The Role of CCL5/RANTES in Regulating Cellular Metabolism in Activated T Cells

The Role of CCL5/RANTES in Regulating Cellular Metabolism in Activated T cells

Olivia Chan

A thesis submitted in conformity with the requirements for the degree of

Master of Science

Graduate Department of Immunology

University of Toronto

Olivia Chan

Master of Science

Graduate Department of Immunology

University of Toronto, 2011

Recruitment of effector T cells to sites of infection is essential for an effective adaptive immune response. The inflammatory chemokine CCL5/RANTES activates its cognate receptor, CCR5, to initiate cellular functions including chemotaxis. This thesis describes the signaling events invoked by CCL5 and its ability to regulate the energy status of activated T cells. CCL5 treatment in ex vivo activated human T cells induced the activation of AMPK and downstream substrates ACC1, PFKFB2 and GSK-3. Evidence is provided that CCL5 treatment is able to induce glucose uptake in an mTOR-dependent manner. Using 2-deoxy-D-glucose, an inhibitor of glucose uptake, and Compound C, an inhibitor of AMPK, evidence is provided that demonstrate that CCL5-mediated chemotaxis is dependent on metabolic events, since these inhibitors perturb chemotaxis in a dose-dependent manner. Collectively, these studies suggest that CCL5 may also influence the metabolic status of activated T cells by simultaneously activating the

AMPK and mTOR pathways.

ACKNOWLEDGEMENTS

I would like to dedicate this thesis to everyone who supported me throughout my graduate studies, and would like to express my gratitude to the individuals whom, without which, none of this could have been possible.

Eleanor, your guidance, without fail, has always pointed me in the right direction. Whenever things looked grim, your encouragement and advice would always put me back on the right path. Thank you for your patience and mentorship throughout my studies – you have been an incredible driving-force that has helped me grow both as a scientist and as an individual. I am especially thankful for the opportunity to have gone to multiple international conferences, and the chance to present my work around the world. I look forward to working with you in the future, as we further embark on the „CCL5-era‟ in the lab!

To my supervisory committee members, Dr. Pam Ohashi and Dr. Juan Carlos Zuniga-Pflucker, I am grateful for your helpful guidance and input into my work. Also, thank you Dr. Shannon Dun for taking the time to discuss my research plans and long term goals.

To past and present Fishies, I offer my sincerest thanks for all your emotional and scientific support (especially your PBMCs). Beata, you‟ve given multi-tasking a whole new meaning, and I look to you as a constant source of inspiration and motivation. You‟ve been the shoulders I could always depend on and a friend I could always turn to. Thank you for holding my hand through some of the toughest times during my studies. Daniel, without a doubt, you‟ve been an invaluable „metabolism-and-protein-translation- buddy‟. Thank you for our many scientific discussions – which have always made the realm of metabolism a little less daunting. Thank you for all your encouragement and friendship throughout the years, and keep working on your chopstick skills! Carole, I‟ve always been determined to answer at least one of your unanswerable questions! Thank you for taking the time to guide and mentor me throughout my project and for letting me pick-your-brain about experimental design. I wish you, Kip and Kaycee all the very best. Leesa, I can‟t help but chuckle whenever I come across or hear about PKR (I think that video is still on my desktop…). Thank you for sharing your incredible energy with the lab, and although there is much work to be done to improve your Chinese accent, there is no doubt that you are well on your way towards obtaining your Ph.D. Ben, thank you for bringing Fail Blog into our daily lunch routine (or was that Craig?). I wish you all the best in your Ph.D. journey, and remember to take advantage of the lab hammock when you truly need it! Craig, I look forward to the day when you and Ben revolutionize IGSA (with karaoke nights!). Thank you for bringing your calming-influence to the lab and I wish you best of luck in your Ph.D. studies. Thomas, Danlin and Ramtin, thank you for your continuous support throughout the years and for always being an email away! You‟ve been brilliant mentors and role models, and I wish you all happiness in your new lives.

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To my Mom, Dad and Oscar, thank you for your constant vote of confidence. Mom and Dad, you give me strength and hope during my rough patches – thank you for believing, before I did, in my ability to achieve something substantial. Oscar, you never fail to cheer me up following rather disappointing experiments. Thank you for your tremendous support and encouragement, and I can‟t wait to see what life has in store for you!

Thanks to all my friends and labmates who have donated blood to „fuel‟ the studies undertaken in my project. None of this would have been possible without your generous donations; and I may owe royalties to many of you.

Finally, a heartfelt thank you goes to my fellow students of the Immunology Department for their support and friendship. Nothing beats sharing a pint of beer together when experiments go awry, and I look forward to working with all of you again in the future.

Table of Contents

Title Page………………………………………………………………………………….. i Abstract…………………………………………………………………………………ii-iii Acknowledgments……………………………………………………………. ………..iv-v Table of Contents……………………………………………………………………….v-vi List of Figures………………………………………………………………………….... vii List of Tables…………………………………………………………………………….viii List of Abbreviations………………………………………………………………….. ix-xi

CHAPTER 1: INTRODUCTION…………………………………………………... 1-52

1.1 Chemokine Superfamily……………………………………………………………….2 1.1.1. Classification……………………………………………………………………2 1.1.2. Chemokine Structure……………………………………………………………4 1.1.3. Chemokine-mediated Signaling………………………………………………... 7 1.1.3.1. Jak-Stat Pathway…………………………………………………………11 1.1.3.2. MAPK Signaling Cascade……………………………………………….12

1.2 Chemokine Receptors……………………………………………………………….. 15 1.2.1. Classification…………………………………………………………………. 15 1.2.2. Chemokine Receptor Structure and Ligand Binding…………………………. 18 1.2.3. Receptor Dimerization and Internalization…………………………………… 19

1.3. Chemokine/ Chemokine Receptor Functions………………………………………. 20 1.3.1. Chemotaxis…………………………………………………………………… 20 1.3.1.1. Cellular Polarization……………………………………………………. 20 1.3.1.2. The Rho Family GTPases in Cytoskeletal Rearrangement…………….. 21 1.3.1.3. Activation of the PI-3‟K Pathway……………………………………… 22 1.3.1.4. The mTOR/4E-BP1 Pathway and Chemotaxis………………………… 24

1.3.2. Role in determining Cellular Fate…………………………………………….. 32 1.3.2.1. T cell Differentiation and Activation…………………………………… 33 1.3.2.2. Role in Cell Death………………………………………………………. 35

1.4. mTOR Signaling and Metabolic Regulation……………………………………...... 36 1.4.1. Growth Factor and Nutrient-Sensing by mTOR……………………………… 36 1.4.1.1. AMPK-regulation of mTOR……………………………………………. 39 1.4.2. mTOR Signaling in Lymphocyte Trafficking………………………………… 40 1.4.3. mTOR-mediated Proliferation………………………………………………... 41

1.5. Energy Metabolism and the T cell Response……………………………………….. 43 1.5.1. Regulation of T lymphocyte Metabolism…………………………………….. 43 1.5.2. Quiescent Cells and Oxidative Phosphorylation……………………………… 44 1.5.3. Proliferating Lymphocytes and Glycolysis…………………………………... 45 1.5.3.1. PI-3‟K Signaling in Aerobic Glycolysis………………………………... 46 1.5.4. Energy Regulation during an Immune Response…………………………….. 48 v

1.6. Thesis Hypothesis and Objectives………………………………………………….. 52

CHAPTER 2: MATERIALS AND METHODS…………………………………. 53-57

2.1. Cells and Reagents ………………………………………………………………….54 2.2. Immunoblotting …………………………………………………………………….55 2.3. Flow Cytometric Analysis ………………………………………………………….55 2.4. Chemotaxis Assay …………………………………………………………………..56 2.5. Glucose Uptake Assay ………………………………………………………………56 2.6. AMPK Antibody Signaling Array ………………………………………………….57 2.7 Statistical Analysis……………………………………………………………………57

CHAPTER 3: RESULTS…………………………………………………………... 58-87

3.1. CCL5 induces phosphorylation of proteins in the AMPK signaling pathway……... 59 3.2. CCL5-mediated glucose uptake is mTOR-dependent……………………………… 69 3.3. CCL5-mediated glucose uptake is not accompanied by changes in the surface expression of nutrient receptors …………………………………...... 74 3.4. Glucose uptake and AMPK signaling are required for efficient CCL5-mediated chemotaxis ………………………………………………………………………………80 3.5. CCL5-induced AMPK signaling phosphorylates the 4E-BP1 repressor of mRNA translation………………………………………………………………………………... 80

CHAPTER 4: DISCUSSION………………………………………………………. 88-97

CHAPTER 5: FUTURE DIRECTIONS………………………………..…………98-101

CHAPTER 6: REFERENCES…………………………………………………...102-118

LIST OF FIGURES

CHAPTER 1

Figure 1.1. Chemokines share similar structure………………………………………….. 5 Figure 1.2. Chemokine-induced signaling pathways……………………………………... 9 Figure 1.3. The MAPK signaling cascade………………………………………………. 13 Figure 1.4. Two-dimension depiction of CCR5 and residues critical for ligand binding and signaling transduction………………………………………………………………. 16 Figure 1.5. mTOR signaling complexes………………………………………………….26 Figure 1.6. Regulation of cap-dependent mRNA translation ……………………………30

CHAPTER 3

Figure 3.1. CCR5 surface expression is induced upon T cell activation in the presence of cytokines………………………………………………………………………………….60 Figure 3.2. CCL5 induces phosphorylation of proteins in the AMPK signaling pathway …………………………………………………………………………………………… 64 Figure 3.3. CCL5 activates the energy-sensing kinase AMPK and downstream substrate GSK-3β………………………………………………………………………………...... 67 Figure 3.4. CCL5-mediated glucose uptake is mTOR-dependent……………………….71 Figure 3.5. CCL5 increases cell surface expression of GLUT-1 and CD98 …………….76 Figure 3.6. Glucose uptake and AMPK signaling are required for efficient CCL5- mediated chemotaxis……………………………………………………………………...81 Figure 3.7. Effects of 2-DG and Compound C on T cell viability ……………………….83 Figure 3.8. CCL5-induced AMPK signaling phosphorylates the 4E-BP1 repressor of mRNA translation…………………………………………………………………………86

CHAPTER 4

Figure 4.1. Illustration of the AMPK and mTOR signaling cascades…………………... 92

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LIST OF TABLES

CHAPTER 1

Table 1.1. The Chemokine Superfamily and Classification………………………….. 3

Table 3.1. List of AMPK Signaling Phospho-Specific Antibodies..…………………62

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LIST OF ABBREVIATIONS

ACC Acetyl CoA carboxylase AICAR 5-aminoomidazole-4-carboxyamide AICD Activation induced cell death AMPK Adenosine monophosphate-activated protein kinase AOP-CCL5 Aminooxypentane-CC chemokine ligand 5 ATP Adenosine-5‟-triphosphate APC Antigen presenting cell Bcl-2 B cell lymphoma 2 C-terminal Carboxy-terminal CCL5 CC chemokine ligand 5 CCR5 CC chemokine receptor 5 CCX-CKR ChemoCentryx chemokine receptor CXCL CXC chemokine ligand CXCR CXC chemokine receptor CX3CL CX3C chemokine ligand CX3CR CX3C chemokine receptor DAD Defender against cell death DAG Diacylglycerol DARC Duffy antigen receptor for chemokines DC Dendritic cell DN Double negative DNA Deoxyribonucleic acid DP Double positive EDTA Ethylenediamine tetra-acetic acid EGTA Ethylene glycol-bis (2-aminoethylether)-N‟N‟N‟N‟-tetra-acetic eIF Eukaryotic translation initiation factor ERK Extracellular signal-related kinase F1,6BP Fructose-1,6-biphosphate F2,6BP Fructose-2,6-biphosphate F6P Fructose-6-phosphate FKBP12 FK506-binding protein 12kDa FOXO Forkhead box class O GAG Glycosaminoglycan GDP Guanosine diphosphate GTP Guanosine triphosphate GLUT Glucose transporter gp120 Glycoprotein of 120 kDa GPCR G-protein coupled receptor GPK G-protein receptor kinase GSK Glycogen synthase kinase HEK Human embryonic kidney HEV High endothelial venules HXK Hexokinase HIV Human immunodeficiency virus ix

IFN Interferon IGF Insulin-like growth factor IL Interleukin IP3 Inositol 1,4,5- phosphate IRS Insulin receptor substrate 1 Jak Janus kinase JNK Jun NH2-terminal protein kinase kDa Kilodalton KLF Kruppel-like factor KRH Krebs-Ringer-HEPES LKB1 Liver kinase B1 LKLF Lung Kruppel-like factor m7GpppN 7-methyl guanosine residue MAPK Mitogen-activated protein kinase MAPKK MAPK kinases MAPKKK MAPKK kinases MAPKAP MAP kinase-activation protein Met-CCL5 Methionine-CC chemokine ligand 5 MDCK Madin-Darby canine kidney μM Micromolar mTOR Mammalian target of rapamycin mTORC Mammalian target of rapamycin complex N-terminal Amino-terminal nM Nanomolar NMR Nuclear magnetic resonance NP-40 Nonidet-40 PAMPs Pathogen-associated molecular patterns PB Peripheral blood PBS Phosphate buffered saline PDK Phosphoinositide-dependent protein kinase PFA Paraformaldehyde PFK Phosphofructokinase PFKFB2 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; or PFK-2 PH Pleckstrin homology PI-3‟K Phosphatidylinositol 3-OH kinase PIKK Phosphatidylinositol kinase-related kinase PIP3 Phosphatidylinositol (3,4,5) triphosphate PIP2 Phosphatidylinositol (3,4) biphosphate PKB Protein kinase B PKC Protein kinase C PLC Phospholipase C PMSF Phenylmethylsulfonylflouride PRR Pattern recognition receptor PTEN Phosphatase and tensin homolog deleted in chromosome ten PTx Pertussis toxin RAG Recombinase-activating gene Raptor Regulatory associated protein of mTOR x

Rheb Ras-homolog enriched in brain Rictor Rapamycin insensitive companion of mTOR rpS6 Ribosomal protein S6 S1P Sphingosine 1-phosphate S6K S6 kinase SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SH2 Src homology 2 Stat Signal transducers and activators of transcription Th T helper TBS Tris buffer saline TCR T cell receptor TNF Tumor necrosis factor TRAF TNF-receptor-associated factor TSC Tuberous sclerosis complex TOP 5‟ tract of oligopyrimidine VLA Very late antigen Vps34 Vacuolar protein sorting 34 XCL XC chemokine ligand XCR XC chemokine receptor 2-DG 2-deoxy-D-glucose 4E-BP 4E-binding protein 4F2HC 4F2 heavy chain

CHAPTER 1

INTRODUCTION

1.1. Chemokine Superfamily

1.1.1. Classification

Chemokines are soluble, low molecular weight (8-14 kDa) chemotactic cytokines that bind to their cognate seven trans-membrane G-protein coupled receptors (GPCRs) to mediate cellular migration. To date, there are over 40 characterized human chemokines, all of which have two to four highly conserved cysteine residues (Table 1.1.) as well as a number of virally encoded chemokine-like proteins (Alcami, 2003). The chemokine superfamily can be separated into four sub-families based on the presence and relative positioning of the first two cysteine residues at the N-terminus. The cysteine residues in the CXC (or α) family are separated by a non-conserved amino acid, while in the CC (or

β) family, these cysteine residues are adjacent to each other. The XC (or γ) chemokines have only a single cysteine residue, while the CX3C (or δ) chemokine, CX3CL1, has three non-conserved amino acids between the first two cysteine residues.

