Contribution of complement receptors to melanoma growth: potential therapeutic targets

Jamileh Alham Nabizadeh

BBiot. Honours

A thesis submitted for the degree of Doctor of Philosophy at

The University of Queensland in 2015

Australian Institute for Bioengineering and Nanotechnology

ABSTRACT

The incidence of melanoma is increasing world-wide, and Australia has the highest rate of melanoma in the world. Hence there is an urgent need for safe and effective treatments. The aim of this thesis was to elucidate our understanding of the role that the , in particular the complement and C5a, plays in tumour development and growth.

Given the mounting evidence for a role for the C5a (C5aR1) in the growth of other tumour types, Chapter 3 explored the role of C5aR signalling in a murine model of melanoma. Immunostaining revealed that B16 murine melanoma cells expressed both receptors for C5a (C5aR1 and C5aR2). Although C5aR1 was capable of ERK activation in response to recombinant mouse C5a, there was no effect on cell proliferation or migration. Melanoma growth was significantly retarded in C5aR1-deficient (C5aR1-/-) mice, and the growth of established melanomas was inhibited by treatment of wild-type mice with a C5aR1 antagonist (C5aR1A), thus suggesting the potential of C5aR1 as a therapeutic target for melanoma. In accord with the premise that C5aR2 acts as a modulator of C5aR1, tumour growth was enhanced in mice lacking C5aR2.

Given that the growth of C5aR-expressing tumour cells was retarded in C5aR1-/- mice, we hypothesised that the protective effect of C5aR1 inhibition could be due to changes in the immune response. This was supported by FACS analysis which showed a significant increase in tumour infiltrating leukocytes in C5aR1-/- mice. Myeloid derived suppressor cells (MDSC) were significantly reduced in both C5aR1-/- and C5aR1A-treated wild-type mice, whereas tumour-infiltrating CD3+ and CD4+ T were increased. Tumour infiltrating leukocyte populations in C5aR2-/- mice were not significantly different from those of their wild-type counterparts. Tumour tissue from C5aR1-deficient mice also showed significantly lower levels of CCL2, CCL4 and CCL5, all known to regulate the movement of MDSC and maintain their immunosuppressive activity.

Chapter 4 investigated whether C3aR, like the C5aRs, also plays a role in regulating tumour growth. Like C5aRs, C3aR was detected at low levels on B16 melanoma cells in vitro and in vivo. Moreover these cells responded to C3a, activating ERK and AKT signalling pathways and enhancing cell migration. The tumour-promoting activity of C3aR signalling was

ii

demonstrated by in vivo studies which showed that B16 melanomas were retarded in C3aR- deficient (C3aR-/-) mice. The growth of established melanomas was arrested by treatment with C3aR antagonist (C3aRA), suggesting that C3aR, like C5aR1, may be a therapeutic target for melanoma. Further, the anti-tumour effect observed in C3aR-/- mice was not augmented by treatment with C5aR1A, implying that in this model the tumour-promoting effects of C3a are at least as potent as those of C5a.

The demonstration that growth of C3aR-expressing B16 cells is retarded in C3aR-deficient mice suggested that, as for C5aR, the tumour-promoting effects of C3a-C3aR signalling are via the host . This was supported by FACS analysis showing alterations in tumour infiltrating leukocyte populations in the absence C3aR signalling. Whereas C5aR was shown to act via induction of MDSC and inhibition of T lymphocytes, C3aR deficiency lead to increased , and CD4+ T-cell subsets (Th1, Th2 and Th17) within the tumour.

The increase in tumour infiltrating neutrophils in C3aR-/- and C3aRA-treated mice is in accord with recent research demonstrating a role for C3a-C3aR signalling in the retention of neutrophils in the bone marrow (Wu et al., 2013). The central role of neutrophils in the tumour inhibitory response observed in C3aR-/- mice was confirmed by depletion experiments which significantly reversed the anti-tumour effects, and returned CD4+ T populations to wild-type levels. The association of C3aR-deficiency with increased tumour infiltrating CD4+ cells suggests the existence of a positive feedback loop whereby the secretion of chemokines and by neutrophils sequestered to the tumour microenvironment promotes CD4+ recruitment and expansion; cytokines produced by CD4+ cells may in turn favour neutrophil recruitment and survival. Similar to previous studies which showed an up-regulation of M1-associated cytokines/chemokines in anti-tumour myeloid cell populations, we show up-regulation of IL-1β and CCL5 in plasma and tumour tissue (respectively) from C3aR-/- mice. Finally, we showed that C3aR deficiency is also protective in murine BRAFV600E mutant melanoma and colon cancer models, suggesting the broader potential of C3aR as a therapeutic target for cancer.

Collectively, the results presented in this thesis demonstrate the key role of complement components C3a and C5a in regulating the anti-tumour immune response to melanoma. They

iii

not only confirm previous reports of a tumour-promoting role for C5aR, but identify a hitherto unknown role for C3a in regulating tumour growth. Whereas C5a appears to exert its effects via induction of MDSC and Tregs, C3a mediates its effects via regulation of neutrophil and CD4+ T lymphocyte populations. Thus by altering the tumour inflammatory milieu, inhibition of either C5aR or C3aR tips the balance towards an effective anti-tumour response.

iv

DECLARATION BY AUTHOR

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

v

PUBLICATIONS DURING CANDIDATURE

None

CONFERENCE ABSTRACTS

Oral Presentations

The Role of Complement in Melanoma Tumour Growth. Presented

to the 6th International Conference on Complement Therapeutics, Kos, Greece, June 2013.

Poster Presentations

The Role of Complement Anaphylatoxin Receptors in Melanoma Tumour Growth. Presented to the International Postgraduate Symposium in Biomedical Sciences, The University of Queensland, Brisbane, Australia. October 15 2013.

The Role of Complement Anaphylatoxin Receptors in Melanoma Tumour Growth. Presented to the NanoBio Australia Conference, The University of Queensland, Brisbane, Australia. July 10 2014.

PUBLICATIONS INCLUDED IN THIS THESIS

No publications included.

CONTRIBUTIONS BY OTHERS TO THE THESIS

In all chapters the names of contributor and their respective contributions are stated clearly in the introductory statements to the corresponding chapters.

Dr. Barbara Rolfe was the primary supervisor while Assoc. Prof. Trent Woodruff was a co- supervisor. Both supervisors provided the primary input in project design, direction and

vi

coordination of the research project in addition to editing and reviewing this thesis. Dr. Helga Manthey provided help with the analysis of FACS data. Dr Derik Steyn assisted with assays and data collection.

STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF ANOTHER DEGREE

None

vii

ACKNOWLEDGEMENTS

The pursuit of knowledge is never obtained alone. It is with this thought in mind that I thank those who have given so generously of themselves to make this dissertation a reality. Foremost, I would like to express my deepest gratitude to my principal advisor, Dr. Barbara Rolfe for her excellent guidance, enthusiasm, patience and providing me with an excellent atmosphere for doing research. Her training and guidance helped me become a better scientist and without her push this thesis would not come to an end. There was never a moment when you could not make time for me and you were always willing to listen patiently as I rambled on about my life or work. Secondly, I would like to express my thanks and appreciation to my co-advisor, Assoc. Prof. Trent Woodruff for the continuous support of my PhD study and research, for his patience, immense knowledge and motivation throughout my study. I am extremely grateful for the massive amount of time that both supervisors have put into reading and editing this thesis. I could not have imagined having better advisors for my PhD study. Besides my advisors, I would like to thank the rest of my thesis committee: Dr. Wenyi Gu, Dr. Glen Boyle, Dr. Angela Jeanes and Assoc. Prof. Christine Wells, for their encouragement, insightful comments, and hard questions.

I would like to thank Drs. Fazrena Mhd Akhir and Helga Manthey who as good friends were always willing to help and give their best suggestions. It would have been a lonely lab without them. Many thanks to my fellow past and present members of the laboratory: Dr. Kelly Hitchens, Dr. Florian Rohart, Dr. Silvia Manzanero, Edward Huang, Eric Chen, Dipti Vijayan, Samah Alharbi, Suad Alarteeq, Yu-Qian Chau and Keith Leong for their friendship, training and stimulating discussions support and for making my time in the lab a lot of fun. Thanks for keeping me company at insane hours and always providing me with coffee. My research would not have been possible without their help. I would like to thank Dr. Helga Manthey for all her help with FACS sorting, analysis and interpretation of my data. I would also like to mention that I was lucky enough to have received the University of Queensland Scholarship from an Australian Postgraduate Award (APA) scholarship, without which, this PhD would not have been possible. I cannot thank my family enough for supporting me spiritually throughout my life.

Special thank goes to my parents, Sakineh Nabizadeh and Mousa Nabizadeh and to all my beloved sisters and brothers, but most importantly to my dearest sister, Batoul Ahlam

viii

Nabizadeh, for your constant and unwavering support and encouragement in all aspects of my life. Without all these people, I would not have been able to complete this journey. Thank you Allah for answering my prayers and giving me the strength to make this happen. I am dedicating this dissertation to my parents, Sakineh and Mousa Nabizadeh. Their continual prayers before God on my behalf have made my career and educational pursuits a reality.

ix

KEYWORDS

Complement component C5a, complement component C3a, complement component (C5aR1 and C5aR2), complement component (C3aR), B16 F0 mouse melanoma tumour, C3aR antagonist (SB290157), C5aR antagonist (PMX53) innate and adaptive .

AUSTRALIAN AND NEW ZEALAND STANDARD RESEARCH CLASSIFICATIONS (ANZSRC)

ANZSRC code: 061107, Immunology, 60%

ANZSRC code: 061115, Pharmacology and Pharmaceutical Sciences, 40%

FIELDS OF RESEARCH (FOR) CLASSIFICATION

FoR code: 1107, Immunology, 60%

FoR code: 1115, Pharmacology and Pharmaceutical Sciences, 40%

x

TABLE OF CONTENTS

ABSTRACT………………………………………………………………...………………….i DECLARATION BY AUTHOR ...... v

PUBLICATIONS DURING CANDIDATURE……………………………………...………vi

CONFERENCE ABSTRACTS………………………………………………………………vi PUBLICATIONS INCLUDED IN THESIS…………………………………………...……..vi CONTRIBUTIONS BY OTHERS TO THE THESIS…….………………….…………….. vii STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF ANOTHER DEGREE……………………………………… …………….…..vii

ACKNOWLEDGEMENTS………………………………………..…………………..……viii KEYWORDS……………………………………………………………………………...…..x AUSTRALIAN AND NEW ZEALAND STANDARD RESEARCH CLASSIFICATIONS (ANZSRC)……………………………………………………………………...……………..x

FIELDS OF RESEARCH (FOR) CLASSIFICATION……………………………………….x

TABLE OF CONTENTS……………………………………………………..………………xi LIST OF FIGURES……………………………………………………………………….…xv LIST OF TABLES………………………………………………………………….……...xvii LIST OF ABBREVIATIONS………………………………………………………...……xviii

CHAPTER 1: INTRODUCTION ...... 1

1.1 GENERAL INTRODUCTION ...... 2

1.2 MELANOMA ...... 2

1.2.1 Epidemiology ...... 2

1.2.2 Environmental factors ...... 3

1.2.3 Genetic mutations ...... 3

xi

1.2.4 Staging of melanoma ...... 5

1.2.5 Treatment ...... 6

1.3 IMMUNE SURVEILLANCE ...... 7

1.3.1 The role of the immune response in tumour development and growth ...... 7

1.4 OVERVIEW OF THE IMMUNE SYSTEM ...... 9

1.4.1 The innate immune system ...... 9

1.4.2 The ...... 16

1.5 CHRONIC AND TUMOUR DEVELOPMENT ...... 21

1.5.1 The complement system ...... 21

1.5.2 Complement components C3a and C5a ...... 24

1.5.3 Peptide and antagonists of anaphylatoxins C3a and C5a ...... 26

1.5.4 The complement anaphylatoxins and tumour growth ...... 28

1.6 CONCLUSIONS AND PROJECT RATIONALE ...... 31

CHAPTER 2: MATERIALS & METHODS ...... 33

2.1 Cell Lines ...... 34

2.2 Drugs ...... 34

2.3 Animals ...... 34

2.4 Tumour Induction and Drug Treatments ...... 35

2.5 Tissue Processing and Histology ...... 35

2.6 Immunofluorescence Staining ...... 36

xii

2.7 Immunohistochemistry ...... 36

2.8 Western analysis ...... 37

2.9 Cell proliferation (MTT assay) ...... 38

2.10 Cell migration assay ...... 38

2.11 Flow cytometric analysis ...... 39

2.12 Cytokine Assay ...... 41

2.13 Statistical Analysis ...... 42

CHAPTER 3: THE OPPOSING EFFECTS OF COMPLEMENT C5A RECEPTORS, C5AR1 AND C5AR2, IN A MOUSE MODEL OF MELANOMA ...... 43

3.1 INTRODUCTION ...... 44

3.2 EXPERIMENTAL OUTLINE...... 46

3.3 RESULTS ...... 47

3.3.1 Evidence for complement activation in human malignant melanoma tissue ...... 47

3.3.2 C5aR1 antagonism inhibits melanoma growth ...... 48

3.3.3 Complement C5a receptor expression by B16 melanoma cells in vitro and in vivo 49

3.3.4 Functional evaluation of C5aR1 expressed by B16 melanoma ...... 51

3.3.5 B16 melanoma growth is altered in C5a receptor-deficient mice ...... 53

3.3.6 Effect of C5aR inhibition on tumour infiltrating leukocytes ...... 56

3.3.7 Effect of C5aR deficiency on cytokine/ expression ...... 66

3.4 DISCUSSION ...... 69

xiii

4 CHAPTER 4: THE COMPLEMENT C3A RECEPTOR CONTRIBUTES TO MELANOMA TUMOURIGENESIS BY INHIBITING NEUTROPHIL AND CD4+ RESPONSES ...... 75

4.1 INTRODUCTION ...... 76

4.2 EXPERIMENTAL OUTLINE...... 77

4.3 RESULTS ...... 79

4.3.1 C3a-C3aR signalling contributes to melanoma development and growth...... 79

4.3.2 Effect of combined C3aR/C5aR1 blockade on melanoma growth ...... 79

4.3.3 C3aR blockade inhibits the growth of established primary melanoma ...... 81

4.3.4 Melanoma cells express functional C3aR ...... 82

4.3.5 Tumour-infiltrating leukocyte populations are altered in C3aR deficient mice .. 84

4.3.6 C3aR antagonism alters tumour infiltrating leukocyte populations ...... 88

4.3.7 Neutrophil depletion rescues the tumour inhibitory effect of C3aR deficiency .. 91

4.3.8 Neutrophils are associated with human melanoma ...... 92

4.3.9 Influence of C3aR deficiency on cytokine/chemokine expression ...... 93

4.3.10 C3aR signalling contributes to the growth of other tumour types ...... 95

4.4 DISCUSSION ...... 96

5 CHAPTER 5: GENERAL DISCUSSION AND FUTURE PROSPECTS ...... 103

5.1 GENERAL DISCUSSION ...... 104

5.2 FUTURE PROSPECTS ...... 109

6 REFERENCES ...... 112

xiv

LIST OF FIGURES

Figure 1.1 The three phases of immunoediting: ...... 8

Figure 1.2. The Complement Cascade ...... 23

Figure 3.1. Immunohistochemical staining of human melanoma tissue array ...... 47

Figure 3.2. C5aR1 antagonism inhibits melanoma growth...... 48

Figure 3.3. expression by mouse melanoma B16 cell lines...... 49

Figure 3.4. Expression of C5aR1 byB16 melanoma cells...... 50

Figure 3.5. C5aR1 expression by B16 melanoma tissue...... 51

Figure 3.7. Tumour development is retarded in C5aR1-deficient mice...... 53

Figure 3.8. Tumour growth is enhanced in C5aR2-deficient mice...... 54

Figure 3.9. Effect of C5aR1 antagonism on growth of established B16 tumours in wild type and C5aR1 deficient mice...... 55

Figure 3.10. Leukocyte infiltration of B16 melanoma tissue...... 56

Figure 3.11. C5aR signalling influences tumour infiltrating leukocyte populations...... 58

Figure 3.12. Flow cytometric analysis of tumour infiltrating leukocyte populations from C5aR1- and C5aR2- deficient mice...... 59

Figure 3.13. Flow cytometric analysis of blood leukocyte populations from mice with and without tumours and comparison with tumour infiltrating populations...... 62

Figure 3.14. Flow cytometric analysis of draining leukocyte populations from mice with and without tumours compared with tumour infiltrating populations...... 64

Figure 3.15. Flow cytometric analysis of leukocyte populations in from mice with and without tumours compared with tumour infiltrating populations...... 66

xv

Figure 3.16. Influence of C5aR deficiency on cytokine and chemokine expression...... 68

Figure 4.1. C3aR deficiency retards B16 melanoma growth...... 80

Figure 4.2. C3aR blockade inhibits the growth of established primary melanoma...... 81

Figure 4.3. B16 melanoma cells express functional C3aR...... 83

Figure 4.4. Leukocyte infiltration of B16 melanomas from C3aR-deficient and wild-type mice...... 84

Figure 4.5. Tumour-infiltrating leukocyte populations are altered in C3aR deficient mice...... 87

Figure 4.6. Leukocyte populations in healthy C3aR deficient and wild-type mice...... 88

Figure 4.7. C3aR antagonism alters tumour infiltrating leukocyte populations...... 90

Figure 4.8. The tumour inhibitory effect of C3aR deficiency is rescued by neutrophil depletion...... 92

Figure 4.9. Influence of C3aR deficiency on cytokine expression...... 93

Figure 4.10. C3aR deficiency influences chemokine expression...... 94

Figure 4.11 C3aR signalling contributes to tumour growth in other tumour models...... 95

xvi

LIST OF TABLES

Table 1.1: Stages of Melanoma………………………………………………………...4 Table 2.1: Primary used for immunostaining……………………………...7 Table 2.2: Secondary antibodies used for immunostaining……………………………7 Table 2.3: Antibodies used for FACS analysis………………………………………..10

xvii

LIST OF ABBREVIATIONS

ANOVA Analysis of variance APC presenting cell ATCC American Type Culture Collection ATM Ataxia Telangiectasia Mutated Bursa of fabricius-derived cell (B lymphocyte) bFGF Basic fibroblast growth factor BRCA1 Breast cancer 1, early onset BRCA2 Breast cancer 2, early onset BSA Bovine serum albumin C1q Subcomponent of complement 1 C3 C3a Complement component 3a C3aR Complement component 3a receptor Complement component 3b C3d Complement component 3d C4d Complement component 4d C5 C5a C5aR Complement component 5a receptor C5L2 Complement component 5a-like receptor 2 Ca2+ Calcium ion CCR5 type 5 CD Cluster of Differentiation CD3+ T lymphocytes expressing CD3 surface antigen CD4+ T lymphocytes expressing CD4 surface antigen CD8+ Cytotoxic T lymphocytes expressing CD8 surface antigen CDH1 Cadherin-1 CHEK2 Checkpoint Kinase 2 ChemR23 Receptor 23 CO2 Carbon dioxide CRP Complement-reactive

xviii

CTL Cytotoxic T lymphocyte CXCR1 Chemokine receptor specific for stromal derived factor 1 CXCR2 Chemokine receptor specific for stromal derived factor 2 DAF Decay accelerating factor DC DMEM Dulbecco’s Modified Eagle Medium DNA Deoxyribonucleic acid DTT Dithiothreitol EDTA Ethylenediamninetetraacetic acid ER Estrogen receptor ERK Extracellular signal-regulated kinases FACS Fluorescence-Activated Cell Sorting FCS Foetal calf serum Fcγ Fragment, crystallisable fMLP Formyl peptide FOXP3 Forkhead box P3 GPCR G protein-coupled receptor HBSS Hank's balanced salt solution HSPC Hematopoietic stem/progenitor cell IgG IgM Immunoglobulin M IL Interleukin KHCO3 Potassium bicarbonate Ki-67 Protein that in humans is encoded by the MKI67 LKB1 Liver Kinase B1 mAbs Monoclonal antibodies MAC Membrane attack complexv MAPK Mitogen-activated protein kinases MASPs Membrane-binding lectin associated serine proteases MBL Mannan-binding lectin MCP Membrane cofactor protein M-CSF colony stimulating factor

xix

MDSC Myeloid derived suppressor cell MMPs Matrix metalloproteinases mRNA Messenger RNA NaCl Sodium chloride NaF Sodium fluoride Na3VO4 Sodium orthovanadate NK NH4Cl Ammonium chloride NP-40 Nonyl phenoxypolyethoxylethanol-40 OCT Optimal cutting temperature PALB2 Partner and Localizer of BRCA2 PAMPs Pathogen-associated molecular patterns PBA Phosphate buffered saline plus sodium azide PBS Phosphate buffered saline PBS-T Phosphate buffered saline plus Tween 20 PDGF Platelet-derived growth factor PFA Paraformaldehyde PMSF Phenylmethanesulphonyl fluoride PR Progesterone receptor RIPA Radioimmunoprecipitation RNA Ribonucleic acid RNS Reactive nitrogen species ROS Reactive oxygen species rt-PCR Real time polymerase chain reaction s.c. Subcutaneous SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SEM Standard error of mean siRNA Small interfering RNA STK11 Serine/Threonine Kinase 11 TAM Tumour-associated macrophage T cell Thymus-derived cell (T lymphocyte) TGF-β Transforming growth factor beta

xx

Th1 Type 1 helper T cell Th2 Type 2 helper T cell TLR Toll-like receptor TN Triple negative TNF Tumour necrosis factor TNM Tumour size, Lymph node, Metastasis TP53 Tumour Protein Treg T regulatory cells VEGF Vascular endothelial growth factor

xxi

xxii

CHAPTER 1

INTRODUCTION

Jamileh Alham Nabizadeh

1

1.1 GENERAL INTRODUCTION

Melanoma is the most dangerous type of skin cancer. It accounts for less than 5% of skin cancer cases, but is responsible for the majority of skin cancer deaths (Kuphal and Bosserhoff, 2009). Australia has the highest rate of melanoma in the world, and in the last 30 years, the incidence has doubled (Garbe and Leiter, 2009). Melanoma is able to spread or metastasise to skin, subcutaneous tissues, distant lymph nodes, lung, liver and brain (Silberman, 1987). There are several options available for treating melanoma, including surgery, immunotherapy (Alexandrescu et al., 2010), radiation therapy and chemotherapy, depending on the extent of the disease (Naylor et al., 2006). However despite recent improvements in therapy, advanced melanoma remains poorly responsive to systemic therapy (Hegde et al., 2011), with a 3 year survival rate of less than 15% for metastatic disease (Eggermont et al., 2014). While numerous studies have investigated immunotherapeutic approaches (Weber, 2011; Yao et al., 2011), to date these have met with limited success, extending survival rate by only a few months (Leach et al., 1996; Pandolfi et al., 2008; Pardoll, 2012). The present project investigates the role of inflammatory mediators, the complement components C3a and C5a, in melanoma growth.

1.2 MELANOMA

Melanoma is a malignant tumour of melanocytes, and is the most serious type of skin cancer. Melanocytes are pigment-producing (melanin) cells responsible for skin colour and protecting the deeper layer of the skin from harmful ultraviolet rays (Noonan et al., 2001). Melanocytes originate from multipotent melanocyte stem cells (MSCs) in the neural crest through the process of melanogenesis (Erickson and Reedy, 1998). During embryonic development, multipotent glial-melanocyte progenitors are generated in the neural crest, and through Wnt signalling produce melanoblasts that develop into melanocytes (Dupin et al., 2000; Ikeya et al., 1997).

1.2.1 Epidemiology Melanoma accounts for less than 5% of skin cancer cases, but is responsible for the majority of skin cancer deaths (Kuphal and Bosserhoff, 2009). According to the World Health Organisation (WHO) the world wide incidence of melanoma is increasing at a faster rate than any other cancer type with approximately 53,000 deaths annually (World Health Organisation 2014) Queensland has the highest rate of melanoma in the world, and in the last 30 years, the

2

incidence has almost doubled in Australia, from 26.7 per 100,000 in 1982 to 48.8 per 100,000 in 2014 (Australian Institute of Health and Welfare 2014). Melanoma is able to metastasise to skin, subcutaneous tissues, distant lymph nodes, lung, liver and brain (Silberman, 1987). Melanoma arises as the result of interactions between environmental, genetic and host factors.

1.2.2 Environmental factors Prolonged exposure to UV rays causes melanocytes to grow abnormally and (in some cases) develop into cancerous cells (melanoma). UV radiation from the sun causes sunburn, skin damage and sometimes cancer. There are three types of ultraviolet rays: UV-A, UV-B and UV-C. UV-A is the most common and most damaging form of radiation, since it is constant throughout the seasons and in contrast to other rays, penetrates the skin layers more deeply, damaging skin connective tissue (Cascinelli, 1989). Exposure to UV-A can cause premature ageing, sunburn and increased cancer risk. UV-B rays are stronger than UV-A but they are more intense during summer, at high altitudes and closer to the equator. Most UV-B rays are absorbed by the ozone layer around the earth, and therefore they are less of a concern. However, the remaining UV-B that reaches the earth also causes ageing, sunburn and loss of skin elasticity. Finally the most dangerous and strongest rays are UV-C, but these are usually filtered out by the ozone layer and generally do not reach the earth (Sugiyama et al., 1985).

1.2.3 Genetic mutations The understanding of molecular pathways and mutations from which melanomas originate are keys to histopathologic diagnosis and targeted mutation-specific treatments. Large scale sequencing of components of the ERK pathway revealed a high frequency mutation in BRAF as the most common genetic lesion in human melanoma (Davies et al., 2002), with 44% of melanomas containing mutations in BRAF, 21% NRAS, 2% KRAS and 1% HRAS (Curtin et al., 2006). The consequence of these mutations is constitutive activation of ERK in melanoma cells. Over 90% of BRAF mutations occurring in melanoma are due to a glutamic acid for valine substitution at position 600 (V600E) which results in a 500-fold increase in BRAF activation (Davies et al., 2002). c-KIT (CD117) is a which in humans is encoded by the KIT gene and upon binding to its stem cell ligand induces downstream signalling pathways regulating cell growth and differentiation (Andre et al., 1997). Point mutations along the KIT gene (in

3

exons 11, 13 and 17) have been found to occur in 20% of acral, mucosal and melanoma patients (Beadling et al., 2008; Curtin et al., 2006; Woodman et al., 2009).

The Cancer Genome Atlas (TCGA) project, begun in 2005, uses a bioinformatics approach to catalogue genetic mutations responsible for cancer. TCGA examination of genomic alterations in primary and/or metastatic melanoma from 331 patients has confirmed four genomic subtypes; BRAF, RAS, NF1 and Triple-WT with considerable expression of immune infiltration markers among the subtypes which is correlated with overall patients survival and potential application for immunotherapy (Cancer Genome Atlas, 2015). This study further illustrated that 76% of primary and 84% of metastatic melanoma samples had a UV signature (ie a high level of C>T and CC>TT transitions). Interestingly samples enriched for associated with lymphocyte infiltration were linked to improved patient survival. Other studies have an mTOR point mutation in the P13K-AKT-mTOR signalling pathway which is harboured by 6.1 % of melanomas (Grabiner et al., 2014). Upregulation and/or variation in the expression of the DNA cytosine deaminase APOBEC3B (an enzyme that provides innate immunity against DNA-based parasitic elements) has also been reported in melanoma (Burns et al., 2013b). Together these findings broaden our understanding of the genomic landscape of cancers and have implications for cancer diagnosis and targeted therapy.

4

1.2.4 Staging of melanoma Based on analysis of data from 17,600 patients, the Melanoma Staging Database was used by The American Joint Committee on Cancer (AJCC) to identify a number of factors that determine melanoma prognosis and staging, such as presence or absence of tumour ulceration, mitotic/proliferation rate, tumour burden and thickness of the melanoma (Balch et al., 2009). These are summarised in Table 1.1.

Table 1.1 Stages of Melanoma Melanoma Description Survival Percentage stage Time survival Stage I Localised melanoma, no distant 10 years 97% metastasis, thickness <1.5 mm IA 93% IB Stage II Localised melanoma IIA Thickness 1.5-4.0mm IIB Thickness >4.0 mm IIC 10 years 39% Stage III Regional metastatic melanoma 5 years 39% IIIA 5 years 78% IIIB 5 years 59% IIIC 5 years 40% Stage IV Distant metastatic melanoma M1a Metastasis in the skin and distant lymph 1 year 62% nodes and normal LDH level. Good prognosis M1b Metastasis to the skin and lung with 1 year 53% normal LDH level. Intermediate prognosis M1c Metastasis to other locations with elevated 1 year 33% LDH level. Poor prognosis

5

1.2.5 Treatment The most effective treatment option for melanoma is surgical intervention, but only for early stage disease which has a greater than 90% 10 year survival rate. However, as the disease progresses, there is an increased risk of recurrence and the survival rate drops significantly. Treatment for metastatic melanoma remains poor with a 5 year survival rate of less than 15% (Balch et al., 2001; Hegde et al., 2011). Despite numerous attempts at immunotherapy for melanoma (Weber, 2011; Yao et al., 2011), these have been largely unsuccessful (Pandolfi et al., 2008). However recent clinical trials have demonstrated potent clinical efficacy for immunotherapeutic antibodies which inhibit immune checkpoints for T cell activation, cytotoxic T-lymphocyte-associated protein 4 (CTL-4; Hodi et al., 2010; Robert et al., 2011) and programmed cell death (PD)-1 (Brahmer et al., 2012; Leach et al., 1996; Topalian et al., 2012a; Topalian et al., 2012b). Pembrolizumab and nivolumab block programmed cell death (PD-1) receptor from binding to its ligand PD- (Brahmer et al., 2012; Hamid et al., 2013; Robert et al., 2014; Robert et al., 2011). However the therapeutic benefits are short-lived, and increase overall survival by only a few months (Sanderson et al., 2005; Yang et al., 2005).

The identification of targetable somatic mutations in genes such as BRAF (McArthur and Ribas, 2013) and NRAS (Jakob et al., 2012) has led to promising oncogene-targeted therapeutic strategies which improve survival of patients with advanced melanoma. The BRAF inhibitor antibodies interrupt signalling through the MAPK pathway, altering cell proliferation and survival and inducing apoptosis of melanoma cells. Clinical studies have reported that anti-BRAF antibodies vemurafenib (formerly PLX4032) and debrafenib (formerly GSK2118436) induce a high frequency of tumour regression among metastatic melanoma patients with BRAF V600E positive mutations, and lead to improved overall survival (Falchook et al., 2012; Flaherty et al., 2010; Sosman et al., 2012). However, in the majority of patients disease progression is resumed after the initial BRAF inhibitor-based therapy due to the tumour rapidly developing resistance to these antibodies. This occurs because of reactivation of the MAPK pathway, up-regulation of the ser/thr MAP kinase kinase kinase (MAP3K8) (Johannessen et al., 2010; Nazarian et al., 2010; Poulikakos et al., 2011; Shi et al., 2012a; Shi et al., 2012b; Wagle et al., 2011) or activation of alternate survival pathways (Nazarian et al., 2010; Straussman et al., 2012; Villanueva et al., 2010).

The discovery of genetic aberrations in the c-KIT gene in some melanoma patients has identified it as an additional therapeutic target for metastatic melanoma. Imatinib is an oral

6

tyrosine kinase inhibitor that has demonstrated increased survival in patients harbouring c- KIT mutation or amplification (Carvajal et al., 2011; Guo et al., 2011). Dasatinib, another tyrosine kinase inhibitor, has been observed to inhibit the proliferation of melanoma cell lines (Woodman et al., 2009) and improve response rates in melanoma patients (Kalinsky et al., 2012). Ongoing clinical trials are investigating the potential of dasatinib as an individual therapy or in combination with immunotherapy.

Further understanding of the role of the immune system in tumour development and growth, and the interaction with other clinical strategies, will lead to improved treatments for melanoma and other cancers.

1.3 IMMUNE SURVEILLANCE

1.3.1 The role of the immune response in tumour development and growth There is overwhelming evidence that the immune system is capable of recognising tumours as ‘altered self’. However the role of the immune system in tumour growth has long been debated, with evidence for both anti-tumour and tumour-promoting effects (Dunn et al., 2002). The concept of tumour ‘immune surveillance’ refers to recognition and, in most cases, destruction of cancer precursors before tumours can develop (Burnet, 1970).

The theory that the immune system is capable of recognising and eliminating cancer cells was first proposed by Ehrlich in 1909. However, evidence that the immune system can be induced to mount an effective anti-tumour response was provided even before this by Coley (1891) who demonstrated that administration of bacterial toxins induced a successful anti-tumour immune response to sarcomas. This idea is supported by studies showing that immunodeficient mice have increased tumour incidence compared to wild-type mice (Street et al., 2001; Stutman, 1975; Trainin et al., 1967), and similarly that immunosuppressed patients have a higher incidence of some tumour types than healthy controls (Koebel et al., 2007). Historically, the presence of leukocytes in and around tumour tissues was thought to indicate an attempt by the host to eradicate the tumour, and the infiltration of pro- inflammatory T-lymphocytes has been associated with an improved prognosis in a range of cancers, including ovarian, colorectal, and breast cancers (Kryczek et al., 2009; Prestwich et al., 2008).

7

Figure 1.1 The three phases of immunoediting: ‘elimination’, ‘equilibrium’ and ‘escape’ (Chikamatsu*, 2013). 1. Immune surveillance leads to successful elimination of the cancer; 2. tumor cells that survive the immune surveillance process are in equilibrium with the host immune system; 3. tumor cells escape detection and elimination by the host immune system.

Despite the evidence that the immune system can recognise tumour cells as foreign and mount a response against them, these defence mechanisms sometimes fail. There are a number of ways that tumours are thought to evade detection by the immune system. For example, tumours can either reduce or modify their expression of tumour-associated , so that immune recognition is reduced (Bai et al., 2003). Moreover, most tumour antigens are poorly immunogenic and elicit weak immune responses, thus preventing an effective anti- tumour response (Whiteside, 2008). Tumour cells produce a variety of soluble factors that suppress the immune response against them (Janke et al., 2000). These include immunoinhibitory cytokines such as transforming growth factor (TGF)- and interleukin

(IL)-10 (Prestwich et al., 2008; Quatromoni and Eruslanov, 2012) which𝛽𝛽 inhibit effective immune responses or induce tumour-promoting responses in cancer patients (Janke et al., 2000; Whiteside, 2008). Other soluble tumour products such as chemokines recruit

8

immunosuppressive inflammatory cells to inhibit the development of an effective adaptive immune response (Shields et al., 2010). This includes tumour-associated (TAM) or myeloid-derived suppressive cells (MDSC) and their secreted cytokines IL-6, tumour necrosis factor (TNF) and IL-1β, all of which have been shown to promote tumour development (Gabrilovich and Nagaraj, 2009; Movahedi et al., 2010). CD4+ T lymphocyte subsets (particularly T helper (Th17) and CD25+ Foxp3+ regulatory (Treg) lymphocytes) have also been associated with poor prognosis and decreased survival of patients with breast and renal carcinomas (DeNardo et al., 2008; Ruffell et al., 2010; Siddiqui et al., 2007).

The ‘immune surveillance’ theory was formulated by Burnet (1970) who proposed that immune cells identify antigens expressed by tumour cells, and then eliminate them before a detectable tumour develops. More recently this theory has been updated as the ‘immunoediting’ hypothesis which describes three phases: 1) ‘immunosurveillance’ which involves active eradication of immunogenic tumour cells; 2) an ‘equilibrium’ phase which occurs if the tumour is not completely eliminated but is prevented from unrestrained growth; 3) an ‘escape’ phase in which tumour cell variants are selected that avoid or suppress the immune system (Bui and Schreiber, 2007; Swann and Smyth, 2007) (Figure 1).

1.4 OVERVIEW OF THE IMMUNE SYSTEM

The immune system can be divided into innate and adaptive (or acquired) responses that are mediated by distinct cell types (Roitt and Delves, 2001). Together these cells defend the host against invading pathogens and damaged cells by recognising and eliminating them, and restoring tissue homeostasis.

1.4.1 The innate immune system The innate immune system is the first line of defence, responding rapidly to infection or tissue injury. It provides an immediate response to injurious stimuli but does not confer memory or lasting protective immunity to the host. It is mediated by a number of cell types including macrophages, mast cells, dendritic cells, natural killer (NK) cells, and (polymorphonuclear neutrophils, basophils and eosinophils) which are recruited to the site of infection (Roitt and Delves, 2001).

Innate immune cells such as macrophages and mast cells continuously patrol their microenvironment for distress signals and are pivotal in initiating inflammation. Upon detecting distress signals, these cells immediately release soluble mediators such as

9

chemokines, cytokines and reactive oxygen species which activate other immune cells and attract them to the site of injury (Whiteside, 1999). This includes phagocytic cells which engulf pathogens and infected or injured cells. Professional include neutrophils, monocytes, macrophages, mast cells and dendritic cells, all of which express receptors such as scavenger receptors, toll-like receptors (TLRs) and pattern recognition (PRRs) capable of recognising foreign objects, damaged cells or danger signals not found in healthy tissues (Erwig and Henson, 2008; Hajishengallis and Lambris, 2011).

1.4.1.1 Neutrophils Neutrophils, also called polymorphonuclear neutrophils (PMNs), constitute the majority of circulating leukocytes (10-25% in mice and 50-70% in humans) (Doeing et al., 2003; Mestas and Hughes, 2004). Daily production of neutrophils can reach up to 2x1011 cells (Borregaard, 2010). Circulating neutrophils have a short half-life of approximately 12.5 hours and 5.4 days in mice and humans, respectively under steady state conditions (Galli et al., 2011; Pillay et al., 2010). However, during inflammation, the survival of activated neutrophils increases by several fold, ensuring the recruitment of neutrophils to the site of injury/inflammation (Colotta et al., 1992; Kim et al., 2011; Summers et al., 2010).

Neutrophils develop in the bone marrow from myeloid precursors via a process called granulopoiesis. The principal regulator of this process is colony stimulating factor (G-CSF; CSF-3) whose effects include progenitor cell commitment to the myeloid lineage, proliferation of granulocytic precursors, and release of mature cells from the bone marrow (Summers et al., 2010). G-CSF is produced in response to the pro-inflammatory cytokine IL-17A which is synthesized by specialized T lymphocytes (Th17) and released in response to IL-23 produced by tissue macrophages and dendritic cells (DC) (Ley et al., 2006). The bone marrow serves as a reservoir for neutrophils, such that less than 2% of mature neutrophils are in the circulation under basal conditions (Semerad et al., 2002). Neutrophil retention and release from the bone marrow is orchestrated by chemokines: stromal derived factor-1 (SDF-1; CXCL12) binding to its CXC chemokine receptor 4 (CXCR4) results in bone marrow neutrophil retention whereas neutrophil release is mediated by CXCR2 produced by bone marrow stromal cells (Eash et al., 2010; Martin et al., 2003).

Upon appropriate stimulation, circulating neutrophils adhere via specific receptors to ligands expressed by endothelial cells lining the blood vessel walls, and migrate between these cells.

10

Once within the tissue, neutrophils are rapidly recruited by chemokines such as IL-8 (CXCL8 in humans, CXCL1/KC in mice) to the site of inflammation, where they eradicate invading pathogens via phagocytosis, release preformed granular enzymes and , and produce reactive oxygen species. Upon resolution of the inflammatory stimulus, neutrophils undergo apoptosis and are cleared by macrophages; this in turn leads to down-regulation of IL-23 and reduction of G-CSF release (Borregaard, 2010; Christopher and Link, 2007; Stark et al., 2005). Senescent neutrophils have also been shown to home back to the bone marrow (Casanova-Acebes et al., 2013).

