T cells. Therefore, we first tested the chemotactic activity of SLC using Hut78 and Hut102. As shown in Fig. 5A, SLC induced vigorous cell migration in both types of cells with a maximal chemotactic index (the ratio of the number of migrated cells toward a chemotactic factor divided by the number of cells migrated toward control medium) of Ͼ100 at 10 -100 nM. A checkerboard-type analysis revealed that the migration of the responder cells toward SLC was chemotactic and not chemokinetic (Fig. 5B). Furthermore, PT completely inhibited the migration of the cells toward SLC (Fig. 5C), suggesting that the SLC receptor is coupled with a trimeric G-protein of the G i class.
We next tested chemotactic activity of SLC on peripheral blood leukocyte subsets (Fig. 6). Freshly isolated PBL showed a vigorous migration toward SLC with a maximal chemotactic index of 3.7 Ϯ 0.5 (mean Ϯ S.E., n ϭ 3) at 0.1 nM. On the other hand, SLC induced no significant migration in neutrophils, monocytes, or a monocytic cell line THP-1 within a dose range from 0.01-100 nM. These cells, however, vigorously responded to respective positive controls, MCP-1 and IL-8. SLC also in- Induction of Calcium Flux by SLC-We next examined induction of calcium flux by SLC. Cells were loaded with Fura-2, a calcium indicator, and the intracellular calcium concentration (presented by R 340/380 ) was measured before and after addition of SLC. Fresh PBL did not show detectable levels of calcium mobilization in response to SLC (not shown). On the other hand, cultured normal T cells expanded with PHA ϩ IL-2 responded vigorously to SLC with an EC 50 of 1 nM (Fig. 7A). SLC, but not RANTES or MCP-1, desensitized cultured T cells to a subsequent stimulation with SLC (Fig. 7B). SLC did not induce calcium flux in neutrophils or monocytes although these types of cells responded vigorously to subsequent treatment with IL-8 or MCP-1, respectively (data not shown).
Specific Binding of SLC to Fresh PBL-Previously, we have characterized specific receptors for novel chemokines, TARC, LARC, and ELC, by using chemokines fused with the secreted form of alkaline phosphatase with the (His) 6 tag (SEAP) (18 -20). To prepare labeled SLC convenient for binding experiments, we employed the same strategy and prepared SLC-SEAP. The alkaline phosphatase allowed quantitative determination of specific binding whereas the (His) 6 tag was useful for one-step affinity purification with a nickel agarose column. In displacement-type experiments where fresh PBL were incubated with a fixed concentration of SLC-SEAP and increasing concentrations of unlabeled SLC, SLC competed with SLC-SEAP with an IC 50 of 0.12 nM (Fig. 8A). In heterologous competition experiments where fresh PBL were incubated with a fixed concentration of SLC-SEAP in the presence of indicated unlabeled chemokines at 200 nM, only SLC, but not any other CC chemokines so far tested, inhibited the binding of SLC-SEAP, indicating that resting PBL express a class of receptors highly specific for SLC (Fig. 8B).
Chromosomal Mapping of the SLC Gene-To determine the chromosomal localization of the SLC gene, we examined a panel of somatic cell hybrids each containing a single human chromosome by PCR using SLC-specific primers. The results showed that the gene was present on chromosome 9 (Fig. 9A). A similar PCR analysis using GeneBridge 4 radiation hybrids further placed the SLC gene near the chromosomal markers D9S1978(WI-8765) and AFM326vd1 that are located at 9p13 (data not shown). Consistently, the analysis on the Stanford G3 panel localized the SLC gene between markers NIB64 and D9S1978 (data not shown). Previously, we have mapped the gene for another novel CC chemokine ELC at the same locus (18). Therefore, we examined mega-YAC clones constituting a contig containing these markers for the presence of the SLC and ELC genes by PCR. Fig. 9B schematically shows the marker contents of each YAC clone. The SLC and ELC genes were found to exist within 1 megabase pair. To further narrow down the region, we examined BAC clones with ϳ120-kb inserts derived from this region by PCR and identified one clone, 235d21, that contains both the SLC and ELC genes. Thus, the two genes are closely linked, probably within 100 kb.

