The Dendritic Cell-Specific CC-Chemokine DC-CK1 Is Expressed by Germinal Center Dendritic Cells and Attracts CD38-Negative Mantle Zone B Lymphocytes

Ernst Lindhout, Joost L. M. Vissers, Franca C. Hartgers, Richard J. F. Huijbens, Nicole M. Scharenborg, Carl G. Figdor, and Gosse J. Adema1

DC-CK1 (CCL18) is a dendritic cell (DC)-specific chemokine expressed in both T and B cell areas of secondary lymphoid organs that preferentially attracts CD45RA؉ T cells. In this study, we further explored the nature of DC-CK1 expressing cells in germinal centers (GCs) of secondary lymphoid organs using a newly developed anti-DC-CK1 mAb. Immunohistochemical analysis dem- onstrated a remarkable difference in the number of DC-CK1 expressing cells in adjacent GCs within one tonsil, implicating that the expression of DC-CK1 in GCs depends on the activation and/or progression stage of the GC reaction. Using immunohistology and RNA analysis, we demonstrated that GCDC are the source of DC-CK1 production in the GCs. Considering the recently described function of GCDC in (naive) B cell proliferation, isotype switching and Ab production, we investigated the ability of DC-CK1 to attract B lymphocytes. Here we demonstrate that DC-CK1 is a pertussis toxin-dependent chemoattractant for B lymphocytes with a preference in attracting mantle zone (CD38؊) B cells. The findings that GCDC produce DC-CK1 and attract mantle zone B cells support a key role for GCDC in the development of GCs and memory B cell formation. The Journal of Immunology, 2001, 166: 3284–3289.

endritic cells (DC)2 are a specific subset of APC that are initiation would occur in a multicell complex consisting of DC and highly capable to initiate primary immune responses, es- T and B lymphocytes. In vitro studies showed that DC can support D pecially the activation of naive (CD45RAϩ) T cells. Im- B cell proliferation, differentiation, and Ab production (7–10). mature, Ag-capturing DC, such as Langerhans cells, mature into a However, how B cells are recruited by DC into such a cluster in T cell activating DC by several proinflammatory products, like vivo is poorly understood. Several B cell-attracting chemokines LPS, TNF-␣, and IL-1␤, and by CD40 triggering. DC maturation expressed in secondary lymphoid tissues have now been described, involves a complex set of processes: 1) migration from periphery but these are involved in B cell homing to secondary lymphoid into secondary lymphoid organs; 2) expression of high numbers of organs (SDF-1␣ (CXCL12), SLC (CCL21), MIP3␤ (CCL19)) MHC-molecules; 3) up-regulation of costimulatory molecules, and (11–14) or to B cell follicles (BLC/BCA-1 (CXCL13)) (15–18) by guest on September 30, 2021. Copyright 2001 Pageant Media Ltd. 4) the release of cytokines and chemokines. DC are able to recruit rather than in recruiting B cells to DC. lymphocytes by producing various chemokines, such as Mip1␣ In this study we define GCDC as the source of DC-CK1 syn- (CCL3), Mip1␤ (CCL4), RANTES (CCL5), MDC (CCL22), thesis in the GCs. In addition, we demonstrate that DC-CK1 acts TARC (CCL17), and MIP3␤ (CCL19) (1, 2). Recently, we have as a chemoattractant for CD38-negative mantle zone (MZ) B lym- cloned a DC-specific chemokine, DC-CK1 (CCL18 (3), expressed phocytes, creating the conditions to establish DC-B cell and/or by DC in the T cell zone and germinal centers (GCs) of tonsils. DC-T-B cell interactions beneficial for inducing a primary immune DC-CK1 (also described as PARC) (4) preferentially attracts naive response. CD45RAϩ T lymphocytes, whereas CD45ROϩ T cells, mono- cytes, or DC are not attracted by DC-CK1. Because of its DC- https://www.jimmunol.org specific expression and its selectivity for naive T cells, DC-CK1 Materials and Methods may be very important in the onset of primary immune responses. Chemokines For initiating a humoral immune response, the recruited and sub- Recombinant DC-CK1 was produced in Escherichia coli strain X156F, sequently activated T cells have to interact with Ag-specific B transformed with the pOMP plasmid containing the DC-CK1 coding region lymphocytes. As suggested by several groups (5, 6), in humans this as described previously (3). After lysis, the periplasmic fraction was pu- rified on Q-Sepharose and S-Sepharose columns (Pharmacia LKB, Upp- sala, Sweden) using a linear NaCl gradient (0–0.1 M). The DC-CK1- Downloaded from enriched fractions were loaded on a reverse-phase column and eluted using Department of Tumor Immunology, University Medical Center, Nijmegen, The Neth- a linear gradient of 2–80% acetonitrile. erlands Received for publication July 24, 2000. Accepted for publication December 28, 2000. Immuno-labeling of cryosections The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Six-micrometer-thick freshly prepared cryosections of human tonsils were with 18 U.S.C. Section 1734 solely to indicate this fact. fixated for 15 min with 3% paraformaldehyde (in PBS) and permeabilized 1 Address correspondence and reprint requests to Dr. Gosse J. Adema, NCMLS, Tu- for 10 min with 0.1% saponin (Sigma, St. Louis, MO). Sections were mor Immunology, University Medical Center, Nijmegen St. Radboud, Geert Groote- stained with primary Abs for1hat37°C, followed by 30 min incubation plein Zuid 30, 6525 GA Nijmegen, The Netherlands. E-mail address: with biotin-conjugated isotype-specific sheep anti-mouse Abs (The Bind- [email protected] ing Site, Birmingham, U.K.), 30-min avidin-biotin-AP or avidin-biotin- 2 Abbreviations used in this paper: DC, dendritic cell(s); GC, germinal center; MZ, HRP complex (Vector Laboratories, Burlingame, CA), and stained with mantle zone; FDC, follicular DC(s); LD, low density; HD, high density; PTX, per- Fast-red, Fast-blue substrate (for alkaline phosphatase) (Vector Laborato- tussis toxin; ISH, in situ hybridization. ries) or AEC substrate (Zymed, San Francisco, CA). Sections were

