CD200: A NOVEL THERAPEUTIC TARGET FOR CHRONIC LYMPHOCYTIC LEUKEMIA

by

Karrie Ka Wai Wong

A thesis submitted in conformity with the requirements for the degree of PhD Institute of Medical Science University of Toronto

© Copyright by Karrie Wong 2012

CD200: a novel therapeutic target for Chronic Lymphocytic Leukemia

Karrie Ka Wai Wong

Doctor of Philosophy

Institute of Medical Science University of Toronto

2012

Abstract

The ability of cancer cells to escape anti-tumor immune responses is acknowledged as one of the hallmarks of cancer. Overexpression of immunoregulatory molecules is one mechanism responsible for the immunsuppressive network that is characteristic of the tumor microenvironment.

In this thesis, we investigated the role of CD200, a potent immunoregulatory molecule, in Chronic Lymphocytic Leukemia. We showed that functional blockade of

CD200 on lymphoma cells or primary CLL cells, both of which express CD200 at high levels, augmented cytotoxic killing of these cells by effector CD8 + T cells in vitro . We

also identified and characterized a previously unrecognized soluble form of CD200,

sCD200, present in elevated levels in CLL plasma when compared to plasma from

controls.

The data reported show that patients with high sCD200 levels have more

aggressive disease, inferring that sCD200 may be a novel prognostic marker for CLL.

The in vivo function of sCD200 was investigated for its ability to support engraftment of

CLL splenocytes in NOD.SCID mice. Infusion of sCD200 hi CLL plasma, but not sCD200 lo normal plasma, enhanced engraftment of CLL-splenocytes in vivo , an effect

ii which was abrogated by depletion of sCD200 from CLL plasma. The prolonged engraftment of CLL cells seen in this model (>6 months) suggests these mice represent a useful pre-clinical model for drug screening. The effect of CD200 blockade was tested in this model, and was found to be as effective in eliminating engrafted CLL cells as rituximab. Investigation of the mechanisms leading to the release of sCD200 from CLL cells showed that sCD200 was produced following ectodomain shedding by ADAM proteases and MMPs.

Results from studies reported in this thesis support the hypothesis that CD200

plays a major role in CLL biology, and suggests it may represent a novel therapeutic

target for CLL.

iii Dedication

To my parents, Chi Ping and Yuk Lin, and my husband, David, for their unconditional love and patience

iv Acknowledgments

The journey from my first day in the lab to now the completion of this thesis has been an exciting ride with the participation of many individuals. I would like to take this opportunity to extend my gratitude and appreciation. To my supervisor and mentor Dr. Reg Gorczynski: I’m eternally grateful for your patience, support, and guidance, both in terms of mentorship and actual technical help. Thank you for believing in me, encouraging me to explore my own ideas, and giving me the freedom to work independently. Your great sense of humor, wisdom, and passion for science has provided me a positive “microenvironment” from where I have been able to thrive as a scientist and a person in the past 5 years. To Dr. David Spaner, who has provided all the clinical materials for this project and whose scientific inputs and suggestions have been instrumental for this work: thank you for your encouragements and optimism. I have learned from you tremendously; your interesting ideas and unyielding enthusiasm for science have been a source of inspiration. To Dr. Andras Kapus and Dr. Li Zhang, my committee members whose helpful comments and suggestions have made this work better: I’m truly grateful for your participation at the PAC meetings and contribution to my work. Thank you for being kind and accommodating. To members of the Gorczynski and Cattrel lab, past and present: thank you for your company and friendship. In particular, to Dr. Ismat Khatri, our indispensible lab manager who has generated the two major rabbit polyclonal antibodies used in studies reported in chapter 4: thank you for making my life in the lab easy in general, and for being there with your listening ears in our morning coffee sessions which have allowed me a fresh start to each day in the past 5 years. To Dr. Jun Diao: thank you for being generous and never saying no whenever I ask to borrow your reagents. I’d also like to extend a special thank you to my summer student Qiang Huo, who has assisted me in some of the experiments reported in chapter 4, and who has tolerated my impatience at times. A tremendous thank you also extends to members of the Spaner lab, particularly Suchinta Shaha and Yonghong Shi, who have prepared the CLL cells and CLL plasma impeccably and maneuvered the Sunnybrook shuttle bus schedule to deliver me the samples each and every time. I’m also grateful for the CIHR-Training Program in Regenerative Medicine for funding my graduate studies and for my department the Institute of Medical Science for all their support. On a personal note, I’d like to thank my parents, who have taught me hard work and perserverance: mom and dad, I hope I have made you proud. To my brother and sister, Erik and Alicia, thank you for your support. Last, but most certainly not least, this work would not have been possible without the unconditional support and love from my husband, David, who has been my rock through thick and thin from the beginning. Thank you for tolerating me on my bad days, sharing in my excitment, traveling to places with me that you wouldn’t otherwise go, and most importantly, for being my IT expert who has solved all my computer issues.

v Table of Contents

Dedication ...... iv

Acknowledgments...... v

Table of Contents ...... vi

List of Tables ...... ix

List of Figures ...... x

List of abbreviations ...... xii

List of CD antigens ...... xvi

Chapter 1: Introduction and literature overview ...... 1

1.1 Chronic Lymphocytic Leukemia ...... 2

1.1.1 Clinical features ...... 2

1.1.2 Biology of CLL cells ...... 20

1.1.3 CLL microenvironment ...... 27

1.1.4 Animal models of CLL ...... 36

1.1.5 Immunotherapy for CLL ...... 40

1.2 CD200 ...... 44

1.2.1 Immunoregulatory molecules in the evasion of tumor immunosurveillance ...... 44

1.2.2 The CD200:CD200R axis ...... 46

1.2.3 CD200 in cancer ...... 47

1.3 Ectodomain shedding ...... 49

1.3.1 The ADAM proteases ...... 50

1.3.2 Regulation of ADAM proteases...... 50

1.3.3 ADAM proteases in CLL ...... 53

1.4 Objectives and hypotheses ...... 55

Chapter 2: The role of CD200 in immunity to B cell lymphoma ...... 58

vi 2.1 Abstract ...... 59

2.2 Introduction ...... 60

2.3 Materials and Methods:...... 62

2.4 Results: ...... 68

2.5 Discussion: ...... 75

2.6 Tables ...... 82

2.7 Figure legends ...... 84

2.8 Figures...... 88

Chapter 3: Soluble CD200 supports in vivo survival of CLL ...... 103

3.1 Abstract ...... 104

3.2 Introduction ...... 105

3.3 Materials and Methods ...... 107

3.4 Results ...... 113

3.5 Discussion ...... 120

3.6 Tables ...... 124

3.7 Figure legends ...... 128

3.8 Figures...... 132

Chapter 4: Ectodomain shedding of CD200 ...... 146

4.1 Abstract ...... 147

4.2 Introduction ...... 148

4.3 Materials and Method ...... 150

4.4 Results ...... 157

4.5 Discussion ...... 165

4.6 Table ...... 171

4.7 Figure legends ...... 172

4.8 Figures...... 176

vii Chapter 5: General discussion ...... 196

5.1 General discussion ...... 197

5.1.1 sCD200 as a prognostic marker in CLL ...... 198

5.1.2 A novel xenograft model for CLL which utilizes sCD200 ...... 199

5.1.3 The role of CD200:CD200R axis in the CLL microenvironment ...... 201

5.1.4 Ectodomain shedding of CD200 ...... 205

5.2 Future directions ...... 206

5.2.1 The role of CD200R+ cells and T cells the in CLL microenvironment ...... 206

5.2.2 The effects of CD200 blockade on T cells...... 207

5.2.3 The Applicability of the xenograft model described in testing novel therapeutics for CLL ...... 208

5.3 Concluding remarks ...... 209

Chapter 6: References ...... 210

viii List of Tables

Table 1.1: Major prognostic markers in CLL ...... 19

Table 2.1: Clinical characteristics of patients used in the study ...... 82

Table 3.1: Clinical characteristics of patients in plasma sCD200 analyses ...... 124

Table 4.1: Correlation between patient plasma sCD200 and sCD200 in corresponding CLL supernatants ...... 171

ix List of Figures

Figure 1-1: CLL pathogenesis model proposed by Kikushige et al (121) ...... 21

Figure 1-2: Cellular components of CLL proliferation center ...... 28

Figure 1-3: Molecular crosstalks between CLL cell and the cellular components in the CLL microenvironment (see section 1.1.3a-f) ...... 29

Figure 1-4: Domain structure of ADAM protease ...... 51

Figure 1-5: Potential role of CD200/sCD200 in the CLL microenvironment ...... 56

Figure 2-1: Expression of CD200 on primary CLL cells and 2 CLL cell lines ...... 88

Figure 2-2: Effect of anti-CD200 mAb 1B9 on induction of CTL by lymphoma cells.... 90

Figure 2-3: Silencing of CD200 expression by specific oligodeoxynucleotides ...... 93

Figure 2-4: The effect of CD200 silencing in the killing of Ly5 cells ...... 94

Figure 2-5: Modulation of cytokine production in MLCs using anti-CD200 mAb or CD200-specific siRNAs...... 95

Figure 2-6: CD200 blockade augments killing of primary CLL cells by allogenic effector PBLs and CD200R expression on CLL-splenocytes ...... 97

Figure 2-7: Expression of CD200 on CLL cells in response to stimulation by PMA, Imiquimod, and IL2 ...... 102

Figure 3-1: Identification of sCD200 and clinical analysis of plasma sCD200 in CLL . 132

Figure 3-2: Characterization of CLL splenocytes harvested from splenectomised patients ...... 135

Figure 3-3: Engraftment of human CLL cells and T cells in NOD.SCID γcnull mice ..... 136

x Figure 3-4: Effects of sCD200 and/or T cell depletion on CLL engraftment in NOD.SCID γcnull mice at day 21 ...... 141

Figure 3-5: Therapeutic efficacy of Rituxan and 1B9 on CLL engraftment in NOD.SCID γcnull mice ...... 143

Figure 4-1: CD200 is constitutively released from CLL cells ...... 176

Figure 4-2: sCD200 is secreted from CLL cells in response to different stimuli ...... 179

Figure 4-3: PMA-induced CD200 shedding is reflected as a loss of CD200 expression from the surface of CLL cells ...... 182

Figure 4-4: Detection of CD200 in membrane extracts from PMA-treated CLL ...... 186

Figure 4-5: Spontaneous and inducible shedding of CD200 in Hek-hCD200 cells ...... 189

Figure 4-6: Absence of epitopes associated with the cytoplasmic domain of CD200 in sCD200 and functional properties of sCD200 ...... 192

Figure 5-1: The in vivo effects of sCD200 on T cell engraftment...... 203

Figure 5-2: Proposed model of CD200:CD200R mediated immunosuppression in the CLL microenvironment ...... 204

xi List of abbreviations

7AAD 7-Amino-actinomycin D ADAM A disintegrin and metalloproteinase ADCC Antigen dependent cellular cytotoxicity AID Activation-enduced (cytidine) deaminase AIHA Autoimmune hemolytic anemia ALC Absolute lymphocyte count ALL Acute lymphoid leukemia AML Acute myeloid leukemia APRIL A proliferation-inducing ligand B7-H1 B7-homolog 1 (PD-L1 or CD274) BAFF B cell-activating factor belonging to the tumor necrosis factor family Bcl6 B-cell lymphoma 6 BCMA B-cell maturation antigen BCR B-cell receptor BTLA B and T lymphocyte attenuator (CD272) CCL C-C motif chemokine ligand CCR7 C-C chemokine receptor CD Cluster of differentiation CDC Complement dependent cytotoxicity CDR Complementarity determining region CIA Collagen-induced arthritis CLL Chronic lymphocyte leukemia CMV Cytomegalovirus CSR Class switch recombination CTL Cytotoxic lymphocyte CTLA-4 Cytotoxic T-lymphocyte antigen 4 (CD152) CXCL C-X-C motif chemokine ligand

xii CXCR4 C-X-C chemokine receptor EAE Experimental autoimmune encephalomyelitis EBV Epstein-Barr virus EGR-1 Early growth response protein 1 ELISA Enzyme-linked immunosorbent assay ERK Extracellular signal-regulated kinase FACS Fluorescence-activated cell sorting Fc γRIIb Fc gamma receptor IIb FDC Follicular dendritic cell Foxp3 Forkhead box P3 G418 Geneticin GAPDH Glyceraldehyde 3-phosphate dehydrogenase GM6001 Galardin GVHD Graft versus host disease CD200v+c Extracellular domain of CD200 HSC Hematopoietic stem cell HVEM Herpesvirus entry mediator I.P. Immunoprecipitation IFN Interferon IgH Immunoglobulin heavy chain IgV Immunoglobulin variable region IgVH Immunoglobulin heavy chain variable region ip Intraperitoneal ITAM An immunoreceptor tyrosine-based activation motif ITIM An immunoreceptor tyrosine-based inhibitory motif iv Intravenous LDT Lymphocyte doubling time LPS Lipopolysaccharide (endotoxin) MAPK Mitogen-activated protein kinase

xiii MBL Monoclonal B-cell lymphocytosis Mcl-1 Myeloid cell leukemia protein M-CLL CLL with mutated IgVH MDR Minimal deletion region MHC Major histocompatibility complex MLC Mix lymphocyte culture MMP Matrix metalloprotease MSC Mesenchymal stromal cell MT-MMP Membrane-type matrix metalloprotease MyD88 Myeloid differentiation primary response gene (88) NFAT Nuclear factor of activated T-cells NF κB Nuclear factor kappa-light-chain-enhancer of activated B cells NLC Nurse-like cell NOD Non-obese diabetic NOD.SCID γcnull IL2-receptor γ-chain allelic mutation in NOD.SCID background P2RX7 P2X purinoceptor 7 PBL Peripheral blood lymphocyte PBMC Periphreal blood mononuclear cell PCR Polymerase chain reaction PD-1 Programmed death-1 PDGF Platelet-derived growth factor PECAM1 Platelet endothelial cell adhesion molecule (CD31) PHA Phytohemagglutinin PKC Protein kinase C PMA Phorbol 12-myristate 13-acetate Pt Patient SCID Severe combined immunodeficiency sCD200 Soluble CD200 SDF-1 Stromal cell-derived factor-1 (CXCL12)

xiv SHM Somatic hypermutation siRNA Small interfering RNA STAT Signal transducer and activator of transcription Syk Spleen tyrosine kinase TAA Tumor-associated antigen TACI Transmembrane activator and CAML interactor TAPI-0 TNF-α Protease Inhibitor-0 TBP TATA-binding protein Tcl1 T cell leukemia/lymphoma 1 TCR T cell receptor TGF Transforming growth factor TH T-helper TIMP Tissue inhibitor of metalloproteases TLR Toll-like receptor TNF Tumor-necrosis factor U-CLL CLL with unmutated IgVH VEGF Vascular endothelial growth factor Zap70 Zeta-chain (TCR)-associated protein kinase 70 β2-M Beta-2 microglobulin

xv List of CD antigens

CD antigens relevent for this thesis

Cellular expression Function

CD3 Mature T, different levels on T activation; regulates TCR thymocytes expression CD4 Thymocyte subsets; T helper, Treg; amplifies TCR signals; HIV entry monocytes/macrophages, DC CD5 Subtypes of B, B-CLL; T T-B interaction; T activation

CD8 Thymocyte subsets; cytotoxic T, Co-receptor for MHC class I NK, DC subsets CD14 Monocytes, macrophages, receptor for LPS and LBP Langerhan CD19 B; FDC BCR co-receptor; signaling

CD20 B; subsets of T B activation and proliferation

CD22 B adhesion; inhibitory receptor for BCR

CD23 B (upon activation); activated low-affinity receptor for IgE macrophages CD25 Activated T, B, and monocytes; IL2R α chain subsets of T CD31 T subsets; monocytes, endothelial PECAM-1; adhesion; CD38 ligand cells CD38 Variable levels on hematopoietic ADP-ribosyl cyclase; activation, cells proliferation, adhesion CD40 B, monocytes/macrophages, DC co-stimulation; B-differentiation and isotype switching CD44 Hematopoietic and non- leukocyte rolling, homing, and hematopoietic cells adhesion CD45 Hematopoietic cells except Leukocyte common antigen, erythrocytes and plasma cells activation CD49d T, B, NK, DC, monocytes, mast α4-integrin; subunit of VLA-4 cells receptor; adhesion, migration, homing CD52 Mature T and B; DC, monocytes, Co-stimulation; molecular target of mast cells Alemtuzumab

xvi CD56 NK, NKT, T subsets NCAM; cell-cell adhesion

CD62L B, T subsets, NK, monocytes L-selectin; leukocyte rolling and homing CD69 Activated leukocytes; NK, co-stimulation; signaling Langerhan CD71 Proliferating cells; reticulocytes; transferin receptor; iron uptake erythroid precurors CD79b B Ig β; subunit of BCR; signaling

CD80 Activated B and T; DC, receptor for CD28 and CD153; co- marcophages stimulation CD83 Activated B and T; mature DC, co-stimulation Langerhans CD86 Activated B and T; DC, monocytes, co-stimulation of T activation and endothelial cells proliferation CD100 Leukocytes migration, T and B activation, angiogenesis CD152 Activated T and B CTLA-4; immunoregulation

CD154 Activated T and monocytes CD40L; co-stimulation

xvii

Chapter 1: Introduction and literature overview

1 This thesis describes studies designed to investigate the role of CD200, an immunoregulatory molecule, in chronic lymphocytic leukemia (CLL). The major topics in CLL relevant for this thesis will be discussed in section 1.1 of this chapter. Literature on CD200 and its role in cancer immunology will be reviewed in section 1.2. Section 1.3 provides a brief overview on ectodomain shedding, a mechanism that is relevant to the release of a functional soluble form of CD200. Lastly, the overall objectives and hypotheses of this study will be discussed in section 1.4.

1.1 Chronic Lymphocytic Leukemia

CLL is the most common adult leukemia in the western world, accounting for 9% of all cancers and 30% of leukemia. The disease is characterized by the accumulation of small, monoclonal B lymphocytes exhibiting mature, antigen-experienced, “anergic” phenotypes in peripheral blood, bone marrow, spleen, and secondary lymphoid organs.

1.1.1 Clinical features

1.1.1.1 Diagnosis and clinical characteristics

According to the 2008 guidelines from WHO and the International Workshop on

Chronic Lymphocytic Leukemia (IWCLL), CLL is diagnosed when a patient presents with ≥ 5x10 9L-1 monoclonal, CD5 + B cells that co-express CD19 and CD23 in peripheral

blood (1). Cell surface markers CD25, CD69, and CD71, which tend to be up-regulated,

and CD22, Fc γRIIb, CD79d, and cell-surface IgD, which are often down-regulated, are

used as further criteria to distinguish CLL from other B cell-malignancies.

In the absence of extramedullary tissue involvement, the clonal expansion of B

cells persists for a minimum of 3 months (2). The requirement of ≥ 5x10 9L-1 monoclonal

2 B cells in circulation distinguishes CLL from the asymptomatic haematological condition known as monoclonal B-cell lymphocytosis (MBL), where monoclonal or oligoclonal populations of CD5 + cells, present often with CLL-like cell surface phenotypes and even with chromosomal abnormalities and other biological properties associated with CLL, are detected in the peripheral blood of otherwise healthy individuals (3, 4). The presence of these B cell clones has been found in a vast majority of patients prior to CLL diagnosis, and may represent an early marker for CLL (5). In a prospective study in which 185 subjects were diagnosed with MBL and were followed subsequently for a median of 6.7 years, 15% were reported to develop progressive CLL (4). The molecular mechanisms dictating the MBL to CLL transition remain elusive.

A large proportion of CLL patients are asymptomatic with diagnosis typically made following routine blood tests. One of the most common manifestations of the disease is repeated infections due to hypogammaglobulinemia, which occurs in 60% of cases (6). Autoimmune-associated phenomena directed against hematopoietic cells are common in CLL and may be present at diagnosis (7). Presentation of autoimmune hemolytic anemia (AIHA), the most common autoimmune cytopenia in CLL, at diagnosis is associated with older age, the male gender, and a higher lymphocyte count

(8).

CLL is a heterogeneous disease with either an aggressive or indolent disease course. Patient survival ranges from months in the former to decades post-diagnosis in the latter. Patients with indolent disease have a favorable clinical course and typically die with the disease rather than from it, with minimal requirement for medical intervention.

In contrast, patients with aggressive disease tend to progress quickly with onset of

3 splenomegaly, lymphadenopathy, and development of AIHA and autoimmune thrombocytopenia. Median survival for these patients is 18 months to 3 years (9).

Currently, despite advances in therapeutic approaches to CLL, CLL largely remains an incurable disease.

Clinical presentations at diagnosis are insufficient predictors of disease course, as some patients with early disease at diagnosis could progress and succumb to disease within a short period of time. A meta-analysis of clinical trials investigating the efficacy of early interventions using conventional chemotherapeutic agents for all early disease patients showed no additional benefit on survival, and supported a “watch and wait” approach to CLL, with no treatment given until symptoms appear (10, 11). However, these earlier studies failed to address the potential benefits of early intervention for the subgroup of patients at high risk of developing aggressive disease. Recently, with advances both in the development of combinational therapeutic approaches and the use of prognostic markers to separate high-risk patients at diagnosis, several clinical studies have shown that early therapeutic intervention is indeed beneficial and improved overall survival in this subgroup of patients (12, 13).

The key to successful early intervention is thus to separate effectively patients with high-risk of developing aggressive disease from those with low-risk at diagnosis, thus delineating these high-risk patients which may benefit from aggressive treatment regimen to control disease progression while avoiding potential unnecessary therapeutic side effects for low-risk patients. The use of prognostic markers to predict disease progression is crucial in this respect.

4 Despite advances in stratifying patients, the use of prognostic markers for prediction of time to first treatment, response to treatment, and complete remission requires further research (14). Identification of new prognostic markers will undoubtedly aid both in deciding the treatment regimen as well as when treatment should begin, thus improving treatment outcome.

The following section discusses some of the more important clinical and biological prognostic markers for CLL against which the value of new markers can be compared. Understanding the role of prognostic markers in CLL progression helps provide insights into mechanisms driving CLL leukemogenesis.

1.1.1.2 Prognostic factors i) Staging systems

The Rai and Binet staging systems, developed more than 3 decades ago, were the first prognostic factors used clinically for assessment and prediction of disease progression (15, 16). Staging in both systems is determined based on physical examination and standard laboratory testing, and is easily applied in clinics.

The Rai staging system uses 5 stages (stage 0, I, II, III, and IV), which are further simplified to 3 groups: low risk (stage 0), intermediate risk (stage I and II), and high risk

(stage III and IV) (17). Assessment is made according to the presence of lymphocytosis, anemia, and thrombocytopenia, as well as the presence or absence of lymphadenopathy or splenomegaly. Patients with lymphocytosis in the peripheral blood or bone marrow

(>30% lymphoid cells) but no other clinical signs are considered to have low risk disease

(stage 0). Patients with lymphocytosis, enlarged lymph nodes, and/or splenomegaly or

5 heptomegaly are defined as having intermediate-risk disease (stage I-II). Patients with disease-related anemia (hemoglobin level <110g/L; stage III disease) or thrombocytopenia (platelet count <100x10 9/L; stage IV disease), with or without

lymphadenopathy or splenomegaly, are included in the high risk disease group (17).

The Binet staging system uses 3 stages: A, B, and C; staging is based on the

number of sites involved with CLL, as defined by lymph nodes >1cm in diameter or

organomegaly, in addition to presence or absence of anemia and/or thrombocytopenia

(16). The areas of involvement considered include head and neck, axilla, groin, palpable

spleen, and liver. Like the Rai staging system, the three Binet stages reflect low (A),

intermediate (B), and high risk disease (C), with high risk patients defined as having

anemia and/or thrombocytopenia, independent of the number of sites involved.

Both the Rai and Binet staging systems are widely applied in clinics worldwide,

with the former more commonly practiced in North America and the latter in Europe.

The staging systems are instrumental in clinical decision making particularly regarding

treatment, which is typically not recommended for patients in the low-risk group (1).

Regardless of the staging system used, the low-risk group (Rai stage 0 or Binet stage A)

generally comprise up to 80% of newly diagnosed patients (18). However, neither

staging systems is adequate to identify the patients within this subgroup that might

proceed to aggressive disease; nor does disease stage alone predict other important

parameters such as time to first treatment and response to treatment (19). Determination

of disease stage at diagnosis remains important, nevertheless, and is one of the criteria for

patient selection for clinical trials (1). Recently, a number of studies have concluded that

disease stage, together with analyses of a combination of several other important

6 prognostic factors (to be discussed below), is the most effective way to predict high-risk patients at an early stage (20-23). ii) Biological prognostic markers

Morphology

The morphology of CLL cells in circulation as determined in a blood smear, or in the bone marrow, is an important prognostic factor for CLL. Atypical, prolymphocytic morphology, mainly large-cell size and cleaved nucleus, is associated with poor prognosis, whereas granular and small-sized lymphocytes predicts good disease outcome

(24). In an analysis of CLL cells from 270 patients at Binet stage A, atypical morphology of CLL cells was shown to predict adverse disease (25, 26).

Absolute Lymphocyte count and lymphocyte Doubling Time (LDT)

Absolute lymphocyte count (ALC) reflects disease burden. In an earlier study,

ALC at presentation was found to be an independent prognostic factor in a univariate analysis with patients having a count of >50x10 9 having worse prognosis than patients

with lower counts (27). In a more recent multivariate analysis of 2146 patients over 20

years, an ALC of >30x10 9 was identified to be an independent predictor of shorter survival (28). Furthermore, in a prospective study conducted by Letestu et al to validate prognostic strength of routine parameters in 339 Binet stage-A patients, lymphocytosis emerged as one of the 4 independent prognostic factors predicting survival for early stage patients (29).

LDT is, by definition, the period of time during which the absolute count of lymphocytes is doubled, and is an indicator of disease activity. The prognostic

7 significance of LDT has been shown in a number of studies, in that a LDT <12 months is associated with poor prognosis and survival, while a LDT >12 months is correlated with favorable disease course and survival (30-33). For early stage patients, LDT was shown to be a useful prognostic marker to predict time to first treatment and overall survival

(23). The 2008 guidelines from IWCLL suggests a LDT of less than 6 months in early stage patients can be used as an indicator for treatment, particularly in patients with ALC

>30x10 9 (1).

IgV mutational status

Diversity and antigen binding specificity of the B-cell receptor (BCR) results from random recombination events of the variable (V), diversity (D), and joining (J) gene segments of the immunoglobulin heavy chain (IgH) and the variable (V) and joining (J) gene segments of the immunoglobulin light chains (34). Upon antigen binding to the

BCR with the adequate specificity and avidity, an immature B cell enters the germinal center in a lymphoid follicle where it rapidly undergoes proliferation and its V genes undergo somatic hypermutation. Somatic hypermutation results in introduction of further mutations into the rearranged VDJ and VJ genes to create a BCR with distinct properties from its original counterpart. Post-somatic hypermutation, cells that have acquired receptors with enhanced antigen binding affinity are selected to survive and further differentiate into a mature phenotype (35, 36). This process generally requires help by T cells, including engagement of T cell-CD40L with CD40 expressed on B cells (37).

Somatic hypermutation can also proceed independent of T cells and in marginal zones

(38-40)

8 In CLL, the mutation status of IgVH genes is one of the strongest predictors of disease progression. The pattern and distribution of IgVH mutations in CLL was shown to be consistent with the canonical somatic hypermutation process involving activation- induced cytidine deaminase (AID) (41, 42). In two independent landmark studies, patients whose CLL cells expressed mutated IgVH (M-CLL) were shown to have favorable disease course, while patients whose cells expressed unmutated IgVH (U-CLL) showed poor response to treatment and had shorter overall survival rate (43, 44). In a multivariate analysis of 205 patients, in which a >98% homology to the germline sequence was used as a cutoff, unmutated IgVH, along with loss of p53, emerged as a prognostic indicator independent of all other prognostic factors tested (45).

Despite the strong prognostic value of IgVH mutation status, most clinical diagnostic laboratories are not equipped to routinely perform IgVH mutation analysis, which is time consuming and expensive. Thus, IgVH mutation analysis has not been incorporated into routine diagnostic testing.

Zap70 expression

Microarray analyses have shown differential expression of over 300 genes between the M-CLL and U-CLL, providing a rationale for the search for surrogate markers for IgVH mutation status to circumvent the difficulty in performing routine

IgVH mutation analysis (46). Zap70, an intracellular protein that is normally expressed on T and NK cells in association with the antigen receptor, but not on normal B cells, was found to be overexpressed in U-CLL at the mRNA level (46). Flow cytometry analyses of CLL cells subsequently showed that Zap70 was expressed in U-CLL at the same level as in T cells (47).

9 In a clinical study using a cutoff of >20% Zap70 + cells as determined by flow cytometry, 100% of patients with Zap70 expression above the cutoff were found to have unmutated IgVH, while 87.5% of patients with low Zap70 expression had mutated IgVH

(48). Generally, depending on adjustments of the cutoff, the concordance rate between

Zap70 expression and IgVH mutation status ranges from 77-95% (49). Moreover, similar to IgVH mutation status, Zap70 expression correlated significantly with time to first treatment (50). In a follow-up analysis with a larger sample size, Zap70 expression was shown to be a superior indicator for treatment than IgVH mutation status (51). The contribution of Zap70 expression to CLL leukemogenesis is linked to the ability of Zap70 to directly enhance signaling through BCR, a topic to be elaborated in section 1.1.2b.

CD38

CD38 is a cell surface glycoprotein normally found on T cells, early B-cell progenitors in bone marrow, activated B cells in germinal centers, and plasma cells (52).

CD38 is absent on naïve and mature B cells in peripheral blood (53). In B cells, CD38 is associated with the B-cell co-receptor complex CD19/CD81, the chemokine receptor

CXCR4, and adhesion molecules such as CD49d in lipid rafts on the cell surface (54).

Using a >30% cutoff to define “high” CD38 expression, patients with U-CLL were found to have high CD38, while patients with M-CLL expressed low levels of CD38 (43).

However, unlike Zap70, CD38 expression and IgHV mutation status are not directly linked, and CD38 expression was unable to predict the two IgVH subgroups correctly in two independent studies (55, 56). Nevertheless, in both studies, CD38 expression was found to be a risk factor that predicted clinical outcome independent of IgVH mutation status.

10 When compared with other prognostic factors, CD38 expression was found to be an independent prognostic factor associated with aggressive disease in a multivariate analysis using 20% as a CD38 expression cutoff (57). Moreover, CD38 expression also identified a subgroup of high risk patients at early disease stage but with progressive clinical course. In a separate analysis where a 30% cutoff was used, CD38 expression was found to predict response to fludarabine and overall survival (58). In this study, the predictive value of CD38 was maintained in a multivariate analysis within the Rai intermediate risk group.

