Vivo and Function on Naive T Lymphocytes in Dynamic Modulation of CCR7 Expression

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

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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. Cells were stained with anti-CCR7 (4B12; a gift from J. FIGURE 1. Flow cytometric analysis of naive (CD62L ) CD4 T Zwirner, Georg August University, Go¨ttingen, Germany; on the online lymphocytes from spleen. The anti-CCR7 Ab recognizes all surface CCR7 datasheet, eBioscience reports that the anti-CCR7 staining quality is im- and CCL19-Fc recognizes CCR7 free of bound CCL19. A, Splenocytes proved by staining at 37°C; in this study stainings were done at 4°C to from wild-type (WT) and Ccr7Ϫ/Ϫ mice were stained on ice with anti- avoid CCR7 modulation by experimental procedures) or the isotype control CCR7, CCL19-Fc or isotype controls. Stainings with isotype controls on anti-keyhole limpet hemocyanin (BioLegend) followed by PE-coupled wild-type and Ccr7Ϫ/Ϫ splenocytes were identical (data not shown). De- donkey anti-rat IgG (Jackson ImmunoResearch Laboratories). Before other picted are histograms and bar plots showing MFI of stainings. Data are Abs were added, a blocking step with 4% normal rat serum was performed. Ϫ Ϫ Murine CCL19-Fc (40) and the control human Fn14-Fc (a gift from P. representative of over 10 wild-type and two Ccr7 / mice. B, Splenocytes Schneider, University of Lausanne, Epalinges, Switzerland) were both fu- were incubated for1hat37°C, cooled on ice for 15 min, and then incu- ␮ sion proteins with a human IgG1 Fc portion. The fusion proteins (8 g/ml) bated for 30 min on ice with different chemokines before staining with were added to the cells and detected using a biotinylated goat anti-human anti-CCR7 or CCL19-Fc. Depicted are histograms and bar plots showing IgG (Jackson ImmunoResearch Laboratories) that had been pretreated for MFI of stainings. The 1 ␮g/ml of the irrelevant chemokine CXCL12 did 30 min with 4% normal mouse serum and normal rat serum. Finally, not inﬂuence the anti-CCR7 staining or the CCL19-Fc binding (data not streptavidin-PE (eBioscience) was added, along with other surface mark- shown). Data are representative of three independent experiments. ers. Other Abs used were anti-CD62L-FITC (eBioscience), anti- CD4-Alexa647 (H129), anti-CD8␣-Alexa647 (53–6.7), and anti-CD8␣-PE (BioLegend). Data were acquired on a FACSCanto ﬂow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star). Cell preparation Chemotaxis assay Cells were isolated from LN (inguinal, axillary, and brachial) and spleen by Murine CCL19, CCL21, and CXCL12 were obtained from PeproTech. meshing the tissues through a 40-␮m cell strainer (BD Biosciences) and Migration of splenic lymphocytes was assessed in Transwell plates (96 collecting the soluble fraction. Special care was taken to always keep cells wells, 5-␮m pores, ChemoTx 101-5; NeuroProbe) according to the man- on ice. Blood, but not spleen or LN cells, were RBC lysed for 3 to 5 min ufacturer’s protocol and using DMEM ϩ Glutamax medium containing at room temperature before putting them again on ice. This step did not 0.5% fatty acid-free BSA (Calbiochem) and 10 mM HEPES. Chemokine change much anti-CCR7 or CCL19-Fc staining, as tested in a control ex- dilutions were added to the plate and incubated at 37°C for at least 15 min periment where LN, spleen, and blood were RBC lysed using the same before loading 250,000 cells per well. Migration assays were run at 37°C conditions. Cells were kept in complete DMEM containing 5% FBS. with 5% CO2. To measure the number of migrated cells, two wells were The Journal of Immunology 7683

A LN B LN C freshly isolated spleen spleen resensitized blood blood 2500 40000 50 2000 30000 40 1500 30 20000 1000 20

CCL19-Fc (MFI) 10000 CCR7 (MFI) 500 10 Migrated of input (%) Migrated 0 0 0 0 30 60 90 120 150 180 0180 0306090120150180 0180 0.01 0.04 0.2 1 0.2 1 4 time (min) time (min) CCL19 CCL21 FIGURE 3. Free CCR7 correlates with increased migratory capacity of T lymphocytes. A and B, MFI values of CCR7 or CCL19-Fc stainings on naive (CD62Lhigh) CD4ϩ T lymphocytes isolated from the indicated organs and incubated at 37°C for various lengths of time before staining (n ϭ 3). CCR7 data for blood cells after 180 min incubation were not obtained. Data are representative of three independent experiments. As a control, IL7R␣ levels were determined which remained constant over the entire incubation period (data not shown). C, Transwell migration of naive (CD62Lhigh) CD4ϩ T cells toward the indicated concentrations (␮g/ml) of CCL19 and CCL21. The cells were either freshly isolated from spleen (and warmed up to 37°C for 5 min) or resensitized for1hat37°C (n ϭ 2). The time for the transwell migration assay was only 45 min to counter the rapid adjustment of cells to the environment. Due to the low efﬁciency in migration, background migration levels toward medium were subtracted. Data are representative of two independent experiments with two to three data points per condition. Downloaded from

pooled, stained for flow cytometry, and cell numbers counted with a FACS- Canto at high flow (for 30 s; normalized using a titration of cells). Adoptive cell transfers A 0.01µg/ml CCL19 B 0.01µg/ml CCL19 Lymphocytes (25 ϫ 106) from spleen and LN of wild-type mice were 1µg/ml CCL19 1µg/ml CCL19 labeled with 20 ␮M CFSE (Molecular Probes) and transferred into recip- 1µg/ml CCL21 1µg/ml CCL21 http://www.jimmunol.org/ ient mice by i.v. injection. Two hours or 2 days later, cells isolated from 120 120 LN were stained for flow cytometry, and the levels of CCR7 and 100 100 CCL19-Fc were analyzed on transferred CFSEϩ and endogenous CFSEϪ cells. 80 80 60 60 Statistical analysis 40 40 Results of experimental points are reported as means Ϯ SD. Statistical 20 20 significance was determined using an unpaired two-tailed Student’s t test 0 0 0102030 0102030 rel. CCR7 expression (%) CCR7 expression rel.