Chemokines are also functionally categorized depending on whether they are constitutively produced or are inducible. Constitutive, or homeostatic, chemokines are involved in basal leukocyte migration and development, whereas inducible, or inflammatory, chemokines control the recruitment of effector leukocytes during an immunological insult (Proudfoot, 2002). It is important to note that this division is not absolute, given that several chemokines cannot be assigned unambiguously to either one of the two functional categories. CXCL9, for instance, is a “dual-function” chemokine that is up-regulated under inflammatory conditions, but also participates in T cell lymphopoiesis (Moser et al., 2004). Most chemokines are secreted from the cell, with the

Table 1.1. The Chemokine Superfamily and Classification

Systematic Names Alternate Names Receptor(s) Expression Profile

CXC Chemokines CXCL1 Groα/MGSAα CXCR2, CXCR1 Inducible CXCL2 Groβ/MGSAβ CXCR2 Inducible CXCL3 Groγ CXCR2 Inducible CXCL4 PF4 CXCR3b Inducible CXCL5 ENA-78 CXCR2 Inducible CXCL6 GCP-2 CXCR1, CXCR2 Inducible CXCL7 NAP-2 CXCR2 Inducible CXCL8 IL-8 CXCR1, CXCR2 Inducible CXCL9 MIG CXCR3, CXCR3b Dual-function CXCL10 IP-10 CXCR3, CXCR3b Dual-function CXCL11 I-TAC CXCR3, CXCR3b, CXCR7 Dual-function CXCL12 SDF-1α/β CXCR4, CXCR7 Constitutive CXCL13 BLC, BCA-1 CXCR5 Constitutive CXCL14 BRAK, Bolekine Unknown Constitutive CXCL15 none Unknown Constitutive CXCL16 none CXCR6 Dual-function CXCL17 DMC Unknown Unknown

CC Chemokines CCL1 I-309 CCR8 Dual-function CCL2 MCP-1 CCR2 Inducible CCL3 MIP-1α/LD78α CCR1, CCR5 Inducible CCL4 MIP-1β CCR5 Inducible CCL5 RANTES CCR1, CCR3, CCR5 Inducible CCL7 MCP-3 CCR1, CCR2, CCR3 Inducible CCL8 MCP-2 CCR1, CCR2, CCR3, CCR5 Inducible CCL11 Eotaxin CCR3 Inducible CCL13 MCP-4 CCR1, CCR2, CCR3 Inducible CCL14 HCC-1 CCR1 Inducible CCL15 HCC-2/LKN1/MIP-1γ CCR1, CCR3 Inducible CCL16 HCC-4/LEC/LCC-1 CCR1, CCR3 Dual-function CCL17 TARC CCR4 Dual-function CCL18 DC-CK1/PARC/AMAC-1 Unknown Constitutive CCL19 MIP-3β/ELC CCR7 Constitutive CCL20 MIP-3β/LARC CCR6 Dual-function CCL21 SLC/6Ckinase CCR7 Constitutive CCL22 MDC/STCP-1 CCR4 Dual-function CCL23 MPIF/CKβ8 CCR1 Constitutive CCL24 Eotaxin-2/ MPIF-2 CCR3 Inducible CCL25 TECK CCR9 Dual-function CCL26 Eotaxin-3 CCR3 Inducible CCL27 CTACK/ILC CCR10 Inducible CCL28 MEC CCR3, CCR10 Inducible

C Chemokines XCL1 Lymphotactin/ SCM-1α XCR1 Inducible XCL2 SCM-1β XCR1 Inducible

CX3C Chemokines CX3CL1 Fractalkine CX3CR1 Inducible 3 exception of CXCL16 and CX3CL1, which are membrane-bound proteins (Bazan et al.,

1997). These proteins can also exist as soluble glycoproteins upon protease cleavage of their trans-membrane stalks. Thus far, 47 human chemokines have been described, many of which bind to several of the 18 described human chemokine receptors. Although there is considerable redundancy in the chemokine system, chemokine ligand-receptor binding does not usually cross the CC versus CXC chemokine boundaries. This redundancy also ensures sufficient levels of robustness within the system such that essential processes are not compromised by chance mutations.

This thesis will review our general understanding of chemokine-chemokine receptor function and signaling, with an emphasis on the CC chemokine CCL5 and its receptor, CCR5 in activated T cells.

1.1.2. Chemokine Structure

Although chemokines have relatively low sequence identity, they share considerable amount of structural homology. The three-dimensional structure of CCL5, for example, is similar to that of CCL2, CCL3, CCL4 and CXCL8, wherein they all have the same monomeric fold. This “chemokine fold” consists of three anti-parallel β-sheets, a carboxy (C) terminal helix and a flexible amino (N) terminal region (Figure 1.1.). Two disulfide bonds exists between the first and third, and the second and fourth cysteine residues to stabilize the conformation. The N-terminal segment of many chemokines, including CCL5, is important for receptor binding. Chemical modifications to the N- terminus of CCL5, such as the addition of amino-oxypentane (AOP-CCL5), methioine

(Met-CCL5), or pharmacophore grafting CCL5 (PSC-CCL5), have been shown to result

Figure 1.1. Chemokines share similar structures

Superimposed structures of CCL2 (yellow), CCL5 (blue) and CCL11 (red) revealing similar structural elements despite low sequence homology.

Adapted from M. Crump et al., J. Biol. Chem 273 (1998)

6 in antagonists for CCR5 (Proudfoot et al., 1996; Simmons et al., 1997; Gaertner et al.,

2008). AOP-CCL5 and Met-CCL5 act as competitive inhibitors of CCL5 and CCL3 by binding to CCR5 with high affinity without inducing signaling.

Chemokine oligomers have been the topic of interest in recent years, as accumulating evidence point out distinct biological functions of chemokine monomers and higher order multimers. While chemokine monomers are thought to be the receptor- binding unit, it has been postulated that chemokine dimers bind to the glycosaminoglycans (GAGs) on cell surfaces to generate a chemical gradient for chemotaxis. CCL5 has the ability to self-aggregate, and more intriguingly, is able to form multimers at high concentrations (Appay et al., 2000). The amino acids involved in this self-aggregation are the negatively charged glutamine residues Glu66 and Glu26. Several positively charged residues are found on the surface of CCL5, making it likely for ionic bonding to occur for the formation of multimers (Appay and Rowland-Jones, 2001). At high concentrations, CCL5 does not behave as a typical chemokine. Not only is it able to induce the activation of T cells independent of antigen (Appay et al., 2000), micromolar concentrations of CCL5 initiate signaling cascades that are distinct from typical monomeric chemokines to induce T cell apoptosis (Section 1.1.3. and Section 1.3.2.2.).

The biological significance of aggregated CCL5 will be further discussed in Section

1.3.2.2.

1.1.3. Chemokine-mediated Signaling

Chemokine-induced signaling is mediated by the binding and activation of a G- protein coupled receptor (GPCR). This binding results in the dissociation of heterotrimeric G-protein into Gi and G subunits and leads to the activation of a 7 number of signaling cascades (Figure 1.2.). For instance, free G can then activate the phosphoinositide-3-OH kinase (PI-3‟K) pathway and/or the pathway that involves phospholipase C (PLC), among others. PLC activation leads to the generation of two messenger molecules, inositol-1,4,5- trisphosphate (IP3) and diacylglycerol (DAG). The first mediates the transient increase in cytosolic calcium, while the second activates protein kinase C (PKC) and phosphorylates a number of serine residues at the C-terminus of CCR5, namely Ser336, Ser337, Ser342 and Ser349 (Mellado et al., 2001). The majority of chemokine-mediated signaling responses are inhibited by pertussis toxin

(PTx), a bacterial toxin that prevents the Gi subunit from interacting with GPRCs. In some studies, however, chemokine receptors have been reported to couple to PTx- insensitive G-proteins, such as Gq or G16, where the receptor/G protein pairings may be cell-specific (Al-Aoukaty et al., 1996; Arai and Charo, 1996).

Figure 1.2. Chemokine-induced signaling pathways

Schematic model depicting the signaling pathways activated following chemokine binding to its receptor. Some of the molecules involved in these pathways and the effects they promote are shown. A detailed description of these signaling pathways is presented throughout this Thesis.

1.1.3.1. Jak-Stat Pathway

Many cytokines and growth factors signal through the Jak-Stat pathway to mediate their biological effects. Upon ligand binding, receptor dimerization occurs and brings two Janus kinases (Jaks) into close proximity to each other. This allows for trans- phosphorylation of the Jaks to occur, as well as the tyrosine phosphorylation of the receptor (Rodriguez-Frade et al., 2001). Active Jaks induce the phosphorylation of signal transducer and activator of transcription (Stat) proteins, subsequently allowing them to dimerize via their SH2-domains, and translocate into the nucleus where they regulate gene transcription. Several studies have established that chemokines can also invoke Jak-

Stat signaling (Rodriguez-Fade et al., 1999; Wong and Fish, 1998; Wong et al., 2001).

Nuclear extracts from Molt-4 and Jurkat T cells treated with CCL3 or CCL5 contained phosphorylated Stat1:Stat1 and Stat1:Stat3 dimers that were bound to the DNA complexes of a Stat-inducible gene, c-fos (Wong and Fish, 1998). Furthermore, CCL5 treatment of PM1 T cells resulted in the rapid phosphorylation/ activation of CCR5, Jak2 and Jak3 in a PTx-insensitive manner. These data are suggestive that these CCL5- mediated phosphorylation events are not dependent on Gi -protein signaling (Wong et al., 2001). Other studies in HEK-293 cells have shown that Jak1, but not Jak2 or Jak3, associate with the CCR5 receptor upon CCL5 treatment, and this association promotes the activation of STAT5b (Rodriguez-Frade et al., 1999). These data indicate that the recruitment of Jaks and Stats to CCR5 upon CCL5 treatment appear to be cell type specific, however, it is clear that CCL5 engages this signaling pathway to activate various biological processes (Wong and Fish, 2003).

1.1.3.2. MAP Kinase Cascade

Signaling through the mitogen-activated protein kinase (MAPK) pathways regulate several different cellular responses including oncogenesis, cell proliferation and inflammation (Johnson and Lapadat, 2002). The three major MAPK signaling pathways include the extracellular signal-regulated protein kinase (ERK 1/2), p38, and c-JUN NH2- terminal protein kinases (JNKs). All MAPKs are activated through a kinase cascade where MAPKK kinases (MAPKKKs) activate MAPK kinases (MAPKKs), which then active MAPKs by phosphorylating the threonine and tyrosine residues within the activation loop. This hierarchy of kinase signaling is shown in Figure 1.3. Chemokine signaling has been reported to active MAPK signaling, and evidence has accumulated for

MAPKs contributing to chemokine-mediated cell migration. CCL5-CCR5 interactions in

PM1 T cells, for example, activate p38 and its downstream substrate MAP kinase- activation protein (MAPKAP) kinase-2 (Wong et al. 2001). Indeed, additional studies have shown that CCR5 activation can also activate ERK to promote cytoskeletal

Figure 1.3. The MAPK signaling cascade

The MAPK pathways activate ERK, JNK and p38 kinases to elicit a wide-range of biological outcomes, including gene expression, proliferation and cellular motility.

14 rearrangement and integrin activity to promote T cell chemotaxis (Brill et al., 2001;

Ganju et al., 1998). The emerging roles of MAPKs in cytoskeletal dynamics will be further discussed in Section 1.3.1.4.

1.2. Chemokine Receptors

1.2.1. Classification

18 human chemokine receptors have been described to date (Table 1.1).

Chemokines exert their biological effects by binding to and activating their cognate

GPCR on target cells. Similar to their chemokine ligands, chemokine receptors can also be sub-divided into four major families, CR, CCR, CXCR and CX3CR. This classification is based on the sub-family of chemokine ligands they are receptors for; therefore CC chemokines bind to CC chemokine receptors (CCRs), CXC chemokines bind to CXC chemokine receptors (CXCR), XC chemokines bind to XC chemokine receptors (XCRs) and CX3CL1 binds to the CX3CR1 receptor (Mellado et al., 2001). The human CC chemokine receptor 5, CCR5, comprises 352 amino acids with a molecular mass of 40.6 kDa, and is the receptor for CCL3, CCL4, and CCL5 (Figure 1.4.). It shares

83% sequence identity with the mouse CCR5 and 71% sequence identity with CCR2

(Appay and Rowland-Jones, 2001; Raport et al., 1996; Samson et al., 1996). Non- functional CCR5 variants exist in the human population, the best-studied being the truncated CCR5Δ32 variant that is not expressed on the cell surface (Smith et al., 1997;

Samson et al., 1996).

Three atypical chemokine receptors which function as interceptors (internalizing receptors) have been described. These receptors are DARC (Duffy Antigen Receptor for

Figure 1.4. Two-dimension depiction of CCR5 and residues critical for ligand binding and signaling transduction

Tyrosine sulfation sites

Disulfide bonds Extracellular domain

7-Trans-membrane domains

Intracellular domain G-protein binding

Serine phosphorylation sites

Adapted from M. Oppermann Cellular Signaling 16 (2004)

Chemokines), CCX-CKR (ChemoCentryx Chemokine Receptor) and D6. Despite structural similarities and high-affinity ligand binding, these receptors fail to invoke signaling cascades observed for other chemokine receptors (Weber et al., 2004). Also, given their predominant expression on non-leukocytic cells, these receptors play a minimal role in directing leukocyte migration. Instead, decoy receptor-like functions have been reported. D6 is able to internalize inflammatory chemokines in a non-specific manner, and target them for degradation (Fra et al., 2003; Graham, 2009). As a scavenger receptor, D6 has an essential role in the regulation of inflammatory responses and limits chemokine circulation following an immune response.

1.2.2. Chemokine Receptor Structure and Ligand Binding

Chemokine receptors are seven trans-membrane receptors with an N-terminal segment, seven hydrophobic trans-membrane domains, and a cytoplasmic C-terminal tail containing multiple serine/threonine and tyrosine phosphorylation residues (Figure 1.4.).

Chemokine receptors have disulfide bridges between cysteine residues in the extracellular loop to stabilize the receptor conformation important for ligand binding (Oppermann

2004). The N-terminal tail of several receptors, including CCR2, CCR3, CCR5 and

CXCR1, is essential for ligand binding. NMR spectroscopy studies have identified that the first 25 amino acid residues of CCR5 are essential for CCL5 binding. In addition, several site-directed mutagenesis studies reveal that these residues are crucial for the viral entry and interactions with the HIV-1 gp120 protein (Duma et al., 2006; Farzan et al.,

1998). CCR5 is also post-translationally modified by O-linked glycosylations and tyrosine sulfations of the N-terminus. These modifications have been reported to be necessary for CCL3 and CCL4 binding, as well as enhancing the usage of CCR5 by HIV- 18

1 as a co-receptor for viral entry (Farzan et al., 1999; Dragic, 2001; Oppermann, 2004).

Upon ligand binding, CCR5 is phosphorylated on conserved serine residues Ser-336, Ser-

337, Ser-342 and Ser-349 by PKC and G-protein receptor kinases (GPK) (Oppermann et al., 1999). With the exception of decoy/scavenger receptors, most chemokine receptors have a conserved DRYLAIV motif in the second intracellular loop that is coupled to the heterotrimeric G-proteins for intracellular signaling (Oppermann, 2004).