For decades, neutrophils have been considered the first responders in acute inflammatory conditions such as lung injury, and are indispensable for protecting from bacterial and fungal pathogens (Grommes and Soehnlein, 2011; Kolaczkowska and Kubes, 2013; Moraes et al., 2006; Williams and Chambers, 2014). However, there is compelling evidence to suggest that neutrophils are more complex cells capable of a vast array of specialised functions. The sustained influx of neutrophils has been observed in inflammatory conditions such as cardiovascular disease, rheumatoid arthritis and chronic obstructive pulmonary disease. Their role in chronic diseases and cancers has just begun to be recognised. The recruitment of neutrophils into the inflammatory site is mediated by chemokines, complement fragment C5a, platelet activating factor and lipid mediators. Upon activation, neutrophils release a range of chemokines as well as toxic contents, such as reactive oxygen species (ROS) and the matrix metalloproteinase (MMP)-9 which contribute to tissue damage and remodelling by degrading extracellular matrix components (Christoffersson et al., 2012). MMP-9 also regulates leukocytosis (Opdenakker et al., 1998) and activates bound growth factors such as vascular endothelial growth factor-A (VEGF-A), a key mediator of angiogenesis (Van den Steen et al., 2002).

Neutrophils contribute to the regulation of inflammatory responses through production of large variety of cytokines and chemokines. Depending on the stimulatory factors, neutrophils can synthesise and release cytokines such as IL-1ß, TNFα, IL-6 and IL-12 (Dorhoi et al., 2013). They also release a range of chemokines for neutrophils, monocytes, NK cells, DCs, helper T (Th1) and Th17 cells (Sadik et al., 2011), including IL-8/CXCL8, KC/CXCL1 (Cassatella et al., 1992; Cassatella et al., 1995; Hachicha et al., 1995; Koch et al., 1995), macrophage inflammatory protein 2 (MIP-2α/CXCL2) (Huang et al., 1992; Xing et al., 1994), MIP-1α/CCL3 and MIP-1ß/CCL4 (Lloyd and Oppenheim, 1992). Expression of these

11

cytokines/chemokines is triggered by activation of cytokine receptors, G protein coupled receptors such as complement receptors and pattern recognition receptors (PRR) such as TLR (Benelli et al., 2002; Cassatella, 1999). Although recent findings have expanded the repertoire of neutrophil effector functions during acute inflammation, their role in cancer remains uncertain (Brandau et al., 2013). Neutrophils have been shown to promote tumour progression, angiogenesis and metastatic seeding via the production of chemokines, cytokines, reactive oxygen species and matrix remodelling enzymes; they also contribute to MDSC populations which inhibit effector T cell responses (Gregory and Houghton, 2011). However, neutrophils can also possess anti-tumour properties (Souto et al., 2011) and are able to kill tumour cells directly or via regulation of adaptive immune responses (Eruslanov et al., 2014).

1.4.1.2 Monocytes Monocytes originate from myelo-monocytic stem cells in primary lymphoid organs such as foetal liver and bone marrow (Robbins et al., 2012) and represent 4% and 10% respectively of total leukocytes. Monocytes extravasate through the endothelial lining of blood vessels to seed tissues such as and lung where they differentiate into macrophages or dendritic cells (Geissmann, 2010; van Furth and Cohn, 1968). In mice, splenic production of monocytes can be induced under inflammatory conditions (Fogg et al., 2006) and their survival, proliferation and differentiation completely depend on colony-stimulating factor 1 (M-CSF; CSF-1) whose effects are mediated via the CSF-1 receptor (CSF-1R) (Cecchini et al., 1994; Dai et al., 2002). In humans, circulating blood monocytes can be divided into two main subsets: classical CD14+ and non-classical CD14low CD16+ monocytes (Passlick et al., 1989). In response to a broad range of microbial cues, CD14+ monocytes secrete inflammatory cytokines and reactive oxygen species and phagocytose foreign particles. CD16+ monocytes are responsible for tissue surveillance and secrete TNF-α, IL-1ß and CCL3 (Cros et al., 2010). In mice, subsets equivalent to human monocytes are Ly6Chi and Ly6Clow respectively (Cros et al., 2010; Ingersoll et al., 2010). In response to injury, Ly6Chi cells are recruited from the circulation to the site of inflammation where they differentiate into mononuclear phagocytes.

12

1.4.1.3 Macrophages Macrophages are a heterogeneous cell population, with different names according to their anatomical location and activation status (Gordon and Taylor, 2005). They are distributed in all tissues throughout the body and play a critical role in host defence, inflammation and tissue remodelling by producing cytokines and phagocytosing foreign particles and cell debris. Macrophages recognize pathogens such as bacteria, fungi and viruses through a variety of cell surface and cytoplasmic receptors, including TLRs (Xu et al., 2007), NOD like receptors, RIG-I like receptor (RLRs), C-type lectin and scavenger receptors (Drickamer and Fadden, 2002; Janeway and Medzhitov, 2002; Zelensky and Gready, 2005). Upon binding of these receptors to pathogen, macrophages activate transcription factors such as signal transducer and activator of transcription (STATs), interferon-regulatory factor (IRFs), nuclear factor (NF)-κB, activator protein (AP) 1, peroxisome proliferator-activated receptor (PPAR)- γ and cAMP-responsive element-binding protein (CREB), which interact with each other to induce expression of a wide range of cytokines including IL-1α and ß, IL-18, IL12, IL-6, IL- 23, α and ß interferons (IFN), IL-4, IL-10 and TGF-ß (Aderem and Ulevitch, 2000; Bopst et al., 1998; Elenkov and Chrousos, 2002; Lawrence and Natoli, 2011; Liu et al., 2014). Although macrophages have the flexibility to switch their phenotype in response to micro- environmental signals (Hagemann et al., 2008; Kawanishi et al., 2010; Mylonas et al., 2009; Rutschman et al., 2001), several macrophage subsets have been described based on specific profiles (Cros et al., 2010). Classically activated (M1) macrophages are induced by Th1 cytokines (IFNγ, TNFα) and granulocyte monocyte-colony stimulating factor (GM-CSF; CSF-2) to express pro-inflammatory mediators such as TNF-α, IL-1, IL-6, as well as reactive nitrogen and oxygen intermediates (Mantovani et al., 2007; Mills et al., 2000; Mosser and Edwards, 2008). Alternatively activated (M2) macrophages are induced following exposure to Th2 cytokines (IL-4 or IL-13); these cells are characterised by expression of arginase1 (Arg1), chitinase 3-like 3 (Ym1), IL-10 and Mrc1 (CD206), and play a role in parasite infestation, tissue remodelling and tumour progression (Gordon and Martinez, 2010).

Although all macrophages were originally thought to differentiate from monocytes, recent studies have shown that most tissue macrophages do not originate and are maintained independently from monocytes (Davies and Taylor, 2015; Hashimoto et al., 2013; Liu et al., 2009; Schlitzer et al., 2013; Varol et al., 2009; Yona et al., 2013). Rather, adult tissue

13

macrophages are derived from embryonic precursors before birth, and are capable of self- renewal (Hashimoto et al., 2013; Yona et al., 2013).

1.4.1.4 Myeloid derived suppressor cells Myeloid derived suppressor cells (MDSC) are a heterogeneous group of myeloid progenitor and immature myeloid cells (IMC) which expand in states of inflammation and cancer to exert suppressive effects on host innate and adaptive immune responses (Gabrilovich and Nagaraj, 2009; Gallina et al., 2006; Marx, 2008). In healthy conditions IMCs primarily reside in bone marrow and differentiate into mature granulocytes, macrophages or dendritic cells (Sawanobori et al., 2008). In pathological conditions including cancer, autoimmune disease, trauma and , IMCs are prevented from full differentiation into mature cells. MDSC have up-regulated expression of arginase1 (which depletes arginine from the tumour microenvironment and impairs T cell signal transduction and function), as well as inducible nitric oxide synthase (iNOS) and nitric oxide (NO) (which limit the efficacy of immune surveillance and inhibit the development of a specific adaptive immune response to the tumour). These cells possess mixed morphologies of granulocytes and monocytes but lack the cell surface markers expressed by them (Ostrand-Rosenberg and Sinha, 2009; Youn et al., 2008). In addition, MDSCs promote tumour vascularization and angiogenesis by secreting high levels of soluble factors such as MMP-9 and VEGF (Balkwill et al., 2005; Torisu et al., 2000; Yang et al., 2004). In mice MDSCs are characterised by expression of Gr-1 (Ly6C/G) and CD11b (also known as αM-) (Kusmartsev et al., 2004), whereas in humans monocytic MDSC are characterized as HLA-DR-, CD11b+, CD33+ and CD14+ (CD11b + Ly6G-/Ly6C+ in mice) and granulocytic MDSC as HLA-DR-, CD11b+, CD33+, CD15+ (CD11b + Ly6G+/Ly6Clow in mice) (Wesolowski et al., 2013).

1.4.1.5 Dendritic cells Dendritic cells (DC) are a specialized, but heterogeneous group of cells that reside mainly in the skin, the inner lining of the nose, the lungs, the stomach and the intestines. DCs are bone marrow derived and, along with macrophages and monocytes, are part of the mononuclear system (Auffray et al., 2009; Fogg et al., 2006; Steinman and Cohn, 1973). They are the most powerful professional antigen presenting cells (APC). describes the process by which phagocytic cells (macrophages and dendritic cells) ‘present’ protein fragments of engulfed foreign materials on their surface for recognition by other cells

14

of the immune system (Steinman et al., 1974). DCs display antigens in the context of major histocompatibility complex (MHC) class I and II molecules, then migrate to the lymphoid tissues where they activate cytotoxic T cells as well as T helper cells to secrete cytokines to induce an immune response (Banchereau and Steinman, 1998; Hashimoto et al., 2011; Steinman, 2007). Although their phenotypic similarity to macrophages makes it difficult to evaluate their exact involvement in immune responses, they can be distinguished by their distinct ability to activate T cells in an antigen-dependent manner (Bogunovic et al., 2009; Ginhoux et al., 2009). The maturation and migration of DCs to draining lymph nodes occur in response to cytokines such as TNF-α that are produced during tissue injury and exposure to microbial products (Banchereau and Steinman, 1998; Grewal and Flavell, 1998). DCs have a major influence on Th1 and Th2 differentiation and tolerance induction (Moser and Murphy, 2000) through activation of STAT4 and secretion of IL-12, IL-18 and IL-23 (Th1 response) (Macatonia et al., 1995) or IL-4 and IL-10 (Th2 polarization and generation of regulatory T cells) (Iwasaki and Kelsall, 1999; Kelleher et al., 1999).

In the context of cancer, DCs play a key role in inducing and maintaining anti-tumour immunity, and hence prolong patient survival (Dieu-Nosjean et al., 2008; Iwamoto et al., 2003; Ladanyi et al., 2007; Nakakubo et al., 2003). However, within the tumour environment their antigen-presenting function may be lost or inefficient. They may also be polarized into immunosuppressive/tolerogenic regulatory DCs by tumour production of cytokines such as IL-10, TGF-ß, VEGF, M-CSF and E2 (PGE2) (Preynat-Seauve et al., 2006; Zou, 2005). This may limit the activity of effector T cells, and lead to enhanced tumour growth and progression (Ma et al., 2014), as has been reported in colorectal cancer (Chaux et al., 1997) and melanoma (Ataera et al., 2011).

1.4.1.6 Natural killer cells Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system, comprising 5-20% of human peripheral blood lymphocytes, and defined as CD3-CD56+ (Ferlazzo and Munz, 2004; Lanier et al., 1983). NK cells were first identified in 1975 as a distinct lymphocyte subset with the ability to kill tumour cells (Herberman et al., 1975a; Herberman et al., 1975b; Kiessling et al., 1975a; Kiessling et al., 1975b). NK cell activation and maturation can be triggered by IL-2, -12, -15, -21 and IFN-α/ß cytokines, thereby augmenting the cytolytic effect against tumour cells (Biron et al., 1999; Nutt et al., 2004; Smyth et al., 2002). Upon activation, cytotoxic NK exocytose granules containing perforin and other

15

enzymes that lead to perforation of virus-infected and tumour cells (Lieberman, 2003; Voskoboinik et al., 2006). NK cells exert their effector mechanisms through coordinate expression of cytokines and chemokines such as IFN-γ, TNF, GM-CSF, MIP-1α and RANTES (regulated upon activation, normal T cell expressed and secreted; CCL5) leading to enhanced depletion of the implanted tumour (Biron et al., 1999; Dorner et al., 2004). NK cell- derived IFN-γ is important for induction of the adaptive T cell response, hence these cells act as an interface between innate and adaptive immunity (Martin-Fontecha et al., 2004; Mocikat et al., 2003).

1.4.2 The adaptive immune system The adaptive immune system provides the ability to recognise and eliminate a specific pathogen or other foreign agent. It also provides long-term and specific immunity (‘immune memory’). It is comprised of highly specialised cells: -producing B lymphocytes which are responsible for ‘humoral’ responses and T lymphocytes (CD4+ and CD8+) which are responsible for ‘cell-mediated’ responses. Lymphocytes arise from haemopoietic progenitors in the bone marrow. The thymus is the primary lymphoid organ where T cell development and maturation take place, whereas foetal liver and postnatal bone marrow are the sites for B cell development. These cells then migrate to the secondary lymphoid organs (spleen, lymph node and mucosa-associated lymphoid tissues) where they differentiate into effector cells (von Andrian and Mackay, 2000) which eradicate altered cells or pathogens, either by secretion of antigen-specific immunoglobulins (B lymphocytes) or direct cell- mediated cytotoxicity (T lymphocytes) (Finch and Crimmins, 2004).

1.4.2.1 B lymphocytes B lymphocytes are antibody-secreting cells responsible for the humoral arm of adaptive immunity, and comprise 7-10% of white blood cells in the circulation. Immature B cells migrate to the spleen and other secondary lymphoid tissues where they mature and differentiate into immunocompetent B cells. Each B cell expresses a unique immunoglobulin on its surface (the B cell receptor) which is capable of recognising and binding a single specific antigen. Upon binding its cognate antigen, the B cell requires an additional signal from a T helper cell before it can further differentiate into either a plasma B cell capable of secreting large amounts of antibody or a long-lived memory B cell (Fuchs and Matzinger, 1992; Ho et al., 1994). B cells can be activated in either a T cell-independent or T cell- dependent manner upon stimulation by foreign antigens. In T cell dependent responses, the

16

B-cell antigen receptor delivers antigen to intracellular sites where it is degraded and returned to the B-cell surface as a peptide complexed with an MHC class II molecule; this complex is recognized by antigen-specific armed helper T cells, which in turn stimulate B cell proliferation and differentiation into antibody-secreting plasma cells (Parker, 1993). Conversely some microbial antigens can activate B cells directly via TLR engagement in the absence of T-cell help, thus providing a rapid response to important pathogens (Vos et al., 2000).

Mizoguchi and colleagues (2002) introduced a new B cell subset known as 'regulatory B cells' which are identified by their IL-10 production. These cells alter the Th1/Th2 balance, by inhibiting T cell proliferation and cytokine production (IFN-γ and TNF-α) by Th1 cells. They also promote infiltration of CD4+CD25+Foxp3+ Treg cells and suppress Th17 cell differentiation. Based on these findings, it is likely that Breg cells play an important role in T- cell plasticity. These cells maintain tolerance by suppressing CD4+ T cell proliferation and production of pro-inflammatory cytokines IFN-γ (Carter et al., 2012) and IL-17 (Carter et al., 2011), as well as promoting the infiltration of Treg cells (Liu et al., 2013). The role of B cells in cancer is complex and controversial, with evidence of both pro- and anti- tumour roles for B cells. For example, in the murine metastatic B16-F0 melanoma model, elimination of CD20+ expressing B cells increased lung tumour burden (Sorrentino et al., 2011) and optimal CD4+ and CD8+ anti-tumour responses have been shown to require B cells. Activated B cells (DiLillo et al., 2010) may also be highly cytotoxic towards tumour cells (Li et al., 2009; Sorrentino et al., 2011). Despite these findings, pro-tumour effects of B cells have also been reported. For example, CD4+ helper T cell-dependent anti-tumour immunity (Qin et al., 1998; Shah et al., 2005) and production of IFN- γ from CD8+ T cells and NK cells (Inoue et al., 2006) was enhanced in the absence of B cells, suggesting that B cell depletion may have therapeutic benefits.

1.4.2.2 T lymphocytes Bone marrow-derived T lymphocytes migrate to the thymus for differentiation and acquire unique cell surface antigen-binding receptor, the T cell receptor (TCR), a heterodimer consisting of two different protein chains, TCRα and TCRß. In contrast to B cells, T cells recognise antigenic peptides in complex with MHC molecules (Moss et al., 1992) on the surface of APCs. Mature T lymphocytes express either CD4 or CD8 molecules and hence form two major sub-populations with helper/regulatory and cytotoxic activities (Stockwin et

17

al., 2000; von Andrian and Mackay, 2000). CD8+ cytotoxic T cells are capable of killing infected cells and tumour cells, whereas CD4+ cells act as ‘helpers’, secreting cytokines to facilitate different types of immune responses. CD4+ cells can be further divided into Th1, Th2, Th17, Treg and follicular helper T subsets based on their distinct gene expression and regulation signatures. Activation of naive CD4+ cells is restricted to antigen presented by MHC II molecules on the surface of APCs such as dendritic cells (Stockwin et al., 2000). Specialised memory cells (either CD4+ or CD8+) maintain immune surveillance, so that on encountering antigen for a second time these cells initiate a more rapid and enhanced response (Roitt and Delves, 2001).

CD8+ cytotoxic T lymphocytes (CTL) are a key component of adaptive immunity, circulating throughout the body and seeking out intracellular pathogens. CTL recognize antigens in the context of MHC class I molecule (expressed on all nucleated cells) (Sykulev et al., 1996) and effectively eliminate intracellular pathogens by direct lysis of antigen-bearing infected or malignant cells (Gullo et al., 2008; Walsh et al., 2008).

CD8+ CTL activation, differentiation and proliferation require various cytokines including IL-2 (Frankenberger et al., 2005), IL-4 (Hiraoka et al., 2005), IL-7 (Bertagnolli and Herrmann, 1990), IL10 (Chen and Zlotnik, 1991), IL-12 (Pardoux et al., 1997), IL-15 (Kanai et al., 1996) and TNF-α (Kasahara et al., 2003). Among these, IL-2 has been shown to be a critical cytokine for inducing specific cytotoxicity (Gately et al., 1994; Ito et al., 2008; Leist et al., 1989; Lin et al., 2007). CTL play a well-defined role in restraining tumour development (Dunn et al., 2004).

CD4+ T lymphocytes are classified on the basis of their cytokine expression profiles (Zhou et al., 2009). To date, five subsets of CD4+ T cells have been identified: Th1, Th2, (Mosmann et al., 1986) and Th17 cells (Ansel et al., 2003; Harrington et al., 2005) that target specific classes of pathogens, regulatory T cells (Tregs) (Wing and Sakaguchi, 2010) that are required to maintain self-tolerance, and follicular helper T cells (TFH) that provide help to B cells for antibody production (Ma et al., 2012).

In response to infection with intracellular pathogens such as bacteria or viruses, specialized dendritic cells present antigen to uncommitted naïve T cells and, with the aid of cytokines IL- 12 and IL-2, induce the differentiation of CD4+ Th1 cells. Th1 cells are characterised by the production of cytokines such as IFN-γ, TNF-α and chemokines such as monocyte

18

chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1, and are thought to activate macrophages to destroy pathogens and enhance CD8+ CTL responses (Ley, 2014). Conversely, IL-4 induces naïve T cells to differentiate to CD4+ T Th2 cells which are characterised by production of IL-4, IL-5, IL-10 and IL-13 (Romagnani, 1991). Th2 cells are required to fight extracellular parasites, but are also involved in allergic inflammatory responses (Robinson et al., 1992). Their main effector cells are eosinophils, basophils, and mast cells as well as B cells, and IL-4/IL-5 CD4 T cells. The generation of Th1 and Th2 cells depends on transcription factors T-bet (Szabo et al., 2002) and GATA-3 (Zheng and Flavell, 1997) respectively, which not only determine expression of cytokines including IFN-γ (Th1) and IL-4 (Th2 cells), but also help to maintain their phenotype.

Both Th1 and Th2 cells have been reported to mediate anti-tumour immunity (Nishimura et al., 1999), although Th1 cells are thought to be more effective. Th1 cells recruit NK cells and macrophages to the tumour site to eradicate it. Production of IL-4 by Th2 cells may also exert anti-tumour effects via mobilisation of innate immune cells such as eosinophils (Tepper et al., 1989). Conversely, Th2 cells have been shown to induce proliferation and inhibit apoptosis of breast cancer cell lines in vitro, while secretion of Th1 (IFNγ) and Th2 (IL-4 and IL-13) cytokines has been reported to promote development of mammary carcinomas (Aspord et al., 2007; DeNardo et al., 2009). Consistent with these findings, Chin and co- workers prepared tumour infiltrating lymphocytes (TIL) from 30 human breast cancers and demonstrated a correlation between a high levels of CD4+ cells and clinical tumour progression and metastasis (Chin et al., 1992; Sharma et al., 2009; Vukmanovic-Stejic et al., 2006). Th2 cells have also been reported to promote neoplastic transformation and growth of pancreatic tumours (Ochi et al., 2012).

Human IL17 producing CD4+ T cells (Th 17) were first described in 1990s in the context of chronic conditions such as airway inflammation and rheumatoid arthritis (Kotake et al., 1999; Teunissen et al., 1998) but were only recognized as a discrete T helper cell subset in 2005 (Harrington et al., 2005; Park et al., 2005). Th17 cells develop in response to IL-6, IL-1 and TGFß, and express the defining transcription factor RORγt (Rorc) (Koenen et al., 2008; Voo et al., 2009). They are also induced through a STAT3-dependent mechanism by IL-9, IL-21 and IL-10 (Zielinski et al., 2012). Th17 cells play an important role in protecting against extracellular pathogens by maintaining the integrity of mucosal surfaces but are also involved in the pathogenesis of many autoimmune diseases (Bettelli et al., 2008; Ouyang et al., 2008;

19

Zielinski et al., 2012). Th17 cells mediate their biological effects through production of signature cytokines IL-17A, IL-17F and IL-21 whose receptors are broadly expressed on epithelial tissues (Acosta-Rodriguez et al., 2007; Dong, 2008; Zielinski et al., 2012). IL-17A induces the expression of chemokines including CCL2, CCL7, CXCL1 and CCL20, as well as matrix metalloproteases (Park et al., 2005). The functional contribution of Th17 to tumour immunity remains controversial, with evidence for both pro- and anti-tumour effects, depending on the tumour model and specific inflammatory conditions within the tumour microenvironment. Most data supporting an anti-tumour role is derived from murine models. For example, Martin-Orozco and colleagues demonstrated in a B16 melanoma model that IL- 17 depletion rendered mice more susceptible to lung metastasis while immunotherapy with Th17 cells induced a strong anti-tumour response mediated by tumour specific CD8+ T cells and DCs (Martin-Orozco et al., 2009b). In contrast, increased infiltration of Th17 cells in SCID mice bearing human non-small cell lung cancer (NSCLC) has been shown to promote angiogenesis and hence tumour growth (Numasaki et al., 2003; Numasaki et al., 2005). In humans also, a high percentage of CD4+ IL17-producing Th17 cells has been reported to correlate with poor prognosis in patients with prostate, colon, ovarian and hepatocellular tumours (Miyahara et al., 2008; Sfanos et al., 2008). .

Follicular helper T (TFH) cells are able to migrate into follicles of secondary lymphoid organs where they promote B cell differentiation (Basso et al., 2010; Klein and Dalla-Favera, 2008). These cells are dependent on expression of the transcription factor Bcl-6 (Yu et al., 2009), and identified by surface expression of CXCR5, inducible T cell co-stimulator (ICOS) and programmed cell death protein (PD)-1 (Breitfeld et al., 2000; Kim et al., 2001; Schaerli et al., 2000). Analysis of tumour infiltrating immune cells in human colorectal and breast cancers has shown a correlation between the presence of TFH and B cells and patient survival (Coppola et al., 2011; Gu-Trantien et al., 2013)

Induced regulatory T cells (iTregs) are immunosuppressive cells that maintain immunological tolerance to self-antigens (Oh and Li, 2013), and play an important role in protecting against autoimmune disease. iTregs develop in the thymus as a functionally mature T cell subset, but can also be induced in the periphery from naive T cells. The transcription factor Foxp3 is expressed by iTregs in response to IL-27 and IFNγ through a STAT1-dependent mechanism (Josefowicz et al., 2012; Sakaguchi et al., 2008).

20

Intra-tumour Tregs have been shown to inhibit effective immune responses against tumour cells, and high Treg:CD8 ratios in tumour tissue correlates with poor prognosis (Predina et al., 2013). There is also evidence that tumour-derived factors can promote the recruitment and proliferation of Tregs to the tumour site (Quezada et al., 2006), and elimination of Tregs has been shown to augment natural and pharmacological immunity (Betts et al., 2007; Tawara et al., 2002).

1.5 CHRONIC INFLAMMATION AND TUMOUR DEVELOPMENT

Rudolf Virchow first proposed in 1863 that chronic inflammation may lead to tumourigenesis, after he observed the presence of leukocytes in tumour tissues. This hypothesis is supported by numerous studies in a wide range of cancers (including melanoma, lung, breast, bladder, head and neck cancer) that demonstrate an association between chronic inflammation and increased risk of tumour development (Mastellos, 2009; Niculescu et al., 1992). Preceding this, Leibovici and Hoenig (1985) found that increased macrophage density was associated with tumour growth and low macrophage numbers with tumour destruction. Further evidence that chronic inflammation promotes tumourigenesis comes from clinical and experimental studies demonstrating that prolonged administration of anti-inflammatory drugs such as COX2 inhibitors leads to a reduced risk of cancer development (Howe et al., 2001). Chronic inflammation is thought to promote tumour onset through mechanisms such as production of ROS (including peroxynitrites) and pro-angiogenic factors including VEGF and matrix metalloproteases which degrade the surrounding extracellular matrix (Balkwill et al., 2005; Coussens and Werb, 2002; Philip et al., 2004; Stutman, 1975).. The close association between inflammation and cancer suggests that pro-inflammatory components of the complement system may have a role in development and/or progression of malignant tumours.

1.5.1 The complement system The complement system is an essential part of the innate immune system, regulating inflammation, facilitating immune defense mechanisms and maintaining tissue homeostasis (Ricklin et al., 2010). The complement system not only mediates innate immune responses to infectious organisms, damaged tissue and other foreign materials (Walport, 2001), but can also contribute to adaptive immunity (Dunkelberger and Song, 2010).

21

The complement system is an enzymatic cascade, comprising more than 30 highly regulated soluble proteins (C1 inhibitor, C4b binding protein, and I, clusterin and vitronectin; synthesised primarily in the liver) and membrane bound receptors (CD35, CD46, CD55 and CD59) (Alper et al., 1969; Bonifati and Kishore, 2007; Morgan, 1999). The complement cascade can be triggered via four pathways (Figure 2), depending on the stimulus: the classical, alternative, lectin and extrinsic protease pathways (Ricklin and Lambris, 2007). The classical pathway is activated via interaction of antigen-antibody complexes with the multimeric collectin C1q (Loveland and Cebon, 2008). The alternative pathway is triggered by the presence of pathogen and interaction with foreign antigens on its surface, and accounts for up to 80% of complement activation (Ricklin and Lambris, 2007). Binding of mannan- binding lectin (MBL) to carbohydrates on the pathogen surface activates the (Guo and Ward, 2005). Finally, the extrinsic pathway can be triggered directly by proteolytic enzymes such as thrombin and kallikrien, which results in cleavage of C3 and C5 respectively (Ricklin and Lambris, 2007). The activation of classical and lectin pathways causes cleavage of C4 into and C4b, of which C4b binds to C2, generating the classical/lectin C3 convertase (C4b2a complex) (Guo and Ward, 2005). The alternative pathway however, proceeds directly through C3 cleavage to generate an alternative pathway convertase (C3bBb). These convertases then lead to production of C3a and C3b which is a major that also forms a C5 convertase.

Formation of membrane attack complexes (MAC) is a mechanism used by the complement system to eliminate foreign or infected cells directly via cell lysis. Initiation of membrane attack complex formation occurs upon cleavage of C5 by the C5 convertases to produce C5b which combines with C6, C7, C8, and multiple units of C9 (Jurianz et al., 1999). Cleavage of C3 and C5 also results in the production of the potent anaphylatoxins C3a and C5a (described in more detail below).

Activation of the complement system is restricted by soluble and membrane bound regulatory proteins which protect both normal and neoplastic cells from damage. Membrane complement regulatory proteins (mCRPs) including CD35, CD46 and CD55 (complement receptor type-1; CR-1), membrane cofactor protein (MCP) and decay-accelerating factor (DAF) respectively regulate C3 activity. Membrane-bound protein, CD59, binds to C8 and C9 to prevent assembly of the MAC (Fishelson et al., 2003).

22

Figure 1.2. The Complement Cascade is activated via four pathways: the Classical, Alternative, Lectin and Extrinsic Pathways. Initiation of all four pathways leads to formation of C3 convertase, a key component in the cascade, which cleaves C3 to its active fragments, C3a and C3b. C3b aids recognition and clearance of foreign material by macrophages (opsonisation) and formation of the membrane attack complex (C5b-9) which destroys invading pathogens. Additional bioactive products are anaphylatoxins C3a and C5a. Serine proteases of the coagulation cascade (Extrinsic Pathway) can also generate C5a. Overall, complement activation elicits a range of pro-inflammatory effects including increased vascular permeability, recruitment of leukocytes to damaged tissues, enhanced phagocytosis and damage to pathogen cell membranes (opsonisation), and cell lysis through the MAC (Ruddy et al., 1972). The ability of complement to participate in host defense is not limited to these innate immune activities however, with complement effector systems also contributing to efficient adaptive immune responses (Baruah et al., 2009; Baudino et al., 2014; Cutler et al., 1998). Although the role of complement proteins in regulating T cell function is still not completely understood, the complement regulator DAF (CD55), which is expressed by most cells throughout the body, has been shown to have a role as a co- stimulator of human T cell proliferation (Capasso et al., 2006; Davis et al., 1988). C1q is

23

known to prevent autoimmune T cell responses (Lalli et al., 2008; Strainic et al., 2008a) while anaphylatoxins C3a and C5a promote T cell activation and proliferation (see Section 1.5.1.1), and thus promote allograft rejection, and responses to infection (Clarke and Tenner, 2014). However excessive complement activity, or deficiency of complement regulators, is known to contribute to a wide range of pathologic conditions including sepsis, asthma, rheumatoid arthritis, and acute respiratory distress syndrome (Guo and Ward, 2005).

Historically, the complement system has been thought to contribute to anti-tumour defence mechanisms via complement dependent cytotoxicity (Ostrand-Rosenberg, 2008) and antibody-dependent cell mediated cytotoxicity (Gelderman et al., 2004). Indeed cancer cells are thought to evade complement-mediated destruction by up-regulating endogenous complement inhibitors (Hauschild et al., 2012; Macor and Tedesco, 2007). However recent studies have implicated the anaphylatoxin C5a for a role in regulating tumour growth (discussed in Section 1.5.4).

1.5.2 Complement components C3a and C5a The complement components C3a and C5a are anaphylatoxins through which the complement system exerts many of its effects. Both C3a and C5a molecules are highly cationic with a core structure of 4 helix bundles stabilised by three disulphide bonds (Klos et al., 2009). C5a is a small peptide comprising 74 amino acids (el-Lati et al., 1994). It is one of the most potent inflammatory proteins known, exerting its activity at low nanomolar concentrations upon binding to specific receptors C5aR1 (CD88) and C5aR2 (C5L2) (Guo and Ward, 2005). C3a consists of 77 amino acids, and has 36% overall homology to the C5a sequence, with higher homology in the C-terminal “active” region of the molecule (Takabayashi et al., 1996). It exerts its main biological effects by binding to a single receptor, C3aR, although other receptors and functions for C3a have been identified (Coulthard and Woodruff, 2015).

All three receptors (C5aR1 and C5aR2 and C3aR) belong to the superfamily of G-protein- coupled receptors and are expressed by myeloid cells including monocytes, eosinophils, basophils and neutrophils (Gerard et al., 1989), as well as lymphocytes (Eden et al., 1973; Okada and Nishioka, 1973; Ross et al., 1973). They can also be expressed by non-myeloid cells, especially in lung and liver (Haviland et al., 1995; Klos et al., 2009; Schieferdecker et al., 2001).

24

C5a binds both of its receptors with high affinity although it exerts most of its described biological activity via binding to C5aR1 (Guo and Ward, 2005). C5aR2 lacks G-protein coupling, and thus was originally thought to be a “decoy” or scavenger receptors binding excess C5a without exerting direct physiological effects (Gao et al., 2005). However there is emerging evidence to suggest that C5aR2 can independently induce and moderate biological functions of C5a through β-arrestin signalling (Bamberg et al., 2010; Croker et al., 2014; Li et al., 2013).

C5a binding to C5aR1 activates signalling pathways such as calcium mobilisation via phospholipase Cβ, adenyl cyclase/cAMP production, PI3K/AKT and MAPK (Klos et al., 2013) to stimulate a range of pro-inflammatory responses. These include chemoattraction of macrophages, neutrophils, activated B and T cells, basophils and mast cells (Guo and Ward, 2005; Woodruff et al., 2011), enhanced phagocytosis (el-Lati et al., 1994) and stimulation of cytokine release. It triggers release from basophils and mast cells (Kretzschmar et al., 1993) which in turn stimulates vasodilation and increased vascular permeability (Ember et al., 1998), as well as production of the angiogenic growth factor, VEGF-A. It can also stimulate angiogenesis directly by promoting the migration of microvascular endothelial cells. C5a stimulation of neutrophil causes the release of toxic mediators and matrix metalloproteinases (Lacy, 2006; Martinelli et al., 2004). In addition, C5a links to the adaptive immune system, influencing the trafficking and migration of B-cell populations (Kupp et al., 1991; Ottonello et al., 1999) and modulating T-cell responses by providing survival signals for naïve CD4+ cells (Strainic et al., 2008b), inhibiting induction and function of Tregs (Strainic et al., 2013) and promoting T cell activation during interaction with APCs in vitro and in vivo (Cravedi et al., 2013). C5aR1 synergises with Toll-like receptor (TLR)-4 to elicit a stronger inflammatory response (Zhang et al., 2007) whereas C5aR1 antagonism has been shown to reduce the CD8 T cell response to influenza A in mice (Kim et al., 2004).

Binding of C3a to C3aR induces intracellular signal transduction pathways, including calcium mobilisation, ERK and AKT (Venkatesha et al., 2005). Compared to C5a, C3a is a much weaker chemoattractant (Fernandez et al., 1978) but has a range of immunomodulatory functions including degranulation of eosinophils, basophils and mast cells (el-Lati et al., 1994; Takafuji et al., 1994) and stimulation of cytokine expression by monocytes/macrophages (Norgauer et al., 1993; Zhang et al., 2007). C3aR is also a key mediator of insulin resistance and functions by modulating macrophage infiltration and

25

activation in adipose tissue (Mamane et al., 2009). It has also been reported to negatively regulate the mobilisation of haematopoietic stem and progenitor cells from the bone marrow (Ratajczak et al., 2004; Reca et al., 2003) while Wu et al (2013) showed that C3aR prevents neutrophil egress into the circulation, thus reducing acute tissue injury after ischemia (Wu et al., 2013). Like C5a, C3a signalling also contributes to the regulation of adaptive immunity. B cells have been reported to express C3aR, and C3a has been shown to negatively regulate antibody responses (Fischer and Hugli, 1997; Morgan et al., 1982). Moreover C3a has been reported to promote T cell activation, with C3aR required for CD4+ and CD8+ T cell responses to Chlamydia psittaci infections (Dutow et al., 2014). C3a may also affect T cell responses indirectly via antigen-presenting cells, with C3a signalling in bone marrow derived dendritic cells shown to enhance IL-23 and IL-6 production and promote Th17 differentiation in a mouse model of allergic asthma (Lajoie et al., 2010). An absence of C3aR and C5aR1 signalling in CD4+ T cells has been shown to lead to enhanced IL10 and TGFβ expression and Foxp3 iTreg-mediated immunosuppression (Strainic et al., 2013) whereas C3aR/C5aR1 signalling abrogates nTreg function (Kwan et al., 2013). Although Strainic et al (2013) showed that a combination of C3aR/C5aR1 inhibition and TGFβ induced the development of functional Tregs(et al., 2013), the effect of C5aR1 signa1ling on the malaria-specific CD4+ T cell response was shown to be indirect via inhibition of DCs (Liu et al., 2013).

Despite their critical role in the development of effective immune responses, excess production of C5a and C3a can lead to up-regulated pro-inflammatory responses, resulting in tissue damage and eventually multi-organ failure (Gao et al., 2005). Indeed the anaphylatoxins have been implicated in a range of inflammatory diseases including rheumatoid arthritis, ischemia-reperfusion injury, sepsis, neurodegenerative diseases and macular degeneration (Bonifati and Kishore, 2007; Guo and Ward, 2005; Manderson et al., 2004; Zipfel et al., 2006). Thus, the regulation of anaphylatoxin receptor activation may have therapeutic benefits in a wide range of diseases. To this end, a number of agonists and antagonists have been developed which may lead to effective therapies for these diseases.

1.5.3 Peptide agonists and antagonists of anaphylatoxins C3a and C5a The development of agonists and antagonists for complement components has provided researchers with important tools to dissect the roles of the complement anaphylatoxins. A number of peptide agonists have been developed based on the native C-terminal sequences of C3a and C5a (Taylor et al., 2001). One of these peptides, termed EP141 (amino acid

26

sequence WWGKKYRASKLGLAR) was developed by Ember and co-workers (1991) and is a highly potent and selective of C3aR. This peptide contains a region with the essential functional site of the molecule, which interacts with the binding site on the C3a receptor(Ember et al., 1991). Another complement anaphylatoxin analogue, EP54 (amino acid sequence YSFKPMPLaR) is selective for C5aR1 and C3aR (Tempero et al., 1997), and demonstrated to act as molecular adjuvant, targeting C5aR1 on dendritic cells (Hegde et al., 2008).

A series of cyclic peptide C5aR1 antagonists have also been developed at The University of Queensland that are selective inhibitors of C5aR1 (Strachan et al., 2000). Two key compounds, termed PMX53 (AcF-[OPdChaWR]) and PMX205 (HC-[OPdChaWR]), are orally active, specific C5aR1 antagonists (March et al., 2004) These compounds prevent C5a-mediated release of pro-inflammatory mediators TNF-α and IL-6 both in vivo and in vitro (Proctor et al., 2004) and have been shown to effectively reduce C5a-mediated inflammatory responses in numerous animal models of disease, including arthritis (Woodruff et al., 2011) ischaemia-reperfusion injury and stroke (Arumugam et al., 2004; Woodruff et al., 2004), inflammatory bowel disease and atherosclerosis (Manthey et al., 2011; Proctor et al., 2004; Woodruff et al., 2003). Although several other C5aR1 antagonists have been reported in the literature (Woodruff et al., 2011), to date the PMX-series of molecules are the only compounds with robust activity in multiple chronic disease models, making them ideal therapeutic tools to study C5aR1 pathobiology in vivo.

SB290157 is a non-peptide arginine analogue that acts as a competitive antagonist with high affinity and specificity for the C3a receptor (Ames et al., 2001). It exhibits anti-inflammatory properties, and has been shown to block C3a-induced internalization of C3aR in human neutrophils and C3a-induced Ca2+ mobilization in basophilic leukaemia RBL-2H3 cells expressing murine, guinea pig, or human C3aR. Anti-inflammatory activity has also been demonstrated, due to effects on neutrophil populations (Proctor et al., 2004). However SB290157 has a short half-life (Denonne et al., 2007), and has been reported to have agonist properties in a number of cellular systems (Mathieu et al., 2005), possibly depending on the level of receptor expression. These data caution against the sole reliance on this compound to assign roles for C3aR; rather additional studies using other supporting methods, such as C3aR-/- animals, are required to definitively ascribe functions to C3aR (Woodruff and

27

Tenner, 2015). Clearly, there is a continuing need for the development of potent and selective C3aR antagonists for interrogating and potentially treating C3a-mediated conditions.