It is important to note that CD38 level may vary over time and during disease progression (56, 57, 59). CD38 expression on CLL cells is also modulated through interactions with non-malignant cells (60). Analysis of markers on CD38 + CLL cells

showed that CD38 expression was associated with an “activated” phenotype and may

represent a more recently activated population of CLL cells (61). This observation is

further supported by reports that CLL cells in peripheral lymphoid tissues and bone

marrow, where CLL cells likely encounter antigens and other activation signals, tend to

express higher levels of CD38 than CLL cells (from the same patient) in the peripheral

blood (62).

Due to this biological feature of CD38, the optimal cutoff value for CD38 for

disease prediction remains controversial. Although a 20-30% cutoff had been used in the

majority of clinical studies, Ghia et al showed that the presence of a subpopulation of

CD38 + CLL cells, regardless of frequency, in patients with levels below an arbitrary

cutoff point was associated with autoimmune manifestations and poor prognosis (63).

11 Indeed, several studies have demonstrated that a cutoff point of 5-7% was more effective in distinguishing the different prognostic groups (64, 65).

CD38 expression has also been identified as a risk factor for the development of

high-grade non-Hodgkin’s lymphoma in CLL patients (Richter’s transformation), and is

the only risk factor shared between Richter’s transformation and CLL progression (66).

Besides expression on the cell surface, a recent study by Aydin et al identified CD38

gene polymorphism, characterized by a C>G variation in intron 1 of the gene, as a

predictor for Ritcher’s transformation.

Recently, CD38 and Zap70 were shown to be functionally linked with CD38

ligation leading to Zap70 phosphorylation (67). In addition, CD38 +Zap70 + CLL cells

were shown to have enhanced migration in response to stromal derived factor-1α (SDF-

1α). Given this functional link between CD38 and Zap70, combined analysis of CD38 and Zap70 expression may enhance identification of high-risk subgroups. In an analysis of 242 patients, the CD38 +Zap70 + subgroup was shown to have shorter overall survival

(30 months) compared with CD38 -Zap70 - patients (130 months) (68). Survival for patients with discordant CD38 and Zap70 expression was found to be 40 months, reflecting intermediate disease progression.

The function of CD38 in CLL biology appears to be multifaceted, with roles in both CLL proliferation and chemotaxis (section 1.1.3).

Cytogenetics

Genomic aberrations can be identified in ~80% of cases by fluorescence in-situ hybridization (FISH) with a disease-specific probe (69). Five major classes of

12 cytogenetic categories that have prognostic significance were identified by Döhner in a seminal study: deletions in 13q, 11q, 17p regions, trisomy 12, and normal karyotype (70).

In this study, trisomy 12, normal karyotype, and 13q deletion were found to be associated with median survival times of 114, 111, and 133 months, respectively, in comparison to the 32 and 79 months median survival associated with 17p and 11q deletions. Of all the cytogenetic lesions reported, 13q deletions are the most common, accounting for cytogenetic aberrations in over 50% patients. Cytogenetic aberrations are not directly correlated with IgVH mutation status, although patients with 13q deletions tend to be overrepresented in the mutated IgVH subgroup, while patients with 11q or 17p deletions tend to have unmutated IgVH; patients with trisomy 12, on the other hand, are distributed equally in both IgVH subgroups (64). Currently, there is increasing evidence that cytogenetic analysis may represent one of the most relevant predictors of treatment outcome, with prognostic value independent of IgVH, and Zap70 and CD38 expression

(1, 49). a) 13q aberration:

Deletions in 13q14 are considered a favorable prognostic marker predicting relatively benign disease course. Patients with 13q14 deletions were reported to show the longest median survival amongst all cytogenetic groups in the Döhner study (70). In a separate study of 159 untreated early stage patients, patients with 13q14 deletions showed a median survival time of 17 years, compared to 13 years for patients with normal karyotype (71). Subsequent studies identified the microRNAs miR-15a and miR-16, both located in the minimal deleted region (MDR) of 13q14, as two major targets that are downregulated as a result of 13q14 deletions (72). Interestingly, miR16 is also

13 downregulated in the New Zealand black mouse, a de novo murine model for indolent,

late-onset CLL, supporting a role for these microRNAs in CLL pathogenesis (73). The

important roles of miR15 and miR16 were further demonstrated by Klein et al, who

showed that targeted deletion of 13q14-MDR, a cluster that contains genes for miR-

15a/16 and DLEU2, in mouse B cells resulted in accelerated proliferation of the B-cell

compartment and development of indolent lymphoproliferative disorders similar to

indolent CLL in human (74). The two micro RNAs appear to affect apoptosis by

negatively regulating Bcl2 at the transcription level (75). More recent studies have also

shown miR-15a and miR-16 to be involved in growth arrest by modulating oncogenes

involving in cell cycle control (76-78). Deletion of DLEU7, a gene also located in the

MDR of 13q14, was recently suggested to contribute to CLL leukemogenesis (79).

DLEU7 was found to encode a protein that suppresses NF κB activities in B cells by

directly inhibiting the receptors for BAFF (B-cell activating factor) and APRIL (a

proliferation inducing ligand), both important survival factors for B cells that are

upregulated in CLL (79, 80).

b) Trisomy 12

Trisomy 12 is the second most common cytogenetic aberration in CLL with

frequencies ranging from 10-20% (81). The Döhner study found patients with trisomy 12

to have comparable median survival to those with normal karyotype (70). In a recent

clinical trial assessing the efficacy of the chemoimmunotherapy regiment fludarabine,

cyclophosphamide, and rituximab (FCR), patients with trisomy 12 showed the best

response to treatment and complete remission rate in the FCR arm amongst all

cytogenetic groups, suggesting that trisomy 12 may identify a group of patients best able

14 to benefit from this regimen (82). The effectiveness of FCR for this group of patients maybe explained by the observation that CLL cells with trisomy 12 tend to express high levels of CD20, the cellular target of rituximab (83). To date, it is generally agreed that trisomy 12 predicts intermediate-risk disease (69). Trisomy 12 is also associated with atypical and prolymphocytic morphology in CLL, which has been shown to be a risk factor for disease progression (25, 84). The genes targeted in trisomy 12 have yet to be confirmed, although some genetic analyses have pointed to STAT6 and p27 as candidates

(85). The Hedgehog signalling pathway also appears to be affected by trisomy 12, as

CLL cells with trisomy 12 were shown to have constitutively activated hedgehog signalling, driven by autocrine secretion of hedgehog ligands (86). c) 11q and 17p deletions

Deletions in 11q22-23 occur at similar frequencies to trisomy 12 in CLL (81).

Patients with 11q22-23 deletions generally are younger, have shorter treatment-free survival, more rapid progression of disease, and shorter median survival times than patients with trisomy 12, normal karyotype, and 13q deletions (70, 87). Deletions in

17p13 are the rarest of the 5 major cytogenetic categories, occurring in 3-8% of patients at diagnosis (69). However, frequencies of 17p13 deletions increase to nearly 30% in the specific subgroups of patients with relapsing or refractory disease, indicating 17p13 deletions may occur as disease progresses (88, 89). Moreover, 17p13 deletions also occur as a secondary cytogenetic lesion during disease progression, which, regardless of the primary aberration, predict poor survival (90). Of all the cytogenetic aberrations reported in CLL, patients with primary 17p13 deletions have the worst prognosis and

15 shortest overall median survival (70, 91). Both 11q and 17p deletions have emerged as independent prognostic markers in several major studies (28).

Genetic analyses have shown that the MDR in 11q22-23 deletions in CLL contains the ATM (Ataxia Telangiectasia Mutated) gene, encoding for the ATM protein, a molecular sensor of oxidative stress with a role in the DNA-damage response (92, 93).

MicroRNAs miR-34b and miR-34c, both part of the p53 tumor suppressor network, can also be lost with 11q22 deletion (94, 95). One of the most important genes in the MDR of 17p13 deletions is TP53, which encodes p53, perhaps the most well known tumor suppressor protein. p53 functions through multiple pathways to control a wide range of cellular responses from carcinogenesis to drug resistance (96, 97). 17p13 deletions are also associated with loss of miR-34c, which is upregulated after DNA damage in the presence of p53 (89). In addition, ATM and TP53 mutations, regardless of the presence of absence of 11q and 17p deletions, are observed in CLL at frequencies ranging from 4-

12% (98-100). Importantly, both ATM and TP53 mutations were identified to be strongly associated with poor progression-free survival and overall survival, independent of 11q and 17p deletions, and IgVH mutation status.

Both 11q22-23 and 17p13 deletions are important risk factors for poor response to the chemoimmunotherapeutic regiments fludarabine + cyclophosphamide (FC) and fludarabine + rituximab (FR), and are associated with a significant lower response rate and shorter progression-free survival and overall survival in several clinical trials (21,

101, 102). Both cytogenetic aberrations were also identified to be a risk factor for early relapse following therapy (21). Deletions in 17p13, in particular, appear to be a predictor of poor response to fludarabine-based regiments, when compared to all other cytogenetics

16 subgroups, including 11q aberrations (82, 103, 104). The poor response associated with

17p deletions is likely associated, at least in part, with the loss of p53 and miR-34a (105-

107).

β2-microglobulin (β2M)

Beta 2-microglobulin ( β2M) is one of the two polypeptide chains that make up the

MHC Class I complex and is necessary for the cell surface expression of MHC class I

and stability of the peptide binding groove (108). β2-M is known to be released by CLL cells on a constitutive basis and is elevated in the serum of CLL patients (109, 110). The release of β2-M is a function of total protein synthesis and is increased after stimulation

(111, 112).

In a number of recent studies conducted to evaluate the prognostic strength of all currently used parameters in early stage patients, β2-M has consistently emerged as an independent prognostic marker of overall survival in multivariate analyses (19, 29, 113,

114). Of the four independent parameters identified in a study by Letestu et al, serum β2-

M > 2.5mg/L showed the highest hazard ratio. Based on its prognostic strength, Letestu et al proposed determination of β2-M level at diagnosis as part of a cost-effective strategy for prognosis in Stage-A (Rai stage 0) patients. In a separate study, the prognostic strength of β2-M was shown to further increase if the β2-M level after glomerular filtration

rate adjustment is used (115).

iii) Prognostic models

Based on the prognostic factors currently known and assessed routinely in the

clinic, a model can be established which incorporates the relative risk contribution of

17 each factor. A prognostic index with 6 prognostic factors identified by multivariate analysis was first proposed by Wierda et al for risk assessment (19). The proposed prognostic index for prediction of 5 and 10 year survival took into account the weight of risk contribution for each factor in the index and was shown to have high predictive power.

In a simpler approach, using prognostic scores assigned based on the presence or absence each of four independent prognostic factors identified in the study, with a score of 1 for each factor present regardless of the risk contribution of the factor, Letestu et al reported that 85% of patients with a score of 0-1 did not show disease progression in 7 years, while patients with a score above 2 showed progression within 20 months (29).

The predictive power of this method was shown to be stronger than that using IgVH mutation status or Zap70 expression, particularly in predicting progression-free survival and treatment-free survival in early stage patients.

Prognostic markers most routinely tested in clinics are summarized in table I. In this thesis, we attempt to elucidate the prognostic value of CD200, an immunorgulatory molecule to be discussed in depth in section 1.2, in CLL. Identification of new prognostic markers and inclusion of these markers into current prognostic models may improve the predictive power of current models. Prognostic models for prediction of treatment response, which may require inclusion of different sets of prognostic factors, remain to be developed. As prognostic markers and CLL biology are intrinsically linked, insights into the major factors driving CLL pathogenesis are instrumental for the discovery of new prognostic markers as well as therapeutics. The next section discusses the important biological features of CLL.

18

Table 1.1: Major prognostic markers in CLL

Prognostic marker Good prognosis Poor prognosis

Rai Stage Early stage: 0-II Late stage: III-IV

LDT >12 months <12 months

CD38 expression <20% >20%

Normal karyotypes, Trisomy 12, 11q22-23 Cytogenetics: 13q14 deletions deletions, 17p13 deletions

Serum β2 Microglobulin <2.5mg/L >2.5mg/L

19 1.1.2 Biology of CLL cells

1.1.2.1 The origin of CLL

Two models for the cellular origin of CLL have been proposed. The first model, based on the observation that CLL cells carry either mutated or unmutated IgVH, proposes that M-CLL and U CLL cells are derived from two distinct populations: M-CLL cells are derived from antigen-experienced B cells expanded through a classical germinal center reaction, where somatic hypermutation occurs, in the presence of T cell-help. U-

CLL cells, in contrast, are derived from marginal zone B cells by T-cell independent processes. The second model proposes a common origin for both M-CLL and U-CLL

(116). Evidence from gene expression profiling supports the second model, as U-CLL and M-CLL cells were shown to have a homogeneous phenotype bearing markers of memory B cells (46, 117).

Based on the observation that CLL cells share phenotypic characteristics of antigen-experienced memory-type cells, the search for a cellular origin of CLL has largely focused on identifying normal counterparts to CLL in the mature B-cell compartments. For example, based on several murine models of CLL, B-1a B cells have been proposed as a candidate population from which CLL is derived (118-120).

However, this view was recently challenged by Kikushige et al, in a study that demonstrated clonal proliferation early in B-cell ontogeny and abnormalities in the hematopoietic stem cell (HSC) compartment in CLL (121). Specifically, transplantation of purified CLL-HSC, but not CLL-pro-B cells or mature CLL cells, into immuncompromised NODSCID/IL2r null mice resulted in de novo generation of

CD19 +CD5 + CLL-like B cell clones with IGH-VDJ combinations distinct from the

20 original donors. Gene analyses showed elevated levels of lymphoid-related lineage specific genes in CLL-HSCs, although no CLL-related cytogenetic abnormality was detected. Based on these results, Kikushige et al propose a new model for the cellular origin of CLL in which CLL leukemogenesis occurs in a stepwise pattern that is initiated at a much earlier stage than had been appreciated. Aberrations in HSC with predisposition for B-cell ontogenesis are suggested to lead to polyclonal expansion at the pro-B cell stage, and then to MBL, with each step leading to acquisition of new malignant properties. According to this model, progression from MBL to CLL requires further oncogenic events (Fig 1.1).

Figure 1-1: CLL pathogenesis model proposed by Kikushige et al (121)

21 However, several issues with this model remain to be resolved. A vast majority of monoclonal B cells generated de novo in the mouse as shown in the study were found to have mutated IgVH, unlike in CLL where unmutated IgVH is found in about 50% of cases (122, 123). The high frequency of mutated IgVH and absence of cytogenetic abnormalities in the engrafted B cells also suggests an MBL-like condition, from which only about 1.1% of patients would go on to develop progressive CLL (4). Moreover, molecular analyses showed distinct IGHV and IGLV gene usage by M-CLL and U-CLL, indicating no conversion from U-CLL to M-CLL (124). Engrafted B cell clones also did not have cytogenetic abnormalities found in both MBL and CLL cells (3, 125). Thus, whether HSCs are the origin from which both M-CLL and U-CLL are derived and the point during the development pathway at which the two distinct diseases diverges then evolves require further investigation.

1.1.2.2 Role of BCR signaling

In B-cell lymphoma, chronic active BCR signaling due to point mutations in

CD79b (the Ig β subunit of the BCR) has been identified as a key pathogenic mechanism

resulting in constitutive NF κB activation (126). The observations from Kikushege et al

that monoclonal B cells generated de novo in the mouse had IGH-VDJ combinations

distinct from the original CLL clones demonstrated in vivo clonal selection based on

BCR specificity and an essential role for BCR signaling. Indeed, several lines of

evidence, in addition to this recent in vivo data, have supported a central role of BCR

signaling in the pathogenesis and progression of CLL.

First, the variable (V) region repertoire of the immunoglobulin heavy chain (IgH)

in CLL cells is highly restricted (122). A number of studies have also demonstrated that

22 a subset of patients, regardless of geographic location, share remarkably similar BCRs with closely related complementarity determing region 3 (CDR3) structural features that suggests the recognition of common antigens (127-131). Structural analyses of CLL-

BCRs with closely homologous (stereotyped) CDR3 showed specificity to apoptotic antigens and carbohydrate determinants of bacterial capsules or viral coats, akin to polyreactive “natural” antibodies, leading to the hypothesis that clonal expansion of CLL cells is driven by antigen-selection (129, 131, 132). A high frequency of stereotyped

HCDR3 was observed particularly in patients with unmutated IgVH (133). Moreover, expression of a particular stereotyped IGHV was shown to be associated with distinct clinical and biological characteristics. For example, cases expressing stereotyped

IGHV4-34/IGKV2-30 were shown to have mostly indolent disease, although a separate study also showed a tendency for CMV and EBV persistence (134, 135). Expression of

IGHV3-21, on the other hand, was associated with poor prognosis regardless of IgVH mutation status (136). Furthermore, a recent study showed extensive intraclonal diversification occurring in a subset of cases with stereotyped IGHV4-34, demonstrating ongoing interactions with antigen (137).

Second, though sharing a gene “signature” profile, M-CLL and U-CLL cells were found to differ in the expression of over 300 genes, many of which are known to play important roles in BCR signaling (46, 117). Interestingly, CLL cells have been shown to have a diminished response to BCR ligation, resembling an “anergic” phenotype (138).

Indeed, BCR ligation by soluble anti-IgM antibodies, which deliver transient signals through BCR, was shown to result in an incomplete BCR activation response. However, sustained BCR ligation by immobilized anti-IgM was able to elicit prolonged phosphorylation of ERK and Akt, resulting in increased levels of the anti-apoptotic

23 protein myeloid cell leukemia-1 (Mcl-1) and protection from fludarabine-induced apoptosis, showing that BCRs on CLL cells were responsive only to sustained stimulation (139, 140). In addition, CLL cells with unmutated IgVH (U-CLL cells) appear to show stronger responses to BCR ligation than CLL cells with mutated IgVH, further supporting the role of BCR signaling in disease activity (141).

Upon BCR ligation, Syk, a key mediator of proximal BCR signaling, is recruited to the BCR complex, which in turn associates with adaptor proteins to phosphorylate downstream signaling intermediates, eventually resulting in the activation of NF κB and several pro-survival pathways, as well as STAT3, an anti-apoptotic transcription factor

(142). Syk was found to be overexpressed and constitutively phosphorylated in CLL cells (143). Moreover, the protection from chemotherapy-induced apoptosis by BCR cross-linking was abrogated by inhibition of Syk (144).

A third line of evidence for an important role of BCR signaling is the association between Zap70 expression and disease progression. Involvement of Zap70 in BCR signaling, likely as an adaptor protein, was demonstrated by ligation of BCR in Zap70 +

CLL cells, which resulted in more phosphorylation of downstream signaling molecules

than in Zap70 - CLL cells (47, 145). Transgenic expression of Zap70 in Zap70 - CLL cells was also shown to be effective in enhancing downstream signaling through IgM-ligation

(146). Proteomic analyses on Zap70 + and Zap70 - CLL cells following IgM ligation established two distinct proteomic profiles of activation based on Zap70 expression that were reflective of the aggressive and indolent disease phenotype (147). Interestingly, through this analysis, a third subgroup was identified, in which CLL cells showed an

24 indolent phenotype (low Zap70 expression) but an activation proteomic profile similar to that of aggressive CLL (147).

The BCR is known to transmit low levels of signals in the absence of antigen

(tonic BCR signaling), a process that is mediated by Syk and has been shown to mediate

B cell survival during B cell maturation as well as survival of B lymphoma cells (145,

148). Tonic BCR signalling is particularly important in the pro-survival effects of BAFF and APRIL, both of which are elevated in CLL (148, 149). However, the role of tonic

BCR signaling in mediating CLL survival has yet to be determined.

In addition to mediating pro-survival signals, sustained BCR signaling mediated through Syk also appears to upregulate a number of adhesion molecules and increase the ability of CLL cells to migrate toward the chemokines CXCL12 (SDF-1) and CXCL13, both important for homing of CLL cells to the tissue microenvironment (150). Following entry into the tissue microenvironment, BCR triggering plays a role in retaining CLL cells in the microenvironment by downregulating CXCR4, a receptor for CXCL12, and

CD62L, preventing re-entry of CLL cells into the blood stream (151). Interestingly, U-

CLL cells or cells from patients with aggressive disease were shown to be more responsive to BCR-mediated retention in tissue microenvironment. The intrinsic link between BCR signaling and the CLL microenvironment is further addressed in section

1.1.3.

1.1.2.3 Proliferative potential of CLL cells

Until recent years, CLL was believed to be a disorder resulting from accumulation of malignant B cell clones defective in apoptosis, rather than from uncontrolled cell growth like in other malignancies. Earlier studies on CLL in vivo kinetics found CLL

25 cells in peripheral blood to be generally arrested at the G0/ G1 phase of cell cycle (152).

However, data from recent studies have challenged this view.

Analysis of telomere length and telomerase activity in CLL cells, which had shorter telomeres than B cells from normal individuals, indicated a prolonged

2 proliferative history of CLL cells (153). Using in vivo deuterium ( H2O) labelling of

CLL cells, Messmer et al showed that CLL cells from each patient in the study had

definable birth rates, ranging from 0.1% to 1% of the entire clone per day, providing

strong evidence that CLL is a more dynamic disease than was previously appreciated

(154). In a more recent study using the same labelling technology, and in which both

CLL birth rates and disappearance rates (death rates) were measured, CLL cells were

shown to have at least two-fold lower cell-turnover rates than the average normal B-cell.

However, CLL cells also disappeared from the circulation at a 10-fold lower rate than

normal B cells, demonstrating that CLL disease reflects a low level of cell proliferation

as well as accumulation by escape from apoptosis (155).

CLL cells with unmutated IgVH were shown to have significantly higher cell

turnover than cells with mutated IgVH, further supporting a significant role of BCR

signaling in CLL, and that CLL proliferation is intrinsically linked to disease activity

(155). CD38 also appears to be an important mediator of CLL proliferation. Treatment

of CLL cells with an activating anti-CD38 mAb in conjunction with IL2 was shown to

induce proliferation in vitro (62). Intraclonal analysis of CLL turnover showed distinct

differences in proliferative potential within a CLL clone, with the subsets of cells

expressing CD38 having higher proliferation rate than the CD38 - clones (156). Given that CD38 is highly expressed on CLL cells in the secondary lymphoid tissues, and that

26 CD38 expression can be modulated by interaction with non-CLL cells, CD38 + CLL cells in circulation may represent a subset of cells that have recently exited the tissue microenvironment, where most of CLL proliferation is believed to occur (60).

1.1.3 CLL microenvironment

One of the hallmarks of CLL cells, whether from aggressive or indolent disease, is their resistance to apoptosis in vivo . However, when cultured in vitro , CLL cells tend to undergo apoptosis spontaneously and generally fail to proliferate without external stimuli (157, 158). The apoptosis of CLL cells in vitro can be rescued by co-culturing with stromal cells or addition of a number of soluble factors mimicking those found in the tissue microenvironments in vivo (159, 160). Indeed, pseudofollicles termed

“proliferation centers” in secondary lymphoid compartments such as lymph nodes and spleen are known to be crucial for CLL disease progression (Fig 2). CLL cells in proliferation centers express markers associated with proliferation, such as CD71 and

Ki67, and higher levels of CD23, as compared to CLL cells from peripheral blood, supporting the hypothesis that the proliferative pool of CLL cells contributing to the dynamics of CLL turnover reside in these tissue microenvironments (161). Constituents of the microenvironment provide proliferative and pro-survival signals to CLL cells both through soluble factors and cell-cell contacts (Fig 3). These pro-survival factors in the

CLL microenvironment play a crucial role in controlling the in vivo dynamics of CLL cells, and are critical in determining the efficacy of chemotherapeutic agents for CLL.

Insight into the CLL microenvironment thus has significant implications in the design of in vitro and in vivo models of CLL for drug screening purposes.

27 Figure 1-2: Cellular components of CLL proliferation center

Follicular Resting Nurse-like dendritic CLL cells cell cell

Proliferating T-cell Mesenchymal CLL cells stem cell

28 Figure 1-3: Molecular crosstalks between CLL cell and the cellular components in the CLL microenvironment (see section 1.1.3a-f)

Antigenic CD38 BCR Ystimulation BAFF-R Syk Zap70 TACI VEGF-R VEGF BCMA BAFF ERK PKC PDGF PDGF-R APRIL

NF κB Plexin-B1 NLC STAT3 Mcl-1 Bcl2 Akt MSC CD100

CXCR5 CLL

CXCL13 CXCR4 CD40 IL4-R CXCL12

CD40L IL4, IL6, etc

CCL3/4

CCR1/ CCR5 T cell

29 1.1.3.1 BCR signaling in CLL microenvironment

One of the key signals that CLL cells likely receive in proliferation centers is antigenic stimulation. Microarray analyses of CLL cells from lymph nodes showed upregulation of genes implicated in ongoing BCR signaling and activation of the NF κB

and NFAT pathways when compared to CLL cells from peripheral blood (162). One of

the genes downstream of BCR induced in CLL cells from lymph nodes is EGR-1, a gene

that is responsible for BCR-induced proliferation in normal B cells. EGR-1 is known to

be essential for marginal zone B cell development, supporting the hypothesis that BCR

ligation contributes significantly to clonal expansion of CLL cells (163). Consistent with

in vitro data which showed activation of anti-apoptotic molecules downstream of BCR

ligation, anti-apoptotic genes including BCL2A1 are also upregulated in CLL cells from

lymph nodes (140, 162, 164).

1.1.3.2 TLR signaling in CLL microenvironment

In addition to BCR ligation, signaling through toll-like receptors (TLRs) is capable of inducing the NF κB pathway in a MyD88-dependent manner (165).

Stimulation of CLL cells by bacterial lipopeptides, ligands for TLR1/2/6, was shown to protect CLL cells from spontaneous apoptosis in vitro via the NF κB pathway, while triggering of TLR9 resulted in proliferation and increased CD38 expression on CLL cells

(166, 167). Importantly, in a whole-genome sequencing analyses conducted by Puente et al, activating mutations in MYD88 leading to a 5-10 fold increase in response to TLR triggering were identified as one of the recurrent mutations prominent in U-CLL (168).

The increased response to TLR triggering was shown to result in elevated production of

IL1 receptor antagonist (IL1RN), IL6, and the chemokines CCL2, CCL3, and CCL4, all

30 of which have been implicated in the recruitment of macrophages and T lymphocytes, and likely contribute to disease progression by establishing a microenvironmental niche favoring the survival and proliferation of CLL cells. The contribution of TLR signaling to CLL progression was further demonstrated in Tcl1 trangenic mice (a murine model of

CLL, to be discussed in section 1.1.4) lacking TIR8, a negative regulator of TLR, which were found to have accelerated disease and shorten life span in comparison to mice with wildtype TIR8 (169).

1.1.3.3 TNFR signaling in CLL microenvironment

Signaling through the tumor necrosis factor family member BAFF, in concert with BCR signaling, has been shown to be required for B-cell survival and the maintenance of B-cell homeostasis (59, 170). BAFF and its closely related homolog

APRIL share the receptors TACI and BCMA; in addition, BAFF also interacts with the

BAFF receptor BAFFR (171). CLL cells, like normal B cells, were shown to express all three BAFF and APRIL receptors at the mRNA level; moreover, addition of soluble

BAFF and APRIL was sufficient to protect CLL cells from spontaneous and drug- induced apoptosis in vitro through activation of the NF κB pathway (172, 173). BAFF and APRIL were shown to be produced by a subpopulation of monocyte-derived CD68 +

cells, termed Nurse-like cells (NLCs), present in the CLL microenvironment (Fig 1.3)

(172, 174, 175). The pathogenic role of BAFF in CLL was also shown in a murine model

generated by crossing Tcl-1 transgenic mice with BAFF transgenic mice. These mice

showed disease development at a significantly younger age and also had more rapid

disease progression (176). APRIL also appears to play a role in CLL pathogenesis, as

APRIL transgenic mice were reported to develop CLL-like conditions spontaneously at

old age (119).

31 1.1.3.4 T cells in CLL microenvironment

Systemic dysregulation in the T-cell compartment has been reported in numerous studies, and contributes to increased susceptibility of CLL patients to bacterial and opportunistic infections (6). Immunophenotyping with absolute-bead calibrated measurements showed down-regulated TCR signaling, impaired expression of T-helper 1

(Th1) cytokines, and increased production of IL-4 by CD4 + helper T cells even at early disease stages (177). CD4 + T cells and CD8 + T cells from CLL patients also showed

increased cell surface expression of the immunoregulatory molecule CTLA-4 and

reduced expression of CD28 upon PHA stimulation (178, 179). Increase in both the

frequency and numbers of regulatory T cells, including the IL-10 +, TGF β+ , and

CD25 +Foxp3 + subsets have been observed in CLL patients (180, 181). Moreover, CLL

patients generally have reduced number of CD4 + T cells in the circulation and increased

infiltration of CD4 + T cells with an activated phenotype in lymph nodes where they form

an integral part of the CLL microenvironment (182).

Activated CD4 + T cells expressing CD40L (CD154) are found to reside in close- proximity and likely come into contact with CLL cells (159). Activation of CD40 by

CD40L protects CLL cells from apoptosis by induction of survivin and activation of the

NF κB pathway (183, 184). Microarray analyses showed that CD40 activation in CLL cells resulted in increased induction of CD27, STAT3, and IL10 receptor, as well as genes involved in resistance to apoptosis when compared to normal B cells (185).

Consistent with these data, CD40 activation was also shown to induce production of cytokines important for CLL survival by CLL cells, including IL-6, IL-8, and IL-10 (186-

188). In addition, interaction between CD40 and CD40L, via cell-cell contacts between

B and T cells, also induces activation-induced cytidine deaminase (AID), an enzyme

32 required for somatic hypermutation and class-switch recombination (CSR) that is significantly elevated in a subset of U-CLL cells (189).

CD4 + T cells from CLL patients appear to produce IL-4 constitutively (177).

PHA-activated CLL-CD4 + T cells were also shown to produce higher levels of IL-4 than

cells from normal individuals in vitro (190, 191). IL-4 is known to protect CLL cells

from apoptosis via a Bcl-2 dependent pathway (192). Supernatants from activated

CD4 +T cells from CLL patients also induced drug resistance in CLL cells, an effect that

was at least in part contributed by IL-4 (193). Furthermore, CD38 expression is

upregulated on CLL cells upon contact with CD4 + T cells, possibly via exposure CD40L and IL4 (60, 194).

Communications between CLL cells and T cells and other cellular components in the microenvironment are multi-directional and are mediated through both cell-cell contacts and secretion of soluble factors (Fig 1.3). Some of the defects observed in CLL-

T cells appear to be induced directly by CLL cells through cell-cell contact (195). In a microarray analysis, Görgun observed reduced NF κB activation and a shift to Th2 differentiation in normal T cells that were co-cultured with CLL cells (196). CLL cells also appear to induce cytoskeletal defects in normal CD4 + T cells leading to defective immunological synapse formation (195). CLL cells affect normal CD8+ cytotoxic T cells by downregulating genes involving in vesicle trafficking and mobilization of effector molecules (196). Furthermore, CLL cells also play a role in the recruitment of T cells into the microenvironment by production of T-cell chemokines CCL3 and CCL4 through a mechanism that involves CD68 + NLCs (197).