1.2.3. Receptor Dimerization and Internalization

Several chemokine receptors, namely CXCR2, CXCR4, CCR2 and CCR5, homo- or hetero-dimerize on the cell surface (Angers et al., 2002). CCR5 dimerization was originally thought to occur only upon ligand binding, as dimerization was postulated to be crucial for the initiation of chemokine signaling (Rodriguez-Frade, 2001). Subsequent studies have shown that CCR5 can homo-dimerize shortly after synthesis in the endoplasmic reticulum, and can form dimers on the cell surface in the absence of ligand

(Issafras et al., 2002; El-Asmar et al., 2005). Interestingly, the simultaneous presence of

CCL2 and CCL5 can induce the formation of a functional CCR2-CCR5 hetero-dimer

(Mellado et al., 2001). In contrast to CCR2 and CCR5 homo-dimers, this complex promoted the recruitment of PTx-insensitive Gq/11, and showed distinct phosphoinositide-

3-OH kinase (PI-3‟K) activation kinetics. These hetero-dimers are as abundant as homo- dimers, and are only able to bind a single chemokine ligand of either cognate receptor at one time (El-Asmar et al., 2005).

Ligand binding to chemokine receptors induces rapid receptor phosphorylation as well as the clathrin-dependent endocytosis of receptors into cellular endosomes. Many chemokine receptors are internalized through the -arrestin/clathrin-mediated pathway, 19 namely CXCR1, CXCR2, CXCR4, CCR5, CCR7 and the decoy receptor D6 (Barlic et al.,

1999; Yang et al., 1999; Signoret et al., 1997; Signoret et al., 2005; Otero et al., 2006;

Bonecchi et al., 2008). Electron microscopy and immunofluorescent studies demonstrated that upon CCL5 binding, -arrestins are recruited to the plasma membrane where they act as scaffold proteins that link the phosphorylated CCR5 together with clathrin-coated pits and target receptors for recycling (Signoret et al., 2005). CCR5 accumulates in peri- nuclear recycling endosomes and soon returns back to the cell surface in a dephosphorylated form. CXCR4, on the other hand, undergoes similar clathrin-dependent endocytosis but is targeted to late endosomal and lysosomal compartments where it is ubiquitinated and degraded (Marchese and Benovic, 2001).

1.3. Chemokine/ Chemokine Receptor Functions

1.3.1. Chemotaxis

Directed cell migration, or chemotaxis, is a highly coordinated process that is crucial for a wide spectrum of biological processes including leukocyte effector functions during an immune response, development and wound healing. Chemotaxis involves the sensing of a chemokine gradient for the generation of a „leading edge‟, rearrangement in the cytoskeleton, and the initiation of the leukocyte adhesion cascade in order for leukocyte trafficking to occur.

1.3.1.1. Cellular Polarization

When cells encounter a migration-promoting agent they quickly become polarized and extend protrusions in the direction of migration. First, filamentous F-actin within the

20 cell becomes concentrated at the lamellipodium, or the leading edge, and a dynamic pseudopod is extended. The pseudopod is an extension of the cell membrane which adheres to the extracellular matrix and acts as a traction site for migration as the cell moves forward. These adhesions disassemble as the cell detaches at the rear (termed the uropod), and this cyclic process begins again with the protrusions at the leading edge moving forward and re-adhering (Ridley et al., 2003). While the front of the cell contains actin filaments which aid in the formation of new pseudopods, the back of the cell is rich in myosin filaments that anchor the cell to the extracellular matrix during migration (Haastert and Devreotes, 2004).

1.3.1.2. The Rho Family GTPases in Cytoskeletal Rearrangement

The ubiquitously expressed Rho family of GTPases are key regulators of cytoskeleton rearrangement, cell polarity, gene expression, microtubule dynamics and vesicular trafficking. While 20 members have been characterized in mammals, the most well known members of the family are Rho, Rac and Cdc42 (Raftopoulou and Hall,

2004). These regulatory proteins act as molecular switches by cycling between GDP- bound, inactive and GTP-bound, active forms to control signal transduction. Once activated, Rac and Cdc42 localize to the front of migrating cells to modulate the polymerization of actin to form pseudopods at the leading edge. In contrast, Rho localizes at the rear of the cell where it regulates the contraction and retraction of the cell

(Raftopoulou and Hall, 2004). It comes as no surprise that the Rho family GTPases are essential for chemokine-triggered migration. In macrophages, CCL5 activates Rac and its downstream substrate PAK2 to induce macrophage polarization and directional migration

(Weiss-Haljiti et al., 2004). Subsequent studies extended these findings and showed that 21 other β-chemokines, CCL3 and CCL4, activate Rac for lamellipodia formation to occur in macrophages (Di Marzio et al., 2005). These studies provide evidence for Rac in mediating cytoskeletal rearrangement and protrusions at the leading edge downstream of

CCR1 and CCR5 ligation. Specifically, chemokine activation of GPCRs and subsequent activation of the PI-3‟K pathway were prerequisites for Rac-induced cytoskeletal modifications. The PI-3‟K signaling pathway is discussed below.

1.3.1.3. Activation of the PI-3’K Pathway

One signaling pathway critical for the regulation of cellular migration is initiated by PI-3‟K and its lipid product phosphatidylinositol (3,4,5) triphosphate (PIP3). The family of PI-3‟Ks have been divided into four classes (class IA, IB, II and III) based on their structure and substrate specificity. Class IA and IB PI-3‟Ks are heterodimeric enzymes that are primarily responsible for the generation of PIP3 from phosphatidylinositol (3,4) biphosphate (PIP2) upon GPCR-signaling. Class IA PI-3‟Ks are comprised of a 110 kDa catalytic subunit and an adaptor regulator subunit. Four catalytic isoforms (α, β, γ, δ) and five regulatory subunits (p85α, p85β, p55α, p55γ and p50α) exist. On the other hand, class IB PI-3‟Ks have a p110γ catalytic subunit that binds one of two regulatory subunits, p101 or p84. The class II PI-3‟Ks, which include PI3K-

C2α, PI3K-C2β and PIK3-C2γ isoforms, poorly phosphorylates PIP2, and their importance in cell signaling, is less clear (Kok et al., 2009). Vacuolar protein sorting 34

(vps34) is the sole class III PI-3‟K, and has been implicated in endocytosis, autophagy and nutrient sensing (Backer, 2008).

During chemotaxis, class I PI-3‟Ks translocate to the leading edge of the cell where they phosphorylate the 3‟-OH position of the inositol ring of PIP2 to generate PIP3. 22

PIP3 is then able to bind to the pleckstrin homology (PH) domains of signaling proteins necessary for chemotaxis and localize them to lamellipodia. Important PIP3–binding proteins include the serine/threonine kinase protein kinase B (PKB, or Akt) and phosphoinositide-dependent protein kinase 1 (PDK1). Co-localizing PDK1 and AKT promote PDK1-dependent phosphorylation/activation of AKT at Thr-308. Once activated,

Akt phosphorylates a number of critical signaling molecules and transcription factors that are involved in cell motility, metabolism, protein translation, and cell survival (Finlay and Cantrell, 2010). The lipid phosphatase PTEN (Phosphatase and Tensin Homolog

Deleted in Chromosome Ten) is a critical negative regulator of PIP3 levels. PTEN primarily localizes at the back of the cell where it dephosphorylates PIP3 to PIP2, ensuring the polarization of PIP3 at the leading edge. Cells that lack PTEN extend multiple pseudopodia simultaneously, which impairs progress towards the chemoattractant and invokes a lack of directionality (Lijima and Devreotes, 2002).

PI-3‟K activity is rapidly stimulated by chemoattractants including CXCL12,

CCL5, CCL19, and CCL21, among others (Ward, 2004). CCL5-induced chemotaxis and polarization of human T lymphocytes were reported to correlate with an increase in PI-

3‟K activity and PIP3 accumulation at the leading edge. Moreover, the chemotactic effects of CCL5 were inhibited by wortmannin, the fungal metabolite which potently inhibits PI-3‟K activity (Turner et al., 1995). The differential roles for PI-3‟K isoforms in mediating chemotaxis have been investigated in studies using gene-specific knockout mice, and it has become clear that PI-3‟Kγ is a key regulator of chemotaxis. PI-3‟Kγ (or p110γ) is activated by G-protein βγ subunits from activated chemokine GPCRs.

Neutrophils and macrophages purified from PI-3‟Kγ-null mice display impaired motility and reduced chemoattractant-mediated migration. Moreover, these neutrophils are unable 23 to generate PIP3 at the leading edge and are unable to activate downstream effector Akt in response to the chemoattractant N-formyl-met-leu-phe (Hannigan et al., 2002). While numerous studies have underscored the importance of p110γ in mediating chemotaxis, evidence has accumulated which indicates other PI-3‟K isoforms are crucial for chemotaxis. Studies conducted by Reif and colleagues demonstrated that B cell homing to Peyer‟s patches and splenic white pulp was impaired only in B cells deficient in p110 and not p110γ. Furthermore, the chemotactic ability of PI-3‟Kγ-deficient macrophages and T cells in response to chemokines is not completely abrogated (Hirsch et al., 2000;

Reif et al., 2004). This is suggestive that other PI-3‟K isoforms and/or additional PI-3‟K- independent pathways may be important for directional migration.

While PI-3‟Ks play a critical role in mediating chemotaxis, the most intriguing aspect of these kinases is their ability to affect an extraordinarily diverse number of cellular functions including cell survival, receptor trafficking, metabolism and differentiation. Their roles in regulating metabolism will be discussed in Section 1.5.2.1.

1.3.1.4.The mTOR/4E-BP1 Pathway and Chemotaxis

The evolutionarily conserved mammalian target of rapamycin (mTOR) is a serine/threonine kinase that is downstream of PI-3‟K activation. Much like PI-3‟K, mTOR senses and integrates a plethora of extrinsic signals to regulate cellular growth, proliferation, differentiation, metabolism and migration (Murooka et al., 2008; Sinclair et al., 2008; Peter et al., 2010). As will be further discussed in Section 1.4., mTOR serves as a central regulator of nutrient-sensing and metabolism which have important ramifications on energy-consuming processes like chemotaxis and protein translation

(discussed below). 24

Protein translation is a highly regulated process that affects development, cell cycle progression and apoptosis. Following transcription in the nucleus, mRNAs are processed and exported to the cytoplasm where they are translated. Ribosomes are recruited to the 5‟ end of an mRNA and begin translation where a start codon sequence

(RNA sequence with AUG) is located. The binding of ribosomes to mRNAs is facilitated in one of two ways. First, eukaryotic translation initiation factors (eIFs) recognize and bind to the 7-methyl guanosine residue (m7GpppN; where „m‟ is a methyl group and „N‟ is any nucleotide) that cap the 5‟ end of all nuclear-transcribed mRNAs – this is termed cap-dependent translation. eIF4E binds the 5‟ cap together with other initiation factors, eIF4G, eIF4A and eIF4B to form the eIF4F-complex that is responsible for unwinding mRNA and allowing for ribosomal binding to occur (Hay and Sonenberg, 2004).

Ribosomes can also be recruited to complex RNA structural elements termed an internal ribosomal entry segment (IRES), and initiate cap-independent translation (Stoneley and

Willis, 2004). Notably, numerous components of cap-dependent translation are regulated by mTOR, namely eIF4B, eIF4G and eIF4E.

mTOR exists in two complexes: mTOR complex 1 (mTORC1), which is sensitive to rapamycin, an anti-fungal macrolide, and mTOR complex 2 (mTORC2), which is rapamycin-insensitive (Figure 1.5) (Hay and Sonenberg, 2004). mTORC1 consists of catalytic mTOR, Raptor (regulatory associated protein of mTOR), mLST8, PRAS40 and

Deptor, while mTORC2 is made up of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSIN1, Protor-1, mLST8 and Deptor (Laplante and Sabatini, 2009).

Although mTORC1 and mTORC2 share several accessory proteins, they regulate distinct signaling pathways and relatively little is known about mTORC2 biology.

Figure 1.5. mTOR signaling complexes

The serine/threonine protein kinase mTOR exist as two complexes (mTORC1 and mTORC2) that are structurally and functionally distinct.

mTORC1 promotes mammalian protein translation via activation of the p70 ribosomal S6 kinase 1 (S6K1) and via inhibition of the eukaryotic initiation factor 4E

(eIF4E)-binding protein 1 (4E-BP1). mTORC1 phosphorylation and activation of S6K1 at Thr389 leads to the subsequent phosphorylation of rpS6 (40S ribosomal protein S6).

These phosphorylation events promote the translation of a subset of mRNAs that contain a 5‟ tract of oligopyrimidine (TOP). These mRNAs encode components of the translation machinery, including ribosomal proteins and elongation factors, wherein translation of these mRNAs up-regulate global translation capacity (Wullschleger et al., 2006). The eIF4E-binding proteins are a family of translational repressor proteins, consisting of three members, 4E-BP1, 4E-BP-2 and 4E-BP3. These compete with eIF4G for the overlapping binding site on eIF4E. The sequestering of eIF4E prevents the formation of a functional initiation complex and mRNA translation is inhibited. Binding of the 4E-BPs to eIF4E is regulated by phosphorylation events: hypo-phosphorylated 4E-BP binds with high affinity to eIF4E, whereas the hyper-phosphorylation of specific serine and threonine residues prevent this interaction. Several phosphorylation sites have been reported on 4E-

BP1, but the most important for eIF4E release are Thr37, Thr46, Ser65 and Thr70 which occur in an ordered, hierarchical manner (Hay and Sonenberg, 2004). Phosphorylation of two priming sites, Thr37/46, by mTORC1 is required for the subsequent phosphorylation of Thr70, Ser65, and finally the release of 4E-BP1 from eIF4E and enabling cap- dependent translation (Hay and Sonenberg, 2004). Taken together, the association of mTORC1 with S6K1 and 4E-BP1 co-ordinate mRNA translation by (1) up-regulating translational machinery through 5‟TOP mRNA translation and (2) directly enabling

28 eIF4E availability for 5‟capped mRNA translation initiation. mTOR-mediated translation is depicted in Figure 1.6.

Many facets of an immune response are influenced by mTOR/4E-BP1-mediated protein translation, including lymphocyte proliferation (Section 1.4.2.), growth and cellular migration. The expression and activity of cytoskeletal regulators, Rho, Rac and

Cdc42, are regulated by mTORC1-modulation of 4E-BP1 and S6K1 activity (Liu et al.,

2010). Inhibition of mTORC1 with rapamycin inhibits F-actin reorganization and cell motility induced by IGF-1 (type I insulin-like growth factor) in a number of tumor cell lines. Specifically, rapamycin inhibits mTORC1-mediated protein synthesis and activity of small GTPases, Rho, Rac and Cdc42, leading to decreased F-actin polymerization, reduced lamellipodium formation, and decreased cell motility. Furthermore, Liu and colleagues reported that mTORC1-mediated 4E-BP1 and S6K1 signaling pathways are involved in rapamycin inhibition of Rho, Rac and Cdc42 expression. Cells transfected with a constitutively hypo-phosphorylated 4E-BP1 bind with high affinity to eIF4E and inhibited its ability to initiated cap-dependent translation. In response to IGF-1 stimulation, these cells also displayed a reduced expression of Rho, Rac and Cdc42, mimicking the effects of rapamycin. Down-regulating S6K1 also impaired the expression of GTPases in different tumor cells lines. Collectively, both 4E-BP1 and S6K1 pathways are essential for mTORC1-regulation of GTPase expression and cell motility.

