Characterization of the chemokine receptor CCR7 and its role in cell migration

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FACHBEREICH BIOLOGIE

Characterization of the chemokine receptor CCR7 and its role in cell

migration

Dissertation

Zur Erlangung des akademischen Grades eines

"Doktors der Naturwissenschaften"

(Dr. rer. nat.)

des Fachbereiches für Biologie an der Universität Konstanz

vorgelegt von

Maria Carolina Otero Acuña

Tag der mündlichen Prüfung: 11.12.2006 1. Referent: Prof. Dr. M. Scheffner 2. Referent: Prof. Dr. M. Groettrup

Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2006/2242/

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With the deepest love to my parents Für meine lieben Eltern

A mis padres con el más profundo amor

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Acknowledgements

First of all I sincerely want to thank Marcus Groettrup for convincing me that Germany is a nice place to live! I also appreciate a lot that he always found time to discuss about experiments, my project and life in science with me. It was really a great opportunity for me to work in his lab!

Thank you to the Swiss Team! I specially want to thank Daniel Legler, who helped me with my experiments, anytime I felt frustrated and anytime I was jumping because an experiment worked. Also he supported me a lot personally. And I also have to thank Michi, for being always open to help me and discuss about whatever I wanted.

Ah! Also for taking me home with his car from time to time. "Merci vielmals!"

Thanks to Petra Eisele, for having an extensive patience with me all the time that I tried to explain an experiment in German and for letting me feel that we could work in a great team together!

I am very thankful and I feel very lucky to have found all the people of this laboratory! Especially the “Tussi-Box” integrated by Tina, who took me a lot of times to the mountains and made me feel not so “homesick” and Jackie who always was very open to hear my stories and my foreigners and Chilean problems. I also want to thank Eva for her friendship and being always there. To Petra Krause for helping me all the time with the “Amaxa Transfection”.

I am fully grateful to Nela Kolb who helped me a lot personally during my stay here in Konstanz. And to the rest of the lab: “The Fat10 people”: Christiane, Sebastian (my computer assistant) and Birte. Thanks also to Marcel and Brigitte. To Elisabeth who was always disposed to help with a smile and Ulli who helped me every time that I moved or every time that I needed something at home. And Gunter, he always was opened to help me when I have problems with my experiments. I also want to thank the people at the BITg.

I also extend my thanks to our laboratory neighbors: Matthias Langhorst for assistance in image processing and to the “Latino mafia group” including Ed and Erik, who also helped me with the corrections of this thesis. To Gerardo for being my friend all the time and for having always a lot of fun at the lab. I also want to thank Christopher who was almost part of the “Latino mafia group”.

Thanks also to my WG, especially to Jens and Dominik, for making me feel that home is not so far away, “my little family”. Thank you Dominik for singing my latino american songs and bringing music to my life! My deepest thanks to Carsten for his patience, affection and for being always with me.

I want to thank with all my heart my parents, who were always present in every moment, for having a great and warm home that is always waiting for me! And to my dear “Oma”, my best friend who made Germany part of my history. I am also very thankful to my friends in Chile who support me all the time, especially Alejandra and Pablo Nelson. Also Andrea Soza who helped me to reach this place.

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This work was supported by the German Research Foundation (DFG, TR-SFB 11), the Thurgauische Stiftung für Wissenschaft und Forschung, and the State Secretariat for Education and Research (§16 law of research).

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Abbreviations

Aa Amino acids

APC Antigen presenting cell Arp2/3 Actin-related protein 2/3

BSA Bovine serum albumin

cAMP Cyclic adenosine monophosphate CCL CC chemokine ligand

CCR CC chemokine receptor CTx-B Cholera toxin B subunit DAG Diacylglycerol

DMEM Dulbecco`s modified eagle medium DMSO Dimethylsulfoxide

DOCK-2 Downstream of Crk-180 homolog-2 protein DRM Detergent resistant membrane

DTT Dithiothreitol

EBI1 Epstein-Barr virus inducible gene 1 (CCR7)

EBV Epstein-Barr virus

EDTA Ethylenediamine-tetraacetic acid EGF Epidermal growth factor

eGFP Enhanced GFP

EGFR Epidermal growth factor receptor ELC EBI1-ligand chemokine (CCL19)

ER Endoplasmic reticulum

ERK Extracellular regulated kinase FACS Fluorescence activated cell sorting FCS Fetal calf serum

FITC Fluorescein isothiocyanat

fMLP Formyl-methionyl-leucyl-phenylalanine FRET Fluorescence resonance energy transfer

GDP Guanosine diphosphate

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GEF Guanine-nucleotide exchange factor GFP Green fluorescent protein

GM1 Ganglioside-monosialic acid 1 GPCR G-protein-coupled receptor GPI Glycosylphosphatidylinositol G protein Guanine nucleotide binding protein

GTP Guanosine triphosphate

GPCR G protein coupled receptor HA Influenza haemagglutinin protein

HEPES N-(2-hydroxyethyl)-piperazine-N’-2-ethanesulfonic acid HEV High endothelial venules

HIV Human immunodeficiency virus

HRP Horseradish peroxidase

IgG Immunoglobulin G IL Interleukin

IMDM Iscove’s modified dubelcco’s medium

IP3 Inositol triphosphate

kDa Kilo dalton

LFA-1 Lymphocyte function-associated antigen-1 LSM Laser scanning microscopy MAPK Mitogen-activated protein kinase MFI Mean fluorescence intensity MHC Major histocompatibility complex MLC Myosin light chain

mM Millimolar

MW Molecular weight

n Number in study or group

NA Numerical aperture

NAD⁺ Nicotinamide-adenine-dinucleotide nm Nanometer

PAGE Polyacylamide gel electrophoresis PBMC Peripheral blood mononuclear cells

PBS Phosphate-buffered saline

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PCR Polymerase chain reaction PE Phycoerythrin

P-ERK ERK phosphorylated at positions T202/Y204 PFA Paraformaldehyde

PGE2 Prostaglandin E2

PH Pleckstrin homology domain

PI3K Phosphatidylinositol-3-phosphat kinase PIP2 Phosphatidylinositol bisphosphate PKC Protein kinase C

PLC Phospholipase C

PTEN Phosphatase and tensin homolog on chromosome 10

RANTES Regulated upon activation, normal T cell expressed and secreted ROCK Rho-associated coiled-coil kinase

RT Room temperature SDS Sodium dodecyl sulfate

SLC Secondary lymphoid tissue chemokine (CCL21) SV40 Simian virus 40

TCA Trichloroacetic acid

TCR T cell receptor

TLR Toll-like receptor

TNF Tumour necrosis factor ub Ubiquitin

µM Micro molar

v/v Volume per volume VSV Vesicular stomatitis virus

WAVE WASP verprolin-homologous protein

w/v Weight per volume

WB Western blot

WT Wildtype

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“The product of mental labor - science - always stands far below its value, because the labor-time necessary to reproduce it has no relation at all to the labor-time required for its original production.”

Karl Marx

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Zusammenfassung

Die Zellwanderung ist eine grundlegende Eigenschaft der Zellen des Immunsystems, die es ihnen ermöglicht, sich zu einem von Pathogenen verursachten Infektionsherd zu bewegen. Dieser Prozess ist essentiell für die komplexe Interaktion zwischen den Immunzellen, welcher die Immunantwort abstimmt und begrenzt. Die Zellwanderung wird durch die Interaktion zwischen Chemokinrezeptoren mit dem dazugehörigen Chemokinen gesteuert. Diese sind verantwortlich dafür, die Zellen exakt an den entsprechenden Ort zu navigieren. Unter allen Chemokinrezeptoren ist CCR7 für das korrekte Rekrutieren von Lymphozyten und reifen dendritischen Zellen in die lymphatischen Gewebe verantwortlich. Dieser Rezeptor wird von zwei verschiedenen Liganden aktiviert: ELC (CCL19) und SLC (CCL21). Beide CC Chemokine werden von Stromazellen produziert, welche sich in den T-Zell Zonen der Lymphknoten befinden.

