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 dem- onstrate 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 in- hibit 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 chemilumi- nescence immunoassay. MLR was an- alyzed 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