33 1.1.3.5 Other cellular components in CLL microenvironment

In addition to the monocyte-derived CD68 + NLCs, co-culturing of CLL cells with

marrow-derived mesenchymal stromal cells (MSCs), and follicular dendritic cells (FDCs)

have all been shown to rescue CLL cells from drug-induced apoptosis in vitro (198-202).

The anti-apoptotic effects of both MSCs and FDCs were shown to involve activation of

Mcl-1 (202). Expression of Plexin-B1, the high affinity receptor for CD100, was

detected in lymph nodes in CLL patients and on BMSCs, FDCs, and T cells, which,

through interaction with CD100 on CLL cells, was shown to induce pro-survival signals

in CLL cells (203).

Bi-directional communication occurs between CLL cells and multiple cellular constituents of the microenvironment. In co-cultures, CLL cells were shown to be capable of turning on pro-survival capacity in normal peripheral blood CD14 + cells in

vitro , which in turn then upregulate their expression of BAFF, APRIL, and PECAM1

(204). CLL cells were also shown to produce Platelet-derived growth factor (PDGF), which binds the PDGF receptor on MSCs leading to activation of the pro-survival Akt pathway and production of VEGF by MSCs (205, 206). VEGF, in turn, is known to induce pro-survival signals and upregulate expression of PKC β, a key mediator

downstream of BCR activation, in CLL cells (207).

1.1.3.6 CLL trafficking to the microenvironment

Due to the importance of microenvironental cues for CLL survival and growth, molecules involved in the trafficking of CLL cells and recruitment of bystander cells to lymphoid tissues or bone marrow play a critical role in driving disease progression. One of the most important chemokine networks involved in CLL homing is the CXCR4-

34 CXCL12 axis, with CXCR4 overexpressed on CLL cells and CXCL12 (SDF-1) produced by MSCs in the tumor microenvironment (174, 208). CXCR4 drives both migration of

CLL cells into lymphoid tissues and adhesion of CLL cells to MSCs, promoting CLL survival and chemo-resistance (209). CD38 also appears to play an important role in this axis, as CLL cells activated with an anti-CD38 mAb were shown to be have increased sensitivity to CXCL12 (210).

Migration through endothelial cell-barriers and basement membranes permitting entry of CLL cells from the blood stream into the tissue microenvironment requires the chemokines CCL21 and CCL19 produced by cells located in high endothelial venules

(HEVs), which acts on the receptor CCR7 on CLL cells (211). This process is aided by matrix metalloprotease 9 (MMP9) on CLL cells, which is involved in the degradation of the extracellular matrix and/or basement membrane (212). Within lymphoid follicles, the

CXCR5-CXCL13 axis appears to promote positioning of CXCR5 + CLL cells (213). In

Acute lymphocytic leukemia (ALL), the CXCR5-CXCL13 axis has also been shown to regulate the interaction between leukemic cells and CD8 + T cells (214).

The hyaluronan receptor CD44 and integrins, particularly those containing the α4 chain (CD49d/VLA-4) are also essential to CLL homing. CD49d, whose expression on

CLL cells has been proposed to predict adverse disease, facilitates migration of CLL cells to the bone marrow (215-217). In addition, engagement of α4β1 integrin results in

MMP9 upregulation (218). CD44, in association with α4β1 integrin, has been shown to constitute a docking complex for MMP9 in mediating CLL migration and survival (219,

220).

35 1.1.4 Animal models of CLL

The search for novel therapeutics for CLL relies largely on assays in which therapeutic agents are tested with CLL cells growing in tissue culture conditions bearing little resemblance to the in vivo microenvironment where CLL cells thrive. This approach leaves an undesirable gap between bench science and clinical relevance.

Animal models, often considered the bridge that links the bench to the bedside, thus have an important role in elucidating the in vivo efficacy of therapeutic agents. For generation of applicable animal models for CLL, two approaches have traditionally been employed: a) the generation of genetically modified mice to produce animals which develop pathology resembling human CLL; b) the generation of humanized xenograft models of

CLL by engraftment of human CLL cells into immunocompromised animals.

1.1.4.1 Murine models of CLL

The APRIL transgenic mice and DLEU2/miR-15a/16 knockout-mice discussed in previous sections develop lymphoproliferative disorders (74, 119). B-cell targeted transgenic expression of the microRNA miR-29, often found in patients with indolent disease, also resulted in clonal expansion of CD5 + B cells and development of frank

leukemia in 10% of the mice (221). These mouse models have been instrumental in

providing insights into CLL pathogenesis; however, their use as pre-clinical drug

screening models is limited since they fail to recapitulate fully the human disease,

particularly aggressive CLL.

The model that most closely mimics human CLL to-date uses E-TCL-1

transgenic mice. TCL-1 is an oncogene whose translocation and inversion are the most

common cytogenetic abnormalities in T-cell leukemias (222). The Tcl-1 protein is

36 predominantly expressed on B cells, and has been shown to be an activator of the PI3K-

Akt oncogenic pathway (223). E-TCL-1 mice, with targeted transgenic expression of

TCL-1 on B cells, showed oligoclonal expansion of CD5 + B cells by 2 months of age. At

16-20 months of age, these mice develop a CLL-like disease akin to aggressive CLL in

human, as manifested by accumulation of monoclonal CD5 + leukemic cells in peripheral blood, splenomegaly, lymph node involvements, and bone marrow infiltration (224).

Like human CLL cells, CD5+CD23 + leukemic cells from these mice express stereotyped

BCRs that react with autoantigens, suggesting antigen involvement in disease development (225). Moreover, most leukemic cells from the peripheral blood of E -

TCL1 transgenic mice were arrested at G 0/G 1 stage and showed in vitro sensitivity to

fludarabine at similar dosage as human CLL (226). T cells from these mice were shown

to recapitulate defects observed in human CLL (227, 228).

Although E-TCL-1 mice develop a leukemic disease that seems to recapitulate aggressive human CLL, limitations to its use remain because of the extended period of time required for disease development and the variability in disease onset in each individual mouse.

1.1.4.2 Xenograft models of CLL

Engraftment of human CLL cells in immunocompromised animals may represent a superior approach to drug screening, since it may allow for a more shortened experimental duration and uses human CLL cells as targets. This approach, however, has been compounded by the resting nature of CLL cells, particularly those from peripheral blood, and the requirement for complex interactions between CLL cells and the various microenvironmental components to sustain CLL survival and growth, which are likely

37 lacking in a xeno-microenvironment. Efforts to date at generating xenograft models for

CLL have resulted in sub-optimal engraftment of CLL cells and generally have failed to recapitulate the human disease.

In early attempts using SCID mice, infusion of PBMC from CLL patients into immunocompromised SCID mice was associated with recovery of only 10% of injected

CLL cells within 48 hours in vivo . The remaining human cells persisted only in the peritoneal cavity with no human CLL migration to spleen or bone marrow (229, 230).

Furthermore, these human cells resulted from de novo EBV transformation of bystander

B cells rather than from the original B-CLL clones (229). In later studies using SCID-

Balb/c chimeric mice, generated by reconstitution of lethally irradiated Balb/c with SCID bone marrow, predominant engraftment of CLL cells was noted when PBMCs from Rai stage IV patients were used. Infusion of PBMCs from early stage patients, in contrast, resulted in engraftment of predominantly T cells (231, 232). In addition, autologous T cells were shown to impede engraftment of CLL cells from both early and late stage patients (233). However, CLL engraftment was only followed up to two weeks in these studies, and again persistence of CLL cells was observed only in peritoneal cavity.

More recently, Dürig J et al showed that a combination of iv and ip injection of

CLL PBMC into NOD.SCID mice resulted in recovery of CD19 +CD5 + CLL cells in both the peritoneal cavity and spleen of recipients at up to 12 weeks post engraftment (234).

The engraftment efficiency of CLL cells in mouse recipients was shown to correlate with disease stage and LDT, but not other clinical parameters, including cytogenetics, CD38 and Zap70 expression, and IgVH mutation status. Although the human CLL cells engrafted in spleen were shown to co-localize with T cells, the absolute cell recovery in

38 both peritoneal cavity and spleen remained low, with a greater than 10-fold decline in cell number observed from 8-12 weeks.

Using the more immunocompromised NOD.SCID/IL2R null mice, in which the IL-

2 receptor γ-chain knockout on the NOD.SCID background results in the absence of NK

cells, Bagnara et al recently found substantially improved engraftment of CLL cells by

pre-conditioning recipient mice with infusion of normal human cord-blood derived

CD34 + cells, or normal human bone marrow derived MSC, prior to transplantation of

CLL cells (235, 236). This approach presumably circumvents the failure of the murine xeno-microenvironment to support CLL growth. Proliferation of CLL cells and detection of CLL cells in mouse blood was observed, two features of human CLL not seen in previous xenograft studies. In contrast to previous reports by Shimoni A et al, autologous

T cells, but not the normal stromal components, were found to be required for the survival and proliferation of CLL cells in this model, at least in the initial phase of engraftment. However, although substantial in vivo proliferation of CLL cells was noted in this model, engraftment of CLL cells did not persist beyond 12-weeks, by which time

T cells eventually suppressed CLL repopulation and the animals subsequently succumbed to GVHD.

An alternative approach to creating xenograft models of CLL involves the use of human cell lines rather than primary CLL cells, which actively proliferate without the need for microenvironment cues. This approach also has limitations. In a study by

Loisel et al, the prolymphocytic leukemia derived JVM-3 cells, but not the CLL-derived

MEC2 cells were shown to establish tumors in SCID animals (237). Recently, a xenograft model using the CLL cell line MEC1 was reported and suggested to represent a

39 useful pre-clinical model for CLL. Persistent engraftment of MEC1 was achieved in

-/- -/- Rag2 γc mice, which lack B, T, and NK cells (238). Both the subcutaneous and intravenous inoculation route resulted in significant organ infiltration of tumor cells and a substantial presence of MEC1 cells in mouse blood accounting for a majority of circulating cells. One caveat to the application of this model for CLL drug screening is that MEC1 cells lack CD5 expression, the most significant marker for CLL that is known to play an important functional role for CLL pathophysiology (116).

Development of a useful xeongraft model for CLL remains elusive. We postulate advances in development of such a model might benefit from further understanding in the biology of CLL cells, their interaction with the microenvironment, and the role of other in vivo factors driving CLL growth in patients.

1.1.5 Immunotherapy for CLL

Although therapeutic responses to chemotherapeutic regiments for CLL have improved substantially in the past decade with the use of purine analogs such as fludarabine, development of drug resistance and refractory disease in a subset of patients posts a major barrier to achievement of maximal disease eradication (239). In recent years, therapeutic monoclonal antibodies have emerged as a promising treatment option for CLL, either alone as a single agent or in conjunction with traditional chemotherapeutic regiments (240). Therapeutic approaches to augment immune responses against leukemic cells could potentially synergize with cytotoxic chemotherapeutic agents and therapeutic monoclonal antibodies to further improve treatment responses.

40 1.1.5.1 Therapeutic monoclonal antibodies

Currently, the two most commonly used therapeutic antibodies in the clinic are

Rituximab and Alemtuzumab. Rituximab, the first therapeutic monoclonal antibody approved for the use as an anti-cancer agent, targets the CD20 antigen commonly expressed on CLL cells, while Alemtuzumab targets cell-surface CD52, found on both B and T cells (241). Therapeutic monoclonal antibodies mediate anti-tumor effects through several distinct mechanisms, including complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and Fc γR-mediated phagocytosis (242). The efficacy of therapeutic antibodies thus depends on the expression level of the targeted antigen on tumor cells, the integrity of apoptotic cascades within tumor cells, and the availability and functional capacity of effector cells.

Rituximab is known to target CLL cells by CDC and ADCC dependent mechanisms, and the therapeutic response can be compromised by low CD20 expression by CLL cells, the presence of p53 mutations, and altered innate immune function (83, 243, 244).

Competent immune-effector cells, in particular, are instrumental for optimal efficacy of many therapeutic antibodies (245).

1.1.5.2 Chemotherapy-elicited immune responses

Traditional cytotoxic chemotherapeutic agents and cytotoxic thereapeutic

antibodies such as Rituximab, in addition to mediating direct killing of tumor cells, can

also elicit anti-tumor immune responses by cross-presentation of tumor antigens from

dying tumor cells (246). The induction of an effective anti-tumor response involves

production of IFN-γ and activation of cytotoxic effector cells, which requires

“immunologic” cell death. Immunologic cell death reflects apoptotic-type cell death

(rather than necrotic cell death) resulting in the release of endoplasmic reticulum-derived

41 vesicles (calreticulin), extracellular ATP, and factors associated with chromatin, all of which have been shown to increase the capacity of CD8 + T cells to produce IFN γ (247-

249). Induction of such effective immune responses through immunologic cell death also involves TLRs and the purinergic receptor P2RX7 on dendritic cells (250, 251). In

CLL, the generation of effective anti-tumor immune responses in this scenario is compounded by the systemic immune dysfunction seen particularly in late stage patients.

Global reduction in naïve CD4 + and CD8 + T cells, coupled with increased frequencies of regulatory T cells, hinders induction of cytotoxic CD8 + T cells (252).

1.1.5.3 Approaches to CLL immunotherapy

Immunotherapeutic approaches to improve effector cell functions and for induction of protective anti-tumor immune responses have to overcome the immunological barriers posed by the immunosuppressive nature of the tumor microenvironment (253). This is challenging particularly in the case of CLL, as in addition to immune dysfunction, CLL cells also lack expression of co-stimulatory molecules and are themselves poorly immunogenic (254).

A number of vaccine approaches have been proposed to improve immune responses to leukemic cells in CLL patients. In a phase I clinical trial, vaccination using autologous CLL cells modified ex vivo to express CD40L was shown to be effective in transiently increasing the number of tumor-specific T cells following treatment (255). In addition, the CD40L-transduced autologous CLL-cell vaccine induced expression of the costimulatory molecules CD80 and CD86 on by-stander CLL cells. In a separate study with 8 patients, vaccination using autologous CLL cells transduced to express CD40L and IL2 ex vivo augmented granzyme B expression and production of IFN-γ by T cells,

42 with concomitant reduction in the sizes of affected lymph nodes in some patients (256).

Interestingly, specific cytotoxic T-cell responses against a subpopulation of CLL cells with increased drug resistance were found in a small subset of patients who received the vaccine (257). Non-gene therapy based approaches have also been proposed to enhance immunogenicity of CLL cells. For example, treatment of CLL cells with a TLR7 agonist was shown to induce expression of co-stimulatory molecules on CLL cells (258, 259). In a follow-up phase I/II clinical trial, however, TLR7 agonist immunotherapy was shown to induce only weak responses in patients in vivo (260).

Another promising approach is the manipulation of T lymphocytes to express tumor-specific T-cell receptors or chimeric antigen receptors. Tumor-specific T cells were successfully derived by genetic modification of T cells to express a chimeric antigen receptor targeting CD19, followed by in vitro expansion and activation on anti-

CD3 and anti-CD28 beads (261). Reduction in lymphadenopathy was observed in 3 out

of 9 patients after infusion of ex vivo modified CD19-targeting autologous T cells (262).

CD19-targeting T cells retrieved from patients at 9 days after infusion showed persistent

cytotoxicity against tumor cells. T cells genetically modified with a chimeric antigen

receptor targeting CD23 have also been reported (263). To enhance further effector

response, Porter et al recently reported the design of a chimeric antigen receptor with

specificity for CD19, coupled with 4-1BB (CD137, a costimulatory receptor) and CD3 ζ signaling domains (264). Importantly, infusion of these modified autologous T cells was shown to achieve complete remission in a patient with refractory disease. The generation of chimeric receptors against CLL-specific antigens coupled with factors to correct T cell defects thus provides a promising approach for T-cell based therapy.

43 Despite these recent advances persistent therapeutic response and disease remission have yet to be achieved by immunotherapeutic approaches. One potential hindrance to effective induction of anti-tumor immune responses may reflect an overexpression of immunoregulatory molecules and receptors by CLL cells, which, due to the systemic nature of the disease, may contribute to systemic immunosuppression.

A molecule whose immunomodulatory function has been documented extensively

in work by us and others, and which is now believed to contribute to the

immunoregulatory phenotye of CLL cells, is CD200. The section which follows provides

an overview on immunoregulatory molecules in the context of cancer immunology, and

reviews the literature on CD200 in different disease models.

1.2 CD200

1.2.1 Immunoregulatory molecules in the evasion of tumor immunosurveillance

In the recent update to a seminal paper on the hallmarks of cancer by Hanahan et al, the ability of tumor cells to evade anti-tumor immune responses is acknowledged as an important factor influencing tumor progression (265). The evasion of tumor immunosurveillance by tumor cells is mediated by an immunosuppressive network that includes tumor cells, tumor-infiltrating immune cells with regulatory phenotypes, and the production of immunomodulatory cytokines and chemokines (266).

Immunoregulation per se can be attributed to one of, or a composite of, several mechanisms, including deletion/inactivation of functional responding lymphocytes, or their suppression by exogenous cells/factors. As far as tumor biology is concerned, there is growing evidence for a role for immunosuppressive molecules released by tumor cells

44 themselves, or whose release is under control of those tumor cells, in antagonizing the activity of anti-tumor immune cells. In addition, infiltration of populations of regulatory cells, including a Treg population, or so-called myeloid-derived-suppressor cells

(MDSCs), has also been suggested to be relevant to suppression of host immunity to tumors.

It is now apparent that the overexpression of immunoregulatory molecules by tumor cells and tumor-infiltrating immune cells is a relatively common feature of the tumor microenvironment. Immunoregulatory molecules and their receptors mediate suppression by delivering signals that dominate and override costimulation (267). A number of immunoregulatory molecules have been shown to be overexpressed on cancer cells of different tissue origins. For example, the co-inhibitory molecule of the B7 family, B7-H1 (PD-L1), is overexpressed in multiple myeloma, leukemia, ovarian, and breast cancer (267). Immunoregulatory molecules such as B7-H1 mediate inhibition through interaction with their corresponding receptors on effector cells. Thus, in ovarian cancer, the inhibitory receptor for B7-H1, PD-1, was found on tumor infiltrating CD8 + T cells carrying TCRs with specificity for tumor-associated antigens but with defective effector functions (268). Another co-inhibitory receptor, BTLA, was found to be expressed at high levels on human melanoma tumor antigen-specific effector CD8 + T cells that were susceptible to inhibition by the BTLA ligand HVEM (269). BTLA +CD8 +

T cells from the tumor microenvironment were shown to produce less IFN γ than their

BTLA - counterparts (270). These recent studies support the hypothesis that immunoregulatory molecules expressed by tumor cells play an important role in modulating anti-tumor immune responses.

45 Monoclonal antibody therapies targeting the B7-H1 receptor PD-1 have improved anti-tumor activity in a phase I clinical trial on patients with metastatic disease (271).

The blockade of CTLA-4, the inhibitory receptor for CD80 and CD86, by the targeting monoclonal antibody ipilimumab, in concert with the chemotherapeutic agent dacarbazine, was recently reported to improve overall survival of metastatic melanoma patients in a phase 3 clinical trial (272). In a separate study in which immunity to a tumor-associated antigen (TAA) was analyzed following ipilimumab treatment, patients with base-line TAA antibody prior to treatment attained even greater benefits from ipilimumab (273). From these prelimnary approaches, immunoregulatory molecule blockade appears to be a novel promising approach to immunotherapy.

1.2.2 The CD200:CD200R axis

CD200 is a type I transmembrane molecule and a member of the immunoglobulin supergene family (274). CD200 has a short cytoplasmic tail with no known signaling motifs, and is thought to mediate its immunregulatory functions through binding to its receptor, CD200R(s) (275). CD200 expression has a relatively broad distribution, and can be found on both hematopoietic and non-hematopoietic cells. In mouse, five CD200 receptors, CD200R1-R5, have been described. Of the five receptors, only two, CD200R1 and R2, are found in human. CD200R1 is the major receptor for CD200 in both mouse and human, and contains the inhibitory ITIM motif in its cytoplasmic tail that is responsible for delivering inhibitory signals downstream (276). Functional properties have been attributed to the alterative receptors CD200R2-R5 in mouse; however, in human, the function of CD200R2 remains unknown (277). Expression of CD200 receptor(s) is restricted to cells of the myeloid lineage, B cells, NK cells, and activated T cells (276, 278).

46 The immunoregulatory function of CD200 has been demonstrated in a number of models. We reported attenuation of collagen-induced arthritis and reduced allo- and xeno- graft rejection by infusion of a soluble recombinant form of CD200, CD200Fc

(279-281). CD200 knockout mice show a normal phenotype but were more susceptible to experimental allergic encephalomyelitis (EAE) (282). Tolerance and long term survival of both skin and cardiac allografts occurred in mice with transgenic overexpression of CD200 (283). Analyses of infiltrating cells in tolerated allografts in

CD200 transgenic mice showed increased presence of Foxp3 + Treg cells and non-

degranulating mast cells, demonstrating a role of CD200 in the regulation of regulatory T

cells (284). At least in mice, engagement of CD200R2 appears to alter differentiation of

DCs into a phenotype that is capable of inducing CD4 +CD25 +Foxp3 + Treg cells (285).

The CD200:CD200R1 axis is implicated also in regulating inflammatory responses (286,

287). Several vial homologs of CD200 have also been identified, indicating that this axis has been exploited by viruses as a mean to control host immune responses (288, 289).

1.2.3 CD200 in cancer

In recent years, CD200 has been identified as an immunoregulatory molecule that is frequently elevated in various types of cancers. In acute myeloid leukemia (AML) and multiple myeloma, elevated expression of cell surface CD200 predicts poor prognosis

(290, 291). A functional role for CD200 on AML cells in suppressing the cytolytic activity and production of IFN γ by NK cells in vitro was reported (292). In a genome- wide gene expression analysis of lymphoblasts from acute lymphoblastic leukemia,

CD200 was identified as a new marker for minimal residual disease (293).

47 Overexpression of CD200 has also been documented in non-hematopoietic tumors (294). CD200 expression in melanoma was found to be a downstream target of the RAS/RAF/MEK/ERK oncogenic pathway and was shown to inhibit T cell activation through DCs (295). Moreover, tumor cells with stem cell markers in prostate, breast, colon, and brain cancers were found to co-express CD200, suggesting a potential role of

CD200 in cancer stem cells (296, 297). Importantly, CD200 expression on tumor cells can be modulated upon immune challenge in vivo . Using a murine breast tumor cell line

EMT6 with no detectable CD200 expression in vitro , we showed that CD200 expression on EMT6 cells was induced in vivo after transplantation into immunocompetent C57B/6 mice (298). CD200 induction in EMT6 appeared to require the presence of a functioning immune system, as EMT6 cells remained CD200 - in immunocompromised NOD.SCID mice and CD200-transgenic mice.

Insights into the contribution of CD200 in tumorigenesis were provided by studies on CD200-knockout mice, which were shown to be resistant to chemical-induced skin carcinogenesis (299). Decreased tumor growth in these mice was accompanied by increased expression of proinflammatory cytokines by DCs in skin-draining lymph nodes, indicating that tumor growth in the presence of CD200 was likely a result of

CD200-induced immunosuppression. It remains unknown whether CD200:CD200R mediated immune evasion generally requires tumor and/or host expression of CD200.

Expression of CD200 on tumor-infiltrating cells in the microenvironment may be sufficient to mediate the immunosuppressive effects of CD200 in the presence of

CD200R-expressing effector cells.

48 Recently, evidence has accumulated to suggest a role for CD200 in tumor metastasis. In an attempt to clone tumor cells which have metastasized to tumor draining lymph nodes, higher frequencies of CD200 + metastatic clones were observed in wildtype

(WT) animals than in immunocompromised mice, even though the latter mice showed

faster tumor growth. Thus, in the presence of an intact immune system, CD200

expression on tumor cells seemed to offer a metastatic advantage (300). In a separate

study, CD200 expression, while absent in primary squamous cell carcinoma, was shown

to be highly induced in metastases to the lymph node and other solid organs (301).

Consistent with the EMT6 model, CD200 + squamous carcinoma cells appeared to have a selective advantage to metastasize, possibly through modulating CD200R+ myeloid cells in the lymph node microenvironment.

1.3 Ectodomain shedding

Studies on CD200 in various disease models have thus far focused on the function of CD200 expressed on the cell surface. However, many cell-surface immunoregulatory molecules are found in soluble form in the serum of cancer patients where they are believed to play significant roles in disease progression. We recently identified a novel soluble form of CD200 that is likely generated by mechanisms of ectodomain shedding, and investigated its potential contribution to immunoregulation in the context in CLL.

This section provides a brief introduction to ectodomain shedding.

Ectodomain shedding involves proteolytic cleavage of a transmembrane protein at the juxta-membrane region, resulting in the release of the free ectodomain into the extrcellular matrix (302). The released cytoplasmic domain in some cases may participate in signal transduction events in the cytoplasm (303). All structural and

49 functional categories of transmembrane proteins are susceptible to ectodomain shedding.

Hence, it is a process that potentially modulates most major cellular processes, from the release of cytokines, growth factors, to cell adhesion and migration.

1.3.1 The ADAM proteases

A disintegrin and metalloprotease (ADAM) family of proteases have been identified as the main mediator of ectodomain shedding responsible for the cleavage of a wide range of functionally and structurally diverse molecules (304). Other proteases, including members of the matrix metalloprotease (MMP) family, are also known to shed transmembrane proteins (305-307). In human, 22 ADAMs have been identified, although of the 22 genes, only 12 are found to possess the catalytic metalloprotease domain that is required for proteolytic cleavage (304). Of all the ADAMs, ADAM8, 10, and 17, and 28 are found on lymphocytes, while ADAM9, 12, and 15 are expressed on cells of epithelial origin (308-311). In particular, ADAM10 appears to play a pivotal role in B-cell development by regulating the notch signaling pathway (312). In human leukemia, including CLL, only expression of ADAM10, 17, and 28 have been documented (313,

314).

1.3.2 Regulation of ADAM proteases

All ADAM proteases are characterized by 7 distinctive domain structures. An N- terminal propeptide region (pro-domain) is followed by the catalytic, metalloprotease domain that is responsible for the cleavage activity of ADAMs. C-terminal to the catalytic domain is the disintegrin domain allowing interaction of ADAMs with integrins, which is in turn followed by a cysteine-rich region containing an EGF-like repeat structure. The transmembrane domain, and cytoplasmic tail which vary widely in length

50 and sequence amongst the ADAMs, comprise the c-terminal end of an ADAM protease

(315) (Fig 1.4). A hypervariable region within the cysteine-rich domain is likely responsible for substrate recognition and binding (Fig 1.4).

Figure 1-4: Domain structure of ADAM protease (304)

Ectodomain shedding by all ADAM proteases occurs at a constitutive level that is dependent on the availability of both sheddases and substrates on the cell surface (316).

Upregulation of expression of several ADAM proteases has been reported in a number of malignancies, at both the mRNA and protein levels (317-321). The precise mechanisms controlling ADAM expression are unknown, although upregulation of both ADAM9 and

15 has been shown to be increased under inflammatory conditions (322). It is known furthermore that the proteolytic activities of ADAMs are subjected to multiple levels of post-translation controls. The pro-domain of ADAMs can block the catalytic activity of the metalloprotease domain, which is critical for the trafficking of ADAMs amongst

51 subcellular compartments (304). Removal of the pro-domain by pro-domain convertases at the level of the Golgi apparatus, or on the cell surface, thus represents one level of regulation of ADAM activity (323).

Availability of ADAMs at the cell surface is a function of control of both intracellular trafficking and compartmentalization of ADAMs. For example, following synthesis, the majority of ADAM17 resides in perinuclear vesicles near the plasma membrane or in the Golgi complex, and is apparently not available at the cell surface in the absence of external stimuli (324). Trafficking of vesicles containing ADAMs to the plasma membrane is thought to be controlled by proteins involved in cytoskeletal remodeling, although the precise mechanisms remain elusive (303).

The cytoplasmic domain of some ADAMs contains motifs that can act as binding sites for a variety of kinases, representing yet another level of control for ADAM activity

(314). One well-characterized pathway that is known to induce ADAM activity is the

PKC pathway, which can be stimulated by phorbol esters, as well as a wide variety of external stimulants (309, 314). ADAM17, known to be responsible for the shedding of many important cytokines and well as growth factors, is activated by PMA, which in turn activates PKC, thus leading to increased ADAM17 activity at the plasma membrane

(325). Several isoforms of PKC are reported to be upregulated in cancers, including

CLL, and this may in turn play a role in the overexpression and increased activity of

ADAM proteases in this disease (326, 327). ADAMs activity can also be induced by stimuli that enhance intracellular Ca 2+ release (328). It is important to note that activity

in different ADAM proteases can often be induced by more than one stimulus, although

one stimulus may predominate. For example, PKC activation stimulates ADAM17 most

52 potently, while ADAM10 is most sensitive to stimulation by intracellular Ca 2+ release

(329-331).

Due to their regulation of many growth factor/receptor signaling pathways,

ADAM proteases are thought to play a crucial role in cancer biology (304). In CLL, both

ADAM10 and ADAM17 contribute to disease progression by mediating the shedding of

several important immunomodulators.

1.3.3 ADAM proteases in CLL

One substrate of the ADAM proteases that is important for both B-cell and CLL biology is CD23. CD23 is a low-affinity receptor for IgE normally expressed on mature

B cells, antigen presenting cells, and platelet. CD23 expression, elevated on most CLL cells, is regulated by Notch2 and is involved in CLL cell-survival and proliferation (332-

334). CD23 is shed in a soluble form, and levels of sCD23 in patient serum have been shown to have powerful prognostic value in CLL disease progression (335, 336). The pro-domains of both ADAM10 and 17 were shown to cleave CD23 in vitro , although

ADAM10 appeared to be the major CD23 sheddase in vivo (337, 338).

Another substrate of ADAM proteases that is important in CLL is CD62L (L-

selectin), which mediates leukocyte-endothelial cell interactions and is involved in CLL

trafficking. Downregulation of CD62L on the cell surface impairs trans-endothelial

migration by CLL cells and prevents their exit from blood stream and lymph nodes (151).

Ectodomain cleavage of CD62L is one mechanism responsible for the downregulation of

CD62L (339). CD62L is known to be shed by both ADAM10 and ADAM17, although

ADAM17 appears to be the dominant sheddase of CD62L (331, 340). CD62L shedding

by ADAM17 can be potently induced by phorbol ester, a global PKC activator (325).

53 Interestingly, PKC β is a downstream mediator in BCR signaling, and is upregulated in

CLL cells (326). It is not known whether the overexpression of PKC in CLL cells has functional consequences in the activity of ADAM proteases.

