CD200: A NOVEL THERAPEUTIC TARGET FOR CHRONIC … · ALL Acute lymphoid leukemia AML Acute myeloid leukemia APRIL A proliferation-inducing ligand B7-H1 B7-homolog 1 (PD-L1 or CD274)

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

ii

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

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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.

iv

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

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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

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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

ix

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

x

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

xi

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

iv Intravenous

LDT Lymphocyte doubling time

LPS Lipopolysaccharide (endotoxin)

MAPK Mitogen-activated protein kinase

xiv

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

xvi

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 non-hematopoietic 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

xvii

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

CD62L B, T subsets, NK, monocytes L-selectin; leukocyte rolling and homing

CD69 Activated leukocytes; NK, 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; co-stimulation

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

1

Chapter 1: Introduction and literature overview

2

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

3

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

4

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.

5

Despite advances in stratifying patients, the use of prognostic markers for

prediction of time to first treatment, response to treatment, and complete remission

requires further research (14). Identification of new prognostic markers will undoubtedly

aid both in deciding the treatment regimen as well as when treatment should begin, thus

improving treatment outcome.

The following section discusses some of the more important clinical and

biological prognostic markers for CLL against which the value of new markers can be

compared. Understanding the role of prognostic markers in CLL progression helps

provide insights into mechanisms driving CLL leukemogenesis.

1.1.1.2 Prognostic factors

i) Staging systems

The Rai and Binet staging systems, developed more than 3 decades ago, were the

first prognostic factors used clinically for assessment and prediction of disease

progression (15, 16). Staging in both systems is determined based on physical

examination and standard laboratory testing, and is easily applied in clinics.

The Rai staging system uses 5 stages (stage 0, I, II, III, and IV), which are further

simplified to 3 groups: low risk (stage 0), intermediate risk (stage I and II), and high risk

(stage III and IV) (17). Assessment is made according to the presence of lymphocytosis,

anemia, and thrombocytopenia, as well as the presence or absence of lymphadenopathy

or splenomegaly. Patients with lymphocytosis in the peripheral blood or bone marrow

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

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

6

heptomegaly are defined as having intermediate-risk disease (stage I-II). Patients with

disease-related anemia (hemoglobin level <110g/L; stage III disease) or

thrombocytopenia (platelet count <100x109/L; stage IV disease), with or without

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

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

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

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

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

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

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

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

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

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

The staging systems are instrumental in clinical decision making particularly regarding

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

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

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

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

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

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

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

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

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

7

prognostic factors (to be discussed below), is the most effective way to predict high-risk

patients at an early stage (20-23).

ii) Biological prognostic markers

Morphology

The morphology of CLL cells in circulation as determined in a blood smear, or in

the bone marrow, is an important prognostic factor for CLL. Atypical, prolymphocytic

morphology, mainly large-cell size and cleaved nucleus, is associated with poor

prognosis, whereas granular and small-sized lymphocytes predicts good disease outcome

(24). In an analysis of CLL cells from 270 patients at Binet stage A, atypical morphology

of CLL cells was shown to predict adverse disease (25, 26).

Absolute Lymphocyte count and lymphocyte Doubling Time (LDT)

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

ALC at presentation was found to be an independent prognostic factor in a univariate

analysis with patients having a count of >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

8

significance of LDT has been shown in a number of studies, in that a LDT <12 months is

associated with poor prognosis and survival, while a LDT >12 months is correlated with

favorable disease course and survival (30-33). For early stage patients, LDT was shown

to be a useful prognostic marker to predict time to first treatment and overall survival

(23). The 2008 guidelines from IWCLL suggests a LDT of less than 6 months in early

stage patients can be used as an indicator for treatment, particularly in patients with ALC

>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)

9

In CLL, the mutation status of IgVH genes is one of the strongest predictors of

disease progression. The pattern and distribution of IgVH mutations in CLL was shown

to be consistent with the canonical somatic hypermutation process involving activation-

induced cytidine deaminase (AID) (41, 42). In two independent landmark studies,

patients whose CLL cells expressed mutated IgVH (M-CLL) were shown to have

favorable disease course, while patients whose cells expressed unmutated IgVH (U-CLL)

showed poor response to treatment and had shorter overall survival rate (43, 44). In a

multivariate analysis of 205 patients, in which a >98% homology to the germline

sequence was used as a cutoff, unmutated IgVH, along with loss of p53, emerged as a

prognostic indicator independent of all other prognostic factors tested (45).

Despite the strong prognostic value of IgVH mutation status, most clinical

diagnostic laboratories are not equipped to routinely perform IgVH mutation analysis,

which is time consuming and expensive. Thus, IgVH mutation analysis has not been

incorporated into routine diagnostic testing.

Zap70 expression

Microarray analyses have shown differential expression of over 300 genes

between the M-CLL and U-CLL, providing a rationale for the search for surrogate

markers for IgVH mutation status to circumvent the difficulty in performing routine

IgVH mutation analysis (46). Zap70, an intracellular protein that is normally expressed

on T and NK cells in association with the antigen receptor, but not on normal B cells, was

found to be overexpressed in U-CLL at the mRNA level (46). Flow cytometry analyses

of CLL cells subsequently showed that Zap70 was expressed in U-CLL at the same level

as in T cells (47).

10

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.

11

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).

12

Indeed, several studies have demonstrated that a cutoff point of 5-7% was more effective

in distinguishing the different prognostic groups (64, 65).

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

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

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

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

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

predictor for Ritcher’s transformation.

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

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

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

1α). Given this functional link between CD38 and Zap70, combined analysis of CD38

and Zap70 expression may enhance identification of high-risk subgroups. In an analysis

of 242 patients, the CD38+Zap70+ subgroup was shown to have shorter overall survival

(30 months) compared with CD38-Zap70- patients (130 months) (68). Survival for

patients with discordant CD38 and Zap70 expression was found to be 40 months,

reflecting intermediate disease progression.

The function of CD38 in CLL biology appears to be multifaceted, with roles in

both CLL proliferation and chemotaxis (section 1.1.3).

Cytogenetics

Genomic aberrations can be identified in ~80% of cases by fluorescence in-situ

hybridization (FISH) with a disease-specific probe (69). Five major classes of

13

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

14

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

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

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

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

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

compartment and development of indolent lymphoproliferative disorders similar to

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

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

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

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

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

DLEU7 was found to encode a protein that suppresses 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

15

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

16

shortest overall median survival (70, 91). Both 11q and 17p deletions have emerged as

independent prognostic markers in several major studies (28).

Genetic analyses have shown that the MDR in 11q22-23 deletions in CLL

contains the ATM (Ataxia Telangiectasia Mutated) gene, encoding for the ATM protein, a

molecular sensor of oxidative stress with a role in the DNA-damage response (92, 93).

MicroRNAs miR-34b and miR-34c, both part of the p53 tumor suppressor network, can

also be lost with 11q22 deletion (94, 95). One of the most important genes in the MDR

of 17p13 deletions is TP53, which encodes p53, perhaps the most well known tumor

suppressor protein. p53 functions through multiple pathways to control a wide range of

cellular responses from carcinogenesis to drug resistance (96, 97). 17p13 deletions are

also associated with loss of miR-34c, which is upregulated after DNA damage in the

presence of p53 (89). In addition, ATM and TP53 mutations, regardless of the presence

of absence of 11q and 17p deletions, are observed in CLL at frequencies ranging from 4-

12% (98-100). Importantly, both ATM and TP53 mutations were identified to be

strongly associated with poor progression-free survival and overall survival, independent

of 11q and 17p deletions, and IgVH mutation status.

Both 11q22-23 and 17p13 deletions are important risk factors for poor response to

the chemoimmunotherapeutic regiments fludarabine + cyclophosphamide (FC) and

fludarabine + rituximab (FR), and are associated with a significant lower response rate

and shorter progression-free survival and overall survival in several clinical trials (21,

101, 102). Both cytogenetic aberrations were also identified to be a risk factor for early

relapse following therapy (21). Deletions in 17p13, in particular, appear to be a predictor

of poor response to fludarabine-based regiments, when compared to all other cytogenetics

17

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

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

107).

β2-microglobulin (β2M)

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 β2-

M > 2.5mg/L showed the highest hazard ratio. Based on its prognostic strength, Letestu

et al proposed determination of β2-M level at diagnosis as part of a cost-effective strategy

for prognosis in Stage-A (Rai stage 0) patients. In a separate study, the prognostic

strength of β2-M was shown to further increase if the β2-M level after glomerular filtration

rate adjustment is used (115).

iii) Prognostic models

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

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

18

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.

19

Table 1.1: Major prognostic markers in CLL

>20%<20%CD38 expression

<12 months>12 monthsLDT

>2.5mg/L

Poor prognosis

<2.5mg/LSerum β2 Microglobulin

Trisomy 12, 11q22-23 deletions, 17p13 deletions

Normal karyotypes, 13q14 deletions

Cytogenetics:

Late stage: III-IVEarly stage: 0-IIRai Stage

Good prognosisPrognostic marker

>20%<20%CD38 expression

<12 months>12 monthsLDT

>2.5mg/L

Poor prognosis

<2.5mg/LSerum β2 Microglobulin

Trisomy 12, 11q22-23 deletions, 17p13 deletions

Normal karyotypes, 13q14 deletions

Cytogenetics:

Late stage: III-IVEarly stage: 0-IIRai Stage

Good prognosisPrognostic marker

20

1.1.2 Biology of CLL cells

1.1.2.1 The origin of CLL

Two models for the cellular origin of CLL have been proposed. The first model,

based on the observation that CLL cells carry either mutated or unmutated IgVH,

proposes that M-CLL and U CLL cells are derived from two distinct populations: M-CLL

cells are derived from antigen-experienced B cells expanded through a classical germinal

center reaction, where somatic hypermutation occurs, in the presence of T cell-help. U-

CLL cells, in contrast, are derived from marginal zone B cells by T-cell independent

processes. The second model proposes a common origin for both M-CLL and U-CLL

(116). Evidence from gene expression profiling supports the second model, as U-CLL

and M-CLL cells were shown to have a homogeneous phenotype bearing markers of

memory B cells (46, 117).

Based on the observation that CLL cells share phenotypic characteristics of

antigen-experienced memory-type cells, the search for a cellular origin of CLL has

largely focused on identifying normal counterparts to CLL in the mature B-cell

compartments. For example, based on several murine models of CLL, B-1a B cells have

been proposed as a candidate population from which CLL is derived (118-120).

However, this view was recently challenged by Kikushige et al, in a study that

demonstrated clonal proliferation early in B-cell ontogeny and abnormalities in the

hematopoietic stem cell (HSC) compartment in CLL (121). Specifically, transplantation

of purified CLL-HSC, but not CLL-pro-B cells or mature CLL cells, into

immuncompromised NODSCID/IL2rnull mice resulted in de novo generation of

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

21

original donors. Gene analyses showed elevated levels of lymphoid-related lineage

specific genes in CLL-HSCs, although no CLL-related cytogenetic abnormality was

detected. Based on these results, Kikushige et al propose a new model for the cellular

origin of CLL in which CLL leukemogenesis occurs in a stepwise pattern that is initiated

at a much earlier stage than had been appreciated. Aberrations in HSC with

predisposition for B-cell ontogenesis are suggested to lead to polyclonal expansion at the

pro-B cell stage, and then to MBL, with each step leading to acquisition of new

malignant properties. According to this model, progression from MBL to CLL requires

further oncogenic events (Fig 1.1).

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

22

However, several issues with this model remain to be resolved. A vast majority

of monoclonal B cells generated de novo in the mouse as shown in the study were found

to have mutated IgVH, unlike in CLL where unmutated IgVH is found in about 50% of

cases (122, 123). The high frequency of mutated IgVH and absence of cytogenetic

abnormalities in the engrafted B cells also suggests an MBL-like condition, from which

only about 1.1% of patients would go on to develop progressive CLL (4). Moreover,

molecular analyses showed distinct IGHV and IGLV gene usage by M-CLL and U-CLL,

indicating no conversion from U-CLL to M-CLL (124). Engrafted B cell clones also did

not have cytogenetic abnormalities found in both MBL and CLL cells (3, 125). Thus,

whether HSCs are the origin from which both M-CLL and U-CLL are derived and the

point during the development pathway at which the two distinct diseases diverges then

evolves require further investigation.

1.1.2.2 Role of BCR signaling

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

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

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

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

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

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

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

signaling in the pathogenesis and progression of CLL.

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

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

23

a subset of patients, regardless of geographic location, share remarkably similar BCRs

with closely related complementarity determing region 3 (CDR3) structural features that

suggests the recognition of common antigens (127-131). Structural analyses of CLL-

BCRs with closely homologous (stereotyped) CDR3 showed specificity to apoptotic

antigens and carbohydrate determinants of bacterial capsules or viral coats, akin to

polyreactive “natural” antibodies, leading to the hypothesis that clonal expansion of CLL

cells is driven by antigen-selection (129, 131, 132). A high frequency of stereotyped

HCDR3 was observed particularly in patients with unmutated IgVH (133). Moreover,

expression of a particular stereotyped IGHV was shown to be associated with distinct

clinical and biological characteristics. For example, cases expressing stereotyped

IGHV4-34/IGKV2-30 were shown to have mostly indolent disease, although a separate

study also showed a tendency for CMV and EBV persistence (134, 135). Expression of

IGHV3-21, on the other hand, was associated with poor prognosis regardless of IgVH

mutation status (136). Furthermore, a recent study showed extensive intraclonal

diversification occurring in a subset of cases with stereotyped IGHV4-34, demonstrating

ongoing interactions with antigen (137).

Second, though sharing a gene “signature” profile, M-CLL and U-CLL cells were

found to differ in the expression of over 300 genes, many of which are known to play

important roles in BCR signaling (46, 117). Interestingly, CLL cells have been shown to

have a diminished response to BCR ligation, resembling an “anergic” phenotype (138).

Indeed, BCR ligation by soluble anti-IgM antibodies, which deliver transient signals

through BCR, was shown to result in an incomplete BCR activation response. However,

sustained BCR ligation by immobilized anti-IgM was able to elicit prolonged

phosphorylation of ERK and Akt, resulting in increased levels of the anti-apoptotic

24

protein myeloid cell leukemia-1 (Mcl-1) and protection from fludarabine-induced

apoptosis, showing that BCRs on CLL cells were responsive only to sustained

stimulation (139, 140). In addition, CLL cells with unmutated IgVH (U-CLL cells)

appear to show stronger responses to BCR ligation than CLL cells with mutated IgVH,

further supporting the role of BCR signaling in disease activity (141).

Upon BCR ligation, Syk, a key mediator of proximal BCR signaling, is recruited

to the BCR complex, which in turn associates with adaptor proteins to phosphorylate

downstream signaling intermediates, eventually resulting in the activation of NFκB and

several pro-survival pathways, as well as STAT3, an anti-apoptotic transcription factor

(142). Syk was found to be overexpressed and constitutively phosphorylated in CLL

cells (143). Moreover, the protection from chemotherapy-induced apoptosis by BCR

cross-linking was abrogated by inhibition of Syk (144).

A third line of evidence for an important role of BCR signaling is the association

between Zap70 expression and disease progression. Involvement of Zap70 in BCR

signaling, likely as an adaptor protein, was demonstrated by ligation of BCR in Zap70+

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

than in Zap70- CLL cells (47, 145). Transgenic expression of Zap70 in Zap70- CLL cells

was also shown to be effective in enhancing downstream signaling through IgM-ligation

(146). Proteomic analyses on Zap70+ and Zap70- CLL cells following IgM ligation

established two distinct proteomic profiles of activation based on Zap70 expression that

were reflective of the aggressive and indolent disease phenotype (147). Interestingly,

through this analysis, a third subgroup was identified, in which CLL cells showed an

25

indolent phenotype (low Zap70 expression) but an activation proteomic profile similar to

that of aggressive CLL (147).

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

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

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

148). Tonic BCR signalling is particularly important in the pro-survival effects of BAFF

and APRIL, both of which are elevated in CLL (148, 149). However, the role of tonic

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

In addition to mediating pro-survival signals, sustained BCR signaling mediated

through Syk also appears to upregulate a number of adhesion molecules and increase the

ability of CLL cells to migrate toward the chemokines CXCL12 (SDF-1) and CXCL13,

both important for homing of CLL cells to the tissue microenvironment (150). Following

entry into the tissue microenvironment, BCR triggering plays a role in retaining CLL

cells in the microenvironment by downregulating CXCR4, a receptor for CXCL12, and

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

CLL cells or cells from patients with aggressive disease were shown to be more

responsive to BCR-mediated retention in tissue microenvironment. The intrinsic link

between BCR signaling and the CLL microenvironment is further addressed in section

1.1.3.

1.1.2.3 Proliferative potential of CLL cells

Until recent years, CLL was believed to be a disorder resulting from accumulation

of malignant B cell clones defective in apoptosis, rather than from uncontrolled cell

growth like in other malignancies. Earlier studies on CLL in vivo kinetics found CLL

26

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

However, data from recent studies have challenged this view.

Analysis of telomere length and telomerase activity in CLL cells, which had

shorter telomeres than B cells from normal individuals, indicated a prolonged

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

expressing CD38 having higher proliferation rate than the CD38- clones (156). Given

that CD38 is highly expressed on CLL cells in the secondary lymphoid tissues, and that

27

CD38 expression can be modulated by interaction with non-CLL cells, CD38+ CLL cells

in circulation may represent a subset of cells that have recently exited the tissue

microenvironment, where most of CLL proliferation is believed to occur (60).

1.1.3 CLL microenvironment

One of the hallmarks of CLL cells, whether from aggressive or indolent disease,

is their resistance to apoptosis in vivo. However, when cultured in vitro, CLL cells tend

to undergo apoptosis spontaneously and generally fail to proliferate without external

stimuli (157, 158). The apoptosis of CLL cells in vitro can be rescued by co-culturing

with stromal cells or addition of a number of soluble factors mimicking those found in

the tissue microenvironments in vivo (159, 160). Indeed, pseudofollicles termed

“proliferation centers” in secondary lymphoid compartments such as lymph nodes and

spleen are known to be crucial for CLL disease progression (Fig 2). CLL cells in

proliferation centers express markers associated with proliferation, such as CD71 and

Ki67, and higher levels of CD23, as compared to CLL cells from peripheral blood,

supporting the hypothesis that the proliferative pool of CLL cells contributing to the

dynamics of CLL turnover reside in these tissue microenvironments (161). Constituents

of the microenvironment provide proliferative and pro-survival signals to CLL cells both

through soluble factors and cell-cell contacts (Fig 3). These pro-survival factors in the

CLL microenvironment play a crucial role in controlling the in vivo dynamics of CLL

cells, and are critical in determining the efficacy of chemotherapeutic agents for CLL.

Insight into the CLL microenvironment thus has significant implications in the design of

in vitro and in vivo models of CLL for drug screening purposes.

28

Figure 1-2: Cellular components of CLL proliferation center

Resting CLL cells

Proliferating CLL cells

Nurse-like cell

Follicular dendriticcell

T-cell Mesenchymalstem cell

29

Figure 1-3: Molecular crosstalks between CLL cell and the cellular components in

the CLL microenvironment (see section 1.1.3a-f)

YSyk

CD40

CD40LIL4, IL6, etc

NLC

T cell

MSC

BAFF

APRIL

BAFF-R TACI BCMA

CCL3/4

NFκB

CD100

Plexin-B1

BCRAntigenic

stimulation

PDGF PDGF-R

VEGFVEGF-R

PKC

Mcl-1 Bcl2STAT3

IL4-R

Akt

CD38

Zap70

ERK

CLL

CCR1/ CCR5

CXCL12

CXCR4

CXCR5

CXCL13

30

1.1.3.1 BCR signaling in CLL microenvironment

One of the key signals that CLL cells likely receive in proliferation centers is

antigenic stimulation. Microarray analyses of CLL cells from lymph nodes showed

upregulation of genes implicated in ongoing BCR signaling and activation of the NFκB

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

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

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

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

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

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

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

lymph nodes (140, 162, 164).

1.1.3.2 TLR signaling in CLL microenvironment

In addition to BCR ligation, signaling through toll-like receptors (TLRs) is

capable of inducing the NFκB pathway in a MyD88-dependent manner (165).

Stimulation of CLL cells by bacterial lipopeptides, ligands for TLR1/2/6, was shown to

protect CLL cells from spontaneous apoptosis in vitro via the NFκB pathway, while

triggering of TLR9 resulted in proliferation and increased CD38 expression on CLL cells

(166, 167). Importantly, in a whole-genome sequencing analyses conducted by Puente et

al, activating mutations in MYD88 leading to a 5-10 fold increase in response to TLR

triggering were identified as one of the recurrent mutations prominent in U-CLL (168).

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

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

31

of which have been implicated in the recruitment of macrophages and T lymphocytes,

and likely contribute to disease progression by establishing a microenvironmental niche

favoring the survival and proliferation of CLL cells. The contribution of TLR signaling

to CLL progression was further demonstrated in Tcl1 trangenic mice (a murine model of

CLL, to be discussed in section 1.1.4) lacking TIR8, a negative regulator of TLR, which

were found to have accelerated disease and shorten life span in comparison to mice with

wildtype TIR8 (169).

1.1.3.3 TNFR signaling in CLL microenvironment

Signaling through the tumor necrosis factor family member BAFF, in concert

with BCR signaling, has been shown to be required for B-cell survival and the

maintenance of B-cell homeostasis (59, 170). BAFF and its closely related homolog

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

BAFF receptor BAFFR (171). CLL cells, like normal B cells, were shown to express all

three BAFF and APRIL receptors at the mRNA level; moreover, addition of soluble

BAFF and APRIL was sufficient to protect CLL cells from spontaneous and drug-

induced apoptosis in vitro through activation of the NFκB pathway (172, 173). BAFF

and APRIL were shown to be produced by a subpopulation of monocyte-derived CD68+

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

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

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

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

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

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

old age (119).

32

1.1.3.4 T cells in CLL microenvironment

Systemic dysregulation in the T-cell compartment has been reported in numerous

studies, and contributes to increased susceptibility of CLL patients to bacterial and

opportunistic infections (6). Immunophenotyping with absolute-bead calibrated

measurements showed down-regulated TCR signaling, impaired expression of T-helper 1

(Th1) cytokines, and increased production of IL-4 by CD4+ helper T cells even at early

disease stages (177). CD4+ T cells and CD8+ T cells from CLL patients also showed

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

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

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

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

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

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

an integral part of the CLL microenvironment (182).

Activated CD4+ T cells expressing CD40L (CD154) are found to reside in close-

proximity and likely come into contact with CLL cells (159). Activation of CD40 by

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

NFκB pathway (183, 184). Microarray analyses showed that CD40 activation in CLL

cells resulted in increased induction of CD27, STAT3, and IL10 receptor, as well as

genes involved in resistance to apoptosis when compared to normal B cells (185).

Consistent with these data, CD40 activation was also shown to induce production of

cytokines important for CLL survival by CLL cells, including IL-6, IL-8, and IL-10 (186-

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

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

33

required for somatic hypermutation and class-switch recombination (CSR) that is

significantly elevated in a subset of U-CLL cells (189).

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

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

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

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

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

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

upregulated on CLL cells upon contact with CD4+ T cells, possibly via exposure CD40L

and IL4 (60, 194).

Communications between CLL cells and T cells and other cellular components in

the microenvironment are multi-directional and are mediated through both cell-cell

contacts and secretion of soluble factors (Fig 1.3). Some of the defects observed in CLL-

T cells appear to be induced directly by CLL cells through cell-cell contact (195). In a

microarray analysis, Görgun observed reduced NFκB activation and a shift to Th2

differentiation in normal T cells that were co-cultured with CLL cells (196). CLL cells

also appear to induce cytoskeletal defects in normal CD4+ T cells leading to defective

immunological synapse formation (195). CLL cells affect normal CD8+ cytotoxic T

cells by downregulating genes involving in vesicle trafficking and mobilization of

effector molecules (196). Furthermore, CLL cells also play a role in the recruitment of T

cells into the microenvironment by production of T-cell chemokines CCL3 and CCL4

through a mechanism that involves CD68+ NLCs (197).

34

1.1.3.5 Other cellular components in CLL microenvironment

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

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

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

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

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

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

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

in CLL cells (203).

