CCR7 Signaling Inhibits T Cell Proliferation

CCR7 Signaling Inhibits T Cell Proliferation Ekkehard Ziegler, Martin Oberbarnscheidt, Silvia Bulfone-Paus, Reinhold Förster, Ulrich Kunzendorf and Stefan Krautwald This information is current as of October 2, 2021. J Immunol 2007; 179:6485-6493; ; doi: 10.4049/jimmunol.179.10.6485 http://www.jimmunol.org/content/179/10/6485 Downloaded from References This article cites 50 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/179/10/6485.full#ref-list-1

Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CCR7 Signaling Inhibits T Cell Proliferation1

Ekkehard Ziegler,* Martin Oberbarnscheidt,* Silvia Bulfone-Paus,† Reinhold Fo¨rster,‡ Ulrich Kunzendorf,2* and Stefan Krautwald*

CCR7 and its ligands, CCL19 and CCL21, are responsible for directing the migration of T cells and dendritic cells into lymph nodes, where these cells play an important role in the initiation of the immune response. Recently, we have shown that systemic application of CCL19-IgG is able to inhibit the colocalization of T cells and dendritic cells within secondary lymphoid organs, resulting in pronounced immunosuppression with reduced allograft rejection after organ transplantation. In this study, we demonstrate that the application of sustained high concentrations of either soluble or immobilized CCL19 and CCL21 elicits an inhibitory program in T cells. We show that these ligands speciﬁcally interfere with cell proliferation and IL-2 secretion of CCR7؉ ,cells. This could be demonstrated for human and murine T cells and was valid for both CD4؉ and CD8؉ T cells. In contrast CCL19 had no inhibitory effect on T cells from CCR7 knockout mice, but CCR7؊/؊ T cells showed a proliferative response upon

TCR-stimulation similar to that of CCL19-treated wild-type cells. Furthermore, the inhibition of proliferation is associated with Downloaded from delayed degradation of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 and the down-regulation of CDK1. This shows that CCR7 signaling is linked to cell cycle control and that sustained engagement of CCR7, either by high concentrations of soluble ligands or by high density of immobilized ligands, is capable of inducing cell cycle arrest in TCR-stimulated cells. Thus, CCR7, a chemokine receptor that has been demonstrated to play an essential role during activation of the immune response, is also competent to directly inhibit T cell proliferation. The Journal of Immunology, 2007, 179: 6485–6493. http://www.jimmunol.org/ ffective T cell priming depends on distinct interactions was shown by the efficacy of FTY720. This drug induces a mi- between T cells and APCs in secondary lymphoid or- gratory block that prevents lymphocytes from exiting the lymph E gans. The contact between both cell types is the result nodes. A notable immunosuppressive effect could be demonstrated of a highly coordinated migration and functional maturation in human solid organ transplantation (6, 7). program in which the chemokine receptor CCR7 and its ho- There is emerging evidence that CCR7 signaling can also influ- meostatic ligands, CCL19 and CCL21, play pivotal roles (1). ence other key steps in the immune response. For example, CCL19 The essential role of CCR7 and its ligands in the migration of costimulates LFA-1 activation (8) by accelerating the movement mature dendritic cells (DCs)3 and naive T cells to lymph nodes of T cells scanning for their Ag (9, 10). Furthermore, subsets of T was largely discovered by studying plt/plt mice, which lack the cells require CCR7 for efficient priming (11), and DC maturation by guest on October 2, 2021 Ϫ/Ϫ CCR7 ligands (2), and CCR7 gene-deficient (CCR7 ) mice and endocytic capacity are facilitated by CCL19 (12–14). (3). Among other mediators, activated DCs produce proinflam- Although these effects are still being investigated, blocking matory cytokines and subsequently up-regulate CCR7, which these coactivation capacities and impairing cellular migration are makes them responsive to CCR7 ligands. This in turn promotes promising targets for suppressing cellular immune responses. DC migration from peripheral tissues to secondary lymphoid In our previous work (15), systemic application of pharmaco- organs, such as lymph nodes and spleen. Furthermore, CCR7 is logical doses of CCL19-IgG in mice was used to disturb this par- also important in mediating colocalization and, thus, Ag-spe- ticular chemokine/chemokine-receptor system. We were able to cific T cell activation (4, 5). show that recirculation and colocalization of T cells and DCs in An efficient way to induce immunosuppression is to inhibit the secondary lymphoid organs were impaired, which prompted us to entry and exit of immune cells in secondary lymphoid organs. test CCL19-IgG in different models of solid organ transplantation. Proof of the principle for this mechanism of immunosuppression Using murine models for allogeneic kidney as well as allogeneic heart transplantation, we were able to show that treatment with CCL19-IgG significantly delayed allograft rejection in vivo. In *Department of Nephrology and Hypertension, University of Kiel, Kiel Germany; particular, we could further demonstrate that treatment with †Department of Immunology and Cell Biology, Research Center Borstel, Borstel, Germany; and ‡Institute of Immunology, Hannover Medical School, Hannover, CCL19-IgG had a significant impact on Ag-induced T cell prolif- Germany eration in vivo. However, because these in vivo experiments can- Received for publication May 1, 2007. Accepted for publication September 1, 2007. not differentiate between effects mediated by impaired migration The costs of publication of this article were defrayed in part by the payment of page and/or potential direct cellular events, we set up this study to test charges. This article must therefore be hereby marked advertisement in accordance the hypothesis that the CCR7 ligands CCL19 and CCL21 are re- with 18 U.S.C. Section 1734 solely to indicate this fact. sponsible for reduced T cell proliferation by a direct cellular effect. 1 This study has been supported by a grant from the Medical Faculty of the University of Kiel, Kiel Germany. In the current study, we present evidence that CCL19 and 2 Address correspondence and reprint requests to Dr. Ulrich Kunzendorf, Department CCL21 are also able to directly inhibit T cell proliferation after of Nephrology and Hypertension, University of Kiel, Schittenhelmstrasse 12, 24105 TCR activation, and we show that CCR7 signaling is linked to the Kiel, Germany. E-mail address: [email protected] regulation of cell cycle progression. 3 Abbreviations used in this paper: DC, dendritic cell; CDK, cyclin-dependent kinase; These data add a new aspect to the multiple capabilities of this CHO, Chinese hamster ovary; hIgG, human IgG. chemokine receptor. We also show that chemokines and their re- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 ceptors are closely linked not only to cell migration but also to the www.jimmunol.org 6486 CHEMOKINE SIGNALING PATHWAYS