Die Rolle von CCR7 im korrekten "homing" von Lymphozyten und dendritischen Zellen wurde ausführlich untersucht. Dennoch gibt es nur wenig Information über die zellbiologische Funktion von CCR7. Deshalb ist der Gegenstand dieser Arbeit die Analyse eines Rezeptors als einzelnes Molekül in einem zellulären Zusammenhang.

Um unsere Fragestellungen zu beantworten, generierten wir einen terminal GFP- markierten CCR7 zur Visualisierung des Rezeptors mittels konfokaler Mikroskopie.

Des Weiteren stellten wir ELC und SLC als Fc-Fusionsproteinen her. Beide rekombinanten Proteine wurden aufgereinigt und zeigten biologische Funktionalität, da sie, ähnlich wie die Wildtypchemokine, einen Kalziumeinstrom ins Zytosol und Wanderung von CCR7 exprimierenden Zellen vermitteln. Mit den Werkzeugen, die während meiner Arbeit entwickelt wurden, war es uns möglich, sowohl das zelluläre

"trafficking" von CCR7, als auch die Aminosäuren, welche für die Funktion und die Verteilung in der Zelle während der Polarisierung verantwortlich sind, zu untersuchen. Dadurch war es uns möglich, eine Lücke in der Biologie von CCR7 und dem Feld der Chemokine zu schließen.

Um einen besseren Überblick der verschiedenen Themen dieser Arbeit zu bekommen, habe ich sie in drei verschiedene Gebiete unterteilt. Das erste Thema

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befasst sich mit der Untersuchung des CCR7 "trafficking" zusammen mit seinem Liganden ELC. In diesen Untersuchungen konnten wir zeigen, dass CCR7 zusammen mit seinem Liganden ELC aufgenommen wird. Im Weiteren wird ELC über Lysosomen abgebaut, wohingegen CCR7 wiederverwendet wird und zurück an die Membran gelangt. Der zweite Teil der Arbeit beschäftigt sich mit der Charakterisierung verschiedener Mutanten des C-Terminus von CCR7 und die mögliche Rolle der Ubiquitylierung auf das "trafficking" und die Wanderung. Die Charakterisierung von GFP gekoppeltem CCR7 in wandernden Zellen zusammen mit der Beteiligung an "lipid rafts" wird im dritten Teil der Arbeit beschrieben.

Diese Ergebnisse bieten eine gute Basis, um funktionelle Domänen von CCR7, das "trafficking" und die Verteilung in wandernden Zellen besser definieren zu können.

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Preface and summary

Cell migration is a fundamental property of immune cells that enables their mobilization to sites of pathogen infection. This process is essential for the complex interaction between immune cells that modulates and restricts the immune response.

Cell migration is coordinated by the interaction of chemokines and their receptors, which orchestrate the entire immune system. The chemokine receptor CCR7 is responsible for the proper recruitment of lymphocytes and mature dendritic cells to lymphoid tissues. This receptor is activated by two different ligands: ELC (CCL19) and SLC (CCL21), both CC chemokines that are mainly expressedby stromal cells in the T cell areas of lymph nodes.

The role of CCR7 in the proper homing of lymphocytes and DCs has been described. However, there is only little information on the function of CCR7 on the single cell level. Therefore, our objective in this work was to analyze the receptor from a cell biological point of view.

In order to facilitate the visualization of the receptor by confocal microscopy under different conditions we created a version of tagged CCR7 with a GFP at the C- terminus. Moreover, we generated ELC and SLC as Fc-fusion proteins. Both recombinant proteins were purified and biologically functional as both mediated calcium flux and migration, similar to wild type chemokines in CCR7 expressing cells. With the tools generated during my thesis, we could demonstrate that CCR7 was internalized together with ELC, but then ELC was degraded in lysosomes whereas CCR7 recycled back to the plasma membrane. Moreover we created different mutants of CCR7 demonstrating that the C-terminus of CCR7 is not ubiquitylated and that CCR7 C-terminus is essential for cell migration but not for receptor recycling or endocytosis. Through time laps microscopy using CCR7-GFP we found that the receptor is predominantly localized at one aspect of a chemokine triggered cell.

Moreover, we identified that CCR7 constitutively partitions within specialized membrane microdomains, termed lipid rafts, which is required for efficient signal transduction.

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These results provide a solid base to better define functional domains of CCR7, its trafficking and its distribution on a migrating cell.

Carolina Otero July 2006

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Chapter 1 Introduction

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“If you don’t know where you are, you don’t know who you are…Migratoriness has its dangers.”

The sense of place, Wallace Stenger (Random House, 1992)

Getting through the complexity of the human organism, the immune system consists of trillions of cells widely dispersed all over the body. When a pathogen invades our body, our organism is able to orchestrate this huge number of cells in a way that the pathogen can be eliminated, being thus a formidable process resulting in perfect organization. Different circulating cells mediate this process. Some of them are migrating passively over long distances in the blood, which facilitates rapid detection and guarantees effective measurements in very different tissue environments. This process must be extremely well regulated in order to avoid inappropriate migration and activation contributing to autoimmune disease (Alt et al., 2002; Cahalan and Gutman, 2006).

But how do cells know where to go? The high degree of specificity demanded in the immune system is achieved in part by the efficient organization and compartmentalization of its cellular constituents. Chemokines are the primary guidance cues used by leukocytes during their controlled trafficking to and from different compartments.

Chemokines are a group of small (8-14kDa), mostly basic heparin bound- proteins whose function is the regulation of leukocyte trafficking mediated by its chemoattractant ability. Approximately 50 different human chemokines have been identified to date (Murphy et al., 2000). The sequence identity between chemokines is relatively low. In spite of that, their three-dimensional structures is quite similar (Proudfoot, 2002). A main characteristic is a flexible amino terminal region that is followed by a conserved motif of four cysteine residues that form two characteristic disulphide bridges. Four classes of chemokines have been defined, based on the arrangement of the conserved cysteine (C) residues in the N-terminal domain of the mature proteins: CXC, CC, C chemokines, and CX3C chemokines (Luster, 1998;

Rollins, 1997). The CXC- and CC-chemokines are the major chemokine subfamilies.

Chemoattractants like C5a (Complement factor 5a) and chemokines act by activating seven transmembrane domain receptors coupled to G protein (the name of which will be derived from its respective ligand: CCR7 for instance binds CC chemokines), thus triggering chemotaxis towards the required destination (Paoletti et al., 2005).

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A more recent classification which include conditions and localization of chemokine production as well as chemokine receptor distribution on different leukocyte subtypes, are “inflammatory” and “homeostatic” chemokines.

Inflammatory chemokines are inducible, produced in inflamed tissues by resident and infiltrating cells upon proinflammatory stimuli such as lipopolysaccharide (LPS), and cytokines such as IL-1 and TNF-α, and which together orchestrate innate and adoptive immune responses. In contrast, the homeostatic groups of chemokines are produced in discrete microenvironments within lymphoid and non-lymphoid tissues such as the skin and mucosa. These constitutively expressed chemokines regulate the traffic and positioning of cells that mainly belong to the adoptive immune system during haematopoiesis, antigen sampling in secondary lymphoid tissue and immune surveillance. There is also one group of overlapping chemokines in which the distinction between inflammatory and homeostatic chemokines is not absolute (Cyster et al., 1999).