Bi-directional communication occurs between CLL cells and multiple cellular

constituents of the microenvironment. In co-cultures, CLL cells were shown to be

capable of turning on pro-survival capacity in normal peripheral blood CD14+ cells in

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

(204). CLL cells were also shown to produce Platelet-derived growth factor (PDGF),

which binds the PDGF receptor on MSCs leading to activation of the pro-survival Akt

pathway and production of VEGF by MSCs (205, 206). VEGF, in turn, is known to

induce pro-survival signals and upregulate expression of PKCβ, a key mediator

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

1.1.3.6 CLL trafficking to the microenvironment

Due to the importance of microenvironental cues for CLL survival and growth,

molecules involved in the trafficking of CLL cells and recruitment of bystander cells to

lymphoid tissues or bone marrow play a critical role in driving disease progression. One

of the most important chemokine networks involved in CLL homing is the CXCR4-

35

CXCL12 axis, with CXCR4 overexpressed on CLL cells and CXCL12 (SDF-1) produced

by MSCs in the tumor microenvironment (174, 208). CXCR4 drives both migration of

CLL cells into lymphoid tissues and adhesion of CLL cells to MSCs, promoting CLL

survival and chemo-resistance (209). CD38 also appears to play an important role in this

axis, as CLL cells activated with an anti-CD38 mAb were shown to be have increased

sensitivity to CXCL12 (210).

Migration through endothelial cell-barriers and basement membranes permitting

entry of CLL cells from the blood stream into the tissue microenvironment requires the

chemokines CCL21 and CCL19 produced by cells located in high endothelial venules

(HEVs), which acts on the receptor CCR7 on CLL cells (211). This process is aided by

matrix metalloprotease 9 (MMP9) on CLL cells, which is involved in the degradation of

the extracellular matrix and/or basement membrane (212). Within lymphoid follicles, the

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

Acute lymphocytic leukemia (ALL), the CXCR5-CXCL13 axis has also been shown to

regulate the interaction between leukemic cells and CD8+ T cells (214).

The hyaluronan receptor CD44 and integrins, particularly those containing the α4

chain (CD49d/VLA-4) are also essential to CLL homing. CD49d, whose expression on

CLL cells has been proposed to predict adverse disease, facilitates migration of CLL cells

to the bone marrow (215-217). In addition, engagement of α4β1 integrin results in

MMP9 upregulation (218). CD44, in association with α4β1 integrin, has been shown to

constitute a docking complex for MMP9 in mediating CLL migration and survival (219,

220).

36

1.1.4 Animal models of CLL

The search for novel therapeutics for CLL relies largely on assays in which

therapeutic agents are tested with CLL cells growing in tissue culture conditions bearing

little resemblance to the in vivo microenvironment where CLL cells thrive. This

approach leaves an undesirable gap between bench science and clinical relevance.

Animal models, often considered the bridge that links the bench to the bedside, thus have

an important role in elucidating the in vivo efficacy of therapeutic agents. For generation

of applicable animal models for CLL, two approaches have traditionally been employed:

a) the generation of genetically modified mice to produce animals which develop

pathology resembling human CLL; b) the generation of humanized xenograft models of

CLL by engraftment of human CLL cells into immunocompromised animals.

1.1.4.1 Murine models of CLL

The APRIL transgenic mice and DLEU2/miR-15a/16 knockout-mice discussed in

previous sections develop lymphoproliferative disorders (74, 119). B-cell targeted

transgenic expression of the microRNA miR-29, often found in patients with indolent

disease, also resulted in clonal expansion of CD5+ B cells and development of frank

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

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

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

particularly aggressive CLL.

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

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

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

37

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

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

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

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

human, as manifested by accumulation of monoclonal CD5+ leukemic cells in peripheral

blood, splenomegaly, lymph node involvements, and bone marrow infiltration (224).

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

BCRs that react with autoantigens, suggesting antigen involvement in disease

development (225). Moreover, most leukemic cells from the peripheral blood of Eµ-

TCL1 transgenic mice were arrested at G0/G1 stage and showed in vitro sensitivity to

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

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

Although Eµ-TCL-1 mice develop a leukemic disease that seems to recapitulate

aggressive human CLL, limitations to its use remain because of the extended period of

time required for disease development and the variability in disease onset in each

individual mouse.

1.1.4.2 Xenograft models of CLL

Engraftment of human CLL cells in immunocompromised animals may represent

a superior approach to drug screening, since it may allow for a more shortened

experimental duration and uses human CLL cells as targets. This approach, however, has

been compounded by the resting nature of CLL cells, particularly those from peripheral

blood, and the requirement for complex interactions between CLL cells and the various

microenvironmental components to sustain CLL survival and growth, which are likely

38

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

CLL have resulted in sub-optimal engraftment of CLL cells and generally have failed to

recapitulate the human disease.

In early attempts using SCID mice, infusion of PBMC from CLL patients into

immunocompromised SCID mice was associated with recovery of only 10% of injected

CLL cells within 48 hours in vivo. The remaining human cells persisted only in the

peritoneal cavity with no human CLL migration to spleen or bone marrow (229, 230).

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

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

Balb/c chimeric mice, generated by reconstitution of lethally irradiated Balb/c with SCID

bone marrow, predominant engraftment of CLL cells was noted when PBMCs from Rai

stage IV patients were used. Infusion of PBMCs from early stage patients, in contrast,

resulted in engraftment of predominantly T cells (231, 232). In addition, autologous T

cells were shown to impede engraftment of CLL cells from both early and late stage

patients (233). However, CLL engraftment was only followed up to two weeks in these

studies, and again persistence of CLL cells was observed only in peritoneal cavity.

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

CLL PBMC into NOD.SCID mice resulted in recovery of CD19+CD5+ CLL cells in both

the peritoneal cavity and spleen of recipients at up to 12 weeks post engraftment (234).

The engraftment efficiency of CLL cells in mouse recipients was shown to correlate with

disease stage and LDT, but not other clinical parameters, including cytogenetics, CD38

and Zap70 expression, and IgVH mutation status. Although the human CLL cells

engrafted in spleen were shown to co-localize with T cells, the absolute cell recovery in

39

both peritoneal cavity and spleen remained low, with a greater than 10-fold decline in cell

number observed from 8-12 weeks.

Using the more immunocompromised NOD.SCID/IL2Rnull mice, in which the IL-

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

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

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

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

CLL cells (235, 236). This approach presumably circumvents the failure of the murine

xeno-microenvironment to support CLL growth. Proliferation of CLL cells and detection

of CLL cells in mouse blood was observed, two features of human CLL not seen in

previous xenograft studies. In contrast to previous reports by Shimoni A et al, autologous

T cells, but not the normal stromal components, were found to be required for the

survival and proliferation of CLL cells in this model, at least in the initial phase of

engraftment. However, although substantial in vivo proliferation of CLL cells was noted

in this model, engraftment of CLL cells did not persist beyond 12-weeks, by which time

T cells eventually suppressed CLL repopulation and the animals subsequently succumbed

to GVHD.

An alternative approach to creating xenograft models of CLL involves the use of

human cell lines rather than primary CLL cells, which actively proliferate without the

need for microenvironment cues. This approach also has limitations. In a study by

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

MEC2 cells were shown to establish tumors in SCID animals (237). Recently, a

xenograft model using the CLL cell line MEC1 was reported and suggested to represent a

40

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

Rag2-/-γc-/- mice, which lack B, T, and NK cells (238). Both the subcutaneous and

intravenous inoculation route resulted in significant organ infiltration of tumor cells and a

substantial presence of MEC1 cells in mouse blood accounting for a majority of

circulating cells. One caveat to the application of this model for CLL drug screening is

that MEC1 cells lack CD5 expression, the most significant marker for CLL that is known

to play an important functional role for CLL pathophysiology (116).

Development of a useful xeongraft model for CLL remains elusive. We postulate

advances in development of such a model might benefit from further understanding in

the biology of CLL cells, their interaction with the microenvironment, and the role of

other in vivo factors driving CLL growth in patients.

1.1.5 Immunotherapy for CLL

Although therapeutic responses to chemotherapeutic regiments for CLL have

improved substantially in the past decade with the use of purine analogs such as

fludarabine, development of drug resistance and refractory disease in a subset of patients

posts a major barrier to achievement of maximal disease eradication (239). In recent

years, therapeutic monoclonal antibodies have emerged as a promising treatment option

for CLL, either alone as a single agent or in conjunction with traditional

chemotherapeutic regiments (240). Therapeutic approaches to augment immune

responses against leukemic cells could potentially synergize with cytotoxic

chemotherapeutic agents and therapeutic monoclonal antibodies to further improve

treatment responses.

41

1.1.5.1 Therapeutic monoclonal antibodies

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

Rituximab and Alemtuzumab. Rituximab, the first therapeutic monoclonal antibody

approved for the use as an anti-cancer agent, targets the CD20 antigen commonly

expressed on CLL cells, while Alemtuzumab targets cell-surface CD52, found on both B

and T cells (241). Therapeutic monoclonal antibodies mediate anti-tumor effects through

several distinct mechanisms, including complement-mediated cytotoxicity (CDC),

antibody-dependent cell-mediated cytotoxicity (ADCC), and FcγR-mediated

phagocytosis (242). The efficacy of therapeutic antibodies thus depends on the

expression level of the targeted antigen on tumor cells, the integrity of apoptotic cascades

within tumor cells, and the availability and functional capacity of effector cells.

Rituximab is known to target CLL cells by CDC and ADCC dependent mechanisms, and

the therapeutic response can be compromised by low CD20 expression by CLL cells, the

presence of p53 mutations, and altered innate immune function (83, 243, 244).

Competent immune-effector cells, in particular, are instrumental for optimal efficacy of

many therapeutic antibodies (245).

1.1.5.2 Chemotherapy-elicited immune responses

Traditional cytotoxic chemotherapeutic agents and cytotoxic thereapeutic

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

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

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

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

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

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

42

vesicles (calreticulin), extracellular ATP, and factors associated with chromatin, all of

which have been shown to increase the capacity of CD8+ T cells to produce IFNγ (247-

249). Induction of such effective immune responses through immunologic cell death

also involves TLRs and the purinergic receptor P2RX7 on dendritic cells (250, 251). In

CLL, the generation of effective anti-tumor immune responses in this scenario is

compounded by the systemic immune dysfunction seen particularly in late stage patients.

Global reduction in naïve CD4+ and CD8+ T cells, coupled with increased frequencies of

regulatory T cells, hinders induction of cytotoxic CD8+ T cells (252).

1.1.5.3 Approaches to CLL immunotherapy

Immunotherapeutic approaches to improve effector cell functions and for

induction of protective anti-tumor immune responses have to overcome the

immunological barriers posed by the immunosuppressive nature of the tumor

microenvironment (253). This is challenging particularly in the case of CLL, as in

addition to immune dysfunction, CLL cells also lack expression of co-stimulatory

molecules and are themselves poorly immunogenic (254).

A number of vaccine approaches have been proposed to improve immune

responses to leukemic cells in CLL patients. In a phase I clinical trial, vaccination using

autologous CLL cells modified ex vivo to express CD40L was shown to be effective in

transiently increasing the number of tumor-specific T cells following treatment (255). In

addition, the CD40L-transduced autologous CLL-cell vaccine induced expression of the

costimulatory molecules CD80 and CD86 on by-stander CLL cells. In a separate study

with 8 patients, vaccination using autologous CLL cells transduced to express CD40L

and IL2 ex vivo augmented granzyme B expression and production of IFN-γ by T cells,

43

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

Interestingly, specific cytotoxic T-cell responses against a subpopulation of CLL cells

with increased drug resistance were found in a small subset of patients who received the

vaccine (257). Non-gene therapy based approaches have also been proposed to enhance

immunogenicity of CLL cells. For example, treatment of CLL cells with a TLR7 agonist

was shown to induce expression of co-stimulatory molecules on CLL cells (258, 259). In

a follow-up phase I/II clinical trial, however, TLR7 agonist immunotherapy was shown to

induce only weak responses in patients in vivo (260).

Another promising approach is the manipulation of T lymphocytes to express

tumor-specific T-cell receptors or chimeric antigen receptors. Tumor-specific T cells

were successfully derived by genetic modification of T cells to express a chimeric

antigen receptor targeting CD19, followed by in vitro expansion and activation on anti-

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

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

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

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

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

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

specificity for CD19, coupled with 4-1BB (CD137, a costimulatory receptor) and CD3ζ

signaling domains (264). Importantly, infusion of these modified autologous T cells was

shown to achieve complete remission in a patient with refractory disease. The generation

of chimeric receptors against CLL-specific antigens coupled with factors to correct T cell

defects thus provides a promising approach for T-cell based therapy.

44

Despite these recent advances persistent therapeutic response and disease

remission have yet to be achieved by immunotherapeutic approaches. One potential

hindrance to effective induction of anti-tumor immune responses may reflect an

overexpression of immunoregulatory molecules and receptors by CLL cells, which, due

to the systemic nature of the disease, may contribute to systemic immunosuppression.

A molecule whose immunomodulatory function has been documented extensively

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

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

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

reviews the literature on CD200 in different disease models.

1.2 CD200

1.2.1 Immunoregulatory molecules in the evasion of tumor immunosurveillance

In the recent update to a seminal paper on the hallmarks of cancer by Hanahan et

al, the ability of tumor cells to evade anti-tumor immune responses is acknowledged as

an important factor influencing tumor progression (265). The evasion of tumor

immunosurveillance by tumor cells is mediated by an immunosuppressive network that

includes tumor cells, tumor-infiltrating immune cells with regulatory phenotypes, and the

production of immunomodulatory cytokines and chemokines (266).

Immunoregulation per se can be attributed to one of, or a composite of, several

mechanisms, including deletion/inactivation of functional responding lymphocytes, or

their suppression by exogenous cells/factors. As far as tumor biology is concerned, there

is growing evidence for a role for immunosuppressive molecules released by tumor cells

45

themselves, or whose release is under control of those tumor cells, in antagonizing the

activity of anti-tumor immune cells. In addition, infiltration of populations of regulatory

cells, including a Treg population, or so-called myeloid-derived-suppressor cells

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

tumors.

It is now apparent that the overexpression of immunoregulatory molecules by

tumor cells and tumor-infiltrating immune cells is a relatively common feature of the

tumor microenvironment. Immunoregulatory molecules and their receptors mediate

suppression by delivering signals that dominate and override costimulation (267). A

number of immunoregulatory molecules have been shown to be overexpressed on cancer

cells of different tissue origins. For example, the co-inhibitory molecule of the B7

family, B7-H1 (PD-L1), is overexpressed in multiple myeloma, leukemia, ovarian, and

breast cancer (267). Immunoregulatory molecules such as B7-H1 mediate inhibition

through interaction with their corresponding receptors on effector cells. Thus, in ovarian

cancer, the inhibitory receptor for B7-H1, PD-1, was found on tumor infiltrating CD8+ T

cells carrying TCRs with specificity for tumor-associated antigens but with defective

effector functions (268). Another co-inhibitory receptor, BTLA, was found to be

expressed at high levels on human melanoma tumor antigen-specific effector CD8+ T

cells that were susceptible to inhibition by the BTLA ligand HVEM (269). BTLA+CD8+

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

BTLA - counterparts (270). These recent studies support the hypothesis that

immunoregulatory molecules expressed by tumor cells play an important role in

modulating anti-tumor immune responses.

46

Monoclonal antibody therapies targeting the B7-H1 receptor PD-1 have improved

anti-tumor activity in a phase I clinical trial on patients with metastatic disease (271).

The blockade of CTLA-4, the inhibitory receptor for CD80 and CD86, by the targeting

monoclonal antibody ipilimumab, in concert with the chemotherapeutic agent

dacarbazine, was recently reported to improve overall survival of metastatic melanoma

patients in a phase 3 clinical trial (272). In a separate study in which immunity to a

tumor-associated antigen (TAA) was analyzed following ipilimumab treatment, patients

with base-line TAA antibody prior to treatment attained even greater benefits from

ipilimumab (273). From these prelimnary approaches, immunoregulatory molecule

blockade appears to be a novel promising approach to immunotherapy.

1.2.2 The CD200:CD200R axis

CD200 is a type I transmembrane molecule and a member of the immunoglobulin

supergene family (274). CD200 has a short cytoplasmic tail with no known signaling

motifs, and is thought to mediate its immunregulatory functions through binding to its

receptor, CD200R(s) (275). CD200 expression has a relatively broad distribution, and

can be found on both hematopoietic and non-hematopoietic cells. In mouse, five CD200

receptors, CD200R1-R5, have been described. Of the five receptors, only two, CD200R1

and R2, are found in human. CD200R1 is the major receptor for CD200 in both mouse

and human, and contains the inhibitory ITIM motif in its cytoplasmic tail that is

responsible for delivering inhibitory signals downstream (276). Functional properties

have been attributed to the alterative receptors CD200R2-R5 in mouse; however, in

human, the function of CD200R2 remains unknown (277). Expression of CD200

receptor(s) is restricted to cells of the myeloid lineage, B cells, NK cells, and activated T

cells (276, 278).

47

The immunoregulatory function of CD200 has been demonstrated in a number of

models. We reported attenuation of collagen-induced arthritis and reduced allo- and

xenograft rejection by infusion of a soluble recombinant form of CD200, CD200Fc

(279-281). CD200 knockout mice show a normal phenotype but were more susceptible

to experimental allergic encephalomyelitis (EAE) (282). Tolerance and long term

survival of both skin and cardiac allografts occurred in mice with transgenic

overexpression of CD200 (283). Analyses of infiltrating cells in tolerated allografts in

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

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

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

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

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

287). Several vial homologs of CD200 have also been identified, indicating that this axis

has been exploited by viruses as a mean to control host immune responses (288, 289).

1.2.3 CD200 in cancer

In recent years, CD200 has been identified as an immunoregulatory molecule that

is frequently elevated in various types of cancers. In acute myeloid leukemia (AML) and

multiple myeloma, elevated expression of cell surface CD200 predicts poor prognosis

(290, 291). A functional role for CD200 on AML cells in suppressing the cytolytic

activity and production of IFNγ by NK cells in vitro was reported (292). In a genome-

wide gene expression analysis of lymphoblasts from acute lymphoblastic leukemia,

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

48

Overexpression of CD200 has also been documented in non-hematopoietic

tumors (294). CD200 expression in melanoma was found to be a downstream target of

the RAS/RAF/MEK/ERK oncogenic pathway and was shown to inhibit T cell activation

through DCs (295). Moreover, tumor cells with stem cell markers in prostate, breast,

colon, and brain cancers were found to co-express CD200, suggesting a potential role of

CD200 in cancer stem cells (296, 297). Importantly, CD200 expression on tumor cells

can be modulated upon immune challenge in vivo. Using a murine breast tumor cell line

EMT6 with no detectable CD200 expression in vitro, we showed that CD200 expression

on EMT6 cells was induced in vivo after transplantation into immunocompetent C57B/6

mice (298). CD200 induction in EMT6 appeared to require the presence of a functioning

immune system, as EMT6 cells remained CD200- in immunocompromised NOD.SCID

mice and CD200-transgenic mice.

Insights into the contribution of CD200 in tumorigenesis were provided by studies

on CD200-knockout mice, which were shown to be resistant to chemical-induced skin

carcinogenesis (299). Decreased tumor growth in these mice was accompanied by

increased expression of proinflammatory cytokines by DCs in skin-draining lymph

nodes, indicating that tumor growth in the presence of CD200 was likely a result of

CD200-induced immunosuppression. It remains unknown whether CD200:CD200R

mediated immune evasion generally requires tumor and/or host expression of CD200.

Expression of CD200 on tumor-infiltrating cells in the microenvironment may be

sufficient to mediate the immunosuppressive effects of CD200 in the presence of

CD200R-expressing effector cells.

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Recently, evidence has accumulated to suggest a role for CD200 in tumor

metastasis. In an attempt to clone tumor cells which have metastasized to tumor draining

lymph nodes, higher frequencies of CD200+ metastatic clones were observed in wildtype

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

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

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

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

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

Consistent with the EMT6 model, CD200+ squamous carcinoma cells appeared to have a

selective advantage to metastasize, possibly through modulating CD200R+ myeloid cells

in the lymph node microenvironment.

1.3 Ectodomain shedding

Studies on CD200 in various disease models have thus far focused on the function

of CD200 expressed on the cell surface. However, many cell-surface immunoregulatory

molecules are found in soluble form in the serum of cancer patients where they are

believed to play significant roles in disease progression. We recently identified a novel

soluble form of CD200 that is likely generated by mechanisms of ectodomain shedding,

and investigated its potential contribution to immunoregulation in the context in CLL.

This section provides a brief introduction to ectodomain shedding.

Ectodomain shedding involves proteolytic cleavage of a transmembrane protein at

the juxta-membrane region, resulting in the release of the free ectodomain into the

extrcellular matrix (302). The released cytoplasmic domain in some cases may

participate in signal transduction events in the cytoplasm (303). All structural and

50

functional categories of transmembrane proteins are susceptible to ectodomain shedding.

Hence, it is a process that potentially modulates most major cellular processes, from the

release of cytokines, growth factors, to cell adhesion and migration.

1.3.1 The ADAM proteases

A disintegrin and metalloprotease (ADAM) family of proteases have been

identified as the main mediator of ectodomain shedding responsible for the cleavage of a

wide range of functionally and structurally diverse molecules (304). Other proteases,

including members of the matrix metalloprotease (MMP) family, are also known to shed

transmembrane proteins (305-307). In human, 22 ADAMs have been identified, although

of the 22 genes, only 12 are found to possess the catalytic metalloprotease domain that is

required for proteolytic cleavage (304). Of all the ADAMs, ADAM8, 10, and 17, and 28

are found on lymphocytes, while ADAM9, 12, and 15 are expressed on cells of epithelial

origin (308-311). In particular, ADAM10 appears to play a pivotal role in B-cell

development by regulating the notch signaling pathway (312). In human leukemia,

including CLL, only expression of ADAM10, 17, and 28 have been documented (313,

314).

1.3.2 Regulation of ADAM proteases

All ADAM proteases are characterized by 7 distinctive domain structures. An N-

terminal propeptide region (pro-domain) is followed by the catalytic, metalloprotease

domain that is responsible for the cleavage activity of ADAMs. C-terminal to the

catalytic domain is the disintegrin domain allowing interaction of ADAMs with integrins,

which is in turn followed by a cysteine-rich region containing an EGF-like repeat

structure. The transmembrane domain, and cytoplasmic tail which vary widely in length

51

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

(315) (Fig 1.4). A hypervariable region within the cysteine-rich domain is likely

responsible for substrate recognition and binding (Fig 1.4).

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

Ectodomain shedding by all ADAM proteases occurs at a constitutive level that is

dependent on the availability of both sheddases and substrates on the cell surface (316).

Upregulation of expression of several ADAM proteases has been reported in a number of

malignancies, at both the mRNA and protein levels (317-321). The precise mechanisms

controlling ADAM expression are unknown, although upregulation of both ADAM9 and

15 has been shown to be increased under inflammatory conditions (322). It is known

furthermore that the proteolytic activities of ADAMs are subjected to multiple levels of

post-translation controls. The pro-domain of ADAMs can block the catalytic activity of

the metalloprotease domain, which is critical for the trafficking of ADAMs amongst

52

subcellular compartments (304). Removal of the pro-domain by pro-domain convertases

at the level of the Golgi apparatus, or on the cell surface, thus represents one level of

regulation of ADAM activity (323).

Availability of ADAMs at the cell surface is a function of control of both

intracellular trafficking and compartmentalization of ADAMs. For example, following

synthesis, the majority of ADAM17 resides in perinuclear vesicles near the plasma

membrane or in the Golgi complex, and is apparently not available at the cell surface in

the absence of external stimuli (324). Trafficking of vesicles containing ADAMs to the

plasma membrane is thought to be controlled by proteins involved in cytoskeletal

remodeling, although the precise mechanisms remain elusive (303).