FIGURE 1. CCL19 and CCL21 inhibit allogeneic MLR. Splenocytes from BALB/c mice (effector cells) were mixed and cocultured for 4 days at a 1:1 ratio with allogeneic C57BL/6 spleen cells (stimulator cells) previously treated with mitomycin C. Proliferation was measured by BrdU incorporation and subsequent detection with a chemiluminescence immunoassay. MLR was analyzed in the presence of the indicated concentrations of recombinant CCL19, CCL19-IgG, and Fc receptor-mutated CCL19-⌬IgG, and CCL21-IgG. PBS (vehicle) and hIgG served as controls. Mean proliferation Ϯ SD from at least p Ͻ ,ء ;three experiments are shown 0.05. rlu, Relative luminescence unit. Downloaded from

homeostasis of the immune response by controlling other cell Murine lymphocytes were isolated from spleen and lymph node sus- functions such as proliferation. pensions of untreated mice. T cells were purified using a MACS system (Miltenyi Biotec) after magnetic labeling and depletion of non-T cells (Pan T cell Ab mixture; Miltenyi Biotec). Human T cells were extracted from Materials and Methods healthy donor blood. Purity levels of Ͼ90% of T cells were confirmed by Generation, expression, and characterization of fusion protein anti-CD3 staining (anti-CD3-PC5, clone UCHT1; Beckman Coulter) and http://www.jimmunol.org/ The CCL19-IgG fusion protein was generated as described previously (15). FACS analysis. FACS analysis was performed on a Coulter Epics XL flow The CCL21-IgG construct was generated by exchanging the CCL19 do- cytometer (Beckman Coulter). Analyses of flow cytometry listmode data main with the CCL21 domain, which was obtained by PCR amplification files were performed using WinMDI 2.8 software. of murine lymph node cDNA. The primers were designed with restriction sites appropriate for ligation into the IgG1-coding vector pCDNA3.1 (sense: 5Ј-CTGCCTAAGCTTTCTGCCATGGCTCAGATGATGACTCT Cell proliferation assays GAG-3Ј; antisense: 5Ј-CACATCTCGGATCCCATCCTCTTGAGGGCT GTGTCTG-3Ј). To perform a MLR, splenocytes from C57BL/6 mice were treated with 50 The chimeric molecules were expressed in suspension Chinese hamster ␮g/ml mitomycin C (Sigma-Aldrich) and mixed (as stimulator cells) with allogeneic BALB/c spleen cells (as responders) at a 1:1 ratio and a final

ovary (CHO) cells that were adapted to grow in serum-free ProCHO4- by guest on October 2, 2021 CDM medium (Cambrex). The culture supernatant containing either se- density of 2 ϫ 105 cells/well. For in vitro T cell proliferation assays, creted CCL19-IgG1 or CCL21-IgG was purified using protein A-Sepha- primary T cells were cultured with either 0.1–3 ␮g/ml plate-bound anti- rose Fast Flow (Amersham Biosciences) as described previously (16). human CD3 (clone OKT3; Janssen-Cilag) or anti-mouse CD3 (clone 145- Protein concentrations were determined with the Bio-Rad protein assay; 2C11; BD Pharmingen) and 1 ␮g/ml anti-human CD28 (clone CD28.2, BD serum concentrations in mice were determined by an ELISA capture assay Pharmingen) or anti-mouse CD28 (clone 37.51, BD Pharmingen), respec- as previously described (17). The biologic activity of the recombinant fu- tively. For Ag-specific T cell priming experiments, primary T cells were sion proteins was routinely checked in a chemotaxis assay. cocultured with activated DCs at a DC/T cell ratio of 1:40. Cells were To exclude an Fc-mediated effect by Fc␥R binding or complement ac- seeded (in quadruplicate) in 96-well round-bottom microtiter plates in the ⌬ tivation, we created a CCL19-control protein (CCL19- IgG). Three amino presence of rCCL19 (R&D Systems), CCL19-IgG, CCL19-⌬IgG, CCL21- ␥ acids within the hinge region critical for Fc R binding (18) at positions 233 IgG, or equimolar ChromePure human IgG (hIgG; Dianova) at 37°C for a 3 to 235 were mutated (ELL PVA) using the QuikChange site-directed total of 4 days. As an additional specificity control 2.5 ␮M eotaxin mutagenesis kit from Stratagene. (CCL11) (R&D Systems) was added in a subset of assays. After 3 days, 10 ␮ Animals, cells, and cell lines M BrdU was added to each well. T cell proliferation was determined after fluorescence labeling with PKH26 (Sigma-Aldrich) and FACS analysis or Female inbred C57BL/6 (H2b), female BALB/c (H2d), and OVA TCR- by using a chemiluminescence immunoassay based on the measurement of transgenic DO11.10 mice were supplied by Charles River Laboratories and BrdU incorporation during DNA synthesis according to the manufacturer’s housed at the central animal facilities of the University of Kiel (Kiel, Ger- instruction (Roche Diagnostics). Signals were quantified using a Victor Ϫ Ϫ many) under conventional conditions. Generation of CCR7 / mice has Light 1420 luminometer (PerkinElmer). been described earlier (19). These animals were maintained at the central Plastic plates were preincubated with CCL19-IgG or CCL21-IgG (2– animal facility of Hannover Medical School (Hannover, Germany). The 200 ␮g/ml) in combination with an anti-CD3 Ab (2 ␮g/ml) to analyze the CCR7-deficient mice have been backcrossed to the BALB/c genetic back- effect of immobilized chemokine ligands. Up to a final concentration of ground for at least seven generations. All mice were maintained under 200 ␮g/ml total protein hIgG or BSA was supplemented to ensure equal specific pathogen-free conditions and were used at 8–12 wk of age in all amounts of plate-bound anti-CD3 while using different concentrations of experiments. chemokine ligands. As a negative control, anti-CD3 was preincubated with HUT78, a CCR7-expressing human T cell lymphoma cell line, and CHO either hIgG or BSA alone (each 200 ␮g/ml) without chemokine ligands. cells were obtained from American Type Culture Collection. Cell lines After2hat37°C,plates were washed twice and cell suspensions con- were grown in RPMI 1640 supplemented with 10% FCS, 2 mM L-glu- ␮ tamine, streptomycin (100 ␮g/ml), and penicillin (100 U/ml). For large- taining anti-CD28 (1 g/ml) were incubated for 3 days on coated plates scale protein production and purification, the adherent transfected CHO before analyzing proliferation using BrdU incorporation. cells were successively adapted to serum-free conditions and maintained in To measure IL-2 production, cells were stimulated as described and ProCHO4-CDM medium. supernatants were obtained after 24 h by centrifugation. IL-2 concentra- Preparation and culture of bone marrow cells to generate DCs has been tions were quantified using an ELISA kit (IL-2 Quantikine; R&D Systems). described previously (20). For DC maturation, 1 ␮g/ml LPS and 1 ␮g/ml Surface marker expression was analyzed by FACS analysis using fluo-

PGE2 (Sigma-Aldrich) were added on day 10 for 24 h. For Ag-speciﬁc T rescence-labeled mAbs (clones anti-CD3 UCHT1, anti-CD4 13B8.2, and cell priming experiments, bone marrow DCs were additionally pulsed with anti-CD8 B9.11, Beckman-Coulter; clones anti-CD69 FN50 and anti- 5 ␮g/ml OVA peptide 323–339 (NeoMPS). CD25 M-A251, BD Pharmingen). The Journal of Immunology 6487 Downloaded from http://www.jimmunol.org/