1.5.4 The complement anaphylatoxins and tumour growth The detection of complement activation products, including C4d (Ajona et al., 2013) and C5a (Gorter and Meri, 1999; Niculescu et al., 1992) in tumour tissue, as well as elevated C5a (Corrales et al., 2012) and C3a (Habermann et al., 2006; Jinong Li, 2002; Kanmura et al., 2010; Maher et al., 2011; Solassol et al., 2010) levels in serum from cancer patients, suggests that the complement system is activated in response to tumours. Indeed tumour cells are thought to escape immune attack by up-regulating complement regulatory proteins (Fishelson et al., 2003; Varela et al., 2008; Watson et al., 2006; Zell et al., 2007). Enhanced expression of complement regulatory proteins has been shown to protect against complement mediated lysis of tumour cells (Gorter and Meri, 1999; Jurianz et al., 1999; Varsano et al., 1998) whereas treatment with antibodies against tumour antigen and the complement lysis inhibitor protectin (CD59) lead to eradication of micro-metastases and small solid tumours from breast carcinoma and ovarian teratocarcinoma cell lines by complement-mediated mechanisms (Hakulinen and Meri, 1998).

To date, the majority of studies investigating the role of complement anaphylatoxins in tumour growth have been directed towards C5a, and these have yielded conflicting results. C5a has been utilised as a molecular adjuvant to enhance the efficacy of antibodies to tumour antigens. For example Fuenmayor et al. (2010) demonstrated that the tumouricidal effect of a against human epidermal growth factor receptor 2 (HER2/neu) was enhanced by fusion with C5a. These fusion proteins facilitated of human granulocytes, which are the primary immune effector cell responsible for facilitating antibody dependent cell mediated cytotoxicity (ADCC).

Kim et al (2005) showed that over-expression of C5a by mouse mammary tumour cells protected against tumour growth . They suggested that C5a can either act directly on tumour cells by inhibiting their growth through apoptosis and cell cycle arrest, or indirectly by activating tumour infiltrating inflammatory cells (macrophages and/or granulocytes) to eliminate the tumour by phagocytosis or via cytokine secretion and production of ROS. The demonstration that mice were immune to subsequent challenge with normal tumour cells (ie not transduced with the C5a construct) suggested the involvement dendritic cells and the adaptive immune response. Further evidence for a protective role for C5a against mouse

28

mammary tumours has been provided by Hezmee (2010) and Akhir (2014) who showed that the growth of mouse mammary tumours could be inhibited by daily treatment with the dual C3a/C5a agonist, EP54.

Studies on other tumour types have implicated C5a for a tumour promoting role. Markiewski et al (2008) convincingly demonstrated that deficiencies in complement components C3 or C5a were associated with retarded tumour growth in a mouse cervical cancer model, while pharmacological blockade of C5aR1 using PMX53 contributed to tumour regression. Their results suggested that this effect was due to the ability of C5a to recruit myeloid derived suppressor cells which in turn suppress the tumour-specific immune response. C5a was also shown to increase the production of reactive oxygen (ROS) and nitrogen (RNS) species by these cells . Similarly Corrales et al. (2012)demonstrated in a murine model of lung cancer that C5aR1 antagonism retarded tumour growth. C5a appeared to promote an immunosuppressive microenvironment, with C5aR1 antagonism attenuating MDSCs, including the granulocytic subpopulation; immunosuppressive molecules, including ARG1, CTLA4, IL-6, IL-10, LAG3 and PDL1, were also down-regulated. C3 and C5aR1 deficiencies have also been reported to profoundly impair growth of a transgenic murine model of epithelial ovarian cancer (Nunez-Cruz et al., 2012). However, in this case, complement inactivation did not alter the inflammatory infiltrate within tumours, but reduced tumour vascularisation via inhibition of specific VEGF isoforms.

Although C5a may have different effects in different tumour types, Gunn and co-workers (2012) have suggested that the concentration of C5a within the tumour microenvironment is critical in determining its effects. They found that mice bearing high C5a-producing syngeneic lymphomas had significantly accelerated tumour progression with more Gr- 1+CD11b+ myeloid cells in spleen and overall decreased CD4+ and CD8+ T cells in tumour, tumour-draining lymph nodes (TDLN), and spleen. Conversely, mice with low C5a- producing lymphomas had significantly reduced tumour burden with increased IFN-γ- producing CD4+ and CD8+ T cells in spleen and TDLN.

Recent studies have provided evidence that C5a may also contribute to development of tumour metastasis. The first direct evidence that C5a promotes tumour metastasis was provided by Vadrevu et al. (2014) who showed in a murine breast cancer model that lung metastasis are reduced in the absence of C5aR1 signalling. They showed that inhibition of C5aR1 signalling results in the down regulation of MDSCs, Treg cells and

29

immunosuppressive cytokines such as TGF-ß and IL-10, but increased levels of CD4+ and anti-tumour CD8+ T-cells in the pre-metastastic niche. Conversely, C5aR1 blockade did not affect primary tumour growth. More recently, Piao and co-workers (2015)have shown that C5a contributes to hepatic metastasis of colon cancer. The absence of C5aR1 reduced the infiltration of macrophages, neutrophils and dendritic cells whereas C5a signalling increased the expression of the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) and anti-inflammatory cytokines IL-10 and TGFβ.

Despite the growing data regarding the effects of C5aR signalling on tumour immunity, the role of C3aR remains elusive. Up-regulated levels of serum C3a have been reported in many cancers including breast, colorectal and oesophageal cancer (Maher et al., 2011; Medina- Echeverz et al., 2014), and gene expression analysis has identified the up-regulation of C3a receptor (C3aR) by some tumours, including melanoma (Xu et al., 2008). A recent report by Cho and co-workers (2014) demonstrated the expression of both C3aR and C5aR by ovarian cancer cells. They further showed that these receptors are capable of signal activation via the P13K/AKT pathway to induce cell proliferation, migration and invasion. Conversely receptor inhibition reduced cell proliferation, leading these researchers to conclude that tumour-derived complement proteins are capable of promoting tumour growth via autocrine mechanisms. They also applied data mining techniques using Cancer Cell Line Encyclopaedia (CCLE) database to identify high level of C3 expression in liver, kidney, lung, endometrial and ovarian cancer cell lines (Cho et al., 2014). In another recent study, Surace et al (2015) have reported that the local production of C3a and C5a are critical to the anti- tumour response induced by radiotherapy. Additional indirect evidence that C3a, as well as C5a, may play a role in tumour growth was provided by an earlier study showing that both C3aR- and C5aR1-deficient mice had reduced levels of neovascularisation, VEGF and blood vessel formation, all critical factors for tumour perfusion and invasion (Nozaki et al., 2006).

30

1.6 CONCLUSIONS AND PROJECT RATIONALE

Although the relationships between inflammation and cancer are well recognized, the roles of key inflammatory mediators (complement components C3a and C5a) are yet to be completely elucidated. The presence of complement activation products and expression of complement receptors by cancerous cells are well established, and in the last few years the contribution of C5a in tumour growth has been demonstrated, albeit with different mechanisms in different tumour types. However, despite evidence that serum C3a levels are up-regulated in many cancer patients, and that C3aR is expressed by some tumour cells, there is little information concerning the role of C3a in tumour growth. Therefore, this thesis investigated the effects of both C3a and C5a in mouse tumour models.

We hypothesised that complement anaphylatoxin receptors C3aR, C5aR1 and C5aR2 are therapeutic targets for melanoma. The specific aims of this study were:

1) To investigate the expression of complement anaphylatoxin receptors in murine and human melanoma cell lines;

2) To identify the roles of C3aR, C5aR1 and C5aR2 in tumour growth in a mouse model of melanoma, and to identify the therapeutic potential of C3aR or C5aR pharmacological antagonism;

3) To investigate the mechanisms underpinning the observed responses, by studying tumour inflammatory cell infiltration and cytokine production in tumour tissue.

31

32

CHAPTER 2

MATERIALS & METHODS

Jamileh Alham Nabizadeh

33

MATERIALS & METHODS

2.1 Cell Lines

The murine melanoma B16-F0 (ATCC® CRL6322™; (Fidler and Nicolson, 1976), B16-F10 (ATCC ® CRL-6475™) and murine J774A.1 macrophage (ATCC® TIB-67™) cell lines (all syngeneic in C57BL/6J mice) were obtained from the American Type Culture Collection (Manassas, VA). Murine SM1WT1 melanoma cells (Knight et al., 2013) and MC38 colon cancer (Rosenberg et al., 1986) cell lines were provided by Prof Mark Smyth (QIMR Berghofer Medical Research Institute), and human metastatic melanoma (MM603, MM608, HT144, MM370 and MM649), BRAFV600E mutant (Mel-RMu) and primary melanoma (MM96L) cell lines by Dr. Glen Boyle (QIMR Berghofer Medical Research Institute). B16, SM1WT1 and MC38 cells were cultivated in high glucose Dulbecco's modified Eagle medium (DMEM; Invitrogen, Auckland, NZ), and human melanoma and J774 cells in RPMI supplemented with 10% heat-inactivated foetal calf serum (FCS; Moregate, Brisbane, Aust) and penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2 in air.

2.2 Drugs

The selective C5aR1antagonist, AcF-[OPdChaWR] (PMX53) was provided by Assoc. Prof. Trent Woodruff from School of Biomedical Science, The University of Queensland, Australia, and prepared in an isotonic 5% glucose/water solution. The C3aR antagonist SB290157, N2-[(2,2-Diphenylethoxy)acetyl]-L-arginine trifluoroacetate (VDM Biochemicals Inc, Bedford Heights, OH) was prepared in 10% ethanol, and diluted in sterile saline.

2.3 Animals

Homozygous C57BL/6J congenic C3aR, C5aR1 and C5aR2 receptor knockout (C3aR -/-, C5aR-/- and C5aR2-/-) or wild-type (WT; C57BL/6J) mice (6-8 weeks old, and sex-matched) were obtained from University of Queensland Biological Resources and fed a normal chow diet ad libitum. All procedures were approved by the University of Queensland Animal Ethics Committee Guidelines (AEC 493/09 and 564/12) and conformed to the Animal Care and Protection Act Qld (2002) and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (8th edition, 2013).

34

2.4 Tumour Induction and Drug Treatments

For induction of primary melanomas, WT, C3aR -/-, C5aR1-/- or C5aR2-/- mice (n=6-8 animals/group) were clipped over the right flank before subcutaneous (s.c.) injection of B16- F0 cells (2.5x105 cells/mouse) or SM1WT1 (1x106 cells/mouse) in 0.05 ml serum-free medium. Colon carcinoma cells (1x106 cells/mouse) were injected s.c. into the left flanks of mice. Tumour growth was monitored daily and measured by Vernier callipers from day 5 after tumour cell injection. For some experiments, C57BL/6J mice received daily i.p. injections (0.05 ml) of either C3aR antagonist (SB290157; 1 mg/kg/day) or vehicle only (10% ethanol in normal saline) control; other mice received s.c. injections of PMX53 (1 mg/kg/day) or vehicle only (5% glucose) control (n=6-8 animals/group), commencing once tumours became palpable (approx. day 7 after tumour cell injection). For most experiments, mice were euthanized at day 14 (or once the largest tumour reached an area of approx 200 mm2), tissue removed for either flow cytometry or histological analysis (n=6-8/group), and tumours weighed. For ‘survival’ studies, the time taken for each tumour to reach maximal size (200 mm2) was recorded, then mice euthanized.

For neutrophil depletion experiments, WT and C3aR-/- mice (n=8/group) were injected i.p. with anti-Ly6G antibody (1A8, selective for neutrophils, 0.1 mg/mouse; Bio X Cell (Daley et al., 2008) or control (rat IgG2a, 2A3) 24 hours prior to s.c.. injection of B16 cells, and then every 3 days until the largest tumour reached 200 mm2. Mice were then euthanized, and blood and tumour tissue removed and weighed.

2.5 Tissue Processing and Histology

Harvested tumour tissue was fixed overnight at 4 °C in 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA), then washed with phosphate buffered saline (PBS) before infusing with 18% sucrose at 4 °C overnight. The sucrose solution was then replaced with a solution containing (O.C.T Compound Sakura Fine technical, Tokyo, Japan) and frozen on liquid nitrogen. Tissue blocks were stored at -20 °C prior to cryosectioning (Hyrax C60, American Optical Corporation, USA), and 5 µm sections dry mounted onto glass slides. Sections were air dried at room temperature for several hours and stored at -20 °C under desiccating conditions until use.

35

2.6 Immunofluorescence Staining

B16, MM96L and J774 cells were seeded (at approx 2.5x104cells/cm2) onto glass cover slips and attached overnight before fixing with 4% PFA for 10 mins. Cells were either non- permeabilised (for membrane staining) or detergent-permeabilised (for intracellular staining) by incubation in 0.1% triton X-100 (Sigma-Aldrich) in PBS for 30 seconds. Cells on coverslips or frozen sections were then incubated with blocking buffer (1% bovine serum albumin (Sigma-Aldrich), 5% sheep serum in PBS) for 30 mins, followed by primary antibodies (Table 2.1) overnight at 4°C. After washing with PBS, cells or tissue sections were incubated with their respective secondary antibodies (Table 2.2) for 1 hour at room temperature in the dark. Alternatively cells or tissues were incubated with directly conjugated primary antibodies for 2 hrs at room temperature. After further washing, nuclei were stained with Hoechst 33342 (1:2000; Life Technologies) for 10 minutes. Cells or tissue sections were washed again, then mounted in ProLong Gold anti-fade reagent (Life Technologies), examined under an Olympus BX-61 microscope (Tokyo, Japan) and photographed with a Retiga EXi cooled CCD camera (Olympus).

2.7 Immunohistochemistry

Formalin-fixed paraffin embedded human melanoma cell pellets or paraffin embedded human melanoma tissue arrays (AccuMax Array, Biomax, Rockville, MD) were de-paraffinised prior to incubation in 0.25% potassium permanganate and 1% oxalic acid for 10 minutes and 1 minute, respectively to bleach the melanin. Sections were then incubated for 10 minutes in 1% glycine solution in PBS to balance any change in charge followed by antigen retrieval by microwaving in 10-2 mol/L citrate buffer, pH6 (Antigen Unmasking Solution, Vector Laboratories; Burlingame, CA) for 20 minutes. This was followed by overnight incubation with primary antibodies (Table 2.1) diluted in blocking buffer (3% goat serum in PBS) at 4 °C. Sections were washed in PBS then incubated for 30 minutes with the appropriate biotinylated secondary antibody, followed by Streptavidin-peroxidase ABC (Vector), and AEC (Vector) as substrate, then once the desired colour developed, sections were lightly counterstained with haematoxylin and mounted with Vectamount (Vector).

Human neutrophils in paraffin embedded tissue were detected using a Naphthol AS-D Chloroacetate (Specific Esterase) Kit (Sigma), according to the manufacturer’s instructions.

36

Images of stained tissue sections were collected on an Aperio Scanscope digital slide scanner (Leica Biosystems, Nussloch, Germany)

Table 2.1 Primary antibodies used for immunostaining

Antibody Host Manufacturer Dilution Anti-mouse C3aR Chicken Bachem 1/100 Anti-mouse C5aR Rat AbD Serotec 1/100 Anti-mouse C5L2 Rabbit Hycult Biotech 1/200 FITC Anti-mouse CD45 Rat eBioscience 1/100 Anti-human C3aR Mouse BioLegend 1/50 Anti-human C4d Rabbit Biomedica 1/50

Table 2.2 Secondary antibodies used for immunostaining Antibody Host Manufacturer Dilution FITC anti-Rat IgG Chicken Millipore 1/200 Alexa Fluor 488 anti-rat IgG Goat Life Technologies 1/100 Alexa Fluor 555 anti-chicken IgG Goat Life Technologies 1/500 Alexa Fluor 488 anti-chicken IgG Goat Sigma-Aldrich 1/600 Biotinylated anti-mouse IgG Goat Life Technologies 1/200 Biotinylated anti- rabbit Goat Life Technologies 1/200

2.8 Western analysis

B16 cells were grown to approx 70% confluence in T-25 culture flasks, followed by serum starvation (in DMEM without FCS) for 24 hours. Cells were then treated with either dual C3a/C5a agonist (EP54), C3a agonist (EP141 at 10 µmol/L) or DMEM alone for the times indicated in the experiment. After treatment, crude protein extracts were obtained by disrupting the cells in RIPA lysis buffer [50 mmol/L Tris HCl pH 7.4, 150 mmol/L NaCl, 1%

NP-40, 0.25% sodium deoxycholate, 1 mmol/L EDTA, 1 mmol/L Na3VO4, 1 mmol/L PMSF, 5 mmol/L NaF, 1 mmol/L DTT, 1X protease inhibitor cocktail (Sigma)]. For each sample, 20 μg of protein (determined by BCA protein assay; Pierce, Rockford, IL) was subjected to

37

SDS-PAGE on 8% polyacrylamide gels (Laemmli, 1970), then separated proteins electrotransferred (Towbin et al., 1979) to a PVDF membrane (Immobilon P; Millipore, Billerica, MA). Membranes were blocked (Blocking Buffer; Li-Cor Biosciences, Lincoln, NE), before incubation with primary antibodies (anti-phospho-AKT and anti-total AKT (Cell Signalling, Danvers, MA) or mouse anti-diphosphorylated ERK1/2 and rabbit anti-total ERK1/2 (Sigma-Aldrich) followed by fluorescently-labelled secondary antibodies (IR Dye 800CW-goat anti-mouse Ig and IR Dye 680LT anti-rabbit Ig; Li-Cor Biosciences; Lincoln, NE). Immunoreactive bands were visualised by scanning on an Odyssey Imaging System (Li- Cor Biosciences) and the percentage phosphorylated protein relative to total protein determined using Odyssey V3.0 image analysis software.

2.9 Cell proliferation (MTT assay)

Cell proliferation was measured using a CyQUANT® NF cell proliferation assay (Life Technologies), according to the manufacturer's instructions. Briefly, cells were seeded at 5x103 cells/well into a 48-well plate, and allowed to attach overnight. After serum-starving (in DMEM + 2% FCS) for 24 hours, cells were treated with PMX53 (1μmol/L in the same medium) or mouse C5a (10-9 mol/L), mouse C3a (10-8 mol/L) for a further 48 hours. Medium was then removed, cells washed with HBSS. Cell proliferation rates were determined by incubation with 10 μL, 12 mmol/L MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide reagent until purple precipitate is visible (approx 2-4 hours), then 100 μL of detergent reagent added and the plates kept in the dark for 2 hours. After mixing each sample, absorbance was read at 570nm.

2.10 Cell migration assay

To investigate the effect of C3aR agonist on B16 migration, cells were seeded at confluence (2x105 cells/cm2) into a 24 well plate, adhered overnight and then confluent monolayers scratched with a pipette tip before being treated for 24 hours with C3aR agonist (10-6 mol/L); control wells were incubated with medium alone or containing 5% FCS. Migration was quantified by measuring cell-free areas at 0 and 24 hours using Image-J software.

38

2.11 Flow cytometric analysis

Blood was collected by cardiac puncture, tumours, spleen, draining lymph nodes (inguinal, axillary and brachial) and femurs excised; tumours were weighed, and femurs flushed with PBA (PBS containing 0.2% BSA, 0.1% sodium azide, 5% rat serum) using a syringe with a 25-gauge needle to obtain bone marrow cells. Single cell suspensions of tumour, spleen and draining lymph nodes were prepared by passing tissues through a 70 μm nylon cell strainer (Becton Dickinson) into PBS containing 0.1% BSA (Sigma-Aldrich, St.Louis, MO) and 0.1% sodium azide (NaN3; Sigma-Aldrich) (PBA). Blood and spleen cell suspensions were incubated in lysis buffer (155 mmol/L NH4CL, 10 mmol/L KHCO3, 0.1 mmol/L EDTA; all from Sigma-Aldrich) for 10 minutes at 4 °C to lyse red blood cells. Cells were washed in PBA and distributed into clear, U-bottom 96 well plates (0.5-2.0x105 cells/well) before centrifugation at 400 g for 5 min at 4 °C. Supernatants were removed and cells pre-incubated with blocking buffer (5% normal rat serum and anti-CD16/32 (TruStainFcX, 1/50; BioLegend, San Diego, CA) in PBA) for 15 minutes to block Fcγ receptor binding prior to surface staining with fluorophore-conjugated monoclonal antibodies (MAb) (Table 2.3) or the appropriate isotype controls at 4 °C for 30 minutes. For staining of Treg cells, cells were stained with antibodies to surface antigens, then fixed and permeabilised with FoxP3 Fix/Perm kit (BioLegend), and incubated for 40 mins with anti-FoxP3. For Th1, Th2, Th17 staining, cells were first stimulated for 4 hours (37 °C) with phorbol myristate acetate (PMA, 50ng/ml; Sigma) and ionomycin (1 µmol/L; Sigma) in the presence of Brefeldin A (5µg/ml; BioLegend). After surface staining (as above), cells were fixed with Fixation Buffer and permeabilised with Permeabilisation/Wash Buffer (BioLegend) before incubation for 40 mins at room temperature with fluorophore-conjugated antibodies to IL-4, IL-17A or IFN-γ (all from BioLegend). After antibody staining, cells were washed twice in PBA, then resuspended in 100 μl PBA. For detection of C3a receptor, cells were incubated with primary anti-human C3aR antibody for 30 minutes followed by 40 minutes incubation with biotinylated anti-mouse IgG, then washed twice in PBS and resuspended in 100 μl PBS for flow cytometry. Compensation was performed using compensation beads (BD Biosciences). All cells were analysed using an Accuri C6 flow cytometer (BD Biosciences) and data analysed using FlowJo software package (Tree Star Inc., Ashland, OR). Gating strategies for different leukocyte populations are illustrated in Figure 2.1.

39

Figure 2.1. Flow cytometry and gating strategies for the tumour inflammatory infiltrate. Representative contour plots show different infiltrating leukocyte subpopulations in tumour tissue. Debris (SSC-A vs. FSC-A) were excluded, then leukocytes (CD45+ cells; A) were sub-gated for monocytes (CD11b+Ly6C+; B), neutrophils (CD11b+Ly6G+; C), MDSC (CD11b+GR1+; D), macrophages (F480+; E) and total T lymphocytes (CD3+; F). Total T lymphocytes were sub-divided into CD4+ (G; red) and CD8+ (G; orange) populations. CD4+ cells were further sub-gated into Tregs (CD4+CD25+FOXP3+; H) , Th1 (CD4+IFNγ+; I), Th2 (CD4+IL4+; J) and Th17 (CD4+IL17+; K). For each sub-gate, the frequency of cells is expressed as percentage of CD45+ or CD4+ cells.

40

Table 2.3 Antibodies used for FACS analysis Antibody Manufacturer Dilution APC/647 rat anti-mouse AbD Serotec 1/100 FITC rat anti-mouse CD45 BioLegend 1/400

PE/Cy7 rat anti-mouse CD45 BioLegend 1/400 PE/Cy7 rat anti-mouse F4/80 BioLegend 1/400 Alexa Fluor 488 rat anti- BioLegend 1/400 PE rat anti-mouse CD11b BioLegend 1/300 FITC rat anti-mouse Gr-1 BioLegend 1/200 FITC rat anti-mouse Ly6G BioLegend 1/300 APC/647 rat anti -mouse BioLegend 1/300 PE/Cy7 rat anti -mouse Ly6C BioLegend 1/300 APC/647 rat anti-mouse BioLegend 1/200 PE rat anti-mouse CD62L BioLegend 1/200 FITC rat anti-mouse CD3 BD Biosciences 1/300 FITC rat anti-mouse CD4 BD Biosciences 1/300 FITC rat anti-mouse CD8a BD Biosciences 1/300

PE rat anti-mouse FoxP3 BioLegend 1/200 PE/Cy7 rat anti-mouse CD25 BioLegend 1/300

PE rat anti-mouse IL4 BioLegend 1/200

PE rat anti-mouse IL17 BioLegend 1/300 APC/647 rat anti-mouse BioLegend 1/300

APC/647 rat anti -mouse BioLegend 1/300

2.12 Cytokine Assay

Blood was taken by cardiac puncture into EDTA tubes, centrifuged at 1800 g for 5 minutes at 4 °C, and plasma collected and stored at -80 °C until assay. Snap frozen tumour tissue was pulverised with a mortar and pestle, then cytoplasmic extracts prepared according to manufacturers’ instructions (Milliplex Cell Lysis Kit; Millipore). Protein concentration was determined by BCA assay, and equal amounts of total protein (0.25 mg/assay) analysed by

41

multiplex ELISA (Mouse Cytokine/Chemokine Panel; Millipore) according to the manufacturer’s instructions.

2.13 Statistical Analysis

Each experiment was performed a minimum of 3 times and statistical analyses were performed using the statistical software package Graph Prism v5.04. Results are expressed as mean ± SD. Tumour growth curves were analysed by Permutation test and survival studies by log- Mantel-Cox test. All other data were analysed by Mann-Whitney test or Kruskal Wallis test followed by Dunn’s multiple comparisons test. For all tests, a P value of <0.05 was considered statistically significant.

42

CHAPTER 3

THE OPPOSING EFFECTS OF COMPLEMENT C5A RECEPTORS, C5AR1 AND C5AR2, IN A MOUSE MODEL OF MELANOMA

Jamileh Alham Nabizadeh

43

3.1 INTRODUCTION

Melanoma is the most aggressive form of skin cancer, with 232,000 new cases and 55,000 deaths reported globally in 2012 (World Health Organisation Report, 2014). Despite recent advances in treatment, sustained responses remain elusive and the 3-year survival rate for metastatic melanoma remains around 15% (Eggermont et al., 2014). There is therefore an urgent need for new therapeutic strategies for advanced melanoma. Given the evidence that the complement system is activated in response to tumour cells (Ajona et al., 2013; Niculescu et al., 1992), along with recent studies demonstrating a role for complement activation product C5a in regulating the growth of some tumour types, this chapter investigated the role of C5a in melanoma.

The complement system is activated via several routes, all of which can ultimately lead to production of the anaphylatoxin C5a (Klos et al., 2013; Markiewski and Lambris, 2007; Mukherji et al., 1986), one of the most potent inflammatory proteins known (el-Lati et al., 1994). Most of the canonical biological activities of C5a are thought to be mediated via the G-protein coupled C5aR1 (Guo and Ward, 2005). Several studies suggest that complement C5a-C5aR1 signalling can promote tumour growth by sustaining chronic inflammation, with C5aR1 antagonism having anti-tumour effects in cervical, lung and ovarian cancer models (Corrales et al., 2012; Markiewski et al., 2008a; Markiewski et al., 2008b; Nunez-Cruz et al., 2012). However these studies suggest differing effects in different tumour types, possibly depending on the levels of C5a (Gunn et al, 2012). Although the cross-talk between C5aR, MDSC and Tregs has been documented in mouse models of cervical, lung and lymphoma (Markiewski et al, 2008; Corrales et al 2012, Gunn et al, 2012), other mechanisms of action have been proposed in other tumour models (Kim et al, 2005; Nunez-Cruz et al, 2012). Thus this thesis investigated possible mechanisms by which C5aR signalling acts on melanoma growth. Moreover, despite emerging evidence that the alternative C5a receptor, C5aR2, can independently induce and modulate biological functions of C5a through β-arrestin signalling (Croker et al., 2014; Li et al., 2013), a role for this receptor, C5aR2, in tumour development has not been reported.

The lack of effective/sustained therapies for melanoma, along with mounting evidence that C5aR1 signalling plays a role in tumour growth, led us to investigate the therapeutic potential of C5aR1 inhibition, and the mechanisms by which C5a influences tumour growth. Given

44

the absence of reports investigating C5aR2 in tumourigenesis, the effects of C5aR1 and C5aR2 deficiency on development of B16 tumours were also compared. The results demonstrate that whilst C5aR1 signalling is pro-tumourigenic, C5aR2 acts to limit tumour growth. Overall, the data provide support for the premise that C5a receptor signalling is a novel therapeutic target for melanoma.

45

3.2 EXPERIMENTAL OUTLINE

To determine whether complement is activated in human melanoma tissue, human melanoma tissue arrays (US BioMax Inc, Rockville, MD) were immunostained for complement activation product C4d (Section 2.7).

To investigate receptor expression, B16 melanoma cells and J774 macrophages (positive control) were cultured on coverslips and immunostained with antibodies specific for C5aR1 and C5aR2 (Section 2.6). Cell suspensions were immunostained with the same antibodies for FACS analysis (Section 2.11). To determine whether cells are capable of activating other signalling pathways, cells were grown to approximately 70% confluence in T-25 culture flasks and treated with recombinant mouse C5a (mC5a; 10 nM) or antagonist (PMX53; 10 μM) for various times, then cell lysates subjected to Western analysis for activated ERK or AKT (Section 2.8). To determine whether C5aR activation stimulates proliferation of B16 melanoma cells, cells were treated with C5a agonist EP54 (1μM) (or medium alone) for 48 hours, then cell proliferation rates determined by MTT assay (Section 2.9).

To investigate the role of C5aR1 signalling in melanoma growth, wild-type C57BL/6J mice were injected s.c. with B16 melanoma cells (2.5 x105 cells/mouse; as described in Section 2.4) followed by daily s.c. injections of either C5aR1 antagonist AcF-[OPdChaWR] (PMX53; 1 mg/kg/day) or vehicle only (saline) control, commencing once tumours became palpable (approx 7 days after injection); tumour areas were measured daily with Vernier callipers (Section 2.4). Once the largest tumours reached 200 mm2 (approx day 14), mice were euthanized, tumour excised and weighed.

In order to further investigate the role of C5aR1 and C5aR2 signalling in melanoma growth, female homozygous C5a receptor knockout (C5aR1 -/- and C5aR2 -/-, both on C57BL/6J background) or wild-type (C57BL/6J) mice were injected s.c. with B16 melanoma cells (2.5 x105 cells/mouse; as described in Section 2.4). Once the largest tumours reached 200 mm2 (approx day 14), mice were euthanized. Blood from each mouse was collected by cardiac puncture, and tumours, draining (axillary and inguinal) lymph nodes, spleen and femurs (for bone marrow cells) removed for FACS analysis (Section 2.11). Tumour tissue from some mice was OCT-embedded for cryosectioning (Section 2.5) and immunostaining (Section 2.6). Plasma and tumour tissue from other mice was snap frozen and protein extracted for multiplex ELISA for chemokines/cytokines (Section 2.12).

46

3.3 RESULTS

3.3.1 Evidence for complement activation in human malignant melanoma tissue Complement activation products have previously been detected in tumour tissue including breast (Niculescu et al., 1992) and lung cancer (Ajona et al., 2013). To investigate whether complement is also activated in human melanoma tissue, melanoma tissue arrays were immunostained for the complement activation product C4d. As shown in Figure 3.1, C4d was detected in melanoma tissue at higher levels than in normal skin. Representative images illustrate high levels of C4d deposition in malignant melanoma stage II (Figure 3.1B), malignant melanoma stage III (Figure 3.1C), and lower levels in normal dermis adjacent to tumour (Figure 3.1D). Of 103 melanoma tissue biopsies examined, C4d staining was evident in 92. However there was no correlation between C4d levels and stage of disease.

Figure 3.1. Immunohistochemical staining of human melanoma tissue array showing C4d deposition (AEC; red) (A). Representative images show malignant melanoma stage II (B) and stage III (C), normal dermis adjacent to tumour (D) and staining with isotype control (E). Arrowheads show C4d (red; scale bar = 100µm).

47

3.3.2 C5aR1 antagonism inhibits melanoma growth Inhibition of C5aR1 using the cyclic hexapeptide antagonist AcF-[OPdChaWR] (PMX53; C5aR1A), developed by Taylor and co-workers (Finch et al., 1999) has previously been demonstrated to be effective at reducing tumour growth in mouse models of cervical (Markiewski et al., 2008a), lung (Corrales et al., 2012; Gu et al., 2013) ovarian (Nunez-Cruz et al., 2012) and metastatic breast cancer (Vadrevu et al., 2014). We therefore first investigated whether pharmacological antagonism of C5a-C5aR1 signalling also has therapeutic effects in the B16 murine melanoma model. For this experiment, B16 melanoma tumours were established in C57Bl/6J mice, and once tumours became palpable (approx. day 7), mice were injected daily with PMX53 (1mg/kg/day). As shown in Fig 3.2, the rate of tumour growth was significantly slowed in PMX53-treated mice (Fig 3.2A; p=0.0001, Permutation test), with excised tumour weight at day 14 reduced to 0.19 ±0.17 g compared with 0.44 ±0.22 g in the vehicle-treated controls (Fig 3.2B; p<0.0001).

Figure 3.2. C5aR1 antagonism inhibits melanoma growth. Tumour areas (A) measured daily in live mice from the time they become palpable, and excised tumour weight (B) following euthanasia on day 14 for wild-type C57BL6/J mice receiving vehicle (saline) or the C5aR1 antagonist (PMX53; 1 mg/kg/ day s.c.) commencing day 7. Representative images (C) show tumours from mice treated with vehicle alone or C5aR1 antagonist. Data expressed as mean ± SD from 3 independent experiments; n=7- 8/group. ** p<0.005 Permutation test; **** p<0.0001, Mann-Whitney test.

48

3.3.3 Complement C5a receptor expression by B16 melanoma cells in vitro and in vivo

3.3.3.1 Immunofluorescence staining of B16 cells In order to understand the mechanism by which C5aR antagonism inhibits tumour growth, we first investigated whether C5a could act directly on tumour cells. Immunostaining demonstrated that both C5a receptors (C5aR1 and C5aR2) (Fig 3.3) are expressed by cultured B16 cells, but at lower levels than found in J774 macrophages (positive control) which are known to express high levels of complement receptors (Chenoweth et al., 1982). The lack of staining with isotype controls shows lack of non-specific binding.

Figure 3.3. Complement receptor expression by mouse melanoma B16 cell lines. Fluorescence micrographs show expression of C5aR1 and C5aR2 (both FITC-labelled; green) by B16 melanoma cells compared with the control (J774 macrophages); nuclei are counterstained with Hoechst 33342 (blue). Isotype control antibodies show lack of non- specific binding. Scale bar = 25 μm.

49

3.3.3.2 FACS analysis of B16 cells The expression of C5aR1 receptor by cultured B16 melanoma cells was confirmed by flow cytometric (Fig 3.4A) and Western (Fig 3.4B) analysis. Again expression was lower than for J774 macrophages. C5aR2 expression was not examined due to lack of a suitable antibody for FACS and Western blotting.

Figure 3.4. Expression of C5aR1 byB16 melanoma cells. Flow cytometric analysis (A) shows C5aR1 (blue) expression by B16 melanoma cells compared with J774 macrophages (positive control) and cells labelled with isotype control antibody (red). Western blot (B) shows C5aR1 expression by B16 melanoma and J774 macrophages.

50

3.3.3.3 Immunohistochemical analysis of B16 melanoma tissue Importantly, C5aR1 expression was also demonstrated in B16 melanoma tissue from C5aR1- /- mice (in which host cells lack C5aR1, but tumour cells express C5aR1) (Fig 3.5), excluding the possibility that receptor expression in tumour tissue is due solely to infiltrating host inflammatory cells.

Figure 3.5. C5aR1 expression by B16 melanoma tissue. Fluorescence micrographs show expression of C5aR1 (FITC; green) by B16 melanoma tissue grown in C57Bl/6J (wild-type) and C5aR1 knockout mice (C5aR1-/-); nuclei are counterstained with Hoechst 33342 (blue). Scale bar = 25 μm.

3.3.4 Functional evaluation of C5aR1 expressed by B16 melanoma Having demonstrated that B16 melanoma cells express C5aR1, we next determined whether these receptors are functionally coupled. Previous studies have demonstrated that Gi protein- coupled anaphylatoxin receptors can activate the MAP kinase pathway (Buhl et al., 1994; Norgauer et al., 1993; Rollins et al., 1991; Rousseau et al., 2006). Given the importance of MAPK signalling to tumour cell proliferation (Beeram et al., 2005; Downward, 2003; Gollob et al., 2006; Pratilas and Solit, 2010; Santarpia et al., 2012), we next examined whether receptors expressed by B16 melanoma cells are capable of activating this pathway. As shown in Fig. 3.6A, stimulation of B16 melanoma cells by recombinant mouse C5a (mC5a) activated p44/p42 ERK kinases at all time-points up to 1 hour, demonstrating that C5aR1 is functionally active in these cells.

51

Since the MAPK pathway is known to regulate cellular processes such as cell proliferation and migration, we next determined the effect of C5a on these processes in B16 cells. However C5a had little or no effect on proliferation (Fig 3.6B) or cell migration (Fig 3.6C). Given the key role of C5a in immunity, and the fact that C5a receptors are expressed at high levels on inflammatory cells (Monk et al., 2007), this suggests that the anti-tumour effects of C5aR1 antagonism as shown above, may be indirect, via the immune system as shown by previous studies (Corrales et al., 2012; Gunn et al., 2012; Markiewski et al., 2008a).

52

3.3.5 B16 melanoma growth is altered in C5a receptor-deficient mice

3.3.5.1 C5aR1 deficiency significantly impairs B16 melanoma growth Although we showed that C5aR1 expressed by B16 cells is capable of signal activation, we were unable to demonstrate any significant effects of C5a on cell proliferation or migration. Thus we next used C5aR1-deficient mice to determine whether the anti-tumour effects of C5aR antagonism may be indirect (via effects on host cells). In this case, the tumour-bearing mice are chimaeras, in which the host cells lack C5aR1, but the injected B16 melanoma cells are wild-type (ie expressing C5a receptors). As shown in Fig 3.7A, tumour growth was markedly reduced in C5aR1-deficient mice (p<0.001, Permutation test). Excised tumour weight at euthanasia (Fig 3.7B) was also significantly reduced in these mice (0.15 ± 0.12 g) compared with the wild-type controls (0.39 ± 0.29 g; p<0.0001, Mann Whitney test).

Figure 3.7. Tumour development is retarded in C5aR1-deficient mice. Tumour area (A) and excised weight at 14 days post-tumour induction (B) in C57BL/6J (wild-type; WT) compared with C5aR1-deficient (C5aR1-/-) mice; images (C) show representative tumours from WT and C5aR1-/- mice. Data expressed as mean ± SD from 4 independent experiments (n=8- 12/group);***P<0.001 Permutation test; ****P<0.0001, Mann-Whitney test.

53

3.3.5.2 C5aR2 deficiency leads to enhanced B16 melanoma growth C5aR2 is the alternate receptor for C5a, but a role for this receptor has not yet been reported in tumour growth. Investigation of B16 melanoma growth in C5aR2-deficient mice showed that, in contrast to the C5aR1-/- mice, in which tumour growth was retarded, tumour growth was enhanced in the absence of C5aR2 (p<0.01; Fig 3.8A); excised tumour weight (0.21 ± 0.16 g) was significantly larger than that for the wild-type controls (0.35 ± 0.19 g; p<0.05; Fig 3.8B).

Figure 3.8. Tumour growth is enhanced in C5aR2-deficient mice. Tumour area (A) and excised weight 14 days post-tumour induction (B) in C57BL/6J (WT) compared with C5aR2 deficient (C5aR2-/-) mice. Images (C) show representative tumours from WT and C5aR2-/-mice. Data expressed as mean ± SD from 3 independent experiments (n=6-8 mice/group); **P<0.01 Permutation test; **P<0.005, Mann-Whitney test.

54

3.3.5.3 C5aR1 antagonist treatment of C5aR1-/- mice does not induce further tumour reduction of B16 melanoma growth We next determined whether the effects of the C5aR1 antagonist (PMX53) are solely due to effects on the host immune system, or whether it also affects tumour cells directly. To do this, we investigated if there was any additional therapeutic effect of C5aR1 antagonism on melanoma growth in C5aR1-/-mice. As in previous experiments, tumour growth was significantly reduced in both C5aR1-/- and wild-type mice treated with C5aR1 antagonist (Fig 3.9). However, daily treatment (from day 7) of tumour-bearing C5aR1-/- mice with C5aR1 antagonist had no additional effect on tumour growth. Since the tumour cells express C5a receptors, this result supports the premise that the tumour promoting effects of C5a are mediated via effects on the host immune system, not by any direct effect on the tumour cells themselves.

