CD200-CD200R Interaction in Tumor Immunity DISSERTATION

CD200-CD200R Interaction in Tumor Immunity

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University

Fatemeh Talebian

Program: Integrated Biomedical Science Graduate Program (IBGP)

* * * * *

The Ohio State University 2012

Dissertation Committee:

Professor Xue-Feng Bai, Advisor Professor Amy Lovett-Racke Professor Ramish Ganju Professor Sujit Basu

Abstract

CD200 is a member of the Ig super family (IgSF) of proteins. It is expressed on cell surface of a variety of normal cells including lymphoid cells and some lineages of cancer cells such as melanoma and ovarian cancer cells. CD200 functions through engaging its specific receptor, CD200R. CD200R is also an IgSF protein, with an inhibitory intracellular NPXY signaling motif. CD200R has a restricted pattern of expression and is mainly detected on cells of the myeloid lineage. CD200-CD200R interaction inhibits the function(s) of myeloid cells. Myeloid cells are the first cells recruited by tumors and are essential in the regulation of tumor initiation, establishment, progression and metastasis.

Tumor associated myeloid cells (TAMCs) are also known to inhibit activation and effector functions of T cells. Therefore, we hypothesized that CD200-CD200R interaction affects tumor formation, metastasis and tumor immunity via inhibiting TAMC functions. The goals of this dissertation thesis are three fold: 1) To investigate

CD200/CD200R expression in the tumor compartments; 2) To determine the role of

CD200-CD200R interaction in tumor formation and metastasis and 3) To determine if targeting CD200R is a feasible approach for cancer immunotherapy.

We investigated CD200/CD200R expression on myeloid cells and T cells under various conditions. We have found that myeloid cells constitutively express CD200 and

CD200R and upregulate both these cell surface molecules when activated. Naive T cells do not express CD200 and CD200R. When activated, T cells upregulate CD200

expression dramatically without upregulating CD200R. In the tumor microenvironment,

myeloid cells express high levels of both CD200 and CD200R. Tumor infiltrating T cells express high levels of CD200, while their expression of CD200R is barely detectable.

We next studied the impact of tumor expression of CD200 on tumor formation and metastasis, using the CD200-positive and CD200-negative B16 melanoma model.

Subcutaneous injection of CD200-positive B16 melanoma cells inhibited tumor formation and growth in C57BL/6 mice but not in Rag1-/-C57BL/6 mice. However, i.v.

injection of CD200-positive B16 melanoma cells dramatically inhibited tumor foci

formation in the lungs of both C57BL/6 and Rag1-/-C57BL6 mice. Flow cytometry

analysis revealed higher expression of CD200R in lung Gr1+ myeloid cells than in

peripheral myeloid cells. In vivo depletion of Gr1+ cells dramatically inhibited tumor foci

formation in the lungs. In addition, treatment with tumor antigen specific CD4 and CD8

T cells or their combination yielded a survival advantage for CD200 positive tumor bearing mice over mice bearing CD200-negative tumors. Analysis of microarray data from human cancer patients revealed that patients with CD200hi tumors have better

prognosis and longer survival time.

To test if CD200R is a suitable target for cancer immunotherapy, we first

generated CD200R-/- mice and found that CD200-positive melanoma cells grow and

metastasize progressively in CD200R-/- mice but not in WT mice. Stimulation of

CD200R with an agonistic antibody dramatically inhibited lung metastasis of CD200- iii

negative melanoma. Use of monoclonal agonistic CD200R antibodies inhibited tumor growth and improved survival time in established tumor models. Finally we found that many cancer cells derived from the myeloid lineage also express CD200R and they are more susceptible to CTL lysis. Blocking CD200 using antibody or knock down of

CD200 gene expression in CTL significantly reduced CTL destruction of CD200R- positive cancer cells. Analysis of microarray data of human myeloma revealed that patients with CD200Rhigh cancer cells had lower relapse rates and a longer survival time.

Taken together, we have found that in the tumor microenvironment there are

highly significant expressions of CD200 and CD200R. CD200-CD200R interaction is

broadly involved in regulating tumor formation, metastasis and tumor immunity.

Targeting CD200R may be a novel approach for the immunotherapy of human cancer.

Dedication

To my Mother, Zahra Taghipour, who gave me life and taught me to have dreams and see the beauty in life. To her who was, is and will be my inspiration at every bend and curve I encounter in life.

To my Father, Mohammad Ali Talebian, whose greatest dreams have always been the success of his children.

Knowing the glowing happiness that would light up your faces when I finished this work was one of the strongest driving forces when times got tough.

To My Husband, Bizhan Matin, who has been my rock, who has kept me going and always been positive and encouraging through the rough patches and the good ones too. It truly would not have been possible without you.

And to my Babies, Mehraneh and Mohammad, who make me realize how valuable life is every morning and how blessed I am every evening.

Acknowledgments It is with profound gratitude that I would like to acknowledge the following people who have been instrumental in my achieving my goal of PhD. Dr. Xue-Feng, My mentor and PI, for giving me a place in his lab to learn and refine my skills, and become a better scientist. Jin-Qing Liu for her assistance with experiments and sharing her experience. Zhenzhen Liu, my lab partner, for her friendship, lively discussions, and assistance with experiments and training. It was a much better journey because we were able to share it. Christine Kerr, for her support and friendship that carried me through some tough times. It would have been much more difficult without her. Dr. Virginia Sanders, for her support, her encouragement and for truly being there for us as students when we needed an advocate. Dr. Amy Lovett-Racke for her advice and her support. You have been Immensely helpful and hopefully will be a lifelong collaborator and colleague. Dr.Sujit Basu, a special thanks to you. Because of everything you have done for me and your unwavering support. Thank you for believing in me and thank you for your time and your refreshing attitude toward a scientist’s life. Dr. Ramesh Ganju, Thank you for your time and purposeful scrutiny. I reserve my deepest appreciation and gratitude for my family, friends and loved ones who gave of their unlimited stores of love and support. Thank you for reminding me “I Can Do It”. Thank you Hassan and Atefeh for bieng the best siblings anyone ever had. For always believing in me. Thank you Bizhan, for I might not have gone the distance without your support. Last but certainly not least, I thank my mother and father, Zahra and Mohammad, for everything you have given me, all you have done for me, and all you still do. Thank you for all the meaning you have infused into my life and all that you keep on giving.

Abbreviations cDNA Complementary DNA

IgSF Immunoglobulin Super Family

Kb Kilo bases

TLR Toll-like receptor

NLR NOD-like receptor

ROS Reactive Oxygen Species

RNS Reactive Nitrogen Species

ITIM Immunoreceptor Tyrosine-based Inhibitory Motif

ERK Extrsacellular signal regulated Kinase

JAK Janus Kinase/Just another kinase

JNK C-Jun N-terminal Kinase

DOK Downstream of Kinase

CNS Central Nervous System

PNS Peripheral Nervous System

NOS Nitric Oxide Synthase

AD Alzheimer’s Disease

MHC Major Histocompatibility Complex

ICAM Intracellular Adhesion Molecule

CIA Collagen Induced Arthritis

RA Rheumatoid Arthritis vii

vCD200 Viral CD200

B-CLL B cell Chronic Lymphoproliferative Leukemia

Hu-SCID Human Severe Combined Immunodefeciency

NK Natural Killer

AML Acute Myeloid Leukemia mRNA messenger RNA

DLN Draining Lymph node

TAMC Tumor Associated Myeloid Cells

MDSC Myeliod Derived Suppressor Cells

TAM Tumor Associated Macrophages

GM-CSF Granulocyte Macrophage Colony Stimulating Factor

STAT Signal Transducers and activators of Transcription

IMC Immature Myeloid Cells

TIL Tumor Infiltrating Lymphocytes

CTL Cytotoxic T Lymphocyte

TCR T-Cell Receptor

ELISA Enzyme Linked Immunosorbent Assay

LPS Lipopolysaccheride

IL Interleukin

TNF-α Tumor Necrosis Factor- α

IFN-γ Interferon-γ viii

ALL Acute Lymphocytic Leukemia

PCM Plasma Cell Myeloma

MCL Mantle Cell Lymphoma

BCLD B cell Lymphoproliferative Disease

VITA

Born: September 1979 Kansas City, Kansas

1996-1998: University of Illinois at Chicago (UIC), Chicago Illinois Bachelor’s of Art: Major 1: Biochemistry Major 2: English Literature

1998-2002: Tehran University, College of Sciences, Tehran Iran Department of Cellular and Molecular Biology; Bachelor’s of Science: Cellular & Molecular Biology

2003-2007: MS Society of Iran Research Assistant in Project: Immunotherapy with Mesenchymal Stem Cells in MS patients

2003-2006: Tehran University of Medical Sciences (TUMS), Tehran, Iran Department of Immunology Master’s of Science: Medical Immunology No.1 in MSc Entrance Exam

2007-2012: Ohio State University, Columbus Ohio USA Department of Pathology Doctor of Philosophy: Integrated Biomedical Sciences Fields of Study: Cancer Biology, Immunology, Molecular Biology, Bioinformatics, immunohistochemistry Publications ¾ F. Talebian, J.Q. Liu, Z. Liu M. Khattabi, Y. He, R. Ganju, X.F. Bai. Melanoma cell expression of CD200 inhibits tumor formation and lung metastasis via inhibition of myeloid cell functions. PLoS One, 2012, 7(2):e31442. ¾ Fatemeh Talebian and Xue-Feng Bai. The role of tumor expression of CD200 in tumor formation, metastasis and susceptibility to T lymphocyte adoptive transfer therapy. Oncoimmunology, 2012 In Press ¾ L.Wang, F. Talebian, J.Q. Liu, M. Khattabi, L.Yu, X.F. Bai Tumor Associated Myeloid Cell-Derived IL-10 Mediates Evasion of Immunotherapy by Cytotoxic T Lymphocytes. Scandinavian Journal of Immunology. 2012, 75: 273-281. x

¾ L. Wang, JQ Liu, F. Talebian, H.Y. El-Omrani, M. Khattabi, L.Yu, X.F. Bai. Tumor Expression of CD200 Inhibits IL-10 production by Tumor-Associated Myeloid Cells and Prevents Tumor Immune Evasion of CTL Therapy. European Journal of Immunology 2010 Sep; 40(9):2569-79. ¾ Z. Liu, JQ Liu, F Talebian, LC Wu, S Li and XF Bai. IL-27 enhances anti-tumor CD8+ T cell responses via multiple mechanisms. Cancer Research 2012 Under Review. ¾ J.Q. Liu, Z. Liu, X. Zhang, Y. Shi, F Talebian et al. Increased Th17 and Treg responses in EBI3-deficient mice lead to marginally enhanced development of autoimmune encephalomyelitis Journal of Immunology 2012 In Press.

Table of Contents

Page

Abstract …………………………………………………………………………. ii

Dedication ………………………………………………………………………. v

Acknowledgments ……………………………………………………………….. vi

Abbreviations……………………………………………………………………… vii

Vita ……………………………………………………………………………….. x

List of Tables …………………………………………………………………….. xvi

List of Figures …………………………………………………………………. xvii

Chapter 1. Introduction ……………………………………………………….. 1

1.1 CD200 and CD200R: what are they? ………………………………………. 1

1.1.1 What is CD200 …..………………………………………………………………… 1

1.1.2 Regulation of CD200 expression ………………………………………………… 2

1.1.3 Identifying a receptor for CD200 ……………………………………………….. 3

1.1.4 CD200-CD200R interaction inhibits functions of myeloid cells……………… 5

1.2 CD200-CD200R interaction in Health and Disease…………………………….. 5

1.2.1. CD200-CD200R in the central nervous system (CNS) and peripheral nervous

system (PNS)……………………………………………………………………………… 5

1.2.2 CD200/CD200R on fetomaternal interface……………………………………… 6

1.2.3 CD200/CD200R interaction in the Lung …………………………………. 6

1.2.4 CD200/CD200R in the gastrointestinal tract ………………………………. 6 xii

1.2.5 CD200 in CNS and PNS diseases ……………………………………………. 7

1.2.6 CD200 and Collagen induced Arthritis ………………………………………… 8

1.2.7 CD200 and Viral immune evasion ……………………………………………… 8

1.2.8 CD200/CD200R and Cancer …………………………………………………….. 11

1.3 The immunosuppressive tumor microenvironment: the place where

CD200-CD200R interaction may play a role ………………………………………… 12

1.3.1 The tumor formation ……………………………………………………… 12

1.3.2 Tumor associated myeloid cells and the suppressive tumor microenvironment…………………………………………………………………………. 14

1.3.3 Tumor infiltrating lymphocytes (TILs) in the tumor microenvironment …… 18

1.4. Hypothesis and goals of the study…………………………………………………. 19

Chapter 2. Materials and methods ………………………………………………… 21

2.1 Mice …………………………………………………………………………. 21

2.2 Generation of CD200-positive and CD200R-positive cancer cells and controls …………………………………………………………………………. 21

2.3 Establishment of subcutaneous and lung metastatic tumors ……………….. 22

2.4 Tumorigenesis and T cell adoptive transfer therapy of mice with established

tumors ………………………………………………………………………….. 23

2.5 Antibodies and flow cytometry ……………………………………………… 24

2.6 Isolation of CD11b+ and Gr1+ cells from spleens or lungs ………………… 25

2.7 51CR-release Assay or the Cytotoxicity assay ……………………………….. 25 xiii

2.8 Cytokine ELISA ……………………………………………………………. 25

2.9 Generation of CD200 positive and negative B16.OVA cells ……………… 26

2.10 Real time PCR………………………………………………………………. 26

2.11 CD200 knockdown on T cells………………………………………………. 27

2.12 Statistics……………………………………………………………………... 27

Chapter 3. Results………………………………………………………………… 28

3.1 CD200-CD200R interaction in the tumor microenvironment……………….. 28

3.1.1 Myeloid cells express and upregulate CD200 ……………………………. 28

3.1.2 CD200R is expressed on myeloid cells and upregulated by activation …..... 30

3.1.3 T cells express high levels of CD200 upon activation ……………………... 33

3.1.4 CD200R expression on T cells is minimal and is not upregulated by activation ……………………………………………………………………… 36 3.1.5 CD200-CD200R expression in the tumor microenvironment 39

3.2 The role of tumor expression of CD200 in tumor formation, metastasis and susceptibility to T cell therapy …………………………………………………… 41

3.2.1 Expression of CD200 on melanoma cells inhibits tumor formation and lung

Metastasis ………………………………………………………………………… 42

3.2.2 Tumor expression of CD200 inhibits melanoma lung metastasis through inhibition

of Gr-1+ myeloid cells …………………………………………………………… 44

3.2.3 Tumor expression of CD200 improves the efficacy of T cell adoptive transfer

Therapy…………………………………………………………………………… 50

xiv

3.2.4 Tumor expression of CD200 is implicated in better prognosis and patient survival …………………………………………………………………… 51

3.3 Targeting CD200R in cancer immunotherapy ………………………………. 54

3.3.1 Tumor development and metastasis is significantly increased in CD200R-/- mice ……………………………………………………………….. 54

3.3.2 Triggering CD200R using a monoclonal antibody inhibits tumor foci formation in the lungs ……………………………………………………………………….. 57

3.3.3 Agonistic CD200R antibody treatment suppresses tumor growth and enhances efficacy of CTL adoptive transfer therapy ……………………………………….. 59

3.3.4 CD200R expression on tumor cells serves as a target for

CTL therapy ……………………………………………………………………… 61

3.3.5 CD200R expression in human cancer cells………………………………. 63

Chapter 4. Discussion ……………………………………………………………. 66

4.1 Abundant CD200 and restricted CD200R expression in the tumor microenvironment …………………………………………………………. 66

4.2 Tumor cell expression of CD200 inhibits tumor formation and metastasis via inhibition of myeloid cell functions ……………………………………………… 68

4.3 Tumor expression of CD200 renders tumor microenvironment more permissive to T cells and predicts better response to T cell therapy ………………………… 69

4.4 Does tumor expression of CD200 predict better prognosis?………………….. 70

4.5 Targeting CD200R may be a novel approach for cancer therapy …………… 71 xv

4.6. Concluding remarks and future directions…………………………………… 73

References ……………………………………………………………………….. 81

xvi

List of Tables

Table 1. vCD200 in viruses and its effect of host immune system ………… 10

xvii

List of Figures

Figure 1. Schematic Depiction of CD200 ……………………………………….. 1 Figure 2. Schematic Depiction of CD200R……………………………………… 3 Figure 3. Schematic representation of the signaling cascades of CD200R……… 4 Figure 4. CD200 is expressed on myeloid cells …………………………………. 29 Figure 5. CD200 is upregulated on activated myeloid cells …………………… 30 Figure 6. CD200R expression on myeloid cells ………………………………… 32 Figure 7. CD200R expression on activated myeloid cells ……………………… 33 Figure 8. CD200 expression on T cell populations …………………………….. 35 Figure 9. The kinetics of CD200 and CD200R expression on CD8+ T cells ……. 36 Figure10. CD200R Expression on resting and activated T cells ……………… 38 Figure 11. CD200 and CD200R expression on TAMC and TIL ……………… 40 Figure 12. CD200 on melanoma cells inhibits tumor formation and lung Metastasis ……………………………………………………………………….. 44 Figure13. CD200 expression on tumor cells inhibits tumor lung metastasis in Rag1-/- C57BL/6 mice …………………………………………………………… 46 Figure 14. Gr1+ myeloid cells express CD200R and mediate lung metastasis …. 47 Figure 15. Tumor expression of CD200 inhibits the functions of Gr1+ myeloid Cells ……………………………………………………………………………… 49 Figure 16. CD200-positive tumors are more susceptible to adoptive T cell Therapy …………………………………………………………………………... 52 Figure 17. CD200 gene expression level and survival analysis of patients with CD200high and CD200low expressing tumors as determined by microarray data … 53 Figure 18. Metastatic tumor growth in CD200R-/- mice ………………………… 56 Figure 19. Triggering CD200R using a mAb inhibits melanoma lung metastasis 58 Figure 20. Treatment of Tumor bearing mice with agonistic CD200R antibody .. 60 Figure 21. CD200R on tumor cells serve as a target for CTL ………………….. 62 xviii

Figure 22. CD200R expression level and Survival analysis of patients with CD200Rhigh and CD200Rlow expressing tumors …………………………………. 64 Figure 23. A model for CD200-CD200R interaction in the tumor Microenvironment ……………………………………………………………….. 67 Figure 24. The impacts of tumor expression of CD200 on melanoma lung metastasis Melanoma lung tumor formation ……………………………………. 76 Figure 25. The impacts of tumor expression of CD200 on melanoma susceptibility to T cell therapy …………………………………………………… 77 Figure26. An agonistic antibody to CD200R inhibits melanoma lung tumor formation and metastasis ………………………………………………………… 78 Figure 27. An agonistic antibody to CD200R enhances the susceptibility of established tumors to T cell therapy …………………………………………….. 79 Figure 28. Monoclonal Agonistic CD200R antibody is a promising candidate for cancer immunotherapy for virtually all tumors ……………………………… 80

xix

Chapter 1. Introduction

1.1 CD200 and CD200R: what are they?

1.1.1 What is CD200?

The cell surface glycoprotein CD200 (first named OX2) was reported in 1979 [4].