for unequal variance. CCL19-Fc binding (%) rel.

time (min) time (min) by guest on September 26, 2021 Results C ** D ** CCL19-Fc binding measures CCR7 occupancy by CCL19 but ** ** not CCL21 120 120 ** * 100 To investigate CCR7 surface expression on naive CD4ϩ T cells, a 100 monoclonal anti-CCR7 Ab and a CCL19-Fc fusion protein were 80 80 used. Because our goal was to get accurate measures of in vivo 60 60 CCR7 expression, cells were not resensitized by incubation at 40 40 37°C, a process known to increase the staining intensity. Both ϩ 20 20 Normalized Migration (%) Normalized Migration

reagents labeled CCR7 on wild-type CD4 T cells but did not stain (%) Normalized Migration Ϫ/Ϫ 0 0 Ccr7 cells, confirming that both reagents were CCR7 specific - - - (Fig. 1A). Because CCL19-Fc but not anti-CCR7 binds to CCR7 Pretreatment Pretreatment (1 µg/ml) CCL19CCL21 CCL19CCL21 (1µg/ml) CCL19CCL21 via the ligand-binding site, we investigated the influence of bound 1 µg/ml 1 µg/ml ϩ Chemotaxis Chemotaxis 0.04µg/ml ligand on the staining intensity. To this end, naive CD4 T cells CCL19 CCL21 CCL19 were incubated with either CCL19 or CCL21 on ice (to minimize FIGURE 4. CCL19 and CCL21 rapidly occupy CCR7 and lead to CCR7 internalization), washed, and then labeled with anti-CCR7 CCR7 internalization and desensitization. A and B, Flow cytometric ␮ ␮ ϩ or CCL19-Fc. Doses of CCL19 (0.01 g/ml) and CCL21 (1 g/ analysis of naive (CD62Lhigh) CD4 T lymphocytes isolated from ml) thought to be physiological (24) did not influence the anti- spleen, incubated at 37°C for 1 h and then incubated for different CCR7 staining (Fig. 1B); however, a superphysiological CCL19 lengths of time with the indicated amounts of CCL19 or CCL21 before dose (1 ␮g/ml) led to a slight reduction in CCR7 staining. As staining with anti-CCR7 (A) or CCL19-Fc (B). MFI values were nor- expected, CCL19 bound to CCR7 diminished CCL19-Fc staining malized to the staining intensity observed on cells not incubated with in a dose-dependent manner. Surprisingly, the presence of a high CCR7-ligands (0 min ϭ 100%) (n ϭ 1). Data are representative of 2 independent experiments. C and D, Transwell migration assay of naive dose of CCL21 did not reduce CCL19-Fc binding. In conclusion, ϩ CCR7 Abs can be used to get an estimate of total CCR7 surface CD4 T lymphocytes from spleen. Cells were incubated for1hat37°C, then for 10 min with 1 ␮g/ml CCL19 or CCL21 at 37°C before washing levels, while CCL19-Fc labels CCR7 that has no CCL19 bound and performing a 45 min transwell migration assay toward CCL19 or and is therefore an indirect readout of CCR7 occupancy by CCL21 at a high (C)orlow(D) concentration (n ϭ 3–4). The number CCL19. of migrated cells was normalized to the migration of control cells that ␮ CCR7 occupancy is high on naive CD4ϩ T cells from SLO had not been pretreated with chemokines. Pretreatment with 1 g/ml CXCL12 did not influence migration (data not shown). Data are repre- During recirculation, lymphocytes are thought to encounter CCR7 sentative of two to three (C)orone(D) independent experiments with .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .ligands in SLO but not in blood or efferent lymph. To see how three to four data points per condition 7684 CCR7 MODULATION ON NAIVE T LYMPHOCYTES IN VIVO

A C transferred endogenous 1200 10000 FIGURE 5. Concentrations of CCR7 1000 ligands present in the environment deter- 8000 800 mine CCR7 surface expression, occu- 6000 pancy, and responsiveness. A and B, Flow 600 4000 cytometric analysis of naive (CD62Lhigh) 400 ϩ CD4 T lymphocytes isolated on ice from 200 2000 CCL19-Fc (MFI) Ϫ Ϫ CCR7 (MFI) LN of wild-type (WT), Ccl19 / or plt/plt CCR7 CCL19-Fc 0 0 mice and stained with anti-CCR7, WT WT WT CCL19-Fc, or isotype controls. A, Repre- Ccl19-/- plt/plt plt/plt Ccl19-/- Ccl19-/- sentative histogram. B, MFI of CCR7 lev- plt/plt els and CCL19-Fc binding on naive isotype control D transferred CD4ϩ T cells from LN and spleen of the ** 1200 endogenous 8000 different mouse models. Indicated is the 1000 fold increase relative to staining levels ob- 6000 served on cells from wild-type mice. Data B LN spleen 800 *** are a compilation of four experiments with 600 4000 ϭ 5 * 5 one to three mice each (n 7–8). C and 400 D, MFI of CCR7 or CCL19-Fc staining 4 4 2000 Downloaded from ϩ * 200 CCL19-Fc (MFI) on naive CD4 T cells from LN at2d(C) 3 * 3 CCR7 (MFI) 0 0 or2h(D) after adoptive transfer of CFSE- 2 2 labeled wild-type lymphocytes into wild- WT WT Ϫ Ϫ 1 1 plt/plt plt/plt type (WT), Ccl19 / and plt/plt mice. As CCR7 increase) (fold Ccl19-/- Ccl19-/- comparison the MFI of endogenous 0 0 CD4ϩ T cells are shown (n ϭ 3 mice per 20 *** 20 E WT group). E, 45 min transwell migration as- Ccl19-/- http://www.jimmunol.org/ 15 15 *** *** say towards the indicated concentrations 60 plt/plt * *** (␮g/ml) of CCL19/21. Naive CD4ϩ T 10 *** 10 *** *** 50 ** *** ** *** cells were freshly isolated from spleen of 5 40 Ϫ/Ϫ 5 ** * ** *** WT, Ccl19 or plt/plt mice (n ϭ 3). CCL19-Fc increase) (fold 30 Data are representative of two to three in- 0 0 ** * 20 ءء Ͻ ء

dependent experiments. , p 0.05; , WT WT 10 plt/plt p Ͻ 0.001. plt/plt ,ءءء ;p Ͻ 0.01 Migrated of input (%) Migrated Ccl19-/- Ccl19-/- 0 - 0.04 0.2 1 0.2 1 CCL19 CCL21 by guest on September 26, 2021