CCL5-mediated chemotaxis is also influenced by mTOR/4E-BP1-mediated protein translation (Murooka et al., 2008). CCL5 induces rapid phosphorylation of mTOR as well as its downstream substrates S6K1, rpS6 and 4E-BP1 in human peripheral blood

(PB) CD4 T cells (Murooka et al., 2008). This CCL5-induced phosphorylation/ deactivation of 4E-BP1 lead to the dissociation of eIF4E and subsequent eIF4F-complex 29

Figure 1.6. Regulation of cap-dependent mRNA translation

4E-BP1 binds to eIF4E and prevents its interaction with eIF4G and other initiation factors, thereby preventing protein translation. mTOR directly phosphorylates 4E-BP1 and results in its release from eIF4E. eIF4E, as part of the eIF4F complex, binds the 5‟ cap of an mRNA and recruits eIF3, 40S ribosomal subunit along with associated complexes necessary for translation.

Adapted from Hay and Sonenberg, Genes Dev.18 (2004)

31 formation. Indeed, CCL5 initiates active mRNA translation, evidenced by the increased presence of high-molecular-weight polysomes which were reduced by rapamycin inhibition. Importantly, CCL5 initiates the translation of chemotaxis-promoting proteins cyclin D1 and MMP-9 (matrix metalloproteinase-9) in an mTOR-dependent manner. The expression of cyclin D1 prevents cell-cell and cell-matrix adhesion and ensures motility during chemotaxis, whereas MMP-9 degrades proteins of the extracellular matrix (Xia et al., 1996; Li et al., 2006). Taken together, CCL5 is able to regulate eIF4E availability and mRNA translation machinery by phosphorylating and inhibiting 4E-BP1 in an mTOR- dependent manner. This enables CCL5-mediated expression of proteins that contribute to efficient chemotaxis.

Eukaryotic protein synthesis can be regulated by transcriptional and translational processes that contribute to a cell‟s decision to grow, proliferate or undergo apoptosis.

The activation of mTORC1 and subsequent signaling of S6K1 and 4E-BP1 promotes the translation of 5‟TOP mRNAs and influences eIF4E availability for 5‟-capped mRNA translation. These events regulate the effectiveness of an immune response by promoting the expression of chemotaxis-related proteins as well as positively controlling proliferation (Section 1.4.2.). Upstream regulators of mTOR activity will be further presented in Section 1.4.1.

1.3.2. Role in determining Cellular Fate

Emerging evidence suggests that chemokines can influence cellular fate during an immune response by affecting cell differentiation, activation and apoptosis (Luther and

Cyster, 2001; Vlahakis et al., 2002). Their ability to induce these changes is critical to the outcome of an infection. 32

1.3.2.1. T cell Differentiation and Activation

Upon activation by APCs, CD4 T cells alter their cytokine production, increase cellular proliferation and acquire different effector functions necessary for an effective adaptive immune response. Interferon γ (IFN-γ)-producing T helper (Th) 1 cells are important for the clearance of intracellular pathogens, while interleukin (IL)-4, IL-5 and

IL-10 producing Th2 cells initiate antibody production by B cells to eliminate extracellular pathogens. Depending on the effector functions acquired, different chemokine receptors are expressed: CCR5 and CXCR3 predominate on Th1 cells, whereas CCR4 and CCR8 are preferentially expressed on Th2 cells (Luther and Cyster,

2001). The polarization of an immune response towards a Th1 or Th2 response is dependent on a combination of host genetic factors, the type and amount of antigen encountered and the cytokines elicited by infectious agents or any other insult

(Romagnani, 1997). Moreover, chemokine receptors and the chemokine milieu can influence T cell fate and determine T helper cell polarization.

For instance, microbial challenge with Toxoplasma gondii is able to induce the production of CCL3, CCL4 and CCL5. These chemokines activate CCR5 and signal the production of IL-12 by CD8α dendritic cells to initiate a Th1 response for clearance of the parasite (Aliberti et al., 2000). Mice deficient in CCR5 and CCR2 have also elucidated the role of chemokine receptors in directing differentiation. When challenged with a colitis-inducing agent or an opportunistic pathogen, CCR5-/- and CCR2-/- mice displayed a Th2-skewed profile compared to their wild-type counterparts. These data indicate the importance of these receptors in mediating a Th1 response (Andres et al.,

2000; Trayner et al., 2002). Taken together, chemokines are able to promote Th1 and Th2 33 differentiation and direct the polarization of an immune response. Distinct chemokine receptors also serve as Th1 versus Th2 markers and ensure that only the appropriate effector T cells are recruited to the sites of inflammation. For instance, rheumatoid arthritis is a Th1 disease characterized by inflammation of the synovial tissue of multiple joints. An abundance of inflammatory chemokines including CCL2, CCL3, CCL4 and

CCL5 are produced, which in turn promote the recruitment of T cells and macrophages that express receptors for these chemokines (CCR1, CCL2 and CCR5) (Luster, 1998) In fact, most of the T cells infiltrating affected rheumatic joints express CCR5 (Suzuki et al.,

1999).

A number of chemokines affect T cell fate by acting as co-stimulatory molecules to promote lymphocyte activation. T cells derived from mice deficient in CCL5 have a reduced capacity to proliferate and secrete inflammatory cytokines IL-2 and IFN-γ in response to antigen or CD3 ligation (Makino et al., 2002). In the context of CD3 T cell stimulation, nanomolar (nM) concentrations of CCL5 results in T cell proliferation and cytokine production. At higher micromolar (M) concentrations, CCL5 induces antigen- independent activation of T cells measurable by increased proliferation, IL-2 receptor expression, and release of IL-2, IL-5, IFN-γ and CCL3 (Bacon et al., 1995; Appay and

Rowland-Jones, 2001). The ability of CCL5 to bypass antigen recognition to activate T cells is also seen in other chemokines including CCL3, CCL4 and CCL2 (Taub et al.,

1996). Their ability to co-stimulate purified human T cells and different T cell clones may be due to their capacity to induce proliferation, IL-2 production, calcium flux, and up-regulation of B7.1 (CD80) on antigen presenting cells (APC). CCL5-induced activation is not restricted to T cells, since activation in monocytes and neutrophils have also been reported (Appay et al., 1999). 34

This mitogen-like property of CCL5 has been attributed to its ability to form multimers at high concentrations. The amino acids involved in CCL5-oligomerization are the negatively charged Glu66 and Glu26. Importantly, CCL5 variants with a Glu26 to alanine mutation (E26A-CCL5), or a Glu66 to serine mutation (E66S-CCL5) were unable to form aggregates at M concentrations and were unable to activate T cells (Appay et al.,

1999). Furthermore, CCL-5 aggregates initiate signaling programs that are distinct from non-aggregated CCL5 to induce T cell activation as well as cellular apoptosis (Section

1.1.3. and Section 1.3.2.2).

1.3.2.2. Role in Cell Death

Different chemokines have been reported to induce pro- and/or anti-apoptotic events when engaging their chemokine receptors. Their ability to protect or enhance cell death depends on the chemokine in question, their concentration and the targeted cell type. CXCL12, for example, is a chemoattractant for T cells and monocytes, but triggers cell death in neurons (Berndt et al., 1998). Subsequent studies have investigated the dichotomy in CXCL12 signaling to address why CXCL12 stimulation of T cells does not result in cell death while in other cell types it does. Vlahakis and colleagues proposed that

CXCL12 is able to simultaneously activate pro-survival and pro-death signals in CD4 T cells, with a net result of cell survival. They provide evidence that CXCL12-CXCR4 signaling results in the activation of ERK 1/2 and PKB/Akt for cell survival, as well as activation of p38 for apoptosis. This model suggests that the default cell fate is apoptosis and CXCL12-induced Akt signaling is able to „override‟ pro-apoptotic events for cell survival. It is inferred that there is a lack of Akt signaling downstream of CCR4 ligation in neurons, which results in cell death. 35

CCL5-CCR5 interactions have also been shown to induce cell death (Murooka et al., 2006). Micromolar (M) concentrations of CCL5 are able to induce apoptosis in PM1,

Molt-4, and activated human PB-T cells in a CCR5-dependent manner. At these high concentrations, CCL5 aggregates and forms multimers to induce cell death through the release of cytochrome c, caspase-9 and caspase-3. Interestingly, CCL5-mediated apoptosis is independent of GPCR-signaling, but rather dependent on tyrosine activity at

Tyr-339 found in the C-terminus of CCR5 (Murooka et al., 2006). Thus, CCL5 is able to induce two distinct signaling pathways in T cells. At nM concentrations, CCL5 acts as a typical chemokine to induce PTx-sensitive GPCR-mediated signaling that is associated with a transient calcium influx resulting in chemotaxis and cell polarization. However, at

M concentrations, CCL5 triggers a tyrosine phosphorylation pathway that leads to prolonged calcium influx and T cell death (Bacon et al., 1995; Appay and Rowland-Jones,

2001; Murooka et al., 2006).

The ability for CXCL12 and CCL5 to induce two distinct signaling outcomes, chemotaxis and apoptosis, may be important for the resolution of an immune response.

At an inflammatory site where high concentrations of CCL5 may be attainable, chemokine-mediated apoptosis may play a regulatory role in resolving an immune response by inducing clonal deletion similar to activation induced cell death (AICD).

1.4. mTOR Signaling and Metabolic Regulation

1.4.1. Growth Factor and Nutrient-sensing by mTOR

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved regulator of cell metabolism, growth, proliferation and survival (Figure 1.5.). Eukaryotic

TOR proteins possess a C-terminal serine/threonine kinase domain that resembles the catalytic domain of PI-3‟K, and belong to a group of kinases known as the phosphatidylinositol kinase-related kinase (PIKK) family (Wullschleger et al., 2006).

Rapamycin is a potent immuno-suppressive agent that forms a complex with intracellular cofactor FKBP12 (FK506-binding protein 12kDa) and inhibits mTORC1 activity by binding to its N-terminus. mTOR positively controls cell growth by integrating signals from growth factors, nutrients and energy status in order to modulate cell metabolism, cell fate (Section 1.4.2.) and different aspects of an immune response, including chemotaxis and protein translation (Section 1.3.1.4.).

Many growth-factor signals converge on the PI-3‟K/Akt pathway to subsequently activate mTOR (Figure 1.2.). The binding of insulin or insulin-like growth factor (IGF) to their receptors leads to the recruitment of IRS1 (insulin receptor substrate 1) together with PI-3‟K and Akt to the plasma membrane. PI-3‟K-mediated activation of Akt subsequently phosphorylates and inactivates the tuberous sclerosis complex (TSC 1/2), an indirect inhibitor of mTOR. It is clear that TSC 1/2 are a complex of tumor suppressor proteins, where mutations in either TSC1 or TSC2 lead to a hyper-active mTOR. These mutations manifest as tumor formations in a number of target organs (Garami et al.,

2003). TSC 1/2 function as a GTPase-activating protein for the small GTPase, Rheb

(Ras-homolog enriched in brain) which is an upstream positive regulator of mTOR. TSC

1/2 negatively regulates Rheb activity by promoting GTP hydrolysis and causing Rheb to remain in its inactive, GDP-bound state (Inoki et al., 2003). In its active, GTP-bound form, Rheb directly interacts with mTOR and stimulates its activity (Long et al., 2005).

Active mTOR can subsequently regulate growth and survival by biosynthesis of proteins, lipids and organelles through S6K1 and 4E-BP1 signaling (Laplante and Sabatini, 2009). 37

Nutrient signals, especially those from amino acids, regulate mTOR activity by inhibiting autophagy and inducing S6K1 activation (Wullschleger et al., 2006).

Withdrawal of the essential amino acid, leucine, results in the rapid dephosphorylation of mTOR effectors S6K and 4E-BP1 (Hay and Sonenberg, 2004). The activation of mTOR by amino acids is TSC1/2-independent, given that S6K1 remained sensitive to amino acid withdrawal in cells lacking TSC1 or TSC2 (Nobukuni et al., 2005). Moreover, levels of

GTP-bound Rheb remain unchanged in cells that were starved of amino acids despite dephosphorylation of mTOR – suggesting that amino acids may invoke signaling in a pathway distinct from the insulin-mediated TSC1/2-Rheb axis (Zhang et al., 2003).

Indeed, studies suggest that the class III PI-3‟K, vps34, signals amino acid availability to mTOR, independent of TSC1-TSC2/Rheb (Nobukuni et al., 2005). Removal of amino acids causes a decrease in vps34 kinase activity and S6K1 phosphorylation levels.

However, the exact role of vps34 in nutrient sensing and how this information is relayed to mTOR remains to be established. mTOR signaling can subsequently up-regulate the surface expression of a number of nutrient receptors. These, among others, include glucose transporters, GLUT-1 and GLUT-4, iron/transferrin receptors, and 4F2HC (4F2 heavy chain; CD98), the invariant heavy chain that associates with different light chains to form the heterodimeric amino acid transporter family (Edinger, 2007). Collectively, nutrient signals are able to stimulate cell growth and proliferation via the mTOR pathway, in part by regulating nutrient transporter expression in an mTOR-dependent manner.

Given that cell growth depends on a high rate of protein synthesis mediated by mTOR signaling, a high level of cellular energy is required to fuel mTOR-mediated processes. The energy status of the cell is signaled to mTORC1 through the AMP-

38 activated protein kinase (AMPK), a master sensor of intracellular energy status and inhibitor of mTORC1 (Wullschleger et al., 2006; Gwinn et al., 2008).

1.4.1.1. AMPK-regulation of mTOR

During nutrient deprivation or environmental stress – such as glucose withdrawal or hypoxia – mammalian cells are able to sense declining intracellular energy levels and attempt to restore homeostasis through AMPK signaling. AMPK is a highly conserved heterotrimeric kinase complex composed of a catalytic α-subunit and two regulatory, β and γ subunits. Under conditions of nutrient deprivation, intracellular ATP levels decline while AMP levels rise and AMPK becomes activated (Shaw, 2009). AMP directly binds to the γ-subunit of AMPK and allosterically activates AMPK. AMPK can also be activated by Thr172 phosphorylation of its activation loop located on the α-subunit. The major upstream regulator of AMPK is the serine/threonine kinase LKB1 (liver kinase B1) which phosphorylates AMPK at its activation loop under low ATP conditions. Once activated, AMPK acts as a metabolic checkpoint – halting cell growth and suppressing

ATP-consuming processes such as protein synthesis while activating ATP-generating processes like fatty acid oxidation.

The inhibitory effects of AMPK ensure that cells do not continue to grow under unfavorable conditions when nutrients are lacking. AMPK activation is able to down- regulate mTORC1-mediated processes, like growth and protein translation, both of which require high levels of cellular energy. Cells deprived of nutrients or treated with the

AMPK activator, AICAR (5-aminoomidazole-4-carboxyamide), exhibit a decrease in mTOR activity where the phosphorylation of S6K1 and 4E-BP1 are reduced (Kimura et al., 2003). Active AMPK can directly phosphorylate TSC2, which contributes to 39 mTORC1 suppression by limiting Rheb activity (Inoki et al., 2003). In addition, AMPK can directly inhibit mTORC1 by phosphorylating the Raptor subunit which induces 14-3-

3-binding of Raptor thereby affecting its conformational state (Gwinn et al., 2008).

Activation of AMPK leads to a metabolic checkpoint where cells will undergo cell-cycle arrest. However, cells that lack components of the AMPK pathway, including upstream activator LKB or downstream effector TSC2, continue cycling and subsequently undergo apoptosis (Inoki et al., 2003; Gwinn et al., 2008). Specifically, the failure to down- regulate mTORC1 under conditions of energy deprivation induces cell death. This metabolic checkpoint function of AMPK has been further emphasized in studies using various cell types under conditions of low glucose, hypoxia and treatment with glycolytic inhibitors (Shaw et al., 2004; Buzzai et al., 2007). These studies underscore the importance for AMPK-mediated inhibition of mTORC1 under conditions of energy stress to halt cell-cycling and prevent cell death.