The mouse CD200 gene is composed of 6 exons and 5 introns spanning 13.7 kb of chromosome 16. It is closely linked with the costimulatory molecules CD80, 86 (B7.1,

B7.2) (similar to that found in humans) [5]. The cDNA sequence of this glycoprotein

suggests a 248 amino acid molecule organized in the form of an Ig super family (IgSF)

light chain anchored to the cell through a single transmembrane portion ending in a 19 amino acid cytoplasmic tail [6]. A monoclonal antibody to CD200 was developed by immunizing mice with a 41-47 KD protein purified from the rat thymocyte [7]. CD200 expression was noted in the brain, thymocytes, endothelium, smooth muscles, blood vessels and a variety of other immune and non-immune cells [8]. In an immunohistochemichal study of rat cerebellular development, high CD200 expression was found during exogenesis [9]. Since Figure1. Schematic Depiction of CD200 CD200 was not expressed ubiquitously, it had no housekeeping role [10], however its widespread distribution and the conserved IgSF-like structural make up suggested a functional role.

Similar expression pattern of CD200 was confirmed to be conserved in chicken

[11] and humans [12]. The human CD200 gene encompasses 8.5 kb, encoded by 4 exons

localized to chromosome 3. The gene displayed 75% homology in the conserved Ig like

domains with up to 90% homology in the transmembrane region to mouse CD200 [13].

For many years after its discovery, CD200 was speculated to be a stop or non-proliferate

signal associated with more “stabilized” or mature cells [7, 14-16].

1.1.2 Regulation of CD200 expression

The molecular and cellular mechanisms of induction and downregulation of

CD200 remain unclear. However, over the years several studies have shed some light on

possible venues of regulation imposed by the environment and mediators. CD200

promoter region is under the transcriptional regulation of C/EBP-β. It can be induced by

TNF-α and IFN-γ in the environment, through NF-κB signaling cascades [17-18].

External factors such as LPS also induce the upregulation of CD200 [19]. LPS exerts its effect through pattern recognition receptors such as TLRs and NLRs [20]. Studies have shown that CD200 expression decreases with age [21]. At this time, IL-4 levels rise [22], which leads to the activation of STAT-6 [23], a molecule involved in anti-inflammatory signaling pathways. Studies on inflammatory conditions have implicated reactive oxygen species (ROS) in the control of CD200 expression [24].

CD200 has a truncated alternative splice variant named CD200tr [25]. Studies

show that transcription of full length CD200 is dependent on direct binding of SF2/ASF

to the exonic splicing enhancer sequence located on exon 2 of the CD200 gene. In the

absence of this DNA binding protein, CD200tr transcription levels are increased and full 2

length CD200 expression is diminished [26]. Thus SF2/ASF is a regulator of CD200

expression.

1.1.3 Identifying a receptor for CD200

Using Recombinant DNA technology, a soluble chimeric protein with the

extracellular domains of CD200 and the 3rd and 4th domain of rat CD4 was produced.

This fusion protein was then coupled to fluorescent beads, allowing for low affinity

binding recognition. The CD200 fusion protein interacted

with a ligand on peritoneal macrophages [4]. A high

affinity monoclonal antibody (OX102) was raised to the

macrophage cell-surface antigen that was able to block

CD200 binding. The obtained protein was cloned,

expressed and found to be a novel protein. It was similar Figure2. Schematic Depiction of CD200R in structure to CD200, containing two extracellular IgSF

domains and a single transmembrane region. However, it was made up of a larger

cytoplasmic tail (67 amino acids) with a few tyrosine residues, a potential for signaling

[27]. Phenotypic analysis showed the receptor for CD200 was mainly restricted to cells of

the myeloid lineage [28]. It was named CD200R. Although an early study [29] claimed

multiple CD200Rs and suggested all of them react with CD200, two other groups [30-31]

have shown the other isoforms do not bind to CD200.

Unlike most of the Ig superfamily receptors, CD200R lacks ITIM domains. The

intracellular tail of CD200R contains 3 conserved tyrosine residues. The third tyrosine is

situated within a NPXY motif, which interacts with the PTB/PID domain present in

signaling adaptor molecules, leading to intracellular signals affecting macrophage

function [32] [kd=2.5microM]. Upon interaction with CD200, the third tyrosine is phosphorylated. This leads to the recruitment of Dok-2 [33] (Figure 3A), which is in turn phosphorylated and associated with RasGAP and SHIP. In mast cells this cascade has been shown to inhibit the phosphorylation of ERK, P38 and JNK [27, 34].

Figure 3. Schematic representation of the signaling cascades of CD200R

Recent work has identified Dok1 as a second target of CD200R tyrosine phosphorylation (Figure 3B). Dok1 phosphorylation is not essential for CD200R

function and it is preceded by dok2 [35]. This work demonstrates that downstream proteins recruited by dok1 and dok2 are different. They suggest that dok1 is a negative

regulator of Dok2.

1.1.4 CD200-CD200R interaction inhibits functions of myeloid cells

CD200 appears to limit autoimmune inflammation in animal models of multiple

sclerosis and arthritis [36] and lung injury caused by viral infection [37], as CD200

deficient mice were found to have a significantly increased disease severity due to hyper

activation of macrophages. CD200R-deficient mice were also shown to be more

susceptible to arthritis, presumably due to enhanced functions of macrophages but not T

cell responses [38]. These findings indicate that CD200-CD200R interactions are

involved in limiting the cellular functions of the myeloid lineage of cells.

1.2 CD200-CD200R interaction in Health and Disease

1.2.1 CD200-CD200R in the central nervous system (CNS) and peripheral nervous

system (PNS)

Although the significance of CD200 in axonal development and neuronal

maturation remains unclear, several studies have documented the abundant expression of

CD200 early in neuronal development and its downregulation and restricted expression at later stages of development. CD200 expression has also been found in the normal CNS

[7-8, 39]. CD200 expression was first noted in the CNS, and today it is recognized as one

of the neuroimmune regulators (NIReg) responsible for maintaining a healthy brain and

restoring normalcy after infection [40]. This regulatory role extends to the peripheral 5

nervous system where Schwann cells and ganglia cells all express CD200 and seem to use it in regulating and maintaining health and balance in the nervous system [41].

1.2.2 CD200-CD200R in fetomaternal interface

CD200 was found on the surface of extraembrynic ectoderm, utrine decedua and

on trophoblasts, in mice and in humans [42]. The greatest density of CD200 expression is

adjacent to the placenta: uterus interface [43]. Administration of CD200Fc fusion protein

decreased the rate of abortion [44]. These data suggest that a prominent expression level

of CD200 may be a condition for successful maintenance of a fetus [45].

1.2.3 CD200-CD200R interaction in the Lung

CD200 expression is high in bronchioles and little in the bronchus [46]. CD200

expression is also detected in pulmonary vein and alveolar capillaries. In an elegant study

Snelgrove et al. [37] demonstrated that alveolar macrophages have a higher response

threshold in part due to higher expression of CD200R on their surface compared to their peers in the spleen or lymph nodes. Since the lung is constantly encountering pathogens, this elevated expression is a manifestation of tissue specific microenvironmental effects.

They observed that CD200R expression is variable and is not lost under inflammatory conditions, but rather upregulated.

1.2.4 CD200-CD200R interaction in the gastrointestinal tract

The largest macrophage population in the body resides in the gastrointestinal

tracts i.e. the intestine and colon [47]. A recent study [48] suggests that intestinal

macrophages are selective in their CD200R expression. They confirmed that similar to 6

alveolar macrophages, intestinal macrophages expressed high levels of CD200R.

1.2.5 CD200 in the CNS and PNS diseases

Patients with nervous system disorders have reduced CD200 expression in affected areas [49-50]. This reduction is primarily due to tissue destruction and thus the absence of intact endothelial layers where CD200 is normally expressed. In CD200-/- mice, microglial cells become hyperactive and NOS+ (a feature not seen in wild type mice) and accelerate the onset of CD4+ T cell mediated diseases such as EAU and EAE

[51]. In 2007 Koning et al. [50] demonstrated that CNS lesions in mice with EAE and

MS patients have reduced CD200 expression. They postulated that CD200 reduction leads to augmented microglia activation in the CNS. This notion was strengthened by another study showing that over expression of CD200 in the CNS was neuroprotective

[52]. On the other hand, CD200R expression levels are elevated under pathological conditions and in alternatively activated microglia [53].

Alzheimer’s disease (AD) is an age related chronic condition brought upon by imbalance in production and secretion of inflammatory mediators. Staining of AD lesions showed lower CD200 expression in the CNS lesions [21].

Parkinson’s disease is also an age associated disease. Two studies suggest that the underlying molecular mechanism is the lower CD200 expression and/or impaired

CD200-CD200R induced silencing of microglia [54-55]. They show a significant inverse correlation between CD200 expression and upregulation of MHCII and ICAM (markers of microglial activation) [56]. 7

1.2.6 CD200 and Collagen induced Arthritis

Collagen induced arthritis (CIA) is an animal model for Rheumatoid Arthritis

(RA) [57]. It is an inflammatory autoimmune disease associated with abundant tissue

specific influx of macrophage, granulocyte and T cells. Macrophages (CD11b+,

CD200R+) and their inflammatory products are an essential element in disease

progression and tissue destruction [36]. CD200-/- mice and CD200R-Ig-treated mice were

more susceptible to CIA development [58]. The enhanced susceptibility can be attributed

to the absence of CD200 and the hyperactivation of CD200R expressing myeloid cells.

When DBA/1 mice induced to develop CIA were treated with either CD200Fc or anti-

CD200R antibody, the mice demonstrated arrest of disease or lower arthritic joint scores

[59]. The reduced clinical and histological presentation of CIA when treated with

CD200Fc was accompanied by a marked decrease in inflammatory cytokines (TNF-α,

IL-1β) and the immunosuppressive cytokine IL-10 [58].

1.2.7 CD200 and Viral immune evasion

Homologues of CD200 have been found in many species of viruses (Table 1)

[60]. It seems that the CD200 gene has been acquired independently by the various viruses over the course of evolution and host-virus adaption. These viral genes have anywhere between 20-60% identity with the human CD200, however they bind the

CD200R with virtually the same affinity as the host CD200 [61]. The pox family of viruses express a truncated vCD200 producing a soluble protein, whereas the herpes viruses produce a cell bound form of the protein [62]. 8

Table1. vCD200 in viruses and its effect of host immune system VCD200 %HOMOLOGY CELL HOMOLO VIRUS WITH HOST LINE(S) MARKERS USED FINDING GUE CD200 USED

IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-13, vCD200 inhibits proinflammatory BC-3, IL-17, G-CSF, GM- cytokine secretion by activated CHO CSF, MCP-1, MIP1β, macrophages via cell-cell interaction with TNF-α. CD200R[61]

human CD200R is predominantly expressed on basophils, activation of K14 is only Ba/F3, Histamine release 40% with identical basophils was down-regulated by expressed NKL, assay HHV-8 affinity for human vCD200 proteins[63] during BC1 CD200R Inhibits FcReR1 dependent activation of Lytic cycle IFN-γ basophils 293T, BJAB, BCBL-1, IL-1β, IL-6, IL-12, K14 induces an activation signal in and JSC- TNF-α, MCP-1 Macrophages[62] 1, U937 myeloid leukemia

56% with rat, binds Cytomegalo host CD200R with REF, iNOS, TNF-α, MHC Downregulates immune responses to the e127 virus the same affinity as NR8383 class II virus[64], an essential virulence factor host protein

monkey kidney (BGMK) cells, iNOS, IFN-γ , TNF-α, rabbit G-CSF, CD8 and Downregulates macrophage activity, kidney Myxovirus CD25 antibodies Less activated T cells (antigen (RK13), Microscopy independent) [65] Rabbit (Count/FOV) CD4T 40% homology M141 lymphoc with host ytes (RL5)

murine TNF-α, G-CSF, iNOS, Downregulates macrophage activity myeloid NF-κB activity (via Myxovirus during the very early stage of virus RAW nuclear NF-κB p65 infection [66] 264.7 measurement)

Rhesus Decreased TNF-α production (>50% rhadinoviru 30% identity with CHO, R15 TNF-α TNFα mRNA levels, >35% TNFα protein s human CD200 THP-1 levels) [67] (RRV17577)

1.2.8 CD200-CD200R interaction in Cancer

Although expression of CD200 has been found in multiple types of cancer cells

[68-70] , the role of CD200-CD200R interaction in tumor immunity is poorly understood.

Reports from various studies are conflicting and thus the role of CD00-CD200R interaction is controversial at present. In an antibody phage display study, CD200 was identified as a highly upregulated molecule on the surface of B-CLL cells [68]. An early report suggests that CD200 expression on tumor cells inhibits activation of tumor specific

T cells [71]. Kretz-Rommel et al [72-73] have demonstrated that anti-CD200 antibody treatment can enhance tumor rejection by T cells and macrophages in a hu-SCID adoptive transfer model for B-CLL. Another recent study suggests the involvement of

CD200 in inhibiting NK activity in AML patients [74]. In two correlation studies, CD200 mRNA expression in malignant cells has been shown to be associated with decreased survival of patients [69-70]. In the case of solid tumors, there are very few studies.

Petermann et al. [75] reported that CD200 expressed on melanoma cells suppressed the ability of dendritic cells to activate T cells. This effect is presumably manifested through

activation of the ERK pathway. Similarly, CD200-positive ovarian cancer cells were

shown to downregulate Th1 cytokine production when co-cultured with allogenic

leukocytes [76]. In two consecutive publications on EMT6, a murine breast cancer cell

line was suggested to be favored by the presence of CD200 to grow [77] and metastasize

[78] to adjacent DLNs. Another work suggested CD200 expression plays a role not in the

initiation and growth but metastasis of squamas cell carcinoma (SCC) [79]. However, 10

these in vitro studies and correlation studies are not conclusive and require further verification in more definitive models. For instance, a group studying development of

Leukemia [80] has demonstrated that lack of CD200-CD200R interaction plays no role in the development or progression of leukemia. Recently, a new study [81] demonstrates that a significant number of tumors that express CD200 at the mRNA level failed to express CD200 protein; while our recent work demonstrates definitively that CD200 expression has a significant role in inhibiting growth and metastasis of tumors [82-83].

1.3 The immunosuppressive tumor microenvironment: the place where CD200-

CD200R interaction may play a role

1.3.1 The tumor formation. Tumor immunology is a war front where the immune system

has lost the initial battle, and now is fighting a “never healing wound” [84]. The tumor

goes through an evolution before it becomes a detective mass. This evolution can be

explained through the Cancer Immune Editing theory which consists of 3 phases: First,

Elimination, as Ehrlich [85] observed. In this stage, innate and adoptive immune systems

come together to a) recognize through innate mechanisms [86], b) mature antigen

presenting cells (APCs), c) generate tumor antigen specific T cells which will then d)

home to the tumor site, attack and kill the tumor cells . When this system fails, an

Equilibrium is achieved. This phase is the result of continuous sculpting of cancerous

cells until resistant cells with reduced immunogenicity arise. This is presumably the

longest phase of cancer development where tumor variants are constantly eliminated and 11

new, selective variants produced [87]. Another way to explain this stage is through the

pathophysiology of inflammation [88]. Initially the response involves the convergence of

innate immunity including macrophages, dendritic cells and natural killer cells [89]. The

mediators released attract leukocytes which will then be activated by the APC in the

vicinity of the tumor mass. The continuous presence of cytokines, ROS, RNS and

chemokines, plus activation of key transcription factors such as NF-kB and STAT-3 [90-

92] leads to genetic instability and mutations in oncogenic and tumor suppressor

pathways such as p53 mutation [93] and aberrant methylations. Eventually, some tumors

develop mechanisms to evade both the innate and the adaptive immune system by

creating a complex local immunosuppressive network. Within this network are the

previously recruited myeloid precursors that have been educated by the tumor

environment to promote cancer progression. The immune suppressive network also

targets the tumor infiltrating lymphocytes (TILs), to either avoid or disable the “killer

cells” [94-95]. Mechanisms such as modulating MHC and costimulatory molecules on

their surface, leading to evasion and ignorance by T cells; expression of Fas and pro- apoptotic molecules, inducing T cell death through FasL [96-97] and other molecular interactions; depletion of tryptophan by secreting indoleamine-pyrrole 2,3-dioxygenase

(IDO); and inhibiting T cells by producing immunosuppressive IL-10, TGF-β and pro-

angiogenic factors such as VEGF [98] are some of the threats the tumor cells and suppressor cells pose for the infiltrating T cells. Tumors also recruit regulatory cells of

the immune system which further promote this suppressive environment [99]. This is

stage three of cancer immune editing or Escape [100]. Here we will briefly review the

pro-tumor and anti-tumor components in the tumor microenvironment, and explain where

CD200-CD200R interactions may play a role.

1.3.2 Tumor associated myeloid cells and the suppressive tumor microenvironment

Cancer formation being a multifactor process involves a myriad of events from genetic mutations to physiological changes. A large body of evidence that suggests chronic inflammation is a major cause of tumor formation and persistence [101]. One study suggests that 25% of cancers are caused and maintained by chronic inflammatory conditions [102]. Consequently, the normal defense mechanisms of the body comprised of inflammatory mediators are present in all tumors. Myeloid cells, being a key component of cancer related inflammation play a major role in encouraging tumorigenesis, progression and metastasis. The direct relation between myeloid cell accumulation and poor prognosis has been overwhelmingly suggested in literature [103-

105].

There is conflicting evidence for the source of accumulating TAMC. Some suggest that chemokine driven entry of inflammatory monocytes maintains the TAMC pool, where they differentiate and become protumor cells [106]. Others [107] believe that tumor resident monocytes represent the bulk of the TAMC population. Blood monocytes are little differentiated cells that are recruited to the tumor via tumor derived chemoattractants such as CCL2 [108] and members of the adhesion molecule family 13

[109]. These early recruited myeloid cells are of the M1 phenotype, or the classically activated macrophages. They release proinflammatory factors that attract NK and promote Th1 cell differentiation and recruitment [110]. This population works to eliminate the tumor cells and inhibit the formation of a tumor microenvironment. As the tumor mass progresses, areas of hypoxia develop and the tumor microenvironment itself becomes more self sustained based on the tumor secreted factors. This defines a new microenvironment where macrophages are polarized to a more M2 phenotype, encouraging Th2 cell differentiation and infiltration [111-112]. The reeducated M2-like myeloid population is pro-tumor and referred to as tumor associated Myeloid cells

(TAMC) [113-114]. Their presence leads to tumor progression and metastasis [115-116] while their depletion correlates with better prognosis and slower tumor growth [117-119].

TAMC subsets (which can make up to 50% of the tumor mass) are responsible for much of tumor sustenance and progression. Two of the main TAMC populations comprise: tumor associated macrophages (TAM) and myeloid derived suppressor cells (MDSC).