Figure 3.9. Effect of C5aR1 antagonism on growth of established B16 tumours in wild type and C5aR1 deficient mice. Tumour areas (A) and weight of excised tumours (B) from WT and C5aR-/- mice treated from day 7 with either saline (Veh) or PMX53 (C5aR1A; 1mg/kg/day). Data expressed as mean ± SD (n= 7/group); *p<0.05, **p<0.01, ***p<0.0005 compared with vehicle-treated wild-type control mice, Permutation test (A) and Kruskal Wallis test followed by Dunn’s multiple comparisons test (B).

55

3.3.6 Effect of C5aR inhibition on tumour infiltrating leukocytes

3.3.6.1 Immunofluorescence staining The demonstration that the growth of C5aR-expressing B16 melanoma is retarded in mice lacking C5aR1 indicated that the observed anti-tumour effects are indirect, likely via the host immune system. Thus we assessed the contribution of immune cells to tumour tissue. Immunofluorescence staining of subcutaneous B16 melanoma tissue demonstrated the presence of infiltrating leukocytes (CD45+ cells). In comparison to wild-type mice, tumour tissue from C5aR1-/- mice contained increased numbers of neutrophils (Ly6G+; green, second panel) and T lymphocytes (CD3+; green, fourth panel); macrophages (F4/80+; red) were also present (Fig 3.10).

Figure 3.10. Leukocyte infiltration of B16 melanoma tissue. Immunostaining of B16 tumours from WT and C5aR1-/- mice shows CD45+ cells (green, first panel) within tumour tissue. Some of these cells stain positive for macrophages (F4/80+; red, second panel), neutrophils (Ly6G+; green, second panel) and T lymphocytes (CD3+; green, fourth panel). Nuclei are stained with Hoechst 33342 (blue); scale bar = 20µm.

3.3.6.2 Flow cytometric analysis of tumour infiltrating leukocytes To investigate the effect of C5aR deficiency/inhibition on tumour infiltrating leukocyte populations, tumour tissues from wild-type (untreated or C5aR1 antagonist-treated) and C5aR-deficient mice were harvested on day 14 and used for FACS analysis (Figure 3.11). Tumour tissue from vehicle-treated wild-type mice showed 2.25 ± 0.87 % of all cells were

56

leukocytes (CD45+). C5aR antagonist treatment of established tumours (commencing day 7 after tumour induction) resulted in a slight increase in leukocyte (CD45+) infiltration (2.66 ± 0.92 % of total cells), although this was not significantly different from the vehicle-treated control. However, tumours from C5aR1-deficient mice contained a significantly higher proportion of tumour infiltrating leukocytes (5.34 ± 3.41% of total cells; p=0.031). While the proportion of neutrophils (CD11b+ Ly6G+) was similar in all groups, the percentage of myeloid derived suppressor cells (CD11b+GR1+) was significantly reduced in tumours from C5aR1-/- (2.48 ± 0.87% of total leukocytes; p=0.008) and C5aR1 antagonist treated mice (2.77 ± 0.8%; p=0.03) compared with vehicle-treated wild-type mice (5.52 ± 2.45 %), a result that is in accord with previous reports (Corrales et al., 2012; Gunn et al., 2012; Markiewski et al., 2008a). Although the percentage of macrophages was not altered in tumours from C5aR1-/-, it was significantly reduced in C5aR1 antagonist-treated mice (4.23 ± 1.09 % compared to 7.33 ± 3.54 % for vehicle-treated controls; p<0.02). There was also a significant increase in tumour-infiltrating CD3+ cells (T lymphocytes) in C5aR1-/- (7.13 ± 1.78% of total leukocytes; p=0.01) compared to wild-type mice (4.92 ± 1.24%); C5aR antagonist treated mice also showed a slight increase in CD3+ cells (6.3 ± 1.46%) compared to the vehicle-treated group (4.92 ± 1.24%), but this was not significant (p=0.09).

The demonstration that T lymphocyte infiltration of tumours was increased in C5aR1-/- mice supports the suggestion by Markiewski and co-workers (2008) that C5a acts to promote MDSCs, which in turn inhibit cytotoxic T cell responses. Given that C5a has been reported to regulate the differentiation of T cell subsets (Fang et al., 2009; Hashimoto et al., 2010; Weaver et al., 2010), we investigated T lymphocyte subsets and showed a significantly higher percentage of tumour-infiltrating CD4+ cells in C5aR1-/- (3.98 ± 2.28% of total leukocytes; p=0.01) compared with vehicle-treated wild type (1.86 ± 0.87%), but no significant difference in C5aR1 antagonist-treated (2.62 ± 1.01%) mice. However, in contrast to Markiewski et al (2008) we found that CD8+ cells were significantly reduced in C5aR1- antagonist treated mice (1.76 ± 0.58 % of total leukocytes; p=0.04) compared with vehicle- treated wild-type mice (3.01 ± 1.4% ).

57

Figure 3.11. C5aR signalling influences tumour infiltrating leukocyte populations. Representative flow cytometric plots show leukocyte sub-populations in B16 melanoma tissue from WT, C5aR1-/- and WT mice treated daily with C5aR1A (1mg/kg/day), commencing from the time tumours became palpable (day 7 after injection of B16 cells). Data is expressed as % positive cells (mean ± SD; n=9/group); results representative of 2 independent experiments; *p< 0.05, **p<0.001 compared with the WT control; Kruskal Wallis test followed by Dunn’s multiple comparisons test.

3.3.6.3 Flow cytometric comparison of tumour-infiltrating leukocyte populations from C5aR1-/- and C5aR2-/- mice. Since C5aR2 inhibition was shown to promote melanoma growth, we next compared tumour infiltrating leukocyte populations from C5aR2-/- mice with those from C5aR1-/- and WT mice. In contrast to C5aR1-deficient mice, tumour infiltrating leukocyte populations in C5aR2-deficient mice were not significantly different from those of their wild-type counterparts (Figure 3.12).

58

Figure 3.12. Flow cytometric analysis of tumour infiltrating leukocyte populations from C5aR1- and C5aR2- deficient mice. Representative flow cytometric plots show leukocyte sub-populations in B16 melanoma tissue from WT, C5aR1-/- and C5aR2-/- mice. Data are expressed as % positive cells (mean ± SD; n=6/group); results representative of 2 independent experiments; *p< 0.05, **p<0.01, ***p<0.001 compared to WT controls; Kruskal Wallis test followed by Dunn’s multiple comparisons test.

3.3.6.4 Leukocyte populations in other tissues Since significant alterations in tumour-infiltrating leukocyte populations were observed in C5aR1-/- mice, we next examined leukocyte populations in blood, spleen and bone marrow (the major leukocyte reservoirs) and draining lymph nodes (the site of T cell maturation/activation) from healthy mice (without tumours) and tumour-bearing mice. Leukocyte populations in these tissues were compared with those within tumour tissue. Analysis of blood from healthy mice (Figure 3.13A) showed differences in frequencies of immune cell populations in C5aR1-/- compared with wild-type mice. Although the percentages of monocytes (CD11b+ Ly6C+), MDSCs (CD11b+GR1+) and macrophages (F480+) were slightly, but not significantly, higher in healthy C5aR1-/- mice, neutrophils (CD11b+ Ly6G+; 10.7 ± 4.98%) were significantly higher than in their wild-type counterparts (5.5 ± 2.4% of total leukocytes; P=0.02). Conversely the percentage of total T lymphocytes (CD3+) was significantly lower in healthy C5aR1-/- mice (10.84 ± 1.17%) compared with

59

wild type mice (25 ± 5.2%; P=0.0002). CD4+ and CD8+ lymphocyte populations were both significantly lower, comprising 3.68 ± 0.54% and 5.3 ± 0.9% respectively compared with 13.9 ± 3.4% (P=0.0002) and 9.5 ± 2.1% (P=0.0002) in wild-type mice. However, examination of CD4+ T cell subsets showed significantly higher percentages of Tregs (CD25+FoxP3+; P=0.02), Th1 (IFNγ+; P=0.0006), Th2 (IL4+; P=0.0006) and Th17 (IL17+; P=0.003) cells in healthy C5aR1-/- mice.

Leukocyte populations in blood of tumour bearing C5aR-/- mice showed a similar trend to their healthy counterparts (compare Fig 3.13 panels A and B), with the percentage monocytes (CD11b+ Ly6C+ P=0.005), neutrophils CD11b+ Ly6G+ (P=0.0012) and MDSCs (CD11b+GR1+ 17.8± 3.01% of total leukocytes; P=0.0002) higher than in tumour bearing wild-type mice. While total T lymphocytes (CD3+), CD4+ and CD8+ T lymphocyte populations in the circulation were significantly lower, all CD4+ subsets were significantly higher, suggesting increased CD4+ cell activation. It is interesting to note that, despite significantly lower levels of T cells in blood of C5aR1-/- mice compared with wild-type, the percentages of tumour infiltrating T cells (CD3+, CD4+) were significantly increased. Moreover, the proportions of tumour infiltrating Tregs were significantly reduced and Th2 cells significantly increased.

These results suggest an inverse relationship between leukocyte populations in the blood and tumour tissue. Whereas C5aR1-deficient mice (with or without tumours) have higher proportions of myeloid cell populations and fewer T lymphocytes, the proportions of monocytes and neutrophils within tumour tissue were similar, and the percentage of MDSCs significantly lower (5.4 ± 2.5% of total leukocyte; P=0.007) compared to wild-type mice (11.4 ± 5.6% of total cells). Importantly, total (CD3+) T lymphocyte infiltration of tumour tissue was significantly higher in C5aR1-/- (5.9 ± 1.4% of total leukocytes) compared with wild type (3.3 ± 0.7%; p=0.0006) mice. CD4+ cells were also significantly higher (P=0.007) but CD8+ cells were not significantly different. Analysis of CD4+ T cell subsets showed the proportion of tumour infiltrating Tregs was significantly lower in tumours from C5aR1-/- mice (P=0.0009), whereas Th2 cells were significantly higher (4.5 ± 2.3% of CD4+; P=0.02) compared to wild type (1.9 ± 0.8% of CD4+) mice. Although Th1 and Th17 cells were slightly (but not significantly) higher, Treg cells were significantly lower in C5aR1-/- mice.

60

61

Figure 3.13. Flow cytometric analysis of blood leukocyte populations from mice with and without tumours and comparison with tumour infiltrating populations. Representative flow cytometric plots show leukocyte sub-populations in blood from WT and C5aR1-/- mice without tumours (NT-WT, NT-C5aR1-/-; A), or bearing B16 melanoma (T-WT, T-C5aR1-/-; B) compared with tumour tissue from WT and C5aR1-/- mice (C). Data are expressed as % positive cells (mean ± SD; n=8/group); *p< 0.05, **p<0.001, Mann-Whitney test.

FACS analysis of draining (axillary and inguinal) lymph nodes (Figure 3.14) showed similar results to blood (Figure 3.13), with the percentage of monocytes, neutrophils and macrophages slightly (but not significantly) higher, but the percentage MDSCs significantly higher (P=0.007) in healthy C5aR1-/- compared with wild-type mice. In contrast to blood, total T lymphocytes (CD3+) were similar in both groups, as were CD8+ cells. CD4+ cells were reduced, but unlike blood in which all CD4+ subsets were significantly higher, only Th1 cells were higher in the lymph nodes of C5aR-deficient mice. Lymph nodes from tumour- bearing mice showed similar trends, but with neutrophils, MDSC and CD4+ subsets (Treg, Th2 and Th17) all significantly higher in C5aR1-/- mice.

62

63

Figure 3.14. Flow cytometric analysis of draining lymph node leukocyte populations from mice with and without tumours compared with tumour infiltrating populations. Representative flow cytometric plots show leukocyte sub-populations in draining lymph nodes from WT and C5aR1-/- mice without tumours (NT-WT, NT-C5aR1-/-; A) or bearing B16 melanoma (T-WT, T-C5aR1-/-; B) compared with tumour tissue from WT and C5aR1-/- mice (C). Data is expressed as % positive cells (mean ± SD; n=8/group); *p< 0.05, **p<0.001, Mann-Whitney test.

Examination of leukocyte populations in spleen showed significantly higher percentages of monocytes, neutrophils and MDSCs in healthy C5aR1-/- mice compared to wild type controls (Fig 3.15). Whereas the proportions of CD3+, CD4+ and CD8+ T cells were significantly lower, CD4+ subsets (Th1, Th2 and Th17) were all significantly higher. Spleens from tumour bearing mice showed a similar pattern to their healthy counterparts, with C5aR-/- mice exhibiting higher percentages of monocytes, neutrophils and MDSC, significantly reduced CD8+ T cells. As for lymph nodes the proportions of CD4+ subsets (Tregs, Th1, Th2 and Th17) were higher in C5aR-/- mice, possibly suggesting increased activation of CD4+ T cells.

64

65

Figure 3.15. Flow cytometric analysis of leukocyte populations in spleens from mice with and without tumours compared with tumour infiltrating populations. Representative flow cytometric plots show leukocyte sub-populations in spleen from wild type (C57BL/6J) mice, C5aR1 deficient mice without tumour (NT-WT, NT-C5aR1-/-; A), or bearing B16 melanoma (T-WT, T-C5aR1-/-; B) compared with tumour tissue from wild type (C57BL/6J) mice and C5aR1 deficient mice (WT, C5aR1-/-; C). Data are expressed as % positive cells (mean ± SD; n=8/group); *p< 0.05, **p<0.001, Mann-Whitney test.

3.3.7 Effect of C5aR deficiency on cytokine/chemokine expression C5a is a potent chemoattractant for leukocytes, but also induces the expression of pro- inflammatory cytokines such as IL-6 and TNF-α (Montz et al., 1991). Tumour tissue is also known to express a range of chemokines and their receptors (Conti and Rollins, 2004). In order to gain insight into how C5a regulates changes in tumour infiltrating populations, we investigated cytokine (Fig 3.16A, B) and chemokine (Fig 3.16C, D) expression in tumour tissue extracts and plasma from wild-type and C5aR-deficient mice. Based on our FACS findings, we chose chemokines previously reported to regulate tumour infiltrating leukocyte populations. Except for IFN-γ which was significantly increased in plasma from C5aR1- deficient mice, and IL12p40 which was significantly reduced in tumour tissue from these mice, expression of other cytokines was not altered in either tumour tissue or plasma from

66

C5aR1- or C5aR2-knockout compared with their wild-type counterparts (Fig 3.16A, B). Analysis of chemokine expression revealed significant reductions in MCP-1 (CCL2; P=0.0003), MIP1α (CCL3; P=0.03), MIP1β (CCL4) (P=0.03), MIP2 (CXCL2; P=0.03) and RANTES (CCL5; P=0.001) in tumour tissue from C5aR1-/- mice; CCL2, CXCL1 and CXCL2 were significantly lower in tumours from C5aR2-/- mice (Fig 3.16C). C5aR2-/- Plasma CCL2 (P=0.01) and CCL4 (P=0.02) were reduced only in C5aR2-/- mice (Fig 3.16D).

67

Figure 3.16. Influence of C5aR deficiency on cytokine and chemokine expression. Cytokine (A, B) and chemokine (C, D) levels in tumour tissue (A, B) and plasma (C, D) from WT (●), C5aR1-/- (○) and C5aR2-/- () mice. Data expressed as pg/ml (for plasma) or pg/mg (for tumour tissue) (mean ± SD, n=6-8/group); *p< 0.05, **p<0.01, Mann Whitney test.

68

3.4 DISCUSSION

The complement system is a key component of the innate immune system, and the activation product anaphylatoxin C5a is a potent chemoattractant and pro-inflammatory mediator responsible for mediating many of the effects of the immune system (Manthey et al., 2009). The present study implicates C5a-C5aR1 signalling for a role in promoting melanoma growth. In accord with previous studies in murine models of cervical, lung and ovarian cancer (Corrales et al., 2012; Markiewski et al., 2008b; Nunez-Cruz et al., 2012), the results show that treatment of mice with the C5aR1 antagonist (PMX53) developed at the University of Queensland (Finch et al., 1997) inhibits the growth of primary B16 melanoma. Similar anti-tumour effects were observed in mice lacking C5aR1, with tumour initiation and growth significantly slower than in their wild-type counterparts. Importantly, this study is the first to show that tumour growth is increased in mice lacking the non-signalling receptor, C5aR2, a result in accord with the postulated role of C5aR2 as an anti-inflammatory modulator of C5aR1 activity (Bamberg et al., 2010; Scola et al., 2009).

Investigation of the mechanisms by which C5a acts on tumour growth showed that B16 melanoma cells express C5a receptors C5aR1 and C5aR2, albeit at lower levels than J774 macrophages. Furthermore, C5aR expressed by melanoma cells is functional, with C5a stimulation leading to signal activation via MAPK pathways, thus indicating that direct effects of C5a on B16 melanoma cells are possible. Similar to the current findings, Hawksworth and co-workers (2014) have recently demonstrated that human embryonic stem cells respond to C5a through ERK and AKT pathways, despite these cells having no detectable calcium mobilisation (et al., 2014). However, our inability to demonstrate any effect of C5a on B16 cell proliferation or migration suggests that the anti-tumour effects of C5aR1 antagonism are predominantly indirect, via the host immune system. This possibility is supported by the demonstration that wild-type (C5aR-expressing) B16 melanomas grow more slowly in C5aR1-deficient mice. Hence this study focussed on how C5a-C5aR signalling influences immune cell populations in tumour-bearing mice.

Examination of leukocyte populations in healthy mice showed that C5aR1 deficiency is associated with intrinsic differences in circulating leukocyte populations. Healthy and tumour-bearing mice showed similar patterns, with higher percentages of myeloid cells but lower lymphocytes (CD3+, CD4+, CD8+) in blood, lymph node and spleen from C5aR1-/-

69

compared with their wild-type counterparts. Interestingly the proportions of CD4+ subpopulations (Treg, Th1, Th2 and Th17) were increased in C5aR1-/- mice. However tumour-infiltrating leukocyte populations showed a different pattern. As previously demonstrated in other cancer models (Corrales et al., 2012; Markiewski et al., 2008a), tumour infiltrating MDSC and macrophages were significantly reduced in C5aR1-deficient mice. MDSCs are potent inhibitors of anti-tumour immunity in vivo, inducing Tregs which down- regulate cell mediated immunity and promote tolerance to tumour antigens (Serafini et al., 2008). In addition to their presence within tumours, MDSCs accumulate in peripheral blood, bone marrow, spleen and lymph nodes (Haile et al., 2008; Kusmartsev et al., 2005; Marigo et al., 2008; Serafini et al., 2008; Sinha et al., 2008).

Although the present study found that C5aR1-/- mice had reduced tumour infiltrating MDSC, these mice (healthy and tumour-bearing) had higher percentages of MDSC in blood, spleen and lymph nodes than their wild-type counterparts, suggesting that the infiltration of these cells into the tumour may be regulated by C5a, a strong chemoattractant for myeloid cells (neutrophils, monocytes and macrophages) (Guo and Ward, 2005). We also demonstrated the down-regulation of several chemokines within the tumour microenvironment of C5aR- deficient mice, including MCP-1 (CCL2), MIP1β (CCL4) and RANTES (CCL5) which are known to regulate the movement of MDSC and maintain their suppressive activity (Boelte et al., 2011; Sevko and Umansky, 2013; Zollo et al., 2012). In particular, CCL2 and its receptors CCR2 and CCR4 have been reported to be responsible for the accumulation of monocytic MDSCs in melanoma and prostate cancer (Lesokhin et al., 2012; Zhang et al., 2010; Zollo et al., 2012), and high levels of CCL2 expression have been associated with poor prognosis in human mammary carcinoma (Sato et al., 1995; Ueno et al., 2000; Valkovic et al., 2002). Others have reported a role for CCL3 and CCL5 in regulating the migration of monocytic MDSC (Connolly et al., 2010; Gama et al., 2012; Sawanobori et al., 2008). The presence of macrophages in melanoma is also positively correlated with aggressiveness of disease (Varney et al., 2005), and the lower levels of CCL2, CCL3 and CCL4 may also explain the reduction in tumour associated macrophages (Roca et al., 2009; Solinas et al., 2009) in C5aR-deficient mice. Although not investigated in the present study, tumour associated macrophages and MDSC are capable of promoting angiogenesis (Leek et al., 1996; Li et al., 2002; Onita et al., 2002; Takanami et al., 1999; Valkovic et al., 2002; Yang et

70

al., 2004), and thus higher expression of CCL2 in melanoma tissue from wild-type mice may contribute to increased angiogenesis (Boelte et al., 2011) and hence enhanced tumour growth.

In contrast to Markiewski and co-workers who showed that the tumour promoting effects are due to inhibition of CD8+ cytotoxic T cells, the present study suggests that the effects of C5a on melanoma growth are due to changes in tumour infiltrating CD4+ T cell sub-populations. Specifically tumours from C5aR1-/- mice showed a significantly lower percentage of Tregs but increased Th2 cells. This result is in contrast to previous reports showing that C5aR signalling reduces Treg function (Kwan et al., 2013; Strainic et al., 2013), but in accord with the findings of Gunn and co-workers (2012) that C5a promotes Treg differentiation in vitro and splenic Treg cells were significantly increased in the presence of high C5a levels. Although Corrales et al (2012) found no effect of C5aR1 antagonism on CD4+, CD8+ or Treg cells, key immunosuppressive molecules associated with Treg activity, including CTLA-4, IL-6, IL-10 and ARG1, were down-regulated. Similar results were obtained by Vadrevu et al (2014)who showed in a murine model of metastatic breast cancer, that C5aR1 signalling suppresses T-cell responses in the lungs via recruitment of MDSCs and generation of Treg cells. In contrast to our results, this group showed that C5aR1 blockade led to increased recruitment of both CD4+ and CD8+ T cells and induction of Th1/Tc1-biased T-cell responses. CD8+ T cells are generally thought to be responsible for cytotoxic anti-tumour responses, whereas Th2-mediated immune responses favour tumour growth through promoting angiogenesis and mediating immunosuppression (Ellyard et al., 2007; Nishimura et al., 1999). However, CD4+ Th2 have been reported to play an important role in the response to MHC class I negative tumours which are resistant to CD8+ cytotoxic T lymphocytes (Mattes and Foster, 2003). Moreover the Th2 response has been shown to be important for immunity against B16 melanoma (Fang et al., 2009). Thus, in the B16 melanoma model, the up-regulation of Th2 cells (and concomitant reduction in CD8+ T cells) in the absence of C5aR1 signalling may be consistent with an effective anti-tumour response. The demonstration that expression of CCR5 ligands CCL3, CCL4 and CCL5 are reduced in tumours from C5aR1-, but not C5aR2-deficient mice suggests that CCR5 may be an important contributor to the effects of C5aR1 on the anti-tumour immune response. CCR5 is expressed on various cell populations including macrophages, dendritic cells and memory T cells (Rottman et al., 1997). CCR5 and its ligands are also expressed by a range of tumours including breast, melanoma, gastric (Fukui et al., 2005), colonic (Cambien et al., 2011) and

71

pancreatic cancers (Vaday et al., 2006). There is evidence that the CCL5/CCR5 axis is associated with melanoma progression due to increased levels of immunosuppressive cells. CCR5 deficiency has been shown to cause apoptosis of melanoma cells (Song et al., 2012), while melanoma growth is delayed in CCR5-deficient mice. CCL5 is a chemoattractant for T cells, macrophages, eosinophils and basophils (Aldinucci and Colombatti, 2014). CCL5 plays a key role in inducing apoptosis of tumour infiltrated lymphocytes (Mellado et al., 2001), with activity up-regulated by the other CCR5 ligands, CCL3 and CCL4. Recently, Schlecker et al. (2012) demonstrated in a mouse model of melanoma, that tumour-infiltrating monocytic-MDSCs directly attract Tregs via CCR5 and that intra-tumoural injection of CCL4 or CCL5 increased tumour-infiltrating Tregs.

In summary, the results presented here suggest the potential of C5aR signalling as a therapeutic target for melanoma. In addition to demonstrating a role for C5aR1 signalling in promoting the growth of murine melanoma, this thesis provides the first evidence for a role of the alternate receptor C5aR2 in modulating the effects of C5a on tumour growth. In accord with previous studies in other tumour types, the results suggest that C5a mediates its effects via the immune system, with tumour infiltrating leukocyte populations altered in C5aR1- deficient mice. Although healthy C5aR-deficient mice are shown to have altered circulating leukocyte populations compared with their wild-type counterparts, the results suggest that leukocyte infiltration into the tumour is tightly regulated – either directly by C5a, or indirectly by other chemokines within the tumour microenvironment.

Although like previous studies, this study demonstrates a tumour promoting role for C5a, further research is required to fully elucidate the mechanisms by which C5a acts. Clearly further investigations are required to determine whether the observed changes in leukocyte populations reflect changes in the anti-tumour response, and whether similar mechanisms apply in human melanoma and other cancers. Research to date suggests that C5a has different effects, depending on the tumour type, possibly due to differences in the immunogenicity of the different tumours, the type of response mounted, the strain of mice used and the levels of C5a present. Indeed Gunn et al (2012) showed that the effect of C5a is concentration-dependent, with high levels promoting tumour growth by accumulation of neutrophilic (CD11b+GR1+) MDSC and reduction of the anti-tumour CD4+ and CD8+ T cell populations, whereas low C5a levels were associated with reduced tumour progression and

72

increased IFN γ producing CD4+ and CD8+ T cells in spleen and tumour draining lymph nodes. These different effects highlight the fact that cancers are many different diseases, with different responses dependent on tumour type, the immune competence of the host and levels of complement activation in the tumour microenvironment. Further research is therefore warranted, in order to progress therapeutic targeting of C5aR in melanoma, to a clinical reality.

73

74

CHAPTER 4

THE COMPLEMENT C3A RECEPTOR CONTRIBUTES TO MELANOMA TUMOURIGENESIS BY INHIBITING NEUTROPHIL AND CD4+ T CELL RESPONSES

Jamileh Alham Nabizadeh

75

4.1 INTRODUCTION

An essential part of the innate immune system, the complement system is comprised of plasma and membrane-bound proteins whose overall function is to regulate inflammation, facilitate immune defense mechanisms and maintain tissue homeostasis (Ricklin et al., 2010). Chapter 3 of this thesis demonstrated a tumour-promoting role for the complement activation product C5a in murine melanoma. In addition to C5a, complement activation leads to the production of another peptide, C3a, which is less well studied, but generally thought to have similar effects (Klos et al., 2013). This simplistic view of C3a however has been challenged recently (Coulthard and Woodruff, 2015).

Up-regulated levels of serum C3a have been reported in many cancers including breast, colorectal and oesophageal cancer (Maher et al., 2011; Medina-Echeverz et al., 2014), and gene expression data identifies the up-regulation of C3a receptor (C3aR) by some tumours, including melanoma (Xu et al., 2008). Despite the evidence for C3a and C3aR expression by tumour tissue, the majority of studies to date have focused on the role of C5a (Corrales et al., 2012; Gunn et al., 2012; Kim et al., 2005; Markiewski et al., 2008a; Nunez-Cruz et al., 2012). Thus, the current study used murine models of melanoma and colonic carcinoma to determine whether C3aR, like C5aR1 and C5aR2, also plays a role in tumourigenesis.

C3aR is expressed by cells of myeloid origin (including neutrophils, mast cells, monocytes, macrophages and dendritic cells) as well as non-immune cells in lung, liver, muscle and other tissues. Activation of C3aR can induce both pro- and anti-inflammatory effects, depending on the cell type and disease context (Coulthard and Woodruff, 2015). This Chapter explored the role of C3aR signalling in melanoma growth and regulation of leukocyte populations in the tumour microenvironment. The results demonstrate that development and growth of syngeneic primary B16 and SM1WT1 (BRAFV600E mutant) murine melanomas are retarded in the absence of C3aR signalling, and that the anti-tumour effects of C3aR inhibition are linked to increased tumour infiltration by neutrophils and enhancement of the adaptive T-cell anti-tumour response. Similar protective effects of C3aR deficiency are demonstrated in a colorectal cancer model. These results suggest the potential of C3aR as a novel therapeutic target for melanoma and potentially other intractable tumours.

76

4.2 EXPERIMENTAL OUTLINE

To investigate the role of C3aR signalling in melanoma growth, C3a receptor knockout (C3aR -/-) or wild-type (WT; C57BL/6J) mice were injected s.c. with murine B16-F0 melanoma (2.5 x105 cells/mouse) or BRAFV600E mutant SM1WT1 melanoma cells (2x106 cells/mouse) (Section 2.4). For some experiments, C57BL/6J mice received daily injections of either C3aR antagonist (SB290157; 1mg/kg/day i.p.), C5aR1 antagonist (PMX53; 1mg/kg/day s.c.) or vehicle only (0.9% saline or 5% glucose solution) control, commencing once tumours became palpable (approx. day 7 after tumour cell injection). For most experiments, mice were euthanized at day 14 (or once the largest tumour reached an area of approx. 200mm2), blood was collected by cardiac puncture, tumours, spleen, draining lymph nodes (inguinal, axillary and brachial) and femurs excised for immunostaining (Section 2.6) or FACS (Section 2.11); tumours were weighed. For ‘survival’ studies (Section 2.4), the time taken for each tumour to reach a pre-determined size (150 mm2) was recorded, then mice euthanized.

To investigate receptor expression, human (MM608, MM370 and MM649) murine (B16-F0) melanoma and J774 macrophages (positive control) cultured on coverslips or OCT-embedded tumour sections were incubated with antibodies specific for human and mouse C3aR (Bachem, Bubendorf, Switzerland) followed by FITC conjugated secondary antibodies (Section 2.6). Cell suspensions stained with the same antibodies were analysed by flow cytometry (Section 2.11). To determine whether these receptors are capable of signal activation, B16 cells were treated with recombinant human C3a (10nM) or the selective mouse C3aR agonist WWGKKYRASKLGLAR (Proctor et al., 2009; Wu et al., 2013) for the times indicated in the experiment, then cell lysates subjected to Western analysis for activation of ERK or AKT (Section 2.8).

To investigate the effect of C3aR agonist on B16 migration, cells were seeded at confluence (2x105 cells/cm2) into a 24 well plate, adhered overnight and then confluent monolayers scratched with a pipette tip before being treated for 24 hours with C3aR agonist (10-6 mol/L); control wells were incubated with medium alone or containing 5% FCS. Migration was quantified by measuring cell-free areas at 0 and 24 hours using Image-J software (Section 2.10). To investigate the effect of neutrophil depletion, WT and C3aR-/- mice were injected i.p. with anti-Ly6G antibody (1A8, selective for neutrophils, 0.1 mg/mouse; Bio X Cell

77

(Daley et al., 2008) or isotype control (rat IgG2a, 2A3) 24 hours prior to s.c.. injection of B16 cells, and then every 3 days until the largest tumour reached 200mm3 (Section 2.4). Mice were then euthanized, and blood and tumour tissue removed and weighed.

To further investigate the role of C3aR signalling in melanoma growth, tissue sections were also incubated with antibodies to leukocyte populations (Section 2.6). Single cell suspensions of blood, tumour, spleen, lymph node and bone marrow cell were prepared for FACS analysis (Section 2.11). Protein extracted from snap frozen tumour and plasma tissues from other mice used for multiplex ELISA for a panel of chemokine/ cytokines (Section 2.12).

To determine whether C3aR signalling contributes to other tumour types, C57BL/6J mice were injected with MC38 colon carcinoma (1x106 cells/mouse), and tumour growth monitored as above.

To determine the relevance to human cancer, neutrophil infiltration in human tissue was examined by staining human melanoma tissue arrays (US BioMax Inc, Rockville, MD) for neutrophil esterase (Sigma-Aldrich; according to the manufacturer’s instructions).

78

4.3 RESULTS

4.3.1 C3a-C3aR signalling contributes to melanoma development and growth To determine whether C3a-C3aR signalling influences melanoma development and growth, we first compared the growth of s.c. implanted B16.F0 melanoma cells in C3aR-deficient (C3aR-/-) and wild-type (WT) C57Bl/6J mice. The rate of tumour growth in C3aR-/- mice was significantly retarded compared with WT mice (Fig 4.1A). Analysis of excised tumour weight at termination (day 14) confirmed that tumours were markedly smaller (23% of control) in C3aR-/- mice (Fig 4.1B, C). Further, survival of C3aR-/- mice (i.e. euthanasia once tumours reached the pre-determined size of 150 mm2) was increased by 70% to 26.6 ± 1.4 days, compared with 15.7 ± 0.7 days for the WT controls (Fig 4.1D). These results suggest that host C3aR signalling promotes B16 tumour growth.

4.3.2 Effect of combined C3aR/C5aR1 blockade on melanoma growth Since the downstream activation product C5a has been demonstrated to promote tumour growth (Corrales et al., 2012; Gunn et al., 2012; Markiewski et al., 2008a), we next investigated whether the tumour inhibitory effects observed in C3aR-/- mice could be augmented by C5a receptor (C5aR1) antagonism. For this experiment, melanomas were induced in WT and C3aR-/- mice. Once tumours became palpable (day 8), mice were administered daily s.c. injections of C5aR1 antagonist (C5aR1A; PMX53) or vehicle alone. As previously shown in murine tumour models (Corrales et al., 2012; Gunn et al., 2012; Markiewski et al., 2008a) including B16 melanoma (Chapter 3), C5aR1 antagonism inhibited melanoma growth in WT mice (Fig 4.1E, F). However, the reduction in tumour growth observed in C3aR-/- mice was not augmented by C5aR1 inhibition, indicating that the contribution of C3aR signalling to melanoma growth is at least as potent as C5aR1 signalling, and that the inhibition of either receptor is sufficient to impair tumour growth.

79

Figure 4.1. C3aR deficiency retards B16 melanoma growth. Tumour area (A) and excised weight (B) 14 days post-tumour induction in C57BL/6J (wild-type; WT ●) and C3aR deficient (C3aR-/- ) mice (data expressed as mean ± SD from 4 independent experiments; n=8/group). Photographs (C) show representative tumours from mice WT and C3aR-/- mice. Kaplan-Meier curve (D) shows increased survival of C3aR-/- compared with WT mice; the terminal event is euthanasia once tumours reach maximal size (n=11/group). Tumour areas (E) and weight (F) of excised tumours from WT or C3aR-/- mice treated from day 7 with either saline (vehicle; WT ●, C3aR-/-) or C5aR antagonist, PMX53 (1mg/kg/day; WT , C3aR-/-) (mean ± SD n= 7/group); **p<0.01, ***p<0.0005, ****p<0.0001, Permutation test (A and B), log-rank (Mantel-Cox) test (D), Mann-Whitney test (E) and Kruskal Wallis test followed by Dunn’s multiple comparisons test (F).

80

4.3.3 C3aR blockade inhibits the growth of established primary melanoma The potent anti-tumour results in the C3aR-/- mice prompted us to determine the potential of C3aR therapeutic inhibition. We therefore next investigated the therapeutic potential of a C3aR antagonist (C3aRA; SB290157) on the growth of established melanoma. As shown in Fig 4.2A, tumour progression was dramatically retarded by daily i.p. injection of C3aRA (commencing day 7), with tumour area remaining relatively constant from this time, and significantly smaller than in the vehicle-treated control group; excised tumour weights at day 14 were also significantly smaller in the C3aRA treatment group (Fig 4.2B, C). Similarly, survival rates were significantly improved by treatment with C3aRA (Fig 4.2D). Together these data suggest C3aR as a potential therapeutic target for the treatment of established melanomas.

Figure 4.2. C3aR blockade inhibits the growth of established primary melanoma. Tumour areas (A) and weight (B) of excised tumours from C57Bl/6J mice treated from day 7 with either saline (vehicle, ●) or selective C3aR antagonist (C3aRA; SB290157, 1mg/kg/day, ). Data expressed as mean ± SD from 3 independent experiments (n=9- 10/group). Photographs (C) show representative tumours from mice treated with vehicle alone or C3aR antagonist. Kaplan-Meier curve (D) shows increased survival of mice treated with C3aRA compared with vehicle alone; the terminal event is euthanasia once tumours reach maximal size; (n=7/group). **p<0.01, ****p<0.0001, Permutation test (A), Mann-Whitney test (B) and log-rank (Mantel-Cox) test (D).

81

4.3.4 Melanoma cells express functional C3aR

4.3.4.1 Immunofluorescence staining In of our results showing C5aR expression by murine melanoma cells (Chapter 3), along with the recent demonstration that tumour-derived complement proteins can promote ovarian tumour growth via autocrine mechanisms (Cho et al., 2014), we determined whether melanoma cells also express C3aR. This was confirmed by immunostaining (Fig 4.3A) and FACS analysis which showed C3aR expression by cultured B16 cells (Fig 4.3C), albeit at lower levels than for J774 macrophages which are known to have high levels of C3aR expression. Human melanoma cell lines also expressed low levels of C3aR (Fig 4.3B, D).

4.3.4.2 Signal activation Although stimulation of B16 cells with recombinant human C3a or a selective C3aR agonist failed to initiate a calcium mobilization response (not shown), ERK and AKT signalling pathways were activated (Fig 4.3E), suggesting that the receptors are functionally coupled. Stimulation of cells with a C3aR agonist had no significant effect on tumour cell proliferation (data not shown), but cell migration was significantly increased (Fig 4.3F), suggesting that C3a can exert effects directly on tumour cells.

82

Figure 4.3. B16 melanoma cells express functional C3aR. Photomicrographs show immunostaining of (A) cultured B16 melanoma cells and J774 macrophages with Alexa- 647-conjugated anti-mouse C3aR (orange), and (B) human melanoma cell lines with anti- human C3aR (green); nuclei are counterstained with Hoechst 33342 (blue); scale bar = 25µm. Representative FACS profiles for (C) cultured B16 melanoma (blue line) and J774 (orange line) cells stained with anti-C3aR or isotype control (red line) and (D) human melanoma cell lines..Representative immunoblots (E) show levels of phosphorylated ERK1/2 or phosphorylated AKT following stimulation of B16 cells with recombinant human C3a (hC3a; 10-8 mol/L) for 0-60 mins. Immunoblotting for total ERK or AKT was used as loading control. Densitometry of bands shows ERK or AKT activation relative to the untreated control (0 mins). Histogram (F) shows B16 cell migration following treatment with DMEM alone or containing C3a agonist (C3a-A; WWGKKYRASKLGLAR, 10-6 mol/L) or 5% FCS for 24 hours. Data expressed as mean ± SD; *p<0.05, ****p<0.0001; one-way ANOVA (F).

83

4.3.5 Tumour-infiltrating leukocyte populations are altered in C3aR deficient mice

4.3.5.1 Immunofluorescence staining of tumour tissue Despite our demonstration that B16 melanoma cells are able to respond to C3a, our in vivo experiments showed that the growth of C3aR-expressing B16 melanoma cells was markedly inhibited in C3aR-deficient mice (Fig 4.1), indicating a predominant role for host-expressed C3aR in the anti-tumour response. To further investigate the effects of host-expressed C3aR on tumour growth, we compared tumour infiltrating leukocyte populations in C3aR-/- and WT mice. Immunofluorescence staining of tumour tissue demonstrated a band of (CD45+) leukocytes encapsulating subcutaneous B16 tumours (Fig 4.4A, B), with cells also infiltrating tumour tissue in both C3aR-/- and WT mice (Fig 4.4C, D). Many tumour infiltrating leukocytes stained positive for neutrophils (Ly6G+; Fig 4.4E, F), with neutrophil numbers apparently increased in C3aR-deficient mice. Macrophages (F4/80+; Fig 4.4G, H) and T + lymphocytes (CD3 ; Fig 4.4I, J) were also evident.

Figure 4.4. Leukocyte infiltration of B16 melanomas from C3aR-deficient and wild- type mice. Immunostaining of B16 tumours from WT and C3aR -/- mice shows CD45+ cells (green) encapsulating the tumour tissue (A, B; dotted line indicates leukocyte ‘capsule’), and within tumour tissue (C, D). Many tumour infiltrating cells are neutrophils (Ly6G+; green; E, F), but macrophages (F4/80+; red; G, H), and T lymphocytes (CD3+; green; I, J), are also present. Tissue stained with an isotype control antibody (K). Nuclei are stained with Hoechst 33342 (blue). Scale bar =20µm.