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 3285

mounted in Kaiser’s glycerin-gelatin solution (Merck, Darmstadt, Germa- The transwell method was done according to Nagira et al. (14). Briefly, ny). For the detection of DC-CK1, we used AZN-CK18 (mouse anti-DC- B lymphocytes (106 per 100 ␮l, total cell fraction) in RPMI 1640/10% FCS CK1, IgG1). This mAb specifically reacts with DC-CK1 (CCL18) in direct were added to the upper compartment. The lower compartments contained and sandwich ELISA and does not cross-react either with MIP1␣ (CCL3), 600 ␮l RPMI 1640 with or without chemokines. Cells were allowed to which has the highest homology with DC-CK1 (CCL18), or with MIP1␤ migrate for4hat37°C and migrated cells were harvested, counted, and (CCL4), HCC-1 (CCL14), TARC (CCL17), MDC (CCL22), MIP3␤ labeled for FACS. (CCL19), MCP-1 (CCL2), RANTES (CCL5), or MCP-3 (CCL7) (E. Lind- hout, R. Torensma, L. Guelen, N. van Berkum, D. Elereld, M. Looman, T. RT-PCR Ruers, C. G. Figdor, and G. J. Adema, manuscript in preparation). Other primary Abs used are: DRC-1 (mouse anti-follicular DC (FDC), IgM; Total RNA was extracted using Trizol Reagent (Life Technologies, Breda, Dako, Glostrup, Denmark), PG-M1 (anti-CD68, IgG3; Dako), Edu-2 The Netherlands). Reverse transcription was performed using random hex- amers and Mo-MLV reverse transcriptase (Life Technologies). The PCR (mouse anti-CD4, IgG2a; NovoCastra, Newcastle, U.K.), B-A1 (mouse ␮ anti-CD4, IgG2a; Diaclone Research, Besanc¸on, France), RPA-T4 (mouse was performed in 50 l Taqman buffer A with cDNA of 25 to 50 ng total RNA, 1.25 U AmpliTaq Gold polymerase (Perkin-Elmer Applied Biosys- anti-CD4, IgG1; PharMingen, San Diego, CA). ␮ tems U.K., Warrington, U.K.), 5 mM MgCl2, 250 M dNTPs, 600 nM B lymphocyte isolation sense, and 600 nM antisense primer using ABI/PRISM 7700 (Perkin-Elmer Applied Biosystems U.K.). Primers used: DC-CK1: forward 5ЈCCTCT Purified tonsil B lymphocytes were isolated according to the method de- GCTCCTGTGCACAAGT-3Ј and reverse 3ЈTGCAGCTCAACAAT scribed by Falkoff et al. (19). Briefly, tonsillar cell suspensions were de- AGAAATCAATT-5Ј, amplifying a 424-bp product and GAPDH: forward pleted of T cells by rosetting with 2-aminoethylisothiouronium bromide- 5ЈGAAGGTGAAGGTCGGAGT-3Ј and reverse 5ЈGAAGATGGTGAT treated (Sigma) SRBC. The rosetted cells were removed by centrifugation GGGATTTC-3Ј, amplifying a 200-bp product. The DC-CK1 primers were on Lymphoprep (1077 mg/ml; Nycomed, Oslo, Norway). The final cell designed as such that they span an intron and do not cross-react with any population contained Ͼ98% CD20-positive cells (B cells) and Ͻ4% CD3- other chemokine. positive cells (T cells). Low-density (LD) and high-density (HD) B cell fractions were obtained Results according to the method of Koopman et al. (20). Briefly, B cells were Localization of DC-CK1 in secondary lymphoid organs centrifuged (15 min, 1200 ϫ g, 4°C) on a Percoll gradient (Pharmacia LKB), consisting of four density layers (1077/1067/1056/1043 mg/ml). Staining of tonsil sections with the anti-DC-CK1 mAb AZN-CK18 Cells at the 1043/1056 interface LD B cells and at the 1067/1077 