TAM are monocytes recruited to the tumor site by tumor derived attractants such as CCL2 [113, 120]. They secrete a slough of factors. Among them are growth factors such as VEGF, PDGF, TGF-β and members of the FGF family [113, 121]; angiogenesis modulating enzymes such as MMP-2, 7, 9 and 12, cycloxgenase-2, and thymidine phosphorylase [102, 122-123]; immunosuppressive factors such as IL-10, TGF-β PGE2,

NO [124-125], and chemoattractants such as CCL17 and CCL22 which preferentially recruit naïve, Th2 and regulatory T cells with little to no cytolytic function [126-127]. 14

Studies suggest that TAM suppress the effector function of T cells in an antigen non-

specific manner [128-129].

MDSCs are a heterogeneous population of immature myeloid cells (IMC)

comprised of hematopoietic progenitor cells and precursors of macrophages, dendritic

cells (DCs) and granulocytes characterized by their cell surface expression of

Gr1+CD11b+ [130-131]. These cells are part of normal hematopoiesis but dramatic expansion of these cells is observed in cancer and other pathological settings [132-134].

MDSCs are especially notorious in suppressing T cell responses [135-136]. Under

chronic pathological conditions, MDSC is markedly recruited by tumor secreted factors

such as IL-10 [132], PGE2 [137], TGF-β [138]MMP9 [131], S100A8/A9, VEGF, GM-

CSF and chemokines CCL2, CXCL5/12 all of which favor tumor progression [139].

Most of the signals that activate and recruit IMC to the pathological site trigger signaling

pathways that converge in the JAK/STAT (especially STAT3) pathway which promote

survival and proliferation of these progenitors. There is now sufficient evidence

demonstrating that unlike the nonspecific manner of T cell tolerance imposed by TAMs

[128-129], MDSC induce Ag-specific CD8 T cell tolerance in cancer [140-141]. MDSC

inhibition is based on cell-cell contact, meaning the effect is mediated either through

molecules expressed on the surface of T cells [142] or short-lived paracrine acting

soluble mediators such as peroxynitrite [143] .

How does the TAMC population work in the tumor microenvironment? Studies

are conflicting but the accumulated evidence strongly suggests that the diverse and plastic 15

myeloid population [144] does not have a single mechanism of action. Their phenotype and activity seems to be dependent on the location within the microenvironment and the type of tumor cells they are working in consort with. For example, one study showed that normoxic areas of a transplanted mammary carcinoma were associated with M1 phenotype TAMs, while the hypoxic area TAMs were of the M2 phenotype [145]. The organs involved are also a determining factor. For example in a squamous epithelium tumor model, FcγRs, B cells and antibodies were responsible for the tumor promoting phenotype of the TAM population [146], while in a mammary carcinoma model Th2 derived IL-4 was the agent that promoted M2 phenotype and lead to metastasis [147].

The fact that myeloid cells are so tuned into their environment and have a reversible phenotype is a great tool for the tumor microenvironment, but it also means there is great hope for therapy, because better understanding of the mechanisms that govern their re-education will allow us to modify them into anti-tumor fighting machines

[148-149].

Given the many fold inhibition various subgroups of TAM/MDSC induce, whether it be recruiting regulatory cells (TAM) or inhibiting T cell function (MDSC), devising novel methods to down-regulate their function is of utmost importance in improving cancer therapy. In this regard, we have shown that TAMCs express high levels of CD200R [82-83], therefore targeting CD200R on TAMC may be a feasible approach for cancer immunotherapy.

1.3.3 Tumor infiltrating lymphocytes (TILs) in the tumor microenvironment 16

Among the array of immune cells in the tumor microenvironment, tumor

infiltrating lymphocytes (TILs) [150], comprised of anti-tumor effector T cells and Treg

cells [151], are important forces determining tumor regression or progression [94-95].

The anti-tumor effector T cells (including CTL [152] and activated CD4 T cells [153-

154]) are usually functionally inhibited to successfully combat the tumor mass and its

associated network of cellular and soluble mediators. To boost the therapeutic potential

of the rare tumor antigen specific T cells, today technologies are available to isolate and

expand those cells ex vivo to a sizable population. Once the ex vivo expanded TILs [155]

are administered back into the cancer patient [156], they can traffic to the tumor bed

[157] and are capable of eradicating the tumor mass. In practice, the success of adoptive

T cell therapy is considerable (~50%), but not complete [158].

To overcome the shortcomings in adoptive T cell therapy, several venues are

being pursued. Some studies have demonstrated that lymphopenia would raise the

response rate to about 70% by providing “expansion space”, reducing competition and

providing a regulatory T cell (Treg) free environment for the effector T cells [159-160].

A poorly explored possibility is the use of antigen specific CD4+ T cells as the

therapeutic agent alone or in conjunction with antigen specific CD8+ T cells. The few

studies that have focused on adoptive CD4+ T cell therapy suggest a) its addition to CD8+

T cell therapy boosts clinical success rates, and b) its efficacy is higher because CD4+ T cells can mount a broader antitumor response by direct and indirect recognition of tumor cells [161-162]. 17

CD4+CD25+FoxP3+ Tregs are naturally occurring CD4+ T cell subsets that play a

key role in restraining antitumor immunity [163]. Studies suggest that Tregs become

dominant in established tumors via a multistep process involving interaction with a

number of immune and stromal cells. They form a self amplifying suppressive circuit that

targets most immune cells. Like MDSC, Treg mediates its effect via cell-cell contact

[164]. The major mechanisms of effector cell suppression mediated by Tregs are as

follows: IL-2 consumption, granzyme mediated cytolysis and cell cycle arrest via

proximal secreted molecules or cell surface proteins (such as galectin-1) [165]. However,

behavior and function of Treg in cancer setting is still mostly a mystery. More studies are

needed to understand the dynamic relationship between Treg and the tumor environment.

Such insights would pave the way for treatment strategies to balance the Treg and

effector T cell populations in diseased conditions. CD200 expression has been implicated

in T cells. Previous studies suggest that CD200R is also present in T cells [37]. However,

no studies have investigated CD200-CD200R interaction in the T cell compartment in a

tumor setting. Thus, investigation of CD200-CD200R interaction in TILs is highly likely

leads to further understanding of how CD200-CD200R interaction regulates T cell immunity in tumors.

1.4. Hypothesis and goals of the study

Myeloid cells are the first cells recruited by tumors and are essential in the regulation of tumor initiation, establishment, progression and metastasis. Tumor associated myeloid 18

cells (TAMCs) are also known to inhibit activation and effector functions of T cells.

Given TAMCs express high levels of CD200R, we hypothesized that CD200-CD200R

interaction affects tumor formation, metastasis and tumor immunity via inhibiting TAMC functions. Our goals for this dissertation thesis were as follows:

1) Determine CD200-CD200R expression in the tumor microenvironment

For this part of our study we used various WT, TCR transgenic and CD200R-/- mouse models to characterize the CD200 and CD200R expression on myeloid/lymphoid cells and their dynamics in normal, activated and established tumor conditions.

2) Investigate the role of tumor expression of CD200 on tumor formation and metastasis

For this aim we used the B16.OVA.Ctl and B16.OVA.CD200 tumor model to study the implications of CD200 expression on tumor cells in local and metastatic models. We also explored the impact of Gr1 depletion and the role myeloid cells play in the CD200-

CD200R axis of the tumor microenvironment.

3) Determine whether targeting CD200R is a promising candidate for Cancer

Immunotherapy

In this section of our work, we examined the implications of using an agonistic CD200R antibody as a therapeutic tool in cancer treatment. We also examined the role of CD200 expression on T cells and their direct effect on CD200R expressing tumors.

Chapter 2. Materials and methods

2.1 Mice

The following mice were used in the presented work

P1CTL Transgenic mice expressing a TCR specific for the tumor rejection antigen H-

2Ld:P1A35-43 complex (P1CTL) has been described (28). P1CTL TCR transgenic mice

were backcrossed with BALB/c mice for at least 15 generations before they were used for

this study.

P1CTL Rag2-/- BALB/c mice with a targeted mutation of the RAG-2 gene and CD200R-

/- mice were purchased from Taconic Farms (Germantown, New York, USA). Through

breeding P1CTL TCR transgenic mice with RAG-2-/-BALB/c mice we have generated

RAG-2-deficient P1CTL TCR transgenic mice (RAG-2-/-P1CTL).

C57BL/6, RAG-1-/- C57BL/6, OT1 (transgenic mice with TCR specific for H-2Kb: OVA

258–265) and OT2 (TCR transgenic mice with TCR specific for I-Ab:OVA 323–339) mice were purchased from Jackson laboratories. All mice were maintained and cared for in OSU laboratory animal facilities which are fully accredited by Institutional Animal

Care and Use Committee.

2.2 Generation of CD200-positive and CD200R-positive cancer cells and controls

The mouse plasmacytoma J558 (BALB/c, H-2Ld) and mastocytoma P815

(DBA/2, H-2d) cells have been previously described [18, 22]. B16.F10 melanoma cells

expressing the full length chicken ovalbumin (referred to as B16.OVA) has also been

described. We have cloned the full-length cDNA of mouse CD200 from a CD200- 20

positive J558 variant cell line (with low expression of CD200) into PCDNA3 (Invitrogen) expression vector and used it to transfect CD200-negative J558 and P815 cells. The resulting G418-resistant J558 and P815 cells were selected for CD200 expression using flow cytometry. The empty PCDNA3 expression vector was also used to transfect J558,

P815 and B16-OVA cells to generate J558-ctrl, P815-ctrl and B16-OVA-ctrl cells. All cell lines were cultured in RPMI 1640 medium containing 5% FCS, 100 μg/ml of penicillin and streptomycin.

P815 mastocytoma cells express the natural tumor peptide P1A [166]. They naturally express CD200R as well. We generated P815 CD200R- cells by sorting out the

rare negative population using Flow sorting. The generated CD200R-positive or

CD200R-negative cells were maintained in RPMI 1640 medium (GIBCO) supplemented

with 5% FBS and 1% Penicillin/ Streptomycin.

In our experiments we have also made use of 3B11, P338D1 and RAW264.7

murine cell lines. These are all tumor cell line derived from mouse macrophages and all

have endogenous expression of CD200R on their cell surface.

2.3 Establishment of subcutaneous and lung metastatic tumors

To establish subcutaneous tumors in vivo, 5x106 of J558 cells or 5x105 P815 cells

were injected into Balb/c or Balb/c Rag2-/- mice. In the case of B16.OVA and 3B11

cells, C57BL/6 and RAG-1-/- mice were injected with 1 x 105 or 5 x 105 for the former

and 5x106 cells/mouse for the later. Development of tumors was monitored and tumors

were measured for length (a) and width (b) every three days using a caliper. Tumor 21

volumes were calculated as ab2/2. To establish tumor lung metastasis, C57BL/6,

C57BL/6.Rag1-/- or CD200R-/- mice were injected with 1 x 105 B16.OVA.CD200 or

B16.OVA.Ctrl cells via the tail vein. Mice were monitored up to 3-4 weeks depending on

symptoms and treatments received. At the end of the experiments, mice were sacrificed

and lungs were collected, weighed and their tumor foci counted.

2.4 Tumorigenesis and T cell adoptive transfer therapy of mice with established

tumors

For CTL therapy of Balb/c and Balb/c.Rag2-/-mice (J558 and P815 Tumors) with

established tumors, pools of spleen and lymph node cells from P1CTL-transgenic mice

were used. For T cell therapy of mice with B16.OVA established tumors, spleen and

lymph node cells of OT1 and/or OT2 mice were used. The single suspension of each

spleen was incubated with a cocktail of mAbs (anti-CD4 mAb GK1.5, anti-FcRmAb

2.4G2 for CD8+ cells OR anti-CD8 mAb TIB210, anti-FcRmAb 2.4G2 for CD4+ cells).

After removal of unbound mAbs, cells were incubated with anti-IgG coated magnetic beads (Dynal Biotech). The antibody-coated cells were removed by a magnet. The unbound cells consisted of more than 90% of the isolated cells. The purified CD8+ /CD4+

T cells (5 x 106/mouse) were injected intravenously (i.v.) into mice bearing established tumors.

For mice bearing 3B11 tumors the antibody was administered intratumorally at a dose of 50 µg/mouse every 3 days, starting on day 0 for up to 7 times. Mice bearing

P815 tumors were given i.v. injections of the antibody at a dose of 100µg/mouse every 4 22

days, starting on day 3 after P1CTL treatment for up to 7 times. For antibody treatment of mice with B16.OVA lung metastatic melanoma, anti-CD200R mAb (OX110,

Biolegend) or an isotype matched control IgG mAb was injected into each mouse i.v. at a dose of 100 µg/mouse every 3 days, starting on day 0 for up to five times. For depletion

Gr1+ cells in mice, each mouse was injected 250 µg of anti-Gr1 antibody i.p. at 4 day intervals, starting on day 0.

2.5 Antibodies and flow cytometry

For CD200 and CD200R staining, PE-labeled anti-CD200 (clone OX-90) and

FITC-labeled anti-CD200R (OX-110) antibodies (Serotech) were used. FITC-, PE-,

APC- or PercP- labeled antibodies to CD4, CD8α, CD11b, Gr1, F4/80, Vα8.3, Vα2,

Vβ5.1/5.2, CD40, MHC II, MHC I H-2Ld, MHC I kb Ly6G, Ly6C, IFN-γ, IL-10 and isotype-matched control antibodies were purchased from BD Biosciences (San Diego,

CA). Cells were incubated with antibodies in 0.1 M PBS (PH7.4) supplemented with 1%

FCS and 0.1% sodium azide on ice for 30 minutes. They were then washed three times and fixed in 1% paraformaldehyde followed by flow cytometry analysis. For detection of intracellular cytokines, cells were stimulated in vitro with PMA (50 ng/ml) and ionomycin (50 ng/ml) for 5 h. GolgiStop (BD Pharmingen) were added (1/1500) during the last 2 h of incubation. The cells were first stained for the cell surface markers such as

Vα8.3, followed by a standard intracellular cytokine staining procedure for IFN-γ. Cells

were analyzed on a FACSCalibur flow cytometer. Data were analyzed using the flowjo

software (Tree Star, Inc., OR).

2.6 Isolation of CD11b+ and Gr1+ cells from spleens or lungs

Monocyte/macrophages were isolated from spleens or lungs by first staining the

cell suspensions with PE-anti-CD11b mAb (BD biosciences) or PE-anti-Gr1 mAb (BD biosciences), followed by magnetic antibody cell separation using anti-PE microbeads

(Miltenyi Biotec). The isolated cells were >90% pure. These cells were co-cultured with

CD200 positive and control tumors (1:1 ratio), with or without LPS stimulation

(100ng/ml). Culture supernatants were collected at 24 and 48 hours for cytokine analysis.

2.7 51CR-release Assay or the Cytotoxicity assay

Splenocytes from P1CTL TCR transgenic mice were stimulated with P1A peptide

(0.1μg/ ml) for 5 days and used as effectors. 51Cr-labeled tumor cells were used as

targets. The effector T cells and the targets were incubated together for 6 h, and the

percentages of specific lysis were calculated based on the following formula:

specific lysis % = 100 Χ (cpmsample−cpmmedium)/(cpmmax−cpmmedium).

2.8 Cytokine ELISA

ELISA kits for the detection of IL-2, IL-6, IL-10, TNF-α and IFN-γ were obtained

from eBiosciences. Standard procedures were followed to detect releases of cytokines in culture supernatants in a variety of settings.

2.9 Generation of CD200 positive and negative B16.OVA cells

B16.F10 melanoma cells expressing the full length chicken ovalbumin (referred to as B16.OVA) has been described [167]. We have cloned the full-length cDNA of mouse CD200 into pcDNA3 (Invitrogen) expression vector and used it to transfect

B16.OVA cells. The resulting hygromycin-resistant cells were selected for CD200 expression using flow cytometry. The empty pcDNA3 expression vector was used to transfect B16.OVA cells to generate B16.OVA.Ctrl cells. The generated CD200-positive or CD200-negative cells were maintained in RPMI 1640 medium (Gibco) supplemented with 5% FBS and 1% Penicillin/ Streptomycin.

2.10 Real time PCR

Quantitative real-time PCR was performed using an ABI 7900-HT sequence system (PE Applied Biosystems) with the QuantiTect SYBR Green PCR kit (Qiagen) in accordance with the manufacturer's instructions. PCR was done using previously determined conditions [168]. The following primers were used for amplifying specific genes: chicken OVA: 5’-ATC TCA AGC TGT CCA TGC AG -3’(forward) and 5’-TGC

GAT GTG CTT GAT ACA GA -3’ (reverse). The HPRT gene was simultaneously amplified as endogenous control. The primers were 5'-

AGCCTAAGATGAGCGCAAGT-3' (forward) and 5'-TTACTAGGCAGATGGCCACA-

3' (reverse). Each sample was assayed in triplicate and the experiments were repeated twice. The relative amount of OVA mRNA was calculated by plotting the Ct (cycle number) and the average relative expression for each group was determined using the

comparative method (2-ΔΔCt).

2.11 CD200 knockdown on T cells

Single cell suspension of splenocytes were prepared and stimulated for 48 hours.

The cells were then collected and resuspended with 300µl of medium in the presence of polybrene and CD200 or control shRNA and spun down at 2500 RPM in room temperature for 2 hours. The suspension was then left over night. On day 3 the cells were

resuspended in medium containing Puromycin (3µg/ml) and IL-2 (5ng/ml). The cells

were left in the medium for 2-3 days after which they were washed and the ficolled. The live population was screened for CD200 expression. The cells were then used to perform the respective experiments.

2.12 Statistics

Student’s t test was used to compare tumor size and number differences between two groups. A chi square (χ2) statistic was used to determine differences for numbers of mice with tumor recurrence. For comparison of mice survival, Kaplan-Meier survival analysis and log-rank test were used (version 10.0, SPSS, Inc., Chicago, IL). A p value less than 0.05 was considered significant.

Chapter 3. Results

3.1 CD200-CD200R interaction in the tumor microenvironment

In the tumor microenvironment, CD200-CD200R interaction could involve a

number of cell types including tumor associated myeloid cells (TAMC), T cells and

tumor cells. However, the expression and regulation of CD200 and CD200R on these cell

types are not well studied. Given CD200-CD200R interaction mainly regulates the

functions of myeloid cells, we hypothesized that in the tumor microenvironment, CD200-

CD200R interactions among TAMCs, tumor cell-TAMC and T-TAMC play important

roles in regulating tumor initiation, metastasis, progression and tumor immunity. To test

this hypothesis, we first investigated the expression of CD200/CD200R in these lineages

of cells under various conditions and in the tumor microenvironment.

3.1.1 Myeloid cells express and upregulate CD200

To understand the CD200 expression pattern and the regulation of its expression

on myeloid lineage of cells, we prepared splenocytes from various strains of mice and

examined CD200 expression on myeloid cells by flow cytometry. As shown in Figure 4,

differential levels of CD200 is expressed on CD11b+ macrophages from BALB/c and

C57BL/6 (Figure 4A), and P1CTL and OT1 TCR transgenic mice (Figure 4B). Myeloid

cells from CD200R-/- and wild type controls displayed similar CD200 expression levels,

with CD11b+Gr-1+ cells have the highest expression, suggesting that the absence of

CD200R has no impact on CD200 expression of these cells. Bystander activation of macrophages using anti-CD3 (Figure 5A), P1A or OVA peptides (Figure 5B), or direct 27

activation of macrophages using LPS (Figure 5C) resulted in dramatic upregulation of

CD200.