54 1.4 Objectives and hypotheses

The previous sections have reviewed literature that strongly supports an important role of immunoregulatory molecules in biology and clinical characeristics of CLL, and their potential as therapeutic targets with an impact on CLL treatment. Preliminary analyses of CD200 expression by us have shown that CD200 is overexpressed on virtually all primary CLL cells and lymphoma cell lines, a result that has since been confirmed by several independent groups (341, 342). Moreover, using a CD200 sandwich ELISA, we have identified a novel soluble form of CD200 (sCD200) that is abundant in CLL plasma. Given our understanding of the immunoregulatory properties of CD200 and its role in cancer immunology, we hypothesized that:

1. CD200 plays an important functional role in CLL (Fig 4)

2. This role of CD200 in CLL can be used to develop a xenograft model of

CLL

3. The CD200:CD200R axis may represent an important novel therapeutic

target for CLL

55 Figure 1-5: Potential role of CD200/sCD200 in the CLL microenvironment

CLL

CD200 sCD200

Pro-CLL factors CD200R that support survival/growth?

T cell/NLC/MSC

56 The objectives of the studies in this thesis were to explore the role of CD200, both its membrane-anchored and soluble forms in CLL. These studies have addressed the following issues:

1. The functional role of CD200 on CLL cells… see Chapter 2

2. The prognostic value of CD200 cell surface expression and plasma

sCD200 levels for CLL… see Chapter 3

3. The in vivo impact of CD200 and sCD200 in developing a xenograft

model of CLL and the potential of CD200 blockade as a novel

therapy…see Chapter 3

4. Analysis of the mechanisms leading to the release of sCD200, and

characterization of the released form…see Chapter 4

57

Chapter 2: The role of CD200 in immunity to B cell lymphoma

A manuscript of the same title has been published in the Journal of Leukocyte Biology, 2010, 88: 361-372 (365)

58 2.1 Abstract

CD200 is a transmembrane protein broadly expressed on a variety of cell types which delivers immunoregulatory signals through binding to receptors (CD200Rs) expressed on monocytes/myeloid cells and T lymphocytes. Signals delivered through the

CD200:CD200R axis have been shown to play an important role in the regulation of anti- tumor immunity, and overexpression of CD200 has been reported in a number of malignancies, including chronic lymphocytic leukemia (CLL), as well as on cancer stem cells.

We investigated the effect of CD200 blockade in vitro on generation of CTL responses against a poorly immunogenic CD200 + lymphoma cell line and fresh cells

obtained from CLL patients using both anti-CD200 mAbs and CD200-specific siRNAs.

Suppression of functional expression of CD200 augmented killing of the CD200 + cells, as well as production of the inflammatory cytokines IFN γ and TNF α by effector PBMCs.

Killing was mediated by CD8 + cytotoxic T cells, while CD4 + T cells play an important role in CD200-mediated suppression of CTL responses. Our data suggest that CD200 blockade may represent a novel approach to clinical treatment of CLL.

59 2.2 Introduction

The differentiation and activation of B cells involves multiple processes which regulate gene rearrangement, proliferation, and apoptosis. When these are disrupted malignancies often occur, including lymphomas and Chronic Lymphocytic Leukemia

(CLL) (36). Complete cure of both diseases with conventional chemotherapy remains extremely rare. While T-cell mediated anti-tumor immune responses have the potential to eliminate tumor cells, CLL and lymphoma cells are inherently poorly immunogenic, rending T-cell based immunotherapies ineffective (343). Various techniques have been used to try to improve immunogenicity of CLL cells including the use of IL2 and TLR agonists (259). Immunoregulatory molecules are known to play critical roles in regulating

T cell-mediated immunotherapy and manipulation of immunoregulatory pathways may be an important alternative method to improve the efficacy of such treatments.

One immunoregulatory molecule, CD200, has been shown to be overexpressed in a number of malignancies, including renal carcinoma, colon carcinoma, ovarian carcinoma, melanoma, acute myeloid leukemia (AML), multiple myeloma (MM), and

CLL (291, 294, 295, 344, 345). In AML, cell surface CD200 expression on malignant cells is correlated with poor prognosis (291). CD200 has also recently been reported to be a cancer stem cell marker (297). The regulatory function of CD200 is delivered through binding to a receptor, CD200R, expressed on cells of the myeloid lineage and T lymphocytes (275).

A regulatory function for CD200 in tumor immunity was suggested following studies which showed that infusion of a soluble form of CD200, CD200Fc, into EL4 thymoma-bearing C57B/6 mice enhanced tumor growth (281). Monoclonal anti-CD200

60 antibodies have recently been reported to abrogate growth of CD200-transduced RAJI and Namalwa cell in NOD.SCID mice (346). In addition, Pallasch et al demonstrated that CD200 expression on CLL cells had inhibitory effects on the proliferation of autologous effector T cells, and CD200 blockade using a rat monoclonal anti-CD200 antibody produced a reduction in the number of CD25+CD4 +Foxp3 + regulatory T cells in vitro (347). In the case of CLL, no correlation has been reported between CD200

surface expression and other CLL prognostic markers such as CD38 expression, IgVH-

mutational status, and Binet staging system (347), and indeed the independent prognostic

value of CD200 expression remains unknown.

In a model system which used either a poorly immunogenic lymphoma cell line

with constitutive CD200 levels, or CD200 + primary CLL cells, we show below that

blockade of CD200 by monoclonal antibodies or downregulation of CD200 by specific

silencers augmented anti-tumor CTL responses in vitro . CD4 +T cells from splenocytes of individual CLL patients expressed CD200R, consistent with the hypothesis that CD200 over-expression on tumor cells themselves may mediate immunosuppression in CLL.

Treatments of primary CLL cells with a TLR7 agonist, alone or in combination with phorbol esters and IL2, have been reported to enhance the immunogeneicity of CLL cells and increase their killing by effector T cells (259, 348). We report below that this treatment also significantly reduced CD200 expression on CLL cells, and imply that downregulation of CD200 expression on tumor cells may improve immunogeneicity of

CLL and lymphoma cells and enhance the efficacy of cell-based immunotherapies.

61 2.3 Materials and Methods:

Cells:

Human PBMC were isolated from heparin treated whole blood of healthy volunteer donors using Ficoll-Paque PLUS gradients (GE Healthcare Bio-Sciences,

Piscataway, NJ). 5 independent volunteer donors were used on multiple occasions throughout the studies described. PBMCs were used in CTL assays immediately after isolation. Two human cell lines propagated from non-Hodgkin’s lymphomas were grown in suspension in AIM-V medium (Invitrogen, Carlsbad, CA) supplemented with 5% FBS

(Hyclone, Logan, UT) (349).

CD5 +CD19 + primary CLL cells were purified from the fresh blood of consenting

CLL patients as described previously (350). CLL spleens were obtained after

splenectomy. Single-cell suspensions from CLL spleens were obtained by standard

protocols. All protocols were approved by institutional review boards.

hCD200 transfected Hek293 cells were obtained from Genetec. Cells were grown

in selection medium DMEM-F12 supplemented with 1µg/ml G418 and 10%FBS.

Antibodies:

The rat anti-hCD200 monoclonal antibodies 1B9 and 5A9 were described

previously (351). 3H4, which showed no immunoreactivity against cell-surface CD200

(data not shown), was used as an isotype control in CTL assays.

A polyclonal rabbit anti-hCD200 serum was generated following immunization of

rabbits with CD200Fc and subsequent boosting x2 with Hek293-hCD200 cell lysate

62 (custom immunization performed by Cedarlane Labs, Hornby, ON). Anti-Fc antibodies in the serum were removed using Fc column absorption (Cedarlane Labs), and immunoreactivity of the sera was confirmed by Western blots.

FACS analyses:

5x10 5 lymphoma cells were washed twice with 1ml FACS buffer (PBS, 1%FBS,

0.1%NaN 3, 5mM EDTA), and incubated with 0.1ug of rat anti-human CD200 mAb (1B9) or isotype control for 45min at 4ºC. Cells were washed x3 with PBS, and incubated with goat anti-rat IgG-FITC antibody (Jackson Labs, Western Grove, PA) at a 1:100 dilution for 30min at 4ºC.

For CD200R1 staining, a mouse anti-hCD200R mAb (R&D Systems) was used at

0.5ug per sample, followed by incubation with a secondary goat anti-mouse IgG-PE antibody (Jackson Labs). The following antibodies were used at concentrations suggested by the supplier (BD Biosciences): CD4-FITC, CD8-PECy7, CD5-PECy5, and

CD19-PE.

In activation experiments, CLL cells were stained with mouse anti-CD83-PE and mouse anti-CD5-FITC antibodiy (BD Biosciences) as per manufacturer’s instruction at

24 and 48 hours after stimulation. All cells were fixed with 1% Paraformaldehyde before being analysed in a Coulter FC500 flow cytometer.

Western Blots:

Cells were lysed in 0.025%SDS, and run on SDS-PAGE gels. After transfer the blots were blocked with 5% milk in TBST overnight at 4ºC. Blots were then probed with the rabbit anti-hC200 serum at a 1:6000 dilution. In siRNA experiments, MAPK was

63 used as a housekeeping protein for reference. Blots were divided at ~40kd, with the top part of the blot (>40kd) probed for CD200, and the bottom part (<40kd) probed with a mouse anti-MAPK mAb, Tag-100 (Qiagen), at a 1:1000 dilution. After thorough washing, blots were probed with either goat anti-rabbit IgG-HRP (1:6000) or goat anti- mouse IgG-HRP (1:2000) (Jackson) for 1 hour at room temperature. Blots were developed using an ECL Western blot detection kit (GE Healthcare Bio-Sciences).

CD200 column absorption:

The rat anti-human CD200 mAb 1B9 was conjugated to CNBr-activated

Sepharose beads (Cedarlane, ON). For CD200 absorption, 1ml of neat serum samples from healthy controls and CLL patients were incubated with 250 l of 1B9 conjugated

beads on a shaker overnight at 4°C. Pre-absorbed serum and CD200-absorbed serum

samples were subsequently used in MLC assays at designated dilutions.

RT-PCR and Real-time PCR:

RNA was extracted from cells using TRIZOL reagent, and cDNA was obtained

using OliogDT primers (Invitrogen). To detect CD200 mRNA level, the following

primer pair was designed to detect ~100bp amplicons:

Forward: AATACCTTTGGTTTTGGGAAGATCT

Reverse: GGTGGTCTTCAGAGAATTTGTAGTGA

Primer mixes for GAPDH and TBP were purchased from Qiagen and used as

housekeeping genes for normalization of CD200 gene expression level.

64 All primers were used in both regular PCR and real-time PCR. For real-time PCR experiments, 50ng cDNA was used per reaction. siRNA transfection:

Three commercial CD200 siRNA, designated CD200 siRNA#1, CD200siRNA

#4, and CD200siRNA#6, were obtained from Qiagen. Two control siRNAs, a positive control GAPDH silencer and a negative control, were purchased from Ambion for use in silencing experiments.

7.5x10 5 lymphoma cells were transfected with 2ug of siRNA using lipofectamine2000 (Invitrogen) as a transfection reagent at a 1:6 ratio. Transfection was performed in triplicate in 12-well plates according to the manufacturer’s instructions.

Cells were harvested for RNA at 48 hours and for protein at 72 hours after transfection.

In some experiments cells were used in CTL assays as stimulators cells 72 hours after transfection.

CTL assays:

1.2x10 6 PBLs were stimulated with mitomycin C treated Ly5 or Ly2 cells at a

15:1 responder to stimulator ratio in 96-well plates. In some wells 8ug of 1B9 rat anti-

hCD200 mAb was added for functional neutralization of CD200 expression.

Supernatants were harvested from each well 18 and 42 hour after stimulation to assay for

cytokines. After 6 days fresh lymphoma cells were labelled overnight with 3HTdR at

37ºC, washed 3 times in PBS, and 1x10 4 cells added to each well in the 96-well plate.

The plate was harvested for 3HTdR analysis at 18hr. All assays were performed in

triplicate, and geometric means used in quantitation of CTL activity. Cytotoxic killing of

65 lymphoma cells was calculated from the 3HTdR remaining in cells with reference to

unstimulated controls and the total counts added in the targets. All results shown were

obtained from a minimum of 3 independent experiments.

Where CD5 +CD19 + primary CLL cells were used as stimulators, 51 Cr release

assays were performed to assess killing. At 7 days after stimulation, with unstimulated

PBL cells set up as negative controls, 51 Cr-labelled CLL cells were added into each well

as killing targets, and 51 Cr release was assessed in supernatant at 6 hours after addition of

51 Cr labeled CLL targets. CLL cells from 3 different patients were used as targets for the

same PBL effectors in 3 independent experiments.

In experiments where CD4 + or CD8 + T cells were depleted, depletion was

performed using EasySep immunomagnetic cell selection kits (StemCell Technologies,

Vancouver, BC) as per the manufacturer’s instructions.

ELISA:

Supernatant samples harvested from CTL assays were assayed for TNF α, IFN γ,

TGF β, IL4, IL6, IL10, and IL12 using ELISA kits purchased from eBioscience (San

Diego, CA), as per the manufacturer’s instruction. A standard curve was obtained in each assay to quantify cytokine present in the supernatant.

Activation of CLL cells:

2x10 6 purified CLL cells were cultured in serum-free AIM-V medium plus 2-ME

(Sigma-Aldrich) in 24-well plates at 37°C in 5% CO 2 in the presence or absence of the

following immunomodulators: TLR-7 agonist Imiquimod (LKT Laboratories, St Paul,

MN), phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), and human-recombinant

66 IL-2. Imiquimod and PMA powders were reconstituted in DMSO as 1mg/ml and

10mg/ml stock solutions. For activation of CLL cells, Imiquimod, PMA, and IL2 were used at a final concentration of 3ug/ml, 30ng/ml, and 500U/ml, respectively. At 24 and

48 hours after stimulation, cells were harvested and cell surface expressions of CD200,

CD83 and CD5 were determined by FACS. Upregulation of CD83 expression was used as an indicator for response to stimulation.

Statistical analyses:

p-values for all experimental data were obtained using the student’s t test to

determine the significance between sample means.

67 2.4 Results:

Comparison of CD200 expression on primary CLL cells and two independent human lymphoma cell lines:

In order to explore the effect of CD200 expression on induction of anti-tumor immunity in vitro , we first characterized expression of CD200 using a number of independent isolates of primary CLL cells and established cell lines using PCR, Western blots, and FACS. Although essentially all primary tumor cells (n=25, table 2.1) analyzed in this study stained positive for CD200 as compared to the respective isotype controls, the mean fluorescent intensity of the staining varied widely (Fig 2-1a). Heterogeneity in

CD200 cell surface expression level was not independently correlated with any of the various clinical parameters of CLL analyzed, including Rai disease stage, CD38 expression, and cytogenetic status. To date there is no data to our knowledge directly addressing the issue of altered CD200 cell surface expression with disease progression. In the two lymphoma cell lines studied, Ly5 cells showed CD200 expression levels comparable to that of primary CLL cells while Ly2 cells failed to stain for CD200 (Fig 2-

1b). Results from FACS studies were confirmed using Western blot analyses, in which

CD200 was detected as a band at around 48kd in lysates of hCD200 transfected Hek293 cells and Ly5 cells, but not Ly2 cells (Fig 2-1c). CD200 expression on Ly5 cells, on the other hand, remained consistently high after prolonged in vitro passage (>20: data not shown). Ly5 and Ly2 cells were used in the functional studies described below designed to investigate the functional consequences of the presence of CD200 expression on tumor cell-induced immunity.

68 The effect of CD200 blockade in the killing of CD200 + Ly5 and CD200 - Ly2 cells

Different epitopes of human CD200 are recognized by independently derived rat

anti-human CD200 mAbs (351). Among this panel of rat anti-human mAbs (all IgG2a),

1B9 and 5A9 recognized the extracellular domain of CD200 (Fig 2-1b), while another

mAb, 3H4 failed to stain any of the CD200 + cells identified by 1B9 (data not shown). We have used 3H4 as an isotype control in the experiments discussed below.

We explored the effect of addition of 1B9, 5A9 or 3H4 in vitro on MLCs using

human PBL from healthy blood donors as effector cells and mitomycin-c treated CD200 +

Ly5 and CD200 - Ly2 cells as stimulators. 3HTdR-based cytotoxicity assays were performed 7 days after stimulation at 3 different effector:target ratios to assess the effect of CD200 blockade on the killing of Ly5 and Ly2 cells by activated effectors. Data shown in the figures below are for E:T ratios of 10:1. Figure 2-2a shows pooled results from 9 independent experiments using PBL stimulators from 5 different donors. Both

Ly5 and Ly2 cells were poorly immunogenic when used alone, with optimal lysis only

~6%. Addition of the 1B9 anti-CD200 mAb, but not of 5A9, produced ~5-fold increase in the killing of CD200 + Ly5 cells, with no significant change in the killing of CD200 -

Ly2 cells (Fig 2-2a and 2-2b). The isotype control antibody 3H4 failed to augment

killing of either Ly5 or Ly2 cells.

The enhanced lysis seen using 1B9 was relatively independent of the PBMC

effector source, and occurred even after pre-treatment of tumor cells (but not PBMC)

with mAb (data not shown), consistent with the primary target being the CD200

expressed on the Ly5 tumor cells themselves. Moreover, the killing of Ly5 cells was

abrogated following depletion of CD8 + cells, suggesting that CD8 + cytotoxic T cells are

69 likely involved in tumor killing in this system (Fig 2-2c). Interestingly, when CD4 + T cells were depleted, lysis of Ly5 cells increased 3-fold even without CD200 blockade

(Fig 2-2c), which may be taken to reflect an intrinsic autoregulatory role for CD4 + cell

subsets.

Functional inhibition of expression of CD200 in Ly5 lymphoma cells by siRNA

As an alternate approach to modifying functional CD200 expression on tumor

cells we used synthetic siRNAs to down-regulate CD200 expression. Three independent

commercial siRNAs were examined for their ability to modify CD200 expression at both

the mRNA (Fig 2-3a) and protein level (Fig 2-3b). Optimal silencing was seen using

siRNAs #4 and #6 (Fig 2-3). Western blotting and FACS analysis were used to monitor

knockdown of CD200 at the protein level following siRNA tranfection. By Western

blots, incomplete silencing was observed (Fig 2-3b). Similarly, cell surface level CD200

expression on Ly5 cells was reduced by >50% at 24 hours after transfection of siRNA #4

and #6 by FACS (data not shown).

We compared the relative increase in induction of CTL by Ly5 cells in vitro using

siRNAs or anti-CD200 mAb to decrease functional CD200 expression on tumor

stimulator cells. As shown in Figure 4, 1B9 and anti-CD200 siRNAs #4 and #6 all

augmented induction of CTL for lymphoma cells in vitro (Fig 2-4, one of 3 such studies).

Consistent with data in Fig 2-2 using CD200 blockade by mAbs, neither of the two

siRNAs modulated the killing of Ly2 cells. These data confirm that the functional

inhibition of CD200 expression on tumor cells, either by mAb or siRNA silencing,

augments the generation of anti-tumor immunity in vitro .

70 Augmented cytokine production in MLCs following decreased CD200 expression

In addition to exploring whether anti-CD200 or CD200siRNAs could alter induction of CTL in vitro in MLCs with tumor cells, we asked whether these same reagents would also alter cytokine production in vitro . Supernatants from PBMCs stimulated with Ly5 or Ly2 cells were collected at 18 and 42 hr after stimulation, with typical data (one of 4 such studies) shown in Figure 2-5 (panel a shows TNF α production; panel b, IFN γ production). Consistent with the data in Figure 2-2, minimal induction of

cytokine production occurred using Ly2 cells as stimulator, with no further augmentation

using anti-CD200 mAb. In contrast, while Ly5 cells induced minimal cytokine

production in the absence of additional manipulation, inclusion of either anti-CD200

mAb or pre-treatment of tumor cells with siRNAs, augmented induction of both TNF α and IFN γ. Again these effects were not seen using control mAbs (3H4) or siRNAs (Fig

2-5). CD200 blockade did not affect the production level of a number of other cytokines, including IL-4, IL-6, IL10, IL-12, and TGF β (data not shown). Moreover, the changes in

TNF α and IFN γ levels were not observed in the absence of stimulation by Ly5 cells.

Augmented killing of primary CLL cells by CD200 blockade

Immunodeficiency is one of the clinical hallmarks of CLL. T cells from CLL

patients generally show Th2 polarization and express low levels of CD80, CD86, and

CD154 (196). Since CLL cells express high levels of CD200, the CD200:CD200R axis

may be an important pathway involved in suppression of T cell activity by CLL cells.

We thus examined the effect of CD200 blockade on the killing of primary CLL cells

using 1B9 (the mAb with the most profound effect in the studies described above).

Effector PBMC from 2 different donors were stimulated with mitomycin C treated

71 primary CLL cells from 3 different CLL patients (see table 1) with killing in this case assessed using a 51 Cr release assay. Interestingly, effector cells derived from donor 1 showed only low levels of killing of all three CLL targets (Fig 2-6a: data are shown as mean+SD for killing of all three CLL targets), whereas effector cells derived from donor

2 killed all targets to a greater degree (data not shown). However, regardless of the

quantitative level of killing, CD200 blockade (but not control 3H4 antibody) increased

killing of all CLL targets, for both effector populations.

As was observed for killing of Ly5 cells, depletion of CD8 + T cells prior to stimulation resulted in minimal killing of CLL targets, indicating involvement of CD8 + effector cells in CLL killing (Fig 2-6b). Interestingly, depletion of CD4 + T cells alone

was sufficient to augment killing of Ly5 targets in the absence of CD200 blockade (Fig

2-2c), while augmented killing of CLL targets was seen only using both CD200 blockade

and depletion of CD4+ T cells (Fig. 2-6b). We speculate that this may reflect the

involvement of different effector populations for the two target populations studied.

As an adjunctive approach, we also investigated whether CLL serum, which we

have found in independent studies to be capable of suppressing human allogeneic CTL

immune responses in vitro lost this suppressive capacity after passage over a CD200-

immunoadsorbent column. Data in Fig 2-6c show that CTL activity (measured in human

allogeneic MLCs at day 6) was inhibited by CLL serum, but that this inhibition was

attenuated following absorption of CD200 from the serum, again consistent with an

important role for CD200 (in this case in soluble form in CLL serum) in suppressing T

cell-mediated immunity.

72 Since CD200 induces immunoregulation following binding to a receptor,

CD200R, receptor expression was examined on cells harvested from the spleens of 2 patients who had undergone splenectomy for clinical reasons associated with disease treatment (patients I and II, see Table 1). >90% of cells were CD19 +CD5 + CLL cells in

the spleen of patient I, whereas T cells constituted >50% of all cells from the spleen of

patient II (Fig 2-6d and e). Despite these differences in cellular constitution, >90% of

CD4 + T cells in both spleen populations stained positive for CD200R, while only a minor

population of CD8 + T cells (>1%) expressed CD200R (Fig 2-6d). Splenic CD5 + CLL

cells, on the other hand, did not show detectable levels of CD200R. A direct comparison

of CD200R expression on splenic and circulating CD4+ T cells and CLL cells in the same patients could not be made as peripheral blood from the two splenectomized patients was not available at the time of study. However, unlike CD200 (Fig. 2-6e), CD200R was never detected on CD5 + CLL cells in spleen (Fig 2-6d) or peripheral blood (data not shown).

Association of down-regulated CD200 expression with increased immunogenicity of CLL cells

Treatments of primary CLL cells with immunomodulators such as TLR7 agonists,

IL2, and PKC agonists have been shown to improve immunogenicity of CLL cells in vitro , potentially by increasing expression of co-stimulatory molecules and rendering them more effective targets for lymphokine activated killer (LAK) cells (259, 348, 350).

Since cell surface expression of CD200 provides immunosuppressive signals that counter the effect of co-stimulatory molecules, we asked whether treatments designed to modulate immunogenicity of CLL cells would have a concomitant effect on CD200 expression. Primary CLL cells from 5 patients (see table 1) at different stages of disease

73 (Rai stage II-IV) were treated with a TLR7 agonist of the imidazoquinoline family,

Imiquimod, alone or in combination with human recombinant IL2 and PMA for 24 hours, and then assessed for cell surface CD200 and CD5 expression by FACS. Data from stimulation of CLL cells from patient 61 is shown below as a representative data set (Fig

2-7).

Both PMA and Imiquimod treatments significantly reduced CD200 expression on

CLL cells in all patients tested, while expression of the CLL surface-marker CD5 remained relatively unchanged (Fig 2-7). Expression of CD83, a co-stimulatory molecule and an activation marker, was also increased in response to both PMA and

Imiquimod, showing that the CLL cells were in “activated” states following treatment.

PMA-induced CD200 down-regulation was observed in all CLL cells, while Imiquimod- induced CD200 down-regulation was observed in cells from 4 out of 5 patients (data not shown). IL2, on the other hand, had no effect on CD200 expression and produced minimal increase in CD83 expression (Fig 2-7). In agreement with previous reports in which CLL cells were shown to exhibit heterogeneous response to PMA and Imiquimod in the upregulation of CD83, CD80, and CD86 expressions, the effect of these two stimuli on CD200 expression also varied among patients (data not shown) (258).

Reduction in CD200 expression was most pronounced by concomitant treatment of PMA,

Imiquimod, and IL2, which, as reported previously, also resulted in the greatest increase in CD83 expression (259). The presence of IL2 in combination with PMA and

Imiquimod, while causing further augmentation of CD83 expression, had no effect on

CD200 downregulation.

74 2.5 Discussion:

Immunomodulatory molecules contributing to negative signaling of T cells are thought to play a pivotal role in regulating anti-tumor responses and tumor progression in human malignancies. As examples, altered expression of immunomodulatory molecules of the B7 family, B7-H1, B7-H3, and B7-H4, have been detected in lung, prostate, ovarian, kidney carcinomas, and neuroblastoma (352). In prostate cancer and clear cell renal carcinoma, B7-H3 overexpression on tumor cells is associated with poor prognosis

(353, 354). In ovarian cancer, serum B7-H4 level has been identified as another marker which predicts poor prognosis (355). Functional blockade of these immunomodulatory molecules might thus provide a novel therapy for such malignancies. Indeed, blockade of

CTLA4, a negative regulator of T cells, using a fully humanized antibody is currently under development in Phase III clinical trials in patients with advanced melanoma and other malignancies (356).

In B cell malignancies, including lymphomas and CLL, the tumor cells themselves are known to be poorly immunogenic, despite the expression of high levels of

MHC molecules and tumor antigens (357, 358). A number of strategies have been investigated to develop clinically applicable methodologies to enhance the immunogenicity of CLL cells (359, 360). For example, transduction of CLL cells with

CD40L has been shown to enhance antigen specific recognition of tumor cells by autologous T cells in vitro (360). Various attempts have also been made to improve the

efficacy of vaccines targeting CLL-specific antigens (361, 362).

Given the dominant nature of immunomodulatory signals, the stimulation of co-

stimulatory molecules on tumor cells alone may not be sufficient to overcome the poor

75 immunogenicity of the tumor. Expression of CD200, a known immunoregulatory molecule, has been reported on CLL and lymphoma cells (363). Although CD200 cell surface expression level does not seem to correlate with other CLL clinical markers, it remains unknown whether CD200 expression levels on CLL cells varies in response to treatment or during disease progression. Our results described herein, and data from other groups, supports the hypothesis that CD200 expression on tumor cells might be one of the contributors for the poor immunogenicity of leukemic/lymphoma cells. Blockade of functional CD200 expression would thus provide a promising approach to enhance immunogenicity of such tumor cells. In support of this hypothesis, Kretz-Rommel et al recently demonstrated that blockade of CD200 using specific humanized mAbs enhanced anti-tumor responses using hPBMCs and tumor cells artificially transfected with a

CD200 lentiviral vector (346).

A drawback to the study reported by Kretz-Rommel et al is that lentiviral transfection often produces protein expression levels not reflective of those seen physiologically. Accordingly we have been interested in the induction of tumor responses directed against primary CLL cells isolated from peripheral blood of patients as well as a B-lymphoma cell line, Ly5, which constitutively expresses CD200 at levels paralleling those expressed by primary CLL cells. A CD200 - cell line, Ly2, was used as a control. CTL assays using these two 2 cell lines as targets showed that while both cell lines were poorly immunogenic, killing of CD200 +Ly5 cells, and indeed of primary CLL cells, but not CD200 - Ly2 cells, was augmented > 5 fold by the presence of a rat anti-

hCD200 mAb 1B9 when compared with an isotype control antibody, 3H4.

76 Although CD200 is expressed on normal B cells and its expression is increased on

T cells upon activation, the effect of CD200 blockade was PBMC-donor independent,

and appears to target CD200 expressed on tumor cells (unpublished observations) (364).

Antibody-mediated CD200 blockade as a mean of enhancing CTL responses was affected

by the CD200 epitopes targeted, since another CD200-specific mAb 5A9, produced much

less augmentation of CTL induction than 1B9, despite equivalent staining of Ly5 cells in

FACS by both 5A9 and 1B9 (Fig 2-1b). This is consistent with previous data indicating

heterogeneity in the activity of different anti-CD200 mAbs in different functional assays

(341). Thus, biochemical and functional characterization of the epitopes recognized by

anti-CD200 MAbs is of crucial importance in the design of CD200-specific mAb

therapies.

CD200 blockade by both 1B9 and CD200 specific siRNAs enhanced production

of TNF α and IFN γ in vitro from effector cells, suggesting that CD200 blockade may

affect anti-tumor immunity through other (cytokine mediated) mechanisms (365). The

data described used two independent CD200 siRNAs (#4 and #6), both of which showed

specific knockdown of CD200 at both RNA and protein levels. Interestingly, CD200

knockdown by siRNA#6 resulted in higher production of both TNF α and IFN γ, despite

similar augmentation of the CTL response to Ly5 cells after transfection with both

silencers (#4 and #6). This may simply reflect the difference in the effector populations

responsible for activity in these two assays.

The in vitro killing of tumor cells in our CTL assays was mediated by CD8 + cytotoxic effector cells, as demonstrated by the minimal CTL response to both Ly5 and primary CLL cells when CD8 + T cells were depleted from responder populations. NK

77 cells, which have been shown to express CD200R, could also potentially play a role in the killing of tumor targets, particularly in assays with CLL targets, where killing was not completely abrogated after CD8 depletion (366). In our hands ~30% of the PBMCs stained with anti-CD56 mAb in FACS analysis before cultures. We were unable to detect statistically significant changes in the % of cells stained with anti-CD56 mAb following culture (immediately before assaying lytic activity), with levels ~2-3% in all groups, while levels of CD4 +/CD8 + cells in non-depleted (by anti-CD4/-CD8) cultures were less

that 10%. We presume this relative insensitivity (of % surviving NK cells) to various cell

depletion strategies reflects the absence of production of mediators (cytokines) from

CD4 + and/or CD8 + cells in these cultures which might contribute to NK survival/growth

in vitro . In addition no significant changes in CD56 + cells were seen in cultures incubated

with anti-CD200 mAb (data not shown). We conclude that the differential killing activity

seen following the manipulations shown in Fig.6 is best explained by our hypothesis that

CD8 + cells are the primary effector population assayed.