During inflammation, chemokine-mediated leukocyte extravasation from the blood into the tissue is a very regulated process involving a series of coordinated complex interactions of adhesion molecules, chemoattractants and their receptors between leukocytes and the endothelial cells as shown in figure 1.1 (Butcher, 1991;

Springer, 1994). Integrin activation by chemokines bound on heparan sulphate proteoglycans at the endothelium wall is a requirement for cytoskeletal rearrangements and cellular polarization of leukocytes for subsequent firm adhesion and extravasation. Within the tissue, cell migration along a chemokine gradient involves the sensing of slight differences in chemokine concentrations and the establishment of cell polarity. Cell polarization is followed by directional cell locomotion via cytoskeletal rearrangements, activation-induced changes in integrin affinity and adhesive interactions with the extracellular matrix. All these processes occur in a coordinated way with extension and retraction of cell membrane protrusions (pseudopods) (Rodriguez-Frade et al., 1999).

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Figure 1.1. Chemokine regulation of leukocyte movement. Chemokines are secreted at sites of inflammation and infection by resident tissue cells, resident and recruited leukocytes and cytokine- activated endothelial cells. Chemokines are locally retained on matrix and cell-surface heparin sulphate proteoglycans, establishing a chemokine concentration gradient surrounding the inflammatory stimulus. Leukocytes rolling on the endothelium in a selectin-mediated process are brought in contact with chemokines retained on cell-surface heparan sulphate proteoglycans. Chemokine signaling induces remodeling of the cytoskeleton such as reorganization of actin filaments, which allows the cells to flatten and attain cellular polarization. Further, integrins are activated, leading to firm adherence and extravasation. The recruited leukocytes are activated by local proinflammatory cytokines and may become desensitized to further chemokine signaling.

Among chemokine receptors, CCR7 is responsible for the correct recruitment of T cells and mature dendritic cells (mDC) to lymph nodes (LNs). An extensive characterization of CCR7-deficient mice has shown that some LNs were devoid of naïve T cells and DCs, whereas the T cell population was expanded in the blood, the red pulp of the spleen and in the bone marrow. In addition, these knock-out mice showed severely delayed kinetics concerning antibody responses and delayed type hypersensitivity reactions. Interestingly, mDCs failed to migrate into the draining lymph nodes (Forster et al., 1999; Ohl et al., 2004). These data clearly show evidence for the important and critical role of this receptor on the primary immune response.

Furthermore, experiments on transgenic mice with a premature CCR7 expression on

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thymocytes resulting in an inappropriate entrance of these cells into the medulla before positive selection has occurred, showing the involvement of CCR7 in medullar migration required for an efficient T cell development (Kwan and Killeen, 2004).

CCR7 is activated by two different ligands, ELC (CCL19, Exodus-3, MIP-3β, CKβ11) and SLC (CCL21, Exodus-2, 6Ckine, TCA-4), both CC chemokines that are highly expressed by stromal cells in the T cell area of lymph nodes (figure 1.2) (Willimann et al., 1998; Luther et al, 2000).In addition, ELC is expressed by mature DCs and is presentedon the luminal side of HEV cells. SLC is expressedby afferent lymphatic endothelial cells and by HEV cells inmice (in humans SLC is only exposed on the lumen, but notexpressed by the HEVs) (Carlsen et al., 2005; Randolph et al., 2005). On the other hand, experiments in plt/plt mice lacking ELC and one of the two genes encoding SLC showed that these ligands are essential for the homing to secondary lymphoid organs (Gunn et al., 1999; Luther et al., 2000). A confirmation of the function of these chemokines in the homing process was achieved in a transgenic mouse model. An ectopic expression of ELC showed that antigen presenting cells were retained, preventing DCs from migration into draining lymph nodes, resulting in a totally impaired immune response (Krautwald et al., 2004).

Figure 1.2. Scheme of stromal cells resident in the T cell zone of lymphoid tissues. Stromal cells produce large amounts of SLC and ELC. CCR7-expressing cells, such as naïve T cells entering from the blood stream and mature dendritic cells entering from the lymph respond to these chemoattractants by migrating into the T cell zone. Figure adapted from Sanjiv Luther web page, Lausanne University.

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As professional antigen presenting cells in peripheral tissues, DCs play a critical role in the detection of pathogens as well as induction of the acquired immune response via stimulation of T-cells in the regional lymph nodes. Two main different stages have been described for DCs: immature and mature DCs. Immature DCs present a high endocytosis rate, resulting in a high antigen-uptake and its migration is regulated by inflammatory chemokine receptors like CCR1, CCR2, CCR5, CCR6, CXCR1 and CXCR2, which guide these cells to sites of inflammation. At the same time, DCs secrete large amounts of proinflammatory chemokines that recruit other immature DCs, monocytes and macrophages (Sallusto et al., 1998). When DCs acquire a mature phenotype due to a danger stimulus such as LPS, cells down-regulate the expression of CCR1, CCR2, CCR5 and CCR6 while CCR7 expression is up- regulated acquiring responsiveness to the chemokines ELC and SLC (figure 1.3) (Ritter et al., 2004; Sallusto et al., 1999; Sozzani et al., 1998; Yanagihara et al., 1998).

Strikingly, CCR7 surface expression on mature monocyte-derived DCs is not sufficient for migration, being PGE2 an essential molecule that has to be present during DC maturation as a key factor for migration (Hopken and Lipp, 2004;

Scandella et al., 2002; Scandella et al., 2004; Legler et al., 2006).

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Figure 1.3. Diagram of DCs maturation. Immature dendritic cells respond to inflammatory chemokines CCL2, CCL3, and CCL5, through their respective chemokine receptors CCR1, CCR2, and CCR5, which enables them to migrate to inflamed tissues. The migratory capacity of iDCs toward inflammatory chemokines is induced upon processing of the extracellular NAD⁺ by the ectoenzyme ADP-Ribosyl Cyclase CD38 producing adenosine diphosphate ribose (ADPR) and cyclic ADP-ribose (cADPR). Lipid mediators such as prostaglandins (PGE2) and sphingolipids (S1P) also promote chemokine responsiveness of iDCs. Triggering of mature dendritic cells with the CD38-derived cADPR and NADPR or cysteinyl leukotrienes (LTC4) renders them competent to migrate toward the chemokines ELC and SLC, which bind to their chemokine receptor CCR7 expressed on mDCs.

Signaling pathways utilized by CD38, lipid mediators, and chemokines are dependent on potent calcium mobilizing activities or the second messengers cADPR, ADPR, and/or IP3 (Hopken and Lipp, 2004).

The process of cell migration has been well characterized on DCs (Arrieumerlou and Meyer, 2005). Some of these studies on mature DCs, after CCR7 stimulation revealed that this chemokine receptor could also induce protrusion formation, indicating that CCR7 can regulate the cytoarchitecture of these cells.

Probably related to the capability of this receptor to regulatethe organization of the actin cytoskeleton (Bardi et al., 2003; Ding et al., 2003). Moreover, studies on T cells have shown that stimulationof CCR7 induces filopodia formation and morphological polarization of T cells (Bardi et al., 2003). Several studies have also reported that plasma membrane microdomains like lipid rafts have been involved in the process of polarization together with migration itself. These microdomains are also termed

“detergent resistant membranes” (DRM) corresponding to a special membrane subtype, which is characterized by its rigidity due to the high amount of cholesterol and its insolubility in cold nonionic detergents such as Triton X-100 (Zajchowski and Robbins, 2002; Manes et al., 1999; Manes and Martinez, 2004). This membrane rafts are relatively large (>50 nm) cholesterol and sphingolipid-rich structures wherein associated proteins are likely to be concentrated (Simons and Toomre, 2000). They also have been described as dynamic assemblies of small size, constituted by components that are preferentially associated with lipids (Anderson and Jacobson, 2002). Today, many reports are available that clearly demonstrate the essential role of this membrane microenvironment on cell migration. Lipid rafts in the cell membrane context are shown in figure 1.4.

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Figure 1.4. Illustration of the fluid mosaic model of membranes incorporating the “raft hypothesis”.