1.4.2. mTOR Signaling in Lymphocyte Trafficking

Naïve T lymphocytes constantly circulate the body through the blood, lymphatics and secondary lymphoid organs by trans-endothelial migration via high endothelial venules (HEVs). Lymphocyte entry depends on a unique set of molecules that are constitutively expressed on naïve T cells, including CCR7, CD62L (L-selectin), and

CXCR4 (Sinclair et al., 2008; Finlay and Cantrell, 2009). Lymphocytes move into secondary lymphoid organs by responding to a gradient of CCR7 ligands, CCL19 and

CCL21, and CD62L that mediate the capture and rolling of naïve lymphocytes on the

HEVs. These molecules ensure the retention of lymphocytes within lymphoid tissues where they interact with APCs until a “match” is found. Upon activation, T cells alter 40 their expression of chemokine receptors and adhesion molecules to facilitate changes in their migratory pattern. Instead of residing in lymphoid tissues, effector T cells migrate to nonlymphoid tissues and sites of inflammation. Activated T cells down-regulate CCR7 and CD62L and up-regulate receptors that aid in the homing to sites in the periphery such as VLA-4 (very late antigen 4), P- and E-selectin ligands, sphingosine 1-phosphate receptor type 1 (S1P1) and inflammatory chemokines CCR5 and CXCR3 (Cyster, 2005;

Mora and von Andrian, 2006). The down-regulation of CCR7 and CD62L is mediated by signaling through PI-3‟Kδ/mTOR, suppressed by LY294002 and rapamycin (Sinclair et al., 2008). Together, the p110δ subunit of PI-3‟K promotes CD62L down-regulation by proteolysis and mTOR regulates the expression of KLF2, a key transcription factor of

CCR7 and CD62L. The most intriguing aspect is the potential link between the PI-

3‟K/mTOR nutrient-sensing pathway, and its ability to affect chemotaxis by regulating chemokine receptor expression. Given that mTOR is a nutrient sensor that promotes cell growth, these data, together with those discussed in Section 1.3.1.4., suggest that mTOR may potentially be able to integrate cellular energy levels and promote trafficking by controlling homing receptor expression together with promoting mRNA translation of proteins important for chemotaxis.

1.4.3. mTOR-mediated Proliferation

As a critical player in metabolic regulation, deregulation of mTOR can manifest as different cancers and metabolic diseases, including diabetes and obesity (Sabatini,

2006; Dann et al., 2007).

Several upstream and downstream components of the mTOR pathway are altered in cancer. Up-regulation and/or mutations of PI-3‟K, Akt, loss of PTEN, mutations of 41

TSC1 and TSC2, among others, have all been identified in different types of cancers

(Cully et al., 2006; Dann et al., 2007). Intriguingly, aberrantly high mTORC1 activity appears to be an underlying cause of different cancers and hamartoma syndromes, which are benign tumors that contain architecturally disorganized cells (Inoki et al., 2005; Tee and Blenis, 2005; Wullscleger et al., 2006). These observations have influenced clinical trials using rapamycin and its derivatives as anti-cancer agents to inhibit growth. Results from clinical trials show that mTOR inhibitors are generally well tolerated and induce tumor regression in a subset of patients (Dancey, 2005; Vignot et al., 2005). Rapamycin derivatives have also been used as immunosuppressive agents in organ transplants

(Wullscleger et al., 2006).

Many cancers are characterized by abnormal chemokine production and chemokine receptor signaling. This allows chemokines to enhance tumor growth, metastasis and survival by initiating angiogenesis or promoting a pro-inflammatory microenvironment (Murooka et al., 2005; Viola and Luster, 2008). For example,

CCL5:CCR5 signaling has been demonstrated to enhance breast cancer progression and proliferation (Murooka et al., 2009). Breast tumor cells expressing lower levels of CCL5 exhibited a decreased growth rate in vitro (Alder et al., 2003). In contrast, CCL5 is highly expressed in high grade tumors and was a predictor of rapid disease progression in breast cancer patients (Yaal-Hahosheney al., 2006). The ability for CCL5 to promote growth and proliferation is, in part, due to its ability to invoke the mTOR/4E-BP1 pathway and up-regulate protein translation (Murooka et al., 2009). Studies in the breast cancer cell line, MCF.7, have demonstrated that CCL5:CCR5 signaling directly promotes proliferation and survival through mTOR signaling. Specifically, CCL5 engages the mTOR/4E-BP1 pathway and promotes the translation of proliferative and survival 42 proteins, namely cyclin D1, c-Myc and defender against cell death-1 (DAD-1) in a rapamycin-sensitive manner (Murooka et al., 2009). These findings suggest that breast cancer cells can exploit chemokine-induced signaling and mTOR-mediated effects to promote proliferation and survival.

1.5. Energy Metabolism and the T cell Response

1.5.1. Regulation of T lymphocyte Metabolism

Upon antigen presentation and activation by APCs, lymphocytes undergo rapid and extensive expansion. Resting lymphocytes shift from a quiescent phenotype to a highly proliferative and secretory state which requires a considerable amount of energy and cellular resources. In order to match the energetic demands of the transcriptional and translational programs that promote growth and effector functions, activated T cells must alter their metabolism to support these activities (Frauwirth and Thompson, 2004; Fox et al., 2005; Pearce, 2010). Interestingly, T cells lack the cell-autonomous ability to control their uptake of metabolites necessary for adenosine-5‟-triphosphate (ATP) generation

(Fox et al., 2005). T cells must therefore rely on external, „instructional‟ signals to access nutrient molecules such as glucose, amino acids and fatty acids from the environment.

Specifically, T cells receive these instructional signals via cytokine, antigen and co- stimulatory receptor signaling. At a fundamental level, it is cellular metabolism which governs T cell function and differentiation, which in turn ultimately influences the outcome of an adaptive immune response.

At different stages of T cell development, different growth-factor signals are required to maintain nutrient uptake. IL-7, for instance, is a survival factor for early

43 thymocytes, resting naïve T cells as well as memory T cells (Ma et al., 2006; Surh and

Sprent, 2008). T cell activation results in the down-regulation of the α-chain of the IL-7 receptor, and effector T cells become more dependent on IL-2. Withdrawal of growth- factors, such as IL-2 or IL-7, results in the decline of cellular metabolism that is characterized by decreased surface expression of nutrient receptors, decreased rate of nutrient uptake and changes in mitochondrial integrity. These changes lead to the release of apoptotic factors into the cytosol and the commitment to cell death by apoptosis (Cory and Adams, 2002). Cytokines, such as IL-4, IL-2 and IL-7 modify the expression and activity of the pro-survival B-cell lymphoma 2 (Bcl-2) proteins and prevent apoptotic events from occurring. It is clear that the decision for T cells to live or die is regulated, in part, by cytokine-dependent effects on T cell metabolism.

1.5.2. Quiescent Cells and Oxidative Phosphorylation

Naïve and memory T cells exist as relatively quiescent cells that consume glucose and other essential nutrients at a low rate to supply energy to maintain normal homeostatic functions (Fox et al., 2005). Their intracellular stores of ATP are largely generated by the combined breakdown (or catabolism) of glucose, amino acids and lipids through oxidative phosphorylation that take place within mitochondria. Oxidative phosphorylation generates large amounts of ATP (36 ATP molecules per glucose molecule) using the energy liberated by the stepwise transfer of free electrons from nutrient intermediates to oxygen.

IL-4 and IL-7 are essential for the survival of naïve T cells (Vella et al., 1997; Fox et al., 2005). Freshly isolated naïve T cells cultured without IL-4 or IL-7 die, even in the presence of high levels of extracellular nutrients. The anti-apoptotic effects of IL-4 and 44

IL-7 have been associated with their ability to maintain levels of survival-promoting Bcl-

2 and Bcl-X1 in immature T cells. Quiescent cells can also rely on autophagy, the break down of intracellular components, to fuel oxidative phosphorylation (Lum et al., 2005;

Lum et al., 2005b). Indeed, in the absence of growth factors, cells will rely on the breakdown of cellular components to maintain survival. It is becoming clear that quiescence is a state that is actively maintained by the expression of transcription factors such as FOXO (Forkhead box class O) and LKLF (lung Kruppel-like factor). IL-7 can induce the expression of these transcriptional factors to regulate genes that inhibit cellular activation, cell-cycle progression and modulate metabolic pathways to maintain the quiescent phenotype (Yusuf and Fruman, 2003; Sinclair et al., 2008). Indeed, quiescence in lymphocytes is an actively maintained state that is under tight transcriptional control rather than a default fate that is determined by a lack of mitogenic signals.

1.5.3. Proliferating Lymphocytes and Glycolysis

Antigenic stimulation of T cells is followed by a metabolic switch from oxidative phosphorylation to glycolysis which is required to support growth, proliferation and effector functions in activated lymphocytes (Frauwirth and Thompson, 2004; Pearce

2010). In mature T cells, ligation of the T cell receptor together with CD28 promotes metabolic pathways that shunt lipids and amino acids into the production (or anabolism) of macromolecules. As a consequence, ATP production becomes dependent on the degradation of glucose by glycolysis. Strikingly, even in the presence of sufficient oxygen to support oxidative phosphorylation, expanding T cells prefer to ferment glucose to meet their energy demands. This phenomenon, known as aerobic glycolysis or the

Warburg effect, is hallmark of many transformed cells (Warburg, 1956; Elstrom et al., 45

2004). In addition, limiting glucose availability to rapidly proliferating lymphocytes will cause cell death, despite the availability of alternative fuels such as fatty acids or amino acids. Taken together, effector T cells rely exclusively on glucose metabolism for ATP production and survival. Although oxidative phosphorylation is the more efficient way of generating ATP compared to aerobic glycolysis (2 ATP molecules per glucose molecule), it remains unclear why proliferating T cells favor this form of metabolism. One explanation is that glycolysis is a process which leaves many cellular metabolites untouched; building blocks like amino acids and fatty acids can then be incorporated into macromolecules to help activated T cells expand and proliferate (Maciver et al., 2008;

Vander Heiden et al., 2009; Pearce, 2010). At the expense of ATP production, effector T cells rely on the hyper-induction of glycolysis initiated by PI-3‟K signaling to support their increased metabolic needs (Frauwirth et al., 2002; Seder et al., 1994).

1.5.3.1. PI-3’K Signaling in Aerobic Glycolysis

Many growth-factors that control aerobic glycolysis bind to their surface receptors and initiate signaling of the PI-3‟K pathway (Frauwirth et al., 2002; Fung et al., 2002;

Wofford et al., 2008). Activated PI-3‟K generates the lipid messenger PIP3 which is required for the recruitment and activation of downstream effectors, including the serine/threonine kinases PKB/Akt and mTOR. Growth-factor-mediated PI-

3‟K/Akt/mTOR activation enhances a number of metabolic activities that support the energy demands of activated T cells.

First, cell surface expression of nutrient receptors can be enhanced to increase nutrient uptake. CD28 co-stimulation together with CD3 ligation induces sustained Akt activation along with an increase in glucose transporter, GLUT-1, surface expression in 46 human peripheral blood T cells (Frauwirth et al., 2002). In contrast, resting T cells express low levels of GLUT-1, the primary glucose transporter of hematopoietic cells. In fact, activation by anti-CD3 or anti-CD28 alone does not change GLUT-1 expression and is unable to sustain Akt phosphorylation. The synergistic/co-operative effects of anti-

CD3/anti-CD28 stimulation of Akt and GLUT-1 are sensitive to PI-3‟K inhibition by

LY294002. Frauwirth and colleagues further demonstrated that CD3/CD28-stimulated T cells exhibit increased glucose uptake that is predominantly converted and secreted as lactate. As much as 90% of the glucose consumed is converted to lactate instead of being processed into macromolecules – indicative of limited oxidative phosphorylation in activated T cells. Subsequent studies using growth-factor-dependent cell lines and ex vivo activated lymphocytes have shown similar results. Specifically, PI-3‟K/Akt signaling is necessary for GLUT-1 translocation from intracellular stores to the cell surface, efficient glucose import and lactate secretion to occur (Bentley et al., 2003; Wieman et al., 2007;

Wofford et al., 2008). Interestingly, the Wieman group noted that mTOR signaling was not required for IL-3-mediated GLUT-1 expression in FL5.12 cells, however, mTOR inhibition with rapamycin greatly diminished glucose uptake. It can be speculated that mTOR signaling may promote GLUT-1 activity rather than receptor levels.

Changes in gene expression and enzymatic activity induced by Akt can also increase the rate of glycolysis. For example, cells with constitutively active Akt have increased total cellular hexokinase (HXK) activity, the enzyme responsible for phosphorylating and trapping glucose in the cell and ensuring its entry into the glycolytic pathway (Rathmell et al., 2003). Moreover, constitutively active Akt is able to induce glucose uptake, maintain HXK activity and prevent cellular apoptosis in the absence of extrinsic factors. Phosphofructokinase (PFK) is the enzyme that catalyzes the rate- 47 limiting step in glycolysis by phosphorylating fructose 6-phosphate to fructose 1,6- biphosphate. This step fully commits glucose to glycolysis. Studies have shown that insulin-activated-Akt can stimulate PFK activity in the heart to up-regulate glycolysis

(Deprez et al., 1997). Taken together, PI-3‟K signaling acts to coordinate energy metabolism by regulating glucose transport and key glycolytic enzymes.

1.5.4. Energy Regulation in an Immune Response

The ability for lymphocytes to alter their metabolism and co-ordinate glycolysis not only affects their decision to proliferate (Section 1.5.3.) or quiesce (Section 1.5.2.), it can also determine lymphocyte function in an immune response. At each stage of development, lymphocyte metabolism is regulated to eventually fuel an effective immune response (Fox et al., 2005).

The development of αβ cells in the thymus is a highly regulated process. The earliest thymocyte precursors lack the expression of CD4 and CD8 co-receptors and are referred to as double-negative (DN) cells. DN precursors can be divided into four populations based on their expression of CD44 and CD25: DN1 (CD44+CD25-), DN2

(CD44+CD25+), DN3 (CD44-CD25+), and DN4 (CD44-CD25-) (Godfrey et al., 1993).

Rearrangement of the T cell receptor (TCR) β locus is catalyzed by recombinase- activating gene 1 (RAG-1) and RAG-2 during the transition of cells from DN2 to DN3.

The β chain of DN3 cells must pair with a surrogate pre-TCR α chain and CD3 molecules in order to form the pre-TCR. Only DN3 cells that generate a functional TCR β chain will further differentiate and enter the first check point of thymopoiesis, β-selection. Both

Notch and IL-7 signaling are important during β-selection for the maintenance of glucose metabolism and survival in thymocytes (Ciofani and Zuniga-Pflucker, 2005; Wofford et 48 al., 2008). Notch signaling not only directs T cell lineage commitment (versus B cell lineage), its presence is also required throughout DN stages for continued differentiation of αβ T cells towards a double positive (DP) CD4+CD8+ T cell (Wolfer et al., 2002). The signaling cascades invoked by Notch signaling during β-selection were studied using DN thymocytes from RAG-2 deficient mice, which fail to produce an endogenous TCR-β chain. Only in DN cells that were retrovirally transduced with TCR-β and cultured on stromal cells in the presence of Notch ligand, Delta-like 1, did cells survive and further differentiate into CD4+CD8+ double positive T cells (Ciofani and Zuniga-Pflucker, 2005).