The cytoplasmic domain of some ADAMs contains motifs that can act as binding

sites for a variety of kinases, representing yet another level of control for ADAM activity

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

PKC pathway, which can be stimulated by phorbol esters, as well as a wide variety of

external stimulants (309, 314). ADAM17, known to be responsible for the shedding of

many important cytokines and well as growth factors, is activated by PMA, which in turn

activates PKC, thus leading to increased ADAM17 activity at the plasma membrane

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

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

ADAM proteases in this disease (326, 327). ADAMs activity can also be induced by

stimuli that enhance intracellular Ca2+ release (328). It is important to note that activity

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

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

53

potently, while ADAM10 is most sensitive to stimulation by intracellular Ca2+ release

(329-331).

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

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

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

several important immunomodulators.

1.3.3 ADAM proteases in CLL

One substrate of the ADAM proteases that is important for both B-cell and CLL

biology is CD23. CD23 is a low-affinity receptor for IgE normally expressed on mature

B cells, antigen presenting cells, and platelet. CD23 expression, elevated on most CLL

cells, is regulated by Notch2 and is involved in CLL cell-survival and proliferation (332-

334). CD23 is shed in a soluble form, and levels of sCD23 in patient serum have been

shown to have powerful prognostic value in CLL disease progression (335, 336). The

pro-domains of both ADAM10 and 17 were shown to cleave CD23 in vitro, although

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

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

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

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

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

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

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

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

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

54

Interestingly, PKCβ is a downstream mediator in BCR signaling, and is upregulated in

CLL cells (326). It is not known whether the overexpression of PKC in CLL cells has

functional consequences in the activity of ADAM proteases.

55

1.4 Objectives and hypotheses

The previous sections have reviewed literature that strongly supports an important

role of immunoregulatory molecules in biology and clinical characeristics of CLL, and

their potential as therapeutic targets with an impact on CLL treatment. Preliminary

analyses of CD200 expression by us have shown that CD200 is overexpressed on

virtually all primary CLL cells and lymphoma cell lines, a result that has since been

confirmed by several independent groups (341, 342). Moreover, using a CD200

sandwich ELISA, we have identified a novel soluble form of CD200 (sCD200) that is

abundant in CLL plasma. Given our understanding of the immunoregulatory properties

of CD200 and its role in cancer immunology, we hypothesized that:

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

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

CLL

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

target for CLL

56

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

CLL

T cell/NLC/MSC

CD200 sCD200

CD200R

Pro-CLL factors that support

survival/growth?

57

The objectives of the studies in this thesis were to explore the role of CD200, both

its membrane-anchored and soluble forms in CLL. These studies have addressed the

following issues:

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

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

sCD200 levels for CLL…see Chapter 3

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

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

therapy…see Chapter 3

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

characterization of the released form…see Chapter 4

58

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

A manuscript of the same title has been published in the Journal of Leukocyte Biology,

2010, 88: 361-372 (365)

59

2.1 Abstract

CD200 is a transmembrane protein broadly expressed on a variety of cell types

which delivers immunoregulatory signals through binding to receptors (CD200Rs)

expressed on monocytes/myeloid cells and T lymphocytes. Signals delivered through the

CD200:CD200R axis have been shown to play an important role in the regulation of anti-

tumor immunity, and overexpression of CD200 has been reported in a number of

malignancies, including chronic lymphocytic leukemia (CLL), as well as on cancer stem

cells.

We investigated the effect of CD200 blockade in vitro on generation of CTL

responses against a poorly immunogenic CD200+ lymphoma cell line and fresh cells

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

Suppression of functional expression of CD200 augmented killing of the CD200+ cells, as

well as production of the inflammatory cytokines IFNγ and TNFα by effector PBMCs.

Killing was mediated by CD8+ cytotoxic T cells, while CD4+ T cells play an important

role in CD200-mediated suppression of CTL responses. Our data suggest that CD200

blockade may represent a novel approach to clinical treatment of CLL.

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2.2 Introduction

The differentiation and activation of B cells involves multiple processes which

regulate gene rearrangement, proliferation, and apoptosis. When these are disrupted

malignancies often occur, including lymphomas and Chronic Lymphocytic Leukemia

(CLL) (36). Complete cure of both diseases with conventional chemotherapy remains

extremely rare. While T-cell mediated anti-tumor immune responses have the potential

to eliminate tumor cells, CLL and lymphoma cells are inherently poorly immunogenic,

rending T-cell based immunotherapies ineffective (343). Various techniques have been

used to try to improve immunogenicity of CLL cells including the use of IL2 and TLR

agonists (259). Immunoregulatory molecules are known to play critical roles in regulating

T cell-mediated immunotherapy and manipulation of immunoregulatory pathways may

be an important alternative method to improve the efficacy of such treatments.

One immunoregulatory molecule, CD200, has been shown to be overexpressed in

a number of malignancies, including renal carcinoma, colon carcinoma, ovarian

carcinoma, melanoma, acute myeloid leukemia (AML), multiple myeloma (MM), and

CLL (291, 294, 295, 344, 345). In AML, cell surface CD200 expression on malignant

cells is correlated with poor prognosis (291). CD200 has also recently been reported to

be a cancer stem cell marker (297). The regulatory function of CD200 is delivered

through binding to a receptor, CD200R, expressed on cells of the myeloid lineage and T

lymphocytes (275).

A regulatory function for CD200 in tumor immunity was suggested following

studies which showed that infusion of a soluble form of CD200, CD200Fc, into EL4

thymoma-bearing C57B/6 mice enhanced tumor growth (281). Monoclonal anti-CD200

61

antibodies have recently been reported to abrogate growth of CD200-transduced RAJI

and Namalwa cell in NOD.SCID mice (346). In addition, Pallasch et al demonstrated

that CD200 expression on CLL cells had inhibitory effects on the proliferation of

autologous effector T cells, and CD200 blockade using a rat monoclonal anti-CD200

antibody produced a reduction in the number of CD25+CD4+Foxp3+ regulatory T cells in

vitro (347). In the case of CLL, no correlation has been reported between CD200

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

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

value of CD200 expression remains unknown.

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

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

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

silencers augmented anti-tumor CTL responses in vitro. CD4+T cells from splenocytes of

individual CLL patients expressed CD200R, consistent with the hypothesis that CD200

over-expression on tumor cells themselves may mediate immunosuppression in CLL.

Treatments of primary CLL cells with a TLR7 agonist, alone or in combination

with phorbol esters and IL2, have been reported to enhance the immunogeneicity of CLL

cells and increase their killing by effector T cells (259, 348). We report below that this

treatment also significantly reduced CD200 expression on CLL cells, and imply that

downregulation of CD200 expression on tumor cells may improve immunogeneicity of

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

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2.3 Materials and Methods:

Cells:

Human PBMC were isolated from heparin treated whole blood of healthy

volunteer donors using Ficoll-Paque PLUS gradients (GE Healthcare Bio-Sciences,

Piscataway, NJ). 5 independent volunteer donors were used on multiple occasions

throughout the studies described. PBMCs were used in CTL assays immediately after

isolation. Two human cell lines propagated from non-Hodgkin’s lymphomas were grown

in suspension in AIM-V medium (Invitrogen, Carlsbad, CA) supplemented with 5% FBS

(Hyclone, Logan, UT) (349).

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

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

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

protocols. All protocols were approved by institutional review boards.

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

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

Antibodies:

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

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

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

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

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

63

(custom immunization performed by Cedarlane Labs, Hornby, ON). Anti-Fc antibodies

in the serum were removed using Fc column absorption (Cedarlane Labs), and

immunoreactivity of the sera was confirmed by Western blots.

FACS analyses:

5x105 lymphoma cells were washed twice with 1ml FACS buffer (PBS, 1%FBS,

0.1%NaN3, 5mM EDTA), and incubated with 0.1ug of rat anti-human CD200 mAb (1B9)

or isotype control for 45min at 4ºC. Cells were washed x3 with PBS, and incubated with

goat anti-rat IgG-FITC antibody (Jackson Labs, Western Grove, PA) at a 1:100 dilution

for 30min at 4ºC.

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

0.5ug per sample, followed by incubation with a secondary goat anti-mouse IgG-PE

antibody (Jackson Labs). The following antibodies were used at concentrations

suggested by the supplier (BD Biosciences): CD4-FITC, CD8-PECy7, CD5-PECy5, and

CD19-PE.

In activation experiments, CLL cells were stained with mouse anti-CD83-PE and

mouse anti-CD5-FITC antibodiy (BD Biosciences) as per manufacturer’s instruction at

24 and 48 hours after stimulation. All cells were fixed with 1% Paraformaldehyde before

being analysed in a Coulter FC500 flow cytometer.

Western Blots:

Cells were lysed in 0.025%SDS, and run on SDS-PAGE gels. After transfer the

blots were blocked with 5% milk in TBST overnight at 4ºC. Blots were then probed with

the rabbit anti-hC200 serum at a 1:6000 dilution. In siRNA experiments, MAPK was

64

used as a housekeeping protein for reference. Blots were divided at ~40kd, with the top

part of the blot (>40kd) probed for CD200, and the bottom part (<40kd) probed with a

mouse anti-MAPK mAb, Tag-100 (Qiagen), at a 1:1000 dilution. After thorough

washing, blots were probed with either goat anti-rabbit IgG-HRP (1:6000) or goat anti-

mouse IgG-HRP (1:2000) (Jackson) for 1 hour at room temperature. Blots were

developed using an ECL Western blot detection kit (GE Healthcare Bio-Sciences).

CD200 column absorption:

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

Sepharose beads (Cedarlane, ON). For CD200 absorption, 1ml of neat serum samples

from healthy controls and CLL patients were incubated with 250µl of 1B9 conjugated

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

samples were subsequently used in MLC assays at designated dilutions.

RT-PCR and Real-time PCR:

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

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

primer pair was designed to detect ~100bp amplicons:

Forward: AATACCTTTGGTTTTGGGAAGATCT

Reverse: GGTGGTCTTCAGAGAATTTGTAGTGA

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

housekeeping genes for normalization of CD200 gene expression level.

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All primers were used in both regular PCR and real-time PCR. For real-time PCR

experiments, 50ng cDNA was used per reaction.

siRNA transfection:

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

#4, and CD200siRNA#6, were obtained from Qiagen. Two control siRNAs, a positive

control GAPDH silencer and a negative control, were purchased from Ambion for use in

silencing experiments.

7.5x105 lymphoma cells were transfected with 2ug of siRNA using

lipofectamine2000 (Invitrogen) as a transfection reagent at a 1:6 ratio. Transfection was

performed in triplicate in 12-well plates according to the manufacturer’s instructions.

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

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

transfection.

CTL assays:

1.2x106 PBLs were stimulated with mitomycin C treated Ly5 or Ly2 cells at a

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

hCD200 mAb was added for functional neutralization of CD200 expression.

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

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

37ºC, washed 3 times in PBS, and 1x104 cells added to each well in the 96-well plate.

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

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

66

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

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

obtained from a minimum of 3 independent experiments.

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

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

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

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

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

same PBL effectors in 3 independent experiments.

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

performed using EasySep immunomagnetic cell selection kits (StemCell Technologies,

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

ELISA:

Supernatant samples harvested from CTL assays were assayed for TNFα, IFNγ,

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

Diego, CA), as per the manufacturer’s instruction. A standard curve was obtained in

each assay to quantify cytokine present in the supernatant.

Activation of CLL cells:

2x106 purified CLL cells were cultured in serum-free AIM-V medium plus 2-ME

(Sigma-Aldrich) in 24-well plates at 37°C in 5% CO2 in the presence or absence of the

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

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

67

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

10mg/ml stock solutions. For activation of CLL cells, Imiquimod, PMA, and IL2 were

used at a final concentration of 3ug/ml, 30ng/ml, and 500U/ml, respectively. At 24 and

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

CD83 and CD5 were determined by FACS. Upregulation of CD83 expression was used

as an indicator for response to stimulation.

Statistical analyses:

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

determine the significance between sample means.

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2.4 Results:

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

In order to explore the effect of CD200 expression on induction of anti-tumor

immunity in vitro, we first characterized expression of CD200 using a number of

independent isolates of primary CLL cells and established cell lines using PCR, Western

blots, and FACS. Although essentially all primary tumor cells (n=25, table 2.1) analyzed

in this study stained positive for CD200 as compared to the respective isotype controls,

the mean fluorescent intensity of the staining varied widely (Fig 2-1a). Heterogeneity in

CD200 cell surface expression level was not independently correlated with any of the

various clinical parameters of CLL analyzed, including Rai disease stage, CD38

expression, and cytogenetic status. To date there is no data to our knowledge directly

addressing the issue of altered CD200 cell surface expression with disease progression. In

the two lymphoma cell lines studied, Ly5 cells showed CD200 expression levels

comparable to that of primary CLL cells while Ly2 cells failed to stain for CD200 (Fig 2-

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

CD200 was detected as a band at around 48kd in lysates of hCD200 transfected Hek293

cells and Ly5 cells, but not Ly2 cells (Fig 2-1c). CD200 expression on Ly5 cells, on the

other hand, remained consistently high after prolonged in vitro passage (>20: data not

shown). Ly5 and Ly2 cells were used in the functional studies described below designed

to investigate the functional consequences of the presence of CD200 expression on tumor

cell-induced immunity.

69

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

Different epitopes of human CD200 are recognized by independently derived rat

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

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

mAb, 3H4 failed to stain any of the CD200+ cells identified by 1B9 (data not shown). We

have used 3H4 as an isotype control in the experiments discussed below.

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

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

Ly5 and CD200- Ly2 cells as stimulators. 3HTdR-based cytotoxicity assays were

performed 7 days after stimulation at 3 different effector:target ratios to assess the effect

of CD200 blockade on the killing of Ly5 and Ly2 cells by activated effectors. Data

shown in the figures below are for E:T ratios of 10:1. Figure 2-2a shows pooled results

from 9 independent experiments using PBL stimulators from 5 different donors. Both

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

~6%. Addition of the 1B9 anti-CD200 mAb, but not of 5A9, produced ~5-fold increase

in the killing of CD200+ Ly5 cells, with no significant change in the killing of CD200-

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

killing of either Ly5 or Ly2 cells.

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

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

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

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

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

70

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.

71

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

72

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 CD200-

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

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

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

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

cell-mediated immunity.

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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

74

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

Imiquimod, alone or in combination with human recombinant IL2 and PMA for 24 hours,

and then assessed for cell surface CD200 and CD5 expression by FACS. Data from

stimulation of CLL cells from patient 61 is shown below as a representative data set (Fig

2-7).

Both PMA and Imiquimod treatments significantly reduced CD200 expression on

CLL cells in all patients tested, while expression of the CLL surface-marker CD5

remained relatively unchanged (Fig 2-7). Expression of CD83, a co-stimulatory

molecule and an activation marker, was also increased in response to both PMA and

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

PMA-induced CD200 down-regulation was observed in all CLL cells, while Imiquimod-

induced CD200 down-regulation was observed in cells from 4 out of 5 patients (data not

shown). IL2, on the other hand, had no effect on CD200 expression and produced

minimal increase in CD83 expression (Fig 2-7). In agreement with previous reports in

which CLL cells were shown to exhibit heterogeneous response to PMA and Imiquimod

in the upregulation of CD83, CD80, and CD86 expressions, the effect of these two

stimuli on CD200 expression also varied among patients (data not shown) (258).

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

Imiquimod, and IL2, which, as reported previously, also resulted in the greatest increase

in CD83 expression (259). The presence of IL2 in combination with PMA and

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

CD200 downregulation.

75

2.5 Discussion:

Immunomodulatory molecules contributing to negative signaling of T cells are

thought to play a pivotal role in regulating anti-tumor responses and tumor progression in

human malignancies. As examples, altered expression of immunomodulatory molecules

of the B7 family, B7-H1, B7-H3, and B7-H4, have been detected in lung, prostate,

ovarian, kidney carcinomas, and neuroblastoma (352). In prostate cancer and clear cell

renal carcinoma, B7-H3 overexpression on tumor cells is associated with poor prognosis

(353, 354). In ovarian cancer, serum B7-H4 level has been identified as another marker

which predicts poor prognosis (355). Functional blockade of these immunomodulatory

molecules might thus provide a novel therapy for such malignancies. Indeed, blockade of

CTLA4, a negative regulator of T cells, using a fully humanized antibody is currently

under development in Phase III clinical trials in patients with advanced melanoma and

other malignancies (356).

In B cell malignancies, including lymphomas and CLL, the tumor cells

themselves are known to be poorly immunogenic, despite the expression of high levels of

MHC molecules and tumor antigens (357, 358). A number of strategies have been

investigated to develop clinically applicable methodologies to enhance the

immunogenicity of CLL cells (359, 360). For example, transduction of CLL cells with

CD40L has been shown to enhance antigen specific recognition of tumor cells by

autologous T cells in vitro (360). Various attempts have also been made to improve the

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

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

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

76

immunogenicity of the tumor. Expression of CD200, a known immunoregulatory

molecule, has been reported on CLL and lymphoma cells (363). Although CD200 cell

surface expression level does not seem to correlate with other CLL clinical markers, it

remains unknown whether CD200 expression levels on CLL cells varies in response to

treatment or during disease progression. Our results described herein, and data from

other groups, supports the hypothesis that CD200 expression on tumor cells might be one

of the contributors for the poor immunogenicity of leukemic/lymphoma cells. Blockade

of functional CD200 expression would thus provide a promising approach to enhance

immunogenicity of such tumor cells. In support of this hypothesis, Kretz-Rommel et al

recently demonstrated that blockade of CD200 using specific humanized mAbs enhanced

anti-tumor responses using hPBMCs and tumor cells artificially transfected with a

CD200 lentiviral vector (346).

A drawback to the study reported by Kretz-Rommel et al is that lentiviral

transfection often produces protein expression levels not reflective of those seen

physiologically. Accordingly we have been interested in the induction of tumor

responses directed against primary CLL cells isolated from peripheral blood of patients as

well as a B-lymphoma cell line, Ly5, which constitutively expresses CD200 at levels

paralleling those expressed by primary CLL cells. A CD200- cell line, Ly2, was used as a

control. CTL assays using these two 2 cell lines as targets showed that while both cell

lines were poorly immunogenic, killing of CD200+Ly5 cells, and indeed of primary CLL

cells, but not CD200- Ly2 cells, was augmented > 5 fold by the presence of a rat anti-

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

77

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

78

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.

79

CLL cell-mediated T cell defects have been well documented. Recently, the

formation of immunological synapses between CLL cells and autologous T cells were

shown to be impaired (195). This impairment appears to be CLL-dependent as

incubation of CLL cells with allogenic T cells also led to failure in formation of normal

immunologic synapse between CLL cells and normal T cells. The expression of

activation markers on T cells was also impaired after incubation with CLL cells, in a

mechanism involving direct cell-cell contact as well as soluble factors secreted by CLL

cells (367). The results of our CTL studies showed that CD200 may be one of the cell-

surface factors contributing to CLL-mediated T cell suppression, as CD200 blockade

resulted in enhanced killing of CLL cells.

Further evidence for an important role of the CD200:CD200R axis in CLL was

supported by the high frequency of CD200R+ CD4+ T cells in the spleen of CLL patients

as detected by FACS analyses. CD8+ T cells and CLL cells, on the other hand, showed

no detectable level of CD200R expression, consistent with the hypothesis that the

primary target for CLL-derived CD200-mediated immunosuppressive signals represents

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

CD200R+CD4+ T cells and CD200+ CLL cells exist in close proximity in vivo in CLL

microenvironments. However, the observation that CD200R-expressing CD4+ T cells

and CD200-expressing CLL cells are present in the same microenvironment (spleen in

this case) supports a model in which CD200-mediated suppression of CD200R+CD4+ T

cells is in part at least responsible for the Th2 cytokine polarization and diminished CD8+

cytotoxic T cell function observed in CLL patients. The ability of anti-CD200 to

augment lysis of fresh CLL cells following CD4 depletion may suggest a role for such

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

80

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

81

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.

2.6 Tables

Table 2.1: Clinical characteristics of patients used in the study

Patient # Sex Age

Years after diagnosis ▪ Rai Stage ♦ WBC ● Treatment %CD38 ○ Cytogenetics

3 M 72 4 IV 175 S/CHOP/CVP 2 13q-/17p- 4 F 84 10 III 200 P/C,S/R 46 13q- 5 M 62 12 IV 121 CP,CVP,S,FC 2 13q- 8 M 74 7 IV 220 FR,splenectomy,S na 17p- 12 M 55 6 IV 23 CHOP,P 81 13q-,17p-

* 14 M 71 6 II 186 none 72 11q-,13q- 16 M 69 6 IV 189 CP 4 normal

22 M 54 6 IV 8 CP,FC,FCR,S 1 normal 27 F 82 11 III 125 none 8 T12 36 M 59 7 IV 10 CP 7 Na 41 F 59 6 III 35 CP,splenectomy 40 normal

* 46 M 55 5 III 121 none 4 T12 57 F 77 10 IV 120 CP na 13q- 60 M 88 6 IV 62 none 1 13q-,17p- ◘ 61 M 81 6 III 89 P 1 normal 65 F 61 26 IV 140 splenectomy 18 13q-,11q- 70 F 78 5 III 33 none 13 13q- 71 M 57 2 II 21 none 1 T12,13q-

72 F 48 2 0 13 none 2 na * 73 F 53 13 IV 65 none 2 13q-

74 M 77 10 III 250 none 1 13q- ◘ 75 F 63 3 II 22 none 71 na ◘ 78 M 72 5 IV 80 CP,FCR 13 normal ◘ 79 M 51 10 IV 140 Cx2 1 13q- ◘ 80 M 51 1 II 88 None 2 13q- ⌂ I M 72 5 IV 96 Cx2, splenectomy 60 17p- ⌂ II M 58 15 IV 77 C, F, FC, S, Splen 65 13q-, 11q-

83

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

84

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 (** p<0.0001) and 5A9 (p>0.05) enhanced

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

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

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

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

All p-values were calculated using % killing obtained from effector PBMC with

stimulation by Ly5 cells as reference.

85

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

Ly5 cells were transfected with 2ug of the CD200 siRNAs #1, #4, or #6. All silencers

were designed by Qiagen. A control using lipofectamine treatment but no siRNA was

included. Cells were assayed ay 48 hours (RNA) or 72hrs (protein) following

transfection. a) CD200 mRNA level as shown by real-time PCR. CD200 silencer #1

showed no effect; b) expression of CD200 protein was decreased after transfection with

silencers #4 and #6, but not #1. a

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

siRNAs for CD200 enhanced cytotoxic killing of Ly5 cells to a similar degree as that

achieved using anti-CD200 mAb 1B9 (** p<0.05 with both CD200 silencers). The

negative control silencer (#1) had no effect.

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

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

1:10 dilution, respectively, for a) TNFα (18 and 42 hr supernatant) and b) IFNγ (18 hr

supernatant). Suppression of functional CD200 expression by mAb 1B9 or the CD200-

specific silencers #4 and #6 augmented production of both TNFα and IFNγ by responder

PBLs. Neither mAb nor siRNAs affected production of TNFα or IFNγ when PBLs were

stimulated with Ly2 cells.

86

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

Effector PBLs were stimulated by mitomycin C treated primary CLL cells in the presence

of either rat IgG or 1B9. Killing was measured 7 days after stimulation by 51Cr release

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

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

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

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

killing. Depletion of CD8+ T cells reduced the killing of CLL cells to levels akin to those

seen with unstimulated PBLs, even in the presence of 1B9. c) Effect of CD200

absorption on immunosuppression in human MLCs using CLL patient serum: CD200

was absorbed from CLL serum by overnight incubation of pooled CLL serum (obtained

from 15 donors) with 1B9-conjugated Sepharose beads. CLL serum before (CLL) or

after (absorbed) CD200 absorption was added to human MLC at the indicated dilutions;

results show % lysis of 51Cr labelled target cells at a 30:1 effector:target ratio in 6hrs.

CLL serum suppressed MLC reactivity in a dose dependent manner compared with

controls (p<0.05), but this inhibition was lost after absorption.

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

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

CLL spleen of both CLL patients, while neither CD8+ T cells nor CLL (CD5+) cells

expressed detectable levels of CD200R.

87

e) Expression of CD200 on splenic CLL cells: CLL populations as determined by the cell

surface markers CD19 and CD5. CD5+CD19+ CLL cells from both CLL spleen

populations express high levels of CD200.

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

Fresh CLL cells were treated with 30ng/ml PMA, 3ug/ml Imiquimod, and 500U/ml

recombinant hIL2, alone or in combination, for 24 hours. CD200 expression (y-axis) on

the surface of CLL cells was reduced in response to PMA and Imiquimod, but not IL2.