FIGURE 2. CCR7 ligands suppress proliferation of both human and murine T cells. A, CCR7ϩ human T cell line HUT78 cells were cultured in the presence of the indicated concentrations of CCL19-IgG, CCL19, or hIgG for 4 days. B, Human lymphocytes were isolated from peripheral blood and T cell separation was performed using negative selection by magnetic separation (MACS). Cells were stimulated with plate-bound anti-CD3 (␣-CD3; 3 ␮ ␣ ␮ g/ml) and anti-CD28 ( -CD28; 1 g/ml) for 4 days in the presence of the indicated concentrations of recombinant CCL19, CCL19-IgG, or CCL21-IgG. by guest on October 2, 2021 Proliferation was measured by BrdU chemiluminescence. C, Murine T cells isolated from splenocytes of BALB/c mice were separated using negative selection by magnetic separation (MACS). Cells were stimulated with plate-bound anti-CD3 at the indicated concentrations. Cells were cultured with1 ␮g/ml anti-CD28 for 4 days in the presence or absence of CCL19-IgG, CCL21-IgG, or eotaxin (each 2.5 ␮M) before proliferation was assessed using BrdU p Ͻ 0.01. D, Human T cells were ﬂuorescence ءء ;p Ͻ 0.05 ,ء ;chemiluminescence. A–C, Mean proliferation Ϯ SD from at least three experiments is shown labeled with PKH26 and stimulated with immobilized anti-CD3/anti-CD28 for 4 days in the presence of 2.5 ␮M CCL19-IgG or hIgG. Cell division was assessed by ﬂuorescence fading using FACS analysis. The proportion of divided cells gated for CD3, CD4, or CD8 is indicated. The graph is representative of three repeated experiments with similar results. rlu, Relative luminescence unit.

Cell cycle analysis accumulation in secondary lymphoid organs, reducing T cell Ag Cultured cells (5 ϫ 105) were stimulated as described above for 4 days and priming. washed with PBS buffer before resuspension in propidium iodide/sodium To determine whether this immunosuppressive effect is also me- citrate/Triton X-100 solution (50 ␮g/ml, 0.1%, and 0.1%, respectively). diated by a direct cellular effect of CCL19 or its second ligand, Cells were incubated at 4°C in the dark for 20 min. Stained cells were CCL21, independently of cell trafficking, we used the MLR with analyzed by flow cytometry. The percentages of cells in different stages of BALB/c splenocytes as effector cells and mitomycin C-treated the cell cycle were determined. C57/B6 splenocytes as stimulator cells in an in vitro alloimmune Western blot analysis response model. Fig. 1 illustrates that the presence of CCL19 sig- Protein levels of p27Kip1, CDK1 (BD Biosciences), and ␤-actin (Cell Sig- nificantly reduced the proliferative response of splenocytes in a naling Technology) were determined by Western blotting (17). concentration-dependent manner. Recombinant CCL19 and equimolar concentrations of CCL19-IgG or CCL21-IgG showed a Statistical analysis comparable effect, whereas control hIgG had no influence on pro- Data are shown as mean Ϯ SD. The difference between groups was tested liferation compared with vehicle treated cells. To exclude the pos- by using Student’s t test for statistical significance with SPSS 12.01 sibility that the inhibitory effect on MLR was influenced by the software. IgG part of the fusion protein, we repeated the experiment with a control fusion protein (CCL19-⌬IgG) carrying a mutation in the Fc Results receptor binding domain (18). The effects mediated by the CCL19- CCR7 ligands suppress MLR Fc-mutated IgG fusion protein and the nonmutated CCL19-IgG We have previously shown that the CCL19-IgG fusion protein is were identical. The presence of either CCL19-IgG or CCL21-IgG able to suppress an alloimmune response in vivo (15). This effect reduced the proliferation of effector cells. Thus, both CCR7 li- can be partially explained by an impairment of T cell and APC gands specifically inhibit Ag-induced splenocyte proliferation. 6488 CHEMOKINE SIGNALING PATHWAYS

CCL19 and CCL21 inhibit T lymphocyte proliferation We next wanted to determine whether CCL19 and CCL21 exert antiproliferative effects directly on T cells. Furthermore, we aimed to clarify whether CCL19 acts as an overall inhibitor of T cell proliferation or speciﬁcally inhibits T cell activation due to TCR engagement. We initially chose the human T cell line HUT78 because these cells constitutively express the chemokine receptor CCR7 at high levels and respond to CCL19 chemotactically (17, 21). Fig. 2A shows that CCL19 strongly inhibited the spontaneous proliferation of this T cell line when used at higher concentrations than those necessary for optimal chemotactic responses in Transwell assays (50–200 nM) (22). At concentrations Ͼ1 ␮M, highly signiﬁcant inhibition of HUT78 cell proliferation was observed in a dose- dependent manner. This effect was not attributable to increased cell death or apoptosis, which were measured by propidium iodide and annexin V staining, respectively (data not shown). This effect could also be demonstrated when CCL21-IgG was present. This

experiment, using a human T cell line, also shows that CCL19 and Downloaded from CCL21 can inhibit proliferation independently of TCR engagement. The concentrations of CCL19, CCL19-IgG, CCL21-IgG, and hIgG from Fig. 1 were used. In addition, it was of interest to determine whether the antiproliferative effect mediated by both CCR7 ligands is also operative in T cells activated by TCR engagement. For this experiment, T cells http://www.jimmunol.org/ were purified from PBMCs or splenocytes and stimulated with plate-bound anti-CD3 and anti-CD28 Abs. In the presence of CCL19, human T cell proliferation was significantly reduced compared with vehicle-treated controls (Fig. 2B). Equimolar concentrations of either CCL19-IgG or CCL21-IgG caused the same inhibitory effect without significant differences between each other. Next, we studied the effect of the fusion protein in purified T lymphocytes stimulated with optimal and suboptimal doses of anti-CD3 and 1 ␮g/ml anti-CD28, respectively (Fig. 2C). CCL19-mediated in- by guest on October 2, 2021 hibition of T cell proliferation was most pronounced under conditions of TCR stimulation with suboptimal doses of anti-CD3 (Յ0.3 ␮g/ml). To exclude effects mediated by unspecific binding of the chemokine ligands to surface receptors and, thus, unspecific signaling, we analyzed the effects of high concentrations of another CC chemokine, eotaxin (CCL11). Eotaxin did not influence the proliferation of T cells. Because this chemokine lacks a specific FIGURE 3. Genetic deficiency for CCR7 reduces T cell proliferation. A, receptor on T cells, the lack of inhibitory effect supports the spec- Murine wild-type (wt) or CCR7Ϫ/Ϫ T cells were separated from spleno- ificity of the effects mediated by CCL19 and CCL21. cytes using MACS and stimulated with immobilized anti-CD3 (3 ␮g/ml)/ In addition, we analyzed the fluorescence fading of labeled lym- anti-CD28 (1 ␮g/ml) for 4 days in the presence of either 2.5 ␮M CCL19- phocytes in purified human CD3ϩ T cells after stimulation with IgG or hIgG. B, Wild-type DCs were stimulated with OVA and LPS for immobilized anti-CD3/anti-CD28 mAbs over time. Fig. 2D shows 24 h followed by cocultivation with murine T cells from either wild-type Ϫ/Ϫ ␮ that activated T cells underwent a reduced in vitro cellular expan- mice or CCR7 mice for another 4 days in the presence of 2.5 M CCL19-IgG or hIgG. Proliferation was measured using BrdU chemilumi- sion of 35.7% in the presence of CCL19-IgG in comparison to nescence. All experiments were repeated twice, and mean proliferation Ϯ 82.9% in the control group. Examination of T cell subsets indicates SD is shown. C, Murine T cells from DO11.10 OVA TCR transgenic mice that the inhibition of cell division is evident in both CD4ϩ and ϩ were stimulated with OVA peptide-pulsed DCs. DCs were generated from CD8 T cells. The proliferative response of CD8 T cells to anti- the bone marrow of wild-type or CCR7 deficient mice as indicated. rlu, ϩ CD3/anti-CD28 was greater than that of CD4 T cells. In the CD8 Relative luminescence unit. T cell subset, the inhibition of cell division seems to be more pronounced. response was significantly reduced in both control and CCL19- treated cells compared with wild-type T cells. Fig. 3B confirms this Genetic knockout of CCR7 confers a hypoproliferative state on finding: CCR7Ϫ/Ϫ T cells exhibit less proliferation than wild-type T cells T cells in DC-mediated stimulation. Again, CCR7Ϫ/Ϫ T cells show To further substantiate the observation that the inhibitory effects markedly reduced proliferation after stimulation independent of are solely mediated by the chemokine receptor CCR7, we analyzed treatment that was more profoundly reduced than that mediated by T cells from CCR7 knockout mice. Fig. 3A shows the anti-CD3-/ CCL19-IgG in wild-type T cells, indicating a robust influence of anti-CD28-induced proliferation of CCR7Ϫ/Ϫ T cells compared CCR7 on proliferation. Compared with TCR cross-linking with with wild-type T cells. The presence of CCL19 had no influence on anti-CD3/anti-CD28, DC-mediated proliferation was reduced to a CCR7 knockout T cell proliferation. In addition, the proliferative lesser extent, albeit still significantly. The Journal of Immunology 6489 Downloaded from