4.3.5.2 Flow cytometric analysis of tumour tissue from C3aR-deficient mice To quantitate potential alterations in the immune response to B16 melanomas, we analysed tumour infiltrating leukocyte populations by flow cytometric analysis (Fig 4.5A). Although the proportion of total leukocytes (CD45+) within tumour tissue was not significantly altered in C3aR knockout mice (5.8 ± 2.7 % versus 4.7 ± 2.9 % in WT), the contribution of

84

CD11b+Ly6G+ cells (neutrophils) was significantly higher (16.6 ± 8.1 % of total leukocytes versus 9.7 ± 4.8 % in WT), a result that is in accord with a prior report that C3aR deficiency causes the egress of neutrophils from bone marrow (Wu et al., 2013). However, although previous studies have demonstrated differences in the activation status (Fridlender et al, 2009; Piccard et al, 2012) of tumour-associated neutrophils, we found no significant difference in neutrophil expression of activation markers CD62L or CXCR4 (not shown). The relative contributions of monocytes (CD11b+Ly6C+) and total T lymphocytes (CD3+) were also significantly higher, but the proportion of macrophages (F4/80+) (Fig 4.5A) B lymphocytes (data not shown) were unaltered. In contrast to previous demonstrations that C5a promotes tumour growth via recruitment of myeloid-derived suppressor cells (MDSC) (Corrales et al., 2012; Gunn et al., 2012; Markiewski et al., 2008a), the percentages of tumour infiltrating CD11b+GR1+ cells) were not significantly different in C3aR-/- compared with WT mice. However, since the GR1 antibody used to identify MDSC recognises a common of Ly6G and Ly6C (Fleming et al., 1993), it is not possible to discriminate between neutrophils and granulocytic MDSC. Investigation of tumour infiltrating CD3+ T lymphocyte subsets showed an increase in the proportion of CD4+ T lymphocytes, but the proportion of CD8+ T lymphocytes was not significantly different between WT and C3aR-/- mice; CD4/CD8 ratios were 2.99 and 2.64 respectively. While the proportion of regulatory T cells (CD4+CD25+Foxp+; Tregs) was unchanged, Th1 (CD4+IFNγ+), Th2 (CD4+IL4+) and Th17 (CD4+IL17A+) subsets were all significantly higher in tumours from C3aR-/- mice (Fig 4.5A).

To investigate the distribution of these immune cell populations, we next analysed leukocyte sub-populations in bone marrow, blood, draining lymph nodes and spleen from tumour- bearing wild-type and knockout mice. Flow cytometric analysis of bone marrow cells (Fig 4.5B) revealed a reduction in monocytes, but no significant difference in the proportion of neutrophils in C3aR-/- mice. Analysis of blood from C3aR-/- and WT mice showed no difference in monocytes or T lymphocyte populations but the percentage of neutrophils was reduced (Fig 4.5C), possibly reflecting the increased sequestration of neutrophils to the tumour site. Although no significant differences were detected in neutrophils, monocytes or total T cell populations, the percentages of Th1, Th2 and Th17 cells were increased in draining lymph nodes of C3aR-/- mice (Fig 4.5D). Within the spleen, the proportions of CD11b+Ly6C+ (monocytes) and CD11b+GR1+ cells were lower in C3aR-/- mice, although

85

CD11b+Ly6G+ (neutrophils) were not altered. Total T lymphocytes were also significantly higher, as was the proportion of CD4+ cells, with Tregs, Th1 and Th17 CD4+ subsets all increased (Fig 4.5E). Analysis of leukocyte populations in healthy mice showed that the proportion of bone marrow (CD11b+Ly6G+) neutrophils was slightly lower in C3aR-deficient compared with their wild-type counterparts (Fig 4.6A), although this was not significant. However there was a significantly higher percentage of circulating (CD11b+Ly6G+) neutrophils (Fig 4.6B). Other leukocyte populations were similar in blood and bone marrow of wild-type and C3aR-deficient mice.

86

Figure 4.5. Tumour-infiltrating leukocyte populations are altered in C3aR deficient mice. Percentages of leukocyte sub-populations in tumour tissue (A), bone marrow (B), blood (C), draining lymph nodes (DLN; D) and spleen (E) from WT and C3aR-/- mice: total leukocytes (CD45+), monocytes (CD11b+Ly6C+), neutrophils (CD11b+Ly6G+), MDSCs (CD11b+GR1+), macrophages (F4/80+), total (CD3+), CD4+ and CD8+ T lymphocytes;. CD4+ T lymphocyte subsets, Treg (CD4+CD25+FoxP3+), Th1 (CD4+IFNγ+), Th2 (CD4+IL4+) and Th17 (CD4+IL17+). Data expressed as % positive cells (mean ± SD); results representative of 2 independent experiments (n=7-8/group). *p< 0.05, **p<0.01, ***p<0.001, ****p<0.0001, Mann Whitney test.

87

Figure 4.6. Leukocyte populations in healthy C3aR deficient and wild-type mice. Percentages of leukocyte sub-populations in bone marrow (A) and blood (B) from WT and C3aR-/- mice: total leukocytes (CD45+), monocytes (CD11b+Ly6C+), neutrophils (CD11b+Ly6G+), MDSCs (CD11b+GR1+), macrophages (F4/80+), total (CD3+), CD4+ and CD8+ T lymphocytes. Data expressed as % positive cells (mean±SD); representative of 2 independent experiments (n=7-8/group). *p< 0.05, Mann Whitney test.

4.3.6. C3aR antagonism alters tumour infiltrating leukocyte populations FACS analysis of tumour infiltrating leukocyte populations in mice treated with SB290157 (C3aRA; Fig 4.7A), showed a similar trend to C3aR-/- animals, with the percentage of tumour infiltrating neutrophils (CD11b+Ly6G+) increased by drug treatment. However, as for C3aR-/- mice, the proportion of CD11b+GR1+ cells was not significantly altered in C3aRA- treated mice, but tumour-associated macrophages were reduced. As observed in C3aR-/- mice, the percentages of total and CD4+ T lymphocytes were higher in tumours following drug treatment; proportions of Th1, Th2 and Th17 subsets were also increased. Bone marrow (Fig 4.7B) showed greater effects on myeloid cell populations than were observed in C3aR knockout mice, with C3aR antagonism causing significant reductions in all myeloid cell populations (identified by Ly6C, Ly6G and GR1 expression). Whereas blood neutrophils were reduced in knockout mice, they were increased in antagonist-treated mice. The proportion of CD11b+GR1+ cells remained unchanged, but macrophages, CD8+ and Treg cells reduced significantly (Fig 4.7C).

88

89

Figure 4.7. C3aR antagonism alters tumour infiltrating leukocyte populations. Percentages of leukocyte sub-populations in tumour tissue (A), bone marrow (B), blood (C) and spleen (D) from WT mice treated with SB209157 (1mg/kg/day) or vehicle commencing once tumours became palpable: total leukocytes (CD45+), monocytes (CD11b+Ly6C+), neutrophils (CD11b+Ly6G+), MDSCs (CD11b+GR1+), macrophages (F4/80+), total (CD3+), CD4+ and CD8+ T lymphocytes; results of 2 independent experiments, n=16-17/group. CD4+ T lymphocyte subsets, Treg (CD4+CD25+FoxP3+), Th1 (CD4+IFNγ+), Th2 (CD4+IL4+) and Th17 CD4+ (CD4+IL17+). Data expressed as % positive cells (mean ± SD; n=9-10/group); *p< 0.05, **p<0.01, ***p<0.001, ****p<0.0001, Mann-Whitney test.

90

Although macrophages were unaltered in draining lymph nodes from C3aRA-treated mice (Fig 4.7D), total T lymphocytes, T lymphocyte subsets (CD4+ and CD8+) and Th2 cells were all significantly increased. Although the proportions of CD11b+Ly6C+ (monocytes), CD11b+Ly6G+ (neutrophils) and F4/80+ (macrophages) were not significantly altered in spleens from C3aRA-treated mice, CD11b+GR1+ cells were significantly lower. Whereas CD8+ T cells were significantly lower, total (CD3+) and CD4+ T lymphocytes were significantly higher in the drug-treated group, coincident with significant increases in Treg and Th2 cells (Fig 4.7E).

4.3.6 Neutrophil depletion rescues the tumour inhibitory effect of C3aR deficiency Given the FACS data showing a clear increase in tumour-infiltrating neutrophils in C3aR- deficient and antagonist-treated mice, we proposed that the influx of neutrophils into the tumour could be one reason for the reduction in tumour growth. To determine whether neutrophils actively contribute to the tumour inhibition observed in the absence of C3aR signalling, we used a monoclonal antibody (α-Ly6G; 1A8) to deplete these cells (Daley et al., 2008). The efficacy of neutrophil depletion from WT and C3aR-/- mice was confirmed by FACS analysis of blood (Fig 4.8A). Neutrophil depletion slowed tumour growth in WT mice, although excised tumour weight was not significantly reduced (Fig 4.8B, C). Conversely, the lack of neutrophils reduced the tumour inhibitory effects observed in C3aR-/- mice, such that excised tumour weight was not significantly different from that of WT mice treated with the isotype control antibody (I.C., 2A3; Fig 4.8C). FACS analysis (Fig 4.8D) also demonstrated that neutrophil depletion was associated with a significant reduction in tumour-infiltrating CD4+ T lymphocytes in C3aR-/- mice, with levels not significantly different to those in isotype control-treated mice, thus suggesting that neutrophils may be regulating the secondary T cell response to tumour. Interestingly, anti-Ly6G treatment also removed the majority of CD11b+GR1+ cells in both wild-type and knockout mice, indicating that in this tumour model the majority of MDSC are granulocytic.

91

4.3.7 Neutrophils are associated with human melanoma Given our finding of a neutrophilic response in mouse melanoma models, we next determined whether neutrophils may also be associated with human melanoma tissue. Staining of melanoma tissue arrays for neutrophil esterase showed neutrophils in some biopsies, thus suggesting the potential to use C3aR-modulating drugs to manipulate neutrophil populations in human melanoma.

Figure 4.8. The tumour inhibitory effect of C3aR deficiency is rescued by neutrophil depletion. FACS results (A) verify neutrophil depletion in blood. Tumour area (B) and excised tumour weights (C) for WT and C3aR-/- mice treated with neutrophil depleting anti-Ly6G (1A8) or isotype control (2A3). Tumour infiltrating leukocyte populations (D) for WT and C3aR-/- mice treated with neutrophil depleting anti-Ly6G (1A8) or isotype control (2A3). Panel (E) shows neutrophils in human melanoma tissue. Data expressed as mean ± SD; results representative of 2 independent experiments (n=6/group); * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Permutation test (B), Kruskal Wallis test followed by Dunn’s multiple comparisons test (A, C, D).

92

4.3.8 Influence of C3aR deficiency on cytokine/chemokine expression To determine how C3a regulates the inflammatory milieu within tumour microenvironment, we analysed plasma and tumour tissue extracts for a panel of cytokines and chemokines previously implicated as regulators of the anti-tumour response. As shown in Fig 4.9, plasma G-CSF was significantly reduced in C3aR-/- mice whereas IL-1β was increased. There were no significant differences in plasma chemokine levels in C3aR-/- compared with WT mice (Fig 4.10). Although most chemokines were slightly increased, only CCL5 (RANTES) showed significant up-regulation in tumour tissue extracts from C3aR-/- mice..

Figure 4.9. Influence of C3aR deficiency on cytokine expression. Cytokine levels in plasma and tumour tissue from WT (●) and C3aR-/- () mice (n=6-8/group). Data expressed as pg/ml (for plasma) or pg/mg (for tumour tissue), mean ± SD (n=7-8/group); *p< 0.05, **p<0.01, Mann Whitney test.

93

Figure 4.10. C3aR deficiency influences chemokine expression. Chemokine levels in plasma and tumour tissue from WT (●) and C3aR-/- () mice (n=6-8/group). Data expressed as pg/ml (for plasma) or pg/mg (for tumour tissue), mean ± SD (n=7-8/group); *p< 0.05, **p<0.01, Mann Whitney test.

94

4.3.9 C3aR signalling contributes to the growth of other tumour types Finally, to determine whether or not the tumour promoting effects of C3aR signalling are restricted to the B16 melanoma model, we investigated the growth in C3aR-/- mice of two other tumour models: the murine melanoma cell line SM1WT1 which bears the BRAFV600E mutation harboured by approximately 50% of human melanomas (Knight et al., 2013) and the colon cancer cell line MC38. Similar to our results with B16 melanoma, the growth of both tumour types was significantly slowed in C3aR-/- mice compared with WT mice (Fig 4.11A, C). Analysis of excised tumour weights at termination confirmed that tumours were smaller in C3aR-/- mice (91% reduction for SM1WT1 and 58% for MC38 tumours; Fig 4.11B, D), thus suggesting the potential of C3aR as a therapeutic target for a range of tumour types.

Figure 4.11 C3aR signalling contributes to tumour growth in other tumour models. Tumour areas (A, C) and excised weights (B, C) for SM1WT1 melanoma (A, B) and MC38 colon carcinoma (C, D) grown in WT (●) and C3aR-/- () mice. Data expressed as mean ± SD (n=6-10/group); ** p<0.01 ***p<0.005, ****p<0.0001, Permutation test (A, C) and Mann-Whitney test (B, D).

95

4.4 DISCUSSION

The complement system, in particular the canonical pro-inflammatory anaphylatoxin C5a, is now widely implicated for a role in tumour growth, with both tumour-promoting and tumour- inhibitory effects reported, depending on tumour type, immune status of the host and C5a levels (Corrales et al., 2012; Gunn et al., 2012; Kim et al., 2005; Markiewski et al., 2008a; Nunez-Cruz et al., 2012). In contrast to C5a, the involvement of the upstream complement activation fragment C3a in tumourigenesis has not been extensively investigated. The present study used both C3aR-deficient mice and C3aR therapeutic inhibition, to identify a hitherto unknown role for C3a-C3aR in regulating tumour growth in murine models of melanoma. The demonstration of similar anti-tumour effects in a murine model of colon carcinoma suggests the wider potential of C3aR as a therapeutic target for a range of cancers. We demonstrated that C3aR is expressed at low levels by B16 melanoma cells in vitro and in vivo, and that these cells are able to respond directly to C3a, activating ERK and AKT signalling pathways and resulting in enhanced migratory capacity. These results are in accord with those of Cho et al (2014) who recently demonstrated the expression of C3aR by murine ovarian cancer cell lines. However, unlike Cho and co-workers, the present study failed to show any effect on cell proliferation. These findings, along with the deposition of complement activation products in human melanoma (Chapter 3), and other tumours (Ajona et al., 2013; Gorter and Meri, 1999; Niculescu et al., 1992), suggests that activation of tumour-expressed C3a receptors in vivo is possible. However the tumour microenvironment also includes inflammatory infiltrates (Gregory and Houghton, 2011) and since inflammatory cells, in particular myeloid cells, are known to express high levels of C3aR (Klos et al., 2009), the anti-tumour effects may instead be indirect, via effects on tumour infiltrating cells. Indeed, our demonstration that growth of C3aR-expressing B16 cells is retarded in C3aR- deficient mice supports the premise that the tumour-promoting effects of C3a-C3aR signalling are mediated via the host immune system.

We also demonstrated the potent arrest of B16 tumour growth in vivo via C3aR antagonism of established tumours. It should be noted that the C3aRA used in this study (SB290157) has been demonstrated to have C3aR agonistic and off-target activity (Woodruff, 2015). However our findings of reduced tumour growth using SB290157 are in accord with our findings in C3aR-/- mice. Thus this compound is unlikely to be exerting C3aR agonistic effects in this

96

model, although we cannot rule out other potential off-target effects of SB290157. Our findings are also in line with those of Markiewski et al (2008) who reported reduced tumour growth in C3-deficient mice, and similarly interpreted the results as being due to changes in the anti-tumour immune response. C3 cleavage produces C3a as well as generating downstream C5a, and the demonstration that tumour growth was reduced in C5aR1 antagonist-treated or C5aR1-deficient mice led these researchers to attribute the tumour- promoting effects solely to C5a. However the present study demonstrates that C3a also impairs an effective anti-tumour response. We further show that the anti-tumour effect observed in C3aR-/- mice is not augmented by treatment with the C5aR1 antagonist PMX53, implying that in this model the tumour promoting effects of C3a are at least as potent as those of C5a. Importantly, our results highlight the fact that these two receptors have discrete functions, affecting different aspects of the immune response.

Evidence that C3a mediates its effects on tumour growth via the immune system is provided by FACS analysis showing alterations in tumour infiltrating leukocyte populations in the absence of C3aR signalling. In contrast to C5aR1 which has been demonstrated to act via induction of MDSC and Tregs and inhibition of T lymphocyte responses (Chapter 3), we show that C3aR inhibition leads to an increase in tumour infiltrating neutrophilic/granulocytic populations, monocytes and CD4+ T-cells. The enhanced neutrophil contribution to tumour inflammatory infiltrates of C3aR-/- and C3aRA-treated mice is in accord with the recently identified role for C3a-C3aR signalling in the retention of neutrophils in the bone-marrow, with C3aR deficiency causing the selective egress of neutrophils in response to ischemic injury or G-CSF administration (Galdiero et al., 2013;Wu et al, 2013). Although Wu and co-workers found that C3aR-deficiency did not affect resting immune cell populations, the present study showed an increase in circulating neutrophils in healthy C3aR-deficient mice. Interestingly cytokine analysis showed a reduction in plasma G-CSF levels in tumour bearing C3aR-/- mice, a result in line with previous studies showing that G-CSF levels are inversely correlated with neutrophil counts, and likely due to negative feedback mechanisms at the transcriptional level (Bugl et al., 2013). In contrast to C5aR1- deficient mice which showed reduced levels of key chemokines including CCL2, CCL3, CCL5 (Chapter 3) in tumour tissue, C3aR-deficient mice tended to have higher chemokine levels, although only CCL5 was significantly increased.

97

Our demonstration that neutrophil depletion significantly reverses the tumour inhibitory effects observed in C3aR deficient mice implicates neutrophils as major contributors to the anti-tumour effects of C3aR deficiency/antagonism. Neutrophils are generally thought to promote tumour cell proliferation, angiogenesis and metastasis via the production of chemokines, cytokines and reactive oxygen species; they also contribute to MDSC populations which inhibit T cell responses (Gregory and Houghton, 2011). Unfortunately there are currently no antibodies available that can effectively discriminate between different neutrophil populations, including granulocytic MDSC. Indeed the demonstration that the majority of tumour infiltrating MDSC are removed by treatment with the anti-Ly6G antibody indicates that in this model, the majority of MDSC are granulocytic. However, there is evidence that under certain conditions neutrophils can exert efficient anti-tumour activity (Souto et al., 2011), and activation of neutrophils by primary tumours has been shown to inhibit metastatic seeding (Granot et al., 2011). These differing roles of neutrophils in tumour growth may be explained by differences in activation status. Fridlender et al (2009) demonstrated in lung cancer models that like macrophages, neutrophils have differential states of activation: a pro-tumourigenic phenotype identified by increased expression of chemokines and growth factors (eg CCL2, CCL5, VEGF-A), and an anti-tumour phenotype associated with increased expression of pro-inflammatory cytokines (eg TNF-α), chemokines (CCL3) and adhesion molecules (ICAM-1). They showed that the presence of TGFβ within the tumour environment induces a population of pro-tumourigenic neutrophils, whereas TGFβ blockade resulted in recruitment and activation of anti-tumour neutrophils. More recently this same group has shown that tumour associated neutrophils have a unique transcriptional profile compared to naïve bone marrow neutrophils, and also differ from granulocytic MDSC (Fridlender et al., 2012). Thus the effects of neutrophils on tumour growth may depend on their activation status, such that a shift in cytokine expression within the tumour microenvironment from acute to chronic inflammation converts anti-tumour effector cells to tumour promoting neutrophils (Souto et al., 2011). This is supported by recent studies showing that neutrophils at the early stages of tumour development are more cytotoxic towards tumour cells (Mishalian et al., 2013) and capable of stimulating effective T cell responses (Eruslanov et al., 2014), but at later stages develop a pro-tumour phenotype. Thus we propose that C3aR inhibition augments the sequestration of naïve neutrophils into the tumour microenvironment, which in turn tip the balance towards an anti-tumour

98

neutrophil response. This may include the recruitment and activation of other anti-tumour effector cells, including T lymphocytes, via neutrophil release of cytokines and chemokines that augment an effector T cell response. Indeed our demonstration that CD4+ T lymphocyte populations are increased in the absence of C3aR signalling, but return to normal (wild-type) levels following neutrophil depletion suggests that these newly mobilised neutrophils may promote effective anti-tumour T cell responses. This hypothesis is in accord with previous reports, for example by Medina-Echeverz et al (2011) who showed that an efficient anti- tumour response can be generated by altering the myeloid cell population within the tumour microenvironment and facilitating effector T cell infiltration. Our demonstration that neutrophils are present in human melanoma tissue suggests the potential to use C3aR- targeting drugs to manipulate neutrophil populations within the tumour, and thus tip the balance towards an anti-tumour response.

Although we were unable to demonstrate significant differences in expression of neutrophil activation markers CD62L and CXCR4 (data not shown), the up-regulation of IL-1β and CCL5 in plasma and tumour tissue (respectively) from C3aR-/- mice is in accord with the reported up-regulation of M1-associated cytokines/chemokines in anti-tumour myeloid cell populations (Medina-Echeverz et al., 2011). As discussed above, CCL5 has been associated with pro-tumourigenic neutrophils, and increased CCL5 levels have been associated with unfavourable outcomes in some cancers (Niwa et al., 2001), it is a predictor of survival in others (Chew et al., 2012; Moran et al., 2002). CCL5 has been implicated for a role in regulating anti-tumour immunity (Johrer et al., 2008; Mule et al., 1996), and shown in a mouse melanoma model to synergize with CXCR3 ligands to attract effector T cells into cutaneous metastases and inhibit tumour growth (Hong et al., 2011). FACS analysis of tumour infiltrating T lymphocyte populations showed C3aR-deficiency/inhibition increased Th1, Th2 and Th17 populations, but had no effect on CD8+ T lymphocytes or Tregs. Although effective anti-tumour responses have been traditionally attributed to CD8+ CTL supported by Th1 cells (Fridman et al., 2012; Ye et al., 2013), this is not always the case. Depending on the tumour type, anti-tumour responses can occur independently of cytotoxic CD8+ T cells, mediated directly by Th1 Th2 or Th17 cells (Chu et al., 2006; Corthay et al., 2005; Kim and Cantor, 2014). Th2 cells have been reported to amplify the innate immune response to tumours by directly recruiting tumouricidal myeloid cells into the tumour (Musiani et al., 1996; Pericle et al., 1994). Moreover, the Th2 response has been reported to

99

play a critical role in immunity to B16 melanoma (Mattes et al., 2003; Zhang et al., 2009). Th17 populations may also be associated with favourable outcomes, for example, in melanoma (Martin-Orozco et al., 2009a; Muranski et al., 2008) and oesophageal (Lv et al., 2011) cancers.

Th17 cells comprise approximately 0.1-0.5% of circulating CD4+ T cells in humans, but significantly higher densities are observed within tumour tissues, including melanoma (Su et al., 2010), prostate, fibrosarcoma, head and neck cancers (Kryczek et al., 2007). However their role in tumour growth is controversial, with tumour infiltrating Th17 cells reported to either contribute to an anti-tumour cytotoxic T cell response or promote tumour growth by facilitating angiogenesis and suppressing the anti-tumour response (Hemdan, 2013). In animal models, induction of Th17 cells has been shown to support anti-tumour immunity. For example, up-regulated expression of IL-6 in a pancreatic tumour model was shown to skew the balance towards Th17 cells, delaying tumour growth and improving survival (Gnerlich et al., 2010) while antigen-specific Th17-polarised cells eradicated established B16 melanoma (Muranski et al., 2008) and metastatic prostate tumours (Kottke et al., 2007). Muranski and co-workers showed that the therapeutic effect of Th17 cells is dependent on their production of IFNγ. Our demonstration of increased tumour infiltrating Th17 cells in the absence of C3aR signalling is in accord with a previous study by Lim et al (2012) who showed in a mouse model of allergic lung inflammation that C3aR deficient mice had higher frequencies of both neutrophils and Th17 cells in the lung. There is also evidence for direct cross-talk between activated neutrophils and Th17 cells, with both cell types producing a range of chemokines and cytokines capable of recruiting the other. Neutrophils mediate the Th17 pathway (Cua and Tato, 2010), via production of IL17 (Li et al., 2010) and chemoattractants such as CCL2, while cytokines produced by Th17 cells (including IFNγ, TNF-α, GM-CSF) favour recruitment, activation and prolonged survival of neutrophils at inflammatory sites (Pelletier et al., 2010). Although the present study found no significant changes in CCL2, IL17 or IFNγ, Th17 cells can also be recruited by CCL5 (Su et al., 2010). Clearly however, the impact of these changes in T cell populations on the anti-tumour response remains to be determined, for example by antibody depletion studies. In summary, this study identifies an important role for C3a/C3aR signalling in promoting the growth of melanoma and colon cancer, and provides evidence that these pro-tumour effects are mediated, at least partly, via the host immune system. Although both C3aR and C5aR1 have

100

tumour promoting effects, our data suggest that they act via different mechanisms. The results show that C3aR deficiency or therapeutic antagonism leads to increased tumour infiltrating leukocyte populations, including neutrophils and CD4+ T lymphocytes. Although neutrophils are generally thought to promote tumour growth, there is considerable evidence in the literature that neutrophils can be manipulated to exert efficient anti-tumour activity. We propose that by enhancing neutrophil infiltration populations, C3aR inhibition may change the inflammatory equilibrium within the tumour environment and tip the balance from a suppressive environment containing a majority of tumour-promoting granulocytic MDSC towards a more effective anti-tumour response. This hypothesis is supported by the demonstration that tumour infiltrating T lymphocytes, specifically CD4+ populations, are also increased. The association of C3aR-deficiency with increased tumour infiltrating CD4+ cells suggests that a positive feedback loop may develop whereby the secretion of chemokines and cytokines by neutrophils sequestered to the tumour microenvironment promotes CD4+ recruitment and expansion; cytokines produced by CD4+ cells may in turn favour neutrophil recruitment and survival. Further studies are required to determine the precise mechanisms by which C3a influences the immune response to melanoma, in particular how C3a deficiency/inhibition influences neutrophil phenotype and function, whether the mechanisms are similar in other tumour types, and the relationship between the effects C3aR and C5aR. The stage of tumour development and commencement of treatment may also be important, and thus future studies should compare the effects of treating advanced and metastatic tumours. Nevertheless the results suggest the potential of C3aR as a therapeutic target for melanoma and, potentially other cancers.

101

102

CHAPTER 5

GENERAL DISCUSSION AND FUTURE PROSPECTS

Jamileh Alham Nabizadeh

103

5.1 GENERAL DISCUSSION

Melanoma is the most deadly form of skin cancer, with 232,000 new cases and 55,000 deaths globally in 2012 (World Health Organisation Report, 2014). The incidence of melanoma is increasing world-wide, and Australia has the highest rate of melanoma in the world, with over 12,500 new cases diagnosed each year. Despite recent advances in treatment of disseminated melanoma including inhibitors of BRAF or MEK (for patients with BRAF mutations) or immunotherapy (anti-CTLA-4 and anti-PD-1 antibodies) (Finn et al., 2012), metastatic melanoma remains an incurable disease with a 3-year survival rate of approx. 15%, and median survival of only 6-9 months (Eggermont et al., 2014). Hence there is an urgent need for new therapies for advanced melanoma. Thus this thesis aimed to investigate one possible approach to the problem – targeting the complement anaphylatoxins C3a and C5a.

The complement system is a key component of the innate immune system, and a major consequence of complement activation is the production of anaphylatoxins C3a and C5a (Dutow et al., 2014; Markiewski and Lambris, 2007; Mukherji et al., 1986). Cancer cells are thought to evade complement-mediated destruction by up-regulating endogenous complement inhibitors (Fishelson et al., 2003; Sato et al., 2010), and complement activation products have been detected in human tumour tissue (Corrales et al., 2012; Lucas et al., 1996; Niculescu et al., 1992). Although the complement system has long been thought to contribute to antibody-dependent responses to tumours (Gelderman et al., 2004), its contribution to other aspects of the anti-tumour response has only recently begun to be investigated. The majority of investigations into the role of complement anaphylatoxins in tumour growth have been confined to complement C5a, with C5a-C5aR1 signalling demonstrated to contribute to tumour growth in cervical, lung, lymphoma and ovarian cancer models (Corrales et al., 2012; Markiewski et al., 2008a; Markiewski et al., 2008b; Nunez-Cruz et al., 2012). Despite evidence that serum C3a levels are up-regulated in many cancer patients (Maher et al., 2011) and that C3aR is expressed by some tumour cells (Cho et al., 2014; Xu et al., 2008) there is currently no information regarding the role of C3a-C3aR signalling in tumourigenesis. Collectively, these gaps in knowledge prompted us to explore and elucidate the contribution of complement anaphylatoxins C3a and C5a to melanoma development and growth. Hence the aim of this thesis was to investigate the hypothesis that complement anaphylatoxin

104

receptors C3aR, C5aR1 and C5aR2 are therapeutic targets for melanoma. The effects of C3aR and C5aR1/2 deficiency or antagonism were investigated in murine models of melanoma.

In agreement with previous reports of complement activation in human tumour tissue (Ajona et al., 2013; Niculescu et al., 1992), the complement activation product C4d was detected in human melanoma tissue, thus demonstrating that the complement system is activated, and may play a role in melanoma progression. Murine B16 melanoma cells were shown to express the complement anaphylatoxin receptors, C3aR, C5aR1 and C5aR2; C3aR was also detected on human melanoma cell lines, albeit at low levels. Further, C3aR and C5aR1 expressed by B16 cells were capable of signal activation via ERK1/2 and AKT pathways, a result that is in accord with the recent study demonstrating C3aR and C5aR1 expression by murine ovarian cancer cell lines (Cho et al., 2014). In contrast to that study however, the present study found no significant effect of C5a on proliferation or migration of B16 cells, although C3a increased cell proliferation.

In accord with previous studies in other tumour models (Corrales et al., 2012; Markiewski et al., 2008a; Nunez-Cruz et al., 2012), this thesis demonstrates a deleterious role for C5aR1 signalling in melanoma growth (Chapter 3). It also demonstrates for the first time, a role for the alternate C5a receptor, C5aR2, in modulating the effects of C5aR1 signalling on tumour growth. Importantly, it is the first to demonstrate a role for C3aR in promoting tumour growth, with C3aR deficiency protecting against B16 melanoma (Chapter 4). Importantly, the effects of C3aR inhibition were at least, if not more, potent as those of C5aR1 inhibition. Moreover, the demonstration that the growth of established tumours can be arrested by treatment with either C5aR or C3aR antagonists highlights the clinical therapeutic potential of C5aR/C3aR targeting drugs. C3aR deficiency was also protective in murine models of BRAFV600E mutant melanoma and colon cancer, suggesting the broader applicability of C3aR as a therapeutic target for cancer.

The results suggest that the effects of both C3aR and C5aR1 are mediated primarily via the immune system, although the mechanisms are different: C5aR1 acts by promoting MDSC and Treg infiltration whereas C3aR acts by regulating neutrophil populations. Despite these different mechanisms, both C3aR and C5aR1 ultimately affect adaptive T cell responses. Interestingly C5aR2 deficiency had no significant effect on tumour infiltrating leukocyte

105

populations. Chapter 3 demonstrated that, as in other tumour types (Corrales et al., 2012; Markiewski et al., 2008a; Nunez-Cruz et al., 2012), the tumour microenvironment of C5aR1 deficient mice contained fewer MDSC and Tregs. A potent chemoattractant for myeloid cells (Guo and Ward, 2005), C5a may act directly to attract MDSC into the tumour. However other chemokines were also down-regulated in these mice, including CCL2, CCL4 and CCL5, which are known to be responsible for regulating the infiltration of MDSC within tumour microenvironments (Boelte et al., 2011; Sevko and Umansky, 2013; Zollo et al., 2012). Among these, CCL2 has been shown to influence infiltration of MDSCs in melanoma and prostate cancer (Lesokhin et al., 2012; Zhang et al., 2010; Zollo et al., 2012), while high expression of CCL2 in human breast cancer is a strong indicator of early relapse (Sato et al., 1995; Ueno et al., 2000; Valkovic et al., 2002). This reduction in CCL2 and CCL4 expression, along with fewer macrophages in tumours from C5aR1-deficient mice, is in accord with Varney and co-workers (Varney et al., 2005) who demonstrated a correlation between macrophage infiltration and melanoma progression. Although not the focus of this thesis, the role of MDSCs and TAMs in promoting tumour growth via angiogenesis (Sawanobori et al., 2008; Varney et al., 2005) requires mention. By promoting infiltration of myeloid cell populations (including MDSC and TAMs), C5a and CCL2 may enhance angiogenesis (Boelte et al., 2011) and hence increase tumour growth.

The changes in tumour infiltrating CD4+ T cell populations in C5aR-deficient mice (fewer Tregs and more Th2 cells) suggests that the tumour-promoting effects of C5a on melanoma growth may be partly due to Treg inhibition of CD4+ effector populations. Despite previous reports that C5aR signalling diminishes Treg function (Kwan et al., 2013; Strainic et al., 2013), Gunn and co-workers (2012)have provided evidence that a high concentration of C5a inhibits Th1 and promotes Treg differentiation. One possibility is that the effect on Tregs is indirect, mediated via tumour infiltrating MDSC which have been shown to attract Tregs via CCR5 ligands, CCL4 and CCL5 (Schlecker et al., 2012). Interestingly healthy C5aR- deficient mice in this study (without tumours) show inherent differences in circulating leukocyte populations: more neutrophils, fewer CD4+ and CD8+ T cell populations, but higher proportions of CD4+ subsets. However, in spite of fewer circulating T cells in C5aR- deficient mice, the proportions of T cells within the tumour microenvironment of these mice were increased, possibly because of the reduced immunosuppression.

106

Chapter 4 shows that C3aR deficiency/antagonism also results in alterations in tumour- infiltrating leukocyte populations, albeit the nature of these changes are completely different. Specifically, the absence of C3aR leads to increased neutrophil and CD4+ T lymphocyte sub- populations within the tumour microenvironment. Given the recent demonstration by Wu and co-workers (2013) that C3aR deficiency results in mobilisation of neutrophils from the bone marrow, we proposed that C3aR inhibition leads to increased infiltration of mature neutrophils into the tumour, and that this in turn tips the balance from a pro- to anti-tumour environment. The central role of neutrophils in the anti-tumour response was confirmed by neutrophil depletion experiments which significantly reversed the tumour inhibitory effects observed in C3aR-deficient mice; the percentage of tumour infiltrating CD4+ T lymphocyte also returned to control levels.

The results presented here suggest the potential of neutrophils as anti-tumour effector cells. Although neutrophils are generally thought to promote tumour growth, by contributing to MDSC populations, angiogenesis and metastatic seeding (Gregory and Houghton, 2011), there is considerable evidence in the literature that neutrophils can be manipulated to exert efficient anti-tumour activity (Eruslanov et al., 2014). Indeed it has been suggested that the effects of neutrophils on tumour growth may depend on their activation status, such that a shift in cytokine expression within the tumour microenvironment from acute to chronic inflammation converts anti-tumour effector cells to tumour-promoting neutrophils and MDSC (Fridlender et al., 2009; Murata and Baldwin, 2009; Souto et al., 2011). Thus by promoting the sequestration of mature neutrophils, C3aR inhibition may change the inflammatory equilibrium within the tumour environment, and tip the balance towards an anti-tumour response. The concomitant increase in tumour infiltrating CD4+ cells suggests that a positive feedback loop may develop whereby chemokines and cytokines secreted by these newly recruited neutrophils act to promote CD4+ recruitment and expansion; cytokines produced by CD4+ cells may in turn favour neutrophil recruitment and survival. Unfortunately the inability of available antibodies to distinguish different neutrophil sub- populations prevented us from confirming our hypothesis at this time. However the demonstrated presence of neutrophils in human melanoma tissue suggests the potential to use C3aR-targeting drugs to manipulate neutrophil populations within human tumours, to promote an effective anti-tumour response. In contrast to healthy C5aR-deficient mice which showed marked differences in circulating leukocyte populations compared to their wild-type

107

counterparts, healthy C3aR-deficient mice in our study showed minor differences, solely in circulating neutrophil levels.

The increased contribution of neutrophils to the anti-tumour response in C3aR-deficient mice may also explain the different pattern of chemokine expression in these tumours. Whereas C5aR-deficiency was associated with significant reductions in key chemokines associated with chemoattraction of MDSC and Tregs, chemokine levels in tumour tissue from C3aR- deficient mice tended to be higher than in their wild-type counterparts, although the differences reached statistical significance only for IL-1β and CCL5. This increase in chemokine levels possibly reflects the increased contribution of neutrophils which are known to secrete a wide range of chemokines (Fleming et al., 1993). CCL5 has been associated with pro-tumourigenic neutrophils (Fridlender et al., 2009) and unfavourable outcomes in some cancers (Niwa et al., 2001). However CCL5 has also been implicated for a role in regulating anti-tumour immunity (Johrer et al., 2008; Mule et al., 1996), and shown in a mouse melanoma model to synergize with CXCR3 ligands to attract effector T cells into cutaneous metastases and inhibit tumour growth (Coppola et al., 2011). In addition, the finding that the tumour inhibitory effects of C3aR deficiency were not augmented by C5aR inhibition suggests that the effects may actually be enhanced by the presence of C5a which may promote infiltration of newly mobilised neutrophils into the tumour.

In contrast to the accepted paradigm that CD8+ T cells are the key mediators of effective anti- tumour responses (Grabiner et al., 2014), the present study found that both C5aR1 and C3aR deficiency lead to changes in CD4+ T cell populations, in particular Th2 cells. Although Th2- mediated immune responses are usually thought to promote tumour growth through promoting angiogenesis and mediating immunosuppression (Ellyard et al., 2007; Nishimura et al., 1999), they have been suggested to play an important role in the response to MHC class I negative tumours which are resistant to CD8+ cytotoxic T lymphocytes. Indeed the Th2 response has been shown to be important for immunity against B16 melanoma (Fang et al., 2009); (Mattes and Foster, 2003), and thus it is possible that the observed effects on T cell populations may be specific to the B16 melanoma model.

In conclusion, this thesis highlights the importance of the complement anaphylatoxins in the immune response to tumour growth, and their potential as therapeutic targets for melanoma, and possibly other tumours. It not only confirms the role of C5aR1 in promoting tumour

108

growth, but identifies hitherto unknown roles for C5aR2 and C3aR. Although C3a and C5a are often thought to have similar effects, this thesis suggests divergent functions. However despite their different effects, both anaphylatoxins create an environment that favours tumour growth. The results presented in this thesis provide convincing evidence that therapeutic inhibition of C3aR and/or C5aR1 is an effective means to tip the balance towards an anti- tumour response.

5.2 FUTURE PROSPECTS

The results presented in this thesis contribute to our understanding of the role of complement effector components C3a and C5a on tumour growth. However, further research is required to characterise these roles in melanoma and other tumours, the mechanisms by which C3a and C5a subvert the anti-tumour response and promote tumour growth, and how this knowledge can be applied clinically.

The pro-tumour effects of C5a have been reported in murine models of cervical, lung, ovarian cancer, lymphoma and melanoma while this thesis is the first to demonstrate pro-tumour effects of C3aR in melanoma and colonic cancer. In particular, further studies are required to examine the effects of C3aR in other cancers. Additional research is also needed to determine the specific role of the complement components in the context of human disease and progression. The stage of tumour development and commencement of treatment may also be important, and thus future studies should compare the effects of treating advanced tumours. Little is known of the role of the complement anaphylatoxins in tumour metastasis, apart from two recent reports that C5a promotes the development of metastatic breast (Vadrevu et al., 2014) and colon cancer (Piao et al., 2015). Given that the majority of cancer deaths are due to metastatic disease, the role of C3aR and C5aR in tumour metastasis is an important question that requires urgent investigation.