interface demonstrates the same expression pattern as previously observed HD B cells were used. LD B cell fractions mainly consist of GC B cells (ϳ70% CD38ϩ, ϳ20% sIgDϩ and CD39ϩ), HD B cell fractions contained with in situ hybridization (ISH); DC-CK1-positive cells were both MZ and GC B cells (50% sIgDϩ and CD39ϩ, 40% CD38ϩ). present in both T and B cell areas of tonsils (Fig. 1b). The spec- Naive MZ B cells were obtained by depletion of CD38ϩ and IgGϩ cells ificity of the AZN-CK18 mAb was further supported by the ab- from the HD B cell fraction by incubating with anti-CD38 (T16; Beckman sence of reactivity against chemokines to which DC-CK1 is most Coulter, Fullerton, CA) and anti-IgG (8a4; Beckman Coulter) followed by homologous in an indirect ELISA (data not shown). In resting depletion of the labeled cells using sheep anti-mouse Ig-coated Dynabeads (Dynal, Oslo, Norway). Purified naive B cells fractions contained Ͼ95% lymph node, expression of DC-CK1 was observed in the T cell IgMϩ, IgDϩ, and CD39ϩ B cells. area (Fig. 1c). In spleen, some DC-CK1-positive cells were found Purified GC B cells were obtained by incubating the LD B cell fraction in the PALS (Fig. 1d). However, DC-CK1-positive cells were most with Abs against sIgD (JA11; Beckman Coulter) and anti-CD39 (AC2; abundant in highly inflamed tonsils. The number of DC-CK1-pos- Beckman Coulter) followed by depletion of the labeled cells using sheep anti-mouse Ig-coated Dynabeads (Dynal). Purified GC B cell fractions con- itive cells observed by immunohistology varied significantly be- sisted of Ͼ98% CD38ϩ cells and Ͻ2% CD39ϩ and sIgDϩ cells. tween tonsils from different donors as was previously observed for by guest on September 30, 2021. Copyright 2001 Pageant Media Ltd. DC-CK1 RNA by ISH, suggesting a strictly regulated expression GCDC isolation of DC-CK1 in vivo. Even within one tonsil, a remarkable differ- GCDC were purified as described by Grouard et al. (21). Briefly, tonsils ence in DC-CK1-positive cells between adjacent GCs was ob- obtained from children undergoing routine tonsillectomy were cut into served (Fig. 1b). small pieces and digested twice with collagenase IV (2 mg/ml; Sigma) and DNase I (0.04 mg/ml; Boehringer Mannheim, Mannheim, Germany) for 30 min at 37°C. Next the cells were washed, resuspended in PBS plus 2 mM EDTA plus 0.5% HSA (PBSe), and centrifuged over a Percoll (Pharmacia LKB) density gradient consisting of layers with densities of 1070, 1060, and 1030 mg/ml, respectively (15 min, 1200 ϫ g). Cells on top of the https://www.jimmunol.org 1060-mg/ml layer were harvested and CD3, CD14 CD19, and CD20-pos- itive cells were depleted using Dynabeads (Dynal). Cells were labeled with FITC-conjugated mouse anti-CD1a (IQP, Groningen, The Netherlands), CD3 (Beckman Coulter), CD16 (Becton Dickinson), CD20 (Dako) and CD34 (Beckman Coulter), PE-conjugated anti-CD11c (Becton Dickinson) and Cy5-conjugated anti-CD4 (Beckman Coulter). FITC-negative, CD4- Cy5 and CD11c-PE double-positive cells were isolated using a Coulter Elite FACSort. Purified GCDC were used for cytospin preparation and Downloaded from mRNA isolation (12.000 GCDC/PCR).