CCR7 reacts to these different conditions, we analyzed CCR7 on CCL19-Fc binding increased 10-fold. All anti-CCR7 and CCL19-Fc lymphocytes isolated from LN, spleen, and blood. Both the levels staining on resensitized CD4ϩ T cells was CCR7-specific (data not of total (anti-CCR7) and “free” CCR7 (CCL19-Fc; CCL19-free) shown). These data corroborate with our in vivo results, suggesting were lowest on LN cells, intermediate on spleen cells, and highest that the absence of CCR7 ligands leads to more total and ligand-free on blood cells (Fig. 2, A and B). The staining levels in each tissue surface CCR7. This appears to occur by re-expression of internalized were homogeneous except in the spleen, where 10–15% of CD4ϩ receptor and to an even greater extent by dissociation of receptor- T cells bound higher amounts of CCL19-Fc (Fig. 2B). Strikingly, ligand complexes at the cell surface. the anti-CCR7 stain in blood was only 30% higher than in LN, To address the question whether different amounts of free CCR7 although CCL19-Fc staining was 400% higher (Fig. 2, A and B). lead to differences in migratory responses, the chemotaxis of The CCR7 dependence of both stainings was confirmed using freshly isolated CD4ϩ T cells was compared with resensitized Ccr7Ϫ/Ϫ cells and argues against the possibility that CCL19-Fc CD4ϩ T cells. Resensitization of splenocytes was performed dur- bound to other molecules present on the T cell surface (Fig. 2C). ing1htoreach a CCR7 phenotype comparable to blood lympho- These findings suggest that a part of surface CCR7 is internalized cytes with ϳ5 times more free CCR7 on the cell surface than in CD4ϩ T cells in LN and becomes re-expressed in cells found in freshly isolated splenocytes (Fig. 3B). Resensitized CD4ϩ T cells blood. An even larger part of CCR7 on LN T cells is occupied with migrated much better than freshly isolated cells to both CCR7 CCL19 and probably becomes liberated on cells found in blood. ligands with a shift in sensitivity of ϳ5-fold when tested in a 45-min chemotaxis assay (Fig. 3C). Therefore, we conclude that CCR7 occupancy determines the potency of the chemotactic high CCR7 occupancy and internalization, as is the case for T cells response within SLO but not blood, dampen the capacity of a T cell to sense To test the hypothesis that entering into a ligand-free environment CCL19/21 and respond by chemotaxis. liberates CCR7 and allows its re-expression on the cell surface, CD4ϩ T lymphocytes from SLO and blood were isolated and in- Both CCL19 and CCL21 can modulate CCR7 function cubated at 37°C for different lengths of time before staining with Given the rapid liberation and re-expression of CCR7 when cells anti-CCR7 or CCL19-Fc. Incubation of LN CD4ϩ T cells led to enter into a chemokine-free environment, we predict the opposite the up-regulation of both total CCR7 (Fig. 3A) and free CCR7 scenario of rapid CCR7 occupation and internalization when T (Fig. 3B). Spleen levels of both markers were reached after 15 min cells transit from blood into the T zone of SLO. To try to mimic and blood levels after 1 h incubation. Over the entire incubation this situation in vitro and test the capacity of the two CCR7 ligands period, the mean fluorescent index (MFI) of CCR7 doubled and to induce these processes, resensitized splenic CD4ϩ T cells were The Journal of Immunology 7685 incubated with either ligand at 37°C for various lengths of time. A Then CCR7 surface expression and occupancy were determined. Within minutes, both CCL19 and CCL21 led to a strong decrease LN in CCR7 surface expression on CD4ϩ T cells (Fig. 4A). CCL19 spleen blood was more potent than CCL21 in inducing CCR7 internalization isotype control consistent with earlier descriptions of human T cells (35, 38). As expected, the amount of free CCR7 decreased even more rapidly and potently upon addition of CCL19. The CCL19-Fc staining on CCR7 CCL19-Fc CD4ϩ T cells decreased 80–90% within 3 min of CCL19 addition (Fig. 4B) and is a combined effect of receptor occupancy and in- B ternalization. Addition of CCL21 also induced a reduction in Ccr7-/- LN CCL19-Fc binding on T cells. As CCL19-Fc can still bind to Ccr7-/- blood WT LN CCR7 in the presence of CCL21, this reduction is largely due WT blood to CCR7 internalization. Also in this setting, the changes in anti- CCR7 and CCL19-Fc staining were CCR7 specific (data not shown). CCR7 CCL19-Fc The increased capacity of CCL19 to cause CCR7 internalization might lead to more pronounced CCR7 desensitization than with C

CCL21. Evidence for such differences have previously been re- Downloaded from WT ported, mainly using human CCR7 ligands (25, 32, 33, 35, 36, 39). Ccl19-/- ϩ To test this hypothesis using murine ligands, resensitized CD4 plt/plt splenocytes were incubated for 10 min at 37°C with either CCL19 isotype control or CCL21 and then tested for their migration potential toward an- other source of CCR7 ligands. Pretreated T cells migrated far less CCR7 CCL19-Fc efﬁciently toward high doses of CCL19 or CCL21 than untreated http://www.jimmunol.org/ cells (Fig. 4C). Typically, CCL19 induced a slightly stronger re- D *** * ceptor desensitization than CCL21. When pretreated cells were 100 tested in a migration assay toward a low dose of CCL19, migration CD4+ *** CD8+ was even more impaired, especially in the case of CCL19 pretreat- 80 ment (Fig. 4D). In summary, pretreatment leads to a loss of sen- *** sitivity toward CCR7 ligands. However, under all conditions 60 ϩ tested, desensitized CD4 T cells kept some responsiveness to 40 high concentrations of CCR7 ligands. 20 by guest on September 26, 2021