CD200 expression is further downregulated when CLL cells were treated with all 3

stimulants, whereas change in expression of the cell surface marker CD5 (x-axis) was

minimal. CD83 expression was also upregulated in response to stimulation by PMA and

Imiquimod (CD83+ cells represented by dark spots). The experiment was repeated with

fresh CLL cells from 5 patients, and typical data from 1 such study is shown.

88

2.8 Figures

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

2-1a)

0.0

50.0

100.0

150.0

200.0

250.0

3 4 5 8 12 14 16 22 27 36 41 46 57 60 61 65 70 71 72 73 74 75 78 79 80

Ly5

Ly2

Nor

MF

I

Mean Fluorescent Intensity (MFI) of CD200 cell surf ace expression

Patient ID

89

2-1b)

1B9

5A9

Ly5 Ly2

2-1c)

a b c a: Hek293-hCD200

b: Ly5 cells

c: Ly2

90

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

2-2a)

% specific lysis of Ly5 cells with or without anti-C D200 mAb

0

5

10

15

20

25

30

35

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

% s

peci

fic ly

sis

% specific lysis of Ly5 cells with or without anti- CD200 mAb

91

2-2b)

0

5

10

15

20

25

30

35

40

Ly2 only Ly2+1B9

% s

peci

fic ly

sis

% specific lysis of Ly2 cells with or without anti-C D200 mAb

92

2-2c)

1B9 only Ly5 only Ly5+1B9

-5

0

5

10

30

40

CD8-deple tion

UntreatedCD4-deple tion

% s

peci

fic ly

sis

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

93

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

2-3a)

0

0.0005

0.001

0.0015

Reagent only CD200 siRNA#1 CD200 siRNA#4 CD200 siRNA#6

Rel

ativ

e m

RN

A le

vel

Relative CD200 RNA levels in Ly5 cells after siRNA t ransfection

2-3b)

CD200

MAPK (House-keeping) protein)

1 2 3 4 5 6

94

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

0

5

10

15

20

25

30

PBL+ly5

PBL+ly5

+1B9

PBL+ly

5+ -v

e siRNA

PBL+ly5

+s iRNA#4

PBL+ly5

+siR

NA#6

% s

peci

fic ly

sis

** **

**

% Specific lysis of Ly5 cells transfected with cont rol or

CD200 specific siRNA

95

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

CD200-specific siRNAs

2-5a)

-10

40

90

140

190

240

290

340

PBL o nly

PBL+ly5

No re

agen

t

PBL+ly5

+1B9

PBL+ly

5 -v

e siR

NA

PBL+ly

5 siR

NA#4

PBL+ly

5 siR

NA#6

Ly5

only

pg/m

l

18h

42h

TNFαααα production 18 and 42hr after stimulation with Ly5

96

2-5b)

IFNγγγγ production 18 hr after stimulation with Ly5

-100

0

100

200

300

400

500

600

700

800

900

PBL onl

y

PBL+ly5 N

o reagen

t

PBL+ly5

+1B9

PBL+ly

5 -ve

siRNA

PBL+ly5

siR

NA#4

PBL+ly5 si

RNA#6

Ly5

only

pg/m

l

IFNγγγγ production 18 hr after stimulation with Ly5

97

Figure 2-6: CD200 blockade augments killing of primary CLL cells by allogenic

effector PBLs and CD200R expression on CLL-splenocytes

2-6a)

of anti -CD200 mAb

0

5

10

15

20

25

30

Rat IgG 1B9

% s

peci

fic ly

sis

% Specific lysis of CLL cells in the presence or ab sence

of anti-CD200 mAb

98

2-6b)

0

5

10

15

20

25

30

No depletion CD4 depletion CD8 depletion

% L

ysis

PBL+CLL

PBL+CLL+control IgG

PBL+CLL+1B9

% Specific lysis of CLL cells in the presence or ab sence

of anti-CD200 mAb

99

CD200-abosrbed serum

2-6c)

Suppression of human MLC response by CLL serum or

CD200-abosrbed serum

100

2-6d) C

D20

0R1

CD5 CD8 CD4

CLL spleen 1

CLL spleen 2

CD5 CD8 CD4

CD

200R

1

0.1%

96.6%

0.6%

99.3% 0.2%

99.0%

0.5%

0.6%

99.0%

0.0%

90.3%

99.5%

101

2-6e) C

D19

CD

19

CD5

CD5

CD5 and CD19 gated CLL splenocytes:Whole CLL splenocytes

CD200

CD200

CLL spleen-1

CLL spleen-2

Whole CLL splenocytes

CD5 and CD19 gated CLL splenocytes:

102

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

Imiquimod, and IL2

30.1%

63.5%

75.2%

27.1%

92.0%

103

Chapter 3: Soluble CD200 supports in vivo survival of CLL

(Manuscript submitted to Cancer Research, April, 2012)

104

3.1 Abstract

CD200, a type I transmembrane molecule overexpressed on most CLL cells, has

been reported to play an important role in modulating tumor immunity. We have

characterized a previously unknown soluble form of CD200 (sCD200) in human plasma.

Levels of sCD200 were elevated in CLL plasma (compared with healthy controls), with a

significant correlation between plasma sCD200 and CLL Rai disease stage.

Infusion of sCD200hi CLL plasma into NOD.SCIDγcnull mice receiving CLL

splenocytes enhanced engraftment of CLL cells in comparison to that seen in mice

receiving sCD200lo normal plasma. CLL cells were detected in both the spleen and

peritoneal cavity of animals for up to 75 days. Engraftment of CLL cells was not

observed following infusion of CLL plasma depleted of sCD200, and was lost following

treatment of mice with anti-CD200 mAb, or OKT3 mAb, suggesting a role for both

sCD200 and T cells in CLL engraftment. Anti-CD200 mAb was as effective as

rituximab in eliminating engrafted CLL cells when given at 21-days post-engraftment.

Our data suggest that sCD200 is a novel prognostic marker and potential therapeutic

target for CLL, and that the humanized mouse model of CLL described here may prove

valuable in studies for screening new treatment regimes.

105

3.2 Introduction

Chronic lymphocytic leukemia is a heterogeneous disease characterized by the

accumulation of malignant CD5+CD19+ B cells in peripheral blood, bone marrow, and

secondary lymphoid organs. Some patients have a benign clinical course while others die

of this disease within a short time from diagnosis. Improved understanding of the

biology of CLL may help identify other variables predicting which patients may have a

poor disease outcome.

In the previous chapter we showed that CD200, overexpressed on most CLL cells,

played a functional role in suppressing cytotoxic killing of CD200+ tumor cells.

Increased expression of CD200R, which is required for signalling mediated following

CD200 engagement, was detected on a subpopulation of CD4+ T cells in the spleen of

CLL patients relative to controls (376). Thus CD200+ CLL cells and CD200R+ CD4+ T

cells appear to co-localize in the tumor microenvironment.

The CLL microenvironment is crucial for CLL survival and proliferation (159).

Non-malignant constituents of the microenvironment, mostly T cells, mesenchymal

stromal cells (MSCs), and CD14+ nurse-like cells (NLCs) provide antigenic signals,

cytokines, and other CLL survival factors such as BAFF, to support CLL survival and

proliferation (162, 172, 190, 191). T cells in the CLL microenvironment are also known

to express CD40L, which, through stimulation of CD40 on CLL cells, induces CLL

proliferation and anti-apoptotic pathways (183). A number of studies have shown that

CLL cells can directly modulate T cell function by expression of cell-surface molecules

and/or production of soluble factors (195, 196). Based on these data, we hypothesized

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that CD200 may be one of the important factors modulating the CLL microenvironment

(290, 291, 295).

A number of membrane-bound molecules with immunomodulatory functions are

known to exist also in soluble forms where they are believed to play a functional role in a

number of disease states, including cancer. Thus soluble forms of CD23, CD44, and

CD14 have been reported to augment CLL survival in vitro (377-379). We found that

CD200 could be shed from CLL cells after stimulation by PMA and TLR7 agonists,

inferring that a soluble form of CD200 (sCD200) might be present in human plasma

including that of CLL patients (chapter 1). The studies described below were designed to

investigate if soluble CD200 levels were increased in CLL patients relative to healthy

controls, and whether those levels were related to disease stage. We also investigated

whether sCD200 present in CLL patient plasma might contribute to growth/survival of

CLL cells in NOD.SCIDγcnull mice.

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3.3 Materials and Methods

Mice:

NOD.SCIDγcnull mice were bred and maintained under sterile conditions at the

Toronto Medical Discovery Tower, MaRs Centre. All mice were used at 8-13 weeks of

age.

Human splenocytes and CLL cells

The spleen from consenting patients undergoing splenectomy at Sunnybrook

Health Science Centre was harvested at surgery and single cell suspensions prepared in

AIMV medium (Invitrogen, Carlsbad, CA). Cells were washed (x2), counted and stored

frozen at -80°C in freezing medium (AIMV+ 40% FBS+ 10% DMSO) at a concentration

of 1x108 cells/ml either in 1.5ml aliquots in cryovials or in 30ml aliquots in 50ml Falcon

tubes. At least 1011 cells in total were harvested from each spleen. An aliquot (107) of

fresh splenocytes was retained for cell surface phenotype analysis in FACS. For in vivo

studies in NOD-SCIDγcnull mice, aliquots of splenocytes were rapidly thawed at 37°C,

washed in PBS, and cell aggregates separated by centrifugation on Ficoll-Paque PLUS

gradients. Cells recovered after centrifugation were resuspended in PBS at appropriate

concentrations for injection.

In some experiments where cells from the peripheral blood of CLL patients were

used for reconstitution, CD19+CD5+ CLL cells were purified from fresh blood as

described previously (348). All protocols were approved by institutional review boards.

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Human plasma

Plasma from CLL patients was obtained at routine clinical follow-up and stored at

-20°C. For in vivo studies in NOD.SCIDγcnull mice, plasma from a group of patients at

late disease stage (Rai Stage III-IV), and/or with high white cell count, were pooled into

batches (>8 donors/batch). The control plasma used was pooled from a group of 10

healthy volunteers. sCD200 levels in all plasma samples were assessed by CD200

ELISA (see below). sCD200 levels in pooled normal plasma were in the range 0.5 +0.2

ng/ml while in various pooled CLL plasma batches levels were consistently ~10-fold

higher (5 ±1.3 ng/ml). Where absorbed plasma was used, the pooled CLL plasma was

absorbed overnight at 4°C with anti-CD200 (1B9)-conjugated CNBr-activated Sepharose

beads (Cedarlane, ON), a method previously shown to be effective in absorbing sCD200

from plasma (376).

Antibodies

The rat anti-hCD200 monoclonal antibodies 1B9 and 3G7 were described

previously (351). 1B9 was previously shown to be effective in blocking CD200 function

in vitro (376). For in vivo use Fab fragments of 1B9 were prepared using a Fab

preparation kit (Thermo Fisher Scientific Inc., Rockford, IL).

The polyclonal rabbit anti-hCD200 serum, absorbed to deplete all anti-Fc

reactivity, was described elsewhere (376). A rabbit polyclonal antibody specific for the

extracellular region (V+C) of CD200 was generated by immunization of rabbits with

protein expressed from CHO cells transfected with an expression vector encoding only

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this extracellular domain of CD200. The immunoreactivity and specificity of both sera

was characterized by Western blot analysis.

Mouse anti-CD200R1-FITC antibody was purchased from R&D systems, while

all other monoclonal antibodies used for cell surface phenotype characterization (CD45,

CD19, CD5, CD20, CD40, CD23, CD38, CD49d, CD4, CD8, CD14, and CD56) were

purchased from eBioscience. Rituxan (Roche Canada, Mississauga, ON) was obtained

from the hospital pharmacy. OKT3, used for depletion of T cells in in vivo studies, was

purchased from Ortho-McNeil Pharmaceuticals (Raritan, NJ).

CD200 sandwich ELISA:

High binding 96-well EIA/RIA plates (Corning Life Sciences) were coated with

the capture anti-CD200 mAb1B9 at 1.25µg/ml overnight at 4°C in Tris-HCI, pH 8.1.

Plates were then blocked for 1 hour at room temperature with the blocking buffer, 5%

FBS in PBS, washed, and different concentrations of either pure CD200Fc (standard

curve) or plasma samples (diluted 1:4 in blocking buffer) were added. Plates were

incubated for 2 hours at room temperature, followed by 2-hours of incubation with the

detection antibody, a rabbit anti-CD200 antiserum at a 1:500 dilution. Goat anti-rabbit

IgG-HRP antibody at a 1:12,500 dilution was added and plates incubated at room

temperature for 30 minutes. 5 washings with wash buffer (PBS+0.01% Tween20) were

performed between each step. After the final wash, TMB substrate was added. All

reactions were stopped by addition of 50µl 0.2M sulfuric acid per well after 10 minute of

incubation at room temperature in the dark. Plates were read at 450nm in a Multiskan

Ascent 96/384 plate reader (MTX Lab Systems, Vienna, VA).

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Engraftment of human CLL splenocytes in NOD-SCIDγcnull mice

On the day of experimentation, mice received 245 rads of γ-irradiation, followed

by 1x108 human splenocytes ip, and 0.8ml of pooled CLL plasma or control plasma, also

given ip. Subsequent infusions of CLL plasma or control plasma were performed bi-

weekly throughout the course of the experiment. Mice were sacrificed at various time

points to assess for CLL engraftment. Spleen and bone marrow were harvested from

individual animals and cells in the peritoneal cavity were recovered by flushing the

peritoneum with 8ml PBS. Single cell suspensions were prepared from all 3

compartments, and cells enumerated in a hemocytometer.

For immunohistochemistry an aliquot of fresh spleen tissue was fixed in 10%

formalin and slides prepared and processed by the Pathology lab at Sunnybrook Health

Science centre. Engraftment of CLL cells was analyzed by multi-color FACS staining

using single cell suspensions and the various mAbs discussed above.

CD200 blockade and T cell depletion in vivo studies

For CD200 blockade experiments, mice were randomly assigned into 3 groups

after infusion of human splenocytes. Two of the three groups received sCD200hi CLL

plasma while a third group received sCD200 absorbed CLL plasma. Of the two groups

that received CLL plasma one group also received 50µg (iv) of Fab anti-CD200 mAb

1B9 the day after spleen cell injection, and on two subsequent occasions at 72 hour

intervals.

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For T cell depletion experiments mice received human splenocytes and sCD200hi

CLL plasma, or sCD200 absorbed CLL plasma. The following day animals were

randomly assigned to receive 20µg OKT3 (anti-CD3) antibody iv, with 2 additional

infusions at 72 hour intervals.

In studies comparing the therapeutic efficiency of 1B9 and Rituxan, mice were

engrafted with human splenocytes along with biweekly infusion of sCD200hi plasma. 21

days following CLL infusion mice were randomly assigned to receive saline, 1B9

(50µg/mouse) or Rituxan (50mg/mouse), all delivered iv in 300µl. All treatments were

repeated at 72-hour intervals for a total of 4 treatments. Animals were sacrificed 8 days

after the last treatment.

For each in vivo experiment, mice of both sexes were used, with at least 3 mice

per group. All experiments were repeated individually with splenocytes from at least two

different patients.

FACS analyses

CD200 cell surface staining was performed using a rat anti-CD200 mAb (3G7) as

previously described (376). Multi-color FACS analyses were performed to characterize

engrafted human cells. The optimal concentration of antibody for staining was

determined individually for each antibody. Single color controls were included in each

experiment for compensation purposes, and all samples were analyzed in a Coulter

FC500 flow cytometer.

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Statistics

Spearman’s Rank Correlation test and a Man-Whitney U test were used to

determine the correlation between sCD200 levels and various clinical markers in CLL.

All clinical analyses were done using SPSS Statistics software. For in vivo studies the

absolute count of each engrafted cell population (CLL or T cells) was calculated from

[total cell count x frequency], with frequency based on FACS staining profiles. Unpaired

t-tests were used to determine significance between sample means. Analysis of in vivo

studies was performed using GraphPad Prism 5.0 software.

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3.4 Results

Identification of sCD200 in plasma from CLL patients

CD200 is normally considered a membrane molecule (275). We have previously

shown that CD200 was shed following activation of CLL cells in vitro, suggesting it might

also exist in a soluble form in the plasma (351, 376). To explore evidence for a plasma form

of CD200 (sCD200), we established a sandwich ELISA using 1B9 as capture antibody and a

rabbit anti-hCD200 detection polyclonal antibody as described in the Materials and Methods.

The sensitivity of this ELISA using pure CD200Fc protein as standard was found to be

0.05ng/ml. CD200Fc was used to generate standard curves in the range 0.05-10ng/ml for

quantitation of sCD200 in samples in all ELISA studies.

sCD200 levels in plasma from 25 healthy controls ranged from 0.4±0.2ng/ml.

These levels were independent of age (20-64 years) or gender (data not shown). Elevated

sCD200 levels were observed in plasma samples from CLL patients across all clinical

stages (Fig 3-1a).

Plasma sCD200 levels are associated with disease stage in CLL

While CD200 expression was detected on CLL cells from all individuals in a

cohort of 25 patients at various stages of disease, the cell surface expression level was not

correlated with either tumor burden or other clinical parameters of disease (376). To

determine whether sCD200 levels correlated with CLL clinical parameters, plasma

samples obtained at diagnosis from 82 CLL patients were tested in the sandwich ELISA

for sCD200 levels. The patient age in this cohort ranged from 38-91 (median age 61).

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Correlation analyses were performed to compare sCD200 levels with other parameters

linked to clinical outcome in CLL (see Table 3.1 for patient details).

Independent of the expression of CD200 on the cell surface, sCD200 levels were

correlated with tumor burden, with patients in later stages of disease (Rai stage III and

IV) having significantly higher sCD200 levels than patients at early stages (Fig 3-1b). In

general clinical treatment of CLL is reserved for patients with late disease and/or rapidly

progressive disease (1). Using the number of treatments received by each patient in our

cohort, regardless of the nature of that treatment, as a surrogate marker of aggressive

disease, we pooled patients into groups who had received two or more courses of

treatments vs. those with no (or only 1 course of) treatment. The former had significantly

higher serum sCD200 levels than those with more indolent disease as defined by no

treatment (p<0.0001, Fig 3-1c) or only 1 treatment (p=0.0027, Fig 3-1c).

Of the conventional prognostic markers for CLL, sCD200 levels correlated most

strongly with serum β2 microglobulin levels (p<0.0001, Fig 3-1d). CD38 expression

levels did not correlate with sCD200 levels (18). The presence of intermediate- to high-

risk cytogenetic abnormalities (trisomy 12 or deletions of regions of chromosomes 11 or

17) also did not correlate with sCD200 levels. However, patients with CLL cells that

exhibited either a normal karyotype or 13q deletions, usually considered to be indicative

of a more benign clinical course, were treated more often if they also had high levels of

sCD200 (Spearman’s r=0.639, p<0.0001, n=37).

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Development of a xenograft model for CLL

Given that sCD200 levels were higher in patients with late stage and/or aggressive

disease, we hypothesized that sCD200 in CLL plasma might play a role in vivo in

fostering CLL growth. In a series of preliminary studies we infused 1x108 purified

circulating PBL-derived CLL cells from 5 individual patients with high white cell counts

into each of 4/group NOD-SCIDγcnull mice, with subsequent bi-weekly infusion of pooled

sCD200hi CLL plasma, or sCD200lo normal plasma pooled from healthy volunteers.

Although animals receiving CLL plasma had greater engraftment of CLL cells than

animals receiving normal plasma, the number of human cells recovered was generally

low (data not shown), consistent with other reports in the literature (230, 231). We

considered that a possible explanation for this poor engraftment might be the absence of

supporting cells that would be present in proliferation centers but not in the blood.

In an attempt to provide a proposed “microenvironment factor”, we next

attempted reconstitution of mice with splenocytes harvested from CLL patients (Table

3.2) (380). The cellular composition of each spleen sample was analyzed by FACS. In

all cases, CD19+CD5+ CLL cells were the predominant cells in the spleen, although the

frequency of CLL cells varied widely in a range from 25%-95% (Fig 3-2a: 4

representative spleens). Expression of CD20 and CD40 on CLL cells also varied, while

CD200 was expressed on CLL cells from all spleens tested (Fig 2a, lower panel; CD40

staining not shown). CD4+ T cells were the next most common population detected, with

frequencies ranging from 3%-20% (Fig 3-2b). CD8+ T cells and CD56+ NK cells were

also detectable (Fig 3-2b, data not shown). Low levels of CD14+ cells, previously

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reported to produce BAFF to support CLL survival in vitro, were found in all spleens

studied (data not shown) (175).

Recapitulating the observation made using CLL cells engrafted from PBL of

patients, superior engraftment of CD19+ CD5+ CLL cells at 28 and 54 days after infusion

of CLL splenocytes was seen in animals receiving CD200hi CLL serum compared with

controls receiving CD200lo normal human serum (Fig 3-3a, day 28; Fig 3-3b, day 54).

This difference was particularly pronounced in the peritoneal cavity compartment

(p=0.003, Fig 3-3b). As noted, splenocytes produced greater engraftment of CLL cells in

both the spleen and peritoneal cavity of NOD-SCIDγcnull recipients compared to

engraftment seen using PBL (data not shown). Substantial patient-to-patient variability

was observed in engraftment of both CLL and T cells (Fig 3-3c, engraftment of spleens 1

and 5 shown). Accordingly in all subsequent studies described detailed analysis was

restricted to use of frozen aliquots of splenocytes from only 3 patients (spleens 1, 5, and

6)

Characterization of CLL in humanized NOD-SCIDγγγγcnull mice

H&E staining of spleen tissue taken from mice at day 54 showed aggregates of

small-lymphocytes resembling proliferation centers by H&E staining that were absent in

control animals (Fig 3-3d, upper panel). CD20+ CLL cells were also more abundant in

the spleen of experimental animals by immunohistochemistry (Fig 3-3d, middle panel).

All CLL cells found in the peritoneal cavity and spleen of both experimental and control

groups expressed CD40, CD38, and CD200 at similar levels to the starting population

(data not shown).

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Splenic CLL cells co-localized with T cells by immunohistochemistry,

reminiscent of the pattern observed in proliferation centers and in the spleen of CLL

patients themselves (Fig 3-2b and 3-3d) (161). Note that T cells were found in the

spleens of mice injected with normal plasma, although CLL cells did not engraft in this

case (Fig 3-3d, lower panel). Interestingly, the ratio of CD4+ and CD8+ T cells engrafted

in the spleen (~2:1 at week 4 –see Fig 2a), is within the range seen in normal individuals,

but not typical of that observed in CLL (~1:3). The explanation for this discrepancy

remains to be determined (see later).

Engraftment of cells other than CLL and T cells was minimal regardless of the

source of plasma. In a study monitoring longer-term engraftment, in which biweekly

infusion of CLL plasma was continued to 1 week before experimental endpoint, we

observed persistence of CLL cells in both peritoneal cavity (PC) and spleen at 75 days

post spleen cell infusion (Fig 3-3e). Cells harvested from these animals were additionally

stained for the proliferative marker ki67, with a finite percentage of ki67+ cells detected

in the peritoneal cavity. The majority of these ki67+ cells (>70%), were CD19+, reflecting

ongoing proliferation in the CLL cells in this locale (Fig 3-3e). By contrast, in the spleen

of the same animals, the majority of ki67+ cells were CD19-CD5+, presumably T cells

(data not shown).

Anti-CD200 and OKT3 mAbs abrogate engraftment of CLL in NOD.SCID mice

To assess whether sCD200 in CLL plasma was an important factor contributing to

engraftment of CLL cells in vivo, animals receiving CLL splenocytes and sCD200hi

serum also received Fab anti-CD200 mAb 1B9. An independent group of mice received

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CLL splenocytes and CLL plasma that had been depleted of CD200 by passage though an

anti-CD200 CNBr column. sCD200 absorption from the serum was confirmed

independently by ELISA (>97% depletion). Both anti-CD200 mAb and depletion of

sCD200 from plasma attenuated the engraftment of CLL cells in vivo (Fig 3-4a).