FIGURE 4. CCL19-mediated reduction of IL-2 secretion is not responsible for the inhibition of T cell proliferation. A, Human T cells were activated with anti-CD3 (␣-CD3)/anti-CD28 (␣-CD28) and cultured for 24 h. Supernatants were analyzed for IL-2 secretion using cytokine-speciﬁc ELISA. CCL19-IgG or hIgG were present at the indicated concentrations. B, A proliferation assay of anti-CD3/anti-CD28-stimulated T cells was performed in the presence or absence of exogenous IL-2. CCL19-IgG or IL-2 was present at the indicated concentrations. C, Expression of http://www.jimmunol.org/ IL2-receptor CD25 and early activation Ag CD69 is not inﬂuenced by CCL19. Human T cells were stimulated by anti-CD3/anti-CD28 in the presence or absence of CCL19-IgG. Surface expression of CD25 and CD69 was analyzed after 24 h by FACS. A representative result of three experiments is shown.

To verify the above results in a more physiological system, we Immobilized CCR7 ligands substantially decrease CD3-mediated used OVA transgenic CD4 T cells stimulated by OVA-pulsed T cell proliferation DCs. Fig. 3C shows that Ag-specific DC-induced T cell prolifer- Recent studies demonstrated that the function of a variety of che- by guest on October 2, 2021 ation using OVA TCR transgenic T cells is also significantly re- mokines is partly dependent on surface presentation and immobi- duced by CCL19-IgG. Moreover, we could not detect a difference Ϫ/Ϫ lization (25, 26). With regard to this physiological condition, we in proliferative response when CCR7 were used as stimulator asked whether immobilization of the CCR7 ligands CCL19 and cells instead of wild-type DCs. This demonstrates that the inhib- CCL21 would influence the demonstrated inhibitory effects. itory effect of CCL19 on the proliferative response is mediated by Therefore, we precoated plastic plates with different amounts of T cell CCR7 and that CCR7 signaling in DCs does not contribute chemokines in combination with anti-CD3 Ab to imitate a surface- to this effect. bound presentation. Subsequently, T cells were stimulated by in- CCL19 leads to a failure of autocrine IL-2 secretion cubation on such a surface. Fig. 5 shows that both CCR7 ligands are also effective in inhibiting the proliferative response to anti- To identify the underlying molecular mechanisms for our findings, CD3/anti-CD28 stimulation when immobilized. Neither immobi- we next analyzed the key steps influencing T cell proliferation, lized hIgG nor BSA was able to inhibit proliferation. The inhibi- cytokine secretion, and cell cycle progression. tory effect was concentration dependent. Because of the pivotal role of IL-2 in T cell activation and pro- When high concentrations of immobilized CCL19 or CCL21 liferation, we analyzed IL-2 production and the expression of IL- were presented on the surface, no further inhibitory effect could be ␣ 2R (CD25) in activated T cells. Fig. 4A indicates that primary elicited by adding soluble CCR7 ligands. Furthermore, soluble human T cells secreted significant amounts of IL-2 after anti-CD3/ CCL19 could not increase the effect when immobilized CC21 in- anti-CD28 stimulation. The presence of CCL19 led to drastically duced the maximal inhibition of T cell proliferation (nor vice ver- reduced IL-2 release, demonstrating a down-regulation of this im- sa; Fig. 5B). This suggests that both CCR7 ligands use identical portant autocrine activation loop by CCL19, which may contribute signaling pathways to inhibit proliferation. to a reduction in T cell proliferation. However, Fig. 4B demonstrates that exogenously adding IL-2 up to 100 ng/ml did not abol- ish the antiproliferative effects of CCL19. In contrast to IL-2 se- CCL19 interferes with cell cycle regulation cretion, we detected no relevant influence of CCL19 on other TCR activation forces lymphocytes to enter the cell cycle (27). activation markers, such as expression of CD25 or CD69, after Because of the reduced cell division and the lack of increased cell TCR stimulation. FACS analysis shows that expression of both death after CCL19 addition, it was intriguing that the cell cycling receptors increased after a 24-h stimulation period with anti-CD3/ of T cells may be influenced by CCL19. Thus, we analyzed the cell anti-CD28, independently of the presence of CCL19 (Fig. 4C). In cycle distribution of antiCD3-/anti-CD28-activated T cells in the addition, phosphorylated target proteins of the early TCR-induced presence or absence of CCL19. We performed time course exper- signaling cascade, like STATs, protein kinase C, and I␬B (23, 24), iments (12, 24, 26, 48, 60, and 72 h of activation) to determine were not significantly affected by CCL19 (data not shown). which phases of the cell cycle were influenced by CCL19. As 6490 CHEMOKINE SIGNALING PATHWAYS Downloaded from http://www.jimmunol.org/