Chemotaxis assays B cell migration was measured using either 48-well chemotaxis chambers (Neuroprobe, Pleasanton, CA) or 5-␮m pore size bare filter Transwell in- serts (Costar, Cambridge, MA). Briefly, chemokines in RPMI 1640 were added to the lower chamber and were separated from 105 B cells in RPMI 1640/10% FCS by a 5-␮m PVP-free polycarbonate membrane (Costar). After incubation for1hat37°C, the membrane was removed and the upper FIGURE 1. Immunohistological characterization of DC-CK1 express- side washed with PBS, scraped to remove residual cells, and washed again. ing cells in tonsil, lymph node and spleen. Isotype control staining of a After methanol fixation and staining with Field’s A and Field’s B (BDH Chemicals, Poole, U.K.), the number of migrated cells was counted mi- tonsillar cryosection (a), anti-DC-CK1 staining (Fast-red) of a tonsillar croscopically in 5 high power fields (ϫ400) per well. Each experiment was cryosection showing positive cells in both T cell area and in GCs (encircled performed in triplicate. Inhibition of DC-CK1 induced migration was done in b), DC-CK1-positive cells in human lymph node (c), cryosection of by treatment with pertussis toxin (PTX; Sigma). For PTX-treatment, cells spleen showing some weak DC-CK1 positive cells (arrowhead) in the were preincubated with PTX (100 ng/ml) for 2 h, 37°C. PALS (d). Magnification ϫ10 (a and b) and ϫ20 (c and d). 3286 DC-CK1 IS EXPRESSED BY GCDC AND ATTRACTS B LYMPHOCYTES

Previously, we and others have reported the expression of DC- (Fig. 2, c and d). Moreover, RT-PCR analysis of mRNA isolated CK1 in GCs. However, the nature of the cell type responsible for from purified GCDC demonstrated the presence of DC-CK1 the DC-CK1 expression has not been identified yet. To define the mRNA in the sorted GCDC-fraction (Fig. 2e). Therefore, these DC-CK1 producing cells inside GCs, we performed double-label- data define GCDC as the DC-CK1 producing cells in the GC. ing experiments with mAb AZN-CK18 to detect DC-CK1 and mAbs DRC-1 or CD68 to detect FDC and tingible body macro- Chemotactic activity of DC-CK1 on B cells phages, respectively. The results of these staining experiments Previously, DC-CK1 was shown to be a potent chemoattractant for showed that both FDC and tingible body macrophages do not ex- freshly isolated naive (CD45RAϩ) T lymphocytes (3), which is press DC-CK1 (Fig. 2, a and b). The immunohistological finding that FDC do not express DC-CK1 was further supported by the finding that FDC, like B and T cells, do not express DC-CK1 mRNA (3) (data not shown). As expected, the DC-CK1 producing cells in the T cell areas do not express CD68 (data not shown). Staining of serial sections with AZN-CK18 and CD4, which is expressed by both GCDC and T cells inside GCs, suggested that GCDC produce DC-CK1. Because CD4/DC-CK1 double staining appeared technically impossible, we isolated GCDC from tonsils by extensive negative selection followed by positive selection for CD4 and the DC marker CD11c, and analyzed them for DC-CK1 expression at protein and mRNA level. Staining of cytospin prep- arations confirmed that isolated GCDC indeed react with anti-DC- CK1 mAb AZN-CK18 but not with an isotype-matched control Ab by guest on September 30, 2021. Copyright 2001 Pageant Media Ltd. https://www.jimmunol.org Downloaded from