(Addison Wesley Longman, 2000)

In addition to the chemotactic function of CCR7 other roles for this receptor have been described. Stimulation of CCR7 in mature DCs protects these cells from apoptosis caused by serum deprivation, suggesting that CCR7may regulate survival (Sanchez-Sanchez et al., 2004). This observation has also been described for the chemokine receptor CXCR4 that may induce protectionfrom IL-10-induced apoptosis in DCs (Zou et al., 2001). Moreover, stimulation of CCR7 may also increase the migratory speed of DCs, indicating that this receptor can regulate DC locomotion (Riol-Blanco et al., 2005). Finally, it has recently been shown that stimulation of CCR7 enhances the mature phenotype of DCs, leading to the secretion of inflammatory cytokines, increase of MHC levels, costimulatory molecules and the potentiation of the abilityof DCs to activate naïve T cells (Marsland et al., 2005).

Now that many other functions have been attributed to CCR7, the question how a receptor can initiate diverse cellular behaviours at temporally and spatially separate stages arises. To master this challenge both ligands seem to be necessary.

Studies using mice lacking ELC and SLC expression have shown that proper localization of DCs was impaired, together with a defect of lymphocyte homing,

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indicating that both ELC and SLC are required for the proper function of CCR7 (Gunn et al., 1999).

In spite of the same affinity of both chemokines to CCR7 (Sullivan et al., 1999), studies on T cells had also revealed that ELC, but not SLC, induces desensitization of the receptor. Additionally, a very interesting finding was the differential receptor phosphorylation, β-arrestin recruitment and 1/2 ERK activation by ELC versus SLC (Kohout et al., 2004). Moreover, ELC triggers CCR7 endocytosis in T cells whereas SLC does not (Bardi et al., 2001). In this regard, and despite the equal capabilityto induce chemotaxis, it is necessary to mentionthat ELC and SLC share only 32% aa identity (Yoshida et al., 1997). Other study showed that amino acids situated at the N-terminus of ELC are responsible for the high affinity binding to CCR7 (Ott et al., 2004).

Concerning the signalling cascade of CCR7, this receptor like most G protein coupled receptors may use G protein-dependent and -independent mechanisms to transduce intracellular signals (Thelen, 2001). G protein-dependent mechanisms involvethe Gα subunit (until now 4 different Gα have been described: αs, αq, αi and α12) and the βγ dimer of the heterotrimeric G proteins. Studies reported that chemotaxis is sensitive to pertussis toxin treatment, indicating that CCR7 is coupled to the Gαi protein (Riol-Blanco et al., 2005). Activation of G protein normally results in the generation of second messengers like Ca²⁺, cAMP and IP3. This involves mainly two enzymes, adenylyl cyclase (AC) (Robison et al., 1968) andphospholipase C β (PLCβ), which are regulated by the active Gα subunit of specific G proteins. AC catalyzes the reaction that forms cAMP from ATP, PLCβ cleaves PIP2 into IP3 and DAG. IP3 mediates an increase in free intracellular Ca²⁺ by releasing Ca²⁺ from stores in the ER. Ca²⁺ binds calmodulin together with others proteins, thus regulating further signalling events that are directed toward the nucleus. DAG retains its membrane association and activates PKC, a family of serine/threonine kinases that induce signalling towards the nucleus as well (Berridge and Irvine, 1984). NF-κB for example, can be activated via this pathway. Some PKC family members also require Ca²⁺ for their activation. Furthermore, Ca²⁺ and/or PIP2 are involved in cytoskeletal remodelling. While actin depolimerization is mediated through Ca²⁺-regulated proteins PIP2-binding proteins like profilin and gelsolin contribute to the opposite process of actin polymerization. After Gαi activation, βγ can also activate the

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PI3K/Akt pathway, together with members of the MAPK family (Sanchez-Sanchez et al., 2004). CCR7 mayalso use adaptors like β-arrestins to relay Gprotein-independent signals from CCR7 (Kohout et al., 2004).

There is no doubt that CCR7 plays a key role for migration of T lymphocytes and DCs, however the cell biology of CCR7 has remained poorly investigated. With the objective to contribute to the chemokine field and elucidate the CCR7 biology, for a better understanding of the immune system under normal, pathological or autoimmune conditions, I chose to study the intracellular trafficking of CCR7 and its function for migration, using different techniques such as confocal and time-lapse microscopy together with biochemical assays. During this study three different aspects of CCR7 were analyzed and accordingly this thesis is divided into three different chapters: in the first part of this work the trafficking of CCR7 and its ligand ELC was described showing opposite fates: CCR7 once internalized, recycled back to the plasma membrane. On the other hand, ELC was internalized together with the receptor being degraded in the lysosomes. Moreover, we also analyzed endocytosis and recycling of CCR7 upon receptor triggering showing profound CCR7 endocytosis and recycling after ELC triggering compared to SLC activation. The process of CCR7 endocytosis was dependent on Dynamin II and Eps15 clearly suggesting clathrin- coated pits internalization pathway. Recycling was independent of de novo protein synthesis and the recycled CCR7 was fully functional. The possible rate of degradation of the CCR7 was also analyzed. Our studies showed no degradation of the receptor even after long exposure with the ligand. This fact suggests that mDCs could reach their destination, even if CCR7 neosynthesis is blocked through viral interference.

In the second part, mutations of the receptor were performed in order to identify which residues are important in signal transduction and trafficking of CCR7.

Through the characterization of these mutants, we could identify that residues at the C-terminus are critical for signal transduction but, interestingly, were not essential for CCR7 endocytosis or recycling. Moreover, a possible role of ubiquitin, through coimmunoprecipitation experiments and site-directed mutagenesis was also analyzed,

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showing that CCR7 is not ubiquitylated even under ligand binding. In the last part, CCR7 was characterized under in vivo migration conditions together with the involvement of lipid raft microdomains, showing that CCR7 is indeed in lipid rafts and is polarized upon activation.

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Chapter 2 Opposite fate of endocytosed CCR7 and its ligands:

recycling versus degradation

Carolina Otero, Marcus Groettrup, and Daniel F. Legler

Journal of Immunology, 2006, 177: 2314–2323

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2.1. Abstract

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play a crucial role for the homing of lymphocytes and dendritic cells to secondary lymphoid tissues. Nevertheless, how CCR7 senses the gradient of chemokines and how migration is terminated is poorly understood. Here we demonstrate that CCR7(-GFP) is endocytosed into early endosomes containing transferrin receptor upon CCL19 binding, but less upon CCL21 triggering. Internalization of CCR7 was independent of lipid rafts but relied on dynamin and Eps15 and was inhibited by hypertonic sucrose, suggesting clathrin-dependent endocytosis. After chemokine removal, internalized CCR7 recycled back to the plasma membrane and was able to mediate migration again. In contrast, internalized CCL19 was sorted to lysosomes for degradation showing opposite fate for endocytosed CCR7 and its ligand.

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2.2. Introduction

Leukocyte traffic is fundamental for immune regulation and hence is highly coordinated. Tissue- and microenvironment-selective leukocyte homing is the basis for this organization. It is now well established that cell migration is mainly orchestrated by chemokines which, together with adhesion molecules, deliver the key signal that allows leukocytes to transmigrate from the bloodstream into the tissue (Campbell et al., 2003; Moser et al., 2004; Muller et al., 2003). All chemokines act through signaling via seven-transmembrane domain G-protein coupled cell surface receptors, but migration properties vary greatly among different cell types. The central role of the chemokine receptor CCR7 and its ligands CCL19 (ELC, Exodus-3, MIP-3β, CKβ11) and CCL21 (SLC, Exodus-2, 6Ckine, TCA-4) in the homing to secondary lymphoid organs is undisputed. CCR7 is highly expressed on naïve T cells and to a lower level on B cells. A transient increase in CCR7 expression is found upon T cell activation (Willimann et al., 1998), whereas T cell differentiation towards effector cells is accompanied by the down-regulation of the receptor on the cell surface (Sallusto et al., 1999). In dendritic cells however, CCR7 expression is induced upon maturation (Gunn, 2003; Sallusto et al., 1998; Sozzani et al., 1998). Mice lacking CCR7 show delayed kinetics in antibody responses, delayed-type hypersensitivity reactions, and morphological abnormalities in secondary lymphoid organs as a consequence of an impaired homing of mature dendritic cells and lymphocytes (Forster et al., 1999; Ohl et al., 2004). The fact that CCR7 ligands are mandatory for the homing to secondary lymphoid organs has been demonstrated in plt/plt mice lacking CCL19 and CCL21 (Gunn et al., 1999; Luther et al., 2000;

Nakano and Gunn, 2001; Vassileva et al., 1999).