This thesis have provided convincing evidence that the effects of C3a and C5a on tumour growth are, at least in part indirect, likely via effects on host immune cells. However, given that complement receptors are expressed by tumour cell lines (Cho et al., 2014; Kim et al., 2005) including human and mouse melanoma cells (Chapters 3 and 4), and that these receptors are capable of signal activation, direct effects on tumour cells are possible. To investigate this possibility, the growth of C3aR/C5aR-deficient tumours in wild-type mice (whose immune cells express complement receptors) should be investigated.

109

As reported previously (Corrales et al., 2012; Gunn et al., 2012; Markiewski et al., 2008a), this thesis suggests that C5aR1 suppresses the anti-tumour response via induction of MDSC and Tregs (Chapter 3). Conversely, the results presented in Chapter 4 suggest that C3aR mediates its effects by regulation of neutrophil populations within the tumour microenvironment. While neutrophil depletion was shown to abrogate the anti-tumour effects in C3aR-deficient mice, further experiments are required to determine the precise mechanisms by which these C3aR-regulated neutrophils influence the anti-tumour response, and how they differ from other neutrophil populations. Despite the absence of clear markers to distinguish the different neutrophil populations, further studies are required to characterise tumour infiltrating neutrophil populations in C3aR-deficient and wild-type mice, for example in regards to expression of chemokine receptors (including CXCR1, CXCR2, CXCR4) and other key genes known to be expressed by tumour associated neutrophils: Ccl2, Ccl3, Ccl5, Vegfa, Mmp9, Cxcr4, Tnf, Icam1 (Fridlender et al, 2009; Mishalian et al, 2013). Experiments to determine the effects of depleting other leukocyte populations, in particular CD4 and CD8 T cell populations, are also required, as well as further analysis of the influence of C3aR/C5aR deficiency on cytokine/chemokines within the tumour environment. Moreover, given the demonstration by Nunez-Cruz and co-workers (2012) that the primary role for C5a in ovarian cancer is to promote tumour neovascularisation, future studies should investigate the potential for C5a/C3a to promote tumour angiogenesis in melanoma.

The present study found that combined C3aR/C5aR inhibition did not augment the anti- tumour response, and suggested that the effects of C3a are as potent as those of C5a. However, further research is needed to establish the relationships between C3a and C5a signalling, and how both mediators can be modulated to achieve improved outcomes. A clear understanding of the precise mechanisms by which C3a and C5a regulate immune cell populations could provide an insight into how these complement components differentially regulate tumour immunity and thus lead to improved anti-tumour therapies. Finally, the potential for interactions with other therapeutic modalities (chemotherapy, radiotherapy and immunotherapy) needs to be investigated. Indeed a recent report by Surace et al (2015) has shown that the local production of C3a and C5a is crucial to achieving an effective tumour response to radiotherapy. This research highlights the complexity of the anti-tumour immune response and the need to fully understand the mechanisms involved before bringing new therapies onto the market. Clearly therapeutic strategies must be developed which take into

110

account these conflicting activities. For example, the timing and dose of complement modulating drugs should be designed to maximise therapeutic benefits.

Clearly, many questions remain to be answered before this research can be translated to the clinic. There is also urgent need for improved C3aR-targeting drugs. While PMX53 has proven to be safe for clinical application (Burns et al., 2013b; Kohl, 2006), the commercially available C3aR antagonist SB290157 is not suitable for administration to humans, and is reported to have agonist as well as off-target effects (Woodruff and Tenner, 2015). Although progress has been made, to date no suitable C3aR inhibitory drug candidate has emerged (Burns et al., 2013a). Despite these notes of caution, the results ultimately support the potential of both C5aR and C3aR as therapeutic targets for melanoma, and potentially other cancers.

111

6 REFERENCES

Acosta-Rodriguez, E.V., Rivino, L., Geginat, J., Jarrossay, D., Gattorno, M., Lanzavecchia, A., Sallusto, F., and Napolitani, G. (2007). Surface phenotype and antigenic specificity of human -producing T helper memory cells. Nat Immunol 8, 639-646.

Aderem, A., and Ulevitch, R.J. (2000). Toll-like receptors in the induction of the innate immune response. Nature 406, 782-787.

Ajona, D., Pajares, M.J., Corrales, L., Perez-Gracia, J.L., Agorreta, J., Lozano, M.D., Torre, W., Massion, P.P., de-Torres, J.P., Jantus-Lewintre, E., et al. (2013). Investigation of complement activation product c4d as a diagnostic and prognostic biomarker for lung cancer. J Natl Cancer Inst 105, 1385-1393.

Akhir, F. (2014). The role of complement anaphylatoxin receptors in mammary tumour growth: identification of new therapeutic targets PhD Thesis, School of Biomedical Science, The University of Queensland.

Aldinucci, D., and Colombatti, A. (2014). The inflammatory chemokine CCL5 and cancer progression. Mediators Inflamm 2014, 292376.

Alexandrescu, D.T., Ichim, T.E., Riordan, N.H., Marincola, F.M., Di Nardo, A., Kabigting, F.D., and Dasanu, C.A. (2010). Immunotherapy for melanoma: current status and perspectives. J Immunother 33, 570-590.

Alper, C.A., Johnson, A.M., Birtch, A.G., and Moore, F.D. (1969). Human C'3: evidence for the liver as the primary site of synthesis. Science 163, 286-288.

Ames, R.S., Lee, D., Foley, J.J., Jurewicz, A.J., Tornetta, M.A., Bautsch, W., Settmacher, B., Klos, A., Erhard, K.F., Cousins, R.D., et al. (2001). Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models. J Immunol 166, 6341-6348.

Andre, C., Hampe, A., Lachaume, P., Martin, E., Wang, X.P., Manus, V., Hu, W.X., and Galibert, F. (1997). Sequence analysis of two genomic regions containing the KIT and the FMS receptor tyrosine kinase genes. Genomics 39, 216-226.

Ansel, K.M., Lee, D.U., and Rao, A. (2003). An epigenetic view of helper T cell differentiation. Nat Immunol 4, 616-623.

Arumugam, T.V., Shiels, I.A., Woodruff, T.M., Granger, D.N., and Taylor, S.M. (2004). The role of the complement system in ischemia-reperfusion injury. Shock 21, 401-409.

Aspord, C., Pedroza-Gonzalez, A., Gallegos, M., Tindle, S., Burton, E.C., Su, D., Marches, F., Banchereau, J., and Palucka, A.K. (2007). Breast cancer instructs dendritic cells to prime interleukin 13-secreting CD4(+) T cells that facilitate tumor development. Journal of Experimental Medicine 204, 1037-1047.

112

Ataera, H., Hyde, E., Price, K.M., Stoitzner, P., and Ronchese, F. (2011). Murine Melanoma- Infiltrating Dendritic Cells Are Defective in Antigen Presenting Function Regardless of the Presence of CD4(+)CD25(+) Regulatory T Cells. PLoS One 6.

Auffray, C., Fogg, D.K., Narni-Mancinelli, E., Senechal, B., Trouillet, C., Saederup, N., Leemput, J., Bigot, K., Campisi, L., Abitbol, M., et al. (2009). CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation. J Exp Med 206, 595-606.

Bai, X.F., Liu, J., Li, O., Zheng, P., and Liu, Y. (2003). Antigenic drift as a mechanism for tumor evasion of destruction by cytolytic T lymphocytes. J Clin Invest 111, 1487-1496.

Balch, C.M., Gershenwald, J.E., Soong, S.J., Thompson, J.F., Atkins, M.B., Byrd, D.R., Buzaid, A.C., Cochran, A.J., Coit, D.G., Ding, S., et al. (2009). Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol 27, 6199-6206.

Balch, C.M., Soong, S.J., Gershenwald, J.E., Thompson, J.F., Reintgen, D.S., Cascinelli, N., Urist, M., McMasters, K.M., Ross, M.I., Kirkwood, J.M., et al. (2001). Prognostic factors analysis of 17,600 melanoma patients: validation of the American Joint Committee on Cancer melanoma staging system. J Clin Oncol 19, 3622-3634.

Balkwill, F., Charles, K.A., and Mantovani, A. (2005). Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7, 211-217.

Bamberg, C.E., Mackay, C.R., Lee, H., Zahra, D., Jackson, J., Lim, Y.S., Whitfeld, P.L., Craig, S., Corsini, E., Lu, B., et al. (2010). The C5a receptor (C5aR) C5L2 is a modulator of C5aR-mediated signal transduction. J Biol Chem 285, 7633-7644.

Banchereau, J., and Steinman, R.M. (1998). Dendritic cells and the control of immunity. Nature 392, 245-252.

Baruah, P., Dumitriu, I.E., Malik, T.H., Cook, H.T., Dyson, J., Scott, D., Simpson, E., and Botto, M. (2009). C1q enhances IFN-gamma production by antigen-specific T cells via the CD40 costimulatory pathway on dendritic cells. Blood 113, 3485-3493.

Basso, K., Saito, M., Sumazin, P., Margolin, A.A., Wang, K., Lim, W.K., Kitagawa, Y., Schneider, C., Alvarez, M.J., Califano, A., and Dalla-Favera, R. (2010). Integrated biochemical and computational approach identifies BCL6 direct target genes controlling multiple pathways in normal germinal center B cells. Blood 115, 975-984.

Baudino, L., Sardini, A., Ruseva, M.M., Fossati-Jimack, L., Cook, H.T., Scott, D., Simpson, E., and Botto, M. (2014). C3 opsonization regulates endocytic handling of apoptotic cells resulting in enhanced T-cell responses to cargo-derived antigens. Proc Natl Acad Sci U S A 111, 1503-1508.

Beadling, C., Jacobson-Dunlop, E., Hodi, F.S., Le, C., Warrick, A., Patterson, J., Town, A., Harlow, A., Cruz, F., 3rd, Azar, S., et al. (2008). KIT gene mutations and copy number in melanoma subtypes. Clin Cancer Res 14, 6821-6828.

113

Beeram, M., Patnaik, A., and Rowinsky, E.K. (2005). Raf: a strategic target for therapeutic development against cancer. J Clin Oncol 23, 6771-6790.

Benelli, R., Morini, M., Carrozzino, F., Ferrari, N., Minghelli, S., Santi, L., Cassatella, M., Noonan, D.M., and Albini, A. (2002). Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation. FASEB J 16, 267-269.

Bertagnolli, M., and Herrmann, S. (1990). IL-7 supports the generation of cytotoxic T lymphocytes from thymocytes. Multiple lymphokines required for proliferation and cytotoxicity. J Immunol 145, 1706-1712.

Bettelli, E., Korn, T., Oukka, M., and Kuchroo, V.K. (2008). Induction and effector functions of T(H)17 cells. Nature 453, 1051-1057.

Betts, G., Twohig, J., Van den Broek, M., Sierro, S., Godkin, A., and Gallimore, A. (2007). The impact of regulatory T cells on carcinogen-induced sarcogenesis. Br J Cancer 96, 1849- 1854.

Biron, C.A., Nguyen, K.B., Pien, G.C., Cousens, L.P., and Salazar-Mather, T.P. (1999). Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 17, 189-220.

Boelte, K.C., Gordy, L.E., Joyce, S., Thompson, M.A., Yang, L., and Lin, P.C. (2011). Rgs2 mediates pro-angiogenic function of myeloid derived suppressor cells in the tumor microenvironment via upregulation of MCP-1. PLoS One 6, e18534.

Bogunovic, M., Ginhoux, F., Helft, J., Shang, L., Hashimoto, D., Greter, M., Liu, K., Jakubzick, C., Ingersoll, M.A., Leboeuf, M., et al. (2009). Origin of the lamina propria dendritic cell network. Immunity 31, 513-525.

Bonifati, D.M., and Kishore, U. (2007). Role of complement in neurodegeneration and neuroinflammation. Mol Immunol 44, 999-1010.

Bopst, M., Haas, C., Car, B., and Eugster, H.P. (1998). The combined inactivation of and interleukin-6 prevents induction of the major acute phase proteins by endotoxin. Eur J Immunol 28, 4130-4137.

Borregaard, N. (2010). Neutrophils, from marrow to microbes. Immunity 33, 657-670.

Brahmer, J.R., Tykodi, S.S., Chow, L.Q., Hwu, W.J., Topalian, S.L., Hwu, P., Drake, C.G., Camacho, L.H., Kauh, J., Odunsi, K., et al. (2012). Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366, 2455-2465.

Brandau, S., Dumitru, C.A., and Lang, S. (2013). Protumor and antitumor functions of neutrophil granulocytes. Seminars in immunopathology 35, 163-176.

114

Breitfeld, D., Ohl, L., Kremmer, E., Ellwart, J., Sallusto, F., Lipp, M., and Forster, R. (2000). Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J Exp Med 192, 1545-1552.

Bugl, S., Wirths, S., Radsak, M.P., Schild, H., Stein, P., Andre, M.C., Muller, M.R., Malenke, E., Wiesner, T., Marklin, M., et al. (2013). Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling. Blood 121, 723-733.

Buhl, A.M., Avdi, N., Worthen, G.S., and Johnson, G.L. (1994). Mapping of the C5a receptor signal transduction network in human neutrophils. Proc Natl Acad Sci U S A 91, 9190-9194.

Bui, J.D., and Schreiber, R.D. (2007). Cancer immunosurveillance, immunoediting and inflammation: independent or interdependent processes? Curr Opin Immunol 19, 203-208.

Burnet, F.M. (1970). The concept of immunological surveillance. Prog Exp Tumor Res 13, 1- 27.

Burns, M.B., Lackey, L., Carpenter, M.A., Rathore, A., Land, A.M., Leonard, B., Refsland, E.W., Kotandeniya, D., Tretyakova, N., Nikas, J.B., et al. (2013a). APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494, 366-370.

Burns, M.B., Temiz, N.A., and Harris, R.S. (2013b). Evidence for APOBEC3B mutagenesis in multiple human cancers. Nature genetics 45, 977-983.

Cambien, B., Richard-Fiardo, P., Karimdjee, B.F., Martini, V., Ferrua, B., Pitard, B., Schmid- Antomarchi, H., and Schmid-Alliana, A. (2011). CCL5 neutralization restricts cancer growth and potentiates the targeting of PDGFRbeta in colorectal carcinoma. PLoS One 6, e28842.

Cancer Genome Atlas, N. (2015). Genomic Classification of Cutaneous Melanoma. Cell 161, 1681-1696.

Capasso, M., Durrant, L.G., Stacey, M., Gordon, S., Ramage, J., and Spendlove, I. (2006). Costimulation via CD55 on human CD4+ T cells mediated by CD97. J Immunol 177, 1070- 1077.

Carter, N.A., Rosser, E.C., and Mauri, C. (2012). Interleukin-10 produced by B cells is crucial for the suppression of Th17/Th1 responses, induction of T regulatory type 1 cells and reduction of collagen-induced arthritis. Arthritis Res Ther 14, R32.

Carter, N.A., Vasconcellos, R., Rosser, E.C., Tulone, C., Munoz-Suano, A., Kamanaka, M., Ehrenstein, M.R., Flavell, R.A., and Mauri, C. (2011). Mice lacking endogenous IL-10- producing regulatory B cells develop exacerbated disease and present with an increased frequency of Th1/Th17 but a decrease in regulatory T cells. J Immunol 186, 5569-5579.

Carvajal, R.D., Antonescu, C.R., Wolchok, J.D., Chapman, P.B., Roman, R.A., Teitcher, J., Panageas, K.S., Busam, K.J., Chmielowski, B., Lutzky, J., et al. (2011). KIT as a therapeutic target in metastatic melanoma. JAMA 305, 2327-2334.

115

Casanova-Acebes, M., Pitaval, C., Weiss, L.A., Nombela-Arrieta, C., Chevre, R., A- Gonzalez, N., Kunisaki, Y., Zhang, D.C., van Rooijen, N., Silberstein, L.E., et al. (2013). Rhythmic Modulation of the Hematopoietic Niche through Neutrophil Clearance. Cell 153, 1025-1035.

Cascinelli, N. (1989). [Sun light (not only UV light), melanoma, skin tumors ... and ethics]. Giornale italiano di dermatologia e venereologia : organo ufficiale, Societa italiana di dermatologia e sifilografia 124, 532-535.

Cassatella, M.A. (1999). Neutrophil-derived proteins: selling cytokines by the pound. Adv Immunol 73, 369-509.

Cassatella, M.A., Bazzoni, F., Ceska, M., Ferro, I., Baggiolini, M., and Berton, G. (1992). IL- 8 production by human polymorphonuclear leukocytes. The chemoattractant formyl- methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway. J Immunol 148, 3216-3220.

Cassatella, M.A., Meda, L., Gasperini, S., D'Andrea, A., Ma, X., and Trinchieri, G. (1995). Interleukin-12 production by human polymorphonuclear leukocytes. Eur J Immunol 25, 1-5.

Cecchini, M.G., Dominguez, M.G., Mocci, S., Wetterwald, A., Felix, R., Fleisch, H., Chisholm, O., Hofstetter, W., Pollard, J.W., and Stanley, E.R. (1994). Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse. Development 120, 1357-1372.

Chaux, P., Favre, N., Martin, M., and Martin, F. (1997). Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats. International Journal of Cancer 72, 619-624.

Chen, W.F., and Zlotnik, A. (1991). IL-10: a novel cytotoxic T cell differentiation factor. J Immunol 147, 528-534.

Chenoweth, D.E., Goodman, M.G., and Weigle, W.O. (1982). Demonstration of a specific receptor for human C5a anaphylatoxin on murine macrophages. J Exp Med 156, 68-78.

Chew, V., Chen, J., Lee, D., Loh, E., Lee, J., Lim, K.H., Weber, A., Slankamenac, K., Poon, R.T., Yang, H., et al. (2012). Chemokine-driven lymphocyte infiltration: an early intratumoural event determining long-term survival in resectable hepatocellular carcinoma. Gut 61, 427-438.

Chikamatsu*, K.S.a.K. (2013). Immune suppression and evasion in patients with head and neck cancer.

Chin, Y., Janseens, J., Vandepitte, J., Vandenbrande, J., Opdebeek, L., and Raus, J. (1992). Phenotypic Analysis of Tumor-Infiltrating Lymphocytes from Human Breast-Cancer. Anticancer Research 12, 1463-1466.

116

Cho, M.S., Vasquez, H.G., Rupaimoole, R., Pradeep, S., Wu, S., Zand, B., Han, H.D., Rodriguez-Aguayo, C., Bottsford-Miller, J., Huang, J., et al. (2014). Autocrine effects of tumor-derived complement. Cell reports 6, 1085-1095.

Christoffersson, G., Vagesjo, E., Vandooren, J., Liden, M., Massena, S., Reinert, R.B., Brissova, M., Powers, A.C., Opdenakker, G., and Phillipson, M. (2012). VEGF-A recruits a proangiogenic MMP-9-delivering neutrophil subset that induces angiogenesis in transplanted hypoxic tissue. Blood 120, 4653-4662.

Christopher, M.J., and Link, D.C. (2007). Regulation of neutrophil homeostasis. Current opinion in hematology 14, 3-8.

Chu, Y., Xia, M., Lin, Y., Li, A., Wang, Y., Liu, R., and Xiong, S. (2006). Th2-dominated antitumor immunity induced by DNA immunization with the genes coding for a basal core peptide PDTRP and GM-CSF. Cancer Gene Ther 13, 510-519.

Clarke, E.V., and Tenner, A.J. (2014). Complement modulation of T cell immune responses during homeostasis and disease. J Leukoc Biol 96, 745-756.

Coley, W.B. (1891). II. Contribution to the Knowledge of Sarcoma. Ann Surg 14, 199-220.

Colotta, F., Re, F., Polentarutti, N., Sozzani, S., and Mantovani, A. (1992). Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood 80, 2012-2020.

Connolly, M.K., Mallen-St Clair, J., Bedrosian, A.S., Malhotra, A., Vera, V., Ibrahim, J., Henning, J., Pachter, H.L., Bar-Sagi, D., Frey, A.B., and Miller, G. (2010). Distinct populations of metastases-enabling myeloid cells expand in the liver of mice harboring invasive and preinvasive intra-abdominal tumor. J Leukoc Biol 87, 713-725.

Conti, I., and Rollins, B.J. (2004). CCL2 (monocyte chemoattractant protein-1) and cancer. Semin Cancer Biol 14, 149-154.

Coppola, D., Nebozhyn, M., Khalil, F., Dai, H., Yeatman, T., Loboda, A., and Mule, J.J. (2011). Unique ectopic lymph node-like structures present in human primary colorectal carcinoma are identified by immune gene array profiling. Am J Pathol 179, 37-45.

Corrales, L., Ajona, D., Rafail, S., Lasarte, J.J., Riezu-Boj, J.I., Lambris, J.D., Rouzaut, A., Pajares, M.J., Montuenga, L.M., and Pio, R. (2012). Anaphylatoxin C5a creates a favorable microenvironment for lung cancer progression. J Immunol 189, 4674-4683.

Corthay, A., Skovseth, D.K., Lundin, K.U., Rosjo, E., Omholt, H., Hofgaard, P.O., Haraldsen, G., and Bogen, B. (2005). Primary antitumor immune response mediated by CD4+ T cells. Immunity 22, 371-383.

Coulthard, L.G., and Woodruff, T.M. (2015). Is the complement activation product C3a a proinflammatory molecule? Re-evaluating the evidence and the myth. J Immunol 194, 3542- 3548.

117

Coussens, L.M., and Werb, Z. (2002). Inflammation and cancer. Nature 420, 860-867.

Cravedi, P., Leventhal, J., Lakhani, P., Ward, S.C., Donovan, M.J., and Heeger, P.S. (2013). Immune cell-derived C3a and C5a costimulate human T cell . Am J Transplant 13, 2530-2539.

Croker, D.E., Halai, R., Kaeslin, G., Wende, E., Fehlhaber, B., Klos, A., Monk, P.N., and Cooper, M.A. (2014). C5a2 can modulate ERK1/2 signaling in macrophages via heteromer formation with C5a1 and beta-arrestin recruitment. Immunol Cell Biol 92, 631-639.

Cros, J., Cagnard, N., Woollard, K., Patey, N., Zhang, S.Y., Senechal, B., Puel, A., Biswas, S.K., Moshous, D., Picard, C., et al. (2010). Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Immunity 33, 375-386.

Cua, D.J., and Tato, C.M. (2010). Innate IL-17-producing cells: the sentinels of the immune system. Nat Rev Immunol 10, 479-489.

Curtin, J.A., Busam, K., Pinkel, D., and Bastian, B.C. (2006). Somatic activation of KIT in distinct subtypes of melanoma. J Clin Oncol 24, 4340-4346.

Cutler, A.J., Botto, M., van Essen, D., Rivi, R., Davies, K.A., Gray, D., and Walport, M.J. (1998). T cell-dependent immune response in C1q-deficient mice: defective interferon gamma production by antigen-specific T cells. J Exp Med 187, 1789-1797.

Dai, X.M., Ryan, G.R., Hapel, A.J., Dominguez, M.G., Russell, R.G., Kapp, S., Sylvestre, V., and Stanley, E.R. (2002). Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood 99, 111-120.

Daley, J.M., Thomay, A.A., Connolly, M.D., Reichner, J.S., and Albina, J.E. (2008). Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol 83, 64-70.

Davies, H., Bignell, G.R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin, H., Garnett, M.J., Bottomley, W., et al. (2002). Mutations of the BRAF gene in human cancer. Nature 417, 949-954.

Davies, L.C., and Taylor, P.R. (2015). Tissue-resident macrophages: then and now. Immunology 144, 541-548.

Davis, L.S., Patel, S.S., Atkinson, J.P., and Lipsky, P.E. (1988). Decay-accelerating factor functions as a signal transducing molecule for human T cells. J Immunol 141, 2246-2252.

DeNardo, D.G., Barreto, J.B., Andreu, P., Vasquez, L., Tawfik, D., Kolhatkar, N., and Coussens, L.M. (2009). CD4(+) T Cells Regulate Pulmonary Metastasis of Mammary Carcinomas by Enhancing Protumor Properties of Macrophages. Cancer Cell 16, 91-102.

DeNardo, D.G., Johansson, M., and Coussens, L.M. (2008). Immune cells as mediators of solid tumor metastasis. Cancer Metastasis Rev 27, 11-18.

118

Denonne, F., Binet, S., Burton, M., Collart, P., Dipesa, A., Ganguly, T., Giannaras, A., Kumar, S., Lewis, T., Maounis, F., et al. (2007). Discovery of new C3aR ligands. Part 1: arginine derivatives. Bioorg Med Chem Lett 17, 3258-3261.

Dieu-Nosjean, M.C., Antoine, M., Danel, C., Heudes, D., Wislez, M., Poulot, V., Rabbe, N., Laurans, L., Tartour, E., de Chaisemartin, L., et al. (2008). Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures. J Clin Oncol 26, 4410-4417.

DiLillo, D.J., Yanaba, K., and Tedder, T.F. (2010). B cells are required for optimal CD4+ and CD8+ T cell tumor immunity: therapeutic B cell depletion enhances B16 melanoma growth in mice. J Immunol 184, 4006-4016.

Doeing, D.C., Borowicz, J.L., and Crockett, E.T. (2003). Gender dimorphism in differential peripheral blood leukocyte counts in mice using cardiac, tail, foot, and saphenous vein puncture methods. BMC clinical pathology 3, 3.

Dong, C. (2008). TH17 cells in development: an updated view of their molecular identity and genetic programming. Nat Rev Immunol 8, 337-348.

Dorhoi, A., Iannaccone, M., Farinacci, M., Fae, K.C., Schreiber, J., Moura-Alves, P., Nouailles, G., Mollenkopf, H.J., Oberbeck-Muller, D., Jorg, S., et al. (2013). MicroRNA-223 controls susceptibility to tuberculosis by regulating lung neutrophil recruitment. J Clin Invest 123, 4836-4848.

Dorner, B.G., Smith, H.R., French, A.R., Kim, S., Poursine-Laurent, J., Beckman, D.L., Pingel, J.T., Kroczek, R.A., and Yokoyama, W.M. (2004). Coordinate expression of cytokines and chemokines by NK cells during murine cytomegalovirus infection. J Immunol 172, 3119-3131.

Downward, J. (2003). Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 3, 11-22.

Drickamer, K., and Fadden, A.J. (2002). Genomic analysis of C-type lectins. Biochem Soc Symp, 59-72.

Dunkelberger, J.R., and Song, W.C. (2010). Complement and its role in innate and adaptive immune responses. Cell Res 20, 34-50.

Dunn, G.P., Bruce, A.T., Ikeda, H., Old, L.J., and Schreiber, R.D. (2002). Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol 3, 991-998.

Dunn, G.P., Old, L.J., and Schreiber, R.D. (2004). The immunobiology of cancer immunosurveillance and immunoediting. Immunity 21, 137-148.

Dupin, E., Glavieux, C., Vaigot, P., and Le Douarin, N.M. (2000). Endothelin 3 induces the reversion of melanocytes to glia through a neural crest-derived glial-melanocytic progenitor. Proc Natl Acad Sci U S A 97, 7882-7887.

119

Dutow, P., Fehlhaber, B., Bode, J., Laudeley, R., Rheinheimer, C., Glage, S., Wetsel, R.A., Pabst, O., and Klos, A. (2014). The complement C3a receptor is critical in defense against Chlamydia psittaci in mouse lung infection and required for antibody and optimal T cell response. J Infect Dis 209, 1269-1278.

Eash, K.J., Greenbaum, A.M., Gopalan, P.K., and Link, D.C. (2010). CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow. J Clin Invest 120, 2423-2431.

Eden, A., Miller, G.W., and Nussenzweig, V. (1973). Human lymphocytes bear membrane receptors for C3b and C3d. J Clin Invest 52, 3239-3242.

Eggermont, A.M., Spatz, A., and Robert, C. (2014). Cutaneous melanoma. Lancet 383, 816- 827.

Ehrlich, P. (1909 ). Über den jetzigen stand der karzinomforschung. Ned. Tijdschr. Geneeskd 5, 273-290. el-Lati, S.G., Dahinden, C.A., and Church, M.K. (1994). Complement peptides C3a- and C5a-induced mediator release from dissociated human skin mast cells. J Invest Dermatol 102, 803-806.

Elenkov, I.J., and Chrousos, G.P. (2002). Stress hormones, proinflammatory and antiinflammatory cytokines, and autoimmunity. Ann N Y Acad Sci 966, 290-303.

Ellyard, J.I., Simson, L., and Parish, C.R. (2007). Th2-mediated anti-tumour immunity: friend or foe? Tissue Antigens 70, 1-11.

Ember, I., Kiss, I., and Malovics, I. (1998). Oncogene and tumour suppressor gene expression changes in persons exposed to ethylene oxide. Eur J Cancer Prev 7, 167-168.

Ember, J.A., Johansen, N.L., and Hugli, T.E. (1991). Designing synthetic superagonists of C3a anaphylatoxin. Biochemistry 30, 3603-3612.

Erickson, C.A., and Reedy, M.V. (1998). Neural crest development: the interplay between morphogenesis and cell differentiation. Current topics in developmental biology 40, 177-209.

Eruslanov, E.B., Bhojnagarwala, P.S., Quatromoni, J.G., Stephen, T.L., Ranganathan, A., Deshpande, C., Akimova, T., Vachani, A., Litzky, L., Hancock, W.W., et al. (2014). Tumor- associated neutrophils stimulate T cell responses in early-stage human lung cancer. J Clin Invest 124, 5466-5480.

Erwig, L.P., and Henson, P.M. (2008). Clearance of apoptotic cells by phagocytes. Cell Death Differ 15, 243-250.

Falchook, G.S., Long, G.V., Kurzrock, R., Kim, K.B., Arkenau, T.H., Brown, M.P., Hamid, O., Infante, J.R., Millward, M., Pavlick, A.C., et al. (2012). Dabrafenib in patients with

120

melanoma, untreated brain metastases, and other solid tumours: a phase 1 dose-escalation trial. Lancet 379, 1893-1901.

Fang, C., Zhang, X., Miwa, T., and Song, W.C. (2009). Complement promotes the development of inflammatory T-helper 17 cells through synergistic interaction with Toll-like receptor signaling and interleukin-6 production. Blood 114, 1005-1015.

Ferlazzo, G., and Munz, C. (2004). NK cell compartments and their activation by dendritic cells. J Immunol 172, 1333-1339.

Fernandez, H.N., Henson, P.M., Otani, A., and Hugli, T.E. (1978). Chemotactic response to human C3a and C5a anaphylatoxins. I. Evaluation of C3a and C5a leukotaxis in vitro and under stimulated in vivo conditions. J Immunol 120, 109-115.

Fidler, I.J., and Nicolson, G.L. (1976). Organ selectivity for implantation survival and growth of B16 melanoma variant tumor lines. J Natl Cancer Inst 57, 1199-1202.

Finch, A.M., Vogen, S.M., Sherman, S.A., Kirnarsky, L., Taylor, S.M., and Sanderson, S.D. (1997). Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity. J Med Chem 40, 877-884.

Finch, A.M., Wong, A.K., Paczkowski, N.J., Wadi, S.K., Craik, D.J., Fairlie, D.P., and Taylor, S.M. (1999). Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a. Journal of Medicinal Chemistry 42, 1965-1974.

Finch, C.E., and Crimmins, E.M. (2004). Inflammatory exposure and historical changes in human life-spans. Science 305, 1736-1739.

Finn, L., Markovic, S.N., and Joseph, R.W. (2012). Therapy for metastatic melanoma: the past, present, and future. BMC Med 10, 23.

Fischer, W.H., and Hugli, T.E. (1997). Regulation of B cell functions by C3a and C3a(desArg): suppression of TNF-alpha, IL-6, and the polyclonal immune response. J Immunol 159, 4279-4286.

Fishelson, Z., Donin, N., Zell, S., Schultz, S., and Kirschfink, M. (2003). Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors. Mol Immunol 40, 109-123.

Flaherty, K.T., Puzanov, I., Kim, K.B., Ribas, A., McArthur, G.A., Sosman, J.A., O'Dwyer, P.J., Lee, R.J., Grippo, J.F., Nolop, K., and Chapman, P.B. (2010). Inhibition of Mutated, Activated BRAF in Metastatic Melanoma. New Engl J Med 363, 809-819.

Fleming, T.J., Fleming, M.L., and Malek, T.R. (1993). Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family. J Immunol 151, 2399-2408.

121

Fogg, D.K., Sibon, C., Miled, C., Jung, S., Aucouturier, P., Littman, D.R., Cumano, A., and Geissmann, F. (2006). A clonogenic bone marrow progenitor specific for macrophages and dendritic cells. Science 311, 83-87.

Frankenberger, B., Pohla, H., Noessner, E., Willimsky, G., Papier, B., Pezzutto, A., Kopp, J., Oberneder, R., Blankenstein, T., and Schendel, D.J. (2005). Influence of CD80, interleukin-2, and interleukin-7 expression in human renal cell carcinoma on the expansion, function, and survival of tumor-specific CTLs. Clin Cancer Res 11, 1733-1742.

Fridlender, Z.G., Sun, J., Kim, S., Kapoor, V., Cheng, G., Ling, L., Worthen, G.S., and Albelda, S.M. (2009). Polarization of tumor-associated neutrophil phenotype by TGF-beta: "N1" versus "N2" TAN. Cancer Cell 16, 183-194.

Fridlender, Z.G., Sun, J., Mishalian, I., Singhal, S., Cheng, G., Kapoor, V., Horng, W., Fridlender, G., Bayuh, R., Worthen, G.S., and Albelda, S.M. (2012). Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils. PLoS One 7, e31524.

Fridman, W.H., Pages, F., Sautes-Fridman, C., and Galon, J. (2012). The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 12, 298-306.

Fuchs, E.J., and Matzinger, P. (1992). B cells turn off virgin but not memory T cells. Science 258, 1156-1159.

Fuenmayor, J., Perez-Vazquez, K., Perez-Witzke, D., Penichet, M.L., and Montano, R.F. (2010). Decreased survival of human breast cancer cells expressing HER2/neu on in vitro incubation with an anti-HER2/neu antibody fused to C5a or C5a desArg. Mol Cancer Ther 9, 2175-2185.

Fukui, R., Nishimori, H., Hata, F., Yasoshima, T., Ohno, K., Nomura, H., Yanai, Y., Tanaka, H., Kamiguchi, K., Denno, R., et al. (2005). Metastases-related genes in the classification of liver and peritoneal metastasis in human gastric cancer. J Surg Res 129, 94-100.

Gabrilovich, D.I., and Nagaraj, S. (2009). Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol 9, 162-174.

Galdiero, M.R., Bonavita, E., Barajon, I., Garlanda, C., Mantovani, A., and Jaillon, S. (2013). Tumor associated macrophages and neutrophils in cancer. Immunobiology 218, 1402-1410.

Galli, S.J., Borregaard, N., and Wynn, T.A. (2011). Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils. Nat Immunol 12, 1035- 1044.

Gallina, G., Dolcetti, L., Serafini, P., De Santo, C., Marigo, I., Colombo, M.P., Basso, G., Brombacher, F., Borrello, I., Zanovello, P., et al. (2006). Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells. J Clin Invest 116, 2777-2790.

122

Gama, L., Shirk, E.N., Russell, J.N., Carvalho, K.I., Li, M., Queen, S.E., Kalil, J., Zink, M.C., Clements, J.E., and Kallas, E.G. (2012). Expansion of a subset of CD14highCD16negCCR2low/neg monocytes functionally similar to myeloid-derived suppressor cells during SIV and HIV infection. J Leukoc Biol 91, 803-816.

Gao, H., Neff, T.A., Guo, R.F., Speyer, C.L., Sarma, J.V., Tomlins, S., Man, Y., Riedemann, N.C., Hoesel, L.M., Younkin, E., et al. (2005). Evidence for a functional role of the second C5a receptor C5L2. FASEB J 19, 1003-1005.

Garbe, C., and Leiter, U. (2009). Melanoma epidemiology and trends. Clin Dermatol 27, 3-9.

Gately, M.K., Warrier, R.R., Honasoge, S., Carvajal, D.M., Faherty, D.A., Connaughton, S.E., Anderson, T.D., Sarmiento, U., Hubbard, B.R., and Murphy, M. (1994). Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo. Int Immunol 6, 157-167.

Geissmann, F. (2010). Development of monocytes, macrophages, and dendritic cells (vol 327, pg 656, 2010). Science 330, 1318-1318.

Gelderman, K.A., Tomlinson, S., Ross, G.D., and Gorter, A. (2004). Complement function in mAb-mediated cancer immunotherapy. Trends Immunol 25, 158-164.

Gerard, N.P., Hodges, M.K., Drazen, J.M., Weller, P.F., and Gerard, C. (1989). Characterization of a receptor for C5a anaphylatoxin on human eosinophils. J Biol Chem 264, 1760-1766.

Ginhoux, F., Liu, K., Helft, J., Bogunovic, M., Greter, M., Hashimoto, D., Price, J., Yin, N., Bromberg, J., Lira, S.A., et al. (2009). The origin and development of nonlymphoid tissue CD103+ DCs. J Exp Med 206, 3115-3130.

Gnerlich, J.L., Mitchem, J.B., Weir, J.S., Sankpal, N.V., Kashiwagi, H., Belt, B.A., Porembka, M.R., Herndon, J.M., Eberlein, T.J., Goedegebuure, P., and Linehan, D.C. (2010). Induction of Th17 cells in the tumor microenvironment improves survival in a murine model of pancreatic cancer. J Immunol 185, 4063-4071.

Gollob, J.A., Wilhelm, S., Carter, C., and Kelley, S.L. (2006). Role of Raf kinase in cancer: therapeutic potential of targeting the Raf/MEK/ERK signal transduction pathway. Seminars in oncology 33, 392-406.

Gordon, S., and Martinez, F.O. (2010). Alternative Activation of Macrophages: Mechanism and Functions. Immunity 32, 593-604.

Gordon, S., and Taylor, P.R. (2005). Monocyte and macrophage heterogeneity. Nat Rev Immunol 5, 953-964.

Gorter, A., and Meri, S. (1999). Immune evasion of tumor cells using membrane-bound complement regulatory proteins. Immunol Today 20, 576-582.

123

Grabiner, B.C., Nardi, V., Birsoy, K., Possemato, R., Shen, K., Sinha, S., Jordan, A., Beck, A.H., and Sabatini, D.M. (2014). A diverse array of cancer-associated MTOR mutations are hyperactivating and can predict rapamycin sensitivity. Cancer discovery 4, 554-563.

Granot, Z., Henke, E., Comen, E.A., King, T.A., Norton, L., and Benezra, R. (2011). Tumor entrained neutrophils inhibit seeding in the premetastatic lung. Cancer Cell 20, 300-314.

Gregory, A.D., and Houghton, A.M. (2011). Tumor-associated neutrophils: new targets for cancer therapy. Cancer Res 71, 2411-2416.

Grewal, I.S., and Flavell, R.A. (1998). CD40 and CD154 in cell-mediated immunity. Annual Review of Immunology 16, 111-135.

Grommes, J., and Soehnlein, O. (2011). Contribution of neutrophils to acute lung injury. Mol Med 17, 293-307.

Gu-Trantien, C., Loi, S., Garaud, S., Equeter, C., Libin, M., de Wind, A., Ravoet, M., Le Buanec, H., Sibille, C., Manfouo-Foutsop, G., et al. (2013). CD4(+) follicular helper T cell infiltration predicts breast cancer survival. J Clin Invest 123, 2873-2892.

Gu, J., Ding, J.Y., Lu, C.L., Lin, Z.W., Chu, Y.W., Zhao, G.Y., Guo, J., and Ge, D. (2013). Overexpression of CD88 predicts poor prognosis in non-small-cell lung cancer. Lung Cancer 81, 259-265.