FIGURE 5. Immobilized chemokines also transmit antiproliferative signals. Murine T cells were stimulated by plate-bound anti-CD3 (␣-CD3) in combination with plate-bound chemokine fusion proteins or control proteins. A, plates were coated with anti-CD3 by simultaneous incubation with control proteins (hIgG and BSA) and/or chemokine fusion proteins at the by guest on October 2, 2021 given concentrations. After washing off unbound proteins, cell suspensions were incubated on coated plates. Proliferation was measured after 4 days. Soluble anti-CD28 was present at a concentration of 1 ␮g/ml. The graph shows a representative result of three experiments. Significance was iden- p Ͻ 0.05). B, Plates were coated ,ء ;p Ͻ 0.01 ,ءء) tical in all experiments simultaneously with anti-CD3 (1 ␮g/ml) and hIgG, CCL19-IgG, or CCL21 FIGURE 6. CCL19 influences mechanisms that regulate the cell cycle. (each 100 ␮g/ml). Cell suspensions containing ␣-CD28 was incubated on A, Human T cells were treated for 4 days with anti-CD3 (␣-CD3)/anti- washed plates. Soluble CCL19-IgG or CCL21-IgG or an identical volume CD28 (␣-CD28) mAbs alone or in the presence of 2.5 ␮M CCL19-IgG. of vehicle was added to the cell suspensions as indicated at a final con- Cell cycle progression was visualized by propidium iodide staining and centration of 2.5 ␮M. Proliferation was determined after 4 days. The graph subsequent FACS analysis. The proportion of cells in the S and G2/M p Ͻ 0.05, denoting phase is indicated. The graph shows representative histograms of three ,ء .shows a representative result of two experiments significance compared with positive control (far left lane, plate coated with experiments with similar results. B, Different cell cycle-regulating proteins hIgG, no soluble chemokine). rlu, Relative luminescence unit. were analyzed by Western blotting. The quantity of the indicated proteins in TCR-stimulated cell lysates demonstrates the effect of CCL19-IgG on proliferating T cells. Blots were stripped and probed with specific Abs. shown in Fig. 6A, resting unstimulated T cells sustained for 4 days were almost exclusively in the G0/G1 phase of the cell cycle in the absence of mitogenic stimulation or CCL19-IgG. Treatment with CCL19 treatment of activated T cells. TCR stimulation alone re- anti-CD3/anti-CD28 for 4 days induced the proliferation of pri- sults in strong up-regulation of CDK1 in proliferating T cells. mary T cells as indicated by a shift of lymphocytes into the S and CCL19-IgG-treatment during TCR activation was associated with Kip1 G2/M phases. The proportion of T cells within the S and G2/M reduced levels of CDK1 and high levels of p27 . phases was significantly increased by CCL19, from 6.54 to 19.9%, consistent with a cell cycle delay or arrest in activated T cells Discussion induced by CCL19. We next determined whether the influence of CCR7 is a prerequisite for immune cells to home into secondary CCL19 on the cell cycle involves previously identified regulatory lymphoid organs. Moreover, evidence has accumulated over recent proteins. We observed that cyclin-dependent kinase (CDK) inhib- years indicating that CCR7-mediated signaling influences more itor p27Kip1 was down-regulated in TCR-stimulated cells (Fig. cell functions than migration. For example, recently it has been 6B). This effect was abolished in the presence of CCL19, which is shown in DCs that CCR7 ligands induce alterations in cytoar- consistent with the increased number of activated T cells in the S chitecture, increase the ability of endocytosis, and promote sur- and G2/M phases (Fig. 6A). Furthermore, CDK1, a serine-threo- vival (14, 28, 29). In T cells, CCR7 is responsible for a variety nine kinase required for entry into mitosis, was down-regulated by of proactivation events such as migratory speed or activation of The Journal of Immunology 6491