Although CCR7 and its ligands are essential for eliciting a potent cellular immune response, CCR7 signaling and its regulation is still sparsely investigated (Bardi et al., 2003; Kohout et al., 2004; Riol-Blanco et al., 2005; Sanchez-Sanchez et al., 2004; Scandella et al., 2004; Tilton et al., 2000). In particular, information on how CCR7-mediated migration is stopped after a cell has arrived at its final destination within the lymph node has remained unclear. CCL19 and CCL21 are both produced by stroma cells within the T cell zone (Luther et al., 2000). Remarkably, CCL21 is transcytosed to high endothelial venules (Carlsen et al., 2005) and mediates LFA-1- mediated arrest of the recruited T lymphocytes (Gunn et al., 1998; Stein et al., 2000;

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Warnock et al., 2000). Thus, T lymphocytes and dendritic cells that home to the T zone of lymph nodes seem first to be recruited to HEV by CCL21 but then the CCL21 signal must be overcome by an attraction signal provided by CCL19/CCL21 derived form the T zone. B cells within the lymph node, that have seen an antigen, migrate directionally towards the B zone-T zone boundary along a gradient of CCL21 (and eventually CCL19) to encounter T cells (Okada et al., 2005).

One way rendering a cell unresponsive to chemokines is receptor internalization.

Chemokine receptor endocytosis is best described for the HIV co-receptors CCR5 and CXCR4 but follow distinct mechanisms (Venkatesan et al., 2003). Remarkably, binding of CXCL12 to CXCR4 leads to the ubiquitylation of the receptor followed by its degradation in lysosomes (Marchese and Benovic, 2001; Marchese et al., 2003). In contrast, endocytosed CCR5 is recycled back to the plasma membrane (Mueller and Strange, 2004; Signoret et al., 2004; Signoret et al., 2000). Strikingly, CCR7 internalization was observed by CCL19 triggering, but not by stimulation with CCL21 (Bardi et al., 2001), although binding affinities and G protein activation are comparable (Kohout et al., 2004; Willimann et al., 1998). Of note, CCR7 desensitization through receptor phosphorylation and β-arrestin binding was enhanced by CCL19 stimulation compared to CCL21 (Kohout et al., 2004), whereas T cell polarization mediated by the chemokines was indistinguishable (Bardi et al., 2003).

However, up to now, the mechanism of CCR7 signaling and trafficking remains largely unclear and there is currently no information on the fate of CCL19 after CCR7 endocytosis.

Cell surface receptors can be internalized by two segregated pathways:

clathrin-dependent and clathrin-independent, lipid raft/caveolae-dependent endocytosis (Le Roy and Wrana, 2005; Pfeffer, 2003). The classical clathrin- dependent pathway is well characterized. Clathrin-coated pits at the plasma membrane bud and pinch off in a dynamin- and adaptor protein (such as Eps15)- dependent manner to form clathrin-coated vesicles. After endocytosis, clathrin-coated vesicles are uncoated and fuse with the early endosomes, the central control organelles for sorting receptors. Either receptors recycle back to the plasma membrane via recycling endosomes or are directed to late endosomes and lysosomes for degradation. Alternatively, receptors can be endocytosed in a lipid raft/caveolae-

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dependent manner. This pathway is ill defined but largely depends on cellular cholesterol (Le Roy and Wrana, 2005; Pfeffer, 2003; Aguilar and Wendland, 2005).

In the present study, we investigated the route of internalization and the trafficking of CCR7 by monitoring a newly generated GFP-tagged CCR7. In addition, we tracked CCL19 after receptor binding by a chemokine-Fc chimera. Analysis of CCR7 endocytosis and investigations on the routes of CCL19 and CCL21 after receptor triggering is critical for a better understanding how immune cells, such as dendritic cells and lymphocytes, sense a chemokine gradient originating in secondary lymphoid organs.

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2.3. Results

2.3.1. Differential endocytosis of CCR7 by CCL19 and CCL21

The homing of lymphocytes and dendritic cells largely depends on the attraction of the cells by the chemokines CCL19 and CCL21. At its final destination, the migratory signal needs to be shut off, which normally occurs by chemokine receptor down-modulation or receptor desensitization. In order to unravel the mechanism how CCR7 is silenced, we investigated the internalization of CCR7 in IL- 2 and PHA activated human peripheral blood T lymphocytes. CCR7 endocytosis by CCL19 was readily observed in a concentration- and time-dependant manner (figure 2.1). Cell surface expression of CCR7 on T cells was already reduced by 15% at a CCL19 concentration of 30 ng/ml. More than 60% of CCR7 was internalized in the presence of 3 µg/ml of CCL19 (figure 2.1 A). Endocytosis was rapid as after 2 min of chemokine addition 30% of CCR7 disappeared from the plasma membrane. Maximal internalization was reached after 30 min of incubation (figure 2.1 B). Interestingly, CCR7 endocytosis by CCL21 was observed only at high chemokine concentrations (above 300 ng/ml) and reached a maximum of about 25% (figure 2.1 A). Our data on CCL19-mediated endocytosis of CCR7 is largely in agreement with a previous study by Bardi and colleagues(Bardi et al., 2001), although they did not find any internalization by CCL21 in T cells at all.

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Figure 2.1. Internalization of CCR7 in human peripheral blood T cells. (A) Human peripheral blood T cells were cultured for 4 to 6 days in the presence of IL-2 and PHA and incubated with graded concentrations of CCL19 (circles) or CCL21 (triangles) for 30 min at 37°C. Cell surface expression of CCR7 was determined by flow cytometry using the monoclonal antibody 3D12 against CCR7 which can still recognize the epitope in the presence of bound ligand. (B) The same T cells were incubated for various time points with 3 µg/ml of chemokines and cell surface expression of CCR7 was determined by flow cytometry. Mean values of three independent experiments are shown.

Error bars are below 1%.

2.3.2. Generation of a fluorescent fully functional CCR7

To further investigate CCR7 localization and trafficking, we fused the enhanced green fluorescent protein (GFP) to the C-terminus of human CCR7. We stably expressed CCR7-GFP in the murine pre-B cell line 300-19, a cell line that does not respond to CCL19 and CCL21 (Legler et al., 1998; Willimann et al., 1998;

Willimann et al., 1998). CCR7-GFP transfected cells readily migrated in response to CCL21, similar to 300-19 cells expressing wild-type CCR7, whereas CCR7-GFP positive cells did not migrate in a Transwell chemotaxis assay in the absence of chemokines (figure 2.2 A). The phosphorylation of the extracellular signal-regulated kinases-1 and -2 (Erk-1/2) is an early and transient event after chemokine triggering (Tilton et al., 2000). To test whether CCR7-GFP is fully functional, we stimulated 300-19 transfected cells for various time points with CCL21 and analyzed the phosphorylation of Erk-1/2 by Western blot analysis. Identical kinetics and potency of Erk-1/2 activation upon CCL21 triggering was observed for cells expressing CCR7- GFP and wild-type CCR7 (figure 2.2 B). 300-19 cells are ideal for testing chemotaxis but not for morphological and trafficking studies. Therefore we stably transfected the human embryonic kidney cell line HEK293 with CCR7-GFP. CCL21 stimulation of HEK293 cells expressing CCR7-GFP resulted in the phosphorylation of Erk-1/2 (figure 2.2 C), comparable to 300-19 transfectants. As expected, CCR7-GFP mainly localized to the plasma membrane of both transfected cell lines as assessed by confocal microscopy (figure 2.2 D). These data provide clear evidence that CCR7- GFP is fully functional.