Balb/c B6 A

C WT + CD11b Wild Type Gr1+ CD200R-/-

Gr1+ CD11b+Gr1+

CD200

P1CTL OT1 Gr1 CD200R-/- B

CD11b+ CD11b+

Gr1+ CD11b CD200

CD200

Figure 4. CD200 is expressed on myeloid cells A. Splenocytes prepared from BALB/c and C57BL/6 mice were stained for CD11b/Gr1/CD200. Figure representative of at least 3 separate experiments with similar results. B. Single cell suspension from spleen of transgenic P1CTL and OT1mice. Cells were stained for CD11b/Gr1/CD200. Figure representative of 3 separate experiments with similar results. C. Single cell suspension of spleen from CD200R-/- mice and their wild type litter mates. CD200R expression is shown for CD11B+/Gr1+/CD11b+Gr1+ populations. Figure representative of 3 separate experiments with similar results.

+ CD11b CD11b+ CD11b+ B A Balb/c P1CTL C

Wild Type

B6 OT1

CD200R-/-

CD200

Figure 5. CD200 is upregulated on activated myeloid cells A. Splenic Balb/c and C57BL/6 cells were stimulated with anti-CD3 antibody. On day 2 cells were stained for CD11b/CD200. Figure representative of 2 separate experiments with similar results. B. Splenic P1CTL and OT1 cells were stimulated with P1A and OVA antigen, respectively. On day 2 cells were stained for CD11b/CD200. Figure representative of 2 separate experiments with similar results. C. Splenic CD200R-/- and wild type cells were stimulated with LPS. On day 2 cells were stained for CD11b/CD200. Figure representative of 2 separate experiments with similar results.

3.1.2 CD200R is expressed on myeloid cells and upregulated by activation

Although there is ample evidence of CD200R expression on myeloid cells, their expression among the various groups and subgroups of the myeloid lineages have not been studied. We set out to characterize CD200R expression on the surface of myeloid cells. For this purpose we prepared single cell suspension from the spleens of Balb/c and

C57BL/6 (Figure 6A), P1CTL and OT1 TCR transgenic mice (Figure 6B) and CD200R-

/- and their wild type litter mates (Figure 6C). We stained the splenocytes for CD11b,

Gr1 and CD200R followed by flow cytometry analysis. Except for cells from CD200R-/- mice, CD200R expression was observed on the surface of all CD11b+, CD11b+Gr1+ and

Gr1+ cells. CD11b+ and CD11b+Gr1+ cells display higher levels of CD200R, while Gr1+ cells generally exhibit lower expression of this molecule. We wanted to know if the absence of CD200R affects the numbers or ratio of myeloid cells. Results from our pooled data (n=7/group) indicate very similar percentages of CD11b+Gr1+ and Gr1+ cells in CD200R-/- and wild type mice. In the case of CD11b+ cells (Figure 6D), our data

indicate a greater number of these myeloid cells are present in the spleen of CD200R-/- mice.

To determine the CD200R expression level on macrophages under activated conditions, we cultured splenocytes from various strains of mice using different stimulators such as anti-CD3 (Figure 7A), P1A or OVA (Figure 7B) and LPS (Figure

7C). Stimulation with all these activators resulted in dramatic upregulation of CD200R on macrophages. Thus, CD200R is constitutively expressed on myeloid cells and upregulated by activation.

Balb/c B6 P1CTL OT1 A B

CD11b CD11b

Gr1 Gr1

CD200R CD200R C WT Wild Type CD200R-/- D Wild Type -/- CD11b+ CD200R 7 P=0.01 6 5 Gr1+ CD200R-/-

Gr1 4 3 2 CD11b+Gr1+

% of cells in Spleen in cells of % 1 0 CD11b CD200R CD11b+ Gr1+ CD11b+ Gr1+

Figure 6. CD200R expression on myeloid cells A. Single cell suspension prepared from wild type Balb/c and C57BL/6 mice were stained for CD11b/Gr1/CD200R. Figure representative of at least 3 separate experiments with similar results. B. Single cell suspension from spleen of transgenic P1CTL and OT1mice. Cells were stained for CD11b/Gr1/CD200R. Figure representative of 3 separate experiments with similar results. C. Single cell suspension of Spleen from CD200R-/- mice and their wild type litter mates. CD200R expression is shown for CD11b+/Gr1+/ CD11b+Gr1+ populations. Figure representative of 3 separate experiments with similar results. D. Quantification of 3 populations of myeloid cells in CD200R-/- mice and wild type litter mates (n=7/group).

AB C CD11b+ CD11b+ CD11b+

Balb/c P1CTL Wild Type

OT1 -/- B6 CD200R

CD200R

Figure 7. CD200R expression on activated myeloid cells A. Splenic Balb/c and C57BL/6 cells were stimulated with 1µg/ml anti-CD3 antibody. On day 2 cells were stained for CD11b/CD200R. Figure representative of 2 separate experiments with similar results. B. Splenic P1CTL and OT1 cells were stimulated with P1A and OVA antigen, respectively. On day 2 cells were stained for CD11b/CD200R. Figure representative of 2 separate experiments with similar results. C. Splenic CD200R-/- and wild type cells were stimulated with LPS. On day 2 cells were stained for CD11b/CD200R. Figure representative of 2 separate experiments with similar results.

3.1.3 T cells express high levels of CD200 upon activation

T cell expression of CD200 has been reported [12, 169-171]. However, it is

unclear if CD200 expression is constitutive or in a regulated pattern. To understand the

dynamics of CD200 expression on the various T cell populations, we first characterized

its expression levels in ex vivo T lymphocytes. We have found that both CD4+ and CD8+

T cells express significant levels of CD200. Interestingly, CD4+CD25+ Treg cells have

higher levels of CD200 expression (Figure 8A). To determine if CD200 expression is

related to the activation status, we examined CD200 expression on different subsets of T

cells. We show that naïve CD4+ and CD8+ T cells (CD62L+) (Figure 8B, C) have undetectable levels of CD200 expression, while effector (CD44+) and memory T cells

(CD44+CD62L+) (Figure 8B, C) show high levels of CD200 expression. These results suggest that CD200 expression is regulated by activation.

To examine the kinetics of CD200 expression on T cells, we stimulated

splenocytes of P1CTL transgenic mice with P1A peptide and measured CD200

expression levels over the course of 5 days (Day0-4) (Figure 9A, B). We found that the

expression level of CD200 begins to elevate after 1 day of in vitro stimulation. It reaches

its highest level on day 3 of in vitro stimulation. On day 4 the level decreases and

becomes relatively stable. Examination of resting T cells (cultured in IL-2 for more than

1 week after in vitro stimulation) shows a lower, but stable expression of CD200.

However, upon restimulation, T cells rapidly upregulated CD200 (Figure 9C). Similar results were obtained when CD4+ T cells were activated. The upregulation of CD4+ and

CD8+ T cells was consistent when cells were activated with other stimulatory agents such

as anti-CD3 or PMA/Ionomycin (data not shown). Thus, CD200 expression on T cells is

strictly regulated by activation.

A B CD62L+

CD8+ CD44+ CD44

+ +

CD8 CD44 CD62L

CD62L CD4+ CD200

+ C CD62L CD25 CD4+CD25+

CD44+ CD44

CD4 CD44+CD62L+

CD200 CD62L

CD200

Figure 8. CD200 expression on T cell populations Results shown are analysis of Flow cytometry data from ex vivo staining of Spleen/tumor infiltrated T cells. Figures represent at least 3 sets of experiments with similar Results. A. Spleen cells were stained for CD4/CD8/CD25/CD200. Results show CD200 expression level on each population.B. Spleen cells were stained for CD4/CD62L/CD44/CD200. Results show CD200 expression level on each subpopulation. C. Spleen cells were stained for CD8/ CD62L/CD44/CD200. Results show CD200 expression level on each subpopulation.

CD200

B 140 1st round

120 Stimulation

100

80 2nd round

60 Stimulation

On CD8+ T cells 40

Mean Florescent Intensity (MFI) 20

0 CD200 0 1 2 3 45 - 16 1718192021 Days

Figure 9. The kinetics of CD200 and CD200R expression on CD8+ T cells P1CTL spleen was stimulated with 0.2µg/ml of P1A peptide. CD200 expression level was obtained by staining the T cells every 24 hours. A. Cells were stained for CD8/Va8.3/CD200. CD200 expression levels shown. B. Mean Florescent intensity (MFI) measures for CD200 expression on P1CTL cells stimulated with P1A, cultured for several days and then restimulated. Shown is the graphical representation or expression pattern in resting, activated, memory and restimulated states, respectively

3.1.4 CD200R expression on T cells is minimal and is not upregulated by activation Some reports have indicated CD200R expression on T cells [172]. We have extensively evaluated CD200R expression on T cells (Figure 10) and their subpopulations (data not shown). First we examined CD200R expression on different subsets

of thymocytes. Both CD4+CD8+ and CD4+ or CD8+ single positive populations have undetectable levels of CD200R (Figure 10A). We quantified the percentages of each T

cell population and observed no difference in the T cell populations in the thymus (data

not shown). WT and CD200R-/- mice have similar T cell populations in the spleen

(Figure 10B). However, CD200R expression on T cells is barely detectable (Figure 10B)

except significant expression of CD200R on CD4+CD25+Foxp3+ cells (Figure 10C). To determine if CD200R expression is upregulated under activation, splenocytes from

CD200R-/- and wild type litter mates were activated by anti-CD3. After 72 hours of

stimulation, we observed no discernible upregulation of CD200R on activated CD4+ and

CD8+ T cells (Figure 10D). Thus, CD200R expression on T cells is minimal and is not

upregulated by activation.

WT A Wild Type CD4+ B CD200R-/- CD4+ Wild Type

CD8+ -/- CD8 CD200R CD8

CD4+CD8+ CD8+ CD200R-/-

CD4 CD200R C CD200R CD4 Wild Type CD4+CD25+ D CD8+ CD4+ CD4+CD25+

Wild Type

-/- CD200R CD4+CD25+FoxP3+ FoxP3 CD25

CD200R-/-

CD4 CD25 CD200R CD200R

Figure10. CD200R Expression on resting and activated T cells A. Percentage and ratio of double positive and single positive T cells and their respective CD200R expression in the thymus of CD200R-/- mice and their wild type litter mates. Flow cytometry results representative of 3 separate experiments with Similar results (n=3) B. Percentage and ratio of CD4+ and CD8+ T cells and their respective CD200R expression in the spleen of CD200R-/- mice and their wild type litter mates. Flow cytometry results representative of at least 4 separate experiments with similar results (n=5) C. Percentage of CD4+CD25+ and CD4+CD25+FOXP3+ T cells and their respective CD200R expression in the spleen of CD200R-/- mice and their wild type litter mates. Flow cytometry results representative of at least 3 separate experiments with similar results (n=4) D. Spleen cells from CD200R-/- mice and their wild type litter mates were cultured with 1µg/ml of anti-CD3 for 72 hours. The activated suspension was then stained for CD4/CD8/CD25/CD200R. Flow cytometry results representative of at least 4 separate experiments with similar results (n=4)

3.1.5 CD200-CD200R expression in the tumor microenvironment

To examine CD200/CD200R expression in the tumor microenvironment, we injected 5 x 106 J558 tumor cells into each BALB/c mouse subcutaneously. When tumors were fully established, we disassociated tumors and examined CD200R expression on different lineages of cells. As shown in Figure 11A, high levels of CD200 and CD200R expression was observed on tumor associated myeloid cells (TAMCs). Tumor infiltrating

CD4+ and CD8+ T cells express high levels of CD200. However, the expression of

CD200R is minimal (Figure 11B).

AB Gr1 CD4

CD11b CD8

Gr1+ CD4+

Gr1+CD11b+

CD11b+ CD8+

CD200 CD200R CD200 CD200R

Figure 11. CD200 and CD200R expression on TAMC and TIL A. Wild Type Balb/c mice were injected with J558 tumor subcutaneously. Established tumors were excised and physically dissociated, strained, lysed and ficolled. Single cell suspensions were then stained for CD11b/Gr1/CD200/CD200R (n=5/group). B. Balb/c.Rag2-/- mice we injected with J558 tumor subcutaneously. Tumors were treated with P1CTL. Several days later tumors were excised and physically dissociated, strained, lysed and ficolled. Single cell suspensions were then stained for CD4/CD8/CD200/CD200R (n=5/group).

3.2 The role of tumor expression of CD200 in tumor formation, metastasis and

susceptibility to T cell therapy

Expression of CD200 has been found in multiple types of cancer [68-70]. It is

generally considered that expression of CD200 on cancer cells has a protumor effect

based on the following evidence. First, in two correlation studies, CD200 mRNA expression in malignant cells has been shown to be associated with decreased survival of patients [69-70]; Second, CD200-expressing melanoma and ovarian cancer cells downregulate Th1 cytokine production when co-cultured with allogenic leukocytes [75,

173] and anti-CD200 antibody treatment can enhance tumor rejection by peripheral blood mononuclear cells in a hu-SCID adoptive transfer model [73, 174]; Third, in a recent study, CD200 expression was found to be positively correlated with the metastatic capacity of squamous cell carcinoma [79]. While human correlation studies remain to be confirmed in other cancer types, studies focusing on regulating immune functions only focused on regulation of dendritic cells. Our recent study [82] has revealed that tumor expression of CD200 has a direct effect on tumor associated myeloid cells (TAMCs).

Myeloid cells are obligate partners for tumor cell migration, invasion and metastasis. Within the tumor microenvironment, TAMCs facilitate angiogenesis and extracellular matrix breakdown, promote tumor cell migration and invasion, and suppress

antitumor immunity; at metastatic sites, TAMCs prepare the target tissue for arrival of

tumor cells [115, 175]. Genetic ablation or depletion of macrophages and inhibition of

macrophage functions have been shown to be effective in inhibiting tumor initiation and 40

growth [176-178]. Since TAMCs are the major lineages of cells expressing CD200R

[82], we hypothesize that tumor expression of CD200 inhibits the functions of TAMCs and thereby affects tumor formation and metastasis.

3.2.1 Expression of CD200 on melanoma cells inhibits tumor formation and lung metastasis

Recent studies have revealed that CD200 is frequently expressed on human

melanoma cells. To test the significance of melanoma expressed CD200 in tumor

formation and metastasis, we generated CD200-positive and CD200-negative B16

melanoma cells by transfecting the B16.OVA cells with either the empty pCDNA3

expression vector or one carrying the murine CD200 cDNA. The resulting cells were

named as B16.OVA.CD200 and B16.OVA.Ctrl, respectively. As shown in Figure 12A,

B16.OVA.CD200 cells expressed significant levels of CD200 while the control cells

were CD200 negative. Both cell types also had similar levels of MHC class I (H-2Kb)

expression (Figure 12A, middle panel) and similar levels of OVA gene expression

(Figure 12A, lower panel). To examine the impact of CD200 expression on tumor

formation and growth, we injected B16.OVA.CD200 or B16.OVA.Ctrl cells into

C57BL/6 mice subcutaneously (s.c.). As shown in Figure 12B and Figure 12C,

expression of CD200 significantly inhibited tumor formation and growth, and promoted

survival of tumor bearing mice. Since B16.F10 tumors are highly metastatic to the lung,

we examined tumor growth in the lung metastasis model. For this purpose, we injected

C57BL/6 mice with 1 x 105 of B16.OVA.CD200 or B16.OVA.Ctrl cells via their tail vein. On day 20, we sacrificed all mice and extracted lungs from all mice and compared their weight and tumor foci formation. As demonstrated in Figure 12D, the lungs from mice who received B16.OVA.Ctrl cells exhibited extensive formation of black foci, characteristic of melanoma metastasis in the lungs. In contrast, the lungs from mice injected with B16.OVA.CD200 cells had much less melanoma foci, and the differences were highly significant in terms of total foci numbers and total lung weight (Figure 12E and Figure 12F). These results suggest that CD200 expression on melanoma cells significantly inhibit tumor formation and lung metastasis.

% max B B16.OVA.Ctrl C 1 ) 3 B16.OVA.CD200 CD200 5000 0.8 P=0.005 4000 0.6 % max 3000 P=0.01 2000 0.4 H-2Kb P=0.01 1000 0.2 1 Tumor Volume (mm

0 Survival of Probability 0 4 7 11 15 19 23 0.1 15 20 25 Days Post Tumor Cell Injection Days Post tumor cell Injection

0.01 rl Ct 00 A. 2 V CD .O . OVA geneexpressionOVA 6 A 1 OV B 6. B1 E 120 F P<0.001 D P<0.001 0.4

B16.OVA.Ctrl 0.3 80

0.2 No. of Foci of No. 40 0.1 Lung weight (gram)

0 0 B16.OVA .Ctrl B16.OVA.CD200 B16.OVA.Ctrl B16.OVA.CD200 B16.OVA.CD200

Figure 12. CD200 on melanoma cells inhibits tumor formation and lung metastasis A. Flow cytometery analysis of B16.OVA.Ctrl and B16.OVA.CD200 cells for CD200, MHC class I H2-Kb expression. qRT-PCR was used to examine OVA gene expression. B. 1 x 105 of B16.OVA.Ctrl or B16.OVA.CD200 cells were injected into each mouse subcutaneously. The tumor growth was observed over time. C. Kaplan-Meier survival analysis and log-rank test were used to analyze mice survival. Mice with a tumor burden of 1.5 x 1.5 cm were sacrificed and counted as dead. Data shown in B and C represent two experiments with similar results. D. C57BL/6 mice were given 1 x 105 B16.OVA.Ctrl or B16.OVA.CD200 cells per mouse via their tail vein. 20 days later mice were sacrificed and tumor growth in the lungs were shown. E. Average number of tumor foci in the lungs from each group of mice shown in D. Error bars represent Mean ± SEM. Student’s two- tailed t test was used for the statistical analysis. F. Average weight of lungs from each group of mice shown in D. Data shown in D-F represent two experiments with similar results.

3.2.2 Tumor expression of CD200 inhibits melanoma lung metastasis through inhibition of Gr-1+ myeloid cells

To understand if CD200-mediated inhibition of tumor formation and metastasis observed in C57BL/6 mice were due to stimulation of adaptive immunity, we did similar

experiments in Rag1-/- C57BL/6 mice. As demonstrated in Figure 13A and Figure 13B,

expression of CD200 on melanoma cells did not significantly affect subcutaneous tumor

formation and growth, and it also did not significantly affect the survival of tumor

bearing mice. However, expression of CD200 on melanoma cells dramatically affected

tumor foci formation in the lungs. As demonstrated in Figure 13C and Figure 13D, the

lungs from mice who received B16.OVA.CD200 cells had much less melanoma foci

compared with the lungs from mice that received B16.OVA.Ctrl cells (Figure 13D). In

addition, the weight of the lungs and the survival times of mice were also significantly

different between the two groups of mice (Figure 13E and Figure 13F). The results

presented in Figure 12 and Figure 13 suggest that while adaptive immunity may play a

role in the subcutaneous tumor model, the difference in tumor lung metastasis was mainly

caused by innate immune components.

