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 sCD200hi CLL plasma, but not sCD200lo 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.

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Dedication

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

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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.

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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

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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

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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.SCIDγcnull mice ..... 136

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Figure 3-4: Effects of sCD200 and/or T cell depletion on CLL engraftment in NOD.SCIDγcnull mice at day 21 ..................................................................................... 141 Figure 3-5: Therapeutic efficacy of Rituxan and 1B9 on CLL engraftment in NOD.SCIDγcnull 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

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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

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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

FcγRIIb

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

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Intravenous

LDT

Lymphocyte doubling time

LPS

Lipopolysaccharide (endotoxin)

MAPK

Mitogen-activated protein kinase

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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

NFκB

Nuclear factor kappa-light-chain-enhancer of activated B cells

NLC

Nurse-like cell

NOD

Non-obese diabetic

NOD.SCIDγcnull

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)

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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

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List of CD antigens CD antigens relevent for this thesis Cellular expression

Function

CD3

Mature T, different levels on thymocytes

T activation; regulates TCR expression

CD4

Thymocyte subsets; T helper, Treg; monocytes/macrophages, DC

amplifies TCR signals; HIV entry

CD5

Subtypes of B, B-CLL; T

T-B interaction; T activation

CD8

Thymocyte subsets; cytotoxic T, NK, DC subsets

Co-receptor for MHC class I

CD14

Monocytes, macrophages, Langerhan

receptor for LPS and LBP

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 macrophages

low-affinity receptor for IgE

CD25

Activated T, B, and monocytes; subsets of T

IL2Rα chain

CD31

T subsets; monocytes, endothelial cells

PECAM-1; adhesion; CD38 ligand

CD38

Variable levels on hematopoietic cells

ADP-ribosyl cyclase; activation, proliferation, adhesion

CD40

B, monocytes/macrophages, DC

co-stimulation; B-differentiation and isotype switching

CD44

Hematopoietic and nonhematopoietic cells

leukocyte rolling, homing, and adhesion

CD45

Hematopoietic cells except erythrocytes and plasma cells

Leukocyte common antigen, activation

CD49d

T, B, NK, DC, monocytes, mast cells

α4-integrin; subunit of VLA-4 receptor; adhesion, migration, homing

CD52

Mature T and B; DC, monocytes, mast cells

Co-stimulation; molecular target of Alemtuzumab

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CD56

NK, NKT, T subsets

CD62L B, T subsets, NK, monocytes

NCAM; cell-cell adhesion L-selectin; leukocyte rolling and homing

CD69

Activated leukocytes; NK, Langerhan

co-stimulation; signaling

CD71

Proliferating cells; reticulocytes; erythroid precurors

transferin receptor; iron uptake

CD79b

B

Igβ; subunit of BCR; signaling

CD80

Activated B and T; DC, marcophages

receptor for CD28 and CD153; costimulation

CD83

Activated B and T; mature DC, Langerhans

co-stimulation

CD86

Activated B and T; DC, monocytes, endothelial cells

co-stimulation of T activation and 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

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Chapter 1: Introduction and literature overview

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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 ≥ 5x109L-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, FcγRIIb, 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 ≥ 5x109L-1 monoclonal

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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

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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.

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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 i)

Prognostic factors

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

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heptomegaly are defined as having intermediate-risk disease (stage I-II). Patients with disease-related anemia (hemoglobin level 50x109 having worse prognosis than patients with lower counts (27). In a more recent multivariate analysis of 2146 patients over 20 years, an ALC of >30x109 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 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 >30x109 (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)

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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 activationinduced 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).

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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.

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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).

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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 (SDF1α). 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 miR15a/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 NFκB 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

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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 412% (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

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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 (105107). β2-microglobulin (β2M) Beta2-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 β2M > 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

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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.

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Table 1.1: Major prognostic markers in CLL

Prognostic marker Rai Stage LDT CD38 expression Cytogenetics: Serum β2 Microglobulin

Good prognosis

Poor prognosis

Early stage: 0-II

Late stage: III-IV

>12 months

40kd) probed for CD200, and the bottom part (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.

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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 CD200Ly2 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

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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.

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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 TNFα production; panel b, IFNγ 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 TNFα and IFNγ. 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 TGFβ (data not shown). Moreover, the changes in TNFα and IFNγ 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

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primary CLL cells from 3 different CLL patients (see table 1) with killing in this case assessed using a 51Cr 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 CD200immunoadsorbent 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.