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Figure 2.2. Characterization of CCR7-GFP. (A) Migration of the murine pre-B cell line 300-19 stably transfected with either human CCR7 wild type (wt) or CCR7-GFP in response to CCL21 (1 µg/ml) was assessed in a Transwell chemotaxis assay. After 3 h of incubation at 37°C, cells in the lower chamber were collected and counted by flow cytometry. Mean values and SD of three independent experiments are depicted as percent of migrated cells. (B) 300-19 cells expressing either CCR7 wt or CCR7-GPF were incubated with 2 µg/ml of CCL21 for indicated time points, lyzed and the activation of Erk-1/2 was determined by Western blot analysis using an antibody recognizing the phosphorylated forms of Erk-1 and Erk-2 (pErk-1/2). An antibody against total Erk-2 (tErk-2) was used to ensure equal protein loading. (C) The activation of Erk-1/2 was confirmed in HEK293 cells expressing CCR7-GFP. (D) Cell surface expression of transfected CCR7-GFP in 300-19 and HEK293 cells was determined by confocal microscopy. Bars indicated correspond to 10 µm.

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2.3.3. CCR7-GFP co-localizes with early and recycling endosomes but not with lysosomes

To investigate the intracellular trafficking of CCR7, we stimulated HEK293 cells expressing CCR7-GFP with CCL19 and CCL21. After 5 min of CCL19 stimulation, CCR7-GFP was endocytosed and appeared as punctuated structures within the cell (figure 2.3 A). Some CCR7-GFP was still present at the plasma membrane, confirming our data from primary T cells. Upon CCL21 stimulation CCR7-GFP mainly remained at the plasma membrane, although intracellular CCR7- GFP spots were reproducibly observed (data not shown) confirming that CCL19 triggers CCR7 internalization more efficiently than CCL21. Internalized G-protein coupled receptors are generally degraded in lysosomes or recycled back to the plasma membrane via early and recycling endosomes. In order to investigate these two possibilities for CCR7, we incubated HEK293-CCR7-GFP cells with CCL19 together with Alexa Fluor 546-labeled transferrin. The trafficking of the iron transport protein transferrin is one of the best studied processes. Upon ligand binding, transferrin receptor is internalized by clathrin-coated pits giving rise to clathrin-coated vesicles.

Endocytosed transferrin receptor, together with transferrin, then fuse with recycling endosomes and are directed back to the plasma membrane (Johnson et al., 1996).

Extensive co-localization of transferrin and CCR7-GFP was observed after 5 min and 3 h of CCL19 and transferrin stimulation (figures 2.3 A and B), suggesting that CCR7-GFP localizes in endosomes. To discriminate recycling from early endosomes, HEK293-CCR7-GFP cells were incubated with CCL19 and Alexa Fluor 546-labeled transferrin for 5 min, washed to remove unbound ligands and further incubated for 15 min at 37°C in the absence of ligands. Confocal microscopy studies revealed that endocytosed CCR7-GFP co-localized with transferrin (data not shown), providing clear evidence that the spotted distribution of CCR7-GFP represent recycling endosomes. Furthermore, we investigated whether CCR7-GFP also resides in lysosomes. To this end, we stimulated CCR7-GFP expressing cells with CCL19 for 3 h. Analysis by confocal microscopy demonstrated that CCR7-GFP did not co-localize with lysotracker, a marker for late endosomes and lysosomes (figure 2.3 C). Also shorter or prolonged incubations with CCL19 (up to 9 h) revealed the same results (data not shown), indicating that CCR7-GFP is not sorted to the degradative pathway.

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Figure 2.3. Internalized CCR7-GFP co-localizes with recycling endosomes but not with lysosomes.

HEK293 cells expressing CCR7-GFP were incubated with 2 µg/ml of CCL19 for 5 min (A) or 3 h (B, C) at 37°C in the presence of either 50 µg/ml of Alexa Fluor 546-labeled transferrin (A, B) or 50 nM of lysotracker (C). Cells were fixed and the fluorescence was analyzed by confocal microscopy. Bars are 10 µm.

2.3.4. Endocytosed CCR7 is recycled and not degraded

To formally prove that CCR7 is indeed recycled, we incubated HEK293 cells expressing CCR7-GFP with CCL19 or CCL21 for 30 min at 37°C. Subsequently, the excess of chemokine was removed and cells were incubated for 1 h in the absence of chemokine at 37°C, to allow receptor recycling. Cell surface expression of CCR7 was determined by flow cytometry using a CCR7 specific monoclonal antibody (figure 2.4 A). As expected, CCR7-GFP surface expression was reduced after incubation of the cells with CCL19, and to a lesser extent also with CCL21. After washing off chemokines, endocytosed CCR7 re-appeared at the plasma membrane (figure 2.4 A),

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demonstrating that CCR7 is either recycled after internalization or de novo synthesized. Similar results were also obtained with 300-19 cells expressing HA- tagged CCR7 (figure 2.4 B). In order to discriminate between recycling and de novo synthesis, we pre-treated 300-19-CCR7-HA cells with cycloheximide for 1 h to prevent protein synthesis followed by CCL19 triggering. As depicted in figure 2.4 B, surface expression of CCR7 after endocytosis and recycling were comparable in untreated and cycloheximide treated cells, providing clear evidence that CCR7 is recycled rather then newly synthesized. To investigate whether recycled CCR7 can mediate chemotaxis, we incubated CEM cells that endogenously express CCR7 with CCL19 for 30 minutes to internalize CCR7 and allowed the receptor to recycle back to the plasma membrane for one hour. As expected, cells with internalized CCR7 did not migrate in response to CCL19, whereas cells with re-expressed CCR7 migrated towards CCL19 (figure 2.4 C). Similar results were obtained with 300-19 cells expressing either CCR7-GFP or CCR7-HA (data not shown). Recycled rather than newly synthesized CCR7 was responsible for chemotaxis as pre-treatment of CEM cells with cycloheximide did not hamper migration (figure 2.4 C). Furthermore, recycled CCR7 elicited the mobilization of cytosolic free calcium upon CCL19 stimulation (data not shown) providing clear evidence that recycled CCR7 is biologically functional.

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Figure 2.4. Internalized CCR7 recycles back to the plasma membrane and can mediate migration. (A) HEK293 cells expressing CCR7-GFP were stimulated with 2 µg/ml of CCL19, CCL21 or medium for 30 min at 37°C and surface expression of CCR7 was determined by FACS analysis using a CCR7 specific antibody (3D12). Where indicated, cells were washed extensively to remove non-bound chemokines and subsequently incubated for 1 h in the absence of chemokines permitting the recycling of CCR7-GFP back to the plasma membrane. Mean values and SEM of 3 independent experiments are shown as percent of CCR7 endocytosis or percent of recycled CCR7. (B) CCR7 endocytosis and recycling in 300-19 cells expressing HA-tagged CCR7 was performed as described in (A). In addition,

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cells were pretreated with 50 µg/ml of cycloheximide for 1 hour and kept in the presence of cycloheximide for the entire experiment to block de novo synthesis of CCR7 (gray bars). (C) CEM T cells were incubated or not for 30 minutes with 2 µg/ml of CCL19 to induce CCR7 endocytosis, washed and subjected to a Transwell chemotaxis assay. In addition, internalized CCR7 was allowed to recycle back to the plasma membrane by incubation for 1 hour in the absence of chemokine prior to the chemotaxis assay. To prevent de novo synthesis of CCR7, CEM cells were pre-incubated for 1 hour and kept in the presence of 50 µg/ml of cycloheximide (gray bars). Cells were allowed to migrate in response to 1 µg/ml of CCL19 for 3 hours and quantified by flow cytometry.