DISCUSSION
In the present study, we have described a novel human CC chemokine designated as SLC from secondary lymphoid-tissue chemokine, which was originally identified in the EST data base (Fig. 1). Even though SLC shows relatively low homologies to other CC chemokines (21-33% identity), SLC retains a number of amino acid residues conserved in most CC chemokines in addition to the four properly spaced cysteine residues (Fig. 2). However, in comparison with other CC chemokines, SLC has a carboxyl-terminal extension of about 30 amino acids which is characterized by an extra pair of cysteine residues and a high content of basic amino acids (Fig. 2). The murine CC chemokine JE is also known to have a carboxyl terminus extended by about 50 amino acids (35). However, the carboxyl-terminal extension of JE is characterized by a high content of serine/ threonine and has no homology with that of SLC.
SLC is expressed constitutively at high levels in lymphoid tissues such as lymph nodes, appendix, and spleen (Fig. 3). The recombinant SLC was a highly efficient chemotactic factor for two human T cell lines, Hut78 and Hut102, and a highly potent one for resting PBL and cultured normal T cells expanded with PHA ϩ IL-2 (Figs. 5 and 6). SLC was, however, not chemotactic for neutrophils, monocytes, or a monocytic cell line THP-1. SLC also induced vigorous transient calcium mobilization in cultured normal T cells expanded with PHA ϩ IL-2 (Fig. 7). Thus, SLC appears to act selectively on lymphocytes and especially T cells. Consistent with its activity on lymphocytes, SLC binds to fresh PBL with a high affinity (Fig. 8). Importantly, the class of receptors expressed on fresh PBL is highly specific for SLC and not shared by any other CC chemokines tested so far (Fig. 8).
In contrast to fresh PBL, cultured normal T cells expanded with PHA ϩ IL-2 did not show a typical bell-shaped doseresponse curve but showed a broad peak in the chemotaxis assay (Fig. 6). A similar tendency was also seen in the chemotactic responses of established T cell lines, Hut78 and Hut102 (Fig. 5A). Furthermore, SLC was much more potent for fresh PBL and cultured normal T cells than for Hut78 and Hut102 (compare Figs. 5 and 6). These discrepancies may indicate that there are at least two (and even more) classes of receptors for SLC with distinct affinities. Thus, fresh PBL may express mainly the high affinity class of receptors for SLC; normal T cells expanded with PHA ϩ IL-2 may express both the high and low affinity classes of receptors for SLC; and Hut78 and Hut102 may express mainly the low affinity class of receptors for SLC. Identification of the SLC receptors will help us examine such possibilities.
Thus far, we have identified four novel CC chemokines, TARC, LARC, ELC, and SLC that appear to act specifically on lymphoid cells. TARC is expressed mainly in the thymus and acts selectively on T cells of the CD4 ϩ lineage especially via its specific receptor CCR4 (15,20). LARC is expressed mainly in the liver and lung and acts selectively on lymphocytes via its specific receptor CCR6 which is expressed on both T and B cells (16,19). ELC is expressed strongly in most lymphoid tissues such as thymus, lymph nodes, and spleen and functions via its specific receptor CCR7/EBI1 which is known to be expressed on activated T and B cells (18). SLC is thus another lymphocytespecific CC chemokine mainly expressed in secondary lymphoid tissues such as lymph nodes, appendix, and spleen and acts via a class of receptors not shared by any other CC chemokines so far tested. Our recent experiments have shown that SLC acts on both T cells and B cells. Identification of the SLC receptors will help us define the exact types of cells responding to SLC.
Even though SLC and ELC are only weakly homologous (32% identity), the presence of their genes at the same chromosomal locus might suggest their functional relationship. In this study, however, the limited supply of recombinant ELC protein (18) precluded experiments such as cross-competition and desensitization between SLC and ELC. Thus, the functional relationship between SLC and ELC remains to be seen.
Collectively, it is tempting to speculate that TARC, LARC, ELC, and SLC represent a new category of CC chemokines that play specific roles in the immune system most probably by regulating migration and homing of particular subsets of lymphocytes within particular lymphoid tissue microenvironments, since selective migration and homing of lymphocytes into and/or within lymphoid tissues have been shown to be highly sensitive to pertussis toxin (36,37) and likely to be regulated by chemokines (3,4).