Interestingly, the killing of both Ly5 and CLL cells were significantly affected by

the absence of CD4 + effectors. While depletion of CD4 + T cells was sufficient to enhance

killing of Ly5 cells (Fig 2-2c), CD4-depletion augmented the killing of CLL cells only in

the presence of CD200 blockade. We interpret these data as suggestive of the

involvement of CD4 + T cells in regulation of killing, either directly (as a regulatory cell

population itself-note we have not independently assessed the effect of depletion of

CD25 + cells in these assays) or indirectly acting to affect the activity of other regulatory cells. The exact mechanism(s) involved remain unexplored.

78 CLL cell-mediated T cell defects have been well documented. Recently, the formation of immunological synapses between CLL cells and autologous T cells were shown to be impaired (195). This impairment appears to be CLL-dependent as incubation of CLL cells with allogenic T cells also led to failure in formation of normal immunologic synapse between CLL cells and normal T cells. The expression of activation markers on T cells was also impaired after incubation with CLL cells, in a mechanism involving direct cell-cell contact as well as soluble factors secreted by CLL cells (367). The results of our CTL studies showed that CD200 may be one of the cell- surface factors contributing to CLL-mediated T cell suppression, as CD200 blockade resulted in enhanced killing of CLL cells.

Further evidence for an important role of the CD200:CD200R axis in CLL was supported by the high frequency of CD200R + CD4 + T cells in the spleen of CLL patients as detected by FACS analyses. CD8 + T cells and CLL cells, on the other hand, showed no detectable level of CD200R expression, consistent with the hypothesis that the primary target for CLL-derived CD200-mediated immunosuppressive signals represents

CD4 + and/or other CD200R + (but non-CD8 +) cells. It remains to be determined whether

CD200R +CD4 + T cells and CD200 + CLL cells exist in close proximity in vivo in CLL microenvironments. However, the observation that CD200R-expressing CD4 + T cells and CD200-expressing CLL cells are present in the same microenvironment (spleen in this case) supports a model in which CD200-mediated suppression of CD200R +CD4 + T cells is in part at least responsible for the Th2 cytokine polarization and diminished CD8 + cytotoxic T cell function observed in CLL patients. The ability of anti-CD200 to augment lysis of fresh CLL cells following CD4 depletion may suggest a role for such

(anti-CD200) therapy in CLL alongside treatment with e.g. fludarabine and

79 alemtuzumab, both of which have a significant ability to kill CD4 + cells (368, 369). A potential concern for immunotherapies targeting the CD200:CD200R pathway is autoimmunity, as this pathway has been shown to play important regulatory roles in a number of autoimmunity models in rodents, including CIA and EAE (280, 370, 371). In vivo models of CLL will be needed to address such safety and efficacy questions. It also remains open to speculation whether CD200 blockade may even enhance treatments such as allogenic bone marrow transplantation, in which killing of tumor cells is mediated by allogenic T cells.

Treatment with imidazoquinolines, a family of TLR7 agonists, along with IL2 and

PKC agonists, has also been considered as a means to improve immunogenicity of CLL cells (343). In vitro treatment of CLL cells with these immunmodulators is effective in transforming CLL cells to a DC-like phenotype with high expression of co-stimulatory molecules, production of inflammatory cytokines, and the ability to stimulate T cell proliferation, at least in vitro (259, 348, 350). We found that expression of CD200 on the surface of CLL cells was downregulated in response to Imiquimod or PMA, with an optimal decrease observed following combined use of Imiquimod and PMA. IL2 treatment did not affect CD200 cell surface expression on CLL cells. Given that reversal of CD200-mediated suppression does not seem to require complete abrogation of CD200 cell surface expression (see Fig 2-3 and 2-4), this reduction of CD200 expression on tumor cells achieved by Imiquimod and PMA may represent a key feature of their immunostimulatory activities. As PMA and Imiquimod are global activators of multiple pathways, further investigations are required to evaluate the contribution of altered

CD200 expression to the biological effects produced by these agents. However, our data provide evidence for the potential of therapies targeting the CD200:CD200R axis, in

80 combination with treatments to enhance immunogenicity of tumor cells, as a mechanism to augment anti-tumor responses.

Whether the downregulation of CD200 expression in response to PMA and

Imiquimod is mediated through transcriptional control or mechanisms involving

ectodomain cleavage by proteases is currently unknown. PMA is a known activator of

the ADAM family of proteases and is responsible for the inducible shedding of a number

of cytokines and chemokines, including TNF, TNFRI, IL6R, and CX3CL1 (372-374). It

is thus possible that CD200 downregulation following PMA stimulation involves

proteolytic cleavage of cell surface CD200, and preliminary observations using in vitro

studies of CLL cells support this hypothesis (375). Such a shedding event might also

contribute to the existence of a soluble immunosuppressive form of CD200 in CLL

serum.

In summary, we have shown that CD200 mediated immunosuppression is an

important mechanism utilized by CLL cells as a mean to inhibit anti-tumor immune

responses. We predict that inhibition of CD200 expression on tumor cells in general may

have important clinical implications in developing novel immunotherapies.

81 2.6 Tables

Table 2.1: Clinical characteristics of patients used in the study Patient Years after # Sex Age diagnosis ▪ Rai Stage ♦ WBC ● Treatment %CD38 ○ Cytogenetics 3 M 72 4 IV 175 S/CHOP/CVP 2 13q-/17p- 4 F 84 10 III 200 P/C,S/R 46 13q- 5 M 62 12 IV 121 CP,CVP,S,FC 2 13q- 8 M 74 7 IV 220 FR,splenectomy,S na 17p- 12 M 55 6 IV 23 CHOP,P 81 13q-,17p- * 14 M 71 6 II 186 none 72 11q-,13q- 16 M 69 6 IV 189 CP 4 normal 22 M 54 6 IV 8 CP,FC,FCR,S 1 normal 27 F 82 11 III 125 none 8 T12 36 M 59 7 IV 10 CP 7 Na 41 F 59 6 III 35 CP,splenectomy 40 normal * 46 M 55 5 III 121 none 4 T12 57 F 77 10 IV 120 CP na 13q- 60 M 88 6 IV 62 none 1 13q-,17p- ◘ 61 M 81 6 III 89 P 1 normal 65 F 61 26 IV 140 splenectomy 18 13q-,11q- 70 F 78 5 III 33 none 13 13q- 71 M 57 2 II 21 none 1 T12,13q- 72 F 48 2 0 13 none 2 na * 73 F 53 13 IV 65 none 2 13q- 74 M 77 10 III 250 none 1 13q- ◘ 75 F 63 3 II 22 none 71 na ◘ 78 M 72 5 IV 80 CP,FCR 13 normal ◘ 79 M 51 10 IV 140 Cx2 1 13q- ◘ 80 M 51 1 II 88 None 2 13q- ⌂ I M 72 5 IV 96 Cx2, splenectomy 60 17p- ⌂ II M 58 15 IV 77 C, F, FC, S, Splen 65 13q-, 11q-

Footnotes to Table 2.1:

* Cells from indicated patients were used as stimulators in CTL assays (Fig 2-6a). ◘ Cells from indicated patients were used in activation experiments (Fig 2-7). Cells from all patients in this table were stained for CD200 (Fig 2-1a), with the exception of patients I and II ( ⌂). ⌂ Spleens were obtained from indicated patients after splenectomy. Corresponding splenocytes were analyzed for CD200R expression (Fig 2-6c). ▪ Rai stage 0: lymphocytosis; I: with adenopathy; II: with hepatosplenomegaly; III: with anaemia; IV: with thrombocytopenia. ♦ WBC: White blood cell count (x10 6 cells/ml) in the peripheral blood. ● CVP, Cyclophosphamide/Vincristine/Prednisone; CHOP, Cyclophosphamide/Oncovin/Prednisone/Doxorubicin; C, Cyclophosphamide; P, Prednisone; F, fludarabine; R, rituximab; S, solumedrol. ○ T12, trisomy 12; na, not available

83

2.7 Figure legends

Figure 2-1: Expression of CD200 on primary CLL cells and 2 CLL cell lines a) Mean Fluorescent Intensity (MFI) of CD200 expression on a panel of primary CLL cells (n=25), 2 lymphoma cell lines (Ly5 and Ly2 cells), and normal B cells. The broken line designates the level of CD200 expression on normal B cells. b) Constitutive cell surface expression of CD200 on Ly5 cells, and absence of expression on Ly2. The monoclonal antibodies 1B9 and 5A9 stained CD200 equally well on Ly5 cells while Ly2 cells showed no CD200 staining. Staining was performed using 0.1ug of 1B9, 5A9, or rat IgG isotype control (shaded histogram); c) detectable CD200 levels in Ly5 cell lysate but not in Ly2 lysate using a rabbit anti-hCD200 serum at a 1:6000 dilution.

Figure 2-2: Effect of anti-CD200 mAb 1B9 on induction of CTL by lymphoma cells

1.2x10 6 hPBLs were stimulated with 8X10 4 mitomycin-C treated Ly5 or Ly2 cells for 7 days in the presence or absence of the rat anti-hCD200 antibodies 1B9 and 5A9, using

3H4 as an isotype control antibody. a) 1B9 (** p< 0.0001) and 5A9 (p>0.05) enhanced

CTL responses to Ly5 cells, while 3H4 showed no significant effect; b) failure of anti-

CD200 monoclonal antibody to augment CTL responses induced by Ly2 cells; c)

Depletion of CD8 + T cells abrogated augmentation of Ly5-lysis by 1B9, while depletion

of CD4 + T cells significantly enhanced Ly5-lysis even in the absence of 1B9 (** p<0.05).

All p-values were calculated using % killing obtained from effector PBMC with stimulation by Ly5 cells as reference.

84

Figure 2-3: Silencing of CD200 expression by specific oligodeoxynucleotides

Ly5 cells were transfected with 2ug of the CD200 siRNAs #1, #4, or #6. All silencers were designed by Qiagen. A control using lipofectamine treatment but no siRNA was included. Cells were assayed ay 48 hours (RNA) or 72hrs (protein) following transfection. a) CD200 mRNA level as shown by real-time PCR. CD200 silencer #1 showed no effect; b) expression of CD200 protein was decreased after transfection with silencers #4 and #6, but not #1. a

Figure 2-4: The effect of CD200 silencing in the killing of Ly5 cells siRNAs for CD200 enhanced cytotoxic killing of Ly5 cells to a similar degree as that achieved using anti-CD200 mAb 1B9 (** p<0.05 with both CD200 silencers). The negative control silencer (#1) had no effect.

Figure 2-5: Modulation of cytokine production in MLCs using anti-CD200 mAb or CD200-specific siRNAs

Supernatants were harvested 18 and 42 hours after stimulation and assayed at 1:5 and

1:10 dilution, respectively, for a) TNF α (18 and 42 hr supernatant) and b) IFN γ (18 hr supernatant). Suppression of functional CD200 expression by mAb 1B9 or the CD200- specific silencers #4 and #6 augmented production of both TNF α and IFN γ by responder

PBLs. Neither mAb nor siRNAs affected production of TNF α or IFN γ when PBLs were stimulated with Ly2 cells.

85

Figure 2-6: CD200 blockade augments killing of primary CLL cells by allogenic effector PBLs and CD200R expression on CLL splenocytes

Effector PBLs were stimulated by mitomycin C treated primary CLL cells in the presence of either rat IgG or 1B9. Killing was measured 7 days after stimulation by 51 Cr release

assay. a) Killing was shown as an average of 3 independent experiments using 3

different CLL targets and one PBL effector. Unstimulated PBLs were used as negative

controls. b) Depletion of CD4 + T cell from MLCs further augmented killing of CLL

targets using CD200 blockade, while CD4 + T cell depletion alone had no effect on CLL

killing. Depletion of CD8 + T cells reduced the killing of CLL cells to levels akin to those seen with unstimulated PBLs, even in the presence of 1B9. c) Effect of CD200 absorption on immunosuppression in human MLCs using CLL patient serum: CD200 was absorbed from CLL serum by overnight incubation of pooled CLL serum (obtained from 15 donors) with 1B9-conjugated Sepharose beads. CLL serum before (CLL) or after (absorbed) CD200 absorption was added to human MLC at the indicated dilutions; results show % lysis of 51 Cr labelled target cells at a 30:1 effector:target ratio in 6hrs.

CLL serum suppressed MLC reactivity in a dose dependent manner compared with controls ( p<0.05), but this inhibition was lost after absorption.

d) Expression of CD200R on cells gated on i) CD5; ii) CD8; and iii) CD4. Of the three

populations, over 90% of CD4 + T cells stained positive for CD200R using cells from

CLL spleen of both CLL patients, while neither CD8 + T cells nor CLL (CD5 +) cells expressed detectable levels of CD200R.

86

e) Expression of CD200 on splenic CLL cells: CLL populations as determined by the cell surface markers CD19 and CD5. CD5 +CD19 + CLL cells from both CLL spleen

populations express high levels of CD200.

Figure 2-7: Expression of CD200 on CLL cells in response to stimulation by PMA, Imiquimod, and IL2

Fresh CLL cells were treated with 30ng/ml PMA, 3ug/ml Imiquimod, and 500U/ml recombinant hIL2, alone or in combination, for 24 hours. CD200 expression (y-axis) on the surface of CLL cells was reduced in response to PMA and Imiquimod, but not IL2.

CD200 expression is further downregulated when CLL cells were treated with all 3 stimulants, whereas change in expression of the cell surface marker CD5 (x-axis) was minimal. CD83 expression was also upregulated in response to stimulation by PMA and

Imiquimod (CD83+ cells represented by dark spots). The experiment was repeated with fresh CLL cells from 5 patients, and typical data from 1 such study is shown.

87

2.8 Figures

Figure 2-1: Expression of CD200 on primary CLL cells and 2 CLL cell lines

2-1a)

Mean Fluorescent Intensity (MFI) of CD200 cell surface expression

250.0

200.0

150.0 M FI 100.0

50.0

0.0 3 4 5 8 12 14 16 22 27 36 41 46 57 60 61 65 70 71 72 73 74 75 78 79 80 Ly5 Ly2 Nor Patient ID

88

2-1b)

Ly5 Ly2

1B9

5A9

2-1c)

a b c a: Hek293-hCD200 b: Ly5 cells c: Ly2

89

Figure 2-2: Effect of anti-CD200 mAb 1B9 on induction of CTL by lymphoma cells

2-2a)

% %specific specific lysis lysis of of Ly5 Ly5 cells cells with with or or without without anti- anti-CCD200D200 mAbmAb 35

30

25

20

15

% specific% lysis 10

5

0 PBL+Ly5 PBL+Ly5+3H4 PBL+Ly5+1B9 PBL+Ly5+5A9

90

2-2b)

% specific lysis of Ly2 cells with or without anti-CD200 mAb

40 35 30 25 20 15

% specific lysis specific % 10 5 0 Ly2 only Ly2+1B9

91

2-2c)

% Specific lysis of Ly5 cells by PBL following depletion of distinct cell subsets

40 Untreated CD4-depletion 30 CD8-depletion

10

5 % specific lysis % specific

0 1B9 only Ly5 only Ly5+1B9

-5

92

Figure 2-3: Silencing of CD200 expression by specific oligodeoxynucleotides

2-3a)

Relative CD200 RNA levels in Ly5 cells after siRNA transfection 0.0015

0.001

0.0005 Relative mRNA level 0 Reagent only CD200 siRNA#1 CD200 siRNA#4 CD200 siRNA#6

2-3b)

1 2 3 4 5 6

CD200

MAPK (House-keeping) protein)

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Figure 2-4: The effect of CD200 silencing in the killing of Ly5 cells

% Specific lysis of Ly5 cells transfected with control or CD200 specific siRNA 30 ** 25 **

20 **

15

10 % specific lysis % specific

5

0

9 A y5 B +l L siRN iRNA#4 iRNA#6 PB e s

5+ -v ly5+ PBL+ly5+1 y PBL+ PBL+ly5+s PBL+l

94

Figure 2-5: Modulation of cytokine production in MLCs using anti-CD200 mAb or CD200-specific siRNAs

2-5a)

TNF ααα production 18 and 42hr after stimulation with Ly5 340

290

240

190 18h

pg/ml 140 42h

90

40

-10 9 6 nt B # nly 1 nly ge + o a 5 5 ly iRNA#4 iRNA re + ve siRNA s s Ly PBL o o L - 5 5 N B 5 ly ly P ly ly5 PBL+ PBL+ PBL+ PBL+

95

2-5b)

γγγ IFN IFN productionγγγ production 18 18 hr hr after after stimulation stimulation with with Ly5Ly5 900 800 700 600 500 400

pg/ml 300 200 100 0 -100 y #6 nl ent A#4 A o g iRNA N N L s e Ly5 only PB 5 siR BL+ly5+1B9 5 -v y5 No rea P L+ly +l L+ly L PBL+ly5 siR PB B PB P

96

Figure 2-6: CD200 blockade augments killing of primary CLL cells by allogenic effector PBLs and CD200R expression on CLL-splenocytes

2-6a)

% Specific lysis of CLL cells in the presence or absence of anti -CD200 mAb 30 of anti-CD200 mAb

25

20

15

10 % specific% lysis

5

0 Rat IgG 1B9

97

2-6b)

% Specific lysis of CLL cells in the presence or absence

of anti-CD200 mAb

30 PBL+CLL PBL+CLL+control IgG 25 PBL+CLL+1B9 20

15 % Lysis 10

5

0 No depletion CD4 depletion CD8 depletion

98

2-6c)

Suppression of human MLC response by CLL serum or CD200-abosrbedCD200-abosrbed serum serum

99

2-6d)

CLL spleen 1

0.1% 0.6%

CD200R1 99.5% 99.3 96.6% % 0.2%

CD5 CD8 CD4

CLL spleen 2

0.0% 0.6% 99.0% CD200R1

90.3 99.0 % % 0.5%

CD5 CD8 CD4

100

2-6e)

CLL spleen-1 CD5 and CD19 gated Whole CLL splenocytes CLL splenocytes: CD19

CD5 CD200 CLL spleen-2 CD5 and CD19 gated Whole CLL splenocytes CLL splenocytes: CD19

CD200 CD5

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Figure 2-7: Expression of CD200 on CLL cells in response to stimulation by PMA, Imiquimod, and IL2

30.1%

63.5%

75.2%

27.1%

92.0%

102

Chapter 3: Soluble CD200 supports in vivo survival of CLL

(Manuscript submitted to Cancer Research, April, 2012)

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3.1 Abstract

CD200, a type I transmembrane molecule overexpressed on most CLL cells, has been reported to play an important role in modulating tumor immunity. We have characterized a previously unknown soluble form of CD200 (sCD200) in human plasma.

Levels of sCD200 were elevated in CLL plasma (compared with healthy controls), with a significant correlation between plasma sCD200 and CLL Rai disease stage.

hi null Infusion of sCD200 CLL plasma into NOD.SCID γc mice receiving CLL splenocytes enhanced engraftment of CLL cells in comparison to that seen in mice receiving sCD200 lo normal plasma. CLL cells were detected in both the spleen and

peritoneal cavity of animals for up to 75 days. Engraftment of CLL cells was not

observed following infusion of CLL plasma depleted of sCD200, and was lost following

treatment of mice with anti-CD200 mAb, or OKT3 mAb, suggesting a role for both

sCD200 and T cells in CLL engraftment. Anti-CD200 mAb was as effective as

rituximab in eliminating engrafted CLL cells when given at 21-days post-engraftment.

Our data suggest that sCD200 is a novel prognostic marker and potential therapeutic

target for CLL, and that the humanized mouse model of CLL described here may prove

valuable in studies for screening new treatment regimes.

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3.2 Introduction

Chronic lymphocytic leukemia is a heterogeneous disease characterized by the accumulation of malignant CD5 +CD19 + B cells in peripheral blood, bone marrow, and

secondary lymphoid organs. Some patients have a benign clinical course while others die

of this disease within a short time from diagnosis. Improved understanding of the

biology of CLL may help identify other variables predicting which patients may have a

poor disease outcome.

In the previous chapter we showed that CD200, overexpressed on most CLL cells,

played a functional role in suppressing cytotoxic killing of CD200 + tumor cells.

Increased expression of CD200R, which is required for signalling mediated following

CD200 engagement, was detected on a subpopulation of CD4 + T cells in the spleen of

CLL patients relative to controls (376). Thus CD200+ CLL cells and CD200R + CD4 + T

cells appear to co-localize in the tumor microenvironment.

The CLL microenvironment is crucial for CLL survival and proliferation (159).

Non-malignant constituents of the microenvironment, mostly T cells, mesenchymal stromal cells (MSCs), and CD14 + nurse-like cells (NLCs) provide antigenic signals, cytokines, and other CLL survival factors such as BAFF, to support CLL survival and proliferation (162, 172, 190, 191). T cells in the CLL microenvironment are also known to express CD40L, which, through stimulation of CD40 on CLL cells, induces CLL proliferation and anti-apoptotic pathways (183). A number of studies have shown that

CLL cells can directly modulate T cell function by expression of cell-surface molecules and/or production of soluble factors (195, 196). Based on these data, we hypothesized

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that CD200 may be one of the important factors modulating the CLL microenvironment

(290, 291, 295).

A number of membrane-bound molecules with immunomodulatory functions are known to exist also in soluble forms where they are believed to play a functional role in a number of disease states, including cancer. Thus soluble forms of CD23, CD44, and

CD14 have been reported to augment CLL survival in vitro (377-379). We found that

CD200 could be shed from CLL cells after stimulation by PMA and TLR7 agonists,

inferring that a soluble form of CD200 (sCD200) might be present in human plasma

including that of CLL patients (chapter 1). The studies described below were designed to

investigate if soluble CD200 levels were increased in CLL patients relative to healthy

controls, and whether those levels were related to disease stage. We also investigated

whether sCD200 present in CLL patient plasma might contribute to growth/survival of

null CLL cells in NOD.SCID γc mice.

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3.3 Materials and Methods

Mice:

null NOD.SCID γc mice were bred and maintained under sterile conditions at the

Toronto Medical Discovery Tower, MaRs Centre. All mice were used at 8-13 weeks of age.

Human splenocytes and CLL cells

The spleen from consenting patients undergoing splenectomy at Sunnybrook

Health Science Centre was harvested at surgery and single cell suspensions prepared in

AIMV medium (Invitrogen, Carlsbad, CA). Cells were washed (x2), counted and stored frozen at -80°C in freezing medium (AIMV+ 40% FBS+ 10% DMSO) at a concentration of 1x10 8 cells/ml either in 1.5ml aliquots in cryovials or in 30ml aliquots in 50ml Falcon tubes. At least 10 11 cells in total were harvested from each spleen. An aliquot (10 7) of fresh splenocytes was retained for cell surface phenotype analysis in FACS. For in vivo

null studies in NOD-SCID γc mice, aliquots of splenocytes were rapidly thawed at 37°C, washed in PBS, and cell aggregates separated by centrifugation on Ficoll-Paque PLUS gradients. Cells recovered after centrifugation were resuspended in PBS at appropriate concentrations for injection.

In some experiments where cells from the peripheral blood of CLL patients were used for reconstitution, CD19 +CD5 + CLL cells were purified from fresh blood as

described previously (348). All protocols were approved by institutional review boards.

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Human plasma

Plasma from CLL patients was obtained at routine clinical follow-up and stored at

null -20°C. For in vivo studies in NOD.SCID γc mice, plasma from a group of patients at late disease stage (Rai Stage III-IV), and/or with high white cell count, were pooled into batches (>8 donors/batch). The control plasma used was pooled from a group of 10 healthy volunteers. sCD200 levels in all plasma samples were assessed by CD200

ELISA (see below). sCD200 levels in pooled normal plasma were in the range 0.5 +0.2

ng/ml while in various pooled CLL plasma batches levels were consistently ~10-fold

higher (5 ±1.3 ng/ml). Where absorbed plasma was used, the pooled CLL plasma was

absorbed overnight at 4°C with anti-CD200 (1B9)-conjugated CNBr-activated Sepharose

beads (Cedarlane, ON), a method previously shown to be effective in absorbing sCD200

from plasma (376).

Antibodies

The rat anti-hCD200 monoclonal antibodies 1B9 and 3G7 were described previously (351). 1B9 was previously shown to be effective in blocking CD200 function in vitro (376). For in vivo use Fab fragments of 1B9 were prepared using a Fab preparation kit (Thermo Fisher Scientific Inc., Rockford, IL).

The polyclonal rabbit anti-hCD200 serum, absorbed to deplete all anti-Fc reactivity, was described elsewhere (376). A rabbit polyclonal antibody specific for the extracellular region (V+C) of CD200 was generated by immunization of rabbits with protein expressed from CHO cells transfected with an expression vector encoding only

108

this extracellular domain of CD200. The immunoreactivity and specificity of both sera was characterized by Western blot analysis.

Mouse anti-CD200R1-FITC antibody was purchased from R&D systems, while all other monoclonal antibodies used for cell surface phenotype characterization (CD45,

CD19, CD5, CD20, CD40, CD23, CD38, CD49d, CD4, CD8, CD14, and CD56) were purchased from eBioscience. Rituxan (Roche Canada, Mississauga, ON) was obtained from the hospital pharmacy. OKT3, used for depletion of T cells in in vivo studies, was purchased from Ortho-McNeil Pharmaceuticals (Raritan, NJ).

CD200 sandwich ELISA:

High binding 96-well EIA/RIA plates (Corning Life Sciences) were coated with the capture anti-CD200 mAb1B9 at 1.25 g/ml overnight at 4°C in Tris-HCI, pH 8.1.

Plates were then blocked for 1 hour at room temperature with the blocking buffer, 5%

FBS in PBS, washed, and different concentrations of either pure CD200Fc (standard

curve) or plasma samples (diluted 1:4 in blocking buffer) were added. Plates were

incubated for 2 hours at room temperature, followed by 2-hours of incubation with the

detection antibody, a rabbit anti-CD200 antiserum at a 1:500 dilution. Goat anti-rabbit

IgG-HRP antibody at a 1:12,500 dilution was added and plates incubated at room

temperature for 30 minutes. 5 washings with wash buffer (PBS+0.01% Tween20) were

performed between each step. After the final wash, TMB substrate was added. All

reactions were stopped by addition of 50 l 0.2M sulfuric acid per well after 10 minute of incubation at room temperature in the dark. Plates were read at 450nm in a Multiskan

Ascent 96/384 plate reader (MTX Lab Systems, Vienna, VA).

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null Engraftment of human CLL splenocytes in NOD-SCID γc mice

On the day of experimentation, mice received 245 rads of γ-irradiation, followed

by 1x10 8 human splenocytes ip, and 0.8ml of pooled CLL plasma or control plasma, also given ip. Subsequent infusions of CLL plasma or control plasma were performed bi- weekly throughout the course of the experiment. Mice were sacrificed at various time points to assess for CLL engraftment. Spleen and bone marrow were harvested from individual animals and cells in the peritoneal cavity were recovered by flushing the peritoneum with 8ml PBS. Single cell suspensions were prepared from all 3 compartments, and cells enumerated in a hemocytometer.

For immunohistochemistry an aliquot of fresh spleen tissue was fixed in 10% formalin and slides prepared and processed by the Pathology lab at Sunnybrook Health

Science centre. Engraftment of CLL cells was analyzed by multi-color FACS staining using single cell suspensions and the various mAbs discussed above.

CD200 blockade and T cell depletion in vivo studies

For CD200 blockade experiments, mice were randomly assigned into 3 groups

after infusion of human splenocytes. Two of the three groups received sCD200 hi CLL plasma while a third group received sCD200 absorbed CLL plasma. Of the two groups that received CLL plasma one group also received 50µg (iv) of Fab anti-CD200 mAb

1B9 the day after spleen cell injection, and on two subsequent occasions at 72 hour

intervals.

110

For T cell depletion experiments mice received human splenocytes and sCD200 hi

CLL plasma, or sCD200 absorbed CLL plasma. The following day animals were

randomly assigned to receive 20 µg OKT3 (anti-CD3) antibody iv, with 2 additional infusions at 72 hour intervals.

In studies comparing the therapeutic efficiency of 1B9 and Rituxan, mice were engrafted with human splenocytes along with biweekly infusion of sCD200 hi plasma. 21 days following CLL infusion mice were randomly assigned to receive saline, 1B9

(50 µg/mouse) or Rituxan (50mg/mouse), all delivered iv in 300 µl. All treatments were

repeated at 72-hour intervals for a total of 4 treatments. Animals were sacrificed 8 days

after the last treatment.

For each in vivo experiment, mice of both sexes were used, with at least 3 mice

per group. All experiments were repeated individually with splenocytes from at least two

different patients.

FACS analyses

CD200 cell surface staining was performed using a rat anti-CD200 mAb (3G7) as previously described (376). Multi-color FACS analyses were performed to characterize engrafted human cells. The optimal concentration of antibody for staining was determined individually for each antibody. Single color controls were included in each experiment for compensation purposes, and all samples were analyzed in a Coulter

FC500 flow cytometer.

111

Statistics

Spearman’s Rank Correlation test and a Man-Whitney U test were used to determine the correlation between sCD200 levels and various clinical markers in CLL.

All clinical analyses were done using SPSS Statistics software. For in vivo studies the absolute count of each engrafted cell population (CLL or T cells) was calculated from

[total cell count x frequency], with frequency based on FACS staining profiles. Unpaired t-tests were used to determine significance between sample means. Analysis of in vivo studies was performed using GraphPad Prism 5.0 software.

112

3.4 Results

Identification of sCD200 in plasma from CLL patients

CD200 is normally considered a membrane molecule (275). We have previously

shown that CD200 was shed following activation of CLL cells in vitro , suggesting it might also exist in a soluble form in the plasma (351, 376). To explore evidence for a plasma form of CD200 (sCD200), we established a sandwich ELISA using 1B9 as capture antibody and a rabbit anti-hCD200 detection polyclonal antibody as described in the Materials and Methods.

The sensitivity of this ELISA using pure CD200Fc protein as standard was found to be

0.05ng/ml. CD200Fc was used to generate standard curves in the range 0.05-10ng/ml for quantitation of sCD200 in samples in all ELISA studies.

sCD200 levels in plasma from 25 healthy controls ranged from 0.4±0.2ng/ml.

These levels were independent of age (20-64 years) or gender (data not shown). Elevated sCD200 levels were observed in plasma samples from CLL patients across all clinical stages (Fig 3-1a).

Plasma sCD200 levels are associated with disease stage in CLL

While CD200 expression was detected on CLL cells from all individuals in a cohort of 25 patients at various stages of disease, the cell surface expression level was not correlated with either tumor burden or other clinical parameters of disease (376). To determine whether sCD200 levels correlated with CLL clinical parameters, plasma samples obtained at diagnosis from 82 CLL patients were tested in the sandwich ELISA for sCD200 levels. The patient age in this cohort ranged from 38-91 (median age 61).

113

Correlation analyses were performed to compare sCD200 levels with other parameters linked to clinical outcome in CLL (see Table 3.1 for patient details).