Since tumor expression of CD200 differentially affect tumor formation in the

subcutaneous model versus the lung metastatic model, we hypothesized that the lung and

peripheral tumor microenvironment decided the differential susceptibility to CD200-

mediated suppression of tumor growth. During tumor initiation, subcutaneous tumors

mainly attract myeloid cells from blood, while in the lung, high numbers of local myeloid

cells exist. We therefore compared splenic myeloid cells and those from the lungs for the

expression of CD200R. As shown in Figure 14A, in the lung and spleens of mice, we

found three populations of myeloid cells in each organ: CD11bhiGr1hi, CD11bhiGr1lo and

CD11bloGr1- cells. In the lungs, CD11bhiGr1hi and CD11bhiGr1lo cells are the cells that 44

express high levels of CD200R, while CD200R expression on CD11bloGr-1- cells were low. In the spleens, CD11bhiGr1lo cells expressed lower levels of CD200R, while the other two populations of cells were essentially CD200R negative. Thus, only one minor subset of systemic myeloid cells express low levels of CD200R, while two subsets of

Gr1+ lung myeloid cells express high levels of CD200R.

A B C B16.OVA.Ctrl

) 8000 1 3 B16.OVA.Ctrl B16.OVA.Ctrl B16.OVA.CD200 0.8 B16.OVA.CD200 6000 0.6 4000 0.4

2000 0.2 Probability of Survival of Probability Tumor Volume (mm Volume Tumor 0 0 4 7 11 15 19 23 26 16 18 20 22 24 B16.OVA.CD200 Days Post Tumor Cell Injection Days Post Tumor Cell Injection

D EF 120 0.3 P=0.001 P=0.001 1 100 0.8 80 0.2 0.6 60 P=0.001 No. of Foci of No. 40 0.1 0.4 Weight (in grams) (in Weight

20 0.2 Probability of Survival of Probability B16.OVA.Ctrl 0 0 B16.OVA.CD200 B16.OVA B16.OVA.CD200 B16.OVA B16.OVA.CD200 0 20 25 30 Days Post Tumor Cell Injection

Figure13. CD200 expression on tumor cells inhibits tumor lung metastasis in Rag1-/- C57BL/6 mice A. 1 x 105 of B16.OVA.Ctrl or B16.OVA.CD200 cells were injected into each Rag1-/- C57BL/6 mouse s.c. The tumor growth was observed over time. B. Kaplan-Meier survival analysis and log-rank test were used to analyze mice survival. Mice with a tumor burden of 1.5 x 1.5 cm were sacrificed and counted as dead. Data shown in A and B represent two experiments with similar results. C. Rag-/- C57BL/6 mice were given 1 x 105 B16.OVA.Ctrl or B16.OVA.CD200 cells per mouse via their tail vein. 20 days later mice were sacrificed and tumor lung metastasis was shown. D. Number of tumor foci in the lungs from mice shown in C were quantified. Error bars represent ± SEM. Student’s two- tailed t test was used for the statistical analysis.

A B 1 12 3 2 Lung B16.OVA.Ctrl/Ctrl Ab 3

B16.OVA.Ctrl/Anti-Gr1 1 1 2 3 2 Max Spleen B16.OVA.CD200/untreated Gr-1 3 %

CD11b CD200R

C E D Gate on live cellsGate on CD11b+ Gate on 1 or 2 P=0.003 P=0.004 1.2 1050 P=0.004 P=0.003 1 1

1 900 Ctrl Ab 2 750 0.8 600 0.6

No. of Foci 450 2 0.4 300 Anti-Gr1 Lung weight (gram) weight Lung 0.2 150 Gr-1 Ly6G Count

0 0 b 1 A r1 ed b r ted trl i-G at rl A ti-G a /C nt tre CD11b Ly6C CD200R Ct n tre trl l/A n rl/ l/A un .C tr /u Ct tr 0/ A .C 00 A. .C 20 OV VA D2 V VA D 6. O .C .O .O .C 1 6. A 16 6 A B 1 V B B1 V B .O 6.O 16 B1 B

Figure 14. Gr1+ myeloid cells express CD200R and mediate lung metastasis. A. Flow cytometry analysis of CD200R expression in mononuclear cells from spleens and lungs. Cells were prepared from spleen and lung of normal C57BL/6 mice and were stained for CD11b, Gr1 and CD200R. The experiment was confirmed in three independent experiments with similar results. B. Two groups of C57BL/6 mice were injected with B16.OVA.Ctrl cells (1 x 105 cells/mouse) via their tail vein. Mice were treated with either 250µg/mouse of anti-Gr-1 (RC57BL/6-8C5, BioXcell) or an isotype-matched control antibody (anti-KLH, BioXcell). An untreated group of mice that received 1 x 105/mouse of B16.OVA.CD200 cells were used for the comparison. 21 days after tumor cell injection, tumor lung metastasis was examined. C. Lung weight in groups of mice shown in B. Error bars represent ± SEM. Student’s two-tailed t test was used for the statistical analysis. D. Average number of tumor foci in the lungs from each group of mice shown in B. Error bars represent ± SEM. Student’s two-tailed t test was used for the statistical analysis. E. Flow cytometry analysis of lung mononuclear cells from anti-Gr-1 treated and control antibody treated mice. C57BL/6 mice received either anti-Gr-1 (250 g x 3 doses per mouse) or isotype-matched control antibody i.p. Data shown represent three experiments with similar results.

Since CD200R-positive cells also co-expressed Gr1, we tested if depletion of Gr1 positive cells could affect melanoma lung metastasis. We injected two groups of Rag1-/-

mice with B16.OVA.Ctrl tumor cells i.v. at a dose of 1 x 105/mouse. One group of the

mice were treated with anti-mouse Gr1 mAb i.v. every 4 days to deplete the Gr1+ cells.

Another group of mice were treated with an isotype-matched control mAb. In parallel, a third group of mice received B16.OVA.CD200 tumor cells i.v. at a dose of 1 x 105/mouse without mAb treatment. As demonstrated in Figure 14B-D, anti-Gr1 treatment dramatically reduced tumor foci in the lungs of mice that received B16.OVA.Ctrl cells.

The lung weight and number of tumor foci in the lungs of anti-Gr1 treated mice were similar to the mice that only received B16.OVA.CD200. To determine if anti-Gr-1 treatment deleted CD200R-positive myeloid cells, we analyzed lung mononuclear cells isolated from anti-Gr-1 treated and control antibody treated mice. As shown in Figure

14E, anti-Gr-1 treatment mainly deleted a large population of Gr-1hiCD11b+ cells, which

were also Ly6G+ and CD200R+. This result suggests that depletion of CD200R-positive

myeloid cells has similar effects to tumor expression of CD200 in inhibiting melanoma tumor metastasis to the lungs.

To determine if tumor cell expression of CD200 directly inhibits the functions of

Gr1+ myeloid cells, we purified Gr1+ cells from spleens or lungs using MACS beads. We

co-cultured purified splenic and lung Gr1+ cells with either B16.OVA.Ctrl or

B16.OVA.CD200 cells in the presence of LPS. The concentrations of IL-6, IL-10 and

TNF-α in the culture supernatants were measured using ELISA. As shown in Figure 15,

cytokine levels were consistently lower in cultures containing CD200-positive tumor cells, while CD200 blockade using an anti-CD200 antibody abrogated the suppression of 48

cytokine production (Figure 15, right panel). These data suggest that tumor expression of

CD200 can directly inhibit the functions of Gr1+ myeloid cells via interaction with

CD200R.

Figure 15. Tumor B16.OVA.Ctrl B16.OVA.CD200/Control Ab expression of CD200 B16.OVA.CD200 B16.OVA.CD200/Anti-CD200 inhibits the functions of A P=0.01 + 700 P=0.003 600 Gr1 myeloid cells 600 + 500 Gr1 cells were isolated 500 P=0.003 400 from spleens and lungs of 400 P=0.04 C57BL/6 mice. The cells 300 300 IL-6 (pg/ml) 200 200 were then co-cultured with 100 100 tumor cells at a 1:1 ratio 0 0 for 48 hours in the Spleen Lung Spleen Lung presence of 100 ng/ml of P=0.004 P=0.003 P=0.01 P=0.04 B 1000 900 LPS (left panel) and 10 900 800 μg/ml of anti-CD200 mAb 800 700 700 600 or an IgG2a isotype control 600 500 mAb (right panel). The 500 400 400 supernatants were collected

IL-10 (pg/ml) IL-10 300 300 from the co-cultures and 200 200 100 100 were examined for the 0 0 Spleen Lung Spleen Lung presence of IL-6, IL-10 and TNF-α. Experiments were P=0.0002 P=0.0006 P=0.001 P=0.05 500 repeated at least 3 times C 700 600 with similar results. Data 400 500 shown are mean ± SEM of 400 300 5 mice. Student’s two- (pg/ml) 300 tailed t test was used for α 200 200 the statistical analysis.

TNF- 100 100

0 0 Spleen Lung Spleen Lung

3.2.3 Tumor expression of CD200 improves the efficacy of T cell adoptive transfer

therapy

The differential effects of tumor expression of CD200 on subcutaneous tumor

growth in C57BL/6 mice versus Rag1-/- mice (Figures 12&13) suggest that CD200

expression on tumor cells affects adaptive immunity. We previously demonstrated that

tumor expression of CD200 inhibits the functions of tumor associated myeloid cells and permits better tumor eradication by CTL [82]. To test if expression of CD200 on melanoma cells could improve the susceptibility to T cell therapy, we injected 5 x 105 of

B16.OVA.Ctrl or B16.OVA.CD200 tumor cells into each Rag-/-C57BL/6 mouse

subcutaneously. The mice were either left untreated or treated with 5 x 106 CD8+ T cells

purified from OT1 mice or CD4+ T cells from OT2 mice or their combination. As shown

in Figure 16, no significant difference was observed between untreated CD200-positive

and CD200-negative tumors (Figure 16A). Adoptive transfer of OVA-specific CD8

(Figure 16B), CD4 (Figure 16C) or their combination (Figure 16D) significantly

improved the efficacy of T cell therapy on CD200-positive, but not on CD200-negative

tumors. The tumor volumes (left panel) of T cell-treated CD200-positive tumors were

significantly smaller and the survival times of mice with CD200-positive tumors were

significantly longer compared to mice with CD200-negtive tumors receiving the same T

cell treatment (right panel). Thus, expression of CD200 on melanoma cells improves

the efficacy of T cell therapy.

3.2.4 Tumor expression of CD200 is implicated in better prognosis and patient

survival

To determine the clinical relevance of our data, we reviewed publically available

microarray data (NCBI website and Oncomine) performed using patient biopsy samples.

As shown in Figure 17 A and B, patient’s whose biopsy samples were high in CD200

expression had significantly higher survival time compared to patients bearing CD200low tumors. In Figure 17C an Oncomine generated graph shows the relative CD200 mRNA expression levels for various subtypes of Acute Myeloid Leukemia (AML) disease.

Interestingly, patients in group 5, which exhibit the highest level of CD200 expression, bear a particular fusion gene and are known to have good prognosis [179]. Taken together, we have shown that CD200 expression on tumor cells is a factor that makes tumor cells more susceptible to immune surveillance and therapy.

Figure 16. CD200-positive tumors are B16.OVA more susceptible to adoptive T cell A B16.OVA.CD200 10000 N.S. therapy. 5 8000 Untreated A. 5 x 10 of tumor cells were injected 6000 into each Rag1-/- C57BL/6 mouse s.c. 4000 Tumor growth (left) and survival of 2000 mice (right) were monitored over time. 0 N = 5 mice per group and data shown B 10000 8 11141720222426 represents two experiments with similar 5 8000 P=0.04 results. B. 5 x 10 of tumor cells were injected into each Rag1-/- C57BL/6 6000 OT1 treated P=0.002 mouse s.c. Mice were then given 5 x 4000 6 +

) 10 /mouse of purified CD8 T cells from 3 2000 OT1 mice i.v. 5 days after tumor cell 0 injection. Five mice per group were used 8 1013151720222426 and data shown represents two 10000 C experiments with similar results. C. 5 x 8000 5 P=0.002 10 of tumor cells were injected into OT2 treated -/- 6000 each Rag1 C57BL/6 mouse s.c. Mice

Tumor Volume (mm Volume Tumor P=0.004 6 P ro b a b ility o fS u rv iv a l 4000 P=0.003 were then given 5 x 10 /mouse of P=0.04 + 2000 purified CD4 T cells from OT2 mice i.v. 5 days after tumor cell injection. 0 Five mice per group were used and data 8 1013151720222426 D shown represents two experiments with 10000 5 8000 similar results. D. 5 x 10 of tumor cells -/- OT1+OT2 treated were injected into each Rag1 C57BL/6 6000 P=0.06 4000 mouse s.c. Mice were then given 5 x P=0.03 6 2000 P=0.01 10 /mouse of purified OT1 and 5 x 0 106/mouse of purified OT2 T cells i.v. 5 8 121416192226 days after tumor cell injection. Five mice Days Post Tumor Cell Injection per group were used.

P = 0.0252 P = 0.0021

Alizadeh et al (2000) CD200 CD200 Pawitan et al (2005) CD200 CD200 low high low high Sample Size (cases) 16 14 Sample Size (cases) 51 51

Median Survival (month) 10.3 32.5 Median Survival (Years) 5.79 7.63

0. No value (452) 1. BCR-ABL1 Gene Fusion (2) 2. DEK-NUP214 Gene Fusion (6) 3. MLL Gene Rearrangements (10) mRNA relative level 4. PML-RARA Gene Fusion (21) 5. RUNX1-RUNX1T1 Gene Fusion (35) CD200 3.

Figure 17. CD200 gene expression level and survival analysis of patients with CD200high and CD200low expressing tumors as determined by microarray data

Survival curve drawn is based on the raw data on Oncomine database. The data was reanalyzed for CD200 expression levels. Patients were divided into three groups: CD200low, CD200intermediate, CD200high.The survival curve is based on average survival time of patients with CD200low vs. CD200high expression in their tumor biopsy samples as determined by mircroarray analysis. A. Data from a study conducted on diffuse large B-cell lymphoma patients. Survival time was given in months [1]. B. Survival curve drawn is based on the raw data available from a study conducted on breast cancer patients. Survival time was given in years [3]. C. Oncomine generated graph showing the median CD200 mRNA expression levels pertaining to the various subtypes of AML. 53

3 Targeting CD200R in cancer immunotherapy

Our results suggest that CD200-CD200R interaction plays important roles in both innate and adaptive components in tumor immunity, therefore targeting CD200-CD200R interaction should provide a potential option for treatment of cancer. CD200 is only expressed in some lineage of tumors, while CD200R is expressed in tumor associated myeloid cells essentially in all solid tumors; thus, targeting CD200R should have broader implication in the treatment of cancer. The experiments shown in this chapter are designed to test this hypothesis.

3.3.1 Tumor development and metastasis is significantly increased in CD200R-/- mice

To determine the importance of CD200R expression and its impact on tumor establishment and metastasis, we injected 1x105 B16.OVA.CD200 melanoma cells into

CD200R-/- and wild type litter mates i.v. After observing the mice for 19 days, we noticed

that 2 of the mice in the CD200R-/- group we paralyzed from the waist down. Another 2 mice had difficulty breathing, two of which also looked bloated and heavy. Therefore, we

the sacrificed mice and examined their liver and lungs. We also examined their

abdominal cavities for metastasis and tumor foci formation. CD200R-/- mice exhibited

dramatically higher tumor foci in the abdominal cavity. There were also many massive

and small metastatic tumor foci all over the mice, including the GI tract, many of which

are not readily detectable (Figure 18A). There were also a lot more tumors in the lung 54

and liver (Figure 18B). One of the CD200R-/- had very little lung and liver metastasis.

However, the abdominal cavity was infested with tumor. Examining this mouse revealed an extensive amount of metastasis to the kidneys of this mouse (Figure 18C). Thus,

CD200R expression is required control and limit melanoma metastasis, to all organs, but

preferentially to the liver and lung.

A Lung Liver Wild Type CD200R-/- B C CD200R-/- 0.3

Wild Type 0.2

Grams 0.1

0 Lung 2 Wild CD200R-/- 1.6 Type 1.2

Grams 0.8 0.4 CD200R-/- 0 Liver 1.2

0.8

Grams 0.4

0 Kidney

Figure 18. Metastatic tumor growth in CD200R-/- mice A. CD200R-/- mice and their wild Type litter mates were injected with 1*105 B16.OVA.CD200 tumor cells. 19 days later, the mice were examined for Tumor formation in the body. Figure shows the abdominal cavity of representative mice. Lung and Liver of tumor bearing mice are also depicted (n =8/group).B. For a more objective analysis the lung and liver of each mouse was weighed. Graph shows the average liver weight difference between the two groups.C. The lung and liver of mouse no.3 in the CD200R-/- group shown in part A has very little metastasis to compared to the rest of its group. The abdominal cavity of this mouse is shown in part C. A huge tumor mass observed is located on the kidney of this mouse. Here we show the size and weight of a normal kidney compared tot eh size and average weight of the kidneys extracted from this mouse.

3.3.2 Triggering CD200R using a monoclonal antibody inhibits tumor foci

formation in the lungs

To test if targeting CD200R can inhibit tumor formation and metastasis, we tested the efficacy of an agonistic anti-CD200R mAb (OX110) [30] in the treatment of lung metastasis of CD200-negative melanoma. To test the efficacy of OX110 on myeloid cells, we isolated CD11b+ cells from Rag1-/- C57BL/6 mice. The cells were stimulated

with LPS in the presence of OX110 (Biolegend) or an isotype-matched control mAb

(Biolegend). As shown in Figure 19A, anti-CD200R mAb significantly diminished

production of cytokines by CD11b+ myeloid cells, suggesting that mAb OX110 can

inhibits the functions of myeloid cells. To test its in vivo effects on tumor foci formation

and lung metastasis, C57BL/6 mice were injected with 1 x 105 B16.OVA.Ctrl cells i.v.

Starting from day 0, we treated mice with 100µg/mouse of OX110 mAb or 100µg/mouse of an isotype-matched control IgG i.v. every 3 days. As shown in Figure 19B, anti-

CD200R treatment dramatically reduced melanoma tumor formation in the lungs compared to treatment with the control antibody. Numbers of melanoma foci (Figure

19C) and lung weights (Figure 19D) were also significantly different between the two groups. Thus, triggering CD200R inhibits tumor foci formation in the lungs and targeting

CD200R by a triggering mAb is feasible for the treatment of CD200-negative tumors.