In order to examine the overall rate of CCR7 degradation, potentially by a non-lysosomal pathway, we performed a degradation assay in the presence of both chemokines. HEK293-CCR7-GFP cells were incubated for up to 9 h with CCL19 or CCL21 in the presence or absence of cycloheximide, to monitor on the one hand the steady state level of the protein and on the other hand the impact of de novo synthesis.

Using flow cytometry we found no evidence for CCR7 degradation, as the fluorescence derived from CCR7-GFP was not reduced upon chemokine triggering (figure 2.5 A). To corroborate this data, we also investigated the degradation of CCR7 by Western blotting. HEK293 cells expressing CCR7-HA were incubated with CCL19 or CCL21 for up to 6 h at 37°C, and the amount of CCR7-HA was determined from total cell lysates using an anti-HA antibody (figure 2.5 B). Again, we found no evidence for CCR7 degradation, suggesting that the half-life of the receptor is very long.

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Figure 2.5. Internalized CCR7 is not degraded. (A) HEK293-CCR7-GFP cells were incubated for different time periods with 2 µg/ml of CCL19 (circles) or CCL21 (triangles) in the presence (open symbols) or absence (closed symbols) of cycloheximide. Total GFP-derived fluorescence was measured by flow cytometry. (B) HEK293 cells stably expressing CCR7-HA were incubated with 2 µg/ml of CCL19 or CCL21 for up to 6 h. Cells were lyzed and the total amount of CCR7-HA was determined by Western blotting using an anti-HA antibody. The α1 proteasome subunit was used as a loading control.

2.3.5. Generation of functional recombinant CCL19-Fc and CCL21-Fc chemokine fusion proteins

In order to monitor the fate of CCL19 and CCL21 once they bound to CCR7, we generated expression constructs encoding for chemokines fused to the Fc part of human IgG1 as there are no good antibodies against the chemokines available. We expressed human CCL19-Fc and human CCL21-Fc in HEK293 cells and purified the recombinant fusion proteins from the supernatants over protein-A columns. Both proteins were purified with a relative mass of about 40 kDa in a monomeric state after reduction and of about 80 kDa in a non-reduced dimeric form as judged by SDS- PAGE followed by Coomassie staining (data not shown). The biological activity of the chemokine Fc fusion proteins was tested by the ability to mobilize intracellular

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free calcium and to induce chemotaxis. 300-19 cells expressing CCR7 were loaded with Fluo-3/AM and subsequently exposed to the chemokines, and the calcium- dependent change in fluorescence was measured over time. Challenging 300-19- CCR7 cells with either CCL19, CCL21 or with the corresponding chemokine-Fc- fusion proteins elucidated comparable transient rises in [Ca²⁺]i (figure 2.6 A), indicating that both CCL19-Fc and CCL21-Fc are functional. No mobilization of [Ca²⁺]i was observed in parental 300-19 cells lacking CCR7, indicating that the rise in [Ca²⁺]i was specific. Additionally, the chemotactic activity of CCL19-Fc and CCL21- Fc were tested in a Transwell chemotaxis assay. As shown in figure 2.6 B, 300-19 cells expressing CCR7 migrated normally in response to CCL19-Fc, CCL19 and CCL21. For an unknown reason, only marginal migration towards CCL21-Fc was observed at different concentrations of chemokine (figure 2.6 B and data not shown).

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Figure 2.6. Generation of functional CCL19-Fc and CCL21-Fc. (A) Parental 300-19 cells (bottom panel) or cells stably expressing CCR7 were loaded with Fluo-3/AM and stimulated with 4 µg/ml of purified CCL19-Fc or CCL21-Fc and chemokine-mediated changes in intracellular free calcium concentrations were recorded over time by flow cytometry. For comparison, [Ca²⁺]i changes in response to 2 µg/ml of untagged CCL19 and CCL21 were measured. The arrowheads indicate the time point of chemokine addition (B) The migration of 300-19 cells expressing CCR7 in response to CCL19, CCL21 (1 µg/ml), and chemokine-Fc fusion proteins (10 µg/ml) was measured in Transwell chemotaxis assays. After 3 h of incubation cells migrated to the lower wells were collected and counted by flow cytometry. As a control, cells migrated in the absence of chemokine was determined.

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2.3.6. CCL19-Fc is internalized together with CCR7 but then sorted to lysosomes for degradation

The functional recombinant proteins enabled us to study the intracellular trafficking and interaction of both chemokines with CCR7. CCL19-Fc induced internalization of CCR7-GFP similar to CCL19; and CCL19-Fc co-localized with CCR7-GFP as shown by confocal microscopy (figure 2.7 A). Consistent with CCL21, CCL21-Fc also induced some internalization of CCR7 (data not shown). Remarkably, after incubation of HEK293 cells expressing CCR7-GFP with CCL19-Fc for 30 min at 37°C, followed by washing off the chemokine and an additional incubation for 6 h in the absence of the chemokine, most of the CCL19-Fc staining disappeared and vaguely co-localized with CCR7-GFP, which recycled back to the plasma membrane (figure 2.7 B). Similar results were also obtained with monobiotinylated CCL19 (data not shown). To address the trafficking of endocytosed chemokine, we stimulated CCR7 transfected HEK293 cells with CCL19-Fc together with Alexa Fluor 546- labeled transferrin. After 30 min of incubation, CCL19-Fc partially co-localized with transferrin (figure 2.7 C), indicating that CCL19-Fc localizes in early endosomes, like CCR7. However, not all intracellular CCL19-Fc spots co-localized with endosomes.

To address the origin of these additional compartments, we stimulated CCR7 expressing cells with CCL19-Fc for 8 h in the presence of lysotracker. As depicted in figure 2.7 D, CCL19-Fc also partially co-localized with lysosomes. To prove that CCL19-Fc is indeed degraded in lysosomes, we stimulated HEK293 cells expressing CCR7-HA with CCL19-Fc for 30 min in the presence or absence of chloroquine.

Cells were washed and further incubated for 3 and 6 h. CCL19-Fc was degraded after 3 and 6 h (figure 2.7 E). Treatment of the cells with the lysosomotrophic agent chloroquine and subsequent incubation with CCL19-Fc significantly inhibited chemokine degradation, providing clear evidence for CCL19-Fc degradation in lysosomes (figure 2.7 E). To exclude that the degradation was due to the Fc part, we repeated the experiments with a chemically synthesized monobiotinylated CCL19, where a single amino acid was biotinylated. In fact, biotinylated CCL19 was also degraded (figure 2.7 E) comparable to CCL19-Fc. Chloroquine treatment abolished the degradation of the biotinylated chemokine leading to an accumulation of CCL19 in intracellular compartments (data not shown).

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Figure 2.7. CCL19-Fc is internalized together with CCR7 and co-localizes with both transferrin and lysosomes. (A) HEK293 cells expressing CCR7-GFP were incubated with 10 µg/ml of CCL19-Fc for 30 min at 37°C. Cells were fixed and permeabilized with Triton X-100. CCL19-Fc was visualized using a biotinylated anti-human IgG antibody and streptavidin-Cy3. The localization of CCR7-GFP and CCL19-Fc was determined by confocal microscopy. (B) HEK293-CCR7-GFP cells were stimulated with CCL19-Fc for 30 min as in (A), washed extensively and cultured in the absence of chemokines for additional 6 h. (C) HEK293 cells stably transfected with VSV-CCR7 were incubated with CCL19-Fc for 30 min at 37°C in the presence of Alexa Fluor 546-labeled transferrin. CCL19-Fc

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was monitored with a biotinylated anti-human IgG antibody and streptavidin-FITC. (D) HEK293 cells expressing VSV-CCR7 were incubated with CCL19-Fc and lysotracker for 8 h. Representative images are shown. Bars indicated correspond to 10 µm. (E) HEK293 cells expressing CCR7-HA were incubated with 10 µg/ml of CCL19-Fc (upper panel) or 3 µg/ml of monobiotinylated CCL19 (lower panel) for 30 min at 37°C in the presence or absence of 200 µM of chloroquine. Cells were washed, incubated for indicated time periods, washed, lyzed and proteins were separated by SDS-PAGE.