Independent of the expression of CD200 on the cell surface, sCD200 levels were correlated with tumor burden, with patients in later stages of disease (Rai stage III and

IV) having significantly higher sCD200 levels than patients at early stages (Fig 3-1b). In general clinical treatment of CLL is reserved for patients with late disease and/or rapidly progressive disease (1). Using the number of treatments received by each patient in our cohort, regardless of the nature of that treatment, as a surrogate marker of aggressive disease, we pooled patients into groups who had received two or more courses of treatments vs. those with no (or only 1 course of) treatment. The former had significantly higher serum sCD200 levels than those with more indolent disease as defined by no treatment ( p<0.0001, Fig 3-1c) or only 1 treatment ( p=0.0027, Fig 3-1c).

Of the conventional prognostic markers for CLL, sCD200 levels correlated most strongly with serum β2 microglobulin levels ( p<0.0001, Fig 3-1d). CD38 expression levels did not correlate with sCD200 levels (18). The presence of intermediate- to high- risk cytogenetic abnormalities (trisomy 12 or deletions of regions of chromosomes 11 or

17) also did not correlate with sCD200 levels. However, patients with CLL cells that exhibited either a normal karyotype or 13q deletions, usually considered to be indicative of a more benign clinical course, were treated more often if they also had high levels of sCD200 (Spearman’s r=0.639, p<0.0001, n=37).

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Development of a xenograft model for CLL

Given that sCD200 levels were higher in patients with late stage and/or aggressive

disease, we hypothesized that sCD200 in CLL plasma might play a role in vivo in

fostering CLL growth. In a series of preliminary studies we infused 1x10 8 purified

circulating PBL-derived CLL cells from 5 individual patients with high white cell counts

null into each of 4/group NOD-SCID γc mice, with subsequent bi-weekly infusion of pooled sCD200 hi CLL plasma, or sCD200 lo normal plasma pooled from healthy volunteers.

Although animals receiving CLL plasma had greater engraftment of CLL cells than

animals receiving normal plasma, the number of human cells recovered was generally

low (data not shown), consistent with other reports in the literature (230, 231). We

considered that a possible explanation for this poor engraftment might be the absence of

supporting cells that would be present in proliferation centers but not in the blood.

In an attempt to provide a proposed “microenvironment factor”, we next

attempted reconstitution of mice with splenocytes harvested from CLL patients (Table

3.2) (380). The cellular composition of each spleen sample was analyzed by FACS. In

all cases, CD19 +CD5 + CLL cells were the predominant cells in the spleen, although the frequency of CLL cells varied widely in a range from 25%-95% (Fig 3-2a: 4 representative spleens). Expression of CD20 and CD40 on CLL cells also varied, while

CD200 was expressed on CLL cells from all spleens tested (Fig 2a, lower panel; CD40 staining not shown). CD4 + T cells were the next most common population detected, with frequencies ranging from 3%-20% (Fig 3-2b). CD8 + T cells and CD56 + NK cells were also detectable (Fig 3-2b, data not shown). Low levels of CD14 + cells, previously

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reported to produce BAFF to support CLL survival in vitro , were found in all spleens studied (data not shown) (175).

Recapitulating the observation made using CLL cells engrafted from PBL of patients, superior engraftment of CD19 + CD5 + CLL cells at 28 and 54 days after infusion of CLL splenocytes was seen in animals receiving CD200 hi CLL serum compared with controls receiving CD200 lo normal human serum (Fig 3-3a, day 28; Fig 3-3b, day 54).

This difference was particularly pronounced in the peritoneal cavity compartment

(p=0.003, Fig 3-3b). As noted, splenocytes produced greater engraftment of CLL cells in

null both the spleen and peritoneal cavity of NOD-SCID γc recipients compared to engraftment seen using PBL (data not shown). Substantial patient-to-patient variability was observed in engraftment of both CLL and T cells (Fig 3-3c, engraftment of spleens 1 and 5 shown). Accordingly in all subsequent studies described detailed analysis was restricted to use of frozen aliquots of splenocytes from only 3 patients (spleens 1, 5, and

6)

Characterization of CLL in humanized NOD-SCID γγγcnull mice

H&E staining of spleen tissue taken from mice at day 54 showed aggregates of

small-lymphocytes resembling proliferation centers by H&E staining that were absent in

control animals (Fig 3-3d, upper panel). CD20 + CLL cells were also more abundant in the spleen of experimental animals by immunohistochemistry (Fig 3-3d, middle panel).

All CLL cells found in the peritoneal cavity and spleen of both experimental and control groups expressed CD40, CD38, and CD200 at similar levels to the starting population

(data not shown).

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Splenic CLL cells co-localized with T cells by immunohistochemistry, reminiscent of the pattern observed in proliferation centers and in the spleen of CLL patients themselves (Fig 3-2b and 3-3d) (161). Note that T cells were found in the spleens of mice injected with normal plasma, although CLL cells did not engraft in this case (Fig 3-3d, lower panel). Interestingly, the ratio of CD4 + and CD8 + T cells engrafted

in the spleen (~2:1 at week 4 –see Fig 2a), is within the range seen in normal individuals,

but not typical of that observed in CLL (~1:3). The explanation for this discrepancy

remains to be determined (see later).

Engraftment of cells other than CLL and T cells was minimal regardless of the

source of plasma. In a study monitoring longer-term engraftment, in which biweekly

infusion of CLL plasma was continued to 1 week before experimental endpoint, we

observed persistence of CLL cells in both peritoneal cavity (PC) and spleen at 75 days

post spleen cell infusion (Fig 3-3e). Cells harvested from these animals were additionally

stained for the proliferative marker ki67, with a finite percentage of ki67 + cells detected in the peritoneal cavity. The majority of these ki67 + cells (>70%), were CD19 +, reflecting

ongoing proliferation in the CLL cells in this locale (Fig 3-3e). By contrast, in the spleen

of the same animals, the majority of ki67 + cells were CD19 -CD5 +, presumably T cells

(data not shown).

Anti-CD200 and OKT3 mAbs abrogate engraftment of CLL in NOD.SCID mice

To assess whether sCD200 in CLL plasma was an important factor contributing to engraftment of CLL cells in vivo , animals receiving CLL splenocytes and sCD200 hi

serum also received Fab anti-CD200 mAb 1B9. An independent group of mice received

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CLL splenocytes and CLL plasma that had been depleted of CD200 by passage though an anti-CD200 CNBr column. sCD200 absorption from the serum was confirmed independently by ELISA (>97% depletion). Both anti-CD200 mAb and depletion of sCD200 from plasma attenuated the engraftment of CLL cells in vivo (Fig 3-4a).

CD200 is known to deliver downstream signals through a receptor CD200R, and we had previously reported that CD200R was detected mostly on splenic CD4 + T cells

but not on CLL cells (275, 376). The absence of CD200R on CLL cells has been

independently confirmed by RT-PCR (data not shown). We investigated the role of T

cells in CLL engraftment by treating mice in the early period post spleen reconstitution

with OKT3 antibody in vivo , analysing CLL and T cells 4 weeks later. As shown in Fig

3-4b, in vivo depletion of T cells abrogated engraftment of CLL cells, despite continuous infusion of sCD200 hi CLL serum.

Comparison of anti-CD200 and rituximab in eliminating engrafted CLL cells

null In a final study we asked whether NOD.SCID γc mice reconstituted with human

CLL-spleen cells could be used as a pre-clinical model to test potential therapy for CLL.

Specifically we compared the efficacy of Rituxan, a clinically approved monoclonal antibody targeting CD20 on CLL cells, with anti-CD200 mAb therapy as treatment of mice with established spleen-cell derived CLL engraftment (381).

At 28 days following CLL splenocyte injection and biweekly sCD200 hi CLL serum infusion, independent groups of mice received Rituxan (50ug/mouse) or Fab anti- mouse CD200 mAb. A total of 4 injections were given, at 84hr intervals over 2 weeks.

Animals were maintained for 10 days after the last dose of treatment, again with ongoing

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injection of CLL serum on a biweekly basis, and were sacrificed at day 45. FACS analyses on cell suspensions harvested from the spleen and peritoneal cavity of these animals showed that both rituxan (p=0.0026) and anti-CD200 mAb (p=0.0057) were

effective in reducing CLL engraftment in both tissue compartments (Fig 3-5a and b)

without affecting engraftment of T cells in either compartment (Fig 3-5c). Essentially all

CLL cells engrafting in the peritoneal cavity were depleted, with significant attenuation

of engraftment in the spleen (>70%).

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3.5 Discussion

The studies described in this chapter implicate a novel soluble form of CD200

(sCD200) in the pathogenesis of CLL. sCD200 is present at high levels in CLL plasma compared with normal plasma and promotes the growth of CLL cells in immunodeficient mice, suggesting it may be a novel therapeutic target for this malignancy.

CD200 expression has been linked with outcome in many malignancies (281, 295,

298). Biochemical analysis has suggested that sCD200 is likely shed from the cell surface by proteolytic cleavage, with the ADAM family of proteases being prime candidates in this mechanism (see chapter 4). Ectodomain shedding mediated by ADAM proteases is an important mechanism by which a number of membrane-bound immunoregulatory molecules are released from the cell surface. CD23, whose levels are used as a prognostic factor for CLL, is shed by members of the ADAM family (336,

338). We found that sCD200 levels (but not surface expression of CD200 on CLL cells) correlated strongly with tumor burden, disease stage, and an aggressive disease course as reflected in the requirement for multiple treatments. Plasma sCD200 was also correlated with plasma β2 microglobulin levels, known to be a predictor of progression-free survival and overall survival in CLL (382, 383). The value of plasma sCD200 as an independent prognostic marker against current clinical prognostic markers remains to be determined.

The hypothesis that sCD200 is important in CLL disease is supported by evidence for its contribution to successful engraftment of spleen (and PBL)-derived CLL cells in

null vivo in NOD.SCID γc mice. PBL-derived CLL cells generally fail to engraft in immunocompromised hosts, although some engraftment was recorded following

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combinations of ip and iv infusion of PBL-derived CLL cells or following injection of a

EBV + transformed CLL cell line (234, 238). Quantitative comparison of engraftment

using spleen or PBL from the same patient has not been performed to assess engraftment

potential of CLL cells from these two tissue sources. Nevertheless, it is clear that

sCD200 hi CLL plasma augments engraftment of CD19 +CD5 + CLL cells in both the peritoneal cavity and spleen, in contrast to the inferior engraftment seen using sCD200 lo

plasma pooled from healthy volunteers with ten-fold lower sCD200 levels (~ 0.5ng/ml).

Those CLL cells which engraft in both sets of animals express the markers CD20, CD40,

CD23, CD38, CD49d and CD200 at levels similar to the CLL cells from the starting

spleen population, suggesting no selection for survival of unique subpopulations occurred in vivo under these conditions (data not shown). Engrafted CLL cells also expressed

CD200.

Engraftment of CLL cells in a xeno-microenvironment often reflects persistence of donor cells, rather than proliferation of CLL cells in the host, and engrafted CLL cells gradually decline with time (230, 231, 234). The use of EBV-transformed CLL cell lines circumvents this issue, though these lines often exhibit very different biologic properties from those of primary cells (237, 238). We did not observe a significant decline in the absolute number of engrafted CLL cells from day 25 to day 75, and indeed, the majority of Ki67 + CD5 + cells in the peritoneal cavity at day 75 were CD19 + CLL cells. We

conclude that CLL engraftment in our model reflects both survival and proliferation of

CLL cells. Since the engrafted CLL cells continue to express CD200 at high levels, it is

possible that sCD200 may be released in ongoing fashion from grafted CLL cells in

vivo —this hypothesis is supported by recent findings from assaying sCD200 levels in

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mice at >6 months post-reconstitution (unpublished data). We have not investigated whether continual infusion of sCD200 hi CLL plasma is needed to sustain CLL

engraftment, or whether the engrafted CLL cells produce sufficient amount of sCD200 in vivo to maintain themselves in the absence of exogenous sCD200.

Normal plasma and CLL plasma differ significantly in their content of multiple molecules, including many proteins and fatty acids. A number of such molecules, including the B cell growth factors BAFF and APRIL, and soluble CD14, have direct effects on CLL cells (377, 384, 385). While we do not rule out a contribution by other factors besides sCD200 in the support of CLL growth, the fact that pre-absorption of sCD200 from CLL plasma minimized CLL engraftment supports the hypothesis that sCD200 is a key contributor to CLL engraftment. Our data also supports a role for T cell involvement in this mechanism of action, since T cell depletion (OKT3 in vivo ) abrogated

CLL engraftment, despite continuous infusion of sCD200 hi CLL plasma. This may help

explain why, in preliminary experiments, infusion of purified CLL cells alone, even with

continuous sCD200 hi CLL serum supplement, produced only minimal engraftment of

CLL cells.

The role of non-malignant T cells in CLL has been investigated by a number of groups (236, 386, 387). T cells harvested from CLL patients differ from normal T cells by their high production of IL4 and reduced expression of co-stimulatory molecules

(387). The observation that CLL cells fail to engraft in the absence of T cells despite sCD200 infusion suggests T cells, a subpopulation of which have previously been shown to express CD200R, may represent an important target of sCD200 (376). Whether

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sCD200-targeted CD200R + T cells affect CLL growth directly, or indirectly through the action of other T or non-T populations, remains unknown.

Studies comparing the efficacy of anti-CD200 mAb or Rituxan as therapy for

null NOD.SCID γc mice reconstituted with CLL splenocytes showed both were effective in

treating pre-existing disease (388). Rituxan induces lysis of CLL cells by complement

dependent and antibody-dependent cell mediated cytotoxicity, while Fab anti-CD200

1B9, likely regulates host resistance to growth (389). Whether there is a role for

synergism in their action remains to be determined.

In summary, our data suggests that sCD200, present in patient plasma, is

associated with disease progression in CLL patients, and its presence can be utilized to

establish a novel xenograft model for CLL which may be useful for preclinical testing.

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3.6 Tables

Table 3.1: Clinical characteristics of patients in plasma sCD200 analyses

▪ Rai % Pt ID Age Sex Stage ♦ WBC CD38+ ◊ β2M ▼ Cytogenetics ●Treatments 1 61 F 4 225 17 7 13q-,17p- Fx2,CP, FC 2 68 M 1 25 9 NA NA None 3 72 M 4 175 2 5.7 13q-/17p- S/CHOP/CVP 4 84 F 3 200 46 NA 13q- P/C,S/R 5 62 M 4 121 2 2.2 13q- CP,CVP,S,FC 6 66 M 0 21 1 1.2 NA None 7 71 M 4 25 2 NA T12, 11q- CVPx2,Splen,FC 8 74 M 4 220 Na NA 17p- FR,splen, S 9 77 M 4 10 40 NA T12 C/P, Splen,FC 10 71 M 4 54 5 NA 13q- CVP 11 72 F 0 32 4 NA 13q-,17p- None 12 55 M 4 23 81 2.3 13q-,17p- CHOP,P 13 57 M 4 22 7 1.4 13q-,17p- CP,CHOP,FCx3,S 14 71 M 2 186 72 NA 11q-,13q- None 15 51 F 4 22 7 NA 17p- Splen 16 69 M 4 189 4 NA Normal CP 17 55 F 3 55 10 NA 13q- CP,FCR 18 88 M 4 35 71 NA 11q-,13q- CVP,FC 19 57 F 4 50 21 8.1 17p- FCR,DHAP, S, R 20 58 F 3 55 9 NA NA CP 21 48 F 4 6 62 NA Monosomy 11, 17p- CP,FC, CHOP 22 54 M 4 8 1 4.3 Normal CP,FC,FCR,S 23 53 M 3 4 10 2.1 T12 CVP,FC 24 73 F 4 100 2 4.6 13q- Cx4,Fx3 25 56 F 4 8 78 NA 13q- Cx3, Fx2, CHOP 26 48 F 3 17 72 NA T12 CP,FCR 27 82 F 3 125 8 NA T12 None 28 62 F 3 110 1 NA 13q- Splen,CP 29 67 M 2 25 1 2.6 13q- None 30 66 M 4 122 54 NA 13q- Splen,CP 31 69 M 1 120 8 2 Normal None 32 51 M 0 18 6 NA Normal None 34 58 M 4 70 19 5.8 Normal FC, S,R 35 64 M 2 20 50 2.8 11q-,13q- FCR 36 59 M 4 10 7 2.7 NA CP 37 58 M 2 25 25 NA Normal None 38 55 M 4 135 18 2.9 T12 CPR 39 74 M 3 185 25 3.7 13q- P 40 52 M 0 42 4 NA 13q- None

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Table 3.1 continued

▪ Rai % Pt ID Age Sex Stage ♦ WBC CD38+ ◊ β2M ▼ Cytogenetics ● Treatments 41 59 F 3 35 40 1.7 Normal CP,Splen 42 89 F 1 25 na NA NA P 44 61 F 3 25 51 2.6 Normal CP 45 60 M 4 100 2 6.1 NA Fx2,CP,Revl,CHOPR 46 55 M 3 121 4 3.1 T12 None 47 61 M 0 33 5 1.4 13q- None 48 70 M 4 25 na NA NA None 49 60 M 4 61 30 NA Normal None 51 80 F 0 15 14 2.8 NA None 52 91 F 0 18 28 2.8 T12 P 53 61 M 3 22 91 NA 13q-,T12 None 54 64 F 2 22 na NA T12 None 55 63 F 0 23 1 1.5 13q- None 56 62 F 0 10 1 1.6 NA None 57 77 F 4 120 na 8.5 13q- CP 58 54 F 4 60 3 2 13q- None 59 68 F 3 85 53 2.4 T12,17p- None 60 88 M 4 62 1 1.7 13q-,17p- None 61 81 M 3 89 1 11.3 Normal P 62 61 F 4 165 1 3.4 13q- CP 63 62 M 4 12 71 4.3 13q-,11q- CP/FC 64 69 F 4 81 na 3.6 Normal CP,CVP,FC 65 61 F 4 140 18 NA 13q-,11q- Splen 66 60 F 3 6 6 5.7 13q-,T12 CP,FC,FCR 67 57 F 3 10 10 NA 13q- CP 68 62 M 1 20 11 1.1 13q- None 69 47 M 2 65 1 NA 13q- Splen 70 78 F 3 33 13 2.2 13q- None 71 57 M 2 21 1 1.9 T12,13q- None 72 48 F 0 13 2 1.5 NA None 73 53 F 4 65 2 NA 13q- None 74 77 M 3 250 1 2.7 13q- None 75 63 F 2 22 71 1.7 NA None 76 58 F 2 17 2 1.1 NA None 77 38 M 2 37 63 NA Normal None 78 72 M 4 80 13 3.8 Normal CP,FCR 79 51 M 4 140 1 3.1 13q- Cx2 80 51 M 2 88 2 2 13q- None 81 64 M 3 160 12 3.3 11q- CPx2 82 74 M 2 45 2 3.5 11q- None

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Table 3.2: Clinical characteristics of patients whose spleens were harvested and used in in vivo studies

Spleen # Pt ID Sex Age Time (yrs) ▪ Rai Stage ♦ WBC % CD38 ◊ β2M ▼ Cytogenetics ◙ Treatments ▲ AIHA

1 102 M 72 5 III 88 40 5 13q CP, steroids Yes

2 113 M 42 3 IV 76 4 3 13q none No

3 114 M 60 10 IV 300 2 4 13q none No

4 125 M 72 5 IV 25 45 6 17p,13q FC, HDP No

5 153 M 71 15 IV 120 44 3 13q CVP, HDP Yes

6 154 M 64 22 III 75 3 3 11q Revl, CP yes

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Footnotes to tables:

Table 3.1:

▪ Rai stage 0: lymphocytosis; I: with adenopathy; II: with hepatosplenomegaly; III: with anaemia; IV: with thrombocytopenia. ♦ WBC: White blood cell count (x10 6 cells/ml) in peripheral blood. ◊ β2M, Plasma β2Microglobulin level, mg/L. ▼ T12, trisomy 12; NA, not available. ● CVP, cyclophosphamide/vincristine/prednisone; CHOP, cyclophosphamide/vincristine/doxorubicin/prednisone; DHAP, dexamethasone/ cytarabine/cisplatin; FC, fludarabine/cyclophosphamide; C, Chlorambucil; P, prednisone; F, fludarabine; R, rituximab; Revl, revlimid (lenalidomide); S, solumedrol; Splen, splenectomy

Table 3.2:

◙ Treatments received before splenectomy: C, Chlorambucil; P, prednisone; CVP, cyclophosphamide/vincristine/prednisone; FC, fludarabine/cyclophosphamide; HDP, high dose prednisone; Revl, revlimid (lenalidomide). ▲ AIHA= documented autoimmune hemolytic anemia

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3.7 Figure legends

Figure 3-1: Identification of sCD200 and clinical analysis of plasma sCD200 in CLL sCD200 levels in plasma samples from age-matched healthy controls (n=27) and CLL patients in clinical cohort (n=77) were measured by ELISA. All p-values were obtained from Mann Whitney U test unless specified otherwise. a) Plasma from CLL patients at various clinical stages of disease showed significantly higher levels of sCD200 than plasma from healthy controls ( p<0.001). b) CLL patients with Rai stage III ( p=0.015) and IV

(p=0.002) diseases showed higher plasma sCD200 levels than patients with early disease

(Stage 0-I). No significant difference in plasma sCD200 level was found between patients at Stage 0-I and Stage II. c) Patients requiring more than 2 courses of treatments had higher plasma sCD200 levels than patients with more indolent disease requiring no ( p<0.0001) or

1 ( p=0.0027) treatment. d) Plasma sCD200 levels strongly correlated with plasma β2 microglobulin levels ( p<0.0001, Spearman’s Rank Correlation test).

Figure 3-2: Characterization of CLL splenocytes harvested from splenectomised patients (Table II)

2x10 6 fresh CLL splenocytes were characterized for relative frequency of CLL (CD20,

CD19, CD5, and CD200) and T cells (CD4 and CD8) in multicolour FACS analyses.

Results from 4 representative spleens are shown (Sp 1, 4, 5, and 6). a) Frequency of

CD19 +CD5 + CLL cells from the 4 spleens ranged from 23%-90% (upper panel).

CD19 +CD5 + CLL cells were gated and analyzed for CD20 and CD200 expression, the results of which are shown in the lower panel. CLL cells from all 4 patients stained brightly for CD200, while CD20 expression varied among the patients (lower panel). b)

Distribution of CD4 + and CD8 + T cells in the same patient spleens as a). Frequency of

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CD4+ T cells ranged from 3%-17% while frequency of CD8+ T cells ranged from less than

0.5% to over 10%.

null Figure 3-3: Engraftment of human CLL cells and T cells in NOD.SCID γc mice

null 8 Irradiated NOD.SCID γc mice at 8-14 weeks of age were infused with 1x10 thawed CLL splenocytes ip, followed up biweekly infusion of sCD200 lo normal plasma, pooled from age-matched healthy volunteers, or sCD200 hi CLL plasma. Mice were scarified at designed time points to assess for CLL and T cell engraftment. p-value was calculated from unpaired t-test. a) FACS analysis of CLL (upper panel) and T cell (lower panel) engraftment in the peritoneal cavity of animals given either sCD200 hi plasma or sCD200 lo normal plasma at day 28. Spleen 1 was used in this experiment; result from 1 representative animal per group is shown. b) Absolute count (x10 5 cells) of CD19 +CD5 +

CLL cells in the peritoneal cavity of animals that received either sCD200 hi plasma or sCD200 lo control plasma, in addition to spleens 1 or 5, at day 54 (data from two independent experiments were pooled). CLL cell counts were obtained by multiplying total cell count with % CD19 +CD5 + cells as found in FACS. Mice that received sCD200hi plasma showed elevated engraftment of CLL cells in comparison to mice that received sCD200 lo control plasma at this time point ( p=0.003). c) Comparison of CLL and T cell engraftment in mouse peritoneal cavity (upper panel) and spleen (lower panel) at day 54 by

CLL splenocytes from Sp1 and Sp5. Sp1 appeared to engraft at higher frequencies than

Sp5 in both compartments. CD4 + and CD8 + T cells from the two different spleens engrafted at similar frequencies. d) Immunohistochemical analysis of mouse spleens at day

54. Upper panel: H&E staining; middle panel: CD20 staining; lower panel: CD3 staining. e) Ki67 staining on CD19 +CD5 + CLL cells and CD19 -CD5 + non-CLL cells engrafted in

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mouse peritoneal cavity at day 75. Ki67 + cells were gated according to CD5 staining: over

70% of Ki67 +CD5 + cells were CD19 + CLL cells; whereas Ki67 +CD5 - cells did not express any human markers (data not shown) and were likely proliferating mouse cells. Results from 1 representative animal are shown in d) and e).

Figure 3-4: Effects of sCD200 and/or T cell depletion on CLL engraftment in null NOD.SCID γc mice at day 21

null 8 a) Irradiated NOD.SCID γc mice were infused with 1x10 CLL splenocytes, and then given either sCD200 hi CLL plasma or sCD200 absorbed CLL plasma. A separate group of animals were given 1B9, an anti-CD200 mAb, in addition to sCD200hi CLL plasma for in vivo sCD200 depletion. Both methods of sCD200 depletion effectively reduced CLL

null engraftment in peritoneal cavity. b) Irradiated NOD.SCID γc mice were infused with

1x10 8 CLL splenocytes, and either sCD200hi CLL plasma or sCD200 absorbed CLL plasma. Both groups of animals were subdivided to receive either OKT3 iv for T cell in vivo depletion, or control saline. OKT3 was given a total of 4 times in two weeks. T cell depletion appeared to abrogate CLL engraftment regardless of the presence of sCD200 in supplemented plasma. Data from 1 representative experiment using sp1, with engraftment of CLL cells in mouse peritoneal cavity, is shown (mouse spleens showed similar engraftment pattern). In both a) and b) CLL engraftment was assessed by CD19, CD5, and

CD200 staining. Error bars represent standard deviation in cell counts within each group (3 animals per group).

Figure 3-5: Therapeutic efficacy of Rituxan and 1B9 on CLL engraftment in null NOD.SCID γc mice

null 8 Irradiated NOD.SCID γc mice were engrafted with 1x10 CLL splenocytes and infused biweekly with sCD200 hi CLL plasma. At day 21, mice were divided into 3 groups and

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given one of rituxan, 1B9, or saline iv 4 times in 2 weeks. Engraftment of CLL and T cells was assessed at day 45. p-value was calculated from unpaired t-test. Both Rituxan and

1B9 were effective in eliminating CLL engraftment in both a) peritoneal cavity ( p=0.0057) and b) spleen without having significant effect on c) CD3+ T cells. Results were pooled from two independent experiments engrafted with splenocytes from Sp1 and 5.

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3.8 Figures

Figure 3-1: Identification of sCD200 and clinical analysis of plasma sCD200 in CLL

3-1a)

sCD200 level in healthy controls vs CLL patients 5

4

3

2 ng/ml sCD200 ng/ml

1

0 Healthy controls CLL patients

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3-1b)

3-1c)

133

3-1d)

β2-Microglobulin

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Figure 3-2: Characterization of CLL splenocytes harvested from splenectomised patients

3-2a)

Sp1 Sp4 Sp5 Sp6 CD5 CD19 CD200

CD20

3-2b)

Sp1 Sp4 Sp5 Sp6 CD4 CD8

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Figure 3-3: Engraftment of human CLL cells and T cells in NOD.SCID γcnull mice

3-3a)

sCD200 lo normal serum sCD200 hi CLL serum CD19 CD20 CD4 CD8

136

3-3b)

Frequency (#) of engrafted CD19+CD5+ CLL cells in peritonael cavity at day54

p=0.003 1.5

1.0 cells 5 x10 0.5

0.0 sCD200 hi CLL plasma sCD200 lo normal plasma

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3-3c)

Sp1

Sp5 CD8-PECy7 CD19-PECy7 CD5-PECy5 CD4-PECy5

138

3-3d)

H&E: CLL plasma Control plasma

CD20:

CD3:

139

3-3e)

Human CD5 + cells CD19 CD5 CD5

Ki67 Human CD5 - cells CD19 CD5

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Figure 3-4: Effects of sCD200 and/or T cell depletion on CLL engraftment in NOD.SCID γcnull mice at day 21

3-4a)

141

3-4b)

142

Figure 3-5: Therapeutic efficacy of Rituxan and 1B9 on CLL engraftment in NOD.SCID γcnull mice

3-5a)

Frequency (#) of engrafted CD19+CD5+ CLL cells in peritonael cavity at day45

p=0.0026

p=0.0057 0.6 Untreated Anti-CD200 Rituxan 0.4 cells 5 x10 0.2

0.0 Untreated Anti-CD200 Rituxan

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3-5b)

Spleen #1 (Pt.102) Spleen #5 (Pt.152)

Untreated

Anti-CD200

Rituxan

144

3-5c)

Spleen #1 (Pt.102) Spleen #5 (Pt.152)

Untreated

Anti-CD200

Rituxan

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Chapter 4: Ectodomain shedding of CD200

(Manuscript submitted to J. Immunology, April, 2012)

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4.1 Abstract

We have previously reported the existence of a soluble form of CD200

(sCD200) in human plasma, and found sCD200 to be elevated in the plasma of CLL patients. In CLL, plasma sCD200 levels correlated with disease stage and response to treatment. We have now explored whether ectodomain shedding mediated by MMPs and

ADAM proteases is at least in part responsible for sCD200 using purified CLL cells and

Hek293 cells stably transfected with CD200. CD200 was released spontaneously from

CLL cells, and this was attenuated by treatment with GM6001, a global protease inhibitor, or with the tissue inhibitors of metalloproteases TIMP1 and TIMP3. PMA stimulation enhanced CD200 shedding by both CLL cells and Hek293 cells stably transfected with

CD200, as shown by a decline in CD200 detected by FACS analysis and Western blotting of membrane extracts from PMA treated cells. The PMA-induced CD200 shedding was inhibited by TAPI-0, a metalloprotease inhibitor, and the loss of cell surface CD200 occurred in parallel with an increase in the detection of sCD200 in the CLL supernatant.

Moreover, Western blot analysis and functional studies using CD200R1 expressing Hek293 cells showed that the shed CD200 as detected in CLL and Hek293-hCD200 supernatants lacked the cytoplasmic domains of CD200 but retained the functional extracellular domain required for binding to CD200R. These data support the hypothesis that CD200 is cleaved from the surface of CLL cells by a process of ectodomain shedding.

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4.2 Introduction

Cancer immunotherapy is limited by the immunosuppressive nature of tumor cells and their microenvironment, often the result of overexpression of immunoregulatory molecules by both tumor cells and tumor-infiltrating effector cells (266). CD200, a type-I transmembrane molecule with potent immunosuppressive functions through interaction with its receptor, CD200R1, is one such molecule whose expression on lymphoma cells has been shown to dampen their killing by allogenic cytotoxic lymphocytes in vitro (376).

In addition to expression on the cell surface, many of these immunoregulatory molecules have also been shown to exist in soluble form (390-392). The soluble form of these cell-surface receptors and ligands may be generated by alternative splicing at the mRNA level, as is in the case of CTLA-4, or by mechanisms of ectodomain cleavage by

MMPs and ADAM family of proteases (393, 394).