LFA-1 or as a costimulatory factor in the priming of T cell this is the first time that a cell cycle-regulating protein has been subtypes (8, 11, 30). associated with the CCR7 signaling pathway. In a previous study, we showed that targeting the CCL19/CCR7 Nevertheless, there are a few examples of a link between system can also result in a profound immunosuppression that can- chemokine receptors and cell cycle regulation in the literature. not be solely explained by the impairment of cell migration (15). The CXC chemokine CXCL4 (PF4) has been identified as a cell In the current study, we present data showing that the CCR7 proliferation blocker in endothelial cells. PF4-induced signaling ligands CCL19 and CCL21 are not only capable of impairing im- resulted in the down-regulation of cell cycle regulators like mune responses by influencing the migratory behavior of T cells p21Cip1/WAF1 (32). Recently, it was shown that CXCR4 influences and DC in vivo but that these ligands can also directly induce a cell cycle-regulating proteins in medulloblastoma cells (33). More- thus far undescribed inhibitory program. As demonstrated by over, the chemokine CCL2 is thought to be a direct inhibitor of the MLR, CCL19 and CCL21 can reduce alloimmune responses. We proliferation of pancreas carcinoma cells (34). CXCR3 was re- could attribute this effect to reduced T cell proliferation. This effect cently be described as a metastasis-promoting receptor (35), but is valid for CCR7-expressing T lymphoma cells and both murine when used in pharmacological concentrations (Ͼ0.5 ␮M) its li- and human T cells. It applies to CD4ϩ as well as CD8ϩ T cells, gands negatively influence the growth of myeloma cells (36). In- and we demonstrate that it is independent of the mode of stimu- terestingly, the connection between chemokine receptors and cell lation, namely TCR cross-linking or DC-mediated activation. cycle control might already be used therapeutically, as Fujino and colleagues suggest (37). They ascribe one of the pleiotropic effects What mechanisms can be responsible for this? of statins to an influence on the cell cycle-regulating proteins p21 The proliferation stimulus in T cells was induced by either Ab- or and cyclin D1, which were associated with the down-regulation of DC-mediated TCR activation. However, even though the immor- CCR2 and CCR5 (37). Downloaded from talized transformed lymphoma cell line HUT78 does not express These examples show that there is expanding evidence that che- CD3 (31), the antiproliferative properties of CCL19 could still be mokine receptors and their ligands, known to mediate trafficking observed. A possible explanation of these observations is a direct events, are also implemented in pivotal cell function control, such effect of CCR7 signaling on cell proliferation that is independent as proliferation. of TCR signals. In agreement with this hypothesis, CCR7Ϫ/Ϫ T In conclusion, we identified the modulation of cell proliferation cells show reduced proliferation upon TCR stimulation. To our as a new function of the chemokine receptor CCR7. CCR7Ϫ/Ϫ http://www.jimmunol.org/ knowledge, there are no published reports that demonstrate an al- cells as well as continuous receptor engagement by high concen- tered proliferation pattern of CCR7Ϫ/Ϫ T cells. In this study we trations of the agonistic ligand CCL19 show reduced proliferation could show for the first time that the genetic knockout of CCR7, caused by cell cycle arrest. independent of CCL19 signaling, is sufficient for a reduced proliferative response of T cells, both after TCR cross-linking as well as after DC-mediated stimulation. As expected, exogenous CCL19 Does the demonstrated inhibitory effect apply to physiological did not have any influence on the proliferation rate of these cells situations? because the specific receptor is missing. Furthermore, the CC che- To date, it has been reported that CCR7 ligands have proactivation by guest on October 2, 2021 mokine eotaxin (CCL11), the receptor of which is not expressed on capabilities. For example, CCL19 facilitates the maturation of DCs lymphocytes, did not have any influence on proliferation. These and thus enhances T cell priming (12). A direct effect on T cell results are convincing evidence that CCR7 ligands specifically ex- proliferation was not observed using concentrations up to 0.1 ␮M ert the demonstrated effects via CCR7 and that expression and CCL19. In this study, we observe inhibitory effects that are not function of CCR7 are linked to proliferation control. DC-mediated because there is no difference when stimulating with wild-type or CCR7Ϫ/Ϫ DC (Fig. 3B). This indicates that the inhi- Which signaling pathways could be involved in proliferation bition of proliferation is due to an influence on proliferation itself control after CCR7 engagement? and not as a consequence of altered priming. Furthermore, we used Because CCR7-mediated effects are independent of TCR activa- concentrations higher than those used for optimal chemotaxis re- tion, the downstream signaling events of CCR7 are likely to be sponse. Consistent with our data, 0.1 ␮M CCL19 did not influence distinct from TCR signaling pathways. This is substantiated by the T cell proliferation directly (12). fact that early TCR-mediated activation signals seem to be unaf- The physiologic concentrations of a chemokine in tissue are fected by CCR7 engagement. For example, the expression profiles hard to determine exactly. Concentrations in certain microenviron- of the very early activation markers CD25 or CD69 were not re- ments might well be higher than what is suggested by an analysis duced by CCL19 treatment. In addition, there were no differences of whole tissue. Local concentrations, e.g., in the center of a lymph in key signals downstream of the TCR, such as STAT or protein node, may well be high enough to result in immune-modulating kinase C activation. Moreover, the effect of CCL19 on T cell di- effects. Recently, published work has confirmed this thesis. Con- vision was cumulative over a 1-wk observation period with in- centrations of 2 to 10 ␮g/ml CCR7 ligands were measured in creasing differences in cell division after longer incubation. Thus, lymph node tissue (9), which is in the range used in our study. For we concluded that early T cell activation is not responsible for the example, on endothelial surfaces even locally higher concentra- demonstrated inhibitory effect of CCL19 and that the sustained tions are discussed, leading to growing evidence that in vivo che- presence of CCL19 is able to continuously decelerate the division mokines are likely to be presented bound to surface structures like of CCR7ϩ T cells, which would be consistent with an impaired glycosaminoglycans (25). Moreover, the functional relevance of cell cycle progression. this surface binding has been demonstrated for a couple of CC The analysis of the distribution of cells within the cell cycle chemokine receptors (26, 38). For example, the CCR2 ligand confirmed this. The cell cycle distribution of T cells cocultured RANTES could not promote migration in an in vivo peritonitis with CCL19 showed a significant increase in G2/S as compared model if its glycosaminoglycan binding domain was mutated, al- with untreated cells. Consistent with this finding, CCL19 induced though this mutated chemokine could induce chemotaxis in vitro an up-regulation of p27Kip1 and a down-regulation of CDK1, both as well as nonmutated RANTES (39). Therefore, to test whether leading to a transitional block at the G2 phase. To our knowledge, the inhibitory effects are influenced by the mode of chemokine 6492 CHEMOKINE SIGNALING PATHWAYS presentation, we tested the inhibitory potency of surface-immobi- also by reducing the growth of these tumors. Alfonso-Perez and lized CCL19 and CCL21. Again, a high density on the surface of colleagues (49) have demonstrated a largely specific lysis of CCL19 and CCL21 was equally effective in inhibiting the prolif- CCR7-positive lymphoma cells in vitro by using Ab-mediated erative response of T cells. complement cytotoxicity. Although we used another approach, this Although these in vitro models cannot assess quantitatively work shows that the high expression of CCR7 in tumor cells makes whether bound CCR7 ligands are more potent than soluble ones in them an easy target for CCR7-directed therapy. One possible ther- terms of inhibition of proliferation, the concentration dependence apeutical approach is to use, as in this study, agonistic ligands in in both settings shows that a sufficient number of CCR7 receptors high concentrations. A combination of traffic impairment, inhibi- have to be engaged to elicit this negative signaling. Although there tion of proliferation by agonistic signaling, and targeting the re- is data that show differing functions of CCL19 and CCL21 (40), ceptor to lyse the cell could indeed be a powerful, clinically both chemokines induced the same effects in all of our experi- usable tool. ments. Furthermore, we could not demonstrate an additional effect In summary, our results indicate that the CCR7 ligands CCL19 if one of them was used in an assay with the other present at the and CCL21 are more than just potent chemoattractants for T cells maximal concentration. This strongly suggests that both ligands but are sufficient to mediate antiproliferative effects via CCR7 by induce the same signaling pathway, leading to inhibition of influencing cell cycle progression. Thus, we show a novel associ- proliferation. ation between chemotaxis and proliferation of T cells and thereby These results could be valid for the physiological environment extend the view on the functions of the receptor CCR7. To date, within lymph nodes in which high densities of surface-bound che- CCR7 is known to mediate LFA-1 up-regulation, providing T cells mokines, high concentrations of soluble chemokines, and T cells with costimulatory signals (8), whereas higher concentrations of with various degrees of activation occur. In such a microenviron- CCL19 or CCL21 suppress the production of the costimulatory Downloaded from ment, bystander T cells that do not receive optimal antigenic stim- cytokine IL-2. Furthermore, CCR7 signals support the phosphor- ulation could be inhibited from proliferating by high concentra- ylation of activating molecules and induce calcium flux (40, 50), tions of CCL19 or CCL21, whereas cells with strong TCR while CCR7 ligands also induce cell cycle arrest and an inhibit T stimulation would proliferate despite CCR7 engagement. This cells from responding to Ags by proliferating. Although the cir- model is consistent with the finding that higher concentrations of cumstances under which these different properties are effective in anti-CD3 or stimulation by DCs were less affected by CCL19- vivo need clarification, the inhibitory function of CCR7 might be http://www.jimmunol.org/ mediated inhibition of proliferation than by suboptimal stimulation of clinical relevance. Indeed, under certain conditions the net effect (Fig. 2C). in vivo is a suppression of the allograft immune response. Thus, As another example, environments rich in chemokine ligands CCR7 features at least two outstanding properties in terms of im- could also occur during lymphoid tissue development and could munosuppression: orchestrating cellular traffic into secondary lym- control organ structure by influencing proliferation. The altered phoid organs and modulating T cell proliferation. The potency of lymphoid structures of CCR7Ϫ/Ϫ mice are indeed a result of both CCL19-IgG to both provide antiproliferative effects and interfere impaired homing (19) and lymphoid organogenesis (41). with the trafficking behavior of CCR7ϩ cells might be used to These examples illustrate that CCR7 ligands may induce anti- extend current immunosuppression therapies. by guest on October 2, 2021 proliferative effects in physiological situations. In addition, the finding that CCR7 is linked to proliferation control offers interest- Acknowledgment ing options for therapeutic interventions independently of the We thank Mareike Newsky for technical assistance. physiological range of in vivo concentrations and the mode of presentation. In vivo use of CCL19-IgG has already proven to mediate im- Disclosures munosuppression at least by impairment of migration and colocal- The authors have no financial conflict of interest. ization. The antiproliferative capacity of CCL19 described here could functionally contribute to this impairment of allograft responses in References mice. For the in vitro proliferation assays, we chose concentrations of 1. Sallusto, F. and A. Lanzavecchia. 2000. Understanding dendritic cell and T- CCR7 ligands comparable to those that were used in the in vivo lymphocyte traffic through the analysis of chemokine receptor expression. Im- munol. Rev. 177: 134–140. transplantation studies (15). The results allow differentiating intra- 2. Luther, S. A., H. L. Tang, P. L. Hyman, A. G. Farr, and J. G. Cyster. 2000. cellular signaling events independently of the influence on recir- Coexpression of the chemokines ELC and SLC by T zone stromal cells and culation or colocalization and show a direct cellular effect by deletion of the ELC gene in the plt/plt mouse. Proc. Natl. Acad. Sci. USA 97: 12694–12699. CCR7-mediated signaling. 3. Beckmann, J. H., S. Yan, H. Luhrs, B. Heid, S. Skubich, R. Forster, and Recently, different reports have already demonstrated that the M. W. Hoffmann. 2004. Prolongation of allograft survival in ccr7-deficient mice. chemokine receptor CCR7 and its ligands can modulate the im- Transplantation 77: 1809–1814. 4. Martin-Fontecha, A., S. Sebastiani, U. E. Hopken, M. Uguccioni, M. Lipp, mune response by participating in signaling events apart from che- A. Lanzavecchia, and F. Sallusto. 2003. Regulation of dendritic cell migration to motaxis and maturation (42, 43), but it has not been previously the draining lymph node: impact on T lymphocyte traffic and priming. J. Exp. demonstrated that CCL19 or CCL21 itself directs CCR7ϩ cells to Med. 198: 615–621. 5. Ohl, L., M. Mohaupt, N. Czeloth, G. Hintzen, Z. Kiafard, J. Zwirner, an antiproliferative status. However, there are a few reports that T. Blankenstein, G. Henning, and R. Forster. 2004. CCR7 governs skin dendritic have already indicated inhibitory effects. One example is that cell migration under inflammatory and steady-state conditions. Immunity 21: ϩ ϩ 279–288. CCL19 is able to inhibit colony forming of human CCR7 CD34 6. Salvadori, M., K. Budde, B. Charpentier, J. Klempnauer, B. Nashan, chronic myelogenous leukemia progenitor cells (44). L. M. Pallardo, J. Eris, F. P. Schena, U. Eisenberger, L. Rostaing, et al. 2006. From a clinical aspect, this newly described capacity of modu- FTY720 versus MMF with cyclosporine in de novo renal transplantation: a 1-year, randomized controlled trial in Europe and Australasia. Am. J. Transplant. lation might render CCR7 an even more valuable target for ther- 6: 2912–2921. apy, not only for the purpose of immunosuppression but also for 7. Kunzendorf, U., E. Ziegler, and D. Kabelitz. 2004. FTY720: the first compound tumor therapy, because CCR7-expressing metastatic tumors have a of a new promising class of immunosuppressive drugs. Nephrol. Dial. Trans- plant. 19: 1677–1681. poor prognosis (45–48). CCR7 targeting might work not only by 8. Stein, J. V., A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn, interfering with tumor cell homing, and thereby metastases, but A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, and U. H. von Andrian. 2000. The The Journal of Immunology 6493

CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lym- 31. Mazerolles, F., C. Barbat, S. Meloche, S. Gratton, M. Soula, R. Fagard, phoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associ- S. Fischer, C. Hivroz, J. Bernier, R. P. Sekaly, et al. 1994. LFA-1-mediated ated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node antigen-independent T cell adhesion is regulated by CD4 and p56lck tyrosine high endothelial venules. J. Exp. Med. 191: 61–76. kinase. J. Immunol. 152: 5670–5679. 9. Okada, T., and J. G. Cyster. 2007. CC chemokine receptor 7 contributes to Gi- 32. Gentilini, G., N. E. Kirschbaum, J. A. Augustine, R. H. Aster, and G. P. Visentin. dependent T cell motility in the lymph node. J. Immunol. 178: 2973–2978. 1999. Inhibition of human umbilical vein endothelial cell proliferation by the 10. Worbs, T., T. R. Mempel, J. Bolter, U. H. von Andrian, and R. Forster. 2007. CXC chemokine, platelet factor 4 (PF4), is associated with impaired downregu- CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo. J. Exp. lation of p21(Cip1/WAF1). Blood 93: 25–33. Med. 204: 489–495. 33. Schuller, U., A. Koch, W. Hartmann, M. L. Garre, C. G. Goodyer, A. Cama, 11. Kursar, M., U. E. Hopken, M. Koch, A. Kohler, M. Lipp, S. H. Kaufmann, and N. Sorensen, O. D. Wiestler, and T. Pietsch. 2005. Subtype-specific expression H. W. Mittrucker. 2005. Differential requirements for the chemokine receptor and genetic alterations of the chemokinereceptor gene CXCR4 in medulloblas- CCR7 in T cell activation during Listeria monocytogenes infection. J. Exp. Med. tomas. Int. J. Cancer 117: 82–89. 201: 1447–1457. 34. Monti, P., B. E. Leone, F. Marchesi, G. Balzano, A. Zerbi, F. Scaltrini, 12. Marsland, B. J., P. Battig, M. Bauer, C. Ruedl, U. Lassing, R. R. Beerli, C. Pasquali, G. Calori, F. Pessi, C. Sperti, et al. 2003. The CC chemokine MCP- K. Dietmeier, L. Ivanova, T. Pfister, L. Vogt, et al. 2005. CCL19 and CCL21 1/CCL2 in pancreatic cancer progression: regulation of expression and potential induce a potent proinflammatory differentiation program in licensed dendritic mechanisms of antimalignant activity. Cancer Res. 63: 7451–7461. cells. Immunity 22: 493–505. 35. Kawada, K., H. Hosogi, M. Sonoshita, H. Sakashita, T. Manabe, Y. Shimahara, 13. Kikuchi, K., Y. Yanagawa, and K. Onoe. 2005. CCR7 ligand-enhanced phago- Y. Sakai, A. Takabayashi, M. Oshima, and M. M. Taketo. 2007. Chemokine cytosis of various antigens in mature dendritic cells-time course and antigen receptor CXCR3 promotes colon cancer metastasis to lymph nodes. Oncogene distribution different from phagocytosis in immature dendritic cells. Microbiol. 26: 4679–4688. Immunol. 49: 535–544. 36. Giuliani, N., S. Bonomini, P. Romagnani, M. Lazzaretti, F. Morandi, S. Colla, 14. Yanagawa, Y., and K. Onoe. 2003. CCR7 ligands induce rapid endocytosis in S. Tagliaferri, L. Lasagni, F. Annunziato, M. Crugnola, and V. Rizzoli. 2006. mature dendritic cells with concomitant up-regulation of Cdc42 and Rac activi- CXCR3 and its binding chemokines in myeloma cells: expression of isoforms and ties. Blood 101: 4923–4929. potential relationships with myeloma cell proliferation and survival. Haemato- 15. Ziegler, E., F. Gueler, S. Rong, M. Mengel, O. Witzke, A. Kribben, H. Haller, logica 91: 1489–1497. U. Kunzendorf, and S. Krautwald. 2006. CCL19-IgG prevents allograft rejection