A 900 P=0.003 P=0.004 200 P=0.001 P=0.002 P=0.003 P=0.002 700 800 600 700 150 600 500 Ctrl IgG (pg/ml) (pg/ml) 500 400 Anti-CD200R

100 α 400 300 300 TNF- IL-6 (pg/ml) IL-6 (pg/ml) (pg/ml) IL-10 200 50 200 100 100 0 0 0 24 hr 48 hr 24 hr 48 hr 24 hr 48 hr

0.3 P=0.04 P=0.04 250 B C D 0.25 Ctrl IgG 200 0.2

150 0.15

100 0.1 Number of of Foci Number

50 (gram) Lung weight 0.05

0 0 G G Anti-CD200R Ig 0R Ig 0R trl 20 trl 0 C C D2 i-CD C nt ti- A An

Figure 19. Triggering CD200R using a mAb inhibits melanoma lung metastasis. A. CD11b+ cells were isolated from the spleen of C57BL/6 mice and were stimulated with LPS (100 ng/ml) in the presence or absence of a monoclonal anti-mouse CD200R antibody (OX110). Supernatants from the cultures were collected at 24 and 48 hours and concentrations of IL-6, IL-10 and TNF-α were examined using ELISA. Data shown represents three independent experiments with similar results. Data shown are mean ± SEM from groups of three mice. Student’s two-tailed t test was used for the statistical analysis. B. 1 x 105 B16.OVA.Ctrl cells were injected into each C57BL/6 mice via their tail vein. Mice were treated with either 100µg/mouse of anti-CD200R (n=5) or an isotype- matched control antibody (n=5) every 3 days starting from day 0. 18 days later mice were sacrificed and melanoma lung metastasis was examined in antibody-treated mice or controls. Data shown represents two experiments with similar results. C. Average number of tumor foci in the lungs from each group of mice. Error bars represent ± SEM. Student’s two-tailed t test was used for the statistical analysis. D. Average weights of lungs from each group of mice. Error bars represent ± SEM. Student’s two-tailed t test was used for the statistical analysis.

3.3.3 Agonistic CD200R antibody treatment suppresses tumor growth and enhances

efficacy of CTL adoptive transfer therapy.

To test the potential of anti-CD200R antibody in inhibiting growth of solid

tumors, we subcutaneously injected 5X106 3B11 tumor cells into C57BL/6.Rag1-/- mice

(n=5/group). The mice were monitored every 3 days and tumor size was measured using

a digital caliper. Once the tumors formed (~4 mm) we administered 50µg of agonistic

anti-CD200R mAb intra tumorally into each mouse every 3 days. As shown in (Figure

20A), injection of agonistic anti-CD200R mAb significantly inhibited tumor growth and

increased survival (Figure 20B) in the treated mice compared to the control group. To

determine if anti-CD200R mAb has a synergistic effect with CTL adoptive transfer

therapy, we injected BALB/c.Rag2-/- mice with 5*105 P815 mastocytoma cells

subcutaneously. The mice were divided into 3 groups (n=6/group). Mice in the first group

were left untreated; mice in the second group were treated with P1CTL cells and mice in

the third group were treated with 5 million/mouse of P1CTL plus anti-CD200R. P815

tumors do not grow into large tumors locally; they metastasize to various organs.

Therefore, we monitored the mice for survival. Mice in the first group had very limited

survival time, P1CTL therapy dramatically increased survival of mice; while mice treated

with anti-CD200R and P1CTL had longest survival time (Figure 20C). Thus, triggering

CD200R not only inhibits tumor formation, but also enhances CTL adoptive transfer

therapy of cancer.

A 2500 P=0.001 Isotype Treatment ** 2000 P=0.01 CD200R Treatment

) * 3 1500

1000

500 C 1 UntreatedUntreated Isotype Treated 0 P1CTL Treated CD200R Ab

Tumor Volume (mm Volume Tumor 0.8 9 12 15 18 21 24 27 TreatedP1CTL+ CD200R Ab Treated Days Post 3B11 Tumor Injection 0.6 B 1 0.4

0.8 0.2 P=0.001 Probability of Survival P<0 P=0.01 0.6 0 .00 P=0.003 Isotype Treatment 1 0.4 0 10203040506070 CD200R Treatment Days Post P815 Tumor Injection Probability of Surviva 0.2

Probability of Survival of Probability 0 20 25 30 DaysDays Post Post 3B11 3B11 tumor Tumor cell Injection Injection

Figure 20. Treatment of Tumor bearing mice with agonistic CD200R antibody A. C57BL/6.Rag1-/- mice were injected with 5*106 3B11 tumor cells. Tumor bearing mice were treated with 50 µg of intratumoral agonistic CD200R or isotype antibody every 3 days. Graph shows tumor growth in the two groups(n=5/group).B. Figure shows survival time of 3B11 tumor bearing mice treated with intratumoral injections of CD200R or isotype antibody.C. Balb/c.Rag2-/- mice were given 5*105 P815 tumor cells i.c. First group was left untreated. Four days later group 2 and 3 mice were treated with tumor antigen specific P1CTL cells intravenously. Group 3 mice were also injected with 100 µg of agonistic CD200R antibody every 4 days post tumor injective via their tail vein. Figure shows the survival time of untreated compared to CTL treated and CTL plus CD200R antibody treated mice(n=6/group).

3.3.4 CD200R expression on tumor cells serves as a target for CTL therapy

We have recently found that some cancer cells, particularly those derived from the myeloid lineage also express CD200R (Figure 21A). Since activated CTL express 60

high levels of CD200, we hypothesize that tumor expression of CD200R can be a direct

target of CTL and impacts target destruction by CTL. To test this hypothesis, we

generated P815CD200R+ or P815CD200R- cells (Figure 21B). These cells expressed

similar levels of MHC class I (Figure 21B) and tumor antigen P1A (not shown). To test

if CD200R expression on tumor cells impacts the destructive potential of CTL cells, we

preformed a 51Cr-release assay using activated P1CTL as effectors, P815CD200R+ or

P815CD200R- cells as targets. The result shown in Figure 21C clearly indicates that

CD200R expression on the tumor cells makes them more susceptible to CTL destruction.

To confirm this observation, we co-cultured P815CD200R+ cells with P1CTL in the

presence or absence of CD200 blocking antibody. Blocking the CD200 on T cells

substantially decreased their effectiveness in destroying the tumor cells (Figure 21D),

indicating that CD200-CD200R interaction plays a direct role in destruction of CD200R

expressing tumor cells. To further confirm this finding, we knocked down CD200 in T

cells using a lentivirus expressing shRNA to CD200. 51Cr-release assay revealed that

shRNA silencing of CD200 in P1CTL significantly reduced their efficacy in destructing

of CD200R-positive P815 cells (Figure 21E). Taken together, these results suggest that

CD200R expression on tumor cells serves as a target for CTL that expresses high levels of CD200.

- + A B P815CD200R P815CD200R 70 P338D1 C P=.05 3B11 60 P=.03 50 40 CD200R 30 P815 Raw264. % of Max 20 P815 CD200R+ 7 P815 CD200R- % Specific Killing 10 0 100 33 11 3 1 0.3 CD200R H2Ld E:T Ratio (CTL:P815 CD200R-/+)

D 60 P<0.05 P<0.01 T+ CTRL Ab E 60 TsiCTRL 50 T+CD200 Ab 50 TsiCD200 P<0.01 40 40 30 30 20 20 % Specific Killing 10 10 % Specific Killing 0 0 10 3 1 0 20 6 2 0.6 E: T Ratio (CTL: P815 CD200R+) E:T Ratio (CTL:P815 CD200R+)

Figure 21. CD200R on tumor cells serve as a target for CTL A. Several myeloid Derived tumor cell lines naturally express CD200R. B. We have generated P815CD200R- and P815CD200R+ tumor cells. Both of these cell lines express the same level of MHC class I molecules on their surface. C. 51Cr- release assay shows that activated CTL (with high CD200 levels) are more effective in killing P815CD200R+ tumor cells. D. 51Cr-release release assay shows that activated CTL (with high CD200 levels) lose their optimal efficacy in killing P815CD200R+ tumor cells when their CD200 surface expression is blocked. E. 51Cr-release release assay shows that CD200 knock down activated CTL (with high CD200 levels) lose their optimal efficacy in killing P815CD200R+ tumor cells compared to wild type T cells expressing CD200. This confirms the results obtained from CD200 blocking experiment.

3.3.5 CD200R expression in human cancer cells

We next examined if CD200R is also expressed in human cancer cells and their

possible significance. We searched the Oncomine database for CD200R expression and its correlation with disease recurrence and patient survival time. Unfortunately the

CD200R probe has only recently been added to the microarray chips therefore the data available is very limited.

A B Recurrence after 1 year

P=0.0005 mRNA relative level Survival Probability (%) CD200R

1.No Recurrence 2.Recurrence Months

Carasco et al (2006) CD200R CD200R low high Sample Size (cases) 23 22

Figure 22. CD200R expression level and Survival analysis of patients with CD200Rhigh and CD200Rlow expressing tumors as determined by microarray analysis A. Oncomine generated graph showing the median CD200R mRNA expression levels of patients with and without recurrent myeloma after 1 year follow up. B. Survival curve drawn is based on the raw data available on Oncomine. The data was reanalyzed for CD200R expression levels. Patients were divided into three groups: CD200Rlow, CD200Rintermediate, CD200high.The survival curve is based on average survival time of patients with CD200Rlow vs. CD200Rhigh expression in their tumor biopsy samples as determined by mircroarray analysis. Data used is from a study conducted on on multiple myeloma patients [2]. Survival time

However, we found one study on multiple myeloma patients. In that study

patients whose myeloma cells have higher CD200R gene expression have a lower rate of

recurrence after treatment (Figure 22A). It also clearly demonstrates that cancer patients

bearing CD200RHigh tumors have a significantly higher survival rate compared to those

with CD200Rlow tumor masses (Figure 22B). Although these data need to be reconfirmed

in definitive studies, our preliminary findings demonstrate a promising potential for using

CD200R expression of tumors as a prognostic toll for identifying patients. Further studies are needed to understand how this novel finding can be applied therapeutically to improve the survival and quality of life in cancer patients.

Chapter 4. Discussion

4.1 Abundant CD200 and restricted CD200R expression in the tumor

microenvironment

In this study we have found that CD200 is constitutively expressed on myeloid

cells. Activation by various approaches (direct activation by LPS, or bystander activation

by T cells) resulted in dramatically upregulated CD200 on myeloid cells. TAMCs from a solid tumor are composed of three populations of cells: CD11b+Gr-1hi, CD11b+Gr-1lo and

CD11b+Gr-1- cells. The first two populations of cells should belong to the MDSC group

[32–33], which represents about 1–5% of total tumor cells, while the latter population of

cells, i.e. TAM, is about 15% of total tumor cells. We found that CD200 expression is

present on all the subsets of TAMCs (Figure 11). Thus, TAMCs are one of the major

sources of CD200. Another source of CD200 comes from activated T cells. In this study

we show that upon activation, tumor antigen specific P1CTL cells dramatically

upregulate CD200. In BALB/c mice bearing established J558 tumors, tumor infiltrating

CD4+ and CD8+ T cells both express high levels of CD200 (Figure 11). Thus, in an

established solid tumor, multiple cell types express CD200.

In contrast to the broader expression of CD200 in a solid tumor, CD200R

expression is mainly limited to TAMCs. We have found that TAMCs express high levels

of CD200R (Figure 11), however, CD200R is low and barely detectable on tumor infiltrating T cells. Based on these observations, we propose the following model (Figure

23) to depict where CD200-CD200R interactions could be involved. We propose that 65

four potential interactions could be regulated by CD200-CD200R interactions. First,

CD200 on tumor cells interacts with CD200R on TAMCs; second, CD200 on T cells interacts with TAMCs; third, CD200 on T cells interacts with CD200R-potive tumors and

fourth, CD200 on TAMC interacts with CD200R on TAMC. In this study we have

demonstrated that the first interaction has dramatic effect on tumor formation and

metastasis. The other three potential interactions and their consequences to tumor

formation, progression, metastasis and tumor immunity remain to be determined.

A •Inhibition of TAMC function •Enhanced tumor susceptibility To destruction •Less suppressive tumor microenvironment by CTL

CTL Tumor C

CD200 TAMC Tumor D

CD200R

B CTL

TAMC

TAMC Inhibitory Signal TAMC

•Enhanced Destruction of TAMC by CTL •Self-limiting immunoregulatory interaction

Figure 23. A model for CD200-CD200R interaction in the tumor microenvironment A. Tumor-TAM interaction. Tumors that express CD200 can directly interact with CD200Rhi TAMC and inhibit their function. This makes the tumor microenvironment less suppressive. It can serve as an indirect mechanism for enhanced CTL function as well.B. CTL-TAMC interaction. CD200hi CTL can directly interact with CD200Rhi TAMC. This cell-cell interaction allows CTL to destroy the tumor antigen presenting TAMC, diminishing the numbers of this protumor compartment. This can indirectly enhance CTL function as well.C. CTL-Tumor interaction. CTL can readily establish a direct cell-cell interaction with tumors that express CD200R on their surface despite all the mechanisms a tumor cell uses to evade CTL recognition. Therefore this subset of tumors is more susceptible to CTL recognition and destruction. This suggests that CD200R expression can be used as a biomarker for identifying tumors with good prognosis and enhanced potential for CTL therapy. D. TAMC-TAMC interaction. TAMC express high levels of CD200 and CD200R. Therefore, they can interact with one another through this pair of molecules. This will serve as a self-limiting mechanism.

4.2 Tumor cell expression of CD200 inhibits tumor formation and metastasis via inhibition of myeloid cell functions

Expression of CD200 has been implicated in a variety of human cancer cells 67

including melanoma cells [75] and has been reported to play pro-tumor effects via inhibiting tumor immunity [75, 79, 174]. However, using CD200-positive and CD200- negative melanoma tumor models, we have revealed a novel role for CD200-CD200R interaction in inhibiting tumor formation and metastasis, i.e. tumor expression of CD200 inhibits tumor formation and metastasis via inhibiting the functions of CD200R+ myeloid cells. Thus, our data challenge the current paradigm that tumor expression of CD200 promotes tumor progression and metastasis.

Myeloid cells are pivotal in tumor initiation, tumor mass formation, tumor progression and metastasis [175]. In the tumor initiation and formation stage, myeloid cells produce an array of factors that promote tumor establishment. During the tumor progression and metastasis stages, myeloid cells provide support for developing tissues through their matrix remodeling capacities, synthesis of growth and angiogenesis factors and capacity in suppressing antitumor immunity. Genetic ablation, depletion of myeloid cells or inhibition of myeloid cell functions have been shown to be effective in inhibiting tumor establishment and tumor progression [176-178, 180]. It has been found that increased number of myeloid cells is strongly associated with shortened survival in patients with classic Hodgkin’s lymphoma [104]. We have recently demonstrated that tumor associated myeloid cells express high levels of CD200R, and they are susceptible to CD200-mediated inhibition [82]. In this study, we have compiled evidence that tumor expressed CD200 can directly interact with myeloid cells to inhibit tumor formation and

metastasis in a CD200R-dependent manner. In in vitro cultures, CD200-positive tumor cells but not CD200-negative tumor cells strongly suppress cytokine production by myeloid cells. Compared to peripheral myeloid cells, Gr1+ lung myeloid cells express

much higher levels of CD200R. This correlated with profound inhibition of tumor

formation and metastasis of CD200+ B16 melanoma in the lung while the effect was

diminished in the periphery. In contrast to our observation, CD200 induction on tumor

cells was recently shown to correlate with more tumor metastasis [79]. However, in that

study it was unclear if expression of CD200 on metastatic tumors was responsible for

tumor metastasis. In our current study, we have clearly shown that depletion of a large

population of CD200R+ myeloid cells (Ly6G+) using anti-Gr1 mAb achieved a similar

effect to CD200 expression on melanoma cells.

4.3 Tumor expression of CD200 renders tumor microenvironment more permissive

to T cells and predicts better response to T cell therapy

Our previous study [82] has revealed that CD200-positive plasmacytoma J558

and mastocytoma P815 tumors are more susceptible to CTL adoptive transfer therapy

presumably due to a more permissive tumor microenvironment. In this study we found

that adoptive transfer of antigen-specific T cells (both CD4+ and CD8+) also significantly

promoted survival of mice with CD200-positive melanoma tumors over CD200-negative tumors. Moreover, significant growth delay of CD200-positive B16 tumors in immune

competent mice but not in immune-deficient Rag1-/- mice suggest that adaptive immunity 69

is also stimulated by CD200-positive tumors. Our data support a model that tumor expression of CD200 inhibits CD200R+ myeloid cells in the tumor microenvironment, which renders the tumor microenvironment more permissive to T cells and confers better tumor destruction. Previous studies have demonstrated that both TAM and MDSC produce high amounts of IL-10 [34–35]. They are known to suppress CTL effector functions while promoting tumor invasion, growth and angiogenesis [25–27]. In addition,

MDSC have been shown to convert M1 macrophages (TNF-α producing) into M2 macrophages (IL-10 producing) through the production of IL-10 [36]. We have confirmed that in CD200-positive tumors, TAMC are converted to a M1 phenotype with lower production of IL-10 and higher production of TNF-α. Since IL-10- deficient

TAMC also had increased production of TNF-α, we consider that the increased TNF-α production by TAMC from CD200-positive tumors is a result of inhibition of IL-10 production. These data coupled with the fact that TAMC express high levels of CD200R explains why they are susceptible to CD200-mediated inhibition [82].

4.4 Does tumor expression of CD200 predict better prognosis?

Microarray analysis suggests increased expression of CD200 in many human cancers [181]. Expression of CD200 has been reported in human Hairy Cell Leukemia

(HCL) [182], lymphoblastic leukemia, large B cell lymphoma, lymphoplasmacytic lymphoma, angioimmunoblastic T cell lymphoma [183-184], acute myeloid leukemia

[70], plasma cell myeloma (PCM) [185] and B-CLL [73, 174]. CD200 was shown to be a 70

reliable marker for differential diagnosis of the various B cell Lymphoproliferative

diseases (BCLDs) [186] and was proposed as a distinguishing marker between the

CD200 positive B-CLL [187-188] and the CD200 negative mantle cell lymphoma (MCL)

[189]. It is generally considered that expression of CD200 on cancer cells has a protumor effect based on the following evidence. First, in two correlation studies, CD200 mRNA expression in malignant cells has been shown to be associated with decreased survival of patients [69-70]; Second, CD200-expressing melanoma and ovarian cancer cells downregulate Th1 cytokine production when co-cultured with allogenic leukocytes [75,

(Figure 17) suggests that tumor expression of CD200 may be beneficial. For instance, patients in group 5 of AML (Figure 17C) exhibit the highest level of CD200 expression are known to have good prognosis [179]. This view point is supported by a more recent study that analyzed correlation of CD200 and PCM subtypes and stages of disease [81]. It was found that the PCM subtypes associated with poor clinical outcome are the ones over-represented in the CD200 negative population. It was also demonstrated that downregulation or lack of CD200 expression is a marker of PCM disease progression. Another finding in this study is that patients with B-ALL that had the strongest CD200 expression carried the subtype of disease associated with excellent clinical outcome. Thus, more 71

human studies are required to establish whether CD200 expression on tumor cells is

beneficial or detrimental. However, to establish the relationship between tumor

expression of CD200 and prognosis based on human biopsy samples could be

complicated by the fact that within a tumor biopsy sample numerous cell types including

regulatory cells express high levels of CD200. Therefore the high expression of CD200 in tumors is the result of a mixed cell types. Hence the elevation of CD200 expression in itself cannot be deemed detrimental or beneficial without specific studies to determine what cell types are the contributor of CD200 expression.