Ectodomain shedding is an important mechanism by which proteolytic cleavage of membrane-anchored molecules at the cell surface leads to the release of a soluble form of the molecule into the extracellular microenvironment (393). Ectodomain shedding plays an important role in the control of immune responses by regulating the release of cytokines, chemokines, cytokine receptors, and many membrane-anchored immunoregulatory molecules (395, 396). CD23, CD62L, and CD44, which are amongst the molecules shed by lymphocytes, are known to be substrates of ADAM8, ADAM10, ADAM17, and MT1-

MMP (337, 397-399). In Chronic Lymphocyte Leukemia, the detection of a soluble form of the NKG2D ligands, CD23, and CD14 in patient plasma has been shown to have prognostic value (400-404).

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In the previous chapter, a novel, soluble form of CD200 was identified in CLL plasma using a CD200 sandwich ELISA. Soluble CD200 (sCD200) was detected in normal human plasma and levels were increased in the plasma of CLL patients, where sCD200 levels were correlated with tumor burden, late stage disease, and disease aggressiveness. A functional significance of high sCD200 levels in CLL plasma was inferred from its ability to enhance

null engraftment of splenic human CLL cells in vivo when NOD.SCID γc mice were supplemented with sCD200 hi CLL plasma. Engraftment was attenuated when sCD200 was pre-absorbed from the plasma or when the mice were treated with an anti-CD200 mAb

(405).

In this chapter, studies designed to explore mechanisms leading to the release of

CD200 from the surface of CLL cells are reported. The data support a role for MMPs and

ADAM proteases in CD200 shedding mediated by ectodomain cleavage.

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4.3 Materials and Method

Cells

Peripheral blood from CLL patients were collected at routine follow-up visits and

CD19 +CD5 + CLL cells were purified using the RosetteSep human B cell enrichment cocktail (StemCell Technologies, Vancouver, BC) as described previously (376). Purified

CLL cells were cultured in AIMV medium (Invitrogen, Carlsbad, CA) supplemented with

5x10 -6M β-mercaptoethanol (2-ME) (Sigma).

Two Hek293 cell-lines permanently transfected with full-length hCD200 (Hek- hCD200) and hCD200R1 (Hek-hR1), respectively, were obtained from Genetec (376).

Cells were grown in a selection medium DMEM-F12 supplemented with 1ug/ml G418 and

10%FBS.

Reagents and antibodies

Phorbal 12-myristate 13-acetate (PMA) and Ionomycin were purchased from Sigma-

Aldrich. PMA was reconstituted to 10mg/ml stocks in DMSO and was further diluted to a working concentration of 40ng/ l in AIMV medium. Imiquimod, a TLR7 agonist, was purchased from LKT Laboratories (St Paul, MN) and reconstituted to 1mg/ml in DMSO.

Recombinant TIMP1, TIMP2, and TIMP3 were purchased from R&D Systems and were reconstituted to working concentrations in AIMV medium. The protease inhibitors

GM6001 and TAPI-0 were purchased from Calbiochem and reconstituted to 10mM and

1mg/ml stock, respectively, in DMSO.

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The monoclonal rat anti-hCD200 antibodies 1B9 and 3G7, and the polyclonal rabbit serum against the extracellular region of CD200 (CD200v+c), were described previously

(376). A polyclonal rabbit serum against the human CD200 receptor (CD200R1) was generated by immunization of rabbits with a fusion protein containing the extracellular region of human CD200R1 with a his-tag at the N-terminal.

Antibodies against CD19 and CD62L used in FACS analyses were purchased from

Biolegend. The apoptosis detection kit for staining of Annexin V and 7AAD was purchased from BD Biosciences. The Pan-Cadherin antibody, used as a plasma membrane marker for loading controls in Western blots, was purchased from Abcam.

Generation of a rabbit polyclonal serum against the cytoplasmic region of CD200

A peptide containing the 19-amino acids of the carboxyl-terminal domain of human

CD200 ( KRHRNQDRGELSQGVQKMT ) was synthesized at the Hospital for Sick Children and used to immunize rabbits (Cedarlane).

The resulting anti-serum (rabbit anti-CD200 c-tail serum) was confirmed to react with the peptide in an ELISA using pre-immune sera as negative control. To confirm specificity for the cytoplasmic domain of CD200, the anti-serum was tested with cell lysates from Hek-hCD200 cells or cells expressing only the extracellular domain of CD200 on Western blots.

Constitutive release of CD200 from CLL cells

To assess constitutive release of CD200, CLL cells were cultured in AIMV medium+2x10 -5 2-ME at 8x10 6/ml in 500 l volume in 24-well plates, with or without the

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global protease inhibitor GM6001 at 20 M final concentration, immediately after isolation from fresh blood. Supernatants were collected at 24 and 48 hours and were analyzed in a

CD200 sandwich ELISA as described below.

Where recombinant TIMPs were used, 1.5x10 6 CLL cells were plated in 96 well- plates in 300 l AIMV medium+2x10 -5 2-ME. All 3 recombinant TIMPs (TIMP1, 2, and 3) were used at a final concentration of 2.5 g/ml.

Stimulation of CLL cells:

Purified fresh CLL cells (8x10 6/ml) were cultured in serum-free AIM-V medium plus

5x10 -5M 2-ME (Sigma-Aldrich) in 24-well plates at 37°C in 5% CO2 in the presence or absence of the following stimulants: TLR-7 agonist, phorbal 12-myristate 13-acetate

(PMA), and Ionomycin. For activation of CLL cells, Imiquimod, PMA, and Ionomycin were used at a final concentration of 3ug/ml, 40ng/ml, and 1 M, respectively. Where the effect of TAPI-0 on PMA-induced shedding was assessed, CLL cells in the same culture conditions were treated with 50 g/ml TAPI-0, with or without PMA stimulation. At 24 and

48-hours after stimulation, cells were harvested and stained for CD200, CD62L, CD19,

AnnexinV, and 7AAD. Tissue culture supernatants were harvested at the same time points to assess for sCD200 concentration in the CD200-sandwich ELISA.

In some PMA-stimulation experiments, membrane proteins were extracted from aliquots of untreated and PMA-stimulated cells using the ProteoJET TM Membrane Protein

Extraction Kit (Fermentas). The protein concentration in membrane extracts was determined by Bradford protein assay (Bio-Rad).

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Serum starvation and stimulation of Hek293-hCD200 cells

Hek-hCD200 cells were seeded in 6-well plates at 2x10 6 cells/ml and grown in serum- containing selection medium for 2 days or until ~80% confluency was reached.

Supernatants were then removed, and after 2 washings in PBS, 1.5ml of serum-free

OPIMEM medium, with or without 40ng/ml PMA, was added per well. Supernatants from untreated and PMA-treated cells were collected at 2, 6, and 24hr time points and sCD200 concentration in each was assessed by ELISA. Supernatants from Hek293-hCD200R1 cells grown under the same conditions were used as negative controls in the ELISA.

FACS

CD200 cell surface staining was performed using 3G7 at 0.005 g per sample in 100 l volume with goat anti-rat IgG-PE (1:100 dilution) as the secondary antibody for detection.

For multi-color staining of CD200, CD62L and CD19, fluorochrome-conjugated CD19 and

CD62L antibodies were added at predetermined optimal concentrations at the same time as the secondary antibody. All antibody-incubations were performed at 4 ◦C. Single color controls were included in each experiment for compensation purposes, and all samples were analyzed in a Coulter FC500 flow cytometer.

CD200 ELISA

The CD200-sandwich ELISA was developed for detection of sCD200 in human plasma, and utilized 1B9 as capture antibody and the rabbit anti-hCD200v+c serum as detection antibody. For optimal detection of sCD200 in CLL supernatants, supernatant samples and CD200Fc standards (0.025ng/ml to 2ng/ml), prepared in AIMV medium, were

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incubated overnight at 4 ◦C on an ELISA plate coated with the capture antibody at

1.25ng/ml and blocked with 5%FBS-PBS. The next day the plate was washed 5 times in

PBS+0.01% Tween20, followed by 2-hr incubation with the detection antibody at a 1:2000 dilution at room temperature. The remainder of the steps were performed as described previously (405). This modified protocol increased sensitivity of the ELISA from

0.05ng/ml, as reported previously, to ~0.01ng/ml.

Immunoprecipitation of sCD200

For immunoprecipitation of sCD200, 1ml of supernatants from untreated or PMA- treated CLL cells or HekhCD200 cells were incubated overnight with 2 g of 1B9 and 50 l of Protein A/G Agarose bead-suspension (Pierce Biochemicals) at 4 ◦C. The following day, after 2 washes in RIPA buffer containing 1 mM Na 3VO 4, the bound immune-complexes were dissociated by boiling in reduced sample buffer containing 0.025% SDS followed by low speed centrifugation. Supernatants containing immune-complexes were loaded directly onto 10% SDS-PAGE gel.

Western blotting

After transfer onto PVDF membranes, the blots were blocked with 5% milk-TBST for 1 hour at room temperature, and then probed with primary antibodies overnight at 4 ◦C.

The following primary antibodies were used in Western blotting experiments: rabbit anti- hCD200v+c serum (1:2000 dilution), rabbit anti-CD200 c-tail serum (1:500 dilution), or rabbit-hCD200R1 serum (1:2000 dilution). Regardless of the primary antibody used, following primary antibody incubation and washings in TBS-T, all blots were probed with goat anti-rabbit IgG-HRP (Jackson) at a 1:10,000 dilution for 45 minutes. After thorough

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washing, blots were developed using an ECL Western blot detection kit (GE Healthcare

Bio-Sciences).

CD200R1 phosphorylation by sCD200

Hek293 cells stably transfected with human CD200R1 (HekhR1) were seeded at

80% confluency in 6-well plates, and serum-starved overnight in OPIMEM medium

(Invitrogen). The following day cells were washed once with PBS and incubated at 37 ◦C for 15 minutes with one of the conditioned supernatants harvested from stimulation experiments: CLL supernatant from untreated or PMA-treated cultures; and HekhCD200 supernatant (in OPIMEM). sCD200 levels in all supernatants were assessed in the CD200 sandwich ELISA. AIMV medium, and supernatant from Hek293 cells in OPIMEM were used as negative controls. For positive control of phosphorylation, a separate well of cells were incubated with 100 M of activated vanadate in OPIMEM medium.

After incubation cells were lysed in 500 l of RIPA buffer containing 50mM NaF,

0.2mM Na 3VO 4, and protease inhibitors. Following centrifugation at 10,000rpm, supernatants were immunoprecipitated with 1 g of 4G10 Anti-Phosphotyrosine (Millipore) in the presence of 50 l of Protein A/G bead suspension overnight at 4 ◦C. Immune complexes were dissociated from Protein A/G beads by boiling in reducing sample buffer.

After quick centrifugation supernatants containing the immune complexes were loaded onto

10% SDS-PAGE gels. Western Blots were performed using the rabbit anti-hCD200R1 serum for detection of CD200R1 in immunoprecipitation (I.P.)-product.

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Statistics

Paired and u npaired t-tests were used to determine significance between sample means in stimulation experiments and were performed on Prism 5.0 software.

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4.4 Results

CD200 is constitutively shed from the surface of CLL cells

To address whether CD200 is released from the surface of CLL cells, primary CLL cells purified from peripheral blood of a cohort of patients (n=6) were cultured in AIM-V medium. Supernatants were collected from cell culture at different time points to assess for sCD200 concentrations using a CD200 sandwich ELISA, with sensitivity of ~0.01ng/ml.

Plasma harvested from patient blood was also analyzed for sCD200 levels.

sCD200 was detectable in most of the CLL supernatants tested at 24 hours (Fig 4-

1a, results from 4 patients shown), with increased levels observed by 48-hr, indicating variable but constitutive release of CD200 by CLL cells (Fig 4-1a). The level of sCD200 detectable in the 48 hour supernatant of CLL cells correlated strongly with sCD200 levels in the corresponding plasma harvested at the same time (Table 4.1; Spearman’s r=0.8857, p=0.0333), inferring that constitutive release of CD200 from CLL cells is as least in part, responsible for sCD200 detected in CLL plasma.

Ectodomain cleavage is thought to be responsible for the constitutive shedding of a number of membrane-anchored molecules (393). We hypothesized that similar mechanisms could be responsible for the release of CD200 from CLL cells. Consistent with this hypothesis, GM6001, a board spectrum metalloprotease inhibitor that inhibits all

MMPs and some ADAM proteases, including ADAM-mediated shedding of CD62L, abolished the release of CD200 from CLL cells (Fig 4-1b) (328, 406, 407).

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To explore further the role of MMPs and ADAM proteases in the constitutive release of CD200, CLL cells from a different cohort of patients were treated with recombinant tissue-inhibitor of metalloproteases (TIMPs) and sCD200 concentrations in

48-hr supernatants were assessed by ELISA (408). Treatment of CLL cells from 3 different patients with the different TIMPs showed that constitutive release of CD200 could be inhibited in different patients by TIMP1 ( p=0.01)and/or TIMP3 ( p=0.04) ( (Fig 4-1c).

CD200 shedding from CLL cells was induced by various stimuli

ADAM protease activity is increased by activation of intraceullular 2 nd messenger systems, such as PKC, or intracellular Ca 2+ pathways, leading to enhanced ectodomain shedding (304). Inflammatory stimuli, such as LPS, inducing downstream signalling through TLRs, have also been shown to induce ectodomain shedding (396, 409, 410). To investigate further whether CD200 is a candidate for ectodomain shedding mediated by

MMPs/ADAM proteases, CLL cells were stimulated with the following agents: phorbol myristate acetate (PMA), which upreguates activities of ADAM proteases, particularly

ADAM17, through the PKC pathway (411); ionomycin, which enhances activities of some

ADAM proteases via intracellular Ca 2+ pathway (331, 412); and Imiquimod, a TLR7 agonist. sCD200 concentrations in the supernatants were assessed at 48 hour time point.

PMA stimulation increased release of CD200 into the supernatant by CLL cells from all patients tested ( p=0.0008, n=6), although the level of response varied amongst patients (Fig 4-2a). Stimulation of CLL cells from the same cohort of patients by ionomycin generally produced enhanced shedding (p =0.0532, n=6), although to a lesser degree than that induced by PMA (Fig 4-2b). Finally, CLL cells from 4 out of the 6

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patients responded to TLR7 stimulation by enhanced release of sCD200 (Fig 4-2c). Once again, as observed for constitutive shedding, the response of CLL cells to these 3 stimuli varied significantly amongst patients.

PMA-induced CD200 shedding was reflected by loss of CD200 from CLL cell surface

In lymphocytes, ectodomain shedding of CD62L by ADAM proteases, which is induced upon PMA stimulation, is reflected as a loss of CD62L from the cell surface by

FACS (413). To explore whether the elevation of sCD200 in the supernatant of PMA treated CLL cells was a function of inducible ectodomain shedding which followed a similar pattern, CLL cells from a different cohort of patients (n=6) were monitored by

FACS for CD200 expression on the cell surface 24h after PMA stimulation (Fig 4-3a-d), and by Western blotting and ELISA using membrane extracts harvested from the same cells

(Fig 4-4a-b). The shedding of CD62L following PMA stimulation as determined by FACS was used as an independent indicator for inducible ectomain shedding (Fig 4-3a and d).

CLL cells were also stained for CD19, which is not reported to be a substrate of membrane sheddases, and Annexin V, and 7AAD to exclude a non-specific response to PMA stimulation (Fig 4-3b-c).

CLL cells from all 6 patients in this cohort responded to PMA stimulation by shedding significant amount of CD62L from the cell surface, as reflected in an average reduction of 86.7% (±17.5%) in CD62L cell surface staining on PMA-stimulated cells compared to untreated cells (Fig 4-3a, data from 3 representative patients shown; Fig 4-3d,

% loss of CD62L and CD200, all patients). CD200 levels were reduced by >20% on the surface of CLL cells from 4 out of the 6 patients following PMA stimulation, with median

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loss at 44.2% and an average loss of 39.3% (±27.5%), supporting the hypothesis that

CD200 was shed from the cell surface following PMA stimulation. Note that CLL cells from all patients shown in Fig 3 shed 60%-98% of CD62L in response to PMA, but only cells from patients 155 and 80 shed CD200 to a similar extent, with cells from patient 47 showing minimal shedding of CD200. CD19 expression on CLL cells remained stable after PMA stimulation (Fig 4-3b). Minimal evidence for apoptosis was seen in all cultures

(Fig 4-3c), negating the contrary hypothesis that non-specific loss of CD200 from dying cells was an explanation for the sCD200 detected in CLL supernatants.

Membrane extracts from PMA-stimulated cells from the 5 patients showing >20%

CD200 shedding by FACS also showed reduced CD200 levels when tested by CD200-

ELISA (Fig 4-4a, 3ug membrane extracts tested, p=0.0391). The reduction in CD200 levels in the membrane extracts after PMA stimulation was further confirmed by Western blotting. 5ug of membrane extracts were run on 10% SDS-PAGE gel, and after transfer,

PVDF blots were probed with the polyclonal rabbit anti-hCD200 v+c serum and a Pan-

Cadherin antibody as control for equal loading (Fig 4-4b). Consistent with previous data, membrane extracts from CLL cells that responded to PMA by shedding CD200 as determined by FACS and ELISA also showed a reduction in CD200 detected by Western

(Fig 4-4b).

Inhibition of PMA-induced CD200 shedding by TAPI-0

As further confirmation that CD200 shedding was mediated by ectodomain cleavage, CLL cells were treated with TAPI-0, a hydroxymate-based inhibitor of metalloproteases (414). CLL cells from 3 different patients were treated with TAPI-0 with

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or without PMA stimulation, and CD200 shedding was assessed at 24-hr by FACS. TAPI-

0 appeared to restore CD200 expression on PMA-treated CLL cells from patient 139 and

158, both of which shed >50% of CD200 in response to PMA (Fig 4-4c). Interestingly,

TAPI-0 failed to inhibit PMA-induced shedding in CLL cells from patient 16, where constitutive shedding of CD200 was also previously shown to be unresponsive to inhibition by TIMP1, 2, or 3 (Fig 4-1c and 4-4c; cells collected on different dates).

CD200 shedding in the epithelial Hek293-hCD200 cells

Given that tissue CD200 expression is relatively ubiquitous, and its overexpression has been reported on a number of solid tumors (301, 345), we next investigated whether

CD200 shedding occurred in cells of epithelial origin using a Hek293 cell line stably transfected with full-length human CD200 (Hek-hCD200) and constitutively expressing

CD200 at the cell surface in high levels (376). To test whether CD200 is shed from Hek- hCD200 cells, cells at ~80% confluency were cultured in the serum-free OPIMEM medium, with or without stimulation by 40ng/ml PMA. Supernatants were collected at 2,

6, and 24, and 48h time-points. Serum-starved Hek-hCD200 cells released CD200 constitutively with detectable levels as early as 6-hrs after serum starvation (Fig 4-5a, data from 1 representative experiment shown). By 24h, sCD200 concentration in the supernatant of Hek-hCD200 cells was measured to be 3.1±1.3ng/ml (Fig 4-5b, average from 4 independent experiments). Importantly, Hek-hCD200 cells also responded to PMA- stimulation by shedding increased amounts of sCD200, with differences already detected at

6 hr (Fig 4-5a). By 24 h, up to 3-fold more sCD200 was present in supernatants from

PMA-stimulated cells compared to supernatants from untreated cells (Fig 4-5a and b; p=0.03, paired-t test).

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To confirm that sCD200 released from Hek-hCD200 cells was a result of ectodomain cleavage, Hek-hCD200 cells were treated with TAPI-0, which inhibited PMA- stimulated CD200 shedding in CLL cells (Fig 4-4c). sCD200 concentration in the supernatant of PMA-stimulated cells was restored to levels seen in untreated cells in the presence of TAPI-0 (Fig 4-5c), indicating that TAPI-0 was effective in inhibiting PMA- induced CD200 shedding in Hek293 cells. Note that TAPI-0 treatment did not affect

CD200 release from serum-starved Hek-hCD200 cells (Fig 4-5c), suggesting that spontaneous shedding of CD200 under serum-free conditions might involve different sheddases and/or additional mechanisms. sCD200 released by CLL and Hek293-hCD200 cells did not contain the cytoplasmic domain of CD200

Ectodomain cleavage by MMPs and/or ADAM family of proteases releases extracellular fragments of the cleaved substrate (393). sCD200 released by CLL and

Hek293-hCD200 cells contained extracellular domains of CD200, as illustrated by its recognition by the two antibodies,1B9 and the polyclonal rabbit anti-hCD200 v+c serum, both of which were raised against the extracellular regions of CD200. To confirm that sCD200 lacked the cytoplasmic domain of full length CD200, we generated a polyclonal rabbit anti-serum to a peptide containing the 19 amino acids that made up the cytoplasmic tail of CD200 (Fig 4-6a). This rabbit anti-c-tail serum reacted only with full-length CD200, but not CD200 v+c, confirming its specificity against the cytoplasmic region of CD200

(Fig 4-6b).

We next investigated whether the sCD200 found in the supernatant of CLL and

Hek-hCD200 cells would react with the rabbit anti-hCD200 c-tail serum. Duplicates of

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supernatants from PMA-stimulated CLL cells and serum-starved Hek293-hCD200 cells were immunoprecipitated with 1B9, and then run on two separate 10% SDS-PAGE gels.

The blots were probed with either the rabbit anti-hCD200 v+c serum or the rabbit anti- hCD200 c-tail serum. Membrane extracts from CLL cells, which expressed full-length

CD200 at high levels, were used as positive controls for both antibodies. As expected, I.P. products from both CLL and Hek293-hCD200 cells were detected by the polyclonal anti- hCD200 v+c serum as a ~47kd band (Fig 4-6c, upper panel). Band intensities of I.P. products from Hek-hCD200 supernatant and CLL supernatant differed substantially but were consistent with quantitation given by ELISAs, which showed that supernatants from serum starved Hek-hCD200 cells generally contained ~8-fold higher concentrations of sCD200 than supernatants from PMA-stimulated CLL cells (Fig 4-2a and Fig 4-4a).

Consistent with the hypothesis that sCD200 were products of ectodomain cleavage, the precipitated products from supernatants were not recognized by the anti-hCD200 c-tail serum, while the full-length CD200 isoform in CLL membrane extract was recognized as a

~48kd band (Fig 4-6c, lower panel). sCD200 in CLL supernatant was capable of interacting with hCD200R1

Phosphorylation of the ITIM-like motif at the cytoplasmic region of CD200R1 upon

CD200 binding is crucial in transmitting signals for mediation of the downstream functions that characterize the CD200:CD200R1 axis of immunoregulation (275). To determine whether sCD200 released from CLL and Hek-hCD200 cells was functionally active, we asked whether sCD200 in CLL and Hek-hCD200 supernatants was capable of binding and phosphorylating CD200R1. Hek293 cells stably transfected with human CD200R1 (Hek- hR1) were seeded in 6-well plates to 80% confluency before overnight serum starvation.

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The cells were then stimulated with supernatants from serum-starved Hek-hCD200 cells and CLL cells (48hr supernatant from untreated cultures) for 15 minutes at 37 ◦C. After stimulation, cells were lysed and immunoprecipitated with an anti-phosphotyrosine antibody. The precipitated products were run on 10% SDS-PAGE gels and probed with the rabbit anti-hCD200R1 serum to determine whether CD200R1 was amongst the phosphorylated proteins immunoprecipitated by the anti-phosphotyrosine antibody.

CD200R1 bands were detected in immunoprecipitate from cells incubated with CLL supernatants or Hek-hCD200 supernatant, but not in cells stimulated with sCD200 - control supernatants (Fig 4-6d). Band intensities of phosphorylated CD200R1 on the Western blots were reflective of the sCD200 concentrations in the supernatants used for stimulation, with supernatant from untreated CLL cells, containing about 10-fold less sCD200 than Hek-293 supernatant (data not shown), giving lower band intensity (Fig 4-6d).

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4.5 Discussion

The soluble form of CD200 (sCD200) is elevated in plasma from CLL patients in comparison to plasma from healthy controls (405). Plasma sCD200 levels correlated significantly with CLL disease stage and tumor burden (405). While CLL cells harvested from patients express CD200 at various levels, sCD200 release is independent of the level of CD200 expression on the surface of corresponding CLL cells harvested at the same time point (unpublished data, Wong et al). We now report that sCD200 detectable in 48-hr supernatants of CLL cells are correlated with sCD200 levels found in corresponding patient plasma, suggesting that: 1) CLL cells release sCD200 constitutively; and 2) differences in the ability of CLL cells to release sCD200 may account, in part at least, for variable sCD200 levels in CLL plasma from different patients (Table 4.1).

Analysis of the human CD200 gene sequence suggests that, unlike CTLA-4, alternative splicing is an unlikely mechanism responsible for the production of sCD200, since the only known isoform of CD200 is a truncated form lacking the first 42 amino acids at the N-terminal, and this form is also membrane bound with different functional properties from full length CD200 (415). Based on this, we hypothesized that sCD200 may reflect active ectodomain shedding.

Constitutive release of sCD200 from CLL cells was inhibited by GM6001, a global inhibitor of metallproteases. In addition, constitutive shedding of CD200 from CLL cells appeared to be sensitive to inhibition by the tissue inhibitor of metalloproteases (TIMPs).

Of the three major TIMPs (TIMP1-3), TIMP3 has been documented extensively to inhibit shedding known to be mediated by ADAM proteases, including CD62L, IL6 receptor, and

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Syndecans, in addition to inhibiting MMPs (413, 416, 417). TIMP1 is known to inhibit

MMPs, and has also been shown to inhibit ADAM10 in vitro (408, 418). TIMP2, on the other hand, inhibits only MMPs with no effect on ADAM proteases (408). The observation that TIMP1 and TIMP3 treatment resulted in reduced sCD200 levels in supernatants from

Pt158 and 139 suggests involvement of ADAM proteases in this process. Note that the sensitivity of CLL cells to inhibition by the different TIMPs is highly heterogeneous and patient-specific. This heterogeneity may reflect potential involvement of multiple ADAMs and MMPs in CD200 shedding, and the differential expression and/or activity of MMPs and ADAM proteases in CLL cells from each patient . It is also important to note that different culture media contain known levels of reducing agents such as antioxidants, as well as calcium, both of which are known to influence ectodomain shedding (329, 419).

Thus, constitutive shedding of sCD200 may itself be under the influence of the tissue culture medium used (AIM-V medium in this case).

sCD200 release from CLL cells, like that of other well-characterized substrates of sheddases such as CD62L and CD44, could be further induced by external and physiological stimuli, including PMA, ionomycin, and TLR7 agonists (398, 420). Of the three shedding stimuli tested in this study, PMA, a potent activator of PKC particularly known for its ability to induce shedding by ADAM17, was the most effective in enhancing

CD200 shedding, as determined by increases in the detection of sCD200 in the supernatant and reduced CD200 expression on the cell surface at the same time point (411, 421).

Moreover, PMA-induced shedding of CD200 was inhibited by TAPI-0, a hydroxymate- based protease inhibitor first developed to inhibit PMA-induced TNF α and CD62L shedding (414, 422). Ionomycin, a calcium ionophore that induces different ADAM

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proteases, most noticeably ADAM10, also enhanced shedding of CD200, though the strength of the response was generally less than that observed with PMA stimulation (329-

331). CLL cells from Patient 82 showed the least increase in CD200 shedding by PMA stimulation while responding strongly to ionomycin by shedding >3-fold more CD200 than cells from the other patients, suggesting the involvement of different proteases in CLL cells from this patient (Fig 4-2b). This heterogeneity in response to the different shedding stimuli by CLL cells from different patients is consistent with that seen for the constitutive shedding of CD200 and supports the notion that CD200 is likely shed following the action of multiple sheddases.

A number of physiological stimuli induce ectodomain shedding by a variety of cell types. LPS, for example, was recently shown to induce ADAM17 activity through TRIF adaptor signalling that involved downstream activation of NADPH oxidase and PKC δ in phagocytes (423). In this study we found that Imiquimod, a TLR7 agonist, could induce

CD200 shedding in some, but not all, patients. The response of CLL cells to other physiologically relevant stimuli, such as cytokines, remains to be explored. In patients, the proliferating pool of CLL cells are known to reside in “proliferation centers” in association with a non-CLL microenvironment which provides additional stimuli either through soluble factors or cell-cell contact that are important in sustaining CLL survival and growth through multiple pathways (424). Given the abundance of external stimuli in these microenvironments, the inherent ability of CLL cells to shed CD200 in response to different stimuli, in addition to their ability to shed CD200 constitutively, could contribute significantly to circulating sCD200 in CLL plasma and to the local effects of CD200 in these environments. Indeed, the ability of CLL cells to shed CD200 in response to PMA

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correlated to some extend with plasma sCD200 levels in patients (Table 4.1, Spearman’s r=0.8286, p=0.0583).

We also explored CD200 shedding in non-CLL cells. In preliminary studies, normal B cells, which expressed CD200 constitutively at low levels, appeared to respond to

PMA by shedding CD200 (unpublished data, Wong et al). CD200 shedding was also observed in the epithelial Hek293 cell line stably transfected with CD200. Hek293 cells appeared to shed CD200 constitutively when cultured in serum-free conditions and, like

CLL cells, also responded to PMA by shedding increased amount of sCD200. The inducible shedding of CD200 in response to PMA by Hek293 cells was inhibited by TAPI-

0, again suggestive of a role for MMPs and/or ADAM proteases in this process.

Interestingly, TAPI-0 did not inhibit constitutive shedding of CD200 by Hek293 cells in serum-free conditions, suggesting mechanisms of CD200 shedding distinct from PMA- induced shedding and spontaneous shedding seen in CLL cells. It is important to note that cells under serum starvation often experience increased oxidative stress and apoptosis, both of which have been shown to be natural stimulants of ectodomain shedding (425-427).

Apoptosis, in particular, has been shown to stimulate shedding of IL6R from neutrophils via mechanisms that are caspase-dependent but ADAM-independent (427). Although we observed no evidence (by FACS) for increased apoptosis as a possible mechanism responsible for increased sCD200 in CLL supernatant, the role of apoptosis in the release of sCD200 by serum-starved Hek293 cells is not known.

Overall, both constitutive and inducible shedding of CD200 in CLL, and consequently the existence of sCD200 in patient plasma, are likely functions of the combination of sheddases expressed by each individual, as well as the presence of different

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stimuli present in the CLL microenvironment. The specific MMPs and/or ADAM proteases responsible for CD200 shedding remains to be elucidated. CLL cells and

HekhCD200 cells express different sets of MMPs and ADAM proteases. CLL cells are known to express and secrete MMP-9, which is associated with tissue invasion (428). In addition, MMP-9 was recently shown to form a macro-molecular complex on the surface of

CLL cells with CD44, CD38, and CD49d (429). Whether MMP-9 acts as a sheddase at the

CLL cell-surface remains to be studied. Preliminary real-time PCR analyses of ADAM proteases in CLL and Hek293 cells found high levels of ADAM 10, 17, and 28 in CLL cells, but not in Hek293 cells (unpublished data). ADAM28, whose catalytic domain has been shown to be capable of shedding CD23 in vitro , is overexpressed in CLL cells in comparison to normal B cells and silencing of this expression decreased sCD200 levels in

CLL supernatants (337, 430).