CCR7 surface expression and occupancy are fine-tuned by the of input (%) Migrated amount of CCL19 and CCL21 present in the in vivo 0 – 0.01 0.04 0.2 1 0.04 0.2 1 4 environment CCL19 (µg/ml) CCL21 (µg/ml) It is unclear whether both CCL19 and CCL21 contribute to the ϩ ϩ FIGURE 6. Reduced migration of CD8 T cells toward CCR7 ligands. CCR7 internalization observed in CD4 T cells in SLO. To ad- A–C, Flow cytometric analysis of anti-CCR7, CCL19-Fc, or isotype con- dress this question, CCR7 levels and CCL19-Fc binding were mea- trol staining of naive (CD62Lhigh) CD8ϩ T lymphocytes isolated on ice ϩ sured on naive CD4 T cells isolated from SLO of wild-type, from LN, spleen, and blood of wild-type mice (A); LN and blood of wild- Ϫ Ϫ Ccl19 / , and plt/plt mice. CCR7 surface levels on cells from type and Ccr7Ϫ/Ϫ mice (B); LN of wild-type, Ccl19Ϫ/Ϫ, and plt/plt mice Ccl19Ϫ/Ϫ and from plt/plt mice were 40 and 200% increased, re- (C). D, Three hours transwell migration assay of naive T cells toward spectively, compared with cells from wild-type mice (Fig. 5A and various concentrations of CCL19 and CCL21. Cells isolated from wild- B), suggesting that both ligands participate in CCR7 internalization type spleen were incubated1hat37°C before migration. Data are repre- in vivo. A 4-fold increase in CCL19-Fc staining was observed for sentative of two independent experiments with three data points per con- .p Ͻ 0.001 ,ءءء ;p Ͻ 0.05 ,ء .ϩ Ϫ Ϫ dition CD4 T cells from Ccl19 / relative to wild-type LN, and cells from plt/plt LN showed a 15-fold increase (Fig. 5, A and B). This correlates with higher total CCR7 levels on T cells in these mice the chemokine-deficient environment (Fig. 5D). In contrast, and the increased availability of CCR7 for CCL19-Fc binding. CCL19-Fc binding studies suggested that wild-type T cells trans- CCL19-Fc staining was intermediate on CD4ϩ T cells from ferred into plt/plt mice retained some CCL19 bound to their CCR7 Ccl19ϩ/Ϫ relative to wild-type and Ccl19Ϫ/Ϫ LN (data not shown), at the 2 h but not the 2 day time point (Fig. 5, C and D). Compa- ϩ suggesting that CCR7 surface and occupancy levels reflect fairly rable results were obtained for CD4 T cells in the spleen (data not precisely the amount of chemokine present in the environment. shown). Together, these results emphasize the great extent and To rule out a developmental defect leading to the high CCR7 rapid dynamics by which the CCR7 ligands expressed within SLO expression on T cells from plt/plt mice, wild-type splenocytes were modulate CCR7 surface expression and occupancy on recirculat- ϩ transferred into plt/plt mice. Two days after transfer, CCR7 ex- ing CD4 T cells in vivo. pression and occupancy on transferred CD4ϩ T cells within LN had completely adapted to the high level of endogenous cells (Fig. Increased CCR7 level on plt/plt T cells conveys higher 5C). Transfer of wild-type cells into wild-type or Ccl19Ϫ/Ϫ mice migratory capacity led to a lower staining level, which was indistinguishable between To test whether the increased level of surface and free CCR7 on transferred and endogenous cells. This indicates that environmen- CD4ϩ T cells isolated from chemokine-deficient mice had a func- tal rather than developmental signals determine CCR7 expression tional consequence, their chemotactic capacity was assessed. In- levels. Already 2 h after transfer, CCR7 levels had adjusted to deed, at all chemokine concentrations tested. splenic CD4ϩ T cells 7686 CCR7 MODULATION ON NAIVE T LYMPHOCYTES IN VIVO from plt/plt mice migrated far better than those from Ccl19Ϫ/Ϫ range of CCL19-Fc vs total CCR7 staining suggests a more im- mice, which in turn migrated better than those from wild-type mice portant role for receptor occupancy than internalization in modu- (Fig. 5E). These results further support the notion that CCR7 func- lating CCR7 responses. tion is critically regulated by the amount of CCR7 expressed at the CCL19-Fc staining allowed us to establish a hierarchy of CCR7 cell surface and its ligand accessibility. occupancy in vivo. In blood, most CCR7 is ligand-free, but in SLO most is occupied. Interestingly, CCR7 occupancy is even higher in Reduced CCR7 levels and migratory response of CD8ϩ ϩ LN than in spleen, in accordance with our previous observation compared with CD4 T cells that CCL19 expression is 2-fold higher in LN than in spleen. Rel- The results reported so far have focused on CD4ϩ T cells. How- ative to CCL21, CCL19 protein levels are more than 100-fold ever, CCR7 expression and function was also assessed for naive lower in both tissues and hard to detect (24). In this light, the high CD8ϩ T cells. Total CCR7 levels were lower on CD8ϩ than CD4ϩ CCR7 occupancy by CCL19 comes as a surprise, as it suggests that T cells, while still being well above background levels of CCR7- T cells migrating within T zones of SLO have continual access to deficient cells (Fig. 6, A and B). CCR7 surface levels were indis- CCL19. tinguishable between CD8ϩ T cells from SLO and blood of wild- CCR7 occupancy by CCL19 was strikingly homogeneous for type mice (Fig. 6A). Even CD8ϩ T cells from wild-type and plt/plt the whole T cell pool within LN. In contrast, splenic T cells could LN showed only small differences (Fig. 6C). The low capacity of be divided into two populations with distinct CCR7 occupancy. CCR7 to be modulated on CD8ϩ T cells was reproduced in re- Although most splenic T cells displayed an intermediate CCR7 sensitization or desensitization assays in vitro (data not shown). In occupancy by CCL19, 10–15% of cells had as much free CCR7 as contrast to the total CCR7 levels, the amount of free CCR7 on cells isolated from blood, indicating that they reside in an envi- Downloaded from CD8ϩ T cells was modulated between SLO and blood of wild-type ronment with little or no CCL19. Recirculating T cells enter the mice (Fig. 6A), indicating that most CCR7 on CD8ϩ T cells in spleen via open arteries, flushing them into the marginal zone of SLO are occupied with CCL19, similar to CD4ϩ T cells. This the white pulp cords. From there they migrate into the T zone of notion is supported by the finding of increased CCL19-Fc staining the white pulp. To exit the spleen, T cells need to access venous on CD8ϩ T cells from Ccl19Ϫ/Ϫ or plt/plt compared with wild- blood vessels found in the red pulp (2). Based on CCL19 mRNA type LN (Fig. 6C). analysis, both the marginal zone and the red pulp are regions lack- http://www.jimmunol.org/ To test whether the difference in CCR7 levels and modulation ing detectable CCL19 expression (31). Therefore, the splenic T between CD8ϩ and CD4ϩ T cells had a functional consequence, cells with lots of free CCR7 are likely to comprise arriving T cells we compared the two T cell subsets in a chemotaxis assay. Both localizing to the marginal zone and exiting T cells that have left the were equally potent in their migratory response to high CCL19 white pulp at least 20 to 30 min ago. concentrations (Fig. 6D). However, at low CCL19 and all CCL21 The homogeneously high CCR7 occupancy on LN T cells sug- concentrations tested, CD8ϩ T cells migrated significantly less gests that upon entry into the chemokine-rich environment of the than CD4ϩ T cells, indicating that the lower CCR7 expression T zone, most CCR7 on T cells is rapidly saturated by CCL19, and level and/or the reduced modulation was associated with a de- possibly CCL21. Therefore, CCR7 occupancy appears to be inde- creased capacity to sense chemokine gradients. pendent of the amount of time a T cell has spent in the T zone or by guest on September 26, 2021 its precise localization. If a T cell would move up a steep CCL19 Discussion gradient within the T zone this should have been detected at the In this study, we showed that CCR7 occupancy and internalization level of CCR7 occupancy. Consistent with the absence of striking are processes occurring continuously in naive T cells in vivo. They chemokine gradients within T zones is the uniform distribution are observed in LN and spleen, where both CCL19 and CCL21 are previously reported for CCL19 and CCL21 transcripts and CCL21 abundantly present and directly affect the capacity of T cells to protein (5, 9, 17, 29–31, 42). Further support for this concept respond to new sources of these ligands. comes from intravital imaging experiments showing that naive T To measure CCR7 expression, we relied on two widely used cells display continuous and non-directional migration along the reagents, which so far have been used interchangeably. We con- TRC network all over the T zone (22, 23, 43). Interestingly, part of firm that anti-CCR7 Ab binding to CCR7 is not influenced by this motility is dependent on CCR7, implying that T cells receive CCL21 (41) or physiological doses of CCL19. The anti-CCR7 Ab continuous CCR7 signals (14–21). Here we provide direct evi- can therefore be used to assess total CCR7 surface expression. dence that, in SLO, most CCR7 on a given T cell is continually The recognition of CCR7 by CCL19-Fc is inhibited by bound occupied by CCL19, and presumably by CCL21, thereby support- CCL19. Although previous studies indicate overlapping binding ing T cell motility. sites for the human CCR7 ligands (25, 36), murine CCL21 did not The notion of continuous CCR7 signaling raises the question of interfere with the binding