CD200 is known to deliver downstream signals through a receptor CD200R, and

we had previously reported that CD200R was detected mostly on splenic CD4+ T cells

but not on CLL cells (275, 376). The absence of CD200R on CLL cells has been

independently confirmed by RT-PCR (data not shown). We investigated the role of T

cells in CLL engraftment by treating mice in the early period post spleen reconstitution

with OKT3 antibody in vivo, analysing CLL and T cells 4 weeks later. As shown in Fig

3-4b, in vivo depletion of T cells abrogated engraftment of CLL cells, despite continuous

infusion of sCD200hi CLL serum.

Comparison of anti-CD200 and rituximab in eliminating engrafted CLL cells

In a final study we asked whether NOD.SCIDγcnull mice reconstituted with human

CLL-spleen cells could be used as a pre-clinical model to test potential therapy for CLL.

Specifically we compared the efficacy of Rituxan, a clinically approved monoclonal

antibody targeting CD20 on CLL cells, with anti-CD200 mAb therapy as treatment of

mice with established spleen-cell derived CLL engraftment (381).

At 28 days following CLL splenocyte injection and biweekly sCD200hi CLL

serum infusion, independent groups of mice received Rituxan (50ug/mouse) or Fab anti-

mouse CD200 mAb. A total of 4 injections were given, at 84hr intervals over 2 weeks.

Animals were maintained for 10 days after the last dose of treatment, again with ongoing

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injection of CLL serum on a biweekly basis, and were sacrificed at day 45. FACS

analyses on cell suspensions harvested from the spleen and peritoneal cavity of these

animals showed that both rituxan (p=0.0026) and anti-CD200 mAb (p=0.0057) were

effective in reducing CLL engraftment in both tissue compartments (Fig 3-5a and b)

without affecting engraftment of T cells in either compartment (Fig 3-5c). Essentially all

CLL cells engrafting in the peritoneal cavity were depleted, with significant attenuation

of engraftment in the spleen (>70%).

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3.5 Discussion

The studies described in this chapter implicate a novel soluble form of CD200

(sCD200) in the pathogenesis of CLL. sCD200 is present at high levels in CLL plasma

compared with normal plasma and promotes the growth of CLL cells in immunodeficient

mice, suggesting it may be a novel therapeutic target for this malignancy.

CD200 expression has been linked with outcome in many malignancies (281, 295,

298). Biochemical analysis has suggested that sCD200 is likely shed from the cell

surface by proteolytic cleavage, with the ADAM family of proteases being prime

candidates in this mechanism (see chapter 4). Ectodomain shedding mediated by ADAM

proteases is an important mechanism by which a number of membrane-bound

immunoregulatory molecules are released from the cell surface. CD23, whose levels are

used as a prognostic factor for CLL, is shed by members of the ADAM family (336,

338). We found that sCD200 levels (but not surface expression of CD200 on CLL cells)

correlated strongly with tumor burden, disease stage, and an aggressive disease course as

reflected in the requirement for multiple treatments. Plasma sCD200 was also correlated

with plasma β2 microglobulin levels, known to be a predictor of progression-free survival

and overall survival in CLL (382, 383). The value of plasma sCD200 as an independent

prognostic marker against current clinical prognostic markers remains to be determined.

The hypothesis that sCD200 is important in CLL disease is supported by evidence

for its contribution to successful engraftment of spleen (and PBL)-derived CLL cells in

vivo in NOD.SCIDγcnull mice. PBL-derived CLL cells generally fail to engraft in

immunocompromised hosts, although some engraftment was recorded following

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combinations of ip and iv infusion of PBL-derived CLL cells or following injection of a

EBV+ transformed CLL cell line (234, 238). Quantitative comparison of engraftment

using spleen or PBL from the same patient has not been performed to assess engraftment

potential of CLL cells from these two tissue sources. Nevertheless, it is clear that

sCD200hi CLL plasma augments engraftment of CD19+CD5+ CLL cells in both the

peritoneal cavity and spleen, in contrast to the inferior engraftment seen using sCD200lo

plasma pooled from healthy volunteers with ten-fold lower sCD200 levels (~ 0.5ng/ml).

Those CLL cells which engraft in both sets of animals express the markers CD20, CD40,

CD23, CD38, CD49d and CD200 at levels similar to the CLL cells from the starting

spleen population, suggesting no selection for survival of unique subpopulations occurred

in vivo under these conditions (data not shown). Engrafted CLL cells also expressed

CD200.

Engraftment of CLL cells in a xeno-microenvironment often reflects persistence

of donor cells, rather than proliferation of CLL cells in the host, and engrafted CLL cells

gradually decline with time (230, 231, 234). The use of EBV-transformed CLL cell lines

circumvents this issue, though these lines often exhibit very different biologic properties

from those of primary cells (237, 238). We did not observe a significant decline in the

absolute number of engrafted CLL cells from day 25 to day 75, and indeed, the majority

of Ki67+ CD5+ cells in the peritoneal cavity at day 75 were CD19+ CLL cells. We

conclude that CLL engraftment in our model reflects both survival and proliferation of

CLL cells. Since the engrafted CLL cells continue to express CD200 at high levels, it is

possible that sCD200 may be released in ongoing fashion from grafted CLL cells in

vivo—this hypothesis is supported by recent findings from assaying sCD200 levels in

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mice at >6 months post-reconstitution (unpublished data). We have not investigated

whether continual infusion of sCD200hi CLL plasma is needed to sustain CLL

engraftment, or whether the engrafted CLL cells produce sufficient amount of sCD200 in

vivo to maintain themselves in the absence of exogenous sCD200.

Normal plasma and CLL plasma differ significantly in their content of multiple

molecules, including many proteins and fatty acids. A number of such molecules,

including the B cell growth factors BAFF and APRIL, and soluble CD14, have direct

effects on CLL cells (377, 384, 385). While we do not rule out a contribution by other

factors besides sCD200 in the support of CLL growth, the fact that pre-absorption of

sCD200 from CLL plasma minimized CLL engraftment supports the hypothesis that

sCD200 is a key contributor to CLL engraftment. Our data also supports a role for T cell

involvement in this mechanism of action, since T cell depletion (OKT3 in vivo) abrogated

CLL engraftment, despite continuous infusion of sCD200hi CLL plasma. This may help

explain why, in preliminary experiments, infusion of purified CLL cells alone, even with

continuous sCD200hi CLL serum supplement, produced only minimal engraftment of

CLL cells.

The role of non-malignant T cells in CLL has been investigated by a number of

groups (236, 386, 387). T cells harvested from CLL patients differ from normal T cells

by their high production of IL4 and reduced expression of co-stimulatory molecules

(387). The observation that CLL cells fail to engraft in the absence of T cells despite

sCD200 infusion suggests T cells, a subpopulation of which have previously been shown

to express CD200R, may represent an important target of sCD200 (376). Whether

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sCD200-targeted CD200R+ T cells affect CLL growth directly, or indirectly through the

action of other T or non-T populations, remains unknown.

Studies comparing the efficacy of anti-CD200 mAb or Rituxan as therapy for

NOD.SCIDγcnull mice reconstituted with CLL splenocytes showed both were effective in

treating pre-existing disease (388). Rituxan induces lysis of CLL cells by complement

dependent and antibody-dependent cell mediated cytotoxicity, while Fab anti-CD200

1B9, likely regulates host resistance to growth (389). Whether there is a role for

synergism in their action remains to be determined.

In summary, our data suggests that sCD200, present in patient plasma, is

associated with disease progression in CLL patients, and its presence can be utilized to

establish a novel xenograft model for CLL which may be useful for preclinical testing.

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3.6 Tables

Table 3.1: Clinical characteristics of patients in plasma sCD200 analyses

Pt ID Age Sex ▪ Rai Stage ♦ WBC

% CD38+ ◊ β2M ▼ Cytogenetics ●Treatments

1 61 F 4 225 17 7 13q-,17p- Fx2,CP, FC 2 68 M 1 25 9 NA NA None 3 72 M 4 175 2 5.7 13q-/17p- S/CHOP/CVP 4 84 F 3 200 46 NA 13q- P/C,S/R 5 62 M 4 121 2 2.2 13q- CP,CVP,S,FC 6 66 M 0 21 1 1.2 NA None 7 71 M 4 25 2 NA T12, 11q- CVPx2,Splen,FC 8 74 M 4 220 Na NA 17p- FR,splen, S 9 77 M 4 10 40 NA T12 C/P, Splen,FC

10 71 M 4 54 5 NA 13q- CVP 11 72 F 0 32 4 NA 13q-,17p- None 12 55 M 4 23 81 2.3 13q-,17p- CHOP,P 13 57 M 4 22 7 1.4 13q-,17p- CP,CHOP,FCx3,S 14 71 M 2 186 72 NA 11q-,13q- None 15 51 F 4 22 7 NA 17p- Splen 16 69 M 4 189 4 NA Normal CP 17 55 F 3 55 10 NA 13q- CP,FCR 18 88 M 4 35 71 NA 11q-,13q- CVP,FC 19 57 F 4 50 21 8.1 17p- FCR,DHAP, S, R 20 58 F 3 55 9 NA NA CP 21 48 F 4 6 62 NA Monosomy 11, 17p- CP,FC, CHOP 22 54 M 4 8 1 4.3 Normal CP,FC,FCR,S 23 53 M 3 4 10 2.1 T12 CVP,FC 24 73 F 4 100 2 4.6 13q- Cx4,Fx3 25 56 F 4 8 78 NA 13q- Cx3, Fx2, CHOP 26 48 F 3 17 72 NA T12 CP,FCR 27 82 F 3 125 8 NA T12 None 28 62 F 3 110 1 NA 13q- Splen,CP 29 67 M 2 25 1 2.6 13q- None 30 66 M 4 122 54 NA 13q- Splen,CP 31 69 M 1 120 8 2 Normal None 32 51 M 0 18 6 NA Normal None 34 58 M 4 70 19 5.8 Normal FC, S,R 35 64 M 2 20 50 2.8 11q-,13q- FCR 36 59 M 4 10 7 2.7 NA CP 37 58 M 2 25 25 NA Normal None 38 55 M 4 135 18 2.9 T12 CPR 39 74 M 3 185 25 3.7 13q- P 40 52 M 0 42 4 NA 13q- None

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Table 3.1 continued

Pt ID Age Sex ▪ Rai Stage ♦ WBC

% CD38+ ◊ β2M ▼ Cytogenetics ● Treatments

41 59 F 3 35 40 1.7 Normal CP,Splen 42 89 F 1 25 na NA NA P 44 61 F 3 25 51 2.6 Normal CP 45 60 M 4 100 2 6.1 NA Fx2,CP,Revl,CHOPR 46 55 M 3 121 4 3.1 T12 None 47 61 M 0 33 5 1.4 13q- None 48 70 M 4 25 na NA NA None 49 60 M 4 61 30 NA Normal None 51 80 F 0 15 14 2.8 NA None 52 91 F 0 18 28 2.8 T12 P 53 61 M 3 22 91 NA 13q-,T12 None 54 64 F 2 22 na NA T12 None 55 63 F 0 23 1 1.5 13q- None 56 62 F 0 10 1 1.6 NA None 57 77 F 4 120 na 8.5 13q- CP 58 54 F 4 60 3 2 13q- None 59 68 F 3 85 53 2.4 T12,17p- None 60 88 M 4 62 1 1.7 13q-,17p- None 61 81 M 3 89 1 11.3 Normal P 62 61 F 4 165 1 3.4 13q- CP 63 62 M 4 12 71 4.3 13q-,11q- CP/FC 64 69 F 4 81 na 3.6 Normal CP,CVP,FC 65 61 F 4 140 18 NA 13q-,11q- Splen 66 60 F 3 6 6 5.7 13q-,T12 CP,FC,FCR 67 57 F 3 10 10 NA 13q- CP 68 62 M 1 20 11 1.1 13q- None 69 47 M 2 65 1 NA 13q- Splen 70 78 F 3 33 13 2.2 13q- None 71 57 M 2 21 1 1.9 T12,13q- None 72 48 F 0 13 2 1.5 NA None 73 53 F 4 65 2 NA 13q- None 74 77 M 3 250 1 2.7 13q- None 75 63 F 2 22 71 1.7 NA None 76 58 F 2 17 2 1.1 NA None 77 38 M 2 37 63 NA Normal None 78 72 M 4 80 13 3.8 Normal CP,FCR 79 51 M 4 140 1 3.1 13q- Cx2 80 51 M 2 88 2 2 13q- None 81 64 M 3 160 12 3.3 11q- CPx2 82 74 M 2 45 2 3.5 11q- None

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Table 3.2: Clinical characteristics of patients whose spleens were harvested and used in in vivo studies

Spleen # Pt ID Sex Age Time (yrs) ▪ Rai Stage ♦ WBC % CD38 ◊ β2M ▼ Cytogenetics ◙ Treatments ▲ AIHA

1 102 M 72 5 III 88 40 5 13q CP, steroids Yes

2 113 M 42 3 IV 76 4 3 13q none No

3 114 M 60 10 IV 300 2 4 13q none No

4 125 M 72 5 IV 25 45 6 17p,13q FC, HDP No

5 153 M 71 15 IV 120 44 3 13q CVP, HDP Yes

6 154 M 64 22 III 75 3 3 11q Revl, CP yes

127

Footnotes to tables:

Table 3.1:

▪ Rai stage 0: lymphocytosis; I: with adenopathy; II: with hepatosplenomegaly; III: with

anaemia; IV: with thrombocytopenia. ♦ WBC: White blood cell count (x106 cells/ml) in

peripheral blood. ◊ β2M, Plasma β2Microglobulin level, mg/L. ▼ T12, trisomy 12; NA,

not available. ● CVP, cyclophosphamide/vincristine/prednisone; CHOP,

cyclophosphamide/vincristine/doxorubicin/prednisone; DHAP, dexamethasone/

cytarabine/cisplatin; FC, fludarabine/cyclophosphamide; C, Chlorambucil; P, prednisone;

F, fludarabine; R, rituximab; Revl, revlimid (lenalidomide); S, solumedrol; Splen,

splenectomy

Table 3.2:

◙ Treatments received before splenectomy: C, Chlorambucil; P, prednisone; CVP,

cyclophosphamide/vincristine/prednisone; FC, fludarabine/cyclophosphamide; HDP, high

dose prednisone; Revl, revlimid (lenalidomide). ▲ AIHA= documented autoimmune

hemolytic anemia

128

3.7 Figure legends

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

sCD200 levels in plasma samples from age-matched healthy controls (n=27) and CLL

patients in clinical cohort (n=77) were measured by ELISA. All p-values were obtained

from Mann Whitney U test unless specified otherwise. a) Plasma from CLL patients at

various clinical stages of disease showed significantly higher levels of sCD200 than plasma

from healthy controls (p<0.001). b) CLL patients with Rai stage III (p=0.015) and IV

(p=0.002) diseases showed higher plasma sCD200 levels than patients with early disease

(Stage 0-I). No significant difference in plasma sCD200 level was found between patients

at Stage 0-I and Stage II. c) Patients requiring more than 2 courses of treatments had higher

plasma sCD200 levels than patients with more indolent disease requiring no (p<0.0001) or

1 (p=0.0027) treatment. d) Plasma sCD200 levels strongly correlated with plasma β2

microglobulin levels (p<0.0001, Spearman’s Rank Correlation test).

Figure 3-2: Characterization of CLL splenocytes harvested from splenectomised patients (Table II)

2x106 fresh CLL splenocytes were characterized for relative frequency of CLL (CD20,

CD19, CD5, and CD200) and T cells (CD4 and CD8) in multicolour FACS analyses.

Results from 4 representative spleens are shown (Sp 1, 4, 5, and 6). a) Frequency of

CD19+CD5+ CLL cells from the 4 spleens ranged from 23%-90% (upper panel).

CD19+CD5+ CLL cells were gated and analyzed for CD20 and CD200 expression, the

results of which are shown in the lower panel. CLL cells from all 4 patients stained

brightly for CD200, while CD20 expression varied among the patients (lower panel). b)

Distribution of CD4+ and CD8+ T cells in the same patient spleens as a). Frequency of

129

CD4+ T cells ranged from 3%-17% while frequency of CD8+ T cells ranged from less than

0.5% to over 10%.

Figure 3-3: Engraftment of human CLL cells and T cells in NOD.SCIDγcnull mice

Irradiated NOD.SCIDγcnull mice at 8-14 weeks of age were infused with 1x108 thawed CLL

splenocytes ip, followed up biweekly infusion of sCD200lo normal plasma, pooled from

age-matched healthy volunteers, or sCD200hi CLL plasma. Mice were scarified at designed

time points to assess for CLL and T cell engraftment. p-value was calculated from

unpaired t-test. a) FACS analysis of CLL (upper panel) and T cell (lower panel)

engraftment in the peritoneal cavity of animals given either sCD200hi plasma or sCD200lo

normal plasma at day 28. Spleen 1 was used in this experiment; result from 1

representative animal per group is shown. b) Absolute count (x105 cells) of CD19+CD5+

CLL cells in the peritoneal cavity of animals that received either sCD200hi plasma or

sCD200lo control plasma, in addition to spleens 1 or 5, at day 54 (data from two

independent experiments were pooled). CLL cell counts were obtained by multiplying total

cell count with % CD19+CD5+ cells as found in FACS. Mice that received sCD200hi

plasma showed elevated engraftment of CLL cells in comparison to mice that received

sCD200lo control plasma at this time point (p=0.003). c) Comparison of CLL and T cell

engraftment in mouse peritoneal cavity (upper panel) and spleen (lower panel) at day 54 by

CLL splenocytes from Sp1 and Sp5. Sp1 appeared to engraft at higher frequencies than

Sp5 in both compartments. CD4+ and CD8+ T cells from the two different spleens

engrafted at similar frequencies. d) Immunohistochemical analysis of mouse spleens at day

54. Upper panel: H&E staining; middle panel: CD20 staining; lower panel: CD3 staining.

e) Ki67 staining on CD19+CD5+ CLL cells and CD19-CD5+ non-CLL cells engrafted in

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mouse peritoneal cavity at day 75. Ki67+ cells were gated according to CD5 staining: over

70% of Ki67+CD5+ cells were CD19+ CLL cells; whereas Ki67+CD5- cells did not express

any human markers (data not shown) and were likely proliferating mouse cells. Results

from 1 representative animal are shown in d) and e).

Figure 3-4: Effects of sCD200 and/or T cell depletion on CLL engraftment in NOD.SCIDγc

null mice at day 21

a) Irradiated NOD.SCIDγcnull mice were infused with 1x108 CLL splenocytes, and then

given either sCD200hi CLL plasma or sCD200 absorbed CLL plasma. A separate group of

animals were given 1B9, an anti-CD200 mAb, in addition to sCD200hi CLL plasma for in

vivo sCD200 depletion. Both methods of sCD200 depletion effectively reduced CLL

engraftment in peritoneal cavity. b) Irradiated NOD.SCIDγcnull mice were infused with

1x108 CLL splenocytes, and either sCD200hi CLL plasma or sCD200 absorbed CLL

plasma. Both groups of animals were subdivided to receive either OKT3 iv for T cell in

vivo depletion, or control saline. OKT3 was given a total of 4 times in two weeks. T cell

depletion appeared to abrogate CLL engraftment regardless of the presence of sCD200 in

supplemented plasma. Data from 1 representative experiment using sp1, with engraftment

of CLL cells in mouse peritoneal cavity, is shown (mouse spleens showed similar

engraftment pattern). In both a) and b) CLL engraftment was assessed by CD19, CD5, and

CD200 staining. Error bars represent standard deviation in cell counts within each group (3

animals per group).

Figure 3-5: Therapeutic efficacy of Rituxan and 1B9 on CLL engraftment in NOD.SCIDγc

null mice

Irradiated NOD.SCIDγcnull mice were engrafted with 1x108 CLL splenocytes and infused

biweekly with sCD200hi CLL plasma. At day 21, mice were divided into 3 groups and

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given one of rituxan, 1B9, or saline iv 4 times in 2 weeks. Engraftment of CLL and T cells

was assessed at day 45. p-value was calculated from unpaired t-test. Both Rituxan and

1B9 were effective in eliminating CLL engraftment in both a) peritoneal cavity (p=0.0057)

and b) spleen without having significant effect on c) CD3+ T cells. Results were pooled

from two independent experiments engrafted with splenocytes from Sp1 and 5.

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3.8 Figures

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

3-1a)

ng/ml sCD200

CLL patientsHealthy controls

5

4

3

2

1

0

sCD200 level in healthy controls vs CLL patients

133

3-1b)

3-1c)

134

3-1d)

β2-Microglobulin

135

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

patients

3-2a)

Sp1 Sp4 Sp5 Sp6

CD20

CD

200

CD19

CD

5

3-2b)

Sp1 Sp4 Sp5 Sp6

CD8

CD

4

136

Figure 3-3: Engraftment of human CLL cells and T cells in NOD.SCIDγcnull mice

3-3a)

CD

19C

D4

CD8

CD20

sCD200lo normal serum sCD200hi CLL serum

137

3-3b)

Frequency (#) of engrafted CD19+CD5+ CLL cells in peritonael cavity at day54

sCD200hi CLL plasma sCD200lo normal plasma0.0

0.5

1.0

1.5

x10

5 cel

ls

p=0.003

138

3-3c)

CD5-PECy5

CD19-PECy7

CD4-PECy5

CD8-PECy7

Sp5

Sp1

139

3-3d)

CLL plasma Control plasmaH&E:

CD20:

CD3:

140

3-3e) C

D5

Ki67C

D19

CD

19

CD5

Human CD5+

cells

Human CD5- cells

CD5

141

Figure 3-4: Effects of sCD200 and/or T cell depletion on CLL engraftment in

NOD.SCIDγcnull mice at day 21

3-4a)

142

3-4b)

143

Figure 3-5: Therapeutic efficacy of Rituxan and 1B9 on CLL engraftment in

NOD.SCIDγcnull mice

3-5a)

Untreated Anti-CD200 Rituxan0.0

0.2

0.4

0.6Untreated

Anti-CD200

Rituxan

x10

5 cel

ls

p=0.0057

p=0.0026

Frequency (#) of engrafted CD19+CD5+ CLL cells in peritonael cavity at day45

144

3-5b)

Spleen #1 (Pt.102) Spleen #5 (Pt.152)

Untreated

Anti-CD200

Rituxan


Untreated

Anti-CD200

Rituxan

145

3-5c)


Untreated

Anti-CD200

Rituxan

146

Chapter 4: Ectodomain shedding of CD200

(Manuscript submitted to J. Immunology, April, 2012)

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4.1 Abstract

We have previously reported the existence of a soluble form of CD200

(sCD200) in human plasma, and found sCD200 to be elevated in the plasma of CLL

patients. In CLL, plasma sCD200 levels correlated with disease stage and response to

treatment. We have now explored whether ectodomain shedding mediated by MMPs and

ADAM proteases is at least in part responsible for sCD200 using purified CLL cells and

Hek293 cells stably transfected with CD200. CD200 was released spontaneously from

CLL cells, and this was attenuated by treatment with GM6001, a global protease inhibitor,

or with the tissue inhibitors of metalloproteases TIMP1 and TIMP3. PMA stimulation

enhanced CD200 shedding by both CLL cells and Hek293 cells stably transfected with

CD200, as shown by a decline in CD200 detected by FACS analysis and Western blotting

of membrane extracts from PMA treated cells. The PMA-induced CD200 shedding was

inhibited by TAPI-0, a metalloprotease inhibitor, and the loss of cell surface CD200

occurred in parallel with an increase in the detection of sCD200 in the CLL supernatant.

Moreover, Western blot analysis and functional studies using CD200R1 expressing Hek293

cells showed that the shed CD200 as detected in CLL and Hek293-hCD200 supernatants

lacked the cytoplasmic domains of CD200 but retained the functional extracellular domain

required for binding to CD200R. These data support the hypothesis that CD200 is cleaved

from the surface of CLL cells by a process of ectodomain shedding.

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4.2 Introduction

Cancer immunotherapy is limited by the immunosuppressive nature of tumor cells

and their microenvironment, often the result of overexpression of immunoregulatory

molecules by both tumor cells and tumor-infiltrating effector cells (266). CD200, a type-I

transmembrane molecule with potent immunosuppressive functions through interaction

with its receptor, CD200R1, is one such molecule whose expression on lymphoma cells has

been shown to dampen their killing by allogenic cytotoxic lymphocytes in vitro (376).