Besides ectodomain cleavage by proteases, shedding of membrane-anchored molecules from the cell surface in the forms of microvesicles is another potential mechanism by which sCD200 is released from the surface of CLL cells to become detectable in the plasma of CLL patients (431, 432). An increased concentration of microvesicles in the plasma of CLL patients has been reported (433). Molecules released in the form of microvesicles are associated with the plasma membrane and thus can exist in their full-length forms (431). Our biochemical analysis argues against this mechanism as an important source of sCD200, since material immunoprecipitated from both CLL and

HekhCD200 supernatants was recognized as a single band only by an antibody against the extracellular domain of CD200 (anti-CD200 v+c), but not by an antibody recognizing specifically the cytoplasmic tail of CD200. This supports the hypothesis that sCD200 does

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not contain the cytoplasmic tail and is cleaved at the cell surface. The exact cleavage site(s) on CD200 remains to be elucidated. The recognition of cleavage substrates by sheddases is thought to involve conformational shapes rather than specific peptide sequences (328, 434). Glycoslyation has also been show to modulate both constitutive and induced ectodomain shedding, and its role in CD200 shedding remains to be explored (435,

436).

Regardless of the mechanism(s) of CD200 shedding, a functional significance for sCD200 was demonstrated by the ability of sCD200 to bind and phosphorylate CD200R1, the major receptor responsible for mediating the downstream immunoregulatory functions of CD200 (275). With a documented functional ability to interact with CD200R, the existence of sCD200 in plasma may have important downstream physiological consequences and could play a role in different pathological conditions.

In conclusion, our data suggest sCD200 is a product of ectodomain cleavage by

ADAM proteases and MMPs. Given the immunoregulatory properties of CD200, the existence of sCD200 in plasma may be an important parameter to measure for both diagnostic and prognostic purposes. Recent data from our laboratory suggests that sCD200 can also be detected in the serum of breast cancer and colonic cancer patients (unpublished data), consistent with the already growing evidence that CD200 itself is reported to be overexpressed in a number of human cancers.

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4.6 Table

Table 4.1: Correlation between patient plasma sCD200 and sCD200 in corresponding CLL supernatants

Spearman’s correlation co-efficient Supernatant with plasma sCD200 level

No treatment 0.8857 (p=0.0333)

PMA 0.8286 (p=0.0583)

IMN 0.6 (p=0.2417)

Imiquimod 0.3143 (p=0.5639)

Footnotes to Table 4.1:

Correlation between patient plasma sCD200 levels and sCD200 levels detected in 48-hr supernatants from corresponding CLL cells with or without external stimuli (see Fig 2; n=6). Plasma sCD200 levels correlated significantly with sCD200 levels in 48-hr supernatants from untreated cells (p=0.03, Spearman r=0.8857)

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4.7 Figure legends

Fig 4-1: CD200 is constitutively released from CLL cells

Supernatants from purified CLL cells cultured in AIMV medium with or without the designated inhibitors were collected at 24 and 48 hrs and sCD200 concentrations were measured in a CD200 sandwich-ELISA. p-values were obtained from paired t-test. a) sCD200 was detected in 24-hr and 48-hr supernatants of untreated CLL cells (n=4), demonstrating continuously release of sCD200 from CLL cells; b) Spontaneous release of

CD200 was inhibited by treatment of CLL cells with 20 M GM6001 in 24-hr supernatants

(n=2); c) CLL cells (n=3) were treated 2.5 g of TIMP1, TIMP2, and TIMP3, and supernatants were collected at 48hr. Results from CLL cells from each of the patients are shown: TIMP1 significantly reduced constitutive CD200 shedding by CLL cells from

Patient 158 ( p=0.01; upper panel); TIMP3 significantly reduced constitutive CD200 shedding by cells from Patient 139 ( p=0.04; lower panel). None of the TIMPs were effective in inhibiting CD200 shedding from Patient 16 (middle panel).

Fig 4-2: sCD200 is secreted from CLL cells in response to different stimuli

CLL cells (n=6) were cultured in AIMV medium and stimulated with a) 40ng/ml PMA; b)

1M Ionomycin; and c) 3g/ml Imiquimod with supernatants collected at 48-hr. At 24-hr, cells were stained for CD62L, CD19, and CD200 expressions by FACS. p-values were obtained from paired t-test: a) CLL cells from all 6 patients responded to PMA by shedding increased amounts of sCD200 ( p=0.0008). The potency of response to PMA varied amongst patients. b) CLL cells from 4 out of 6 patients showed a modest response to Ionomycin ( p=0.0532). 2 out of 6 patients showed no response to Ionomycin treatment.

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c) CLL cells from 4 out of 6 patients showed a modest response to Imiquimod, although the induction of CD200 shedding was not statistically significant (p>0.05).

Figure 4-3: PMA-induced CD200 shedding is reflected as a loss of CD200 expression from the surface of CLL cells

CLL cells from 5 patients were treated or untreated with 40ng/ml PMA and cells were harvested at 24 hour to assess for a) CD200 and CD62L; b) CD200 and CD19; and c)

Apoptosis markers AnnexinV, and 7AAD by FACS. Data from 3 representative patients were shown. d) Median and average % loss of CD200 and CD62L from the surface of CLL cells from all 5 patients in the cohort as determined by FACS. Both CD62L and CD200 expressions were normalized to that of CD19 on the same cells. Median loss of CD200 and

CD62L from CLL cells: 44.25% and 95.5%, respectively. Mean loss of CD200 and

CD62L from CLL cells: 39.3± 27.5% and 86.7±17.5%.

Figure 4-4: Detection of CD200 in membrane extracts from PMA-treated CLL

Membrane proteins from aliquots of treated and untreated cells from experiments in Fig3 were extracted and analyzed by a) ELISA, and b) Western blotting for CD200. Both methods showed loss of CD200 from the membrane fraction in PMA-treated cells from patients that responded to PMA stimulation as determined by FACS (patients not shedding

CD200 in response to PMA as assessed by FACS are highlighted in italic). c) CLL cells from another cohort of patients (n=3) were cultured in AIMV medium, with or without

PMA stimulation, and treated with 50 g/ml of TAPI-0. Cells were harvested at 24-hr and stained for CD200 and CD19. TAPI-0 restored cell surface expression of CD200 on PMA- treated cells from Patient 139 and 158 (upper panels). CLL cells from Patient 16 showed

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no response to TAPI-0 although they did respond to PMA by shedding a low level of

CD200 (bottom panel).

Fig 4-5: Spontaneous and inducible shedding of CD200 in Hek-hCD200 cells

Hek-hCD200 cells were seeded in 6-well plates to 80% confluency in serum-containing medium. Medium was then replaced with serum-free OPIMEM medium with or without

40ng/ml PMA stimulation. Supernatants were collected at different time points and assessed for sCD200 concentration by ELISA. a) sCD200 was detectable in supernatants from untreated cells at 6-hr after serum starvation with sCD200 concentrations remaining relatively stable to 24-hr. PMA-treated cells released two-fold more sCD200 at 6-hr and by

24-hr showed 4-fold higher concentration of sCD200. Data from 1 out of 4 experiments is shown. b) Hek-hCD200 consistently shed increased amounts of sCD200 in response to

PMA as detected in 24-hr supernatants from 4 independent experiments ( p=0.03, paired t- test). c) Serum-starved Hek-hCD200 cells, with or without PMA stimulation, were treated with 50 g/ml TAPI-0, and supernatants were harvested at 24-hr. TAPI-0 inhibited PMA- induced CD200 shedding by Hek-hCD200 cells.

Fig 4-6: Absence of epitopes associated with the cytoplasmic domain of CD200 in sCD200 and functional properties of sCD200 a) Amino acid sequence of full-length human CD200. The peptide sequence used for generation of rabbit anti-CD200 cytoplasmic-tail antibody is highlighted in bold. b)

Characterization of rabbit anti-CD200 c-tail serum; the antiserum recognized lysates from

Hek-hCD200 cells, which expressed full-length CD200, but not lysates from Hek293 cells transfected with hCD200v+c or pure hCD200v+c, indicating specificity for the cytoplasmic domain of CD200. c) Membrane extracts from CLL cells were recognized by both rabbit

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anti-hCD200v+c serum and rabbit anti-CD200 c-tail serum. sCD200 I.P. from CLL and

Hek-hCD200 supernatants was recognized only by rabbit anti-hCD200v+c serum, but not by rabbit anti-CD200 c-tail serum, indicating that sCD200 released from both cell types lacked the cytoplasmic domain of CD200. d) CD200R1 cells were immunoprecipitated with anti-phosphotyrosine antibody following incubation of sCD200-containing CLL and

Hek-hCD200 supernatants and cell lysis. I.P. products were subsequently run on 10%

SDS-PAGE gel and probed with the rabbit anti-hCD200R1 serum. CD200R1 was phosphorylated by both CLL and Hek-hCD200 supernatants, but not AIMV medium or supernatants from Hek-hCD200R1 cells, both devoid of sCD200, indicating that sCD200 was capable of binding to, and causing phosphorylation of CD200R1.

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4.8 Figures

Figure 4-1: CD200 is constitutively released from CLL cells

4-1a)

0.2 Patient 58 0.18 Patient 81 0.16 Patient 43 0.14 Patient 82 0.12 0.1 ng/ml 0.08 0.06 0.04 0.02

0

24h 48h

176

4-1b).

0.06

0.05

Pt14 0.04 Pt90

0.03

ng/ml sCD200 ng/ml 0.02

0.01

0 Untreated GM6001

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4-1c)

Patient 158 0.12 p=0.01

0.1

0.08

0.06

0.04

0.02

0

0.14 Patient 16 0.12 0.1 0.08 0.06 0.04

ng/ml sCD200 0.02 0 Patient 139 0.25

0.2 p=0.04

0.15

0.1

0.05

0 No treatment TIMP1 TIMP2 TIMP3

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Figure 4-2: sCD200 is secreted from CLL cells in response to different stimuli

4-2a)

0.6 Pt. 7

p=0.0008 Pt. 16

0.4 Pt. 43 Pt. 58

Pt. 81

0.2 Pt. 82

ng/ml sCD200

0.0 No treatment PMA

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4-2b)

0.8 p=0.0532 Pt. 7 Pt.16 0.6 Pt.43 Pt.58 0.4 Pt.81 Pt.82 0.2 ng/ml sCD200 ng/ml

0.0 No treatment Ionomycin

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4-2c)

0.5 Pt. 7 0.4 Pt.16 Pt.43 0.3 Pt.58 Pt.81 0.2 Pt.82

ng/ml sCD200 ng/ml 0.1

0.0 No treatment TLR7 agonist

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Figure 4-3: PMA-induced CD200 shedding is reflected as a loss of CD200 expression from the surface of CLL cells

4-3a)

No treatment PMA

Pt. 47:

Pt. 155:

Pt. 80: CD200 CD62L

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4-3b)

No treatment PMA

Pt. 47:

Pt. 155:

Pt. 80: CD200 CD19

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4-3c)

No treatment PMA

Pt. 47:

Pt. 155:

Pt. 80: AnnexinV 7AAD

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4-3d)

% CD200 loss: median=44.25%; mean=39.3± 27.5

% CD62L loss: median=95.5%; mean=86.7±17.5 150 % CD200 loss % CD62L loss

100

50 untreated controls % loss after relative to 0

4 0 55 56 80 15 t.1 1 1 t. Pt.47 . P t. t. P Pt P P

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Figure 4-4: Detection of CD200 in membrane extracts from PMA-treated CLL

4-4a)

180 3ug membrane extract/well

160

140 No treatment 120 PMA treated (40ng/ml)

100

80

60 pgwell) CD200 (per 40

20

0 Pt.47 Pt.154 Pt.10 Pt.155 Pt.156 Pt.80 Pt.85 Pt.157

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4-4b)

Pt. 47 Pt. 156 Pt. 80 Pt.154 Pt.10 Pt.155 Pt. 85 Pt. 157

PMA -- + -- + -- + -- + -- + -- + -- + -- +

Cadherin

CD200

5ug membrane extract loaded

Primary antibody: rabbit anti-hCD200v+c

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4-4c)

No treatment PMA TAPI-0 PMA+TAPI-0

Pt.139

Pt.158

Pt.16 CD200 CD19

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Figure 4-5: Spontaneous and inducible shedding of CD200 in Hek-hCD200 cells

4-5a)

10 OPIMEM 8 OPIMEI+PMA

6

4 ng/ml sCD200 2

0 0 10 20 30 Time (hr)

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4-5b)

15 p=0.03 No treatment PMA

10

5

ng/ml sCD200

0 No treatment PMA

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4-5c)

7

6 No TAPI-0 5 +100uM TAPI-0 4

3

ng/ml sCD200 2

1

0 OPIMEM OPIMEM+PMA

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Figure 4-6: Absence of epitopes associated with the cytoplasmic domain of CD200 in sCD200 and functional properties of sCD200

4-6a)

V-like domain (domain I)------QVQVVTQDEREQLYT PASLKCSLQNAQ EALIVTWQKKKAVSPENMVTF SE

NHGVVIQ PAYKDKINITQLGLQNSTITFWNITLEDEGCYMC LF

c-like domain (domain II)------NTFGFGKISGT ACLTVYVQPIVSLHYKFSEHHLNITCSATARPAPMVFWKV

PRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLH

Transmembrane region------Cytoplasmic tail---- LGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYW KRHRNQDRGEL

SQGVQKMT

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4-6b)

1 2 3 1 2 3 47kDa 47kDa

38kDa

Ab to hCD200 v+c Ab to hCD200 cytoplasmic tail

Lanes: 1. Human CD200v+c (cell lysate) 2. Human CD200v+c (supernatant) 3. Full-length human CD200 (cell lysate)

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4-6c)

Sup Membrane Sup Pt.158 Pt.71 Hek Hek hCD200 hR1 PMA + -- -- + -- -- Anti-CD200 v+c

Anti-CD200 c-tail

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4-6d)

Supernatant in AIM-V Supernatant in OPIMEM

Cell- Hek- Hek- Cell- Vana- free CLL hCD200 hR1 free date

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Chapter 5: General discussion

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5.1 General discussion

The overexpression of immunoregulatory molecules, which deliver inhibitory signals that generally dominate over stimulatory signals, on tumor cells and tumor- infiltrating immune cells results in an immunosuppressive tumor microenvironment that is characteristic of many cancers (437). A role for the CD200:CD200R axis of immunoregulation in the control of anti-tumor immune responses was first postulated by us based on studies that showed infusion of a recombinant form of CD200 (CD200Fc) enhanced growth of EL4 thymoma cells in vivo (281). The overexpression of CD200 has since been reported in several cancers, including CLL, as well as on cancer stem cells

(438).

In the context of B-cell malignancies, Kretz-Rommel et al showed that CD200 blockade attenuated rejection of lymphoma cells transduced to overexpress CD200 by allogenic hPBMC in vivo (346). The ability of CD200 to suppress killing of CD200 + tumor cells by hPBMC was recapitulated in our in vitro model using a cell line that naturally express CD200 at high levels and primary CLL cells (chapter 2). Results from these in vitro and in vivo studies by us and others support a functional, immunosuppressive role for

CD200 on lymphoma and CLL cells. Moreover, we established that T cells are the likely effector targets of CD200-mediated suppression in this in vitro model.

In our efforts to elucidate the role of CD200 in CLL, we identified a previously unknown, soluble form of CD200 (sCD200). We found sCD200 to be elevated in the plasma of CLL patients, and investigated the functional properties of sCD200, its relevance in CLL prognostics and biology, and the mechanisms leading to its release. In particular,

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we explored the nature of sCD200/CD200:CD200R interactions in association with non-

CLL cellular components of the CLL microenvironment, which, by modulating survival and growth of CLL cells, are known to be key players in CLL disease progression. To our knowledge, these are the first studies that have addressed the clinical and biological role of membrane-bound and sCD200 in CLL. The following sections summarize our findings.

5.1.1 sCD200 as a prognostic marker in CLL

In our retrospective analysis of plasma samples from 75 CLL patients at diagnosis, we found that patients who had high plasma sCD200 levels at diagnosis tend to go on to develop aggressive disease as reflected by progression to late disease stage (Rai stage III and IV) and requirement for multiple treatments (chapter 2). We identified a correlation between sCD200 and β2-microglobulin levels, one of the prognostic markers strongly associated with adverse disease (113, 114). Due to limitations in the availability of clinical tests, we were unable to explore the association between sCD200 levels, IgVH mutation status, and Zap70 expression. Given that sCD200 levels in patient plasma are correlated with levels seen following spontaneous and/or inducible shedding of CD200 from CLL cells in vitro (chapter 4), plasma sCD200 levels may reflect the activation status of CLL cells and thus disease activity.

Results from our univariate analysis suggest high plasma sCD200 levels may be a marker for poor prognosis and provide strong rationale for continuous investigation of sCD200 as a clinical prognostic marker. Retrospective and prospective studies with larger sample sizes for multivariate analysis are required to determine the value of sCD200 levels as an independent prognostic factor, or as a prognostic index to be incorporated into current prognostic models, for clinical assessment at diagnosis (19). Correlation analysis of

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sCD200 levels and cytogenetic abnormalities showed that patients with 13q14 deletions or a normal karyotype, both generally markers for a benign disease course, who subsequently developed aggressive disease, tended to have high sCD200 levels at diagnosis (70). This suggests that sCD200 levels may identify a subpopulation of patients with unique disease characteristics and clinical course, and warrants further investigation in a large study for its clinical significance.

5.1.2 A novel xenograft model for CLL which utilizes sCD200

In investigating the in vivo function of sCD200 in CLL plasma, we identified a novel approach to prolong and improve engraftment of CLL cells in immunocompromised mice (chapter 3). Infusion of sCD200 hi CLL plasma, but not sCD200 lo normal plasma,

null significantly enhanced engraftment of CLL cells in NOD-SCID γc mice. Pre-absorption of sCD200 from CLL plasma, or in vivo depletion of sCD200 by mAb blockade, attenuated the enhancing effects of CLL plasma, indicating that sCD200 is an important component in

CLL plasma sustaining CLL survival in vivo . Engraftment of CLL cells is further improved by the use of CLL-splenocytes, containing a mixture of CLL cells and non-CLL cells that form the CLL microenvironment, rather than purified CLL cells. Thus, we proposed the use of [sCD200hi CLL plasma + CLL splenocytes] for optimal engraftment of

CLL cells.

In this model, CD19 +CD5 + CLL cells are found to engraft predominantly in the peritoneal cavity as well as in the spleen of the murine hosts. In the spleen, where T-cell engraftment predominates, CLL cells co-localize with T cells in follicular structures akin to those observed in CLL proliferation centers in the secondary lymphoid tissues of CLL patients. In previous reports on xenograft models of CLL, engraftment of CLL cells

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typically declines drastically after 2 months (234, 236). Importantly, in the model described, we detected ki67 + CLL cells in the peritoneal cavity at over 3 months, indicating persistence of CLL cells with in vivo proliferation. Preliminary studies on long-term engraftment of CLL cells showed detection of CLL cells in both compartments at over 9 months post-engraftment (unpublished observation), further suggesting long-term engraftment of CLL cells.

T cells appear to be required for the engraftment of CLL cells in this model, as in vivo T-cell depletion abrogated CLL engraftment despite continuous infusion of sCD200 hi

CLL plasma. Immunophenotyping showed persistent engraftment of both CD4 + and CD8 +

T cells in vivo throughout the different experimental time points used in our studies without evidence of GVHD in the host.

As CLL is a complex disease with significant contribution to both disease progression and drug resistance by the non-malignant CLL microenvironment, an optimal, pre-clinical animal model of CLL for drug-screening purposes should ensure modeling of these microenvironmental components. To this end, the stable and persistent engraftment of T cells, which contribute to CLL in vivo survival and growth in the xeno- microenvironment, as well as of CLL cells, supports the hypothesis that this model is a relevant one for pre-clinical testing of novel CLL therapeutics. Analysis of the engraftment of T cells also allows for assessment of non-specific effects on bystander cells.

We have tested and compared the efficacy of anti-CD200 blockade and rituximab, a clinically approved mAb therapeutic for CLL in attenuating CLL engraftment, using this model, and found both to be effective. While rituximab kills CLL cells by ADCC and

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CDC, anti-CD200 blockade targets the CD200:CD200R axis in the CLL microenvironment, and potentially mediates its therapeutic effect via different mechanisms

(242). These results illustrate one potential application of our xenograft model for CLL.

Several issues remain to be addressed. CLL cells appear to shed CD200 on a constitutive basis in vitro . Whether ectodomain shedding of CD200 occurs in vivo once

CLL cells are engrafted in the xeno-microenvironment, and whether in vivo shedding of

CD200 provides sufficient level of sCD200 to sustain CLL engraftment has yet to be determined. It is also not known whether the improved engraftment of CLL cells using

[sCD200hi CLL plasma + CLL splenocytes] is, at least in part, due to the intrinsic differences between CLL cells from the peripheral blood, a majority of which are known to be arrested at the G 0/G 1 phase of the cell cycle, and CLL cells from the splenic microenvironment.

5.1.3 The role of CD200:CD200R axis in the CLL microenvironment

The immunosuppressive function of CD200 is mediated through binding to a receptor, CD200R, resulting in the phosphorylation of the ITIM motif in the cytoplasmic tail of CD200R (275). Some of the downstream functional consequences following

CD200:CD200R engagement include inhibition of T-cell activation, reduction in IFN-γ and

TNF-α production by macrophages, and polarization to production of the TH-2 type cytokines (439). sCD200 appears to function in a similar fashion to membrane-bound

CD200, as demonstrated by its ability to bind and phosphorylate CD200R (chapter 4).

CD200R is known to be expressed on activated T cells, NK cells, and cells of the myeloid lineage, but not CLL cells (278). We have shown expression of CD200R on spleen-derived

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CD4 + T cells from CLL patients. It is not known whether other cells in the CLL microenvironment, including CD14 + NLCs (myeloid lineage), express CD200R.

In studies where the in vivo effect of sCD200 hi CLL plasma was compared with that of sCD200 lo normal plasma, we found that infusion of sCD200 lo normal plasma resulted in predominant T-cell engraftment in all compartments with minimal engraftment of CLL cells. Infusion of sCD200 hi CLL plasma, in contrast, resulted in engraftment of both CLL and T cells. This dichotomy indicates that in the absence of sCD200, T cells which engraft in vivo do not favor CLL engraftment, while in the presence of sCD200 it seems the engrafted population produces “pro-CLL” factors that support in vivo survival of CLL cells

(see Fig 1). We have hypothesized that sCD200 affects CLL engraftment indirectly through its effects on T cells.

Based on the knowledge that CD200R is required for the CD200-axis of immunoregulation, we propose a model whereby CD200R+ cells are the targets of CD200- mediated immunosuppression in the CLL microenvironment (see Fig 2). In the absence of

CD200/sCD200, CD200R + cells, including a subpopulation of T cells, survive in preference to CLL cells, through either direct mechanisms which negatively affect CLL survival, or indirectly by depletion of in vivo resources for survival and growth of CLL cells. In the presence of sCD200, CD200R + cells receive regulatory signals, which may or may not be associated with a switch to the production of the so-called Th-2 type cytokines characteristic of the CD200:CD200R axis, resulting in a microenvironment which favors

CLL survival and growth (Fig 2). Note that we have not observed engraftment of cells other than CLL and T cells in the mouse; however, in human, we do not rule out the contribution of CD200R + cells other than T cells in the CLL microenvironment.

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Figure 5-1: The in vivo effects of sCD200 on T cell engraftment

Normal plasma (sCD200 lo ) CLL plasma (sCD200 hi )

Pro- T cells CLL CLL CLL T cells

Favors T cell engraftment with minimal
sCD200 in CLL plasma is important in survival of CLL cells supporting CLL in vivo survival and growth in vivo
T cells that are selected to engraft under
T cells that are selected to engraft under these conditions are required for CLL these conditions do not support CLL growth engraftment and appear to play a positive role in supporting CLL in vivo growth

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Figure 5-2: Proposed model of CD200:CD200R mediated immunosuppression in the CLL microenvironment

Pro-CLL factors CD200R - CLL “Pro-CLL” T cell /non-T cells

CD200

“TH2” switch; ITIM production of

ITIM Suppression “Th2” type cytokines CD200R + T cell/non-T cells

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5.1.4 Ectodomain shedding of CD200

We have shown that CD200 is a novel target of ectodomain shedding, leading to the release of sCD200 from CLL cells. CLL cells shed CD200 on a constitutive basis, and respond to stimulation by external stimuli by shedding increased amount of CD200. The level of constitutive shedding by CLL cells is correlated with sCD200 levels in the corresponding patient plasma, suggesting that basal activity of CD200-sheddases on CLL cells may contribute significantly to CLL biology.

Of the external stimuli tested, stimulation of CLL cells by PMA, an activator of

PKC, resulted in the strongest shedding response by CLL cells (328). It is important to note that PKC, some isoforms of which are overexpressed in CLL, is a common mediator of multiple signaling pathways relating to CLL survival, including antigenic stimulation and signaling through BCR, an important component of the CLL microenvironment (440).

Thus, the response of CLL cells to PMA stimulation may also have physiological significance in CLL.

ADAM proteases are known to be the main mediators of ectodomian shedding. Of the ADAM proteases, PMA is known to stimulate ADAM17 (331). The observation that

PMA stimulation resulted in the strongest shedding response by CLL cells indicates the involvement, at least in inducible shedding, of ADAM17. Some CLL cells also respond to stimulation by ionomycin, which stimulates intracellular Ca 2+ release and is known to stimulate shedding by ADAM10 (331). Both ADAMs are detectable in CLL cells at the mRNA level (unpublished data). In addition, CLL cells also express ADAM28, often at significantly higher levels than ADAM10 and ADAM17 (Twito et al, manuscript in

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preparation). ADAM28 has been shown to possess sheddase activity in vitro , and silencing of ADAM28 in CLL cells reduces constitutive shedding of CD200 by CLL cells (Twito et al, manuscript in preparation).

The precise contribution of each of these ADAMs in the constitutive and inducible shedding of CD200 remains to be explored. The use of specific inhibitors for ADAM10 and ADAM17 may help elucidate the involvement the two ADAMs in CD200 shedding.

Further insights into the protease(s) responsible for CD200 shedding may have implications in other disease models in which CD200 plays a role.

5.2 Future directions

Studies described in this thesis provide some answers to the questions that were raised in the introductory chapter. Nevertheless, additional questions remain. Amongst these are:

5.2.1 The role of CD200R+ cells and T cells the in CLL microenvironment

Our proposed model of CD200:CD200R axis in the CLL microenvironment envisions a major role for CD200R + cells as the effector targets for CD200. However, the precise characteristics of CD200R + cells and their function after CD200 engagement in the

CLL microenvironment remain elusive. One study to address this issue would involve functional blockade of CD200R, by mAb or receptor antagonists, or in vivo depletion of

CD200R + cells, to assess the proposed role of CD200R+ cells in the model.

The precise subpopulation(s) of T cells critical for CLL engraftment in vivo remains to be identified. In vivo depletion of CD4 + and CD8 + T cells could help distinguish the

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roles of these two major populations of T cells in vivo . As sCD200 seems to have direct effects on the in vivo engraftment of T cells, detailed analysis of T cell subtypes engrafted in vivo , including regulatory T cells and Th17 cells, might provide further insight into the role of T cells in the CLL microenvironment.

5.2.2 The effects of CD200 blockade on T cells

The effects of sCD200 on engraftment of T cells suggest CD200 blockade might impact the T-cell compartment. Insight into the effect of CD200 blockade on T cells would not only help to delineate further the role of CD200 in the CLL microenvironment, but might have particular importance for assessment of CD200 blockade as a useful therapeutic agent in the clinic.

In vitro studies have shown CD200 blockade to be effective in augmenting anti- tumor killing of CD200 + lymphoma cells and primary CLL cells by effector CD8 T cells.

Whether CD200 blockade has a similar efficacy in vivo in an autologous setting has not been addressed. Since CD8 + T cells engraft in the xenograft model, the effector function of

CD8 + T cells against autologous CLL cells, with or without in vivo CD200 blockade, could be assessed by in vitro functional assays following harvesting of CD8 + T cells from the mouse. Preliminary data from our laboratory suggest that human cells harvested from mice reconstituted with CLL splenocytes do indeed have cytolytic activity to autologous CLL cells after in vitro or in vivo CD200 blockade. Assessment of CD8 effector activity post- anti-CD200 treatment in such a model may also assist in evaluating the therapeutic efficacy of novel drugs.

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5.2.3 The Applicability of the xenograft model described in testing novel therapeutics for CLL

The utility of our xenograft CLL model has been tested by comparing the effect of

CD200 blockade with rituximab as therapy in NOD.SCID mice. Both antibodies at high dose were effective in attenuating CLL engraftment. Recent data have suggested both may have a role in human CLL (441). In recent years, combination therapies such as the FCR regimen (fludarabine, cyclophosphamide, and rituximab) have shown improved efficacy for

CLL (240). We consider it important to investigate whether CD200 blockade could synergize with current conventional therapies using the xenograft model described. For example, studies to explore the therapeutic efficacy of CD200 blockade in combination with other immunomodulatory agents such as cyclophosphamide or lenalidomide, or with cytotoxic agents such as rituximab or fludarabine, would provide practical information for the translation of CD200 blockade into clinics.

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5.3 Concluding remarks

Studies reported in this thesis provide novel insights into the role of

CD200:CD200R in CLL, and argue that the modulation of the CD200:CD200R axis may represent a novel immunotherapeutic approach for CLL. Given the dominant role of immunoregulatory molecules such as CD200, therapeutic blockade of CD200 may complement current treatment regiments in its ability to modulate T-cell responses. CD200 blockade may also benefit other approaches used to stimulate anti-tumor effector responses, including cancer vaccines, by removing inhibitory elements which might negatively affect vaccination outcome.

The identification of the existence of a soluble form of CD200, sCD200, and elucidation of its functional role in augmenting engraftment of CLL cells in immunocompromised animals, fostered the development of a xenograft model useful in pre-clinical screening of novel therapeutics for CLL. The correlation between sCD200 levels and clinical markers of aggressive disease in CLL provides a rationale for the search for similar correlations in other cancers, particularly those with documented CD200 overexpression. Preliminary work from our laboratory has shown elevated sCD200 levels in the plasma of breast and colonic carcinoma patients and suggested a correlation of sCD200 levels with disease status.

In the larger context of immune responses controlled by CD200, the existence of sCD200 in plasma and its detection may have implication in organ transplantation, as well as inflammatory and autoimmune diseases.

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Chapter 6: References

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