of the murine CCL19-Fc-fusion. This whether T cells within SLO maintain responsiveness toward CCR7 may be because the higher affinity of CCL19 compared with ligands by continuously internalizing and recycling receptor. Re- CCL21 for CCR7 (25–27), allows CCL19-Fc to displace bound ceptor internalization upon ligand binding has been demonstrated CCL21. CCL19-Fc is therefore a readout of CCR7 occupancy in vitro for human CCR7 as well as for several other chemokine by CCL19 and provides information different from the anti-CCR7 receptors (35, 37, 38). We demonstrate that CCR7 internalization staining. indeed also occurs in vivo. CCR7 on CD4ϩ T cells from LN and Total CCR7 levels and levels of free CCR7 were higher on spleen was down-regulated 35% and 10%, respectively, when CD4ϩ T cells from blood compared with SLO, both in wild-type compared with blood. The moderate level of internalized CCR7 and Ccl19Ϫ/Ϫ tissues. This higher CCL19-Fc staining level was observed for T cells ex vivo may be a reflection of the fast kinetics due to a combination of higher surface CCR7 expression and de- of receptor internalization and re-expression preventing complete creased CCR7 occupancy. Importantly, it correlated with a higher CCR7 desensitization. capacity of “blood-phenotype” cells to sense chemotactic gradi- Down-regulation and desensitization of human CCR7 was much ents. These results suggest that the level of free CCR7 and there- more pronounced upon CCL19 than CCL21 binding (35, 38). Both fore responsiveness is regulated both at the level of receptor in- ligands showed a similar capacity to induce the internalization of ternalization/re-expression and occupancy. The higher dynamic murine CCR7 when taking into account the 100-fold difference in The Journal of Immunology 7687 expression of CCL19 and CCL21 protein within SLO (24). The in SLO: T zone vivo role of CCL19 in CCR7 down-regulation was indicated by the blood lymph or 40% increase in CCR7 expression on T cells from CCL19-defi- TRC CCL21 CCL19 blood cient tissues. The precise role of the much more abundant CCL21 in CCR7 modulation could not be addressed as Ccl21Ϫ/Ϫ mice have not been reported. However, we obtained some evidence that CCL21 may contribute to this process in vivo as CD4ϩ T cells T T transferred into plt/plt mice increased their CCR7 expression much more strongly than cells transferred into Ccl19Ϫ/Ϫ mice. This CCR7 expression level is considerably higher than on T cells from minutes hours wild-type blood or LN T cells resensitized in vitro for 3 h, sug- time gesting that de novo production of CCR7 might contribute. Im- ϩ ϩ FIGURE 7. Model proposing how CCR7 activity is regulated on CD4 portantly, the 3-fold higher CCR7 level on CD4 T cells from T cells recirculating through SLO and blood. Blood CD4ϩ T lymphocytes plt/plt relative to wild-type correlated with a much higher chemo- have high CCR7 surface levels and most of it is ligand-free. Consequently, tactic response. A role for CCL21 in modulating CCR7 expres- they are highly sensitive for the CCL21 displayed by HEV and efficiently sion and function is also supported by data from transgenic home into the LN by CCR7-dependent transendothelial migration. Once studies where superphysiological CCL21 concentrations led to inside the LN, T cells crawl along the 3D-network of TRC throughout the reduced CCR7 levels on T cells and consequently reduced re- T zone while continually receiving CCR7 signals that increase their mo- sponsiveness (44, 45). tility. This motility is thought to improve the efficiency of encounters be- Downloaded from CD4ϩ T cells isolated from SLO showed a striking reduction in tween naive T cells and Ag-presenting DCs bound to TRC. The CCR7 their capacity to migrate toward CCR7 ligands, similar to T cells ligands are produced in large amounts by TRC and are likely to be associated with proteoglycans at the TRC surface where migrating T cells can preincubated with CCR7 ligands. Incubation at 37°C for1hwas easily pick them up. Once the ligand has bound to the receptor, this com- sufficient to restore responsiveness. In fact, resensitization by in- plex is eventually endocytosed, the ligand degraded and the receptor re- cubation at 37°C is a common practice in laboratories working on cycled back to the cell surface. The cycling of CCR7 prevents complete tissue lymphocytes and empirically known to improve the effi- desensitization of the cell, but the decreased surface expression of CCR7 http://www.jimmunol.org/ ciency of CCR7 staining and chemotaxis (29, 46). Here we provide diminishes ligand sensitivity and increases the propensity to exit the LN. mechanistic insight into this resensitization process. It appears to Once returned to the efferent lymph or the blood circulation where little involve both re-expression of internalized and liberation of occu- CCL19/21 is found, all CCR7 recycles back to the cell surface. At this pied CCR7, as CD4ϩ T cells isolated from SLO and incubated in stage the T cell has regained the maximal sensitivity toward CCR7 ligands, medium reach blood levels of free and total surface CCR7 within allowing it to respond potently to CCL19/21 presented on HEV. The du- 30 min to 1 h. The kinetic is comparable to studies of CCR7 re- ration needed for full CCR7 resensitization may influence how long T cells stay inside the blood. In that sense the cyclical modulation of CCR7 ac- expression on human cells (35, 38). We provide evidence suggest- tivity appears to be opposite to sphingosine-1-phosphate receptor 1, that is ing similar kinetics of CCR7 re-expression in vivo: 1) Upon entry not expressed on the surface of blood T cells but becomes gradually ex- ϩ by guest on September 26, 2021 into the blood circulation, CD4 T cells swiftly increased their pressed on T cells during their stay in SLO (47). CCR7 expression relative to cells in SLO and displayed homogeneously high CCR7 levels; and 2) CCR7 on adoptively transferred ϩ CD4 T cells rapidly adjusted to the environment, most drastically ϩ chemokinetic cell migration. While migrating, CD4 T cells in- shown in the highly increased CCR7 expression on wild-type cells ternalize and degrade CCL19 (and possibly CCL21). The internal- that had been transferred into plt/plt mice. Importantly, the re- ized receptor returns to the cell surface to prevent complete de- expression of internalized CCR7 and liberation of occupied recep- sensitization. The lower CCR7 level on T cells in SLO may reduce tor were associated in both settings with a strongly increased mi- cell retention and prepare them for the sphingosine-1-phosphate gratory response. Therefore, we think that these processes are receptor 1-mediated exit (4). Once T cells have emigrated from critical for the maintenance of CCL19/21 sensitivity and continu- SLO into the circulation, CCR7 signaling stops and most internal- ous cell motility within T zones. ized receptors are re-expressed. This process may be critical for Although CCR7 on human CD4ϩ and CD8ϩ T cells is similarly efficient exit from blood into LN, when T cells need to be at their modulated by its ligands in vitro (35), we found that CCR7 surface highest sensitivity toward CCR7 ligands displayed on HEVs. expression on murine CD4ϩ T cells is higher and more dynamic than on CD8ϩ T cells. In contrast, CCL19-Fc staining on CD8ϩ T ϩ Acknowledgments cells varied considerably among tissues, similar to CD4 T cells. ϩ We thank J. Cyster, B. Marsland, P. Schneider, and J. Zwirner for provid- Therefore, CCR7 function on CD8 T cells seems to be regulated ing reagents; S. Favre and T. Vogt for technical help; J. Cyster, almost entirely at the level of occupancy. The lower CCR7 surface ϩ ϩ P. Schneider, and M. Sixt for critical reading of the manuscript; and all expression of CD8 vs CD4 T cells correlated with a lower sen- members of the Luther laboratory for discussions. sitivity of the former cells in chemotaxis assays. These findings may provide mechanistic insight into similar differences observed Disclosures by others in chemotaxis assays (29, 31, 36) and suggest that CCR7 The authors have no financial conflict of interest. internalization is not a stringent requirement for efficient T cell migration toward CCL19 and CCL21 in vivo. References In summary, we propose the following model for CCR7 regu- 1. von Andrian, U. H., and T. R. Mempel. 2003. Homing and cellular traffic in ϩ lation on recirculating CD4 T cells (Fig. 7). During recirculation, lymph nodes. Nat. Rev. Immunol. 3: 867–878. T cells pass through two environments that are fundamentally dif- 2. Cyster, J. G. 2005. Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu. Rev. Immunol. 23: 127–159. ferent in their chemokine concentration – no or little CCL19/21 in 3. Forster, R., A. C. Davalos-Misslitz, and A. Rot. 2008. CCR7 and its ligands: blood and splenic red pulp vs high levels of CCL19/21 in T zones balancing immunity and tolerance. Nat. Rev. Immunol. 8: 362–371. 4. Pham, T. H., T. Okada, M. Matloubian, C. G. Lo, and J. G. Cyster. 2008. S1P1 of SLO – and T cells rapidly adjust to them. Within T zones, CCR7 receptor signaling overrides retention mediated by G ␣ i-coupled receptors to is engaged by ligands, leading to continuous CCR7 signaling and promote T cell egress. Immunity 28: 122–133. 7688 CCR7 MODULATION ON NAIVE T LYMPHOCYTES IN VIVO