CCL19-Fc was detected by Western blotting using an anti-human IgG antibody coupled to HRP.

Biotinylated CCL19 was detected using streptavidin^HRP.

2.3.7. CCR7 endocytosis is mediated by clathrin-coated pits

Two principal pathways of membrane receptor internalization are known (Le Roy and Wrana, 2005). The best-studied pathway is clathrin-dependent endocytosis with the respective machinery of adaptor proteins and GTPases. The other pathway depends mainly on cholesterol-rich membrane microdomains, also termed lipid rafts, and referred to as clathrin-independent endocytosis or in cells expressing caveolin also caveolae-dependent endocytosis (Pfeffer, 2003). Both pathways can be specifically inhibited. The formation of clathrin-coated pits can be blocked under hypertonic conditions using 0.4 M sucrose (Heuser and Anderson, 1989). Clathrin- independent endocytosis can be inhibited by sequestering cellular cholesterol by methyl-β-cyclodextrin (MCD) or filipin (Harder et al., 1997; Keller and Simons, 1998; Orlandi and Fishman, 1998). The pathway of CCR7 internalization has not yet been investigated. To do so, we pretreated IL-2 and PHA activated peripheral blood T cells with filipin, MCD or sucrose, incubated the cells with chemokines for 30 min at 37°C and measured the surface expression of CCR7 by flow cytometry. Blocking the clathrin-independent pathway by filipin had no effect on CCR7 endocytosis by CCL19 or CCL21 (figure 2.8). These data were corroborated by treatment with MCD (figure 2.8) at a concentration that hampered T cell receptor signaling (data not shown). Inhibition of the clathrin-dependent pathway by sucrose abolished CCR7 endocytosis by CCL19 and CCL21 (figure 2.8), suggesting that CCR7 endocytosis is mediated by clathrin-coated pits.

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Figure 2.8. Endocytosis of CCR7 in human T cells is prevented by sucrose treatment but not by cholesterol depletion. IL-2 and PHA activated peripheral blood T cells were treated or not with 5 µg/ml filipin III, 2 mg/ml MCD or 0.4 M sucrose. After 1 h of incubation at 37°C cells were stimulated for 30 min with 3 µg/ml of CCL19 or CCL21 or were left untreated. Cells were transferred to 4°C, washed and the surface expression of CCR7 was determined by flow cytometry. Mean values and SD of at least 3 independent experiments are shown.

To further characterize the endocytic pathway, we investigated on the role of dynamin II and Eps15 in CCR7 internalization by confocal microscopy. Dynamin II is involved in the formation of both clathrin-coated and caveolar vesicles (De Camilli et al., 1995; Oh et al., 1998). As expected, a normal rate of CCR7 endocytosis was detected after CCL19-Fc triggering in HEK293-CCR7 cells transiently transfected with GFP-tagged dynamin II (figure 2.9 A). Expression of a GFP-tagged dominant- negative mutant of dynamin II (dyn II K44A), however, abolished the internalization of CCR7 (figure 2.9 B). Eps15, on the other hand, has, until very recently (Chen and De Camilli, 2005; Sigismund et al., 2005), only been implicated in clathrin-coated pits assembly (Benmerah et al., 1999). Internalization of CCR7 was readily observed after CCL19-Fc-mediated stimulation of CCR7 positive cells transfected with Eps15-GFP (figure 2.9 C). Remarkably, over-expressing a GFP-tagged dominant-negative form of Eps15 (Eps15 E∆95/295) completely inhibited CCR7 internalization (figure 2.9 D).

These data in conjunction with the finding that sucrose treatment abolished endocytosis, strongly suggests that CCR7 is internalized through the clathrin- dependent pathway.

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Figure 2.9. CCR7 internalization is dynamin II and Eps15-dependent. HEK293 cells stably expressing VSV-CCR7 were transiently transfected with GFP-tagged Dyn II wt (A), Dyn II K44A (B), Eps15 wt (C) and Eps15 mt (D). After 16 h of transfection, the cells were incubated with 10 µg/ml of CCL19-Fc for 30 min at 37°, fixed and permeabilized. CCL19-Fc was visualized by a biotinylated anti human IgG antibody and streptavidin-Cy3. CCR7 internalization by CCL19-Fc in GFP positive and negative cells was analyzed by confocal microscopy. Bars indicated correspond to 10 µm.

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Taken together, we demonstrate that CCL19 is more efficient than CCL21 in CCR7 internalization. CCR7 and its ligands are most likely endocytosed together through clathrin-coated pits. In early endosomes the CCR7-ligand complex may dissociates and CCR7 and its ligand follow different routes. The chemokine, which is no longer used, is eliminated by lysosomal degradation. The receptor, however, recycles back to the plasma membrane, ready to bind a new ligand, permitting cell migration towards the source of chemokine within the draining lymph node.

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2.4. Discussion

There is no doubt that CCR7 and its ligands are essential for the homing of dendritic cells and T lymphocytes to lymph nodes, Peyer’s Patches and the spleen (Amara et al., 1997; Campbell et al., 2003; Forster et al., 1999; Moser and Loetscher, 2001; Ohl et al., 2004). Antigen-loaded DCs and circulating T lymphocytes enter secondary lymphoid organs by sensing CCL21 presented on high endothelial venules.

Thereafter, they migrate to the T zone where CCL19, as well as CCL21, are expressed, facilitating the contact between naïve T lymphocytes and antigen-loaded DCs and hence the priming of T cells. For this event fine tuning of cell migration may be critical.

One important way of modulating chemokine receptor responsiveness is receptor endocytosis after ligand binding. Studies on chemokine receptor endocytosis moved into focus because chemokine-induced internalization of cell surface receptors was a major defense mechanism of chemokine-mediated inhibition of HIV infection.

Thus endocytosis has been most intensively studied on the HIV co-receptors CCR5 and CXCR4. CXCR4 internalization is induced by its ligand CXCL12, but also by phorbol esters (Amara et al., 1997; Signoret et al., 1997). CXCL12-mediated endocytosis occurs via clathrin-coated pits and depends on Rab5 and Eps15 (Venkatesan et al., 2003). After ligand binding, CXCR4 is mono-ubiquitylated, endocytosed and subsequently sorted to lysosomes for degradation (Marchese and Benovic, 2001). Interestingly, receptor mutants that are not ubiquitylated internalize normally (Marchese and Benovic, 2001), but CXCR4 ubiquitylation by AIP4 is required for sorting to lysosomes and its degradation (Marchese et al., 2003).

However, CXCR4 was also shown to recycle back to the plasma membrane (Amara et al., 1997; Venkatesan et al., 2003). In contrast, CCR5 internalization does not occur by phorbol esters (Signoret et al., 1998), but only by its ligands (Venkatesan et al., 2003). However, data on the routes of CCR5 internalization are controversial. CCR5 endocytosis was shown to be clathrin dependent (Signoret et al., 2005; Signoret et al., 2000). Furthermore, intracellular CCR5 co-localized with fluorescent-labelled transferrin (Mack et al., 1998) and β-arrestin (Kraft et al., 2001; Mueller et al., 2002).

In contrast, cholesterol depletion by nystatin and filipin affected CCR5 endocytosis and CCR5 was found to co-localize with caveolin, suggesting a role of caveolae/lipid

Characterization of the chemokine receptor CCR7 and its role in cell migration