Dynamic Modulation of CCR7 Expression and Function on Naive T Lymphocytes In Vivo This information is current as Mirjam R. Britschgi, Alexander Link, Tonje Katrine A. of September 26, 2021. Lissandrin and Sanjiv A. Luther J Immunol 2008; 181:7681-7688; ; doi: 10.4049/jimmunol.181.11.7681 http://www.jimmunol.org/content/181/11/7681 Downloaded from References This article cites 47 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/181/11/7681.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on September 26, 2021 *average 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Dynamic Modulation of CCR7 Expression and Function on Naive T Lymphocytes In Vivo1 Mirjam R. Britschgi, Alexander Link, Tonje Katrine A. Lissandrin, and Sanjiv A. Luther2 The chemokine receptor CCR7 is critical for the recirculation of naive T cells. It is required for T cell entry into secondary lymphoid organs (SLO) and for T cell motility and retention within these organs. How CCR7 activity is regulated during these processes in vivo is poorly understood. Here we show strong modulation of CCR7 surface expression and occupancy by the two CCR7 ligands, both in vitro and in vivo. In contrast to blood, T cells in SLO had most surface CCR7 occupied with CCL19, presumably leading to continuous signaling and cell motility. Both ligands triggered CCR7 internalization in vivo as shown in Ccl19؊/؊ and plt/plt mice. Importantly, CCR7 occupancy and down-regulation led to strongly impaired chemotactic responses, an effect reversible by CCR7 resensitization. Therefore, during their recirculation, T cells cycle between states of free CCR7 with high ligand sensitivity in blood and occupied CCR7 associated with continual signaling and reduced ligand sensitivity within SLO. We Downloaded from propose that these two states of CCR7 are important to allow the various functions CCR7 plays in T cell recirculation. The Journal of Immunology, 2008, 181: 7681–7688. aive T lymphocytes continually patrol the body in search promote the motility of naive T cells that crawl along the three- of Ags. They use blood and lymph to travel between dimensional network of TRC (14–21). This is thought to enhance secondary lymphoid organs (SLO),3 such as lymph N their chances of encountering DCs attached to TRC and to lead to http://www.jimmunol.org/ nodes (LN) and spleen. Within SLO, T cells spend several hours more efficient T cell priming (22, 23). scanning the Ags presented to them by dendritic cells (DCs). The Several differences in expression and function have been de- recirculation of T lymphocytes is guided by various receptors rec- scribed for CCL19 and CCL21. TRC in LN and spleen produce ognizing chemokines, adhesion molecules, or sphingolipids (1, 2). ϳ10 times more transcripts and 100 times more protein of CCL21 In this study, we focus on the chemokine receptor CCR7 that plays compared with CCL19 (5, 24). CCL19 binds with slightly higher a key role in the entry of T lymphocytes into SLO, in their mi- affinity than CCL21 to human CCR7 (25–27). Although both li- gration within, as well as in their exit from these organs (2–4). 2ϩ gands induce Ca mobilization, chemotaxis, and integrin-medi- CCR7 is highly expressed on naive T cells and mature DCs ated adhesion, CCL19 appears to be more potent at low concen- while its two ligands, CCL19 and CCL21, are constitutively ex- by guest on September 26, 2021 trations (25, 28–35). Sequential stimulation with the two ligands pressed by T zone reticular cells (TRC) within SLO. In addition, led to cross-desensitization of CCR7, with CCL19 being more ef- CCL21 is expressed by high endothelial venules (HEV) and lym- phatic vessels (2, 3, 5). The importance of CCR7 and its ligands in fective than CCL21 (25, 32, 33, 35, 36). Incubation of activated vivo has been demonstrated in Ccr7Ϫ/Ϫ and in “paucity of lymph human peripheral blood lymphocytes or CCR7-transfected cells node T cell” ( plt/plt) mice that lack the Ccl19 and Ccl21 genes with CCL19 induced CCR7 internalization whereas CCL21 had a expressed in lymphoid organs (6–10). Both mice have severe de- much weaker effect (35, 37, 38). After internalization, CCL19 was fects in T cell and DC migration and positioning as well as im- degraded and CCR7 recycled back to the plasma