5. Link, A., T. K. Vogt, S. Favre, M. R. Britschgi, H. Acha-Orbea, B. Hinz, 28. Campbell, J. J., J. Hedrick, A. Zlotnik, M. A. Siani, D. A. Thompson, and J. G. Cyster, and S. A. Luther. 2007. Fibroblastic reticular cells in lymph nodes E. C. Butcher. 1998. Chemokines and the arrest of lymphocytes rolling under regulate the homeostasis of naive T cells. Nat. Immunol. 8: 1255–1265. flow conditions. Science 279: 381–384. 6. Nakano, H., S. Mori, H. Yonekawa, H. Nariuchi, A. Matsuzawa, and T. Kakiuchi. 29. Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, and 1998. A novel mutant gene involved in T-lymphocyte-specific homing into pe- L. T. Williams. 1998. A chemokine expressed in lymphoid high endothelial ripheral lymphoid organs on mouse chromosome 4. Blood 91: 2886–2895. venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. 7. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, and Natl. Acad. Sci. USA 95: 258–263. M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing 30. Nagira, M., T. Imai, R. Yoshida, S. Takagi, M. Iwasaki, M. Baba, Y. Tabira, functional microenvironments in secondary lymphoid organs. Cell 99: 23–33. J. Akagi, H. Nomiyama, and O. Yoshie. 1998. A lymphocyte-specific CC che- 8. Vassileva, G., H. Soto, A. Zlotnik, H. Nakano, T. Kakiuchi, J. A. Hedrick, and mokine, secondary lymphoid tissue chemokine (SLC), is a highly efficient che- S. A. Lira. 1999. The reduced expression of 6Ckine in the plt mouse results from moattractant for B cells and activated T cells. Eur. J. Immunol. 28: 1516–1523. the deletion of one of two 6Ckine genes. J. Exp. Med. 190: 1183–1188. 31. Ngo, V. N., H. L. Tang, and J. G. Cyster. 1998. Epstein-Barr virus-induced 9. Luther, S. A., H. L. Tang, P. L. Hyman, A. G. Farr, and J. G. Cyster. 2000. molecule 1 ligand chemokine is expressed by dendritic cells in lymphoid tissues Coexpression of the chemokines ELC and SLC by T zone stromal cells and and strongly attracts naive T cells and activated B cells. J. Exp. Med. 188: deletion of the ELC gene in the plt/plt mouse. Proc. Natl. Acad. Sci. USA 97: 181–191. 12694–12699. 32. Willimann, K., D. F. Legler, M. Loetscher, R. S. Roos, M. B. Delgado, 10. Nakano, H., and M. D. Gunn. 2001. Gene duplications at the chemokine locus on I. Clark-Lewis, M. Baggiolini, and B. Moser. 1998. The chemokine SLC is ex- mouse chromosome 4: multiple strain-specific haplotypes and the deletion of pressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts secondary lymphoid-organ chemokine and EBI-1 ligand chemokine genes in the activated T cells via CCR7. Eur. J. Immunol. 28: 2025–2034. plt mutation. J. Immunol. 166: 361–369. 33. Sullivan, S. K., D. A. McGrath, D. Grigoriadis, and K. B. Bacon. 1999. Phar- 11. Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, and macological and signaling analysis of human chemokine receptor CCR-7 stably H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ che- expressed in HEK-293 cells: high-affinity binding of recombinant ligands mokine have defects in lymphocyte homing and dendritic cell localization. MIP-3␤ and SLC stimulates multiple signaling cascades. Biochem. Biophys. Res. J. Exp. Med. 189: 451–460. Commun. 263: 685–690. 12. Mori, S., H. Nakano, K. Aritomi, C. R. Wang, M. D. Gunn, and T. Kakiuchi. 34. Stein, J. V., A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn, 2001. Mice lacking expression of the chemokines CCL21-ser and CCL19 (plt A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, and U. H. von Andrian. 2000. The Downloaded from mice) demonstrate delayed but enhanced T cell immune responses. J. Exp. Med. CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lym- 193: 207–218. phoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associ- 13. Junt, T., E. Scandella, R. Forster, P. Krebs, S. Krautwald, M. Lipp, ated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node H. Hengartner, and B. Ludewig. 2004. Impact of CCR7 on priming and distri- high endothelial venules. J. Exp. Med. 191: 61–76. bution of antiviral effector and memory CTL. J. Immunol. 173: 6684–6693. 35. Bardi, G., M. Lipp, M. Baggiolini, and P. Loetscher. 2001. The T cell chemokine 14. Kaiser, A., E. Donnadieu, J. P. Abastado, A. Trautmann, and A. Nardin. 2005. receptor CCR7 is internalized on stimulation with ELC, but not with SLC. Eur. CC chemokine ligand 19 secreted by mature dendritic cells increases naive T cell J. Immunol. 31: 3291–3297.