In the absence of Notch ligand, thymocytes underwent apoptosis with increased caspase 3 activity and a loss of mitochondrial membrane potential. In addition, Notch signaling was required to maintain GLUT-1 expression and glucose metabolism in a PI-3‟K/Akt- dependent manner to promote survival in these pre-T cells. Constitutively active Akt was also sufficient to maintain thymocyte glucose metabolism for β-selection in the absence of Notch. Collectively, these data provide evidence that glucose metabolism is a means for Notch to promote survival during β-selection. By controlling thymocyte metabolism,

Notch regulates the development and selection of T lymphocytes.

Another important response regulated by lymphocyte metabolism is the generation of memory CD8 T cells. In response to an infection CD8 T cells undergo expansion to become antigen-specific effector cells – a process that is accompanied by a metabolic conversion from oxidative phosphorylation to rapid glycolysis (Section 1.5.3.).

Long-lived memory CD8 T cells arise when effector T cells contract and once again become quiescent cells that rely on oxidative phosphorylation after an infection. The exact mechanism which regulates the transition to the memory phenotype remains unclear; however a successful metabolic transition may be required for the differentiation 49 to CD8 memory T cells after an infection (Pearce et al., 2009; Araki et al., 2009). To investigate the mechanisms underlying memory T cell development, Pearce and colleagues studied mice with a T cell-specific deletion of the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). TRAF6 is an adaptor protein in the TNF-receptor and interleukin-1R/Toll-like receptor superfamily and is a known negative regulator of T cell activation. Following bacterial infection, TRAF6-deficient T cells mounted normal antigen-specific effector CD8 T cell responses, but fewer memory CD8 T cells were detected 60 days after infection. In addition, these mice failed to respond robustly to re- infection, indicative that a lack of memory T cells was generated in the T-cell specific

TRAF6-deficient mice. Most interestingly, microarray analyses that compared gene expression between wild-type and TRAF6-deficient CD8 T cells indicated that TRAF- deficient cells displayed defects in the expression of genes in several metabolic pathways, including fatty acid metabolism (Pearce et al., 2009). The oxidation of fatty acids, similar to autophagy, can occur when growth-factor signals are withdrawn and glycolysis becomes compromised (Rathmell et al., 2005). Although TRAF6-deficient CD8 T cells were able to initiate glycolysis during activation, they had a reduced capacity to oxidize fatty acids when IL-2 was withdrawn. Taken together, TRAF6-deficient CD8 T cells fail to persist as long-lived memory cells due to their inability to engage fatty acid oxidation pathways. This switch may be imperative for their survival when growth factors such as

IL-2 become limiting following the peak of an immune response. The conversion in metabolism during the contraction phase may be required in memory T cell development for a successful and accelerated secondary response.

It is becoming clear that changes in T cell metabolism can determine T cell fate and further influence the outcome of an immune response. The ability for growing 50 thymocytes to acquire nutrients will determine their survival and selection in the thymus.

Similarly, the conversion between differing metabolic states is required for effective generation of a given T cell fate.

1.6. Hypothesis and Objective

Hypothesis: CCL5-mediated mTOR activation modulates cellular metabolism by directly regulating nutrient uptake and glucose metabolism in activated T cells.

Given that mTOR is a central regulator of nutrient sensing and processes that are induced by CCL5, including mRNA translation and chemotaxis, that are energy-taxing,

CCL5/mTOR signaling may influence cellular metabolism to match energy demands of activated T cells.

Objective: Examine the role of CCL5-CCR5 mediated signaling in regulating T cell metabolism in the context of chemotaxis.

CHAPTER 2

MATERIALS AND METHODS

2.1. Cells and Reagents

Human peripheral blood (PB)-derived T cells were isolated from consenting healthy donors, approved by the UHN REB. Cells were maintained in RPMI 1640 supplemented 10% dialyzed fetal calf serum (Sigma), 100 units/ml penicillin, 100 mg/ml streptomycin and 2mM L-glutamine (Invitrogen). CD3+ T cells were purified using the

StemSep T cell enrichment cocktail, according to the manufacturer‟s specifications

(StemCell Technologies). T cells were subsequently activated in the presence of 10

µg/ml anti-CD3 antibody (eBiosciences), 5 µg/ml anti-CD28 antibody (eBiosciences), and 5ng/ml hrIL-12 (Bioshop, Canada) for 2 days, and further expanded in culture supplemented with 100U/ml (10ng/ml) hrIL-2 (Bioshop, Canada) for 3 days. T cell purity and CCR5 expression were confirmed at day 6 by flow cytometric analysis using anti-human CCR5 antibody (2D7; BD Pharmingen), anti-human CD3, anti-human CD4 and anti-human CD8 antibodies (eBiosciences). Antibodies for phospho-AMPKα (Thr-

172), AMPKα, phospho-GSK-3β (Ser-9), phospho-4E-BP1 (Thr-37/46) and 4E-BP1, were purchased from Cell Signaling Technology. Mouse monoclonal anti-tubulin antibody was purchased from R & D Systems. Purified mouse anti-human CD98

(4F2HC) and GLUT-1 antibodies were obtained from Santa Cruz Biotechnology Inc. and

R & D Systems, respectively. Rapamycin, Compound C and AICAR were obtained from

Calbiochem. 2-deoxy-D-glucose was purchased from Sigma. Toxicity studies were performed on splenocytes derived from C57Cl/6 mice purchased from Jackson

Laboratory (Bar Harbor, ME). CCL5/RANTES was a generous gift from Dr. Amanda

Proudfoot (Geneva Research Centre, Merck Serono International). The CCR5 antagonist,

TAK-779, was kindly provided by Dr. Clifford Lingwood (University of Toronto,

Sickkids Hospital). 54

2.2. Immunoblotting

Cells were incubated with 10 nM CCL5 for the times indicated, collected, washed twice with ice-cold PBS and lysed in 100 l lysis buffer (1% Triton X-100, 0.5% NP-40,

150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 10

µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A). For all experiments using inhibitors or activators, cells were pretreated for 1 hr with the amount indicated prior to

CCL5 treatment. Protein concentration was determined using the Bio-Rad DC protein assay kit (BioRad laboratories). 50 g of protein lysate were denatured in 5x sample reducing buffer and proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to a nitrocellulose membrane followed by blocking with 5% BSA (w/v) in 1x TBST (0.1% Tween-20) for

1hr at room temperature. Membranes were probed with the specified antibodies overnight in 5% BSA (w/v) in TBST at 4°C and the respective proteins visualized using the ECL detection system (Pierce).

2.3 Flow Cytometric Analysis

1 x 106 cells were incubated with mouse anti-human CCR5 antibody for 30 minutes on ice and washed twice with ice-cold FACS buffer (PBS/2% FCS). Cells were then incubated with AlexaFlour 488-conjugated anti-mouse IgG antibody (eBiosciences).

As control, cells were incubated with AlexaFlour 488-conjugated antibody alone. T cell purity was determined by incubating cells with a PE-conjugated anti-human CD3 antibody. As isotype control, cells were incubated with PE-labeled isotype control IgG 55 antibody (eBiosciences). For GLUT-1 and CD98 (4F2HC) surface expression, cells were collected and washed twice with ice-cold FACS buffer and fixed with 2% PFA at room temperature for 20 minutes. Cells were then washed twice with FACS buffer and incubated with mouse anti-human GLUT-1 or mouse anti-human CD98 for 30 minutes on ice. Cells were then washed twice and incubated with AlexaFlour 488-conjugated anti-mouse IgG antibody. Cells were analyzed using the FACSCalibur and FlowJo software (BD Biosciences).

2.4 Chemotaxis Assay

T cell chemotaxis was assayed using 24-well Transwell chambers with 5 µm pores (Corning). A total of 1 x 105 cells in 100µl chemotaxis buffer (RPMI 1640/0.5%

BSA) were placed in the upper chambers. CCL5, diluted in 600µl chemotaxis buffer, was placed in the lower wells and the chambers incubated for 2 hrs at 37ºC. Migrated cells located in the bottom wells were collected, and counted with a hemocytometer. All experiments were conducted in triplicate. In experiments involving inhibitors, cells were pretreated for 1 hr at the indicated inhibitor concentrations and placed in the upper chambers. Cell viability, as measured by PI staining, was not affected by any of the doses of inhibitors used in this study.

2.5 Glucose Uptake Assay

3-5 x 106 cells were washed with PBS and resuspended in 500µl of Krebs-Ringer-

HEPES (KRH) (at pH 7.4, 136 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.25mM

3 MgSO4, and 10 mM HEPES). 2-Deoxy-D-[H ] glucose (2Ci/ reaction; Perkin Elmer) was added in the presence of CCL5 and the reaction mixture incubated at 37C. 56

Reactions were quenched by the addition of ice-cold 200 M phloretin (Calbiochem) followed by immediate centrifugation through an oil layer (1:1 phthalic acid and dibutlylpthalate from Sigma-Aldrich). Cell pellets were washed and solubilized in 1M

NaOH for 1 hr, and radioactivity was measured using a scintillation counter. In experiments involving inhibitors, cells were pretreated for 1h before the addition of 2-

Deoxy-D-[H3] glucose and CCL5.

2.6 AMPK Signaling Antibody Array

Phosphorylation events in the AMPK signaling pathway were examined using the

Full Moon BioSystems Antibody Microarray, according to the manufacturer‟s specifications (Full Moon BioSystems, Inc.). Briefly, 5 x 106 cells were stimulated with

CCL5 for 10 minutes, washed with ice-cold PBS, and lyzed with 200 µl of Extraction

Buffer. Protein samples were biotinylated and were added to a microscope slide chambers that had specific antibodies bound to its surface. Cy3-streptavidin was added, and fluorescence was detected using the Axon GenePix 400A Microarray Scanner at

PMT voltages between 300-400 (Molecular Devices).

2.7 Statistical Analysis

Statistical significance was analyzed with repeated-measures analysis of variance

(ANOVA). A level of p<0.05 was chosen to identify significant differences. All data are expressed as mean ± S.E.M. (standard error of means).

CHAPTER 3

RESULTS

O.C. performed all experiments and analyzed the data

Dr. E.N.F. designed research and analyzed the data

3.1. CCL5 induces phosphorylation of proteins in the AMPK signaling pathway

To investigate potential metabolic changes induced by CCL5, studies were conducted to examine phosphorylation events in the energy-sensing, AMPK signaling pathway. The phosphorylation/activation of AMPK is an indication of a decrease in intracellular ATP levels – which may initiate processes that attempt to restore homeostasis by up-regulating ATP-generating processes, such as glycolysis, and suppressing ATP-consuming processes, such as cell cycling (Shaw et al., 2004).

Ex vivo cytokine activation of peripheral blood (PB) CD3+ T cells (as described in Materials and Methods) induced the surface expression of CCR5 (Figure 3.1.A). T cell populations used in all experiments were consistently 70% - 75% CCR5 positive, >90%

CD3 positive, and were a heterogeneous population of CD4+ and CD8+ T cells (Figure

3.1.B and Figure 3.1.C). To avoid confounding data attributed to IL-2 effects PB T cell cultures that were used for CCL5 treatment experiments were only stimulated with IL-2 on days 2 and 4, and then CCL5 treated on day 6. IL-2 treated cultures that served as positive controls were stimulated with IL-2 on days 2, 4 and 6.

At the onset, a global screening approach was undertaken to examine signaling events in the AMPK pathway. The Antibody Microarray platform from Full Moon

BioSystems measures the phosphorylation of upstream and downstream substrates of

AMPK. The different phospho-specific antibodies in each array are listed in Table 3.1.

Activated PB T cells either left untreated (control) or treated with 10 nM CCL5 for 10 minutes, were lysed and proteins biotinylated. Biotinylated proteins were then introduced on to the microarray slide chambers which were conjugated with antibodies specific for the AMPK signaling cascade. T cell bound proteins were identified using a Cy3- streptavidin detection system. 59

Figure 3.1. CCR5 surface expression is induced upon T cell activation in the presence of cytokines

(A) PB T cells were activated in the presence of IL-12 and IL-2 to induce the surface expression of CCR5. (B) CD3 expression was measured with anti-CD3 antibody staining

(black), using the appropriate isotype control (grey). Representatives of three independent experiments are shown. (C) T cell populations were a heterogeneous mix of CD4+ T cells and CD8+ T cells; majority of which were CD4+.

Figure 3.1

Table 3.1. List of AMPK Signaling Phospho-Specific Antibodies

Phosphorylation Phosphorylation Phospho-antibody Phospho-antibody Sites Sites Ser65 Thr45 Ser2448 4E-BP1 mTOR Thr36 Thr70 Ser2481 6-phosphofructo-2-kinase/fructose-2,6- Thr2446 Ser483 biphosphatase 2 (PFKFB2) p21Cip1 ACC1 Ser79 Thr145 Ser80 Thr308 Ser15 Ser392 AKT Ser473 Tyr326 Ser20 Ser46 Ser124 Thr72 Ser315 Ser6 AKT1 Ser246 Tyr474 p53 Ser33 Ser9 Thr450 Ser366 Thr18 AKT1S1 Thr246 Ser37 Thr81 AKT2 Ser474 Ser378 AMPK1 Thr174 Ser411 p70 S6 Kinase AMPK1/AMPK2 Ser485/491 Ser424 AMPK/beta 1 Ser182 Ser371 Thr389 CaMK1-alpha Thr177 p70S6K Ser418 Thr421 CaMK2 Thr305 Thr229 CaMK2-beta/gamma/delta Thr287 p70S6k beta Ser423 CaMK4 Thr196/200 PI3-kinase p85-alpha Tyr607 CaMKII Thr286 PI3-kinase p85-subunit alpha/gamma Tyr467/199 Ser126 PKA CAT Thr197 Cyclin B1 Ser147 PKA-R2B Ser113 EEF2 Thr56 PLC-beta Ser1105 eEF2K Ser366 PLC-beta3 Ser537 Ser1177 RapGEF1 Tyr504 eNOS Ser615 SREBP-1 Ser439 Thr495 Ser939 Tuberin/TSC2 HNF4 alpha Ser304 Thr1462 HSF1 Ser303 Ser552/563 HSL Ser554 LKB1 Ser428 Thr189 Thr312 MEF2A Ser408 Thr319 MEF2C Ser396 MEF2D Ser444

The microarray slide images generated are shown in Figure 3.2.A. and phosphorylation events were quantitated and represented as fold induction in Figure 3.2.B. PB T cells treated with 10 nM CCL5 for 10 minutes induced the rapid phosphorylation of a number of signaling effectors in the AMPK signaling pathway, as well as effectors in the PI-

3‟K/Akt and mTOR/4E-BP1 cascades. Notable proteins phosphorylated by are indicated with red arrows. These included phosphorylation of PFKFB-2 (6-phosphofructo-2- kinase/fructose-2,6-biphosphatase 2 or PFK-2), a positive regulator of glycolysis, ACC-1

(acetyl-CoA carboxylase 1), an enzyme important for fatty acid synthesis and inhibitor of fatty acid oxidation, and the master regulators of energy status LKB1, AMPK1/AMPK2 and mTOR.

Figure 3.2. CCL5 induces phosphorylation of proteins in the AMPK signaling pathway

(A) The AMPK Signaling Phospho-Specific Antibody Array includes six replicates

(vertical columns) of phospho-specific antibodies and their non-phospho pairs, targeted against proteins in the AMPK signaling pathway. These antibodies are covalently immobilized on microscope slides. Biotinylated protein lysates were added to microscope slide chambers and fluorescence from Cy3-streptavidin was measured with the Axon

GenePix 400A Microarray Scanner (B) The extent of protein phosphorylation (mean fluorescence intensity, MFI) was normalized within each slide and compared between untreated control and CCL5 treated cells. The data are represented as fold CCL5- induction relative to untreated controls. Important proteins phosphorylated by CCL5 are indicated (red arrows).