Gullo, C.A., Hwang, W.Y., Poh, C.K., Au, M., Cow, G., and Teoh, G. (2008). Use of ultraviolet-light irradiated multiple myeloma cells as to generate tumor-specific cytolytic T lymphocytes. Journal of immune based therapies and vaccines 6, 2.

Gunn, L., Ding, C., Liu, M., Ma, Y., Qi, C., Cai, Y., Hu, X., Aggarwal, D., Zhang, H.G., and Yan, J. (2012). Opposing roles for complement component C5a in tumor progression and the tumor microenvironment. J Immunol 189, 2985-2994.

Guo, J., Si, L., Kong, Y., Flaherty, K.T., Xu, X., Zhu, Y., Corless, C.L., Li, L., Li, H., Sheng, X., et al. (2011). Phase II, open-label, single-arm trial of imatinib mesylate in patients with metastatic melanoma harboring c-Kit mutation or amplification. J Clin Oncol 29, 2904-2909.

Guo, R.F., and Ward, P.A. (2005). Role of C5a in inflammatory responses. Annu Rev Immunol 23, 821-852.

Habermann, J.K., Roblick, U.J., Luke, B.T., Prieto, D.A., Finlay, W.J.J., Podust, V.N., Roman, J.M., Oevermann, E., Schiedeck, T., Homann, N., et al. (2006). Increased serum levels of complement C3a anaphylatoxin indicate the presence of colorectal tumors. Gastroenterology 131, 1020-1029.

Hachicha, M., Naccache, P.H., and Mccoll, S.R. (1995). Inflammatory Microcrystals Differentially Regulate the Secretion of Macrophage Inflammatory Protein-1 and Interleukin- 8 by Human Neutrophils - a Possible Mechanism of Neutrophil Recruitment to Sites of Inflammation in Synovitis. Journal of Experimental Medicine 182, 2019-2025.

124

Hagemann, T., Lawrence, T., McNeish, I., Charles, K.A., Kulbe, H., Thompson, R.G., Robinson, S.C., and Balkwill, F.R. (2008). "Re-educating" tumor-associated macrophages by targeting NF-kappaB. J Exp Med 205, 1261-1268.

Haile, L.A., von Wasielewski, R., Gamrekelashvili, J., Kruger, C., Bachmann, O., Westendorf, A.M., Buer, J., Liblau, R., Manns, M.P., Korangy, F., and Greten, T.F. (2008). Myeloid-derived suppressor cells in inflammatory bowel disease: a new immunoregulatory pathway. Gastroenterology 135, 871-881, 881 e871-875.

Hajishengallis, G., and Lambris, J.D. (2011). Microbial manipulation of receptor crosstalk in innate immunity. Nat Rev Immunol 11, 187-200.

Hakulinen, J., and Meri, S. (1998). Complement-mediated killing of microtumors in vitro. Am J Pathol 153, 845-855.

Hamid, O., Robert, C., Daud, A., Hodi, F.S., Hwu, W.J., Kefford, R., Wolchok, J.D., Hersey, P., Joseph, R.W., Weber, J.S., et al. (2013). Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 369, 134-144.

Harrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy, T.L., Murphy, K.M., and Weaver, C.T. (2005). Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 6, 1123-1132.

Hashimoto, D., Chow, A., Noizat, C., Teo, P., Beasley, M.B., Leboeuf, M., Becker, C.D., See, P., Price, J., Lucas, D., et al. (2013). Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity 38, 792-804.

Hashimoto, D., Miller, J., and Merad, M. (2011). Dendritic cell and macrophage heterogeneity in vivo. Immunity 35, 323-335.

Hashimoto, M., Hirota, K., Yoshitomi, H., Maeda, S., Teradaira, S., Akizuki, S., Prieto- Martin, P., Nomura, T., Sakaguchi, N., Kohl, J., et al. (2010). Complement drives Th17 cell differentiation and triggers autoimmune arthritis. J Exp Med 207, 1135-1143.

Hauschild, A., Grob, J.J., Demidov, L.V., Jouary, T., Gutzmer, R., Millward, M., Rutkowski, P., Blank, C.U., Miller, W.H., Jr., Kaempgen, E., et al. (2012). Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 380, 358-365.

Haviland, D.L., McCoy, R.L., Whitehead, W.T., Akama, H., Molmenti, E.P., Brown, A., Haviland, J.C., Parks, W.C., Perlmutter, D.H., and Wetsel, R.A. (1995). Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR on nonmyeloid cells of the liver and lung. J Immunol 154, 1861-1869.

Hawksworth, O.A., Coulthard, L.G., Taylor, S.M., Wolvetang, E.J., and Woodruff, T.M. (2014). Brief report: complement C5a promotes human embryonic stem cell pluripotency in the absence of FGF2. Stem cells 32, 3278-3284.

125

Hegde, G.V., Meyers-Clark, E., Joshi, S.S., and Sanderson, S.D. (2008). A conformationally- biased, response-selective agonist of C5a acts as a molecular adjuvant by modulating antigen processing and presentation activities of human dendritic cells. Int Immunopharmacol 8, 819- 827.

Hegde, U.P., Chakraborty, N., Mukherji, B., and Grant Kels, J.M. (2011). Metastatic melanoma in the older patient: immunologic insights and treatment outcomes. Expert Rev Pharmacoecon Outcomes Res 11, 185-193.

Hemdan, N.Y. (2013). Anti-cancer versus cancer-promoting effects of the interleukin-17- producing T helper cells. Immunol Lett 149, 123-133.

Herberman, R.B., Nunn, M.E., Holden, H.T., and Lavrin, D.H. (1975a). Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells. Int J Cancer 16, 230-239.

Herberman, R.B., Nunn, M.E., and Lavrin, D.H. (1975b). Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic acid allogeneic tumors. I. Distribution of reactivity and specificity. Int J Cancer 16, 216-229.

Hezmee, M.N.M. (2010). The role of complement factor C5a and its receptor; C5aR in the development of canine mammary tumours PhD Thesis, School of Veterinary Science, The University of Queensland.

Hiraoka, S., Takeuchi, N., Bian, Y., Nakahara, H., Kogo, M., Dunussi-Joannopoulos, K., Wolf, S., Ono, S., and Fujiwara, H. (2005). B7.2-Ig fusion proteins enhance IL-4-dependent differentiation of tumor-sensitized CD8+ cytotoxic T lymphocyte precursors. Int Immunol 17, 1071-1079.

Ho, W.Y., Cooke, M.P., Goodnow, C.C., and Davis, M.M. (1994). Resting and anergic B cells are defective in CD28-dependent costimulation of naive CD4+ T cells. J Exp Med 179, 1539-1549.

Hodi, F.S., O'Day, S.J., McDermott, D.F., Weber, R.W., Sosman, J.A., Haanen, J.B., Gonzalez, R., Robert, C., Schadendorf, D., Hassel, J.C., et al. (2010). Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363, 711-723.

Hong, M., Puaux, A.L., Huang, C., Loumagne, L., Tow, C., Mackay, C., Kato, M., Prevost- Blondel, A., Avril, M.F., Nardin, A., and Abastado, J.P. (2011). Chemotherapy induces intratumoral expression of chemokines in cutaneous melanoma, favoring T-cell infiltration and tumor control. Cancer Res 71, 6997-7009.

Howe, L.R., Subbaramaiah, K., Brown, A.M., and Dannenberg, A.J. (2001). Cyclooxygenase-2: a target for the prevention and treatment of breast cancer. Endocr Relat Cancer 8, 97-114.

126

Huang, S., Paulauskis, J.D., Godleski, J.J., and Kobzik, L. (1992). Expression of Macrophage Inflammatory Protein-2 and Kc Messenger-Rna in Pulmonary Inflammation. American Journal of Pathology 141, 981-988.

Ikeya, M., Lee, S.M., Johnson, J.E., McMahon, A.P., and Takada, S. (1997). Wnt signalling required for expansion of neural crest and CNS progenitors. Nature 389, 966-970.

Ingersoll, M.A., Spanbroek, R., Lottaz, C., Gautier, E.L., Frankenberger, M., Hoffmann, R., Lang, R., Haniffa, M., Collin, M., Tacke, F., et al. (2010). Comparison of gene expression profiles between human and mouse monocyte subsets. Blood 115, e10-19.

Inoue, S., Leitner, W.W., Golding, B., and Scott, D. (2006). Inhibitory effects of B cells on antitumor immunity. Cancer Res 66, 7741-7747.

Ito, H., Ando, K., Ishikawa, T., Nakayama, T., Taniguchi, M., Saito, K., Imawari, M., Moriwaki, H., Yokochi, T., Kakumu, S., and Seishima, M. (2008). Role of Valpha14+ NKT cells in the development of Hepatitis B virus-specific CTL: activation of Valpha14+ NKT cells promotes the breakage of CTL tolerance. Int Immunol 20, 869-879.

Iwamoto, M., Shinohara, H., Miyamoto, A., Okuzawa, M., Mabuchi, H., Nohara, T., Gon, G., Toyoda, M., and Tanigawa, N. (2003). Prognostic value of tumor-infiltrating dendritic cells expressing CD83 in human breast carcinomas. Int J Cancer 104, 92-97.

Iwasaki, A., and Kelsall, B.L. (1999). Freshly isolated Peyer's patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells. J Exp Med 190, 229-239.

Jakob, J.A., Bassett, R.L., Jr., Ng, C.S., Curry, J.L., Joseph, R.W., Alvarado, G.C., Rohlfs, M.L., Richard, J., Gershenwald, J.E., Kim, K.B., et al. (2012). NRAS mutation status is an independent prognostic factor in metastatic melanoma. Cancer 118, 4014-4023.

Janeway, C.A., Jr., and Medzhitov, R. (2002). Innate immune recognition. Annu Rev Immunol 20, 197-216.

Janke, J., Schluter, K., Jandrig, B., Theile, M., Kolble, K., Arnold, W., Grinstein, E., Schwartz, A., Estevez-Schwarz, L., Schlag, P.M., et al. (2000). Suppression of tumorigenicity in breast cancer cells by the microfilament protein profilin 1. J Exp Med 191, 1675-1686.

Jinong Li, Z.Z., Jason Rosenzweig, Young Y. Wang, and Daniel W. Chan* (2002). Proteomics and Bioinformatics Approaches for Identification of Serum Biomarkers to Detect Breast Cancer. Clinical chemistry 48, 1296-1304.

Johannessen, C.M., Boehm, J.S., Kim, S.Y., Thomas, S.R., Wardwell, L., Johnson, L.A., Emery, C.M., Stransky, N., Cogdill, A.P., Barretina, J., et al. (2010). COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968-U370.

127

Johrer, K., Pleyer, L., Olivier, A., Maizner, E., Zelle-Rieser, C., and Greil, R. (2008). Tumour-immune cell interactions modulated by chemokines. Expert Opin Biol Ther 8, 269- 290.

Josefowicz, S.Z., Lu, L.F., and Rudensky, A.Y. (2012). Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol 30, 531-564.

Jurianz, K., Ziegler, S., Garcia-Schuler, H., Kraus, S., Bohana-Kashtan, O., Fishelson, Z., and Kirschfink, M. (1999). Complement resistance of tumor cells: basal and induced mechanisms. Mol Immunol 36, 929-939.

Kalinsky, K., Lee, S.J., Lawrence, D.P., Iafrate, A.J., Borger, D.R., Averbook, B.J., Tarhini, A.A., and Kirkwood, J.M. (2012). A phase II trial of dasatinib in patients with unresectable locally advanced or stage IV mucosal, acral, and solar melanomas: An Eastern Cooperative Oncology Group study (E2607). Journal of Clinical Oncology 30.

Kanai, T., Thomas, E.K., Yasutomi, Y., and Letvin, N.L. (1996). IL-15 stimulates the expansion of AIDS virus-specific CTL. J Immunol 157, 3681-3687.

Kanmura, S., Uto, H., Sato, Y., Kumagai, K., Sasaki, F., Moriuchi, A., Oketani, M., Ido, A., Nagata, K., Hayashi, K., et al. (2010). The complement component C3a fragment is a potential biomarker for hepatitis C virus-related hepatocellular carcinoma. J Gastroenterol 45, 459-467.

Kasahara, S., Ando, K., Saito, K., Sekikawa, K., Ito, H., Ishikawa, T., Ohnishi, H., Seishima, M., Kakumu, S., and Moriwaki, H. (2003). Lack of tumor necrosis factor alpha induces impaired proliferation of hepatitis B virus-specific cytotoxic T lymphocytes. J Virol 77, 2469-2476.

Kawanishi, N., Yano, H., Yokogawa, Y., and Suzuki, K. (2010). Exercise training inhibits inflammation in adipose tissue via both suppression of macrophage infiltration and acceleration of phenotypic switching from M1 to M2 macrophages in high-fat-diet-induced obese mice. Exercise immunology review 16, 105-118.

Kelleher, P., Maroof, A., and Knight, S.C. (1999). Retrovirally induced switch from production of IL-12 to IL-4 in dendritic cells. Eur J Immunol 29, 2309-2318.

Kiessling, R., Klein, E., Pross, H., and Wigzell, H. (1975a). "Natural" killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Characteristics of the killer cell. Eur J Immunol 5, 117-121.

Kiessling, R., Klein, E., and Wigzell, H. (1975b). "Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol 5, 112-117.

Kim, A.H., Dimitriou, I.D., Holland, M.C., Mastellos, D., Mueller, Y.M., Altman, J.D., Lambris, J.D., and Katsikis, P.D. (2004). Complement C5a receptor is essential for the optimal generation of antiviral CD8+ T cell responses. J Immunol 173, 2524-2529.

128

Kim, C.H., Rott, L.S., Clark-Lewis, I., Campbell, D.J., Wu, L., and Butcher, E.C. (2001). Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center- localized subset of CXCR5+ T cells. J Exp Med 193, 1373-1381.

Kim, D.Y., Martin, C.B., Lee, S.N., and Martin, B.K. (2005). Expression of complement protein C5a in a murine mammary cancer model: tumor regression by interference with the cell cycle. Cancer Immunol Immunother 54, 1026-1037.

Kim, H.J., and Cantor, H. (2014). CD4 T-cell subsets and tumor immunity: the helpful and the not-so-helpful. Cancer immunology research 2, 91-98.

Kim, M.H., Granick, J.L., Kwok, C., Walker, N.J., Borjesson, D.L., Curry, F.R., Miller, L.S., and Simon, S.I. (2011). Neutrophil survival and c-kit(+)-progenitor proliferation in Staphylococcus aureus-infected skin wounds promote resolution. Blood 117, 3343-3352.

Klein, U., and Dalla-Favera, R. (2008). Germinal centres: role in B-cell physiology and malignancy. Nat Rev Immunol 8, 22-33.

Klos, A., Tenner, A.J., Johswich, K.O., Ager, R.R., Reis, E.S., and Kohl, J. (2009). The role of the anaphylatoxins in health and disease. Mol Immunol 46, 2753-2766.

Klos, A., Wende, E., Wareham, K.J., and Monk, P.N. (2013). International Union of Pharmacology. LXXXVII. Complement peptide C5a, C4a, and C3a receptors. Pharmacological reviews 65, 500-543.

Knight, D.A., Ngiow, S.F., Li, M., Parmenter, T., Mok, S., Cass, A., Haynes, N.M., Kinross, K., Yagita, H., Koya, R.C., et al. (2013). Host immunity contributes to the anti-melanoma activity of BRAF inhibitors. J Clin Invest 123, 1371-1381.

Koch, A.E., Kunkel, S.L., Shah, M.R., Hosaka, S., Halloran, M.M., Haines, G.K., Burdick, M.D., Pope, R.M., and Strieter, R.M. (1995). Growth-Related Gene-Product-Alpha - a Chemotactic Cytokine for Neutrophils in Rheumatoid-Arthritis. Journal of Immunology 155, 3660-3666.

Koebel, C.M., Vermi, W., Swann, J.B., Zerafa, N., Rodig, S.J., Old, L.J., Smyth, M.J., and Schreiber, R.D. (2007). Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450, 903-907.

Koenen, H.J., Smeets, R.L., Vink, P.M., van Rijssen, E., Boots, A.M., and Joosten, I. (2008). Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood 112, 2340-2352.

Kohl, J. (2006). Drug evaluation: the C5a PMX-53. Curr Opin Mol Ther 8, 529-538.

Kolaczkowska, E., and Kubes, P. (2013). Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13, 159-175.

129

Kotake, S., Udagawa, N., Takahashi, N., Matsuzaki, K., Itoh, K., Ishiyama, S., Saito, S., Inoue, K., Kamatani, N., Gillespie, M.T., et al. (1999). IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 103, 1345- 1352.

Kottke, T., Sanchez-Perez, L., Diaz, R.M., Thompson, J., Chong, H., Harrington, K., Calderwood, S.K., Pulido, J., Georgopoulos, N., Selby, P., et al. (2007). Induction of hsp70- mediated Th17 autoimmunity can be exploited as immunotherapy for metastatic prostate cancer. Cancer Res 67, 11970-11979.

Kretzschmar, T., Jeromin, A., Gietz, C., Bautsch, W., Klos, A., Kohl, J., Rechkemmer, G., and Bitter-Suermann, D. (1993). Chronic myelogenous leukemia-derived basophilic granulocytes express a functional active receptor for the anaphylatoxin C3a. Eur J Immunol 23, 558-561.

Kryczek, I., Wei, S., Szeliga, W., Vatan, L., and Zou, W. (2009). Endogenous IL-17 contributes to reduced tumor growth and metastasis. Blood 114, 357-359.

Kryczek, I., Wei, S., Zou, L., Altuwaijri, S., Szeliga, W., Kolls, J., Chang, A., and Zou, W. (2007). Cutting edge: Th17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment. J Immunol 178, 6730-6733.

Kuphal, S., and Bosserhoff, A. (2009). Recent progress in understanding the pathology of malignant melanoma. J Pathol 219, 400-409.

Kupp, L.I., Kosco, M.H., Schenkein, H.A., and Tew, J.G. (1991). Chemotaxis of germinal center B cells in response to C5a. Eur J Immunol 21, 2697-2701.

Kusmartsev, S., Nagaraj, S., and Gabrilovich, D.I. (2005). Tumor-associated CD8+ T cell tolerance induced by bone marrow-derived immature myeloid cells. J Immunol 175, 4583- 4592.

Kusmartsev, S., Nefedova, Y., Yoder, D., and Gabrilovich, D.I. (2004). Antigen-specific inhibition of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species. J Immunol 172, 989-999.

Kwan, W.H., van der Touw, W., Paz-Artal, E., Li, M.O., and Heeger, P.S. (2013). Signaling through C5a receptor and C3a receptor diminishes function of murine natural regulatory T cells. J Exp Med 210, 257-268.

Lacy, P. (2006). Mechanisms of degranulation in neutrophils. , asthma, and clinical immunology : official journal of the Canadian Society of Allergy and Clinical Immunology 2, 98-108.

Ladanyi, A., Kiss, J., Somlai, B., Gilde, K., Fejos, Z., Mohos, A., Gaudi, I., and Timar, J. (2007). Density of DC-LAMP(+) mature dendritic cells in combination with activated T lymphocytes infiltrating primary cutaneous melanoma is a strong independent prognostic factor. Cancer Immunol Immunother 56, 1459-1469.

130

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Lajoie, S., Lewkowich, I.P., Suzuki, Y., Clark, J.R., Sproles, A.A., Dienger, K., Budelsky, A.L., and Wills-Karp, M. (2010). Complement-mediated regulation of the IL-17A axis is a central genetic determinant of the severity of experimental allergic asthma. Nat Immunol 11, 928-935.

Lalli, P.N., Strainic, M.G., Yang, M., Lin, F., Medof, M.E., and Heeger, P.S. (2008). Locally produced C5a binds to T cell-expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis. Blood 112, 1759-1766.

Lanier, L.L., Le, A.M., Phillips, J.H., Warner, N.L., and Babcock, G.F. (1983). Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J Immunol 131, 1789-1796.

Lawrence, T., and Natoli, G. (2011). Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nature Reviews Immunology 11, 750-761.

Leach, D.R., Krummel, M.F., and Allison, J.P. (1996). Enhancement of antitumor immunity by CTLA-4 blockade. Science 271, 1734-1736.

Leek, R.D., Lewis, C.E., Whitehouse, R., Greenall, M., Clarke, J., and Harris, A.L. (1996). Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res 56, 4625-4629.

Leibovici, J., and Hoenig, S. (1985). Lysis and growth stimulation of a murine melanoma determined by density of macrophage populations. Anticancer Res 5, 545-551.

Leist, T.P., Kohler, M., Eppler, M., and Zinkernagel, R.M. (1989). Effects of treatment with IL-2 receptor specific monoclonal antibody in mice. Inhibition of cytotoxic T cell responses but not of T help. J Immunol 143, 628-632.

Lesokhin, A.M., Hohl, T.M., Kitano, S., Cortez, C., Hirschhorn-Cymerman, D., Avogadri, F., Rizzuto, G.A., Lazarus, J.J., Pamer, E.G., Houghton, A.N., et al. (2012). Monocytic CCR2(+) myeloid-derived suppressor cells promote immune escape by limiting activated CD8 T-cell infiltration into the tumor microenvironment. Cancer Res 72, 876-886.

Ley, K. (2014). The second touch hypothesis: T cell activation, homing and polarization. F1000Research 3, 37.

Ley, K., Smith, E., and Stark, M.A. (2006). IL-17A-producing neutrophil-regulatory Tn lymphocytes. Immunol Res 34, 229-242.

Li, C., Shintani, S., Terakado, N., Nakashiro, K., and Hamakawa, H. (2002). Infiltration of tumor-associated macrophages in human oral squamous cell carcinoma. Oncology reports 9, 1219-1223.

131

Li, L., Huang, L., Vergis, A.L., Ye, H., Bajwa, A., Narayan, V., Strieter, R.M., Rosin, D.L., and Okusa, M.D. (2010). IL-17 produced by neutrophils regulates IFN-gamma-mediated neutrophil migration in mouse kidney ischemia-reperfusion injury. J Clin Invest 120, 331- 342.

Li, Q., Teitz-Tennenbaum, S., Donald, E.J., Li, M., and Chang, A.E. (2009). In vivo sensitized and in vitro activated B cells mediate tumor regression in cancer adoptive immunotherapy. J Immunol 183, 3195-3203.

Li, R., Coulthard, L.G., Wu, M.C., Taylor, S.M., and Woodruff, T.M. (2013). C5L2: a controversial receptor of complement anaphylatoxin, C5a. FASEB J 27, 855-864.

Lieberman, J. (2003). The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat Rev Immunol 3, 361-370.

Lim, H., Kim, Y.U., Drouin, S.M., Mueller-Ortiz, S., Yun, K., Morschl, E., Wetsel, R.A., and Chung, Y. (2012). Negative regulation of pulmonary Th17 responses by C3a anaphylatoxin during allergic inflammation in mice. PLoS One 7, e52666.

Lin, C.T., Tsai, Y.C., He, L., Yeh, C.N., Chang, T.C., Soong, Y.K., Monie, A., Hung, C.F., and Lai, C.H. (2007). DNA vaccines encoding IL-2 linked to HPV-16 E7 antigen generate enhanced E7-specific CTL responses and antitumor activity. Immunol Lett 114, 86-93.

Liu, K., Victora, G.D., Schwickert, T.A., Guermonprez, P., Meredith, M.M., Yao, K., Chu, F.F., Randolph, G.J., Rudensky, A.Y., and Nussenzweig, M. (2009). In vivo analysis of dendritic cell development and homeostasis. Science 324, 392-397.

Liu, Y., Chen, Y., Li, Z., Han, Y., Sun, Y., Wang, Q., Liu, B., and Su, Z. (2013). Role of IL- 10-producing regulatory B cells in control of cerebral malaria in Plasmodium berghei infected mice. Eur J Immunol 43, 2907-2918.

Liu, Y., Mei, C., Liu, H., Wang, H., Zeng, G., Lin, J., and Xu, M. (2014). Modulation of cytokine expression in human macrophages by endocrine-disrupting chemical Bisphenol-A. Biochem Biophys Res Commun 451, 592-598.

Lloyd, A.R., and Oppenheim, J.J. (1992). Poly's lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response. Immunol Today 13, 169-172.

Loveland, B.E., and Cebon, J. (2008). Cancer exploiting complement: a clue or an exception? Nat Immunol 9, 1205-1206.

Lucas, S.D., Karlsson-Parra, A., Nilsson, B., Grimelius, L., Akerstrom, G., Rastad, J., and Juhlin, C. (1996). Tumor-specific deposition of immunoglobulin G and complement in papillary thyroid carcinoma. Hum Pathol 27, 1329-1335.

132

Lv, L., Pan, K., Li, X.D., She, K.L., Zhao, J.J., Wang, W., Chen, J.G., Chen, Y.B., Yun, J.P., and Xia, J.C. (2011). The accumulation and prognosis value of tumor infiltrating IL-17 producing cells in esophageal squamous cell carcinoma. PLoS One 6, e18219.

Ma, C.S., Deenick, E.K., Batten, M., and Tangye, S.G. (2012). The origins, function, and regulation of T follicular helper cells. J Exp Med 209, 1241-1253.

Ma, J., Han, H., Ma, L., Liu, C., Xue, X., Ma, P., Li, X., and Tao, H. (2014). The immunostimulatory effects of retinoblastoma cell supernatant on dendritic cells. Protein & cell 5, 307-316.

Macatonia, S.E., Hosken, N.A., Litton, M., Vieira, P., Hsieh, C.S., Culpepper, J.A., Wysocka, M., Trinchieri, G., Murphy, K.M., and O'Garra, A. (1995). Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J Immunol 154, 5071-5079.

Macor, P., and Tedesco, F. (2007). Complement as effector system in cancer immunotherapy. Immunol Lett 111, 6-13.

Maher, S.G., McDowell, D.T., Collins, B.C., Muldoon, C., Gallagher, W.M., and Reynolds, J.V. (2011). Serum proteomic profiling reveals that pretreatment complement protein levels are predictive of esophageal cancer patient response to neoadjuvant chemoradiation. Ann Surg 254, 809-816; discussion 816-807.

Mamane, Y., Chung Chan, C., Lavallee, G., Morin, N., Xu, L.J., Huang, J., Gordon, R., Thomas, W., Lamb, J., Schadt, E.E., et al. (2009). The C3a anaphylatoxin receptor is a key mediator of insulin resistance and functions by modulating adipose tissue macrophage infiltration and activation. Diabetes 58, 2006-2017.

Manderson, A.P., Botto, M., and Walport, M.J. (2004). The role of complement in the development of systemic lupus erythematosus. Annu Rev Immunol 22, 431-456.

Manthey, H.D., Thomas, A.C., Shiels, I.A., Zernecke, A., Woodruff, T.M., Rolfe, B., and Taylor, S.M. (2011). Complement C5a inhibition reduces atherosclerosis in ApoE-/- mice. FASEB J 25, 2447-2455.

Manthey, H.D., Woodruff, T.M., Taylor, S.M., and Monk, P.N. (2009). Complement component 5a (C5a). Int J Biochem Cell Biol 41, 2114-2117.

Mantovani, A., Sica, A., and Locati, M. (2007). New vistas on macrophage differentiation and activation. European Journal of Immunology 37, 14-16.

March, D.R., Proctor, L.M., Stoermer, M.J., Sbaglia, R., Abbenante, G., Reid, R.C., Woodruff, T.M., Wadi, K., Paczkowski, N., Tyndall, J.D., et al. (2004). Potent cyclic antagonists of the complement C5a receptor on human polymorphonuclear leukocytes. Relationships between structures and activity. Mol Pharmacol 65, 868-879.

133

Marigo, I., Dolcetti, L., Serafini, P., Zanovello, P., and Bronte, V. (2008). Tumor-induced tolerance and immune suppression by myeloid derived suppressor cells. Immunol Rev 222, 162-179.

Markiewski, M.M., DeAngelis, R.A., Benencia, F., Ricklin-Lichtsteiner, S.K., Koutoulaki, A., Gerard, C., Coukos, G., and Lambris, J.D. (2008a). Modulation of the antitumor immune response by complement. Nat Immunol 9, 1225-1235.

Markiewski, M.M., DeAngelis, R.A., and Lambris, J.D. (2008b). Complexity of complement activation in sepsis. J Cell Mol Med 12, 2245-2254.

Markiewski, M.M., and Lambris, J.D. (2007). The role of complement in inflammatory diseases from behind the scenes into the spotlight. Am J Pathol 171, 715-727.

Martin-Fontecha, A., Thomsen, L.L., Brett, S., Gerard, C., Lipp, M., Lanzavecchia, A., and Sallusto, F. (2004). Induced recruitment of NK cells to lymph nodes provides IFN-gamma for T(H)1 priming. Nat Immunol 5, 1260-1265.

Martin-Orozco, N., Muranski, P., Chung, Y., Yang, X.O., Yamazaki, T., Lu, S., Hwu, P., Restifo, N.P., Overwijk, W.W., and Dong, C. (2009a). T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity 31, 787-798.

Martin-Orozco, N., Muranski, P., Chung, Y., Yang, X.X.O., Yamazaki, T., Lu, S.J., Hwu, P., Restifo, N.P., Overwijk, W.W., and Dong, C. (2009b). T Helper 17 Cells Promote Cytotoxic T Cell Activation in Tumor Immunity. Immunity 31, 787-798.

Martin, C., Burdon, P.C., Bridger, G., Gutierrez-Ramos, J.C., Williams, T.J., and Rankin, S.M. (2003). Chemokines acting via CXCR2 and CXCR4 control the release of neutrophils from the bone marrow and their return following senescence. Immunity 19, 583-593.

Martinelli, S., Urosevic, M., Daryadel, A., Oberholzer, P.A., Baumann, C., Fey, M.F., Dummer, R., Simon, H.U., and Yousefi, S. (2004). Induction of genes mediating interferon- dependent extracellular trap formation during neutrophil differentiation. J Biol Chem 279, 44123-44132.

Marx, J. (2008). Cancer immunology. Cancer's bulwark against immune attack: MDS cells. Science 319, 154-156.

Mastellos, D.C. (2009). Targeting innate immune pathways in cancer immunotherapy: state of the art. J BUON 14 Suppl 1, S123-130.

Mathieu, M.C., Sawyer, N., Greig, G.M., Hamel, M., Kargman, S., Ducharme, Y., Lau, C.K., Friesen, R.W., O'Neill, G.P., Gervais, F.G., and Therien, A.G. (2005). The C3a receptor antagonist SB 290157 has agonist activity. Immunol Lett 100, 139-145.

Mattes, J., and Foster, P.S. (2003). Regulation of eosinophil migration and Th2 cell function by IL-5 and eotaxin. Curr Drug Targets Inflamm Allergy 2, 169-174.

134

Mattes, J., Hulett, M., Xie, W., Hogan, S., Rothenberg, M.E., Foster, P., and Parish, C. (2003). Immunotherapy of cytotoxic T cell-resistant tumors by T helper 2 cells: an eotaxin and STAT6-dependent process. J Exp Med 197, 387-393.

McArthur, G.A., and Ribas, A. (2013). Targeting Oncogenic Drivers and the Immune System in Melanoma. Journal of Clinical Oncology 31, 499-506.

Medina-Echeverz, J., Aranda, F., and Berraondo, P. (2014). Myeloid-derived cells are key targets of tumor immunotherapy. Oncoimmunology 3, e28398.

Medina-Echeverz, J., Fioravanti, J., Zabala, M., Ardaiz, N., Prieto, J., and Berraondo, P. (2011). Successful colon cancer eradication after chemoimmunotherapy is associated with profound phenotypic change of intratumoral myeloid cells. J Immunol 186, 807-815.

Mellado, M., de Ana, A.M., Moreno, M.C., Martinez, C., and Rodriguez-Frade, J.M. (2001). A potential immune escape mechanism by melanoma cells through the activation of chemokine-induced T cell death. Current biology : CB 11, 691-696.

Mestas, J., and Hughes, C.C. (2004). Of mice and not men: differences between mouse and human immunology. J Immunol 172, 2731-2738.

Mills, C.D., Kincaid, K., Alt, J.M., Heilman, M.J., and Hill, A.M. (2000). M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol 164, 6166-6173.

Mishalian, I., Bayuh, R., Levy, L., Zolotarov, L., Michaeli, J., and Fridlender, Z.G. (2013). Tumor-associated neutrophils (TAN) develop pro-tumorigenic properties during tumor progression. Cancer Immunol Immunother 62, 1745-1756.

Miyahara, Y., Odunsi, K., Chen, W.H., Peng, G.Y., Matsuzaki, J., and Wang, R.F. (2008). Generation and regulation of human CD4+ IL-17-producing T cells in ovarian cancer. P Natl Acad Sci USA 105, 15505-15510.

Mizoguchi, A., Mizoguchi, E., Takedatsu, H., Blumberg, R.S., and Bhan, A.K. (2002). Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity 16, 219-230.

Mocikat, R., Braumuller, H., Gumy, A., Egeter, O., Ziegler, H., Reusch, U., Bubeck, A., Louis, J., Mailhammer, R., Riethmuller, G., et al. (2003). Natural killer cells activated by MHC class I(low) targets prime dendritic cells to induce protective CD8 T cell responses. Immunity 19, 561-569.

Monk, P.N., Scola, A.M., Madala, P., and Fairlie, D.P. (2007). Function, structure and therapeutic potential of complement C5a receptors. Br J Pharmacol 152, 429-448.

Montz, H., Koch, K.C., Zierz, R., and Gotze, O. (1991). The role of C5a in interleukin-6 production induced by lipopolysaccharide or interleukin-1. Immunology 74, 373-379.

135

Moraes, T.J., Zurawska, J.H., and Downey, G.P. (2006). Neutrophil granule contents in the pathogenesis of lung injury. Current opinion in hematology 13, 21-27.

Moran, C.J., Arenberg, D.A., Huang, C.C., Giordano, T.J., Thomas, D.G., Misek, D.E., Chen, G., Iannettoni, M.D., Orringer, M.B., Hanash, S., and Beer, D.G. (2002). RANTES expression is a predictor of survival in stage I lung adenocarcinoma. Clin Cancer Res 8, 3803-3812.

Morgan, B.P. (1999). Regulation of the complement membrane attack pathway. Crit Rev Immunol 19, 173-198.

Morgan, E.L., Weigle, W.O., and Hugli, T.E. (1982). Anaphylatoxin-mediated regulation of the immune response. I. C3a-mediated suppression of human and murine humoral immune responses. J Exp Med 155, 1412-1426.

Moser, M., and Murphy, K.M. (2000). Dendritic cell regulation of TH1-TH2 development. Nat Immunol 1, 199-205.

Mosmann, T.R., Cherwinski, H., Bond, M.W., Giedlin, M.A., and Coffman, R.L. (1986). Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136, 2348-2357.

Moss, P.A., Rosenberg, W.M., and Bell, J.I. (1992). The human T cell receptor in health and disease. Annu Rev Immunol 10, 71-96.

Mosser, D.M., and Edwards, J.P. (2008). Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8, 958-969.

Movahedi, K., Laoui, D., Gysemans, C., Baeten, M., Stange, G., Van den Bossche, J., Mack, M., Pipeleers, D., In't Veld, P., De Baetselier, P., and Van Ginderachter, J.A. (2010). Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes. Cancer Res 70, 5728-5739.

Mukherji, B., Wilhelm, S.A., Guha, A., and Ergin, M.T. (1986). Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro cytotoxic immune response. J Immunol 136, 1888-1892.

Mule, J.J., Custer, M., Averbook, B., Yang, J.C., Weber, J.S., Goeddel, D.V., Rosenberg, S.A., and Schall, T.J. (1996). RANTES secretion by gene-modified tumor cells results in loss of tumorigenicity in vivo: role of immune cell subpopulations. Hum Gene Ther 7, 1545-1553.

Muranski, P., Boni, A., Antony, P.A., Cassard, L., Irvine, K.R., Kaiser, A., Paulos, C.M., Palmer, D.C., Touloukian, C.E., Ptak, K., et al. (2008). Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood 112, 362-373.

Murata, K., and Baldwin, W.M., 3rd (2009). Mechanisms of complement activation, C4d deposition, and their contribution to the pathogenesis of antibody-mediated rejection. Transplantation reviews 23, 139-150.

136

Musiani, P., Allione, A., Modica, A., Lollini, P.L., Giovarelli, M., Cavallo, F., Belardelli, F., Forni, G., and Modesti, A. (1996). Role of neutrophils and lymphocytes in inhibition of a mouse mammary adenocarcinoma engineered to release IL-2, IL-4, IL-7, IL-10, IFN-alpha, IFN-gamma, and TNF-alpha. Lab Invest 74, 146-157.

Mylonas, K.J., Nair, M.G., Prieto-Lafuente, L., Paape, D., and Allen, J.E. (2009). Alternatively activated macrophages elicited by helminth infection can be reprogrammed to enable microbial killing. J Immunol 182, 3084-3094.

Nakakubo, Y., Miyamoto, M., Cho, Y., Hida, Y., Oshikiri, T., Suzuoki, M., Hiraoka, K., Itoh, T., Kondo, S., and Katoh, H. (2003). Clinical significance of immune cell infiltration within gallbladder cancer. Brit J Cancer 89, 1736-1742.

Naylor, M.F., Chen, W.R., Teague, T.K., Perry, L.A., and Nordquist, R.E. (2006). In situ photoimmunotherapy: a tumour-directed treatment for melanoma. Br J Dermatol 155, 1287- 1292.

Nazarian, R., Shi, H.B., Wang, Q., Kong, X.J., Koya, R.C., Lee, H., Chen, Z.G., Lee, M.K., Attar, N., Sazegar, H., et al. (2010). Melanomas acquire resistance toB-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468, 973-U377.

Niculescu, F., Rus, H.G., Retegan, M., and Vlaicu, R. (1992). Persistent complement activation on tumor cells in breast cancer. Am J Pathol 140, 1039-1043.

Nishimura, T., Iwakabe, K., Sekimoto, M., Ohmi, Y., Yahata, T., Nakui, M., Sato, T., Habu, S., Tashiro, H., Sato, M., and Ohta, A. (1999). Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo. J Exp Med 190, 617-627.

Niwa, Y., Akamatsu, H., Niwa, H., Sumi, H., Ozaki, Y., and Abe, A. (2001). Correlation of tissue and plasma RANTES levels with disease course in patients with breast or cervical cancer. Clin Cancer Res 7, 285-289.

Noonan, F.P., Recio, J.A., Takayama, H., Duray, P., Anver, M.R., Rush, W.L., De Fabo, E.C., and Merlino, G. (2001). Neonatal sunburn and melanoma in mice - Severe sunburn in newborn, but not adult, mice is linked with melanoma in later life. Nature 413, 271-272.

Norgauer, J., Dobos, G., Kownatzki, E., Dahinden, C., Burger, R., Kupper, R., and Gierschik, P. (1993). Complement fragment C3a stimulates Ca2+ influx in neutrophils via a pertussis- toxin-sensitive G protein. Eur J Biochem 217, 289-294.

Nozaki, M., Raisler, B.J., Sakurai, E., Sarma, J.V., Barnum, S.R., Lambris, J.D., Chen, Y., Zhang, K., Ambati, B.K., Baffi, J.Z., and Ambati, J. (2006). Drusen complement components C3a and C5a promote choroidal neovascularization. Proc Natl Acad Sci U S A 103, 2328- 2333.

Numasaki, M., Fukushi, J., Ono, M., Narula, S.K., Zavodny, P.J., Kudo, T., Robbins, P.D., Tahara, H., and Lotze, M.T. (2003). Interleukin-17 promotes angiogenesis and tumor growth. Blood 101, 2620-2627.

137

Numasaki, M., Watanabe, M., Suzuki, T., Takahashi, H., Nakamura, A., McAllister, F., Hishinuma, T., Goto, J., Lotze, M.T., Kolls, J.K., and Sasaki, H. (2005). IL-17 enhances the net angiogenic activity and in vivo growth of human non-small cell lung cancer in SCID mice through promoting CXCR-2-dependent angiogenesis. Journal of Immunology 175, 6177- 6189.