4.5 Targeting CD200R may be a novel approach for cancer therapy

In this study we have made a number of observations that support a role of

CD200R as target of cancer therapy. 1) Tumor development and metastasis is significantly increased in CD200R-/- mice (Figure 18); 2) Triggering CD200R using a

monoclonal antibody inhibits tumor foci formation in the lung and suppresses growth of a

solid tumor in mice (Figure 19). Moreover, anti-CD200R treatment also enhanced

efficacy of CTL adoptive transfer therapy (Figure 20). 3) CD200R expression was found

on some lineages of cancer cells, which could be a direct target of CD200 on tumor

antigen specific CTL (Figure 21). Analysis of microarray data suggests that CD200R

expression on human cancer cells may be beneficial (Figure 22). Because of the restricted

expression of CD200R on myeloid cells and the importance of these cells in essentially all tumor

types, targeting CD200R should be an ideal option for myeloid cell targeted therapy of cancer.

Our successful treatment of CD200-negative tumors using a triggering anti-CD200R mAb proves that this approach is feasible.

4.6. Concluding remarks and future directions

This study was performed to test the hypothesis that CD200-CD200R interaction affects tumor formation, metastasis and tumor immunity via inhibiting TAMC functions. The goals of this dissertation thesis were: a) To investigate CD200-CD200R expression in the tumor compartments; b) To determine the role of CD200-CD200R interaction in tumor formation and metastasis and c) To determine if targeting CD200R is a feasible approach for cancer immunotherapy. Our research has lead to the following observations and conclusions.

1) We demonstrated that CD200 and CD200R are constitutively expressed on resting myeloid lineage of cells. Upon activation, these cells dramatically upregulated

CD200 and CD200R. In the tumor microenvironment, both CD200 and CD200R are

expressed on TAMCs at high levels. This result suggests that CD200-CD200R interaction

among TAMCs may play important roles in regulating tumor growth and immunity, and

modulating CD200-CD200R interaction between TAMCs may have therapeutic potential

for human cancer.

2) Naïve T cells do not express CD200 and CD200R. Upon activation, T cells

rapidly upregulate CD200, while CD200R expression on T cells is barely detectable. In

the tumor microenvironment, CD200 is highly expressed on tumor infiltrating T

lymphocytes (TILs). The high CD200 expression on TILs indicates that CD200 on T 73

cells may potentially regulate TAMC functions. CD200 on T cells may also regulate

CD200R-potive tumor cells.

3) In this study we have found that CD200 on melanoma cells inhibits tumor formation and lung metastasis, mainly through inhibition of myeloid cell functions.

CD200-CD200R interaction between tumor cells and TAMCs may modulate tumor microenvironment, which in turn enhances the effector function of tumor infiltrating

CTL. Hence, CD200-positive B16 melanoma were more susceptible to T cell adoptive transfer therapy. More recent studies and our microarray data analysis suggest that high

CD200 expression on human cancer cells may lead to longer survival time.

4) Since TAMCs are the major pro-tumor immune compartment in the tumor microenvironment and they are highly positive for CD200R, targeting CD200R should be a useful approach for cancer immunotherapy. Indeed, using an agonistic monoclonal antibody to CD200R, we have found that melanoma lung metastasis to the lung was dramatically blocked. This result suggests that targeting CD200R is a highly feasible approach. Given almost all solid tumors have a TAMC compartment, anti-CD200R should be used for the treatment of other types of cancer.

5) We have shown that some cancer cells, particularly those derived from myeloid lineage of cells express CD200R. Moreover, CD200R-positive cancer cells show increased susceptibility to CTL destruction compared to CD200R-negative cancer cells.

Blocking CD200-CD200R interaction between CTL and cancer cells abrogated the increased susceptibility to CTL destruction. Human myeloma microarray data analysis 74

suggests that cancer patients whose myeloma cells express higher CD200R have a longer survival time. Thus, CD200R expression on cancer cells may be more susceptible to immune surveillance and immunotherapy.

Given the important roles of CD200-CD200R interaction in regulating tumor associated myeloid cells and in inhibiting tumor formation and metastasis, targeting

CD200-CD200R interaction should provide an option for the immunotherapy of human cancer. Our successful treatment of CD200-negative tumors using a triggering anti-

CD200R mAb proves that this approach is feasible.

Although certain cellular and molecular mechanisms regarding the role of

CD200-CD200R interaction in tumor immunity has been uncovered, much of the biological functions of CD200-CD200R interaction in the tumor setting remain to be addressed and deciphered. For example, the roles of CD200-CD200R interaction among

TAMC-TAMC, T-TAMC and T-Tumor cells remain to be determined. Future work will be centered around the investigation of these interactions.

Chapter 5. Visual Review of Thesis

ABCCytokines Growth factors

TAMC Enzymes TAMC TAMC ROS/RNS

Chemokines

Cancer Cancer Cancer Cell Cell Cell

Figure 24. The impacts of tumor expression of CD200 on melanoma lung metastasis. Melanoma lung tumor formation depicted in the absence (A) and presence (B) of CD200 expression on tumor cells. CD200R-/- reinforces melanoma lung tumor formation and metastasis even in the presence of CD200 expressing tumors (C).

Inhibitors (IL-10) ABInhibitors (IL-10)

TAMC T cell TAMC T cell

Cancer Cell

Figure 25. The impacts of tumor expression of CD200 on melanoma susceptibility to T cell therapy. T cell therapy of mice with established tumors whose tumor cells are negative for CD200 (A) or positive for CD200 (B) shows that CD200 expression renders tumors more susceptible to this treatment.

With out Treatment

Agonistic anti- CD200R Antibody

TAMC

Cytokines Growth factors With Treatment Chemokines Cancer Cell Enzymes ROS/RNS

Figure26. An agonistic antibody to CD200R inhibits melanoma lung tumor formation and metastasis

Inhibitors(IL-10) With out Treatment

TAMC CTL

With Treatment

Cancer Cell

Figure 27. An agonistic antibody to CD200R enhances the susceptibility of established tumors to T cell therapy.

Cancer Cell Cancer Cell

TAMC T cells

CD200

Reduced Cytokine Production CD200R

Inhibition of Tumor Growth Inhibition of Tumor invasion Better response to Therapy Inhibition/Reduction of Angiogenesis?

Figure 28. Monoclonal Agonistic CD200R antibody is a promising candidate for cancer immunotherapy for virtually all tumors.

References:

1. Alizadeh, A.A., et al., Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature, 2000. 403(6769): p. 503-11. 2. Carrasco, D.R., et al., High-resolution genomic profiles define distinct clinico- pathogenetic subgroups of multiple myeloma patients. Cancer Cell, 2006. 9(4): p. 313-25. 3. Pawitan, Y., et al., Gene expression profiling spares early breast cancer patients from adjuvant therapy: derived and validated in two population-based cohorts. Breast Cancer Res, 2005. 7(6): p. R953-64. 4. Preston, S., et al., The leukocyte/neuron cell surface antigen OX2 binds to a ligand on macrophages. Eur J Immunol, 1997. 27(8): p. 1911-8. 5. Borriello, F., et al., Characterization and localization of Mox2, the gene encoding the murine homolog of the rat MRC OX-2 membrane glycoprotein. Mamm Genome, 1998. 9(2): p. 114-8. 6. Barclay, A.N., M.J. Clark, and G.W. McCaughan, Neuronal/lymphoid membrane glycoprotein MRC OX-2 is a member of the immunoglobulin superfamily with a light-chain-like structure. Biochem Soc Symp, 1986. 51: p. 149-57. 7. Barclay, A.N. and H.A. Ward, Purification and chemical characterisation of membrane glycoproteins from rat thymocytes and brain, recognised by monoclonal antibody MRC OX 2. Eur J Biochem, 1982. 129(2): p. 447-58. 8. Webb, M. and A.N. Barclay, Localisation of the MRC OX-2 glycoprotein on the surfaces of neurones. J Neurochem, 1984. 43(4): p. 1061-7. 9. Morris, R.J. and J.N. Beech, Sequential expression of OX2 and Thy-1 glycoproteins on the neuronal surface during development. An immunohistochemical study of rat cerebellum. Dev Neurosci, 1987. 9(1): p. 33- 44. 10. Barclay, A.N., Different reticular elements in rat lymphoid tissue identified by localization of Ia, Thy-1 and MRC OX 2 antigens. Immunology, 1981. 44(4): p. 727-36. 11. Viertlboeck, B.C., et al., Characterization of the chicken CD200 receptor family. Mol Immunol, 2008. 45(7): p. 2097-105. 12. Wright, G.J., et al., The unusual distribution of the neuronal/lymphoid cell surface CD200 (OX2) glycoprotein is conserved in humans. Immunology, 2001. 102(2): p. 173-9.

13. McCaughan, G.W., M.J. Clark, and A.N. Barclay, Characterization of the human homolog of the rat MRC OX-2 membrane glycoprotein. Immunogenetics, 1987. 25(5): p. 329-35. 14. Chen, Z., H. Zeng, and R.M. Gorczynski, Cloning and characterization of the murine homologue of the rat/human MRC OX-2 gene. Biochim Biophys Acta, 1997. 1362(1): p. 6-10. 15. Bukovsky, A., J. Presl, and M. Holub, The ovarian follicle as a model for the cell- mediated control of tissue growth. Cell Tissue Res, 1984. 236(3): p. 717-24. 16. Bukovsky, A., et al., The localization of Thy-1.1, MRC OX 2 and Ia antigens in the rat ovary and fallopian tube. Immunology, 1983. 48(3): p. 587-96. 17. Chen, Z., P.A. Marsden, and R.M. Gorczynski, Cloning and characterization of the human CD200 promoter region. Mol Immunol, 2006. 43(6): p. 579-87. 18. Chen, Z., P.A. Marsden, and R.M. Gorczynski, Role of a distal enhancer in the transcriptional responsiveness of the human CD200 gene to interferon-gamma and tumor necrosis factor-alpha. Mol Immunol, 2009. 46(10): p. 1951-63. 19. Ko, Y.C., et al., Endothelial CD200 is heterogeneously distributed, regulated and involved in immune cell-endothelium interactions. J Anat, 2009. 214(1): p. 183- 95. 20. Mukhopadhyay, S., et al., Immune inhibitory ligand CD200 induction by TLRs and NLRs limits macrophage activation to protect the host from meningococcal septicemia. Cell Host Microbe, 2010. 8(3): p. 236-47. 21. Walker, D.G., et al., Decreased expression of CD200 and CD200 receptor in Alzheimer's disease: a potential mechanism leading to chronic inflammation. Exp Neurol, 2009. 215(1): p. 5-19. 22. Lyons, A., et al., CD200 ligand receptor interaction modulates microglial activation in vivo and in vitro: a role for IL-4. J Neurosci, 2007. 27(31): p. 8309- 13. 23. Hebenstreit, D., et al., Signaling mechanisms, interaction partners, and target genes of STAT6. Cytokine Growth Factor Rev, 2006. 17(3): p. 173-88. 24. Snelgrove, R.J., et al., An absence of reactive oxygen species improves the resolution of lung influenza infection. Eur J Immunol, 2006. 36(6): p. 1364-73. 25. Chen, Z., et al., Identification of an expressed truncated form of CD200, CD200tr, which is a physiologic antagonist of CD200-induced suppression. Transplantation, 2008. 86(8): p. 1116-24. 26. Chen, Z., et al., Alternative splicing of CD200 is regulated by an exonic splicing 82

enhancer and SF2/ASF. Nucleic Acids Res, 2010. 38(19): p. 6684-96. 27. Zhang, S., et al., Molecular mechanisms of CD200 inhibition of mast cell activation. J Immunol, 2004. 173(11): p. 6786-93. 28. Wright, G.J., et al., Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity, 2000. 13(2): p. 233-42. 29. Gorczynski, R.M., et al., Structural and functional heterogeneity in the CD200R family of immunoregulatory molecules and their expression at the feto-maternal interface. Am J Reprod Immunol, 2004. 52(2): p. 147-63. 30. Wright, G.J., et al., Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol, 2003. 171(6): p. 3034-46. 31. Hatherley, D., et al., Recombinant CD200 protein does not bind activating proteins closely related to CD200 receptor. J Immunol, 2005. 175(4): p. 2469-74. 32. Forman-Kay, J.D. and T. Pawson, Diversity in protein recognition by PTB domains. Curr Opin Struct Biol, 1999. 9(6): p. 690-5. 33. Mihrshahi, R., A.N. Barclay, and M.H. Brown, Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells. J Immunol, 2009. 183(8): p. 4879-86. 34. Zhang, S. and J.H. Phillips, Identification of tyrosine residues crucial for CD200R-mediated inhibition of mast cell activation. J Leukoc Biol, 2006. 79(2): p. 363-8. 35. Mihrshahi, R. and M.H. Brown, Downstream of tyrosine kinase 1 and 2 play opposing roles in CD200 receptor signaling. J Immunol, 2010. 185(12): p. 7216- 22. 36. Hoek, R.M., et al., Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science, 2000. 290(5497): p. 1768-71. 37. Snelgrove, R.J., et al., A critical function for CD200 in lung immune homeostasis and the severity of influenza infection. Nat Immunol, 2008. 9(9): p. 1074-83. 38. Simelyte, E., et al., CD200R1 regulates the severity of arthritis but has minimal impact on the adaptive immune response. Clin Exp Immunol, 2010. 162(1): p. 163-8. 39. Bartolome, M.V., M.A. Ibanez-Olias, and P. Gil-Loyzaga, Transitional expression of OX-2 and GAP-43 glycoproteins in developing rat cochlear nerve fibers. Histol Histopathol, 2002. 17(1): p. 83-95.

40. Hoarau, J.J., et al., Activation and control of CNS innate immune responses in health and diseases: a balancing act finely tuned by neuroimmune regulators (NIReg). CNS Neurol Disord Drug Targets, 2011. 10(1): p. 25-43. 41. Chang, C.Y., et al., Novel distribution of cluster of differentiation 200 adhesion molecule in glial cells of the peripheral nervous system of rats and its modulation after nerve injury. Neuroscience, 2011. 183: p. 32-46. 42. Clark, D.A., et al., Placental trophoblast from successful human pregnancies expresses the tolerance signaling molecule, CD200 (OX-2). Am J Reprod Immunol, 2003. 50(3): p. 187-95. 43. Clark, D.A., et al., Procoagulants in fetus rejection: the role of the OX-2 (CD200) tolerance signal. Semin Immunol, 2001. 13(4): p. 255-63. 44. Clark, D.A., et al., Fgl2 prothrombinase expression in mouse trophoblast and decidua triggers abortion but may be countered by OX-2. Mol Hum Reprod, 2001. 7(2): p. 185-94. 45. Lin, Y., et al., Murine CD200+ CK7+ trophoblasts in a poly (I:C)-induced embryo resorption model. Reproduction, 2005. 130(4): p. 529-37. 46. Jiang-Shieh, Y.F., et al., Distribution and expression of CD200 in the rat respiratory system under normal and endotoxin-induced pathological conditions. J Anat, 2010. 216(3): p. 407-16. 47. Hume, D.A., The mononuclear phagocyte system. Curr Opin Immunol, 2006. 18(1): p. 49-53. 48. Bain, C.C. and A.M. Mowat, CD200 receptor and macrophage function in the intestine. Immunobiology, 2011. 49. Koning, N., et al., Restoring immune suppression in the multiple sclerosis brain. Prog Neurobiol, 2009. 89(4): p. 359-68. 50. Koning, N., et al., Downregulation of macrophage inhibitory molecules in multiple sclerosis lesions. Ann Neurol, 2007. 62(5): p. 504-14. 51. Broderick, C., et al., Constitutive retinal CD200 expression regulates resident microglia and activation state of inflammatory cells during experimental autoimmune uveoretinitis. Am J Pathol, 2002. 161(5): p. 1669-77. 52. Chitnis, T., et al., Elevated neuronal expression of CD200 protects Wlds mice from inflammation-mediated neurodegeneration. Am J Pathol, 2007. 170(5): p. 1695-712. 53. Koning, N., et al., Expression of the inhibitory CD200 receptor is associated with alternative macrophage activation. J Innate Immun, 2010. 2(2): p. 195-200. 84

54. Luo, X.G., et al., Altered regulation of CD200 receptor in monocyte-derived macrophages from individuals with Parkinson's disease. Neurochem Res, 2010. 35(4): p. 540-7. 55. Zhang, S., et al., CD200-CD200R dysfunction exacerbates microglial activation and dopaminergic neurodegeneration in a rat model of Parkinson's disease. J Neuroinflammation, 2011. 8: p. 154. 56. Wang, X.J., et al., Impaired CD200-CD200R-mediated microglia silencing enhances midbrain dopaminergic neurodegeneration: roles of aging, superoxide, NADPH oxidase, and p38 MAPK. Free Radic Biol Med, 2011. 50(9): p. 1094- 106. 57. Williams, R.O., Collagen-induced arthritis as a model for rheumatoid arthritis. Methods Mol Med, 2004. 98: p. 207-16. 58. Simelyte, E., et al., CD200-Fc, a novel antiarthritic biologic agent that targets proinflammatory cytokine expression in the joints of mice with collagen-induced arthritis. Arthritis Rheum, 2008. 58(4): p. 1038-43. 59. Gorczynski, R.M., et al., Anti-CD200R ameliorates collagen-induced arthritis in mice. Clin Immunol, 2002. 104(3): p. 256-64. 60. Afonso, C.L., et al., The genome of swinepox virus. J Virol, 2002. 76(2): p. 783- 90. 61. Foster-Cuevas, M., et al., Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor. J Virol, 2004. 78(14): p. 7667-76. 62. Chung, Y.H., et al., Kaposi's sarcoma-associated herpesvirus OX2 glycoprotein activates myeloid-lineage cells to induce inflammatory cytokine production. J Virol, 2002. 76(10): p. 4688-98. 63. Shiratori, I., et al., Down-regulation of basophil function by human CD200 and human herpesvirus-8 CD200. J Immunol, 2005. 175(7): p. 4441-9. 64. Foster-Cuevas, M., et al., Cytomegalovirus e127 protein interacts with the inhibitory CD200 receptor. J Virol, 2011. 85(12): p. 6055-9. 65. Zhang, J., et al., Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 replication and transcription activator regulates viral and cellular genes via interferon-stimulated response elements. J Virol, 2005. 79(9): p. 5640-52. 66. Zhang, L., et al., Inhibition of macrophage activation by the myxoma virus M141 protein (vCD200). J Virol, 2009. 83(18): p. 9602-7. 67. Langlais, C.L., et al., Rhesus rhadinovirus R15 encodes a functional homologue 85

of human CD200. J Virol, 2006. 80(6): p. 3098-103. 68. McWhirter, J.R., et al., Antibodies selected from combinatorial libraries block a tumor antigen that plays a key role in immunomodulation. Proc Natl Acad Sci U S A, 2006. 103(4): p. 1041-6. 69. Moreaux, J., et al., CD200 is a new prognostic factor in multiple myeloma. Blood, 2006. 108(13): p. 4194-7. 70. Tonks, A., et al., CD200 as a prognostic factor in acute myeloid leukaemia. Leukemia, 2007. 21(3): p. 566-8. 71. Gorczynski, R.M., et al., Nonspecific and specific immunosuppression in tumour- bearing mice by soluble immune complexes. Nature, 1975. 254(5496): p. 141-3. 72. Kretz-Rommel, A. and K.S. Bowdish, Rationale for anti-CD200 immunotherapy in B-CLL and other hematologic malignancies: new concepts in blocking immune suppression. Expert Opin Biol Ther, 2008. 8(1): p. 5-15. 73. Kretz-Rommel, A., et al., Blockade of CD200 in the presence or absence of antibody effector function: implications for anti-CD200 therapy. J Immunol, 2008. 180(2): p. 699-705. 74. Coles, S.J., et al., CD200 expression suppresses natural killer cell function and directly inhibits patient anti-tumor response in acute myeloid leukemia. Leukemia, 2011. 25(5): p. 792-9. 75. Petermann, K.B., et al., CD200 is induced by ERK and is a potential therapeutic target in melanoma. J Clin Invest, 2007. 117(12): p. 3922-9. 76. Siva, A., et al., Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200. Cancer Immunol Immunother, 2008. 57(7): p. 987-96. 77. Gorczynski, R.M., et al., Breast cancer cell CD200 expression regulates immune response to EMT6 tumor cells in mice. Breast Cancer Res Treat, 2010. 123(2): p. 405-15. 78. Gorczynski, R.M., et al., Role of CD200 expression in regulation of metastasis of EMT6 tumor cells in mice. Breast Cancer Res Treat, 2011. 130(1): p. 49-60. 79. Stumpfova, M., et al., The immunosuppressive surface ligand CD200 augments the metastatic capacity of squamous cell carcinoma. Cancer Res, 2010. 70(7): p. 2962-72. 80. Rijkers, E.S., et al., Ligation of CD200R by CD200 is not required for normal murine myelopoiesis. Eur J Haematol, 2007. 79(5): p. 410-6.