Figure 3.2

Control 10 nM CCL5

To validate the antibody array findings for AMPK phosphorylation, Western immunoblot time-course studies (0-60 minutes) were performed. PB T cells treated with

10 nM CCL5 induced the maximal phosphorylation and activation of AMPK-α on

Thr172 by 10 minutes. Phosphorylation at this activation loop threonine residue is absolutely required for AMPK activation (Zhou et al., 2001; Gwinn et al., 2008). CCL5 also induced the phosphorylation of GSK-3β (glycogen synthase kinase 3 β), a downstream substrate of AMPK, on Ser9, with peak phosphorylation detected at 10 minutes post-CCL5 treatment (Figure 3.3.). GSK-3β is a constitutively active serine/threonine kinase that regulates glycogen synthesis, gene transcription, protein translation and cell proliferation (Doble and Woodgett, 2003; Jope and Johnson, 2004). It is in its phosphorylated/ inactive form that GSK-3β is able to de-repress downstream signaling mediated by glycogen synthase, eIF2B, NF-κB, and other downstream substrates. Thus, the inhibitory effect of CCL5 treatment on GSK-3β may not only affect glycogen metabolism, but may also promote energy storage in activated T cells.

Figure 3.3. CCL5 activates the energy-sensing kinase AMPK and downstream substrate GSK-3β

Activated PB T cells were either left untreated, or treated with 10 nM CCL5 for the indicated times. Cells were harvested and protein lysates resolved by SDS-PAGE and immunoblotted with anti-phospho-AMPKα (Thr 172) or anti-phospho-GSK-3β (Ser 9) antibodies. Membranes were stripped and re-probed for loading. Relative phosphorylation is shown as signal intensity over loading control. Data are representative of two independent experiments. *p<0.01

Figure 3.3

3.2. CCL5-mediated glucose uptake is mTOR-dependent

The preceding data suggest that CCL5 may regulate the metabolic state of T cells through AMPKα activation. AMPK acts as a “fuel gauge” to initiate pathways for ATP regeneration and is able to induce glucose uptake in a variety of cell types during energy stress (Fujii et al., 2006; Gwinn et al., 2008). The nutrient-sensitive mTOR is also able to respond to extracellular nutrient signals to regulate growth and glycolysis (Wullschleger et al., 2006). Glucose uptake was therefore examined to determine if the signaling events invoked by CCL5 could lead to active glucose uptake in activated T cells. For these experiments, cells were resuspended in Kreb‟s Ringer HEPES (KRH) buffer, a salt solution that maintains pH and osmotic balance and provides cells with water and essential inorganic ions. This buffer is free of glucose. As shown in Figure 3.4.A, CCL5 treatment was able to stimulate glucose uptake in a dose-dependent manner, with maximal uptake using 10 nM CCL5 (1.2-1.4 fold increase). This effect was abrogated by pretreatment with the glucose analogue and anti-cancer agent, 2-deoxy-D-glucose (2-DG).

2-DG is structurally similar to glucose, differing at the second carbon by a substitution of hydrogen for a hydroxyl group (Aft et al., 2002). 2-DG is transported into cells through glucose transporters and is phosphorylated by hexokinase (HXK), but is not metabolized any further (Figure 3.4.C.). The accumulation of 2-DG in the cell interferes with glycolysis and subsequent glucose uptake by inhibiting the activity of glycolytic enzymes

(Ralser et al., 2008). In agreement with previous studies, IL-2 treatment results in a 1.5-

1.9 fold increase in glucose uptake (Wofford et al., 2008).

Next, the specific contribution of mTOR signaling to the CCL5-mediated increase in glucose uptake was examined using rapamycin. Indeed, inhibiting mTOR effectively reduced CCL5 mediated glucose uptake by 1.5 fold (Figure 3.4.B.), supporting the 69 hypothesis that CCL5-mediated mTOR activation promotes the up-regulation of nutrient uptake and glucose metabolism in activated T cells.

Finally, to confirm that CCL5 specifically induces glucose uptake through CCR5 activation, the CCR5 antagonist TAK 779 was employed. TAK 779 is a small chemical molecule that binds to CCR5 trans-membrane helices 1, 2, 3, and 7 to induce conformational changes in CCR5. These conformational changes disrupt ligand binding and inhibit ligand-mediated signaling (Baba et al., 1999; Dragic et al., 2000). A marked reduction in glucose uptake was observed in T cells pretreated with TAK 779 (Figure

3.4.C.). These data indicate that CCL5 binding to CCR5, and not CCR1 or CCR3, is required for glucose uptake.

Figure 3.4. CCL5-mediated glucose uptake is mTOR-dependent

(A) Activated PB T cells were suspended in KRH buffer and were either left untreated, treated with 20 ng/ml IL-2, or different doses of CCL5 for 2hr. In parallel, cells were pretreated with 10 mM of 2-DG for 1hr prior to treatment with 10 nM CCL5. At time 0, 2

μCi/rxn of 2-deoxy-D-[3H] glucose was added to the cultures. Reactions were quenched with ice-cold phloretin, cells solubilized with NaOH and radioactivity measured with a scintillation counter. Data are representative of three independent studies. (B) Cells were pretreated with either DMSO (carrier) or 50 nM of rapamycin for 1hr prior to treatment with 10 nM CCL5. Tritiated-glucose uptake was measured as in (A). Data are representative of two independent studies. (C) Cells were pretreated with CCR5 antagonist, TAK-779 for 1hr prior to treatment with 10 nM CCL5. Tritiated-glucose uptake was measured as in (A). Data are representative of two independent studies. (D)

Schematic model depicting the pathway in which glucose is imported through glucose transporters and metabolized by enzymes hexokinase (HXK) and phosphofructokinase-1

(PFK-1). 2-DG is taken up into cells through glucose transporters and trapped within cells once phosphorylated by HXK.

Figure 3.4

3.3. CCL5-mediated glucose uptake is not accompanied by changes in the surface

expression of nutrient receptors

The ability of CCL5 to stimulate glucose uptake may be facilitated through enhanced surface expression of nutrient receptors. Glucose uptake is mediated by a family of facilitative integral membrane glucose transporters (GLUTs) that are expressed on the cell surface. In lymphocytes, facilitated diffusion is primarily controlled by

GLUT-1, a ubiquitously expressed glucose transporter that is highly up-regulated upon

CD3/CD28 ligation (Frauwirth and Thompson, 2004; Maciver et al., 2008). Activated lymphocytes also increase expression of the insulin-sensitive GLUT-3 and GLUT-4 receptors. Numerous growth signals mediate cell-surface trafficking and expression of

GLUT-1 through the PI-3‟K/Akt pathway, thereby increasing glucose uptake and glycolytic flux (Frauwirth et al., 2002; Bentley et al., 2003; Wofford et al., 2008).

Another key nutrient receptor that is regulated by this pathway is CD98, a critical component of the amino acid-transporter complex. Accordingly, we undertook studies to examine the ability of CCL5 to regulate the surface expression of GLUT-1 and CD98.

Whereas naïve T cells express low levels of GLUT-1 and CD98 (Figure 3.5.A. and Figure 3.5.B.), their cell surface expression is strongly induced upon T cell activation. In time course studies, CCL5 treatment did not further increase GLUT-1 expression at 2, 4, 6 and 8 hours post CCL5 treatment (Figure 3.5.C.), with evidence of enhanced expression only by 24 hours post-treatment. Notably, neither IL-2 nor CCL5 led to increased GLUT-1 expression at earlier time points that corresponded with enhanced glucose uptake. Given that GLUT-1 surface expression is strongly induced upon T cell activation the effects of CCL5 on glucose uptake in the absence of a concomitant increase in GLUT-1 expression might be anticipated. Indeed, published 74 studies using different diabetes models have demonstrated that alterations in glucose uptake are not necessarily accompanied by changes in GLUT-1 or GLUT-4 expression

(Pedersen et al., 1990; Kahn et al., 1991). Thus, CCL5-mediated mTOR signaling may increase the functionality or intrinsic activity of GLUT-1 to induce glucose uptake without altering surface expression. The increase in GLUT-1 expression seen at 24 hours may also reflect changes in transcriptional activation induced by CCL5 treatment.

Certainly, insulin is able to promote GLUT-1 protein expression by increasing GLUT-1 mRNA levels and GLUT1 gene transcription through Akt activation (Barthel et al., 1999).

Similar to the late induction of GLUT-1, CCL5 likewise resulted in enhanced

CD98 expression at 8 hour and 24 hour post-treatment, but not at earlier time points

(Figure 3.5.D.). Amino acid transport is mediated by CD98 when it forms disulfide- linked hetero-dimers with other membrane-spanning light chains on the cell surface

(Edinger, 2007). CD98 is a ubiquitously expressed membrane protein that is strongly induced following T cell activation, and also following IL-2, IL-15 or insulin treatment

(Deves and Boyd, 2000; Cornish et al., 2006; Edinger, 2007). In agreement with these studies, CCL5 is also able to up-regulate the surface expression of CD98, albeit not to the same extent as IL-2.

Figure 3.5. CCL5 increases cell surface expression of GLUT-1 and CD98

(A) Surface GLUT-1 levels and (B) CD98 levels were determined by FACs on freshly isolated T cells or T cells activated with α-CD3 and α-CD28 for 2 days. (C) Activated

PB T cells were treated with 10 nM CCL5 or 20 ng/mL IL-2 for the indicated times.

Cells were fixed with 2% PFA and stained for cell surface GLUT-1 or (D) CD98 expression and analyzed by FACS. Data are representative of three independent studies.

Figure 3.5.

A B

C GLUT-1

CD98

3.4. Glucose uptake and AMPK signaling are required for efficient CCL5-mediated chemotaxis

To investigate whether CCL5-mediated glucose uptake is important for mediating

T cell chemotaxis, inhibition studies were performed using the glucose uptake inhibitor,

2-DG. As shown in Figure 3.6.A., pre-treatment with 2-DG reduced CCL5-mediated T cell chemotaxis. These data suggest that efficient chemotaxis requires a steady supply of glucose, and that glucose metabolism may contribute to CCL5-mediated cellular migration of T cells. Next, the role of AMPK signaling was evaluated in chemokine- induced chemotaxis. We examined the effects of the AMPK inhibitor, Compound C, on

CCL5-mediated T cell migration. The data reveal that AMPK inhibition reduced CCL5- inducible T cell chemotaxis (Figure 3.6.B). The reduction in CCL5-mediated chemotaxis by the inhibitors, 2-DG and Compound C, at the doses employed, was not due to any cytotoxic effects (Figure 3.7.)

3.5. CCL5-induced AMPK signaling phosphorylates the 4E-BP1 repressor of mRNA translation

The preceding data have suggested that CCL5 may activate both AMPK and mTOR signaling simultaneously, despite AMPK being regarded as an inhibitor of mTOR activity. Thus, the role of AMPK signaling in CCL5/mTOR-mediated phosphorylation of

4E-BP1 was examined. mTOR-mediated phosphorylation of 4E-BP1 is required for the release of the initiation factor, eIF4E, and the de-repression of mRNA translation (Hay and Sonenberg, 2004). In PB T cells, CCL5 induces the phosphorylation of 4E-BP1 in a

PI-3‟K/mTOR-dependent manner (Murooka et al., 2008). The role of AMPK signaling in

CCL5-dependent 4E-BP1 phosphorylation was determined using AICAR, an AMPK 80

Figure 3.6. Glucose uptake and AMPK signaling are required for efficient CCL5- mediated chemotaxis

(A) Activated PB T cells were either left untreated or pretreated with 2-DG at the doses indicated for 1 hr. A total of 1 x 105 cells in 100 μl chemotaxis buffer were then placed in the upper chamber of Transwell chambers. CCL5-mediated chemotaxis was measured using 10 nM CCL5. Data are presented as % migration, with the number of migrated cells at 10 nM CCL5 taken as 100%. Data are representative of three independent experiments. (B) Activated PB T cells were pretreated with either DMSO (carrier) or different doses of Compound C for 1 hr. CCL5-mediated chemotaxis was measured as in

(A). Data are representative of three independent experiments. *p<0.01

Figure 3.6

Figure 3.7. Effects of 2-DG and Compound C on T cell viability

Activated T cells were either left untreated, treated with DMSO (carrier), 2-DG (A) or

Compound C (B) for the indicated times. Cell viability was determined by propidium iodide staining analyzed by FACS. Cells negative for PI stain were considered viable.

Figure 3.7.

84 activator, and Compound C, an inhibitor of AMPK. As shown in Figure 3.8., pretreatment of PB T cells with Compound C reduced 4E-BP1 phosphorylation of

Thr37/46, which indicates AMPK signaling to be required for CCL5-mediated 4E-BP1 deactivation. Given 4E-BP1 to be an important downstream substrate of mTOR, these data suggest that AMPK activation does not strictly inhibit mTOR activity and that biological events invoked by CCL5, such as protein translation and migration, may require both AMPK and mTOR to be active simultaneously.

Figure 3.8. CCL5-induced AMPK signaling phosphorylates the 4E-BP1 repressor of mRNA translation

Activated PB T cells were pre-treated with either DMSO (carrier), 1 mM AICAR or 10

μM Compound C for an hour prior to 10 min treatment with 10 nM CCL5. Cells were harvested and protein lysates resolved by SDS-PAGE and immunoblotted with anti- phospho-4E-BP1 (Thr 37/46) antibodies. Membranes were stripped and re-probed for

4E-BP1 as loading control. Relative phosphorylation is shown as signal intensity over loading control. Data are representative of two independent experiments. *p<0.05

Figure 3.8.

CHAPTER 4

DISCUSSION

Recruitment of immune cells to a site of infection is imperative for an effective immune response. The infiltration of antigen-specific effector T cells is a highly organized process that is co-ordinated by chemokines, and contributes to the clearance of foreign pathogens. Activated T cells are able to navigate to sites of infection with the help of inflammatory chemokines such as CCL5 that are deposited on the GAGs of endothelial cells. T cell migration along a CCL5 gradient establishes cell polarization and promotes directional migration through cytoskeletal rearrangements when CCL5 activates its cognate receptor, CCR5. In addition to promoting lymphocyte trafficking,

CCL5 also regulates a number of cellular processes including cell proliferation, protein translation and T cell fate.

Previous studies have shown that CCL5/CCR5 signaling in primary T cells activate the mTOR/4E-BP1 pathway to directly modulate mRNA translation (Murooka et al., 2008). The ability of CCL5 to regulate protein translation is mediated by the inhibition of 4E-BP1, an inhibitor of translation, and de-repression of the initiation factor, eIF4E. Moreover, CCL5-mediated mTOR activation influences T cell chemotaxis by initiating the translation of chemotaxis-related proteins, including MMP-9 and cyclin D1.

Taken together, CCL5 up-regulation of chemotaxis-related proteins may “prime” T cells for efficient migration.

CCL5-mediated chemotaxis and mRNA translation, two processes that are affected by mTOR, consume high levels of cellular energy (Hay and Sonenberg, 2004).

Indeed, mTOR is a central regulator of nutrient sensing, cell size and glycolysis. This raised the possibility that CCL5 may be linked to cellular metabolism by activating

89 mTOR and directly regulating nutrient uptake. Its ability to do so may be required to meet energy demands of migration and protein translation in activated T cells.