membrane (38). mune response and tolerance induction (2, 3, 6, 7, 11–13). In con- Consistent with these findings, CCL19 but not CCL21 binding led trast, Ccl19Ϫ/Ϫ mice have no gross abnormality in T cell migration to strong CCR7 phosphorylation and desensitization through ␤-ar- and positioning (5). Recently, it has been found that CCR7 ligands restin binding (39). Currently, little is known about whether CCR7 internalization is relevant in vivo and whether CCL19 and CCL21 have different effects on CCR7-expressing T cells. Department of Biochemistry, University of Lausanne, Epalinges, Switzerland In this study we investigated CCR7 expression and function on Received for publication July 18, 2008. Accepted for publication September 17, 2008. naive murine T cells both in vitro and in vivo. We show that both The costs of publication of this article were defrayed in part by the payment of page CCR7 ligands regulate receptor occupancy, internalization and re- charges. This article must therefore be hereby marked advertisement in accordance expression at the cell surface. As a consequence, T cells within LN with 18 U.S.C. Section 1734 solely to indicate this fact. are much less responsive to CCR7 ligands than T cells in blood 1 This work was supported by the Swiss National Science Foundation (PPOOA-68805 and PPOOA-116896 to S.A.L.). where little ligand is present. M.R.B. did all experiments and wrote the manuscript; A.L. set up the chemotaxis assay and helped with the transfer experiments; T.K.A.L. set up the CCR7 and CCL19-Fc staining protocols; S.A.L. designed and directed the study and wrote the manuscript; and all authors critically reviewed the manuscript. Materials and Methods 2 Address correspondence and reprint requests to Dr. Sanjiv A. Luther, Department of Mice Biochemistry, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Ϫ/Ϫ Switzerland. E-mail address: [email protected] C57BL/6 mice were obtained from Janvier. Ccl19 (5) and plt/plt (6) Ϫ/Ϫ 3 mice were backcrossed 12 and Ccr7 mice (7) were backcrossed 9 gen- Abbreviations used in this paper: SLO, secondary lymphoid organs; LN, lymph erations, onto C57BL/6 background. All mice were maintained in patho- nodes; DCs, dendritic cells; TRC, T zone reticular cells; HEV, high endothelial venules; plt, paucity of lymph node T cell; MFI, mean fluorescent index. gen-free conditions and were age- and sex-matched for experiments. All mouse experiments were authorized by the Swiss Federal Veterinary Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 Office. www.jimmunol.org 7682 CCR7 MODULATION ON NAIVE T LYMPHOCYTES IN VIVO A A 1500 * WT * LN 1200 Ccr7-/- spleen 900 control on WT blood 600 0 isotype control 200 400 600 300 CCR7 (MFI) CCR7 (MFI) 0 CCR7 LN Sp Bl CCR7 WT B *** Ccr7-/- 10000 control on WT LN 8000 spleen 0 6000 ** blood 1000 2000 3000 4000 5000 4000 isotype control CCL19-Fc (MFI) 2000 CCL19-Fc (MFI) CCL19-Fc 0 LN Sp Bl CCL19-Fc WT Ccr7-/- C isotype control on WT Downloaded from Ccr7-/- LN Ccr7-/- blood B 1µg/ml CCL21 WT LN 1µg/ml CCL19 WT blood 0.01µg/ml CCL19 no chemokine isotype control CCR7 CCL19-Fc http://www.jimmunol.org/ 0 FIGURE 2. In vivo modulation of CCR7 by receptor occupancy and ϩ 1000 2000 3000 internalization. Flow cytometric analysis of naive (CD62Lhigh) CD4 T CCR7 CCR7 (MFI) lymphocytes. A and B, Histograms and bar plots showing MFI of stainings with anti-CCR7 (A) CCL19-Fc (B) or isotype controls on cells isolated on 1µg/ml CCL21 ice from LN, spleen (Sp), and blood (Bl) of wild-type mice (n ϭ 3). Data 1µg/ml CCL19 are representative of four to five independent experiments with one to three p Ͻ ,ءء ;p Ͻ 0.05 ,ء .0.01µg/ml CCL19 mice each. The values of p are relative to LN cells p Ͻ 0.001. C, Histograms of anti-CCR7 or CCL19-Fc stainings ,ءءء ;no chemokine 0.01 Ϫ/Ϫ isotype control 321 on cells isolated on ice from LN and blood of wild-type or Ccr7 mice. Ϫ/Ϫ 0 Data are representative of over 10 wild-type and two Ccr7 mice. by guest on September 26, 2021 CCL19-Fc 10000 20000 30000 CCL19-Fc (MFI) 1µg/ml CCL21 1µg/ml CCL19 Flow cytometry 0.01µg/ml CCL19 no chemokine Stainings were performed as described previously (5) for 25 min per in- isotype control cubation step, with the exception that cells were blocked with 2% normal high ϩ mouse serum.