81. Alapat, D., et al., Diagnostic usefulness and prognostic impact of CD200 expression in lymphoid malignancies and plasma cell myeloma. Am J Clin Pathol, 2012. 137(1): p. 93-100. 82. Wang, L., et al., Tumor expression of CD200 inhibits IL-10 production by tumor- associated myeloid cells and prevents tumor immune evasion of CTL therapy. Eur J Immunol, 2010. 40(9): p. 2569-79. 83. Talebian, F., et al., Melanoma Cell Expression of CD200 Inhibits Tumor Formation and Lung Metastasis via Inhibition of Myeloid Cell Functions. PLoS One, 2012. 7(2): p. e31442. 84. Schafer, M. and S. Werner, Cancer as an overhealing wound: an old hypothesis revisited. Nat Rev Mol Cell Biol, 2008. 9(8): p. 628-38. 85. Kim, R., M. Emi, and K. Tanabe, Cancer immunoediting from immune surveillance to immune escape. Immunology, 2007. 121(1): p. 1-14. 86. Dunn, G.P., et al., A critical function for type I interferons in cancer immunoediting. Nat Immunol, 2005. 6(7): p. 722-9. 87. Dunn, G.P., L.J. Old, and R.D. Schreiber, The three Es of cancer immunoediting. Annu Rev Immunol, 2004. 22: p. 329-60. 88. Muller-Hubenthal, B., et al., Tumour Biology: tumour-associated inflammation versus antitumor immunity. Anticancer Res, 2009. 29(11): p. 4795-805. 89. Chechlinska, M., M. Kowalewska, and R. Nowak, Systemic inflammation as a confounding factor in cancer biomarker discovery and validation. Nat Rev Cancer, 2010. 10(1): p. 2-3. 90. Van Waes, C., Nuclear factor-kappaB in development, prevention, and therapy of cancer. Clin Cancer Res, 2007. 13(4): p. 1076-82. 91. Aggarwal, B.B., R.V. Vijayalekshmi, and B. Sung, Targeting inflammatory pathways for prevention and therapy of cancer: short-term friend, long-term foe. Clin Cancer Res, 2009. 15(2): p. 425-30. 92. Yu, H., D. Pardoll, and R. Jove, STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer, 2009. 9(11): p. 798-809. 93. Schetter, A.J., N.H. Heegaard, and C.C. Harris, Inflammation and cancer: interweaving microRNA, free radical, cytokine and p53 pathways. Carcinogenesis, 2010. 31(1): p. 37-49. 94. Drake, C.G., E. Jaffee, and D.M. Pardoll, Mechanisms of immune evasion by tumors. Adv Immunol, 2006. 90: p. 51-81.

95. Whiteside, T.L., Immune suppression in cancer: effects on immune cells, mechanisms and future therapeutic intervention. Semin Cancer Biol, 2006. 16(1): p. 3-15. 96. Niehans, G.A., et al., Human lung carcinomas express Fas ligand. Cancer Res, 1997. 57(6): p. 1007-12. 97. Song, E., et al., Soluble Fas ligand released by colon adenocarcinoma cells induces host lymphocyte apoptosis: an active mode of immune evasion in colon cancer. Br J Cancer, 2001. 85(7): p. 1047-54. 98. Leen, A.M., C.M. Rooney, and A.E. Foster, Improving T cell therapy for cancer. Annu Rev Immunol, 2007. 25: p. 243-65. 99. Rabinovich, G.A., D. Gabrilovich, and E.M. Sotomayor, Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol, 2007. 25: p. 267- 96. 100. von Bernstorff, W., et al., Systemic and local immunosuppression in pancreatic cancer patients. Clin Cancer Res, 2001. 7(3 Suppl): p. 925s-932s. 101. Grivennikov, S.I., F.R. Greten, and M. Karin, Immunity, inflammation, and cancer. Cell, 2010. 140(6): p. 883-99. 102. Mantovani, A., et al., Cancer-related inflammation. Nature, 2008. 454(7203): p. 436-44. 103. Bingle, L., N.J. Brown, and C.E. Lewis, The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol, 2002. 196(3): p. 254-65. 104. Steidl, C., et al., Tumor-associated macrophages and survival in classic Hodgkin's lymphoma. N Engl J Med, 2010. 362(10): p. 875-85. 105. Kurahara, H., et al., Indicators of complications and drain removal after pancreatoduodenectomy. J Surg Res, 2011. 170(2): p. e211-6. 106. Movahedi, K., et al., Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes. Cancer Res, 2010. 70(14): p. 5728-39. 107. Ajami, B., et al., Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci, 2007. 10(12): p. 1538-43. 108. Qian, B.Z., et al., CCL2 recruits inflammatory monocytes to facilitate breast- tumour metastasis. Nature, 2011. 475(7355): p. 222-5. 109. Weber, C. and R.R. Koenen, Fine-tuning leukocyte responses: towards a

chemokine 'interactome'. Trends Immunol, 2006. 27(6): p. 268-73. 110. Krausgruber, T., et al., IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses. Nat Immunol, 2011. 12(3): p. 231-8. 111. Martinez, F.O., L. Helming, and S. Gordon, Alternative activation of macrophages: an immunologic functional perspective. Annu Rev Immunol, 2009. 27: p. 451-83. 112. Pollard, J.W., Trophic macrophages in development and disease. Nat Rev Immunol, 2009. 9(4): p. 259-70. 113. Pollard, J.W., Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer, 2004. 4(1): p. 71-8. 114. Talmadge, J.E., M. Donkor, and E. Scholar, Inflammatory cell infiltration of tumors: Jekyll or Hyde. Cancer Metastasis Rev, 2007. 26(3-4): p. 373-400. 115. Condeelis, J. and J.W. Pollard, Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell, 2006. 124(2): p. 263-6. 116. Pollard, J.W., Macrophages define the invasive microenvironment in breast cancer. J Leukoc Biol, 2008. 84(3): p. 623-30. 117. Zeisberger, S.M., et al., Clodronate-liposome-mediated depletion of tumour- associated macrophages: a new and highly effective antiangiogenic therapy approach. Br J Cancer, 2006. 95(3): p. 272-81. 118. Balkwill, F. and A. Mantovani, Inflammation and cancer: back to Virchow? Lancet, 2001. 357(9255): p. 539-45. 119. Bottazzi, B., et al., Regulation of the macrophage content of neoplasms by chemoattractants. Science, 1983. 220(4593): p. 210-2. 120. Leek, R.D., et al., Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res, 1996. 56(20): p. 4625-9. 121. Bingle, L., et al., Macrophages promote angiogenesis in human breast tumour spheroids in vivo. Br J Cancer, 2006. 94(1): p. 101-7. 122. DeNardo, D.G., M. Johansson, and L.M. Coussens, Immune cells as mediators of solid tumor metastasis. Cancer Metastasis Rev, 2008. 27(1): p. 11-8. 123. Hotchkiss, K.A., et al., Mechanisms by which tumor cells and monocytes expressing the angiogenic factor thymidine phosphorylase mediate human endothelial cell migration. Cancer Res, 2003. 63(2): p. 527-33. 124. Zou, W., Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer, 2005. 5(4): p. 263-74. 89

125. Mantovani, A., et al., The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol, 2004. 25(12): p. 677-86. 126. Balkwill, F., Cancer and the chemokine network. Nat Rev Cancer, 2004. 4(7): p. 540-50. 127. Mantovani, A., et al., Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol, 2002. 23(11): p. 549-55. 128. Muller, A.J. and G.C. Prendergast, Indoleamine 2,3-dioxygenase in immune suppression and cancer. Curr Cancer Drug Targets, 2007. 7(1): p. 31-40. 129. Gallina, G., et al., Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells. J Clin Invest, 2006. 116(10): p. 2777-90. 130. Bronte, V., et al., Identification of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progenitor capable of activating or suppressing CD8(+) T cells. Blood, 2000. 96(12): p. 3838-46. 131. Gabrilovich, D.I. and S. Nagaraj, Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol, 2009. 9(3): p. 162-74. 132. Delano, M.J., et al., MyD88-dependent expansion of an immature GR- 1(+)CD11b(+) population induces T cell suppression and Th2 polarization in sepsis. J Exp Med, 2007. 204(6): p. 1463-74. 133. Kerr, E.C., et al., Analysis of retinal cellular infiltrate in experimental autoimmune uveoretinitis reveals multiple regulatory cell populations. J Autoimmun, 2008. 31(4): p. 354-61. 134. Youn, J.I., et al., Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol, 2008. 181(8): p. 5791-802. 135. Srivastava, M.K., et al., Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. Cancer Res, 2010. 70(1): p. 68-77. 136. Sinha, P., et al., Prostaglandin E2 promotes tumor progression by inducing myeloid-derived suppressor cells. Cancer Res, 2007. 67(9): p. 4507-13. 137. Yang, L., et al., Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis. Cancer Cell, 2008. 13(1): p. 23-35. 138. Melani, C., et al., Amino-biphosphonate-mediated MMP-9 inhibition breaks the tumor-bone marrow axis responsible for myeloid-derived suppressor cell expansion and macrophage infiltration in tumor stroma. Cancer Res, 2007. 90

67(23): p. 11438-46. 139. Kusmartsev, S., S. Nagaraj, and D.I. Gabrilovich, Tumor-associated CD8+ T cell tolerance induced by bone marrow-derived immature myeloid cells. J Immunol, 2005. 175(7): p. 4583-92. 140. Nagaraj, S., et al., Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer. Nat Med, 2007. 13(7): p. 828-35. 141. Movahedi, K., et al., Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity. Blood, 2008. 111(8): p. 4233-44. 142. Cobbs, C.S., et al., Inactivation of wild-type p53 protein function by reactive oxygen and nitrogen species in malignant glioma cells. Cancer Res, 2003. 63(24): p. 8670-3. 143. Ha, T.Y., The role of regulatory T cells in cancer. Immune Netw, 2009. 9(6): p. 209-35. 144. Mosser, D.M. and J.P. Edwards, Exploring the full spectrum of macrophage activation. Nat Rev Immunol, 2008. 8(12): p. 958-69. 145. Geissmann, F., et al., Development of monocytes, macrophages, and dendritic cells. Science, 2010. 327(5966): p. 656-61. 146. Andreu, P., et al., FcRgamma activation regulates inflammation-associated squamous carcinogenesis. Cancer Cell, 2010. 17(2): p. 121-34. 147. DeNardo, D.G., et al., CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer Cell, 2009. 16(2): p. 91-102. 148. Watkins, S.K., et al., Rapid release of cytoplasmic IL-15 from tumor-associated macrophages is an initial and critical event in IL-12-initiated tumor regression. Eur J Immunol, 2009. 39(8): p. 2126-35. 149. Mantovani, A., et al., Cancer-promoting tumor-associated macrophages: new vistas and open questions. Eur J Immunol, 2011. 41(9): p. 2522-5. 150. Sasada, T. and S. Suekane, Variation of tumor-infiltrating lymphocytes in human cancers: controversy on clinical significance. Immunotherapy, 2011. 3(10): p. 1235-51. 151. Zamarron, B.F. and W. Chen, Dual roles of immune cells and their factors in cancer development and progression. Int J Biol Sci, 2011. 7(5): p. 651-8. 152. Janssen, E.M., et al., Distinct roles of cytolytic effector molecules for antigen-

restricted killing by CTL in vivo. Immunol Cell Biol, 2010. 88(7): p. 761-5. 153. Stevanovic, S., et al., Human allo-reactive CD4+ T cells as strong mediators of anti-tumor immunity in NOD/scid mice engrafted with human acute lymphoblastic leukemia. Leukemia, 2012. 26(2): p. 312-22. 154. Kennedy, R. and E. Celis, Multiple roles for CD4+ T cells in anti-tumor immune responses. Immunol Rev, 2008. 222: p. 129-44. 155. Junker, N., et al., Tumor associated antigen specific T-cell populations identified in ex vivo expanded TIL cultures. Cell Immunol, 2012. 273(1): p. 1-9. 156. Yee, C., Adoptive therapy using antigen-specific T-cell clones. Cancer J, 2010. 16(4): p. 367-73. 157. Ottobrini, L., et al., In vivo imaging of immune cell trafficking in cancer. Eur J Nucl Med Mol Imaging, 2011. 38(5): p. 949-68. 158. Disis, M.L., H. Bernhard, and E.M. Jaffee, Use of tumour-responsive T cells as cancer treatment. Lancet, 2009. 373(9664): p. 673-83. 159. Berger, C., et al., Adoptive transfer of virus-specific and tumor-specific T cell immunity. Curr Opin Immunol, 2009. 21(2): p. 224-32. 160. Muranski, P. and N.P. Restifo, Adoptive immunotherapy of cancer using CD4(+) T cells. Curr Opin Immunol, 2009. 21(2): p. 200-8. 161. Perez-Diez, A., et al., CD4 cells can be more efficient at tumor rejection than CD8 cells. Blood, 2007. 109(12): p. 5346-54. 162. Wieder, T., et al., T cell-mediated help against tumors. Cell Cycle, 2008. 7(19): p. 2974-7. 163. Qin, F.X., Dynamic behavior and function of Foxp3+ regulatory T cells in tumor bearing host. Cell Mol Immunol, 2009. 6(1): p. 3-13. 164. Shevach, E.M., Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity, 2009. 30(5): p. 636-45. 165. Garin, M.I., et al., Galectin-1: a key effector of regulation mediated by CD4+CD25+ T cells. Blood, 2007. 109(5): p. 2058-65. 166. Bai, X.F., et al., Different lineages of P1A-expressing cancer cells use divergent modes of immune evasion for T-cell adoptive therapy. Cancer Res, 2006. 66(16): p. 8241-9. 167. Zhou, Q., et al., Blockade of programmed death-1 pathway rescues the effector function of tumor-infiltrating T cells and enhances the antitumor efficacy of lentivector immunization. J Immunol, 2010. 185(9): p. 5082-92. 92

168. Bai, X.F., et al., Antigenic drift as a mechanism for tumor evasion of destruction by cytolytic T lymphocytes. J Clin Invest, 2003. 111(10): p. 1487-96. 169. Hatherley, D. and A.N. Barclay, The CD200 and CD200 receptor cell surface proteins interact through their N-terminal immunoglobulin-like domains. Eur J Immunol, 2004. 34(6): p. 1688-94. 170. Clark, D.A., Cell-surface CD200 may predict efficacy of paternal mononuclear leukocyte immunotherapy in treatment of human recurrent pregnancy loss. Am J Reprod Immunol, 2009. 61(1): p. 75-84. 171. Rygiel, T.P., et al., Lack of CD200 enhances pathological T cell responses during influenza infection. J Immunol, 2009. 183(3): p. 1990-6. 172. Rijkers, E.S., et al., The inhibitory CD200R is differentially expressed on human and mouse T and B lymphocytes. Mol Immunol, 2008. 45(4): p. 1126-35. 173. Siva, A., et al., Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200. Cancer Immunol Immunother, 2007. 174. Kretz-Rommel, A., et al., CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy. J Immunol, 2007. 178(9): p. 5595-605. 175. Qian, B.Z. and J.W. Pollard, Macrophage diversity enhances tumor progression and metastasis. Cell, 2010. 141(1): p. 39-51. 176. Lin, E.Y., et al., Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp Med, 2001. 193(6): p. 727-40. 177. Luo, Y., et al., Targeting tumor-associated macrophages as a novel strategy against breast cancer. J Clin Invest, 2006. 116(8): p. 2132-2141. 178. Pekarek, L.A., et al., Inhibition of tumor growth by elimination of granulocytes. J Exp Med, 1995. 181(1): p. 435-40. 179. Grimwade, D., et al., The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties. Blood, 1998. 92(7): p. 2322-33. 180. Ahn, G.O., et al., Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment. Proc Natl Acad Sci U S A, 2010. 107(18): p. 8363-8. 181. Moreaux, J., et al., CD200: a putative therapeutic target in cancer. Biochem Biophys Res Commun, 2008. 366(1): p. 117-22. 93

182. Brunetti, L., et al., CD200/OX2, a cell surface molecule with immuno-regulatory function, is consistently expressed on hairy cell leukaemia neoplastic cells. Br J Haematol, 2009. 145(5): p. 665-7. 183. Dorfman, D.M. and A. Shahsafaei, CD200 (OX-2 membrane glycoprotein) expression in b cell-derived neoplasms. Am J Clin Pathol, 2010. 134(5): p. 726- 33. 184. Dorfman, D.M. and A. Shahsafaei, CD200 (OX-2 membrane glycoprotein) is expressed by follicular T helper cells and in angioimmunoblastic T-cell lymphoma. Am J Surg Pathol, 2011. 35(1): p. 76-83. 185. Olteanu, H., et al., CD200 expression in plasma cell myeloma. Br J Haematol, 2011. 153(3): p. 408-11. 186. El Desoukey, N.A., et al., CD200 expression in B-cell chronic lymphoproliferative disorders. J Investig Med, 2012. 60(1): p. 56-61. 187. Pallasch, C.P., et al., Disruption of T cell suppression in chronic lymphocytic leukemia by CD200 blockade. Leuk Res, 2009. 33(3): p. 460-4. 188. Rawstron, A.C., et al., Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders. Haematologica, 2008. 93(3): p. 431-8. 189. Palumbo, G.A., et al., CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia. Leuk Res, 2009. 33(9): p. 1212-6.