Initial studies investigated the activation of AMPK, the energy-sensing kinase that is activated under conditions of energy stress. CCL5 induced the rapid phosphorylation/activation of AMPK at its activation loop, in addition to the phosphorylation of a number of downstream substrates including ACC1, PFKFB2 and

GSK-3β. The ability of CCL5 to induce AMPK activation suggests that intracellular levels of ATP may be declining due to energy-taxing processes invoked by CCL5. As a result, CCL5 may simultaneously initiate processes that attempt to increase intracellular nutrient and energy levels, while suppressing cell growth and biosynthetic processes via

AMPK activation. Previous studies in various tissues have demonstrated that AMPK acutely inhibits fatty acid and cholesterol synthesis by phosphorylating and inactivating metabolic enzymes ACC1, SREBP1 and HMG-CoA reductase (Marsin et al., 2000; Li et al., 2011). ACC1 is an enzyme that catalyzes the formation of essential substrates necessary for fatty acid synthesis and is also a potent inhibitor of lipid oxidation

(Brownsey et al., 2006). AMPK-mediated phosphorylation of ACC1 allosterically inactivates it and prevents the biosynthesis of fatty acids. Fat and liver cells treated with adrenaline or glucagon – hormones released into the bloodstream during exercise, stress and starvation – leads to the rapid phosphorylation and inactivation of ACC1 which is predominately mediated by AMPK. Several RNA interference studies have also demonstrated the importance of ACC1-mediated lipogenesis for the growth of various tumor cell lines (Brusselmans et al., 2005; Chajes et al., 2006). Here, CCL5-mediated phosphorylation of ACC1 may prevent lipid biosynthesis as a means to conserve energy and limit cellular growth. 90

AMPK activity is also able to stimulate glycolysis through glucose transporter translocation and phosphorylation/activation of the bi-functional enzyme, PFKFB2

(Marsin et al., 2000; Fujii et al., 2006). The first irreversible and commitment step in glycolysis is the conversion of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate

(F1,6BP) which is catalyzed by 6-phosphofructo-1-kinase (PFK-1). A potent stimulator of PFK-1 is fructose-2,6-bisphosphate (F2,6BP), the concentration of which is controlled by PFKFB2. PFKFB2 is a bi-functional enzyme that can act as a kinase and phosphorylate F6P to F2,6BP or act as a phosphatase and dephosphorylate F2,6BP to F6P.

This kinase:phosphatase activity is regulated, in part, by the phosphorylation of Ser466 and Ser483 on PFKFB2 (Bertrand et al., 1999; Marsin et al., 2000; Carlet et al., 2010).

Specifically, phosphorylation of these sites synergistically promotes PFKFB2 affinity for

F6P and catalyzes F6P conversation to F2,6BP, whereas dephosphorylation of the same residues increases the phosphatase activity. In both skeletal and cardiac muscles, insulin, epinephrine, and ischemia are able to increase glycolysis and F2,6BP concentrations by activating PFKFB2. CCL5-induced activation of PFKFB2 may contribute to ATP production by increasing F2,6BP levels and, as a consequence, PFK-1 activity and thus glycolysis.

CCL5 was also able to promote glucose uptake in an mTOR-dependent manner, although this increase in nutrient uptake is not accompanied by changes in GLUT-1 nor

CD98 surface expression. Glucose transport across the plasma membrane of lymphocytes is mediated by specific GLUT proteins: GLUT-1 is responsible for basal glucose transport, while GLUT-3 and GLUT-4 mediate glucose uptake in response to insulin stimulation (Calder et al., 2007). Upon activation during an immune response, lymphocytes increase glucose utilization and rely heavily on glycolysis over oxidative 91 phosphorylation for the generation of ATP (Frauwirth and Thompson, 2004; Fox et al.,

2005). CD3/CD28 ligation is able to stimulate glucose transport, increase GLUT-1 surface expression and promote glycolysis via PI-3‟K/Akt signaling (Frauwirth et al.,

2002; Frauwirth and Thompson, 2004). Intriguingly, increased glucose transport can be detected well before increased GLUT-1 expression, the major GLUT isoform expressed on lymphocytes, suggestive that enhanced nutrient uptake is not necessarily accompanied by increased transporter expression. Several studies in muscle cells, adipose tissues and diabetic models have also demonstrated that hormone-induced changes in glucose uptake can occur without affecting glucose transporter expression and translocation (Pedersen et al., 1990; Kahn et al., 1991; Sweeny et al., 2001; Somwar et al., 2002). The CCL5- stimulated glucose uptake in the absence of enhanced GLUT-1 expression that we observe suggests that CCL5 may promote GLUT-1 intrinsic activity in order to promote glucose uptake. CCL5 stimulation of activated PB T cells induced an up-regulation in the surface expression of CD98, the heavy chain of the hetero-dimeric amino acid transporter complex. Despite only a modest increase induced by CCL5, up-regulation of CD98 expression could subsequently modulate amino acid uptake and amino acid incorporation into proteins. Given its ability to promote mTOR-dependent mRNA translation, CCL5 may also modulate the pool of amino acid „building blocks‟ required to fuel protein synthesis by regulating CD98 expression.

As mentioned, mTORC1 integrates numerous intracellular and extracellular signals and is a central regulator of metabolism, growth, cellular migration and protein synthesis. Studies using the specific mTOR inhibitor, rapamycin, have underscored the importance of mTORC1 signaling for glucose uptake (Wieman et al., 2007; Buller et al.,

2008). The Wieman group demonstrated that IL-3 dependent hematopoietic FL5.12 cells 92 activate the PI-3‟K/Akt/mTOR pathway following IL-3 treatment, to stimulate glucose uptake and GLUT-1 trafficking. Interestingly, mTORC1 activity was not required to maintain surface expression of GLUT-1, although inhibition of mTORC1 by rapamycin greatly diminished IL-3 mediated glucose uptake. These data suggest that mTOR signaling may only be required to promote GLUT-1 intrinsic activity to enhance glucose uptake. We provide evidence that CCL5 is also able to induce glucose uptake in an mTOR-dependent manner. Although rapamycin reduced CCL5-mediate glucose uptake, this reduction in glucose uptake was less than that observed for 2-DG. It may be that other mTORC1-independent mechanisms are also responsible for regulating metabolism in activated T cells, including the MAPKs p38 (Somwar et al., 2002), ERK1/2 (Carr et al.,

2010) and other AMPK-signaling effector molecules (Finlay and Cantrell, 2011).

To investigate whether glucose uptake contributed to efficient CCL5-mediate chemotaxis, the non-metabolized glucose analog, 2-DG, was employed. 2-DG is a potent inhibitor of glucose metabolism and ATP production, and has been examined as a chemotherapeutic agent, given that cancerous cells exhibit an increased rate of glucose uptake (Aft et al., 2002). Prolonged 2-DG treatment in various cancer cell lines interferes with glycolysis, contributing to decreased cell growth, decreased clonogenictiy and enhanced apoptosis through caspase-3 release. Notably, our chemotactic studies using 2-

DG avoided prolonged drug exposure to avoid toxicity and cell death. Glucose uptake inhibition by 2-DG pre-treatment reduced the ability of T cells to migrate towards a

CCL5 gradient, in a dose-dependent manner. Proliferating lymphocytes depend on growth factor signals to promote glucose uptake to maintain survival (Fox et al., 2005).

Even in the presence of alternative energy sources, such as glutamine, T cells maintained in glucose-free medium fail to proliferate, underscoring the essential and non-redundant 93 role of glucose in supporting T cell viability. In the present study, the inability of effector

T cells to take up glucose had an impact on migration. Previous studies have demonstrated that the ability of tumor cells to metastasize to secondary sites in response to a chemoattractant is also dependent on active glycolysis (Beckner et al., 1990;

Kroemer and Pouyssegur, 2008).

For optimal T cell migration orchestrated by CCL5, we hypothesized that AMPK stimulation of ATP-generating processes may be necessary. CCL5-mediated T cell chemotaxis was examined following AMPK inhibition by Compound C. Indeed,

Compound C pre-treatment reduced CCL5-mediated chemotaxis in a dose-dependent manner, suggesting that T cell migration in response to CCL5 is partially dependent on

AMPK signaling. Importantly, while AMPK is most well known for its role as an energy sensor, AMPK signaling also regulates a number of non-metabolic processes, including cell division, cell polarity and directional cell migration (Williams and Brenman, 2008;

Nakano et al., 2010). Recent studies have demonstrated that AMPK activation and signaling to downstream substrates are able to regulate microtubule dynamics and cell polarization to promote migration in the 293 T cell line (Nakano et al., 2010). Moreover,

F-actin polymerization and Rho GTPase activation were reported following AMPK activation in epithelial MDCK cells (Miranda et al., 2010). AMPK inhibition by

Compound C may prevent processes that directly promote CCL5-mediated chemotaxis or indirectly affect ATP-generation. Collectively, these data suggest that both glucose metabolism and AMPK signaling have roles in efficient T cell migration.

Studies undertaken in this thesis have identified AMPK as a novel downstream substrate of CCL5 signaling in activated T cells. In addition, the present studies have identified a role for CCL5-mediated mTOR signaling in modulating glucose metabolism 94 by enhancing glucose uptake. Taken together, CCL5 may simultaneously induce signaling events in both the mTORC1 and AMPK pathways. Intriguingly, whereas

AMPK is active under nutrient-poor conditions, mTOR is active during energy-rich coniditions. More importantly, the current literature indicates that AMPK activation during energy deprivation indirectly suppresses mTOR activity by phosphorylating/activating TSC2 (Inoki et al., 2003), or directly inactivates mTOR by targeting its Raptor subunit (Gwinn et al., 2008). Data generated herein suggest that

CCL5 is able to activate both pathways simultaneously in order to maintain homeostasis: mTOR-dependent processes such as protein translation and chemotaxis are energy taxing

(Hay and Sonenberg, 2004), which may require AMPK signaling to initiate ATP- generating processes. AMPK-mediated inhibition of fatty acid biosynthesis together with its ability to stimulate glycolysis may generate the energy needed to fuel CCL5-mediated processes during an immune response.

Chemokines are critical for the successful recruitment of leukocytes to sites of inflammation during an immune response. Certainly, the mTOR signaling cascade orchestrates aspects of cellular migration including mRNA translation of chemotaxis- related proteins (Murooka et al., 2008) and the expression of lymph node homing receptors, CD62L, CCR7 and CXCR4 (Sinclair et al., 2008). The present studies addressed the cross-talk between lymphocyte migration and cellular metabolism and have shown that the inflammatory chemokine, CCL5, exhibits functions beyond that of a chemotactic cytokine. In addition to its ability to direct PB T cell migration, CCL5- mediated mTOR activation is also able to modulate cellular metabolism by directly regulating glucose uptake in order to match the energy demands of chemotaxis.

Figure 4.1. Illustration of the AMPK and mTOR signaling cascades

AMPK activation upon energy stress leads to the activation of ATP-generating processes such as glycolysis, and the inhibition of ATP-consuming processes such as fatty acid synthesis. CCL5-mediated PI‟3-K/Akt/mTOR signaling results in the phosphorylation of p70 S6K1 and 4E-BP1. Hyper-phosphorylation of 4E-BP1 leads to the release of eIF4E and the formation of the initiation complex for protein translation. A possible model for

CCL5-mediated metabolic changes in activated T cells may be through the simultaneous activation of both the AMPK and mTOR signaling pathway.

Figure 4.1.

CHAPTER 5

FUTURE DIRECTIONS

A complex network of chemokines and chemokine receptors influence the growth and progression of many cancers. Tumor-associated chemokines are able to promote tumor growth directly by stimulating proliferation and/or survival, or indirectly by initiating angiogenesis (Vicari and Caux, 2002; Balkwill, 2004; Murooka et al., 2009).

CCL5 is highly expressed in the microenvironment of breast and prostate cancers. Plasma levels of CCL5 are correlated with breast cancer disease severity, where patients with a more advanced disease express increased levels of CCL5 compared to patients who are in clinical remission (Adler et al., 2003; Yaal-Hahoshen et al., 2006). Various aspects of tumorigenesis are regulated by CCL5, including leukocyte infiltration into primary tumors, metastasis, tumor growth and survival (Niwa et al., 2001; Azenshtein et al., 2002;

Murooka et al., 2009). The ability of CCL5/CCR5 signaling to modulate breast cancer cell metabolism has not been addressed. Previous studies have demonstrated that CCR5 expression on the breast cancer cell line, MCF-7, is able to enhance mTOR-dependent proliferation of cells when cultured in the presence of CCL5 (Murooka et al., 2009).

Moreover, CCL5-mediated mTOR signaling also enhanced mRNA translation of pro- survival proteins cyclin D1, c-Myc and defender against cell death-1 (DAD-1).

Collectively, the proto-oncogenic role of CCL5 can be attributed, in part, to its ability to promote mTOR-dependent mRNA translation in breast cancer cells. Further studies are required to determine whether CCL5-mediated mTOR signaling can affect cancer cell metabolism and nutrient uptake.

To elucidate CCL5-mediated metabolic changes in breast cancer cells, CCR5 expressing MCF-7 cells will be employed to measure changes in glucose uptake invoked by CCL5. We hypothesize that CCL5 may provide a proliferative advantage in cancer 99 cells by stimulating nutrient uptake and promoting glucose metabolism to fuel the high rate of proliferation. The role of CCL5-CCR5 mediated PI-3‟K/Akt/mTOR and AMPK signaling in glucose uptake will be addressed using the appropriate pharmacological inhibitors. If, indeed, CCL5 can promote enhanced glucose uptake in breast cancer cells, it would be intriguing to determine if this can further support breast cancer metastasis and invasion. Cell invasion studies using Matrigel-coated Transwell chambers, together with glucose uptake inhibitors and signaling inhibitors will address if CCL5-mediated glucose uptake is necessary for MCF-7 invasion.

We report herein the ability of CCL5 to up-regulate glucose uptake in PB CD3+ T cells and its importance in mediating chemotaxis. Further studies are required to determine whether CCL5 can regulate the catalytic activity of key glycolytic enzymes in order to promote glucose metabolism and ATP generation for effector T cell functions.

Specifically, enzymatic activity of hexokinase (HXK) and phosphofructokinase-1 (PFK-

1) will be measured to access if CCL5 can enhance glycolysis in addition to glucose uptake. Furthermore, the rates of oxygen consumption and glycolysis can be measured using the Seahorse Bioscience platform. Seahorse Bioscience provides a means of measuring cellular metabolism and metabolic activity in real-time. This platform is a non-invasive and non-destructive way of determining the rate of oxygen consumption, which is a measure of oxidative phosphorylation, and the rate of acidification or lactate production, which is a measure of glycolysis. The ability of CCL5 to modulate cellular metabolism of PB CD3+ T cells and MCF-7 can be determined, in real-time, with the XF

Analyser.

Finally, we have demonstrated the ability of CCL5 to simultaneously activate mTOR and AMPK signaling pathways in PB CD3+ T cells, even though the current 100 literature indicates that AMPK activation is able to suppress mTOR signaling (Inoki et al.,

2003; Gwinn et al., 2008). We propose that homeostasis is maintained when both mTOR and AMPK are activated in conjunction. However, these homeostatic signaling cascades may be disrupted in the context of breast cancer cells that have a proliferative advantage in response to CCL5. The activation/signaling profile of mTOR and AMPK in CCL5- stimulated breast cancer cells can be addressed using Western immunoblotting studies.

These studies will address if CCL5, a chemokine prevalent in the microenvironment of breast cancer tumors, can enhance proliferation by altering signaling pathways that regulate nutrient uptake and growth.

101

CHAPTER 6

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