scanning behavior and their response to rare cognate antigen. J. Immunol. 175: 36. Campbell, J. J., E. P. Bowman, K. Murphy, K. R. Youngman, M. A. Siani, http://www.jimmunol.org/ 2349–2356. D. A. Thompson, L. Wu, A. Zlotnik, and E. C. Butcher. 1998. 6-C-kine (SLC), 15. Bajenoff, M., J. G. Egen, L. Y. Koo, J. P. Laugier, F. Brau, N. Glaichenhaus, and a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is R. N. Germain. 2006. Stromal cell networks regulate lymphocyte entry, migra- an agonist for the MIP-3␤ receptor CCR7. J. Cell Biol. 141: 1053–1059. tion, and territoriality in lymph nodes. Immunity 25: 989–1001. 37. Breitfeld, D., L. Ohl, E. Kremmer, J. Ellwart, F. Sallusto, M. Lipp, and R. Forster. 16. Stachowiak, A. N., Y. Wang, Y. C. Huang, and D. J. Irvine. 2006. Homeostatic 2000. Follicular B helper T cells express CXC chemokine receptor 5, localize to lymphoid chemokines synergize with adhesion ligands to trigger T and B lym- B cell follicles, and support immunoglobulin production. J. Exp. Med. 192: phocyte chemokinesis. J. Immunol. 177: 2340–2348. 1545–1552. 17. Asperti-Boursin, F., E. Real, G. Bismuth, A. Trautmann, and E. Donnadieu. 2007. 38. Otero, C., M. Groettrup, and D. F. Legler. 2006. Opposite fate of endocytosed CCR7 ligands control basal T cell motility within lymph node slices in a phos- CCR7 and its ligands: recycling versus degradation. J. Immunol. 177: phoinositide 3-kinase-independent manner. J. Exp. Med. 204: 1167–1179. 2314–2323. 18. Huang, J. H., L. I. Cardenas-Navia, C. C. Caldwell, T. J. Plumb, C. G. Radu, 39. Kohout, T. A., S. L. Nicholas, S. J. Perry, G. Reinhart, S. Junger, and