37. Fujino, M., S. Miura, Y. Matsuo, H. Tanigawa, A. Kawamura, and K. Saku. 2006. Downloaded from by impairment of immune cell trafficking. J. Am. Soc. Nephrol. 17: 2521–2532. Pitavastatin-induced downregulation of CCR2 and CCR5 in monocytes is asso- 16. Kunzendorf, U., T. Pohl, S. Bulfone-Paus, H. Krause, M. Notter, A. Onu, ciated with the arrest of cell-cycle in S phase. Atherosclerosis 187: 301–308. G. Walz, and T. Diamantstein. 1996. Suppression of cell-mediated and humoral 38. Ali, S., A. C. Palmer, B. Banerjee, S. J. Fritchley, and J. A. Kirby. 2000. Exam- immune responses by an interleukin-2-immunoglobulin fusion protein in mice. ination of the function of RANTES, MIP-1␣, and MIP-1␤ following interaction J. Clin. Invest. 97: 1204–1210. with heparin-like glycosaminoglycans. J. Biol. Chem. 275: 11721–11727. 17. Krautwald, S., E. Ziegler, R. Forster, L. Ohl, K. Amann, and U. Kunzendorf. 39. Proudfoot, A. E., T. M. Handel, Z. Johnson, E. K. Lau, P. LiWang, 2004. Ectopic expression of CCL19 impairs alloimmune response in mice. Im- I. Clark-Lewis, F. Borlat, T. N. Wells, and M. H. Kosco-Vilbois. 2003. Glycos- munology 112: 301–309. aminoglycan binding and oligomerization are essential for the in vivo activity of 18. Isaacs, J. D., J. Greenwood, and H. Waldmann. 1998. Therapy with monoclonal certain chemokines. Proc. Natl. Acad. Sci. USA 100: 1885–1890. http://www.jimmunol.org/ antibodies, II: the contribution of Fc ␥ receptor binding and the influence of C 1 H 40. Kohout, T. A., S. L. Nicholas, S. J. Perry, G. Reinhart, S. Junger, and and C 3 domains on in vivo effector function. J. Immunol. 161: 3862–3869. H R. S. Struthers. 2004. Differential desensitization, receptor phosphorylation, ␤-ar- 19. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, and restin recruitment, and ERK1/2 activation by the two endogenous ligands for the M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing CC chemokine receptor 7. J. Biol. Chem. 279: 23214–23222. functional microenvironments in secondary lymphoid organs. Cell 99: 23–33. 41. Ohl, L., G. Henning, S. Krautwald, M. Lipp, S. Hardtke, G. Bernhardt, O. Pabst, 20. Lutz, M. B., M. Schnare, M. Menges, S. Rossner, M. Rollinghoff, G. Schuler, and and R. Forster. 2003. Cooperating mechanisms of CXCR5 and CCR7 in devel- A. Gessner. 2002. Differential functions of IL-4 receptor types I and II for den- opment and organization of secondary lymphoid organs. J. Exp. Med. 197: dritic cell maturation and IL-12 production and their dependency on GM-CSF. 1199–1204. J. Immunol. 169: 3574–3580. 21. Schweickart, V. L., C. J. Raport, R. Godiska, M. G. Byers, R. L. Eddy, Jr., 42. Flanagan, K., D. Moroziewicz, H. Kwak, H. Horig, and H. L. Kaufman. 2004. T. B. Shows, and P. W. Gray. 1994. Cloning of human and mouse EBI1, a The lymphoid chemokine CCL21 costimulates naive T cell expansion and Th1 polarization of non-regulatory CD4ϩ T cells. Cell. Immunol. 231: 75–84. lymphoid-specific G-protein-coupled receptor encoded on human chromosome by guest on October 2, 2021 17q12–q21.2. Genomics 23: 643–650. 43. Sanchez-Sanchez, N., L. Riol-Blanco, and J. L. Rodriguez-Fernandez. 2006. The 22. Debes, G. F., C. N. Arnold, A. J. Young, S. Krautwald, M. Lipp, J. B. Hay, and multiple personalities of the chemokine receptor CCR7 in dendritic cells. J. Im- E. C. Butcher. 2005. Chemokine receptor CCR7 required for T lymphocyte exit munol. 176: 5153–5159. from peripheral tissues. Nat. Immunol. 6: 889–894. 44. Hromas, R., L. Cripe, G. Hangoc, S. Cooper, and H. E. Broxmeyer. 2000. The 23. Berridge, M. J. 1997. Lymphocyte activation in health and disease. Crit. Rev. exodus subfamily of CC chemokines inhibits the proliferation of chronic my- Immunol. 17: 155–178. elogenous leukemia progenitors. Blood 95: 1506–1508. 24. Ellery, J. M., and P. J. Nicholls. 2002. Alternate signalling pathways from the 45. Sancho, M., J. M. Vieira, C. Casalou, M. Mesquita, T. Pereira, B. M. Cavaco, interleukin-2 receptor. Cytokine Growth Factor Rev. 13: 27–40. S. Dias, and V. Leite. 2006. Expression and function of the chemokine receptor 25. Proudfoot, A. E. 2006. The biological relevance of chemokine-proteoglycan in- CCR7 in thyroid carcinomas. J. Endocrinol. 191: 229–238. teractions. Biochem. Soc. Trans. 34: 422–426. 46. Schimanski, C. C., R. Bahre, I. Gockel, T. Junginger, N. Simiantonaki, 26. Mantovani, A., R. Bonecchi, and M. Locati. 2006. Tuning inflammation and S. Biesterfeld, T. Achenbach, T. Wehler, P. R. Galle, and M. Moehler. 2006. immunity by chemokine sequestration: decoys and more. Nat. Rev. Immunol. 6: Chemokine receptor CCR7 enhances intrahepatic and lymphatic dissemination of 907–918. human hepatocellular cancer. Oncol. Rep. 16: 109–113. 27. Wells, A. D. 2003. Cell-cycle regulation of T-cell responses: novel approaches to 47. Zlotnik, A. 2006. Chemokines and cancer. Int. J. Cancer 119: 2026–2029. the control of alloimmunity. Immunol. Rev. 196: 25–36. 48. Cabioglu, N., M. S. Yazici, B. Arun, K. R. Broglio, G. N. Hortobagyi, J. E. Price, 28. Yanagawa, Y., and K. Onoe. 2002. CCL19 induces rapid dendritic extension of and A. Sahin. 2005. CCR7 and CXCR4 as novel biomarkers predicting axillary murine dendritic cells. Blood 100: 1948–1956. lymph node metastasis in T1 breast cancer. Clin. Cancer Res. 11: 5686–5693. 29. Sanchez-Sanchez, N., L. Riol-Blanco, G. de la Rosa, A. Puig-Kroger, 49. Alfonso-Perez, M., S. Lopez-Giral, N. E. Quintana, J. Loscertales, J. Garcia-Bordas, D. Martin, N. Longo, A. Cuadrado, C. Cabanas, A. L. Corbi, et P. Martin-Jimenez, and C. Munoz. 2006. Anti-CCR7 monoclonal antibodies as a al. 2004. Chemokine receptor CCR7 induces intracellular signaling that inhibits novel tool for the treatment of chronic lymphocyte leukemia. J. Leukocyte Biol. apoptosis of mature dendritic cells. Blood 104: 619–625. 79: 1157–1165. 30. Kaiser, A., E. Donnadieu, J. P. Abastado, A. Trautmann, and A. Nardin. 2005. 50. Sallusto, F., E. Kremmer, B. Palermo, A. Hoy, P. Ponath, S. Qin, R. Forster, CC chemokine ligand 19 secreted by mature dendritic cells increases naive T cell M. Lipp, and A. Lanzavecchia. 1999. Switch in chemokine receptor expression scanning behavior and their response to rare cognate antigen. J. Immunol. 175: upon TCR stimulation reveals novel homing potential for recently activated T 2349–2356. cells. Eur. J. Immunol. 29: 2037–2045.