In addition to expression on the cell surface, many of these immunoregulatory

molecules have also been shown to exist in soluble form (390-392). The soluble form of

these cell-surface receptors and ligands may be generated by alternative splicing at the

mRNA level, as is in the case of CTLA-4, or by mechanisms of ectodomain cleavage by

MMPs and ADAM family of proteases (393, 394).

Ectodomain shedding is an important mechanism by which proteolytic cleavage of

membrane-anchored molecules at the cell surface leads to the release of a soluble form of

the molecule into the extracellular microenvironment (393). Ectodomain shedding plays an

important role in the control of immune responses by regulating the release of cytokines,

chemokines, cytokine receptors, and many membrane-anchored immunoregulatory

molecules (395, 396). CD23, CD62L, and CD44, which are amongst the molecules shed

by lymphocytes, are known to be substrates of ADAM8, ADAM10, ADAM17, and MT1-

MMP (337, 397-399). In Chronic Lymphocyte Leukemia, the detection of a soluble form

of the NKG2D ligands, CD23, and CD14 in patient plasma has been shown to have

prognostic value (400-404).

149

In the previous chapter, a novel, soluble form of CD200 was identified in CLL plasma

using a CD200 sandwich ELISA. Soluble CD200 (sCD200) was detected in normal human

plasma and levels were increased in the plasma of CLL patients, where sCD200 levels were

correlated with tumor burden, late stage disease, and disease aggressiveness. A functional

significance of high sCD200 levels in CLL plasma was inferred from its ability to enhance

engraftment of splenic human CLL cells in vivo when NOD.SCIDγcnull mice were

supplemented with sCD200hi CLL plasma. Engraftment was attenuated when sCD200 was

pre-absorbed from the plasma or when the mice were treated with an anti-CD200 mAb

(405).

In this chapter, studies designed to explore mechanisms leading to the release of

CD200 from the surface of CLL cells are reported. The data support a role for MMPs and

ADAM proteases in CD200 shedding mediated by ectodomain cleavage.

150

4.3 Materials and Method

Cells

Peripheral blood from CLL patients were collected at routine follow-up visits and

CD19+CD5+ CLL cells were purified using the RosetteSep human B cell enrichment

cocktail (StemCell Technologies, Vancouver, BC) as described previously (376). Purified

CLL cells were cultured in AIMV medium (Invitrogen, Carlsbad, CA) supplemented with

5x10-6M β-mercaptoethanol (2-ME) (Sigma).

Two Hek293 cell-lines permanently transfected with full-length hCD200 (Hek-

hCD200) and hCD200R1 (Hek-hR1), respectively, were obtained from Genetec (376).

Cells were grown in a selection medium DMEM-F12 supplemented with 1ug/ml G418 and

10%FBS.

Reagents and antibodies

Phorbal 12-myristate 13-acetate (PMA) and Ionomycin were purchased from Sigma-

Aldrich. PMA was reconstituted to 10mg/ml stocks in DMSO and was further diluted to a

working concentration of 40ng/µl in AIMV medium. Imiquimod, a TLR7 agonist, was

purchased from LKT Laboratories (St Paul, MN) and reconstituted to 1mg/ml in DMSO.

Recombinant TIMP1, TIMP2, and TIMP3 were purchased from R&D Systems and were

reconstituted to working concentrations in AIMV medium. The protease inhibitors

GM6001 and TAPI-0 were purchased from Calbiochem and reconstituted to 10mM and

1mg/ml stock, respectively, in DMSO.

151

The monoclonal rat anti-hCD200 antibodies 1B9 and 3G7, and the polyclonal rabbit

serum against the extracellular region of CD200 (CD200v+c), were described previously

(376). A polyclonal rabbit serum against the human CD200 receptor (CD200R1) was

generated by immunization of rabbits with a fusion protein containing the extracellular

region of human CD200R1 with a his-tag at the N-terminal.

Antibodies against CD19 and CD62L used in FACS analyses were purchased from

Biolegend. The apoptosis detection kit for staining of Annexin V and 7AAD was

purchased from BD Biosciences. The Pan-Cadherin antibody, used as a plasma membrane

marker for loading controls in Western blots, was purchased from Abcam.

Generation of a rabbit polyclonal serum against the cytoplasmic region of CD200

A peptide containing the 19-amino acids of the carboxyl-terminal domain of human

CD200 (KRHRNQDRGELSQGVQKMT) was synthesized at the Hospital for Sick Children

and used to immunize rabbits (Cedarlane).

The resulting anti-serum (rabbit anti-CD200 c-tail serum) was confirmed to react

with the peptide in an ELISA using pre-immune sera as negative control. To confirm

specificity for the cytoplasmic domain of CD200, the anti-serum was tested with cell

lysates from Hek-hCD200 cells or cells expressing only the extracellular domain of CD200

on Western blots.

Constitutive release of CD200 from CLL cells

To assess constitutive release of CD200, CLL cells were cultured in AIMV

medium+2x10-5 2-ME at 8x106/ml in 500µl volume in 24-well plates, with or without the

152

global protease inhibitor GM6001 at 20µM final concentration, immediately after isolation

from fresh blood. Supernatants were collected at 24 and 48 hours and were analyzed in a

CD200 sandwich ELISA as described below.

Where recombinant TIMPs were used, 1.5x106 CLL cells were plated in 96 well-

plates in 300µl AIMV medium+2x10-5 2-ME. All 3 recombinant TIMPs (TIMP1, 2, and 3)

were used at a final concentration of 2.5µg/ml.

Stimulation of CLL cells:

Purified fresh CLL cells (8x106/ml) were cultured in serum-free AIM-V medium plus

5x10-5M 2-ME (Sigma-Aldrich) in 24-well plates at 37°C in 5% CO2 in the presence or

absence of the following stimulants: TLR-7 agonist, phorbal 12-myristate 13-acetate

(PMA), and Ionomycin. For activation of CLL cells, Imiquimod, PMA, and Ionomycin

were used at a final concentration of 3ug/ml, 40ng/ml, and 1µM, respectively. Where the

effect of TAPI-0 on PMA-induced shedding was assessed, CLL cells in the same culture

conditions were treated with 50µg/ml TAPI-0, with or without PMA stimulation. At 24 and

48-hours after stimulation, cells were harvested and stained for CD200, CD62L, CD19,

AnnexinV, and 7AAD. Tissue culture supernatants were harvested at the same time points

to assess for sCD200 concentration in the CD200-sandwich ELISA.

In some PMA-stimulation experiments, membrane proteins were extracted from

aliquots of untreated and PMA-stimulated cells using the ProteoJETTM Membrane Protein

Extraction Kit (Fermentas). The protein concentration in membrane extracts was

determined by Bradford protein assay (Bio-Rad).

153

Serum starvation and stimulation of Hek293-hCD200 cells

Hek-hCD200 cells were seeded in 6-well plates at 2x106 cells/ml and grown in serum-

containing selection medium for 2 days or until ~80% confluency was reached.

Supernatants were then removed, and after 2 washings in PBS, 1.5ml of serum-free

OPIMEM medium, with or without 40ng/ml PMA, was added per well. Supernatants from

untreated and PMA-treated cells were collected at 2, 6, and 24hr time points and sCD200

concentration in each was assessed by ELISA. Supernatants from Hek293-hCD200R1 cells

grown under the same conditions were used as negative controls in the ELISA.

FACS

CD200 cell surface staining was performed using 3G7 at 0.005µg per sample in 100µl

volume with goat anti-rat IgG-PE (1:100 dilution) as the secondary antibody for detection.

For multi-color staining of CD200, CD62L and CD19, fluorochrome-conjugated CD19 and

CD62L antibodies were added at predetermined optimal concentrations at the same time as

the secondary antibody. All antibody-incubations were performed at 4◦C. Single color

controls were included in each experiment for compensation purposes, and all samples

were analyzed in a Coulter FC500 flow cytometer.

CD200 ELISA

The CD200-sandwich ELISA was developed for detection of sCD200 in human

plasma, and utilized 1B9 as capture antibody and the rabbit anti-hCD200v+c serum as

detection antibody. For optimal detection of sCD200 in CLL supernatants, supernatant

samples and CD200Fc standards (0.025ng/ml to 2ng/ml), prepared in AIMV medium, were

154

incubated overnight at 4◦C on an ELISA plate coated with the capture antibody at

1.25ng/ml and blocked with 5%FBS-PBS. The next day the plate was washed 5 times in

PBS+0.01% Tween20, followed by 2-hr incubation with the detection antibody at a 1:2000

dilution at room temperature. The remainder of the steps were performed as described

previously (405). This modified protocol increased sensitivity of the ELISA from

0.05ng/ml, as reported previously, to ~0.01ng/ml.

Immunoprecipitation of sCD200

For immunoprecipitation of sCD200, 1ml of supernatants from untreated or PMA-

treated CLL cells or HekhCD200 cells were incubated overnight with 2µg of 1B9 and 50µl

of Protein A/G Agarose bead-suspension (Pierce Biochemicals) at 4◦C. The following day,

after 2 washes in RIPA buffer containing 1 mM Na3VO4, the bound immune-complexes

were dissociated by boiling in reduced sample buffer containing 0.025% SDS followed by

low speed centrifugation. Supernatants containing immune-complexes were loaded directly

onto 10% SDS-PAGE gel.

Western blotting

After transfer onto PVDF membranes, the blots were blocked with 5% milk-TBST

for 1 hour at room temperature, and then probed with primary antibodies overnight at 4◦C.

The following primary antibodies were used in Western blotting experiments: rabbit anti-

hCD200v+c serum (1:2000 dilution), rabbit anti-CD200 c-tail serum (1:500 dilution), or

rabbit-hCD200R1 serum (1:2000 dilution). Regardless of the primary antibody used,

following primary antibody incubation and washings in TBS-T, all blots were probed with

goat anti-rabbit IgG-HRP (Jackson) at a 1:10,000 dilution for 45 minutes. After thorough

155

washing, blots were developed using an ECL Western blot detection kit (GE Healthcare

Bio-Sciences).

CD200R1 phosphorylation by sCD200

Hek293 cells stably transfected with human CD200R1 (HekhR1) were seeded at

80% confluency in 6-well plates, and serum-starved overnight in OPIMEM medium

(Invitrogen). The following day cells were washed once with PBS and incubated at 37◦C

for 15 minutes with one of the conditioned supernatants harvested from stimulation

experiments: CLL supernatant from untreated or PMA-treated cultures; and HekhCD200

supernatant (in OPIMEM). sCD200 levels in all supernatants were assessed in the CD200

sandwich ELISA. AIMV medium, and supernatant from Hek293 cells in OPIMEM were

used as negative controls. For positive control of phosphorylation, a separate well of cells

were incubated with 100µM of activated vanadate in OPIMEM medium.

After incubation cells were lysed in 500µl of RIPA buffer containing 50mM NaF,

0.2mM Na3VO4, and protease inhibitors. Following centrifugation at 10,000rpm,

supernatants were immunoprecipitated with 1µg of 4G10 Anti-Phosphotyrosine (Millipore)

in the presence of 50µl of Protein A/G bead suspension overnight at 4◦C. Immune

complexes were dissociated from Protein A/G beads by boiling in reducing sample buffer.

After quick centrifugation supernatants containing the immune complexes were loaded onto

10% SDS-PAGE gels. Western Blots were performed using the rabbit anti-hCD200R1

serum for detection of CD200R1 in immunoprecipitation (I.P.)-product.

156

Statistics

Paired and unpaired t-tests were used to determine significance between sample means

in stimulation experiments and were performed on Prism 5.0 software.

157

4.4 Results

CD200 is constitutively shed from the surface of CLL cells

To address whether CD200 is released from the surface of CLL cells, primary CLL

cells purified from peripheral blood of a cohort of patients (n=6) were cultured in AIM-V

medium. Supernatants were collected from cell culture at different time points to assess for

sCD200 concentrations using a CD200 sandwich ELISA, with sensitivity of ~0.01ng/ml.

Plasma harvested from patient blood was also analyzed for sCD200 levels.

sCD200 was detectable in most of the CLL supernatants tested at 24 hours (Fig 4-

1a, results from 4 patients shown), with increased levels observed by 48-hr, indicating

variable but constitutive release of CD200 by CLL cells (Fig 4-1a). The level of sCD200

detectable in the 48 hour supernatant of CLL cells correlated strongly with sCD200 levels

in the corresponding plasma harvested at the same time (Table 4.1; Spearman’s r=0.8857,

p=0.0333), inferring that constitutive release of CD200 from CLL cells is as least in part,

responsible for sCD200 detected in CLL plasma.

Ectodomain cleavage is thought to be responsible for the constitutive shedding of a

number of membrane-anchored molecules (393). We hypothesized that similar

mechanisms could be responsible for the release of CD200 from CLL cells. Consistent

with this hypothesis, GM6001, a board spectrum metalloprotease inhibitor that inhibits all

MMPs and some ADAM proteases, including ADAM-mediated shedding of CD62L,

abolished the release of CD200 from CLL cells (Fig 4-1b) (328, 406, 407).

158

To explore further the role of MMPs and ADAM proteases in the constitutive

release of CD200, CLL cells from a different cohort of patients were treated with

recombinant tissue-inhibitor of metalloproteases (TIMPs) and sCD200 concentrations in

48-hr supernatants were assessed by ELISA (408). Treatment of CLL cells from 3 different

patients with the different TIMPs showed that constitutive release of CD200 could be

inhibited in different patients by TIMP1 (p=0.01)and/or TIMP3 (p=0.04) ( (Fig 4-1c).

CD200 shedding from CLL cells was induced by various stimuli

ADAM protease activity is increased by activation of intraceullular 2nd messenger

systems, such as PKC, or intracellular Ca2+ pathways, leading to enhanced ectodomain

shedding (304). Inflammatory stimuli, such as LPS, inducing downstream signalling

through TLRs, have also been shown to induce ectodomain shedding (396, 409, 410). To

investigate further whether CD200 is a candidate for ectodomain shedding mediated by

MMPs/ADAM proteases, CLL cells were stimulated with the following agents: phorbol

myristate acetate (PMA), which upreguates activities of ADAM proteases, particularly

ADAM17, through the PKC pathway (411); ionomycin, which enhances activities of some

ADAM proteases via intracellular Ca2+ pathway (331, 412); and Imiquimod, a TLR7

agonist. sCD200 concentrations in the supernatants were assessed at 48 hour time point.

PMA stimulation increased release of CD200 into the supernatant by CLL cells

from all patients tested (p=0.0008, n=6), although the level of response varied amongst

patients (Fig 4-2a). Stimulation of CLL cells from the same cohort of patients by

ionomycin generally produced enhanced shedding (p=0.0532, n=6), although to a lesser

degree than that induced by PMA (Fig 4-2b). Finally, CLL cells from 4 out of the 6

159

patients responded to TLR7 stimulation by enhanced release of sCD200 (Fig 4-2c). Once

again, as observed for constitutive shedding, the response of CLL cells to these 3 stimuli

varied significantly amongst patients.

PMA-induced CD200 shedding was reflected by loss of CD200 from CLL cell surface

In lymphocytes, ectodomain shedding of CD62L by ADAM proteases, which is

induced upon PMA stimulation, is reflected as a loss of CD62L from the cell surface by

FACS (413). To explore whether the elevation of sCD200 in the supernatant of PMA

treated CLL cells was a function of inducible ectodomain shedding which followed a

similar pattern, CLL cells from a different cohort of patients (n=6) were monitored by

FACS for CD200 expression on the cell surface 24h after PMA stimulation (Fig 4-3a-d),

and by Western blotting and ELISA using membrane extracts harvested from the same cells

(Fig 4-4a-b). The shedding of CD62L following PMA stimulation as determined by FACS

was used as an independent indicator for inducible ectomain shedding (Fig 4-3a and d).

CLL cells were also stained for CD19, which is not reported to be a substrate of membrane

sheddases, and Annexin V, and 7AAD to exclude a non-specific response to PMA

stimulation (Fig 4-3b-c).

CLL cells from all 6 patients in this cohort responded to PMA stimulation by

shedding significant amount of CD62L from the cell surface, as reflected in an average

reduction of 86.7% (±17.5%) in CD62L cell surface staining on PMA-stimulated cells

compared to untreated cells (Fig 4-3a, data from 3 representative patients shown; Fig 4-3d,

% loss of CD62L and CD200, all patients). CD200 levels were reduced by >20% on the

surface of CLL cells from 4 out of the 6 patients following PMA stimulation, with median

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loss at 44.2% and an average loss of 39.3% (±27.5%), supporting the hypothesis that

CD200 was shed from the cell surface following PMA stimulation. Note that CLL cells

from all patients shown in Fig 3 shed 60%-98% of CD62L in response to PMA, but only

cells from patients 155 and 80 shed CD200 to a similar extent, with cells from patient 47

showing minimal shedding of CD200. CD19 expression on CLL cells remained stable

after PMA stimulation (Fig 4-3b). Minimal evidence for apoptosis was seen in all cultures

(Fig 4-3c), negating the contrary hypothesis that non-specific loss of CD200 from dying

cells was an explanation for the sCD200 detected in CLL supernatants.

Membrane extracts from PMA-stimulated cells from the 5 patients showing >20%

CD200 shedding by FACS also showed reduced CD200 levels when tested by CD200-

ELISA (Fig 4-4a, 3ug membrane extracts tested, p=0.0391). The reduction in CD200

levels in the membrane extracts after PMA stimulation was further confirmed by Western

blotting. 5ug of membrane extracts were run on 10% SDS-PAGE gel, and after transfer,

PVDF blots were probed with the polyclonal rabbit anti-hCD200 v+c serum and a Pan-

Cadherin antibody as control for equal loading (Fig 4-4b). Consistent with previous data,

membrane extracts from CLL cells that responded to PMA by shedding CD200 as

determined by FACS and ELISA also showed a reduction in CD200 detected by Western

(Fig 4-4b).

Inhibition of PMA-induced CD200 shedding by TAPI-0

As further confirmation that CD200 shedding was mediated by ectodomain

cleavage, CLL cells were treated with TAPI-0, a hydroxymate-based inhibitor of

metalloproteases (414). CLL cells from 3 different patients were treated with TAPI-0 with

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or without PMA stimulation, and CD200 shedding was assessed at 24-hr by FACS. TAPI-

0 appeared to restore CD200 expression on PMA-treated CLL cells from patient 139 and

158, both of which shed >50% of CD200 in response to PMA (Fig 4-4c). Interestingly,

TAPI-0 failed to inhibit PMA-induced shedding in CLL cells from patient 16, where

constitutive shedding of CD200 was also previously shown to be unresponsive to inhibition

by TIMP1, 2, or 3 (Fig 4-1c and 4-4c; cells collected on different dates).

CD200 shedding in the epithelial Hek293-hCD200 cells

Given that tissue CD200 expression is relatively ubiquitous, and its overexpression

has been reported on a number of solid tumors (301, 345), we next investigated whether

CD200 shedding occurred in cells of epithelial origin using a Hek293 cell line stably

transfected with full-length human CD200 (Hek-hCD200) and constitutively expressing

CD200 at the cell surface in high levels (376). To test whether CD200 is shed from Hek-

hCD200 cells, cells at ~80% confluency were cultured in the serum-free OPIMEM

medium, with or without stimulation by 40ng/ml PMA. Supernatants were collected at 2,

6, and 24, and 48h time-points. Serum-starved Hek-hCD200 cells released CD200

constitutively with detectable levels as early as 6-hrs after serum starvation (Fig 4-5a, data

from 1 representative experiment shown). By 24h, sCD200 concentration in the

supernatant of Hek-hCD200 cells was measured to be 3.1±1.3ng/ml (Fig 4-5b, average

from 4 independent experiments). Importantly, Hek-hCD200 cells also responded to PMA-

stimulation by shedding increased amounts of sCD200, with differences already detected at

6 hr (Fig 4-5a). By 24 h, up to 3-fold more sCD200 was present in supernatants from

PMA-stimulated cells compared to supernatants from untreated cells (Fig 4-5a and b;

p=0.03, paired-t test).

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To confirm that sCD200 released from Hek-hCD200 cells was a result of

ectodomain cleavage, Hek-hCD200 cells were treated with TAPI-0, which inhibited PMA-

stimulated CD200 shedding in CLL cells (Fig 4-4c). sCD200 concentration in the

supernatant of PMA-stimulated cells was restored to levels seen in untreated cells in the

presence of TAPI-0 (Fig 4-5c), indicating that TAPI-0 was effective in inhibiting PMA-

induced CD200 shedding in Hek293 cells. Note that TAPI-0 treatment did not affect

CD200 release from serum-starved Hek-hCD200 cells (Fig 4-5c), suggesting that

spontaneous shedding of CD200 under serum-free conditions might involve different

sheddases and/or additional mechanisms.

sCD200 released by CLL and Hek293-hCD200 cells did not contain the cytoplasmic domain of CD200

Ectodomain cleavage by MMPs and/or ADAM family of proteases releases

extracellular fragments of the cleaved substrate (393). sCD200 released by CLL and

Hek293-hCD200 cells contained extracellular domains of CD200, as illustrated by its

recognition by the two antibodies,1B9 and the polyclonal rabbit anti-hCD200 v+c serum,

both of which were raised against the extracellular regions of CD200. To confirm that

sCD200 lacked the cytoplasmic domain of full length CD200, we generated a polyclonal

rabbit anti-serum to a peptide containing the 19 amino acids that made up the cytoplasmic

tail of CD200 (Fig 4-6a). This rabbit anti-c-tail serum reacted only with full-length CD200,

but not CD200 v+c, confirming its specificity against the cytoplasmic region of CD200

(Fig 4-6b).

We next investigated whether the sCD200 found in the supernatant of CLL and

Hek-hCD200 cells would react with the rabbit anti-hCD200 c-tail serum. Duplicates of

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supernatants from PMA-stimulated CLL cells and serum-starved Hek293-hCD200 cells

were immunoprecipitated with 1B9, and then run on two separate 10% SDS-PAGE gels.

The blots were probed with either the rabbit anti-hCD200 v+c serum or the rabbit anti-

hCD200 c-tail serum. Membrane extracts from CLL cells, which expressed full-length

CD200 at high levels, were used as positive controls for both antibodies. As expected, I.P.

products from both CLL and Hek293-hCD200 cells were detected by the polyclonal anti-

hCD200 v+c serum as a ~47kd band (Fig 4-6c, upper panel). Band intensities of I.P.

products from Hek-hCD200 supernatant and CLL supernatant differed substantially but

were consistent with quantitation given by ELISAs, which showed that supernatants from

serum starved Hek-hCD200 cells generally contained ~8-fold higher concentrations of

sCD200 than supernatants from PMA-stimulated CLL cells (Fig 4-2a and Fig 4-4a).

Consistent with the hypothesis that sCD200 were products of ectodomain cleavage, the

precipitated products from supernatants were not recognized by the anti-hCD200 c-tail

serum, while the full-length CD200 isoform in CLL membrane extract was recognized as a

~48kd band (Fig 4-6c, lower panel).

sCD200 in CLL supernatant was capable of interacting with hCD200R1

Phosphorylation of the ITIM-like motif at the cytoplasmic region of CD200R1 upon

CD200 binding is crucial in transmitting signals for mediation of the downstream functions

that characterize the CD200:CD200R1 axis of immunoregulation (275). To determine

whether sCD200 released from CLL and Hek-hCD200 cells was functionally active, we

asked whether sCD200 in CLL and Hek-hCD200 supernatants was capable of binding and

phosphorylating CD200R1. Hek293 cells stably transfected with human CD200R1 (Hek-

hR1) were seeded in 6-well plates to 80% confluency before overnight serum starvation.

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The cells were then stimulated with supernatants from serum-starved Hek-hCD200 cells

and CLL cells (48hr supernatant from untreated cultures) for 15 minutes at 37◦C. After

stimulation, cells were lysed and immunoprecipitated with an anti-phosphotyrosine

antibody. The precipitated products were run on 10% SDS-PAGE gels and probed with the

rabbit anti-hCD200R1 serum to determine whether CD200R1 was amongst the

phosphorylated proteins immunoprecipitated by the anti-phosphotyrosine antibody.