P. N. Rocha, T. Wilder, J. S. Bromberg, B. N. Cronstein, M. Sitkovsky, et al. R. S. Struthers. 2004. Differential desensitization, receptor phosphorylation, ␤-ar- by guest on September 26, 2021 2007. Requirements for T lymphocyte migration in explanted lymph nodes. J. Im- restin recruitment, and ERK1/2 activation by the two endogenous ligands for the munol. 178: 7747–7755. CC chemokine receptor 7. J. Biol. Chem. 279: 23214–23222. 19. Okada, T., and J. G. Cyster. 2007. CC chemokine receptor 7 contributes to Gi- 40. Hargreaves, D. C., P. L. Hyman, T. T. Lu, V. N. Ngo, A. Bidgol, G. Suzuki, dependent T cell motility in the lymph node. J. Immunol. 178: 2973–2978. Y. R. Zou, D. R. Littman, and J. G. Cyster. 2001. A coordinated change in 20. Woolf, E., I. Grigorova, A. Sagiv, V. Grabovsky, S. W. Feigelson, Z. Shulman, chemokine responsiveness guides plasma cell movements. J. Exp. Med. 194: T. Hartmann, M. Sixt, J. G. Cyster, and R. Alon. 2007. Lymph node chemokines 45–56. promote sustained T lymphocyte motility without triggering stable integrin ad- 41. Ritter, U., F. Wiede, D. Mielenz, Z. Kiafard, J. Zwirner, and H. Korner. 2004. hesiveness in the absence of shear forces. Nat. Immunol. 8: 1076–1085. Analysis of the CCR7 expression on murine bone marrow-derived and spleen 21. Worbs, T., T. R. Mempel, J. Bolter, U. H. von Andrian, and R. Forster. 2007. dendritic cells. J. Leukocyte Biol. 76: 472–476. CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo. J. Exp. 42. Katakai, T., T. Hara, J. H. Lee, H. Gonda, M. Sugai, and A. Shimizu. 2004. A Med. 204: 489–495. novel reticular stromal structure in lymph node cortex: an immuno-platform for 22. Bajenoff, M., J. G. Egen, H. Qi, A. Y. Huang, F. Castellino, and R. N. Germain. interactions among dendritic cells, T cells, and B cells. Int. Immunol. 16: 2007. Highways, byways and breadcrumbs: directing lymphocyte traffic in the 1133–1142. lymph node. Trends Immunol. 28: 346–352. 43. Cahalan, M. D., and I. Parker. 2008. Choreography of cell motility and interac- 23. Worbs, T., G. Bernhardt, and R. Forster. 2008. Factors governing the intranodal tion dynamics imaged by two-photon microscopy in lymphoid organs. Annu. Rev. migration behavior of T lymphocytes. Immunol. Rev. 221: 44–63. Immunol. 26: 585–626. 24. Luther, S. A., A. Bidgol, D. C. Hargreaves, A. Schmidt, Y. Xu, J. Paniyadi, 44. Unsoeld, H., K. Mueller, U. Schleicher, C. Bogdan, J. Zwirner, D. Voehringer, M. Matloubian, and J. G. Cyster. 2002. Differing activities of homeostatic che- and H. Pircher. 2007. Abrogation of CCL21 chemokine function by transgenic mokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruit- over-expression impairs T cell immunity to local infections. Int. Immunol. 19: ment and lymphoid neogenesis. J. Immunol. 169: 424–433. 1281–1289. 25. Yoshida, R., M. Nagira, M. Kitaura, N. Imagawa, T. Imai, and O. Yoshie. 1998. 45. Christopherson, K. W., 2nd, J. J. Campbell, and R. A. Hromas. 2001. Transgenic Secondary lymphoid-tissue chemokine is a functional ligand for the CC chemo- overexpression of the CC chemokine CCL21 disrupts T-cell migration. Blood 98: kine receptor CCR7. J. Biol. Chem. 273: 7118–7122. 3562–3568. 26. Pilkington, K. R., I. Clark-Lewis, and S. R. McColl. 2004. Inhibition of gener- 46. Brandes, M., D. F. Legler, B. Spoerri, P. Schaerli, and B. Moser. 2000. Activa- ation of cytotoxic T lymphocyte activity by a CCL19/macrophage inflammatory tion-dependent modulation of B lymphocyte migration to chemokines. Int. Im- protein (MIP)-3␤ antagonist. J. Biol. Chem. 279: 40276–40282. munol. 12: 1285–1292. 27. Ott, T. R., F. M. Lio, D. Olshefski, X. J. Liu, N. Ling, and R. S. Struthers. 2006. 47. Lo, C. G., Y. Xu, R. L. Proia, and J. G. Cyster. 2005. Cyclical modulation of The N-terminal domain of CCL21 reconstitutes high affinity binding, G protein sphingosine-1-phosphate receptor 1 surface expression during lymphocyte re- activation, and chemotactic activity, to the C-terminal domain of CCL19. Bio- circulation and relationship to lymphoid organ transit. J. Exp. Med. 201: chem. Biophys. Res. Commun. 348: 1089–1093. 291–301.