FIGURE 3. DC-CK1 (CCL18) is a potent chemoattractant for freshly

isolated tonsillar B lymphocytes and is dependent on a G␣i-coupled recep- FIGURE 2. GCDC are the DC-CK1-expressing cells inside GCs. Dou- tor. Migration of total B cell population (a) or purified MZ (Ⅺ)vsGC(‚) ble staining of a GC with anti-DC-CK1 (blue) and DRC-1 (red) clearly B cells (b) are indicated as the number of migrated cells per 5 high power showing that FDC do not express DC-CK1 (a). Double staining of a GC fields minus the number of migrated cells in the medium control vs the with CD68 (red) and DC-CK1 (blue) indicating that macrophages (tingible amount of chemokine added to the lower well of a modified Boyden mi- body macrophages) also do not express DC-CK1 (b). Immunohistological gration chamber. The medium control values indicated as the mean of staining of GCDC cytospins with isotype control Abs (c) or anti-DC-CK1 duplicates for the total B cells, the CD38Ϫ and CD38ϩ B cells are 19, 24, Abs (d) clearly demonstrate DC-CK1 expression in GCDC. Original mag- and 7 per cells 5 high power fields, respectively. Five-micrometer pore size nifications: a and b, ϫ10; c and d, ϫ40. e, RT-PCR analysis of mRNA filters were used. c, Migration of MZ B cells to 1 ng/ml DC-CK1 (CCL18) isolated from 12.000 purified GCDC (CD11c- and CD4-positive). M ϭ is inhibited by pretreatment with PTX. Results are shown as mean of du- 100-bp marker, C ϭ negative control, D ϭ DC-CK1, G ϭ GAPDH plicates from a representative experiment of five. The Journal of Immunology 3287

FIGURE 4. DC-CK1 (CCL18) predominantly at- tracts CD38-negative B lymphocytes. Total human ton- sillar B cells were subjected to migration using transwell chemotaxis assays with 5-␮M pore size polycarbonate filter inserts and DC-CK1 at an optimal concentration of 1ng/ml. a, Input cells and migrated cells were harvested and phenotyped by flow cytometry with a FITC-conju- gated anti-CD38. b, Percentage of cells migrated to me- dium alone (Ϫ) or to medium (ϩ) DC-CK1. Data are given as mean (Ϯ SEM) from 13 independent experiments.