CD200R1 bands were detected in immunoprecipitate from cells incubated with CLL

supernatants or Hek-hCD200 supernatant, but not in cells stimulated with sCD200- control

supernatants (Fig 4-6d). Band intensities of phosphorylated CD200R1 on the Western blots

were reflective of the sCD200 concentrations in the supernatants used for stimulation, with

supernatant from untreated CLL cells, containing about 10-fold less sCD200 than Hek-293

supernatant (data not shown), giving lower band intensity (Fig 4-6d).

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4.5 Discussion

The soluble form of CD200 (sCD200) is elevated in plasma from CLL patients in

comparison to plasma from healthy controls (405). Plasma sCD200 levels correlated

significantly with CLL disease stage and tumor burden (405). While CLL cells harvested

from patients express CD200 at various levels, sCD200 release is independent of the level

of CD200 expression on the surface of corresponding CLL cells harvested at the same time

point (unpublished data, Wong et al). We now report that sCD200 detectable in 48-hr

supernatants of CLL cells are correlated with sCD200 levels found in corresponding patient

plasma, suggesting that: 1) CLL cells release sCD200 constitutively; and 2) differences in

the ability of CLL cells to release sCD200 may account, in part at least, for variable

sCD200 levels in CLL plasma from different patients (Table 4.1).

Analysis of the human CD200 gene sequence suggests that, unlike CTLA-4,

alternative splicing is an unlikely mechanism responsible for the production of sCD200,

since the only known isoform of CD200 is a truncated form lacking the first 42 amino acids

at the N-terminal, and this form is also membrane bound with different functional

properties from full length CD200 (415). Based on this, we hypothesized that sCD200 may

reflect active ectodomain shedding.

Constitutive release of sCD200 from CLL cells was inhibited by GM6001, a global

inhibitor of metallproteases. In addition, constitutive shedding of CD200 from CLL cells

appeared to be sensitive to inhibition by the tissue inhibitor of metalloproteases (TIMPs).

Of the three major TIMPs (TIMP1-3), TIMP3 has been documented extensively to inhibit

shedding known to be mediated by ADAM proteases, including CD62L, IL6 receptor, and

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Syndecans, in addition to inhibiting MMPs (413, 416, 417). TIMP1 is known to inhibit

MMPs, and has also been shown to inhibit ADAM10 in vitro (408, 418). TIMP2, on the

other hand, inhibits only MMPs with no effect on ADAM proteases (408). The observation

that TIMP1 and TIMP3 treatment resulted in reduced sCD200 levels in supernatants from

Pt158 and 139 suggests involvement of ADAM proteases in this process. Note that the

sensitivity of CLL cells to inhibition by the different TIMPs is highly heterogeneous and

patient-specific. This heterogeneity may reflect potential involvement of multiple ADAMs

and MMPs in CD200 shedding, and the differential expression and/or activity of MMPs

and ADAM proteases in CLL cells from each patient. It is also important to note that

different culture media contain known levels of reducing agents such as antioxidants, as

well as calcium, both of which are known to influence ectodomain shedding (329, 419).

Thus, constitutive shedding of sCD200 may itself be under the influence of the tissue

culture medium used (AIM-V medium in this case).

sCD200 release from CLL cells, like that of other well-characterized substrates of

sheddases such as CD62L and CD44, could be further induced by external and

physiological stimuli, including PMA, ionomycin, and TLR7 agonists (398, 420). Of the

three shedding stimuli tested in this study, PMA, a potent activator of PKC particularly

known for its ability to induce shedding by ADAM17, was the most effective in enhancing

CD200 shedding, as determined by increases in the detection of sCD200 in the supernatant

and reduced CD200 expression on the cell surface at the same time point (411, 421).

Moreover, PMA-induced shedding of CD200 was inhibited by TAPI-0, a hydroxymate-

based protease inhibitor first developed to inhibit PMA-induced TNFα and CD62L

shedding (414, 422). Ionomycin, a calcium ionophore that induces different ADAM

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proteases, most noticeably ADAM10, also enhanced shedding of CD200, though the

strength of the response was generally less than that observed with PMA stimulation (329-

331). CLL cells from Patient 82 showed the least increase in CD200 shedding by PMA

stimulation while responding strongly to ionomycin by shedding >3-fold more CD200 than

cells from the other patients, suggesting the involvement of different proteases in CLL cells

from this patient (Fig 4-2b). This heterogeneity in response to the different shedding

stimuli by CLL cells from different patients is consistent with that seen for the constitutive

shedding of CD200 and supports the notion that CD200 is likely shed following the action

of multiple sheddases.

A number of physiological stimuli induce ectodomain shedding by a variety of cell

types. LPS, for example, was recently shown to induce ADAM17 activity through TRIF

adaptor signalling that involved downstream activation of NADPH oxidase and 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

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correlated to some extend with plasma sCD200 levels in patients (Table 4.1, Spearman’s

r=0.8286, p=0.0583).

We also explored CD200 shedding in non-CLL cells. In preliminary studies,

normal B cells, which expressed CD200 constitutively at low levels, appeared to respond to

PMA by shedding CD200 (unpublished data, Wong et al). CD200 shedding was also

observed in the epithelial Hek293 cell line stably transfected with CD200. Hek293 cells

appeared to shed CD200 constitutively when cultured in serum-free conditions and, like

CLL cells, also responded to PMA by shedding increased amount of sCD200. The

inducible shedding of CD200 in response to PMA by Hek293 cells was inhibited by TAPI-

0, again suggestive of a role for MMPs and/or ADAM proteases in this process.

Interestingly, TAPI-0 did not inhibit constitutive shedding of CD200 by Hek293 cells in

serum-free conditions, suggesting mechanisms of CD200 shedding distinct from PMA-

induced shedding and spontaneous shedding seen in CLL cells. It is important to note that

cells under serum starvation often experience increased oxidative stress and apoptosis, both

of which have been shown to be natural stimulants of ectodomain shedding (425-427).

Apoptosis, in particular, has been shown to stimulate shedding of IL6R from neutrophils

via mechanisms that are caspase-dependent but ADAM-independent (427). Although we

observed no evidence (by FACS) for increased apoptosis as a possible mechanism

responsible for increased sCD200 in CLL supernatant, the role of apoptosis in the release of

sCD200 by serum-starved Hek293 cells is not known.

Overall, both constitutive and inducible shedding of CD200 in CLL, and

consequently the existence of sCD200 in patient plasma, are likely functions of the

combination of sheddases expressed by each individual, as well as the presence of different

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stimuli present in the CLL microenvironment. The specific MMPs and/or ADAM

proteases responsible for CD200 shedding remains to be elucidated. CLL cells and

HekhCD200 cells express different sets of MMPs and ADAM proteases. CLL cells are

known to express and secrete MMP-9, which is associated with tissue invasion (428). In

addition, MMP-9 was recently shown to form a macro-molecular complex on the surface of

CLL cells with CD44, CD38, and CD49d (429). Whether MMP-9 acts as a sheddase at the

CLL cell-surface remains to be studied. Preliminary real-time PCR analyses of ADAM

proteases in CLL and Hek293 cells found high levels of ADAM 10, 17, and 28 in CLL

cells, but not in Hek293 cells (unpublished data). ADAM28, whose catalytic domain has

been shown to be capable of shedding CD23 in vitro, is overexpressed in CLL cells in

comparison to normal B cells and silencing of this expression decreased sCD200 levels in

CLL supernatants (337, 430).

Besides ectodomain cleavage by proteases, shedding of membrane-anchored

molecules from the cell surface in the forms of microvesicles is another potential

mechanism by which sCD200 is released from the surface of CLL cells to become

detectable in the plasma of CLL patients (431, 432). An increased concentration of

microvesicles in the plasma of CLL patients has been reported (433). Molecules released

in the form of microvesicles are associated with the plasma membrane and thus can exist in

their full-length forms (431). Our biochemical analysis argues against this mechanism as

an important source of sCD200, since material immunoprecipitated from both CLL and

HekhCD200 supernatants was recognized as a single band only by an antibody against the

extracellular domain of CD200 (anti-CD200 v+c), but not by an antibody recognizing

specifically the cytoplasmic tail of CD200. This supports the hypothesis that sCD200 does

170

not contain the cytoplasmic tail and is cleaved at the cell surface. The exact cleavage

site(s) on CD200 remains to be elucidated. The recognition of cleavage substrates by

sheddases is thought to involve conformational shapes rather than specific peptide

sequences (328, 434). Glycoslyation has also been show to modulate both constitutive and

induced ectodomain shedding, and its role in CD200 shedding remains to be explored (435,

436).

Regardless of the mechanism(s) of CD200 shedding, a functional significance for

sCD200 was demonstrated by the ability of sCD200 to bind and phosphorylate CD200R1,

the major receptor responsible for mediating the downstream immunoregulatory functions

of CD200 (275). With a documented functional ability to interact with CD200R, the

existence of sCD200 in plasma may have important downstream physiological

consequences and could play a role in different pathological conditions.

In conclusion, our data suggest sCD200 is a product of ectodomain cleavage by

ADAM proteases and MMPs. Given the immunoregulatory properties of CD200, the

existence of sCD200 in plasma may be an important parameter to measure for both

diagnostic and prognostic purposes. Recent data from our laboratory suggests that sCD200

can also be detected in the serum of breast cancer and colonic cancer patients (unpublished

data), consistent with the already growing evidence that CD200 itself is reported to be

overexpressed in a number of human cancers.

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4.6 Table

Table 4.1: Correlation between patient plasma sCD200 and sCD200 in corresponding

CLL supernatants

Supernatant Spearman’s correlation co-efficient

with plasma sCD200 level

No treatment 0.8857 (p=0.0333)

PMA 0.8286 (p=0.0583)

IMN 0.6 (p=0.2417)

Imiquimod 0.3143 (p=0.5639)

Footnotes to Table 4.1:

Correlation between patient plasma sCD200 levels and sCD200 levels detected in 48-hr

supernatants from corresponding CLL cells with or without external stimuli (see Fig 2;

n=6). Plasma sCD200 levels correlated significantly with sCD200 levels in 48-hr

supernatants from untreated cells (p=0.03, Spearman r=0.8857)

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4.7 Figure legends

Fig 4-1: CD200 is constitutively released from CLL cells

Supernatants from purified CLL cells cultured in AIMV medium with or without the

designated inhibitors were collected at 24 and 48 hrs and sCD200 concentrations were

measured in a CD200 sandwich-ELISA. p-values were obtained from paired t-test. a)

sCD200 was detected in 24-hr and 48-hr supernatants of untreated CLL cells (n=4),

demonstrating continuously release of sCD200 from CLL cells; b) Spontaneous release of

CD200 was inhibited by treatment of CLL cells with 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.

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c) CLL cells from 4 out of 6 patients showed a modest response to Imiquimod, although the

induction of CD200 shedding was not statistically significant (p>0.05).

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

CLL cells from 5 patients were treated or untreated with 40ng/ml PMA and cells were

harvested at 24 hour to assess for a) CD200 and CD62L; b) CD200 and CD19; and c)

Apoptosis markers AnnexinV, and 7AAD by FACS. Data from 3 representative patients

were shown. d) Median and average % loss of CD200 and CD62L from the surface of CLL

cells from all 5 patients in the cohort as determined by FACS. Both CD62L and CD200

expressions were normalized to that of CD19 on the same cells. Median loss of CD200 and

CD62L from CLL cells: 44.25% and 95.5%, respectively. Mean loss of CD200 and

CD62L from CLL cells: 39.3± 27.5% and 86.7±17.5%.

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

Membrane proteins from aliquots of treated and untreated cells from experiments in Fig3

were extracted and analyzed by a) ELISA, and b) Western blotting for CD200. Both

methods showed loss of CD200 from the membrane fraction in PMA-treated cells from

patients that responded to PMA stimulation as determined by FACS (patients not shedding

CD200 in response to PMA as assessed by FACS are highlighted in italic). c) CLL cells

from another cohort of patients (n=3) were cultured in AIMV medium, with or without

PMA stimulation, and treated with 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 PMA-

treated cells from Patient 139 and 158 (upper panels). CLL cells from Patient 16 showed

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no response to TAPI-0 although they did respond to PMA by shedding a low level of

CD200 (bottom panel).

Fig 4-5: Spontaneous and inducible shedding of CD200 in Hek-hCD200 cells

Hek-hCD200 cells were seeded in 6-well plates to 80% confluency in serum-containing

medium. Medium was then replaced with serum-free OPIMEM medium with or without

40ng/ml PMA stimulation. Supernatants were collected at different time points and

assessed for sCD200 concentration by ELISA. a) sCD200 was detectable in supernatants

from untreated cells at 6-hr after serum starvation with sCD200 concentrations remaining

relatively stable to 24-hr. PMA-treated cells released two-fold more sCD200 at 6-hr and by

24-hr showed 4-fold higher concentration of sCD200. Data from 1 out of 4 experiments is

shown. b) Hek-hCD200 consistently shed increased amounts of sCD200 in response to

PMA as detected in 24-hr supernatants from 4 independent experiments (p=0.03, paired t-

test). c) Serum-starved Hek-hCD200 cells, with or without PMA stimulation, were treated

with 50µg/ml TAPI-0, and supernatants were harvested at 24-hr. TAPI-0 inhibited PMA-

induced CD200 shedding by Hek-hCD200 cells.

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

a) Amino acid sequence of full-length human CD200. The peptide sequence used for

generation of rabbit anti-CD200 cytoplasmic-tail antibody is highlighted in bold. b)

Characterization of rabbit anti-CD200 c-tail serum; the antiserum recognized lysates from

Hek-hCD200 cells, which expressed full-length CD200, but not lysates from Hek293 cells

transfected with hCD200v+c or pure hCD200v+c, indicating specificity for the cytoplasmic

domain of CD200. c) Membrane extracts from CLL cells were recognized by both rabbit

175

anti-hCD200v+c serum and rabbit anti-CD200 c-tail serum. sCD200 I.P. from CLL and

Hek-hCD200 supernatants was recognized only by rabbit anti-hCD200v+c serum, but not

by rabbit anti-CD200 c-tail serum, indicating that sCD200 released from both cell types

lacked the cytoplasmic domain of CD200. d) CD200R1 cells were immunoprecipitated

with anti-phosphotyrosine antibody following incubation of sCD200-containing CLL and

Hek-hCD200 supernatants and cell lysis. I.P. products were subsequently run on 10%

SDS-PAGE gel and probed with the rabbit anti-hCD200R1 serum. CD200R1 was

phosphorylated by both CLL and Hek-hCD200 supernatants, but not AIMV medium or

supernatants from Hek-hCD200R1 cells, both devoid of sCD200, indicating that sCD200

was capable of binding to, and causing phosphorylation of CD200R1.

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4.8 Figures

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

4-1a)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

24h 48h

ng/m

l

Patient 58Patient 81Patient 43Patient 82

177

4-1b).

0

0.01

0.02

0.03

0.04

0.05

0.06

Untreated GM6001

ng/m

l sC

D20

0

Pt14

Pt90

178

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0

0.02

0.04

0.06

0.08

0.1

0.12

0

0.05

0.1

0.15

0.2

0.25

4-1c)

Patient 16

p=0.01

ng/m

l sC

D20

0

No treatment TIMP2

Patient 139

p=0.04

TIMP1 TIMP3

Patient 158

179

No treatment PMA0.0

0.2

0.4

0.6Pt. 7Pt. 16Pt. 43Pt. 58Pt. 81Pt. 82

ng/m

l sC

D20

0Figure 4-2: sCD200 is secreted from CLL cells in response to different stimuli

4-2a)

p=0.0008

180

No treatment Ionomycin0.0

0.2

0.4

0.6

0.8Pt. 7

Pt.16

Pt.43

Pt.58

Pt.81

Pt.82

ng/m

l sC

D20

0

4-2b)

p=0.0532

181

4-2c)

No treatment TLR7 agonist0.0

0.1

0.2

0.3

0.4

0.5Pt. 7

Pt.16

Pt.43

Pt.58

Pt.81

Pt.82

ng/m

l sC

D20

0

182

Figure 4-3: PMA-induced CD200 shedding is reflected as a loss of CD200 expression

from the surface of CLL cells

4-3a)

No treatment PMA

Pt. 47:

Pt. 155:

Pt. 80:

CD62L

CD

200

183

4-3b)

CD19

CD

200

Pt. 47:

Pt. 155:

Pt. 80:

No treatment PMA

184

4-3c)

7AAD

Ann

exin

V

Pt. 47:

Pt. 155:

Pt. 80:

No treatment PMA

185

4-3d)

Pt.47

Pt.154

Pt.10

Pt.155

Pt.156

Pt.80

0

50

100

150% CD200 loss

% CD62L loss

% lo

ss a

fter

rel

ativ

e to

untr

eate

d co

ntro

ls% CD200 loss: median=44.25%; mean=39.3± 27.5

% CD62L loss: median=95.5%; mean=86.7±17.5

186

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

4-4a)

0

20

40

60

80

100

120

140

160

180

pg C

D20

0 (p

er w

ell)

No treatment

PMA treated (40ng/ml)

3ug membrane extract/well

Pt.47 Pt.154 Pt.10 Pt.155 Pt.156 Pt.80 Pt.85 Pt.157

187

4-4b)

Pt. 80Pt. 156Pt. 47

-- + -- + -- +PMA

Pt.155Pt.10Pt.154

-- + -- + -- + -- + -- +

Pt. 85 Pt. 157

CD200

Cadherin

5ug membrane extract loaded

Primary antibody: rabbit anti-hCD200v+c

Pt. 80Pt. 156Pt. 47

-- + -- + -- +PMA

Pt.155Pt.10Pt.154

-- + -- + -- + -- + -- +

Pt. 85 Pt. 157

CD200

Cadherin

5ug membrane extract loaded

Primary antibody: rabbit anti-hCD200v+c

188

4-4c)

No treatment PMA TAPI-0 PMA+TAPI-0

Pt.139

Pt.158

Pt.16

CD19

CD

200

189

0 10 20 300

2

4

6

8

10OPIMEMOPIMEI+PMA

Time (hr)

ng/m

l sC

D20

0Figure 4-5: Spontaneous and inducible shedding of CD200 in Hek-hCD200 cells

4-5a)

190

No treatment PMA0

5

10

15No treatment

PMA

ng/m

l sC

D20

0

4-5b)

p=0.03

191

4-5c)

0

1

2

3

4

5

6

7

ng/m

l sC

D20

0

No TAPI-0

+100uM TAPI-0

OPIMEM OPIMEM+PMA

192

Figure 4-6: Absence of epitopes associated with the cytoplasmic domain of CD200 in

sCD200 and functional properties of sCD200

4-6a)

QVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSE

NHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLF

NTFGFGKISGTACLTVYVQPIVSLHYKFSEHHLNITCSATARPAPMVFWKV

PRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLH

LGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDRGEL

SQGVQKMT

V-like domain (domain I)------

c-like domain (domain II)------

Transmembrane region------ Cytoplasmic tail----

193

4-6b)

Ab to hCD200 v+c

38kDa

47kDa

Ab to hCD200 cytoplasmic tail

47kDa

1 2 3 1 2 3

Lanes:

1. Human CD200v+c (cell lysate)

2. Human CD200v+c (supernatant)

3. Full-length human CD200 (cell lysate)

Ab to hCD200 v+c

38kDa

47kDa

Ab to hCD200 cytoplasmic tail

47kDa

1 2 3 1 2 3

Lanes:

1. Human CD200v+c (cell lysate)

2. Human CD200v+c (supernatant)

3. Full-length human CD200 (cell lysate)

194

4-6c)

Anti-CD200 v+c

Anti-CD200 c-tail

PMA + -- +--

Hek

hCD200

Hek

hR1Pt.158

Sup

Pt.71

Membrane

-- --

Sup

195

4-6d)

Cell-free CLL

Supernatant in AIM-V

Hek-hCD200

Hek-hR1

Cell-free

Vana-date

Supernatant in OPIMEM

196

Chapter 5: General discussion

197

5.1 General discussion

The overexpression of immunoregulatory molecules, which deliver inhibitory

signals that generally dominate over stimulatory signals, on tumor cells and tumor-

infiltrating immune cells results in an immunosuppressive tumor microenvironment that is

characteristic of many cancers (437). A role for the CD200:CD200R axis of

immunoregulation in the control of anti-tumor immune responses was first postulated by us

based on studies that showed infusion of a recombinant form of CD200 (CD200Fc)

enhanced growth of EL4 thymoma cells in vivo (281). The overexpression of CD200 has

since been reported in several cancers, including CLL, as well as on cancer stem cells

(438).

In the context of B-cell malignancies, Kretz-Rommel et al showed that CD200

blockade attenuated rejection of lymphoma cells transduced to overexpress CD200 by

allogenic hPBMC in vivo (346). The ability of CD200 to suppress killing of CD200+ tumor

cells by hPBMC was recapitulated in our in vitro model using a cell line that naturally

express CD200 at high levels and primary CLL cells (chapter 2). Results from these in

vitro and in vivo studies by us and others support a functional, immunosuppressive role for

CD200 on lymphoma and CLL cells. Moreover, we established that T cells are the likely

effector targets of CD200-mediated suppression in this in vitro model.

In our efforts to elucidate the role of CD200 in CLL, we identified a previously

unknown, soluble form of CD200 (sCD200). We found sCD200 to be elevated in the

plasma of CLL patients, and investigated the functional properties of sCD200, its relevance

in CLL prognostics and biology, and the mechanisms leading to its release. In particular,

198

we explored the nature of sCD200/CD200:CD200R interactions in association with non-

CLL cellular components of the CLL microenvironment, which, by modulating survival

and growth of CLL cells, are known to be key players in CLL disease progression. To our

knowledge, these are the first studies that have addressed the clinical and biological role of

membrane-bound and sCD200 in CLL. The following sections summarize our findings.

5.1.1 sCD200 as a prognostic marker in CLL

In our retrospective analysis of plasma samples from 75 CLL patients at diagnosis,

we found that patients who had high plasma sCD200 levels at diagnosis tend to go on to

develop aggressive disease as reflected by progression to late disease stage (Rai stage III

and IV) and requirement for multiple treatments (chapter 2). We identified a correlation

between sCD200 and β2-microglobulin levels, one of the prognostic markers strongly

associated with adverse disease (113, 114). Due to limitations in the availability of clinical

tests, we were unable to explore the association between sCD200 levels, IgVH mutation

status, and Zap70 expression. Given that sCD200 levels in patient plasma are correlated

with levels seen following spontaneous and/or inducible shedding of CD200 from CLL

cells in vitro (chapter 4), plasma sCD200 levels may reflect the activation status of CLL

cells and thus disease activity.

Results from our univariate analysis suggest high plasma sCD200 levels may be a

marker for poor prognosis and provide strong rationale for continuous investigation of

sCD200 as a clinical prognostic marker. Retrospective and prospective studies with larger

sample sizes for multivariate analysis are required to determine the value of sCD200 levels

as an independent prognostic factor, or as a prognostic index to be incorporated into current

prognostic models, for clinical assessment at diagnosis (19). Correlation analysis of

199

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

200

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 xeno-

microenvironment, as well as of CLL cells, supports the hypothesis that this model is a

relevant one for pre-clinical testing of novel CLL therapeutics. Analysis of the engraftment

of T cells also allows for assessment of non-specific effects on bystander cells.

We have tested and compared the efficacy of anti-CD200 blockade and rituximab, a

clinically approved mAb therapeutic for CLL in attenuating CLL engraftment, using this

model, and found both to be effective. While rituximab kills CLL cells by ADCC and

201

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

202

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 CD200-

mediated immunosuppression in the CLL microenvironment (see Fig 2). In the absence of

CD200/sCD200, CD200R+ cells, including a subpopulation of T cells, survive in preference

to CLL cells, through either direct mechanisms which negatively affect CLL survival, or

indirectly by depletion of in vivo resources for survival and growth of CLL cells. In the

presence of sCD200, CD200R+ cells receive regulatory signals, which may or may not be

associated with a switch to the production of the so-called Th-2 type cytokines

characteristic of the CD200:CD200R axis, resulting in a microenvironment which favors

CLL survival and growth (Fig 2). Note that we have not observed engraftment of cells

other than CLL and T cells in the mouse; however, in human, we do not rule out the

contribution of CD200R+ cells other than T cells in the CLL microenvironment.