Nunez-Cruz, S., Gimotty, P.A., Guerra, M.W., Connolly, D.C., Wu, Y.Q., DeAngelis, R.A., Lambris, J.D., Coukos, G., and Scholler, N. (2012). Genetic and pharmacologic inhibition of complement impairs endothelial cell function and ablates ovarian cancer neovascularization. Neoplasia 14, 994-1004.

Nutt, S.L., Brady, J., Hayakawa, Y., and Smyth, M.J. (2004). Interleukin 21: a key player in lymphocyte maturation. Crit Rev Immunol 24, 239-250.

Ochi, A., Nguyen, A.H., Bedrosian, A.S., Mushlin, H.M., Zarbakhsh, S., Barilla, R., Zambirinis, C.P., Fallon, N.C., Rehman, A., Pylayeva-Gupta, Y., et al. (2012). MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells. J Exp Med 209, 1671-1687.

Oh, S.A., and Li, M.O. (2013). TGF-beta: guardian of T cell function. J Immunol 191, 3973- 3979.

Okada, H., and Nishioka, K. (1973). Complement receptors on cell membranes. I. Evidence for two complement receptors. J Immunol 111, 1444-1449.

Onita, T., Ji, P.G., Xuan, J.W., Sakai, H., Kanetake, H., Maxwell, P.H., Fong, G.H., Gabril, M.Y., Moussa, M., and Chin, J.L. (2002). Hypoxia-induced, perinecrotic expression of endothelial Per-ARNT-Sim domain protein-1/hypoxia-inducible factor-2alpha correlates with tumor progression, vascularization, and focal macrophage infiltration in bladder cancer. Clin Cancer Res 8, 471-480.

Opdenakker, G., Fibbe, W.E., and Van Damme, J. (1998). The molecular basis of leukocytosis. Immunol Today 19, 182-189.

Organization, W.H. (2014). World Cancer Report. Ch. 5.14.

Ostrand-Rosenberg, S. (2008). Cancer and complement. Nat Biotechnol 26, 1348-1349.

Ostrand-Rosenberg, S., and Sinha, P. (2009). Myeloid-derived suppressor cells: linking inflammation and cancer. J Immunol 182, 4499-4506.

Ottonello, L., Corcione, A., Tortolina, G., Airoldi, I., Albesiano, E., Favre, A., D'Agostino, R., Malavasi, F., Pistoia, V., and Dallegri, F. (1999). rC5a directs the in vitro migration of human memory and naive tonsillar B lymphocytes: implications for B cell trafficking in secondary lymphoid tissues. J Immunol 162, 6510-6517.

Ouyang, W.J., Kolls, J.K., and Zheng, Y. (2008). The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 28, 454-467.

138

Pandolfi, F., Cianci, R., Lolli, S., Dunn, I.S., Newton, E.E., Haggerty, T.J., Boyle, L.A., and Kurnick, J.T. (2008). Strategies to overcome obstacles to successful immunotherapy of melanoma. Int J Immunopathol Pharmacol 21, 493-500.

Pardoll, D.M. (2012). The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 12, 252-264.

Pardoux, C., Asselin-Paturel, C., Chehimi, J., Gay, F., Mami-Chouaib, F., and Chouaib, S. (1997). Functional interaction between TGF-beta and IL-12 in human primary allogeneic cytotoxicity and proliferative response. J Immunol 158, 136-143.

Park, H., Li, Z., Yang, X.O., Chang, S.H., Nurieva, R., Wang, Y.H., Wang, Y., Hood, L., Zhu, Z., Tian, Q., and Dong, C. (2005). A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 6, 1133-1141.

Parker, D.C. (1993). T cell-dependent B cell activation. Annu Rev Immunol 11, 331-360.

Passlick, B., Flieger, D., and Ziegler-Heitbrock, H.W. (1989). Identification and characterization of a novel monocyte subpopulation in human peripheral blood. Blood 74, 2527-2534.

Pelletier, M., Maggi, L., Micheletti, A., Lazzeri, E., Tamassia, N., Costantini, C., Cosmi, L., Lunardi, C., Annunziato, F., Romagnani, S., and Cassatella, M.A. (2010). Evidence for a cross-talk between human neutrophils and Th17 cells. Blood 115, 335-343.

Pericle, F., Giovarelli, M., Colombo, M.P., Ferrari, G., Musiani, P., Modesti, A., Cavallo, F., Di Pierro, F., Novelli, F., and Forni, G. (1994). An efficient Th2-type memory follows CD8+ lymphocyte-driven and eosinophil-mediated rejection of a spontaneous mouse mammary adenocarcinoma engineered to release IL-4. J Immunol 153, 5659-5673.

Philip, M., Rowley, D.A., and Schreiber, H. (2004). Inflammation as a tumor promoter in cancer induction. Semin Cancer Biol 14, 433-439.

Piao, C., Cai, L., Qiu, S., Jia, L., Song, W., and Du, J. (2015). Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-mediated Inflammatory Cell Infiltration. J Biol Chem 290, 10667-10676.

Pillay, J., den Braber, I., Vrisekoop, N., Kwast, L.M., de Boer, R.J., Borghans, J.A., Tesselaar, K., and Koenderman, L. (2010). In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days. Blood 116, 625-627.

Poulikakos, P.I., Persaud, Y., Janakiraman, M., Kong, X.J., Ng, C., Moriceau, G., Shi, H.B., Atefi, M., Titz, B., Gabay, M.T., et al. (2011). RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 480, 387-U144.

Pratilas, C.A., and Solit, D.B. (2010). Targeting the mitogen-activated protein kinase pathway: physiological feedback and drug response. Clin Cancer Res 16, 3329-3334.

139

Predina, J., Eruslanov, E., Judy, B., Kapoor, V., Cheng, G., Wang, L.C., Sun, J., Moon, E.K., Fridlender, Z.G., Albelda, S., and Singhal, S. (2013). Changes in the local tumor microenvironment in recurrent cancers may explain the failure of vaccines after surgery. Proc Natl Acad Sci U S A 110, E415-424.

Prestwich, R.J., Errington, F., Hatfield, P., Merrick, A.E., Ilett, E.J., Selby, P.J., and Melcher, A.A. (2008). The immune system--is it relevant to cancer development, progression and treatment? Clin Oncol (R Coll Radiol) 20, 101-112.

Preynat-Seauve, O., Schuler, P., Contassot, E., Beermann, F., Huard, B., and French, L.E. (2006). Tumor-infiltrating dendritic cells are potent antigen-presenting cells able to activate T cells and mediate tumor rejection. Journal of Immunology 176, 61-67.

Proctor, L.M., Arumugam, T.V., Shiels, I., Reid, R.C., Fairlie, D.P., and Taylor, S.M. (2004). Comparative anti-inflammatory activities of antagonists to C3a and C5a receptors in a rat model of intestinal ischaemia/reperfusion injury. Br J Pharmacol 142, 756-764.

Proctor, L.M., Moore, T.A., Monk, P.N., Sanderson, S.D., Taylor, S.M., and Woodruff, T.M. (2009). Complement factors C3a and C5a have distinct hemodynamic effects in the rat. Int Immunopharmacol 9, 800-806.

Qin, Z., Richter, G., Schuler, T., Ibe, S., Cao, X., and Blankenstein, T. (1998). B cells inhibit induction of T cell-dependent tumor immunity. Nat Med 4, 627-630.

Quatromoni, J.G., and Eruslanov, E. (2012). Tumor-associated macrophages: function, phenotype, and link to prognosis in human lung cancer. American journal of translational research 4, 376-389.

Quezada, S.A., Peggs, K.S., Curran, M.A., and Allison, J.P. (2006). CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells. J Clin Invest 116, 1935-1945.

Ratajczak, J., Reca, R., Kucia, M., Majka, M., Allendorf, D.J., Baran, J.T., Janowska- Wieczorek, A., Wetsel, R.A., Ross, G.D., and Ratajczak, M.Z. (2004). Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow. Blood 103, 2071-2078.

Reca, R., Mastellos, D., Majka, M., Marquez, L., Ratajczak, J., Franchini, S., Glodek, A., Honczarenko, M., Spruce, L.A., Janowska-Wieczorek, A., et al. (2003). Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1. Blood 101, 3784-3793.

Ricklin, D., Hajishengallis, G., Yang, K., and Lambris, J.D. (2010). Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11, 785-797.

Ricklin, D., and Lambris, J.D. (2007). Complement-targeted therapeutics. Nat Biotechnol 25, 1265-1275.

140

Robbins, C.S., Chudnovskiy, A., Rauch, P.J., Figueiredo, J.L., Iwamoto, Y., Gorbatov, R., Etzrodt, M., Weber, G.F., Ueno, T., van Rooijen, N., et al. (2012). Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions. Circulation 125, 364-374.

Robert, C., Ribas, A., Wolchok, J.D., Hodi, F.S., Hamid, O., Kefford, R., Weber, J.S., Joshua, A.M., Hwu, W.J., Gangadhar, T.C., et al. (2014). Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial. Lancet 384, 1109-1117.

Robert, C., Thomas, L., Bondarenko, I., O'Day, S., Weber, J., Garbe, C., Lebbe, C., Baurain, J.F., Testori, A., Grob, J.J., et al. (2011). Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 364, 2517-2526.

Robinson, D.S., Hamid, Q., Ying, S., Tsicopoulos, A., Barkans, J., Bentley, A.M., Corrigan, C., Durham, S.R., and Kay, A.B. (1992). Predominant TH2-like bronchoalveolar T- lymphocyte population in atopic asthma. N Engl J Med 326, 298-304.

Roca, H., Varsos, Z.S., Sud, S., Craig, M.J., Ying, C., and Pienta, K.J. (2009). CCL2 and interleukin-6 promote survival of human CD11b+ peripheral blood mononuclear cells and induce M2-type macrophage polarization. J Biol Chem 284, 34342-34354.

Roitt, I.M., and Delves, P.J. (2001). Roitt's essential immunology, 11th edn (Oxford, UK ; Malden, MA: Blackwell Science).

Rollins, T.E., Siciliano, S., Kobayashi, S., Cianciarulo, D.N., Bonilla-Argudo, V., Collier, K., and Springer, M.S. (1991). Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex. Proc Natl Acad Sci U S A 88, 971- 975.

Romagnani, S. (1991). Type 1 T helper and type 2 T helper cells: functions, regulation and role in protection and disease. International journal of clinical & laboratory research 21, 152- 158.

Rosenberg, S.A., Spiess, P., and Lafreniere, R. (1986). A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233, 1318-1321.

Ross, G.D., Polley, M.J., Rabellino, E.M., and Grey, H.M. (1973). Two different complement receptors on human lymphocytes. One specific for C3b and one specific for C3b inactivator- cleaved C3b. J Exp Med 138, 798-811.

Rottman, J.B., Ganley, K.P., Williams, K., Wu, L., Mackay, C.R., and Ringler, D.J. (1997). Cellular localization of the chemokine receptor CCR5. Correlation to cellular targets of HIV- 1 infection. Am J Pathol 151, 1341-1351.

Rousseau, S., Dolado, I., Beardmore, V., Shpiro, N., Marquez, R., Nebreda, A.R., Arthur, J.S., Case, L.M., Tessier-Lavigne, M., Gaestel, M., et al. (2006). CXCL12 and C5a trigger

141

cell migration via a PAK1/2-p38alpha MAPK-MAPKAP-K2-HSP27 pathway. Cell Signal 18, 1897-1905.

Ruddy, S., Gigli, I., and Austen, K.F. (1972). The complement system of man. 4. N Engl J Med 287, 642-646.

Ruffell, B., DeNardo, D.G., Affara, N.I., and Coussens, L.M. (2010). Lymphocytes in cancer development: polarization towards pro-tumor immunity. Cytokine Growth Factor Rev 21, 3- 10.

Rutschman, R., Lang, R., Hesse, M., Ihle, J.N., Wynn, T.A., and Murray, P.J. (2001). Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production. J Immunol 166, 2173-2177.

Sadik, C.D., Kim, N.D., and Luster, A.D. (2011). Neutrophils cascading their way to inflammation. Trends Immunol 32, 452-460.

Sakaguchi, S., Yamaguchi, T., Nomura, T., and Ono, M. (2008). Regulatory T cells and . Cell 133, 775-787.

Sanderson, K., Scotland, R., Lee, P., Liu, D., Groshen, S., Snively, J., Sian, S., Nichol, G., Davis, T., Keler, T., et al. (2005). Autoimmunity in a phase I trial of a fully human anti- cytotoxic T-lymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma. J Clin Oncol 23, 741-750.

Santarpia, L., Lippman, S.M., and El-Naggar, A.K. (2012). Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy. Expert Opin Ther Targets 16, 103-119.

Sato, K., Kuratsu, J., Takeshima, H., Yoshimura, T., and Ushio, Y. (1995). Expression of monocyte chemoattractant protein-1 in meningioma. J Neurosurg 82, 874-878.

Sato, T., Nakashima, A., Guo, L., Coffman, K., and Tamanoi, F. (2010). Single amino-acid changes that confer constitutive activation of mTOR are discovered in human cancer. Oncogene 29, 2746-2752.

Sawanobori, Y., Ueha, S., Kurachi, M., Shimaoka, T., Talmadge, J.E., Abe, J., Shono, Y., Kitabatake, M., Kakimi, K., Mukaida, N., and Matsushima, K. (2008). Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice. Blood 111, 5457- 5466.

Schaerli, P., Willimann, K., Lang, A.B., Lipp, M., Loetscher, P., and Moser, B. (2000). CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. J Exp Med 192, 1553-1562.

Schieferdecker, H.L., Schlaf, G., Jungermann, K., and Gotze, O. (2001). Functions of anaphylatoxin C5a in rat liver: direct and indirect actions on nonparenchymal and parenchymal cells. Int Immunopharmacol 1, 469-481.

142

Schlecker, E., Stojanovic, A., Eisen, C., Quack, C., Falk, C.S., Umansky, V., and Cerwenka, A. (2012). Tumor-infiltrating monocytic myeloid-derived suppressor cells mediate CCR5- dependent recruitment of regulatory T cells favoring tumor growth. J Immunol 189, 5602- 5611.

Schlitzer, A., McGovern, N., Teo, P., Zelante, T., Atarashi, K., Low, D., Ho, A.W., See, P., Shin, A., Wasan, P.S., et al. (2013). IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses. Immunity 38, 970-983.

Scola, A.M., Johswich, K.O., Morgan, B.P., Klos, A., and Monk, P.N. (2009). The human complement fragment receptor, C5L2, is a recycling decoy receptor. Mol Immunol 46, 1149- 1162.

Semerad, C.L., Liu, F., Gregory, A.D., Stumpf, K., and Link, D.C. (2002). G-CSF is an essential regulator of neutrophil trafficking from the bone marrow to the blood. Immunity 17, 413-423.

Serafini, P., Mgebroff, S., Noonan, K., and Borrello, I. (2008). Myeloid-derived suppressor cells promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells. Cancer Res 68, 5439-5449.

Sevko, A., and Umansky, V. (2013). Myeloid-derived suppressor cells interact with tumors in terms of myelopoiesis, tumorigenesis and immunosuppression: thick as thieves. Journal of Cancer 4, 3-11.

Sfanos, K.S., Bruno, T.C., Maris, C.H., Xu, L., Thoburn, C.J., DeMarzo, A.M., Meeker, A.K., Isaacs, W.B., and Drake, C.G. (2008). Phenotypic analysis of prostate-infiltrating lymphocytes reveals T(H)17 and T-reg skewing. Clinical Cancer Research 14, 3254-3261.

Shah, S., Divekar, A.A., Hilchey, S.P., Cho, H.M., Newman, C.L., Shin, S.U., Nechustan, H., Challita-Eid, P.M., Segal, B.M., Yi, K.H., and Rosenblatt, J.D. (2005). Increased rejection of primary tumors in mice lacking B cells: inhibition of anti-tumor CTL and TH1 cytokine responses by B cells. Int J Cancer 117, 574-586.

Sharma, M.D., Hou, D.Y., Liu, Y., Koni, P.A., Metz, R., Chandler, P., Mellor, A.L., He, Y., and Munn, D.H. (2009). Indoleamine 2,3-dioxygenase controls conversion of Foxp3+ Tregs to TH17-like cells in tumor-draining lymph nodes. Blood 113, 6102-6111.

Shi, H.B., Moriceau, G., Kong, X.J., Koya, R.C., Nazarian, R., Pupo, G.M., Bacchiocchi, A., Dahlman, K.B., Chmielowski, B., Sosman, J.A., et al. (2012a). Preexisting MEK1 Exon 3 Mutations in (V600E/K)BRAF Melanomas Do Not Confer Resistance to BRAF Inhibitors. Cancer discovery 2, 414-424.

Shi, H.B., Moriceau, G., Kong, X.J., Lee, M.K., Lee, H., Koya, R.C., Ng, C., Chodon, T., Scolyer, R.A., Dahlman, K.B., et al. (2012b). Melanoma whole-exome sequencing identifies B-V600E-RAF amplification-mediated acquired B-RAF inhibitor resistance. Nature communications 3.

143

Shields, J.D., Kourtis, I.C., Tomei, A.A., Roberts, J.M., and Swartz, M.A. (2010). Induction of lymphoidlike stroma and immune escape by tumors that express the chemokine CCL21. Science 328, 749-752.

Siddiqui, S.A., Frigola, X., Bonne-Annee, S., Mercader, M., Kuntz, S.M., Krambeck, A.E., Sengupta, S., Dong, H., Cheville, J.C., Lohse, C.M., et al. (2007). Tumor-infiltrating Foxp3- CD4+CD25+ T cells predict poor survival in renal cell carcinoma. Clin Cancer Res 13, 2075- 2081.

Silberman, A.W. (1987). Malignant melanoma. Practical considerations concerning prophylactic regional lymph node dissection. Ann Surg 206, 206-209.

Sinha, P., Okoro, C., Foell, D., Freeze, H.H., Ostrand-Rosenberg, S., and Srikrishna, G. (2008). Proinflammatory S100 proteins regulate the accumulation of myeloid-derived suppressor cells. J Immunol 181, 4666-4675.

Smyth, M.J., Hayakawa, Y., Takeda, K., and Yagita, H. (2002). New aspects of natural- killer-cell surveillance and therapy of cancer. Nat Rev Cancer 2, 850-861.

Solassol, J., Rouanet, P., Lamy, P.J., Allal, C., Favre, G., Maudelonde, T., and Mange, A. (2010). Serum protein signature may improve detection of ductal carcinoma in situ of the breast. Oncogene 29, 550-560.

Solinas, G., Germano, G., Mantovani, A., and Allavena, P. (2009). Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J Leukoc Biol 86, 1065-1073.

Song, J.K., Park, M.H., Choi, D.Y., Yoo, H.S., Han, S.B., Yoon do, Y., and Hong, J.T. (2012). Deficiency of C-C chemokine receptor 5 suppresses tumor development via inactivation of NF-kappaB and upregulation of IL-1Ra in melanoma model. PLoS One 7, e33747.

Sorrentino, R., Morello, S., Forte, G., Montinaro, A., De Vita, G., Luciano, A., Palma, G., Arra, C., Maiolino, P., Adcock, I.M., and Pinto, A. (2011). B cells contribute to the antitumor activity of CpG-oligodeoxynucleotide in a mouse model of metastatic lung carcinoma. Am J Respir Crit Care Med 183, 1369-1379.

Sosman, J.A., Kim, K.B., Schuchter, L., Gonzalez, R., Pavlick, A.C., Weber, J.S., McArthur, G.A., Hutson, T.E., Moschos, S.J., Flaherty, K.T., et al. (2012). Survival in BRAF V600- Mutant Advanced Melanoma Treated with Vemurafenib. New Engl J Med 366, 707-714.

Souto, J.C., Vila, L., and Bru, A. (2011). Polymorphonuclear neutrophils and cancer: intense and sustained neutrophilia as a treatment against solid tumors. Med Res Rev 31, 311-363.

Stark, M.A., Huo, Y., Burcin, T.L., Morris, M.A., Olson, T.S., and Ley, K. (2005). Phagocytosis of apoptotic neutrophils regulates granulopoiesis via IL-23 and IL-17. Immunity 22, 285-294.

144

Steinman, R.M. (2007). Dendritic cells: understanding immunogenicity. Eur J Immunol 37 Suppl 1, S53-60.

Steinman, R.M., and Cohn, Z.A. (1973). Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 137, 1142-1162.

Steinman, R.M., Lustig, D.S., and Cohn, Z.A. (1974). Identification of a novel cell type in peripheral lymphoid organs of mice. 3. Functional properties in vivo. J Exp Med 139, 1431- 1445.

Stockwin, L.H., McGonagle, D., Martin, I.G., and Blair, G.E. (2000). Dendritic cells: immunological sentinels with a central role in health and disease. Immunol Cell Biol 78, 91- 102.

Strachan, A.J., Woodruff, T.M., Haaima, G., Fairlie, D.P., and Taylor, S.M. (2000). A new small molecule C5a receptor antagonist inhibits the reverse-passive Arthus reaction and endotoxic shock in rats. J Immunol 164, 6560-6565.

Strainic, M.G., Liu, J., Huang, D., An, F., Lalli, P.N., Muqim, N., Shapiro, V.S., Dubyak, G.R., Heeger, P.S., and Medof, M.E. (2008a). Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells. Immunity 28, 425-435.

Strainic, M.G., Liu, J.B., Huang, D.P., An, F., Lalli, P.N., Muqim, N., Shapiro, V.S., Dubyak, G.R., Heeger, P.S., and Medof, M.E. (2008b). Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4(+) T cells. Immunity 28, 425-435.

Strainic, M.G., Shevach, E.M., An, F., Lin, F., and Medof, M.E. (2013). Absence of signaling into CD4(+) cells via C3aR and C5aR enables autoinductive TGF-beta1 signaling and induction of Foxp3(+) regulatory T cells. Nat Immunol 14, 162-171.

Straussman, R., Morikawa, T., Shee, K., Barzily-Rokni, M., Qian, Z.R., Du, J., Davis, A., Mongare, M.M., Gould, J., Frederick, D.T., et al. (2012). Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 487, 500-504.

Street, S.E., Cretney, E., and Smyth, M.J. (2001). Perforin and interferon-gamma activities independently control tumor initiation, growth, and metastasis. Blood 97, 192-197.

Stutman, O. (1975). Immunodepression and malignancy. Adv Cancer Res 22, 261-422.

Su, X., Ye, J., Hsueh, E.C., Zhang, Y., Hoft, D.F., and Peng, G. (2010). Tumor microenvironments direct the recruitment and expansion of human Th17 cells. J Immunol 184, 1630-1641.

145

Sugiyama, M., Akiu, S., Hidaka, T., and Ogura, R. (1985). [Membrane fluidity of B-16 melanoma cells exposed to ultraviolet light UV-B]. Nihon Hifuka Gakkai zasshi. The Japanese journal of dermatology 95, 1043-1047.

Summers, C., Rankin, S.M., Condliffe, A.M., Singh, N., Peters, A.M., and Chilvers, E.R. (2010). Neutrophil kinetics in health and disease. Trends Immunol 31, 318-324.

Surace, L., Lysenko, V., Fontana, A.O., Cecconi, V., Janssen, H., Bicvic, A., Okoniewski, M., Pruschy, M., Dummer, R., Neefjes, J., et al. (2015). Complement is a central mediator of radiotherapy-induced tumor-specific immunity and clinical response. Immunity 42, 767-777.

Swann, J.B., and Smyth, M.J. (2007). Immune surveillance of tumors. J Clin Invest 117, 1137-1146.

Sykulev, Y., Joo, M., Vturina, I., Tsomides, T.J., and Eisen, H.N. (1996). Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response. Immunity 4, 565-571.

Szabo, S.J., Sullivan, B.M., Stemmann, C., Satoskar, A.R., Sleckman, B.P., and Glimcher, L.H. (2002). Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science 295, 338-342.

Takabayashi, T., Vannier, E., Clark, B.D., Margolis, N.H., Dinarello, C.A., Burke, J.F., and Gelfand, J.A. (1996). A new biologic role for C3a and C3a desArg: regulation of TNF-alpha and IL-1 beta synthesis. J Immunol 156, 3455-3460.

Takafuji, S., Tadokoro, K., Ito, K., and Dahinden, C.A. (1994). Degranulation from human eosinophils stimulated with C3a and C5a. Int Arch Allergy Immunol 104 Suppl 1, 27-29.

Takanami, I., Takeuchi, K., and Kodaira, S. (1999). Tumor-associated macrophage infiltration in pulmonary adenocarcinoma: association with angiogenesis and poor prognosis. Oncology 57, 138-142.

Tawara, I., Take, Y., Uenaka, A., Noguchi, Y., and Nakayama, E. (2002). Sequential involvement of two distinct CD4+ regulatory T cells during the course of transplantable tumor growth and protection from 3-methylcholanthrene-induced tumorigenesis by CD25- depletion. Jpn J Cancer Res 93, 911-916.

Taylor, S.M., Sherman, S.A., Kirnarsky, L., and Sanderson, S.D. (2001). Development of response-selective agonists of human C5a anaphylatoxin: conformational, biological, and therapeutic considerations. Curr Med Chem 8, 675-684.

Tempero, R.M., Hollingsworth, M.A., Burdick, M.D., Finch, A.M., Taylor, S.M., Vogen, S.M., Morgan, E.L., and Sanderson, S.D. (1997). Molecular adjuvant effects of a conformationally biased agonist of human C5a anaphylatoxin. J Immunol 158, 1377-1382.

Tepper, R.I., Pattengale, P.K., and Leder, P. (1989). Murine interleukin-4 displays potent anti-tumor activity in vivo. Cell 57, 503-512.

146

Teunissen, M.B., Koomen, C.W., de Waal Malefyt, R., Wierenga, E.A., and Bos, J.D. (1998). Interleukin-17 and interferon-gamma synergize in the enhancement of proinflammatory cytokine production by human keratinocytes. J Invest Dermatol 111, 645-649.

Topalian, S.L., Drake, C.G., and Pardoll, D.M. (2012a). Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Immunol 24, 207-212.

Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S.N., Smith, D.C., McDermott, D.F., Powderly, J.D., Carvajal, R.D., Sosman, J.A., Atkins, M.B., et al. (2012b). Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443-2454.

Torisu, H., Ono, M., Kiryu, H., Furue, M., Ohmoto, Y., Nakayama, J., Nishioka, Y., Sone, S., and Kuwano, M. (2000). Macrophage infiltration correlates with tumor stage and angiogenesis in human malignant melanoma: possible involvement of TNFalpha and IL- 1alpha. Int J Cancer 85, 182-188.

Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350-4354.

Trainin, N., Linker-Israeli, M., Small, M., and Boiato-Chen, L. (1967). Enhancement of lung adenoma formation by neonatal thymectomy in mice treated with 7,12- dimethylbenz(a)anthracene or urethan. Int J Cancer 2, 326-336.

Ueno, T., Toi, M., Saji, H., Muta, M., Bando, H., Kuroi, K., Koike, M., Inadera, H., and Matsushima, K. (2000). Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res 6, 3282-3289.

Vaday, G.G., Peehl, D.M., Kadam, P.A., and Lawrence, D.M. (2006). Expression of CCL5 (RANTES) and CCR5 in prostate cancer. Prostate 66, 124-134.

Vadrevu, S.K., Chintala, N.K., Sharma, S.K., Sharma, P., Cleveland, C., Riediger, L., Manne, S., Fairlie, D.P., Gorczyca, W., Almanza, O., et al. (2014). Complement c5a receptor facilitates cancer metastasis by altering T-cell responses in the metastatic niche. Cancer Res 74, 3454-3465.

Valkovic, T., Dobrila, F., Melato, M., Sasso, F., Rizzardi, C., and Jonjic, N. (2002). Correlation between vascular endothelial growth factor, angiogenesis, and tumor-associated macrophages in invasive ductal breast carcinoma. Virchows Arch 440, 583-588.

Van den Steen, P.E., Dubois, B., Nelissen, I., Rudd, P.M., Dwek, R.A., and Opdenakker, G. (2002). Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9). Critical reviews in biochemistry and molecular biology 37, 375-536. van Furth, R., and Cohn, Z.A. (1968). The origin and kinetics of mononuclear phagocytes. J Exp Med 128, 415-435.

147

Varela, J.C., Imai, M., Atkinson, C., Ohta, R., Rapisardo, M., and Tomlinson, S. (2008). Modulation of protective T cell immunity by complement inhibitor expression on tumor cells. Cancer Res 68, 6734-6742.

Varney, M.L., Johansson, S.L., and Singh, R.K. (2005). Tumour-associated macrophage infiltration, neovascularization and aggressiveness in malignant melanoma: role of monocyte chemotactic protein-1 and vascular endothelial growth factor-A. Melanoma Res 15, 417-425.

Varol, C., Vallon-Eberhard, A., Elinav, E., Aychek, T., Shapira, Y., Luche, H., Fehling, H.J., Hardt, W.D., Shakhar, G., and Jung, S. (2009). Intestinal lamina propria dendritic cell subsets have different origin and functions. Immunity 31, 502-512.

Varsano, S., Rashkovsky, L., Shapiro, H., Ophir, D., and Mark-Bentankur, T. (1998). Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro, and an insight into mechanism(s) of resistance. Clin Exp Immunol 113, 173-182.

Venkatesha, R.T., Berla Thangam, E., Zaidi, A.K., and Ali, H. (2005). Distinct regulation of C3a-induced MCP-1/CCL2 and RANTES/CCL5 production in human mast cells by extracellular signal regulated kinase and PI3 kinase. Mol Immunol 42, 581-587.

Villanueva, J., Vultur, A., Lee, J.T., Somasundaram, R., Fukunaga-Kalabis, M., Cipolla, A.K., Wubbenhorst, B., Xu, X., Gimotty, P.A., Kee, D., et al. (2010). Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 18, 683-695.

Virchow, R. (1863). Die krankhaften Geschwulste.

von Andrian, U.H., and Mackay, C.R. (2000). T-cell function and migration. Two sides of the same coin. N Engl J Med 343, 1020-1034.

Voo, K.S., Wang, Y.H., Santori, F.R., Boggiano, C., Wang, Y.H., Arima, K., Bover, L., Hanabuchi, S., Khalili, J., Marinova, E., et al. (2009). Identification of IL-17-producing FOXP3+ regulatory T cells in humans. Proc Natl Acad Sci U S A 106, 4793-4798.

Vos, Q., Lees, A., Wu, Z.Q., Snapper, C.M., and Mond, J.J. (2000). B-cell activation by T- cell-independent type 2 antigens as an integral part of the humoral immune response to pathogenic microorganisms. Immunol Rev 176, 154-170.

Voskoboinik, I., Smyth, M.J., and Trapani, J.A. (2006). Perforin-mediated target-cell death and immune homeostasis. Nat Rev Immunol 6, 940-952.

Vukmanovic-Stejic, M., Zhang, Y., Cook, J.E., Fletcher, J.M., McQuaid, A., Masters, J.E., Rustin, M.H., Taams, L.S., Beverley, P.C., Macallan, D.C., and Akbar, A.N. (2006). Human CD4+ CD25hi Foxp3+ regulatory T cells are derived by rapid turnover of memory populations in vivo. J Clin Invest 116, 2423-2433.

148

Wagle, N., Emery, C., Berger, M.F., Davis, M.J., Sawyer, A., Pochanard, P., Kehoe, S.M., Johannessen, C.M., MacConaill, L.E., Hahn, W.C., et al. (2011). Dissecting Therapeutic Resistance to RAF Inhibition in Melanoma by Tumor Genomic Profiling. Journal of Clinical Oncology 29, 3085-3096.

Walport, M.J. (2001). Complement. First of two parts. N Engl J Med 344, 1058-1066.

Walsh, K.B., Lanier, L.L., and Lane, T.E. (2008). NKG2D receptor signaling enhances cytolytic activity by virus-specific CD8+ T cells: evidence for a protective role in virus- induced encephalitis. J Virol 82, 3031-3044.

Watson, N.F., Durrant, L.G., Madjd, Z., Ellis, I.O., Scholefield, J.H., and Spendlove, I. (2006). Expression of the membrane complement regulatory protein CD59 (protectin) is associated with reduced survival in colorectal cancer patients. Cancer Immunol Immunother 55, 973-980.

Weaver, D.J., Jr., Reis, E.S., Pandey, M.K., Kohl, G., Harris, N., Gerard, C., and Kohl, J. (2010). C5a receptor-deficient dendritic cells promote induction of Treg and Th17 cells. Eur J Immunol 40, 710-721.

Weber, J. (2011). Immunotherapy for melanoma. Curr Opin Oncol 23, 163-169.

Wesolowski, R., Markowitz, J., and Carson, W.E., 3rd (2013). Myeloid derived suppressor cells - a new therapeutic target in the treatment of cancer. Journal for immunotherapy of cancer 1, 10.

Whiteside, T.L. (1999). Signaling defects in T lymphocytes of patients with malignancy. Cancer Immunol Immunother 48, 346-352.

Whiteside, T.L. (2008). The tumor microenvironment and its role in promoting tumor growth. Oncogene 27, 5904-5912.

Williams, A.E., and Chambers, R.C. (2014). The mercurial nature of neutrophils: still an enigma in ARDS? American journal of physiology. Lung cellular and molecular physiology 306, L217-230.

Wing, K., and Sakaguchi, S. (2010). Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat Immunol 11, 7-13.

Woodman, S.E., Trent, J.C., Stemke-Hale, K., Lazar, A.J., Pricl, S., Pavan, G.M., Fermeglia, M., Gopal, Y.N., Yang, D., Podoloff, D.A., et al. (2009). Activity of dasatinib against L576P KIT mutant melanoma: molecular, cellular, and clinical correlates. Mol Cancer Ther 8, 2079- 2085.

Woodruff, T.M., Arumugam, T.V., Shiels, I.A., Reid, R.C., Fairlie, D.P., and Taylor, S.M. (2003). A potent human C5a receptor antagonist protects against disease pathology in a rat model of inflammatory bowel disease. J Immunol 171, 5514-5520.

149

Woodruff, T.M., Arumugam, T.V., Shiels, I.A., Reid, R.C., Fairlie, D.P., and Taylor, S.M. (2004). Protective effects of a potent C5a receptor antagonist on experimental acute limb ischemia-reperfusion in rats. J Surg Res 116, 81-90.

Woodruff, T.M., Nandakumar, K.S., and Tedesco, F. (2011). Inhibiting the C5-C5a receptor axis. Mol Immunol.

Woodruff, T.M., and Tenner, A.J. (2015). A Commentary On: "NFkappaB-Activated Astroglial Release of Complement C3 Compromises Neuronal Morphology and Function Associated with Alzheimer's Disease". A cautionary note regarding C3aR. Frontiers in immunology 6, 220.

Woodruff, T.M., Tenner, A. (2015). A Commentary on: NF-ΚΒ-Activated Astroglial Release of Complement C3 Compromises Neuronal Morphology and Function Associated with Alzheimer's Disease. A Cautionary Note Regarding C3aR. Front. Immunol.

Wu, M.C., Brennan, F.H., Lynch, J.P., Mantovani, S., Phipps, S., Wetsel, R.A., Ruitenberg, M.J., Taylor, S.M., and Woodruff, T.M. (2013). The receptor for complement component C3a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization. Proc Natl Acad Sci U S A 110, 9439-9444.

Xing, Z., Jordana, M., Kirpalani, H., Driscoll, K.E., Schall, T.J., and Gauldie, J. (1994). Cytokine Expression by Neutrophils and Macrophages in-Vivo - Endotoxin Induces Tumor- Necrosis-Factor-Alpha, Macrophage Inflammatory Protein-2, Interleukin-1-Beta, and Interleukin-6 but Not Rantes or Transforming Growth Factor-Beta(1) Messenger-Rna Expression in Acute Lung Inflammation. Am J Resp Cell Mol 10, 148-153.

Xu, L., Shen, S.S., Hoshida, Y., Subramanian, A., Ross, K., Brunet, J.P., Wagner, S.N., Ramaswamy, S., Mesirov, J.P., and Hynes, R.O. (2008). Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases. Molecular cancer research : MCR 6, 760-769.

Xu, Y., Jagannath, C., Liu, X.D., Sharafkhaneh, A., Kolodziejska, K.E., and Eissa, N.T. (2007). Toll-like receptor 4 is a sensor for autophagy associated with innate immunity. Immunity 27, 135-144.

Yang, J.C., Beck, K.E., Blansfield, J.A., Tran, K.Q., Lowy, I., and Rosenberg, S.A. (2005). Tumor regression in patients with metastatic renal cancer treated with a monoclonal antibody to CTLA4 (MDX-010). Journal of Clinical Oncology 23, 166s-166s.

Yang, L., DeBusk, L.M., Fukuda, K., Fingleton, B., Green-Jarvis, B., Shyr, Y., Matrisian, L.M., Carbone, D.P., and Lin, P.C. (2004). Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell 6, 409-421.

150

Yao, H., Ng, S.S., Huo, L.F., Chow, B.K., Shen, Z., Yang, M., Sze, J., Ko, O., Li, M., Yue, A., et al. (2011). Effective Melanoma Immunotherapy with Interleukin-2 Delivered by a Novel Polymeric Nanoparticle. Mol Cancer Ther.

Ye, J., Livergood, R.S., and Peng, G. (2013). The role and regulation of human Th17 cells in tumor immunity. Am J Pathol 182, 10-20.

Yona, S., Kim, K.W., Wolf, Y., Mildner, A., Varol, D., Breker, M., Strauss-Ayali, D., Viukov, S., Guilliams, M., Misharin, A., et al. (2013). Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 79-91.

Youn, J.I., Nagaraj, S., Collazo, M., and Gabrilovich, D.I. (2008). Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol 181, 5791-5802.

Yu, D., Rao, S., Tsai, L.M., Lee, S.K., He, Y., Sutcliffe, E.L., Srivastava, M., Linterman, M., Zheng, L., Simpson, N., et al. (2009). The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment. Immunity 31, 457-468.

Zelensky, A.N., and Gready, J.E. (2005). The C-type lectin-like domain superfamily. Febs J 272, 6179-6217.

Zell, S., Geis, N., Rutz, R., Schultz, S., Giese, T., and Kirschfink, M. (2007). Down- regulation of CD55 and CD46 expression by anti-sense phosphorothioate oligonucleotides (S-ODNs) sensitizes tumour cells to complement attack. Clin Exp Immunol 150, 576-584.

Zhang, J., Patel, L., and Pienta, K.J. (2010). CC chemokine ligand 2 (CCL2) promotes prostate cancer tumorigenesis and metastasis. Cytokine Growth Factor Rev 21, 41-48.

Zhang, S., Bernard, D., Khan, W.I., Kaplan, M.H., Bramson, J.L., and Wan, Y. (2009). CD4+ T-cell-mediated anti-tumor immunity can be uncoupled from autoimmunity via the STAT4/STAT6 signaling axis. Eur J Immunol 39, 1252-1259.

Zhang, X., Kimura, Y., Fang, C., Zhou, L., Sfyroera, G., Lambris, J.D., Wetsel, R.A., Miwa, T., and Song, W.C. (2007). Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo. Blood 110, 228-236.

Zheng, W., and Flavell, R.A. (1997). The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587-596.

Zhou, L., Chong, M.M., and Littman, D.R. (2009). Plasticity of CD4+ T cell lineage differentiation. Immunity 30, 646-655.

Zielinski, C.E., Mele, F., Aschenbrenner, D., Jarrossay, D., Ronchi, F., Gattorno, M., Monticelli, S., Lanzavecchia, A., and Sallusto, F. (2012). Pathogen-induced human TH17 cells produce IFN-gamma or IL-10 and are regulated by IL-1beta. Nature 484, 514-518.

151

Zipfel, P.F., Heinen, S., Jozsi, M., and Skerka, C. (2006). Complement and diseases: defective alternative pathway control results in kidney and eye diseases. Mol Immunol 43, 97-106.

Zollo, M., Di Dato, V., Spano, D., De Martino, D., Liguori, L., Marino, N., Vastolo, V., Navas, L., Garrone, B., Mangano, G., et al. (2012). Targeting monocyte chemotactic protein- 1 synthesis with bindarit induces tumor regression in prostate and breast cancer animal models. Clin Exp Metastasis 29, 585-601.

Zou, W. (2005). Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 5, 263-274.

152