consistent with its expression in T cell areas of tonsil and lymph Discussion nodes. Because DC-CK1 is also expressed in B cells areas (GCs) For the initiation of primary immune responses, a sequence of of secondary lymphoid organs, we investigated its chemotactic ac- interactions among DC, T lymphocytes, and B lymphocytes have tivity toward B lymphocytes. Therefore, B lymphocytes were iso- to take place. Interaction between DC and T cells is the major by guest on September 30, 2021. Copyright 2001 Pageant Media Ltd. lated from human tonsils and analyzed for their ability to migrate event necessary to activate naive T cells. Recently, we have cloned in response to different concentrations of DC-CK1 in chemotaxis a DC-specific chemokine, DC-CK1 (3), that preferentially attracts assays. The results showed that DC-CK1 chemoattracts B cells in CD45RAϩ naive T cells and, therefore, may have an important a dose-dependent manner (Fig. 3a). To investigate whether a par- function in the attraction of (naive) T cells to Ag-presenting DC. ticular subpopulation of B cells is preferentially attracted by DC- ISH studies showed that DC-CK1 is expressed both in T cell areas CK1, isolated B cells were (negatively) separated into MZ and GC as well as in B cell follicles of secondary lymphoid organs (3, 4). B cell subsets and subjected to chemotaxis assays. Strikingly, we In this study, we further explored the expression of DC-CK1 by observed that DC-CK1 preferentially attracts MZ B cells (CD38Ϫ, DC in secondary lymphoid organs using DC-CK1-specific mAbs. IgGϪ) at an optimal concentration of 0.1–1 ng/ml DC-CK1 but not https://www.jimmunol.org Ϫ Ϫ Staining of tonsillar cryosections showed that labeling with DC- the GC B cells (CD39 , IgD ) (Fig. 3b). Furthermore, the specific CK1-Abs closely resembled the staining pattern previously ob- migration of B cells to DC-CK1 was completely abolished after served via ISH. Strikingly, a remarkable difference in the number pretreatment of the B cells with PTX, indicating the involvement of DC-CK1-expressing cells in adjacent GCs was observed (Fig. ofaG␣i-coupled receptor (Fig. 3c). Checkerboard analysis dem- onstrated that the effect of DC-CK1 on B cells is chemotactic 1), indicating that within one organ the expression of DC-CK1 in rather than chemokinetic (data not shown). GCs is likely depending on the activation and/or progression stage Downloaded from The preference of DC-CK1 in attracting MZ B cells could also of the GC reaction. In addition, in spleen and lymph node, expres- be demonstrated with total B cell populations in a transwell mi- sion of DC-CK1 could easily be detected. However, the number of gration assay using bare polycarbonate 5-␮m pore size filter in- positive cells was much lower than in inflamed tonsils. This indi- serts. Using this experimental setup, migrated cells could be col- cates that next to a basal constitutive level, DC-CK1 expression is lected and were subject to immunophenotyping. Therefore, the highly increased under inflammatory conditions. This conclusion transmigrated cells were collected, counted, and labeled with a is further supported by the finding that DC-CK1 secretion in- panel of mAbs. FACScan analysis showed that, in line with the creases 30-fold upon maturation of in vitro generated immature data obtained with the B cell subsets, the transmigrated population DC (32). A recent article by Reape et al. (22), showing the pres- was enriched for CD38-negative cells (Fig. 4a). On average 1.2% ence of DC-CK1 mRNA in atherosclerotic plaques, also supports of the total B cells migrated into the lower compartment under this finding. influence of DC-CK1 (ϳ3% of CD38-negative cells) compared Inside GCs, the DC-CK1-expressing cells were negative for with a nonspecific migration of 0.3% (Fig. 4b). These data dem- DRC-1 and CD68 (Fig. 2), implying that in contrast to the sug- onstrate that DC-CK1, next to being chemoattractive for naive T gestion of Hieshima et al. (4), FDC do not express DC-CK1. Fur- cells, is a chemoattractant for CD38-negative MZ B cells. thermore, also tingible body macrophages and macrophages in the 3288 DC-CK1 IS EXPRESSED BY GCDC AND ATTRACTS B LYMPHOCYTES

T cell area do not express DC-CK1. Because staining of serial the GCDC FACS sorting, and our colleagues in the Department of Oto- sections for DC-CK1 and CD4 suggested that GCDC are the DC- rhinolaryngology for providing us with tonsils. CK1-producing cells in GCs, we purified GCDC from tonsils. Analysis of isolated GCDC indicated that they indeed express DC- References CK1 at both the RNA and protein level (Fig. 2). Considering the 1. Sallusto, F., B. Palermo, D. Lenig, M. Miettinen, S. Matikainen, I. Julkunen, R. recently described function of GCDC in (naive) B cell prolifera- Fo¨rster, R. Burgstahler, M. Lipp, and A. Lanzavecchia. 1999. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur. J. Im- tion, isotype switching, and Ab production (23), we investigated munol. 29:1617. the ability of DC-CK1 to attract B lymphocytes. Using two dif- 2. Caux, C., S. Lebecque, Y. J. Liu, and J. Banchereau. 1998. Developmental path- ferent migration assays, DC-CK1 showed to be a (PTX-dependent) ways of human myeloid dendritic cells. In Dendritic Cells. M. T. Lotze and A. W. Thomson, eds. Academic Press, San Diego, pp. 63. chemoattractant for B lymphocytes with a preference for naive 3. Adema, G. J., F. Hartgers, R. Verstraten, E. De Vries, G. Marland, S. Menon, Ϫ ϩ (CD38 , IgM ) B cells (Figs. 3 and 4). The unresponsiveness of J. S. Foster, Y. Xu, P. Nooyen, T. McClanahan, K. B. Bacon, et al. 1997. A GC B cells to DC-CK1 could be due to down-regulation of the dendritic-cell-derived C-C chemokine that preferentially attracts naive T cells. Nature 387:713. putative DC-CK1R. A more likely explanation is a markedly im- 4. Hieshima, K., T. Imai, M. Baba, K. Shoudai, K. Ishizuka, T. Nakagawa, J. Tsuruta, M. Takeya, Y. Sakaki, K. Takatsuki, et al. 1997. 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