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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

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(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 Imiquimodinduced 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.

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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 costimulatory 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 antihCD200 mAb 1B9 when compared with an isotype control antibody, 3H4.

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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 TNFα and IFNγ 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 TNFα and IFNγ, 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.

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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 cellsurface 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 #

Sex

Age

Years after diagnosis

3 4 5 8 12 * 14 16

M F M M M M M

72 84 62 74 55 71 69

4 10 12 7 6 6 6

22 27 36 41 * 46 57 60 ◘ 61 65 70 71 72 * 73 74 ◘ 75 ◘ 78 ◘ 79 ◘ 80 ⌂I ⌂ II

M F M F M F M M F F M F F M F M M M M M

54 82 59 59 55 77 88 81 61 78 57 48 53 77 63 72 51 51 72 58

6 11 7 6 5 10 6 6 26 5 2 2 13 10 3 5 10 1 5 15

▪ Rai Stage

♦ WBC

● Treatment

%CD38

○ Cytogenetics

IV III IV IV IV II IV IV III IV III III IV IV III IV III II 0 IV III II IV IV II IV IV

175 200 121 220 23 186 189

2 46 2 na 81 72 4

13q-/17p13q13q17p13q-,17p11q-,13qnormal

8

S/CHOP/CVP P/C,S/R CP,CVP,S,FC FR,splenectomy,S CHOP,P none CP CP,FC,FCR,S

125 10 35 121 120 62 89 140 33 21

none CP CP,splenectomy none CP none P splenectomy none none

1 8 7 40 4 na 1 1 18

normal T12 Na normal T12 13q13q-,17pnormal 13q-,11q-

13 65

none none

250 22 80 140 88 96 77

none none CP,FCR Cx2 None Cx2, splenectomy C, F, FC, S, Splen

13 1 2 2 1 71 13 1 2 60 65

13qT12,13qna 13q13qna normal 13q13q17p13q-, 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 (x106 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 1.2x106 hPBLs were stimulated with 8X104 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 (** p0.05) enhanced CTL responses to Ly5 cells, while 3H4 showed no significant effect; b) failure of antiCD200 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 (** p3-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 PKCδ 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

167

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 TAPI0, 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 PMAinduced 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 168

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

169

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.

170

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)

171

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 20µM GM6001 in 24-hr supernatants (n=2); c) CLL cells (n=3) were treated 2.5µg 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) 1µM Ionomycin; and c) 3µg/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.

172

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 50µg/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 PMAtreated cells from Patient 139 and 158 (upper panels). CLL cells from Patient 16 showed

173

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 ttest). c) Serum-starved Hek-hCD200 cells, with or without PMA stimulation, were treated with 50µg/ml TAPI-0, and supernatants were harvested at 24-hr. TAPI-0 inhibited PMAinduced 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

174

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.

175

4.8 Figures Figure 4-1: CD200 is constitutively released from CLL cells 4-1a) 0.2 0.18 0.16

ng/ml

0.14

Patient 58 Patient 81 Patient 43 Patient 82

0.12 0.1 0.08 0.06 0.04 0.02 0

24h

48h

176

4-1b).

0.06 0.05 ng/ml sCD200

Pt14

0.04

Pt90

0.03 0.02 0.01 0 Untreated

GM6001

177

4-1c)

0.12

p=0.01

Patient 158

0.1 0.08 0.06 0.04 0.02 0

Patient 16

ng/ml sCD200

0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

Patient 139

0.25

p=0.04

0.2 0.15 0.1 0.05 0

No treatment

TIMP1

TIMP2

TIMP3

178

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

ng/ml sCD200

0.6

Pt. 7 Pt. 16 Pt. 43 Pt. 58 Pt. 81 Pt. 82

p=0.0008

0.4

0.2

0.0

No treatment

PMA

179

4-2b)

0.8

Pt. 7 Pt.16 Pt.43 Pt.58 Pt.81 Pt.82

ng/ml sCD200

p=0.0532

0.6

0.4

0.2

0.0

No treatment

Ionomycin

180

4-2c)

ng/ml sCD200

0.5

Pt. 7 Pt.16 Pt.43 Pt.58 Pt.81 Pt.82

0.4 0.3 0.2 0.1 0.0

No treatment

TLR7 agonist

181

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:

CD200

Pt. 80:

CD62L

182

4-3b) No treatment

PMA

Pt. 47:

Pt. 155:

CD200

Pt. 80:

CD19

183

4-3c)

No treatment

PMA

Pt. 47:

Pt. 155:

AnnexinV

Pt. 80:

7AAD

184

4-3d)

% CD200 loss: median=44.25%; mean=39.3± 27.5 % CD62L loss: median=95.5%; mean=86.7±17.5

% CD200 loss % CD62L loss

100

50

.8 0 Pt

Pt .1 56

Pt .1 55

.1 0 Pt

Pt .1 54

.4 7

0 Pt

% loss after relative to untreated controls

150

185

Figure 4-4: Detection of CD200 in membrane extracts from PMA-treated CLL 4-4a)

3ug membrane extract/well

180 160

pg CD200 (per well)

140

No treatment PMA treated (40ng/ml)

120 100 80 60 40 20 0

Pt.47

Pt.154

Pt.10

Pt.155

Pt.156

Pt.80

Pt.85

Pt.157

186

4-4b)

Pt. 47

PMA --

+

Pt. 156

--

+

Pt. 80

--

+

Pt.154

Pt.10

Pt.155

Pt. 85

--

--

--

--

+

+

+

Pt. 157

+ -- + Cadherin CD200

5ug membrane extract loaded Primary antibody: rabbit anti-hCD200v+c

187

4-4c) No treatment

PMA

TAPI-0

PMA+TAPI-0

Pt.139

Pt.158

CD200

Pt.16

CD19

188

Figure 4-5: Spontaneous and inducible shedding of CD200 in Hek-hCD200 cells 4-5a)

ng/ml sCD200

10

OPIMEM OPIMEI+PMA

8 6 4 2 0 0

10

20

30

Time (hr)

189

4-5b)

ng/ml sCD200

15

No treatment PMA

p=0.03

10

5

0

No treatment

PMA

190

4-5c)

7

ng/ml sCD200

6 5

No TAPI-0 +100uM TAPI-0

4 3 2 1 0 OPIMEM

OPIMEM+PMA

191

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)------

QVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSE NHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLF c-like domain (domain II)------

NTFGFGKISGTACLTVYVQPIVSLHYKFSEHHLNITCSATARPAPMVFWKV PRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLH Transmembrane region------

Cytoplasmic tail----

LGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDRGEL SQGVQKMT

192

4-6b)

1

2

3

1

2

47kDa

3 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)

193

4-6c)

Sup

Membrane

Pt.158

PMA

+

--

Sup

Pt.71

--

+

Hek hCD200

--

Hek hR1

--

Anti-CD200 v+c Anti-CD200 c-tail

194

4-6d)

Supernatant in AIM-V Cellfree

CLL

Supernatant in OPIMEM HekhCD200

HekhR1

Cellfree

Vanadate

195

Chapter 5: General discussion

196

5.1 General discussion The overexpression of immunoregulatory molecules, which deliver inhibitory signals that generally dominate over stimulatory signals, on tumor cells and tumorinfiltrating 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,

197

we explored the nature of sCD200/CD200:CD200R interactions in association with nonCLL 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 198

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 sCD200hi CLL plasma, but not sCD200lo normal plasma, significantly enhanced engraftment of CLL cells in NOD-SCIDγcnull 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 199

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 sCD200hi 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 xenomicroenvironment, 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

200

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 G0/G1 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

201

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 sCD200hi CLL plasma was compared with that of sCD200lo normal plasma, we found that infusion of sCD200lo normal plasma resulted in predominant T-cell engraftment in all compartments with minimal engraftment of CLL cells. Infusion of sCD200hi 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 CD200mediated 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. 202

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

Normal plasma (sCD200lo)

T cells CLL


Favors T cell engraftment with minimal survival of CLL cells


T cells that are selected to engraft under these conditions do not support CLL growth

CLL plasma (sCD200hi)

CLL

ProCLL T cells


sCD200 in CLL plasma is important in supporting CLL in vivo survival and growth in vivo
T cells that are selected to engraft under these conditions are required for CLL 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

CLL

CD200R“Pro-CLL” T cell /non-T cells

ITIM

ITIM

CD200

Suppression

“TH2” switch; production of “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 Ca2+ 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

205

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 antitumor 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 postanti-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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