203

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

Normal plasma (sCD200 lo)

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

Pro-CLL T cells

CLL plasma (sCD200 hi)

� 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

204

Figure 5-2: Proposed model of CD200:CD200R mediated immunosuppression in the

CLL microenvironment

CD200R+ T cell/non-T cells

CD200

ITIM

ITIM

Suppression

CD200R-

“Pro-CLL” T cell /non-T cells

Pro-CLL factors

CLL

“TH2” switch; production of

“Th2” type cytokines

205

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

206

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

207

roles of these two major populations of T cells in vivo. As sCD200 seems to have direct

effects on the in vivo engraftment of T cells, detailed analysis of T cell subtypes engrafted

in vivo, including regulatory T cells and Th17 cells, might provide further insight into the

role of T cells in the CLL microenvironment.

5.2.2 The effects of CD200 blockade on T cells

The effects of sCD200 on engraftment of T cells suggest CD200 blockade might

impact the T-cell compartment. Insight into the effect of CD200 blockade on T cells would

not only help to delineate further the role of CD200 in the CLL microenvironment, but

might have particular importance for assessment of CD200 blockade as a useful therapeutic

agent in the clinic.

In vitro studies have shown CD200 blockade to be effective in augmenting anti-

tumor killing of CD200+ lymphoma cells and primary CLL cells by effector CD8 T cells.

Whether CD200 blockade has a similar efficacy in vivo in an autologous setting has not

been addressed. Since CD8+ T cells engraft in the xenograft model, the effector function of

CD8+ T cells against autologous CLL cells, with or without in vivo CD200 blockade, could

be assessed by in vitro functional assays following harvesting of CD8+ T cells from the

mouse. Preliminary data from our laboratory suggest that human cells harvested from mice

reconstituted with CLL splenocytes do indeed have cytolytic activity to autologous CLL

cells after in vitro or in vivo CD200 blockade. Assessment of CD8 effector activity post-

anti-CD200 treatment in such a model may also assist in evaluating the therapeutic efficacy

of novel drugs.

208

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.

209

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.

210

Chapter 6: References

211

1. Hallek, M., B. D. Cheson, D. Catovsky, F. Caligaris-Cappio, G. Dighiero, H. Dohner, P. Hillmen, M. J. Keating, E. Montserrat, K. R. Rai, and T. J. Kipps. 2008. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood 111: 5446-5456.

2. Hallek, M., and N. Pflug. 2010. Chronic lymphocytic leukemia. Ann Oncol 21 Suppl 7: vii154-164.

3. Fazi, C., L. Scarfo, L. Pecciarini, F. Cottini, A. Dagklis, A. Janus, A. Talarico, C. Scielzo, C. Sala, D. Toniolo, F. Caligaris-Cappio, and P. Ghia. 2011. General population low-count CLL-like MBL persists over time without clinical progression, although carrying the same cytogenetic abnormalities of CLL. Blood 118: 6618-6625.

4. Rawstron, A. C. 2009. Monoclonal B-cell lymphocytosis. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology: 430-439.

5. Landgren, O., M. Albitar, W. Ma, F. Abbasi, R. B. Hayes, P. Ghia, G. E. Marti, and N. E. Caporaso. 2009. B-cell clones as early markers for chronic lymphocytic leukemia. The New England journal of medicine 360: 659-667.

6. Wadhwa, P. D., and V. A. Morrison. 2006. Infectious complications of chronic lymphocytic leukemia. Seminars in oncology 33: 240-249.

7. Hamblin, T. J., D. G. Oscier, and B. J. Young. 1986. Autoimmunity in chronic lymphocytic leukaemia. Journal of clinical pathology 39: 713-716.

8. Mauro, F. R., R. Foa, D. Giannarelli, I. Cordone, S. Crescenzi, E. Pescarmona, R. Sala, R. Cerretti, and F. Mandelli. 1999. Clinical characteristics and outcome of young chronic lymphocytic leukemia patients: a single institution study of 204 cases. Blood 94: 448-454.

9. Riches, J. C., A. G. Ramsay, and J. G. Gribben. 2011. Chronic lymphocytic leukemia: an update on biology and treatment. Current oncology reports 13: 379-385.

10. 1999. Chemotherapeutic options in chronic lymphocytic leukemia: a meta-analysis of the randomized trials. CLL Trialists' Collaborative Group. Journal of the National Cancer Institute 91: 861-868.

11. Dighiero, G., and J. L. Binet. 2000. When and how to treat chronic lymphocytic leukemia. The New England journal of medicine 343: 1799-1801.

212

12. Zent, C. S., T. G. Call, T. D. Shanafelt, R. C. Tschumper, D. F. Jelinek, D. A. Bowen, C. R. Secreto, B. R. Laplant, B. F. Kabat, and N. E. Kay. 2008. Early treatment of high-risk chronic lymphocytic leukemia with alemtuzumab and rituximab. Cancer 113: 2110-2118.

13. Parikh, S. A., M. J. Keating, S. O'Brien, X. Wang, A. Ferrajoli, S. Faderl, J. Burger, C. Koller, Z. Estrov, X. Badoux, S. Lerner, and W. G. Wierda. 2011. Frontline chemoimmunotherapy with fludarabine, cyclophosphamide, alemtuzumab, and rituximab for high-risk chronic lymphocytic leukemia. Blood 118: 2062-2068.

14. Mougalian, S. S., and S. O'Brien. 2011. Adverse prognostic features in chronic lymphocytic leukemia. Oncology (Williston Park, N.Y 25: 692-696, 699.

15. Rai, K. R., A. Sawitsky, E. P. Cronkite, A. D. Chanana, R. N. Levy, and B. S. Pasternack. 1975. Clinical staging of chronic lymphocytic leukemia. Blood 46: 219-234.

16. Binet, J. L., A. Auquier, G. Dighiero, C. Chastang, H. Piguet, J. Goasguen, G. Vaugier, G. Potron, P. Colona, F. Oberling, M. Thomas, G. Tchernia, C. Jacquillat, P. Boivin, C. Lesty, M. T. Duault, M. Monconduit, S. Belabbes, and F. Gremy. 1981. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer 48: 198-206.

17. Rai, K. R., and E. Montserrat. 1987. Prognostic factors in chronic lymphocytic leukemia. Seminars in hematology 24: 252-256.

18. Van Bockstaele, F., B. Verhasselt, and J. Philippe. 2009. Prognostic markers in chronic lymphocytic leukemia: a comprehensive review. Blood reviews 23: 25-47.

19. Wierda, W. G., S. O'Brien, X. Wang, S. Faderl, A. Ferrajoli, K. A. Do, J. Cortes, D. Thomas, G. Garcia-Manero, C. Koller, M. Beran, F. Giles, F. Ravandi, S. Lerner, H. Kantarjian, and M. Keating. 2007. Prognostic nomogram and index for overall survival in previously untreated patients with chronic lymphocytic leukemia. Blood 109: 4679-4685.

20. Kay, N. E., S. M. O'Brien, A. R. Pettitt, and S. Stilgenbauer. 2007. The role of prognostic factors in assessing 'high-risk' subgroups of patients with chronic lymphocytic leukemia. Leukemia 21: 1885-1891.

21. Grever, M. R., D. M. Lucas, G. W. Dewald, D. S. Neuberg, J. C. Reed, S. Kitada, I. W. Flinn, M. S. Tallman, F. R. Appelbaum, R. A. Larson, E. Paietta, D. F. Jelinek, J. G. Gribben, and J. C. Byrd. 2007. Comprehensive assessment of genetic and molecular features predicting outcome in patients with chronic lymphocytic leukemia: results from the US Intergroup Phase III Trial E2997. J Clin Oncol 25: 799-804.

22. Rassenti, L. Z., S. Jain, M. J. Keating, W. G. Wierda, M. R. Grever, J. C. Byrd, N. E. Kay, J. R. Brown, J. G. Gribben, D. S. Neuberg, F. He, A. W. Greaves, K. R.

213

Rai, and T. J. Kipps. 2008. Relative value of ZAP-70, CD38, and immunoglobulin mutation status in predicting aggressive disease in chronic lymphocytic leukemia. Blood 112: 1923-1930.

23. Pepper, C., A. Majid, T. T. Lin, S. Hewamana, G. Pratt, R. Walewska, S. Gesk, R. Siebert, S. Wagner, B. Kennedy, F. Miall, Z. A. Davis, I. Tracy, A. C. Gardiner, P. Brennan, R. K. Hills, M. J. Dyer, D. Oscier, and C. Fegan. 2012. Defining the prognosis of early stage chronic lymphocytic leukaemia patients. British journal of haematology 156: 499-507.

24. Vallespi, T., E. Montserrat, and M. A. Sanz. 1991. Chronic lymphocytic leukaemia: prognostic value of lymphocyte morphological subtypes. A multivariate survival analysis in 146 patients. British journal of haematology 77: 478-485.

25. Matutes, E., D. Oscier, J. Garcia-Marco, J. Ellis, A. Copplestone, R. Gillingham, T. Hamblin, D. Lens, G. J. Swansbury, and D. Catovsky. 1996. Trisomy 12 defines a group of CLL with atypical morphology: correlation between cytogenetic, clinical and laboratory features in 544 patients. British journal of haematology 92: 382-388.

26. Oscier, D. G., E. Matutes, A. Copplestone, R. M. Pickering, R. Chapman, R. Gillingham, D. Catovsky, and T. J. Hamblin. 1997. Atypical lymphocyte morphology: an adverse prognostic factor for disease progression in stage A CLL independent of trisomy 12. British journal of haematology 98: 934-939.

27. Karmiris, T., A. Z. Rohatiner, S. Love, M. Carter, R. K. Ganjoo, J. Amess, A. J. Norton, and T. A. Lister. 1994. The management of chronic lymphocytic leukemia at a single centre over a 24-year period: prognostic factors for survival. Hematological oncology 12: 29-39.

28. Tsimberidou, A. M., S. Wen, S. O'Brien, P. McLaughlin, W. G. Wierda, A. Ferrajoli, S. Faderl, J. Manning, S. Lerner, C. V. Mai, A. M. Rodriguez, M. Hess, K. A. Do, E. J. Freireich, H. M. Kantarjian, L. J. Medeiros, and M. J. Keating. 2007. Assessment of chronic lymphocytic leukemia and small lymphocytic lymphoma by absolute lymphocyte counts in 2,126 patients: 20 years of experience at the University of Texas M.D. Anderson Cancer Center. J Clin Oncol 25: 4648-4656.

29. Letestu, R., V. Levy, V. Eclache, F. Baran-Marszak, D. Vaur, D. Naguib, O. Schischmanoff, S. Katsahian, F. Nguyen-Khac, F. Davi, H. Merle-Beral, X. Troussard, and F. Ajchenbaum-Cymbalista. 2010. Prognosis of Binet stage A chronic lymphocytic leukemia patients: the strength of routine parameters. Blood 116: 4588-4590.

30. Montserrat, E., J. Sanchez-Bisono, N. Vinolas, and C. Rozman. 1986. Lymphocyte doubling time in chronic lymphocytic leukaemia: analysis of its prognostic significance. British journal of haematology 62: 567-575.

214

31. Vinolas, N., J. C. Reverter, A. Urbano-Ispizua, E. Montserrat, and C. Rozman. 1987. Lymphocyte doubling time in chronic lymphocytic leukemia: an update of its prognostic significance. Blood cells 12: 457-470.

32. Molica, S., and A. Alberti. 1987. Prognostic value of the lymphocyte doubling time in chronic lymphocytic leukemia. Cancer 60: 2712-2716.

33. Dhodapkar, M., A. Tefferi, J. Su, and R. L. Phyliky. 1993. Prognostic features and survival in young adults with early/intermediate chronic lymphocytic leukemia (B-CLL): a single institution study. Leukemia 7: 1232-1235.

34. Roth, D. B. 2003. Restraining the V(D)J recombinase. Nature reviews 3: 656-666.

35. Kelsoe, G. 1994. B cell diversification and differentiation in the periphery. The Journal of experimental medicine 180: 5-6.

36. Shaffer, A. L., A. Rosenwald, and L. M. Staudt. 2002. Lymphoid malignancies: the dark side of B-cell differentiation. Nature reviews 2: 920-932.

37. Monson, N. L., S. J. Foster, H. P. Brezinschek, R. I. Brezinschek, T. Dorner, and P. E. Lipsky. 2001. The role of CD40-CD40 ligand (CD154) interactions in immunoglobulin light chain repertoire generation and somatic mutation. Clinical immunology (Orlando, Fla 100: 71-81.

38. de Vinuesa, C. G., M. C. Cook, J. Ball, M. Drew, Y. Sunners, M. Cascalho, M. Wabl, G. G. Klaus, and I. C. MacLennan. 2000. Germinal centers without T cells. The Journal of experimental medicine 191: 485-494.

39. Weller, S., A. Faili, C. Garcia, M. C. Braun, F. F. Le Deist, G. G. de Saint Basile, O. Hermine, A. Fischer, C. A. Reynaud, and J. C. Weill. 2001. CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proceedings of the National Academy of Sciences of the United States of America 98: 1166-1170.

40. Toellner, K. M., W. E. Jenkinson, D. R. Taylor, M. Khan, D. M. Sze, D. M. Sansom, C. G. Vinuesa, and I. C. MacLennan. 2002. Low-level hypermutation in T cell-independent germinal centers compared with high mutation rates associated with T cell-dependent germinal centers. The Journal of experimental medicine 195: 383-389.

41. Degan, M., R. Bomben, M. D. Bo, A. Zucchetto, P. Nanni, M. Rupolo, A. Steffan, V. Attadia, P. F. Ballerini, D. Damiani, C. Pucillo, G. D. Poeta, A. Colombatti, and V. Gattei. 2004. Analysis of IgV gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery. British journal of haematology 126: 29-42.

215

42. Messmer, B. T., E. Albesiano, D. Messmer, and N. Chiorazzi. 2004. The pattern and distribution of immunoglobulin VH gene mutations in chronic lymphocytic leukemia B cells are consistent with the canonical somatic hypermutation process. Blood 103: 3490-3495.

43. Damle, R. N., T. Wasil, F. Fais, F. Ghiotto, A. Valetto, S. L. Allen, A. Buchbinder, D. Budman, K. Dittmar, J. Kolitz, S. M. Lichtman, P. Schulman, V. P. Vinciguerra, K. R. Rai, M. Ferrarini, and N. Chiorazzi. 1999. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 94: 1840-1847.

44. Hamblin, T. J., Z. Davis, A. Gardiner, D. G. Oscier, and F. K. Stevenson. 1999. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 94: 1848-1854.

45. Oscier, D. G., A. C. Gardiner, S. J. Mould, S. Glide, Z. A. Davis, R. E. Ibbotson, M. M. Corcoran, R. M. Chapman, P. W. Thomas, J. A. Copplestone, J. A. Orchard, and T. J. Hamblin. 2002. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood 100: 1177-1184.

46. Rosenwald, A., A. A. Alizadeh, G. Widhopf, R. Simon, R. E. Davis, X. Yu, L. Yang, O. K. Pickeral, L. Z. Rassenti, J. Powell, D. Botstein, J. C. Byrd, M. R. Grever, B. D. Cheson, N. Chiorazzi, W. H. Wilson, T. J. Kipps, P. O. Brown, and L. M. Staudt. 2001. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia. The Journal of experimental medicine 194: 1639-1647.

47. Chen, L., G. Widhopf, L. Huynh, L. Rassenti, K. R. Rai, A. Weiss, and T. J. Kipps. 2002. Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia. Blood 100: 4609-4614.

48. Crespo, M., F. Bosch, N. Villamor, B. Bellosillo, D. Colomer, M. Rozman, S. Marce, A. Lopez-Guillermo, E. Campo, and E. Montserrat. 2003. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. The New England journal of medicine 348: 1764-1775.

49. Cramer, P., and M. Hallek. 2011. Prognostic factors in chronic lymphocytic leukemia-what do we need to know? Nature reviews. Clinical oncology 8: 38-47.

50. Wiestner, A., A. Rosenwald, T. S. Barry, G. Wright, R. E. Davis, S. E. Henrickson, H. Zhao, R. E. Ibbotson, J. A. Orchard, Z. Davis, M. Stetler-Stevenson, M. Raffeld, D. C. Arthur, G. E. Marti, W. H. Wilson, T. J. Hamblin, D. G. Oscier, and L. M. Staudt. 2003. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood 101: 4944-4951.

216

51. Rassenti, L. Z., L. Huynh, T. L. Toy, L. Chen, M. J. Keating, J. G. Gribben, D. S. Neuberg, I. W. Flinn, K. R. Rai, J. C. Byrd, N. E. Kay, A. Greaves, A. Weiss, and T. J. Kipps. 2004. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. The New England journal of medicine 351: 893-901.

52. Malavasi, F., S. Deaglio, R. Damle, G. Cutrona, M. Ferrarini, and N. Chiorazzi. 2011. CD38 and chronic lymphocytic leukemia: a decade later. Blood 118: 3470-3478.

53. Malavasi, F., S. Deaglio, A. Funaro, E. Ferrero, A. L. Horenstein, E. Ortolan, T. Vaisitti, and S. Aydin. 2008. Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology. Physiological reviews 88: 841-886.

54. Deaglio, S., T. Vaisitti, R. Billington, L. Bergui, P. Omede, A. A. Genazzani, and F. Malavasi. 2007. CD38/CD19: a lipid raft-dependent signaling complex in human B cells. Blood 109: 5390-5398.

55. Jelinek, D. F., R. C. Tschumper, S. M. Geyer, N. D. Bone, G. W. Dewald, C. A. Hanson, M. J. Stenson, T. E. Witzig, A. Tefferi, and N. E. Kay. 2001. Analysis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukaemia. British journal of haematology 115: 854-861.

56. Hamblin, T. J., J. A. Orchard, R. E. Ibbotson, Z. Davis, P. W. Thomas, F. K. Stevenson, and D. G. Oscier. 2002. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 99: 1023-1029.

57. Ibrahim, S., M. Keating, K. A. Do, S. O'Brien, Y. O. Huh, I. Jilani, S. Lerner, H. M. Kantarjian, and M. Albitar. 2001. CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 98: 181-186.

58. Del Poeta, G., L. Maurillo, A. Venditti, F. Buccisano, A. M. Epiceno, G. Capelli, A. Tamburini, G. Suppo, A. Battaglia, M. I. Del Principe, B. Del Moro, M. Masi, and S. Amadori. 2001. Clinical significance of CD38 expression in chronic lymphocytic leukemia. Blood 98: 2633-2639.

59. Chang, C. C., and R. P. Cleveland. 2002. Conversion of CD38 and/or myeloid-associated marker expression status during the course of B-CLL: association with a change to an aggressive clinical course. Blood 100: 1106.

60. Patten, P. E., A. G. Buggins, J. Richards, A. Wotherspoon, J. Salisbury, G. J. Mufti, T. J. Hamblin, and S. Devereux. 2008. CD38 expression in chronic lymphocytic leukemia is regulated by the tumor microenvironment. Blood 111: 5173-5181.

217

61. Pittner, B. T., T. D. Shanafelt, N. E. Kay, and D. F. Jelinek. 2005. CD38 expression levels in chronic lymphocytic leukemia B cells are associated with activation marker expression and differential responses to interferon stimulation. Leukemia 19: 2264-2272.

62. Deaglio, S., A. Capobianco, L. Bergui, J. Durig, F. Morabito, U. Duhrsen, and F. Malavasi. 2003. CD38 is a signaling molecule in B-cell chronic lymphocytic leukemia cells. Blood 102: 2146-2155.

63. Ghia, P., G. Guida, S. Stella, D. Gottardi, M. Geuna, G. Strola, C. Scielzo, and F. Caligaris-Cappio. 2003. The pattern of CD38 expression defines a distinct subset of chronic lymphocytic leukemia (CLL) patients at risk of disease progression. Blood 101: 1262-1269.

64. Krober, A., T. Seiler, A. Benner, L. Bullinger, E. Bruckle, P. Lichter, H. Dohner, and S. Stilgenbauer. 2002. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 100: 1410-1416.

65. Hock, B. D., J. L. McKenzie, L. McArthur, S. Tansley, K. G. Taylor, and L. J. Fernyhough. 2010. CD38 as a prognostic marker in chronic lymphocytic leukaemia at a single New Zealand centre: patient survival in comparison to age- and sex-matched population data. Internal medicine journal 40: 842-849.

66. Rossi, D., M. Cerri, D. Capello, C. Deambrogi, F. M. Rossi, A. Zucchetto, L. De Paoli, S. Cresta, S. Rasi, V. Spina, S. Franceschetti, M. Lunghi, C. Vendramin, R. Bomben, A. Ramponi, G. Monga, A. Conconi, C. Magnani, V. Gattei, and G. Gaidano. 2008. Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome. British journal of haematology 142: 202-215.

67. Deaglio, S., T. Vaisitti, S. Aydin, L. Bergui, G. D'Arena, L. Bonello, P. Omede, M. Scatolini, O. Jaksic, G. Chiorino, D. Efremov, and F. Malavasi. 2007. CD38 and ZAP-70 are functionally linked and mark CLL cells with high migratory potential. Blood 110: 4012-4021.

68. Schroers, R., F. Griesinger, L. Trumper, D. Haase, B. Kulle, L. Klein-Hitpass, L. Sellmann, U. Duhrsen, and J. Durig. 2005. Combined analysis of ZAP-70 and CD38 expression as a predictor of disease progression in B-cell chronic lymphocytic leukemia. Leukemia 19: 750-758.

69. Zenz, T., D. Mertens, H. Dohner, and S. Stilgenbauer. 2011. Importance of genetics in chronic lymphocytic leukemia. Blood reviews 25: 131-137.

70. Dohner, H., S. Stilgenbauer, A. Benner, E. Leupolt, A. Krober, L. Bullinger, K. Dohner, M. Bentz, and P. Lichter. 2000. Genomic aberrations and survival in chronic lymphocytic leukemia. The New England journal of medicine 343: 1910-1916.

218

71. Shanafelt, T. D., T. E. Witzig, S. R. Fink, R. B. Jenkins, S. F. Paternoster, S. A. Smoley, K. J. Stockero, D. M. Nast, H. C. Flynn, R. C. Tschumper, S. Geyer, C. S. Zent, T. G. Call, D. F. Jelinek, N. E. Kay, and G. W. Dewald. 2006. Prospective evaluation of clonal evolution during long-term follow-up of patients with untreated early-stage chronic lymphocytic leukemia. J Clin Oncol 24: 4634-4641.

72. Calin, G. A., C. D. Dumitru, M. Shimizu, R. Bichi, S. Zupo, E. Noch, H. Aldler, S. Rattan, M. Keating, K. Rai, L. Rassenti, T. Kipps, M. Negrini, F. Bullrich, and C. M. Croce. 2002. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America 99: 15524-15529.

73. Raveche, E. S., E. Salerno, B. J. Scaglione, V. Manohar, F. Abbasi, Y. C. Lin, T. Fredrickson, P. Landgraf, S. Ramachandra, K. Huppi, J. R. Toro, V. E. Zenger, R. A. Metcalf, and G. E. Marti. 2007. Abnormal microRNA-16 locus with synteny to human 13q14 linked to CLL in NZB mice. Blood 109: 5079-5086.

74. Klein, U., M. Lia, M. Crespo, R. Siegel, Q. Shen, T. Mo, A. Ambesi-Impiombato, A. Califano, A. Migliazza, G. Bhagat, and R. Dalla-Favera. 2010. The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer cell 17: 28-40.

75. Cimmino, A., G. A. Calin, M. Fabbri, M. V. Iorio, M. Ferracin, M. Shimizu, S. E. Wojcik, R. I. Aqeilan, S. Zupo, M. Dono, L. Rassenti, H. Alder, S. Volinia, C. G. Liu, T. J. Kipps, M. Negrini, and C. M. Croce. 2005. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proceedings of the National Academy of Sciences of the United States of America 102: 13944-13949.

76. Calin, G. A., A. Cimmino, M. Fabbri, M. Ferracin, S. E. Wojcik, M. Shimizu, C. Taccioli, N. Zanesi, R. Garzon, R. I. Aqeilan, H. Alder, S. Volinia, L. Rassenti, X. Liu, C. G. Liu, T. J. Kipps, M. Negrini, and C. M. Croce. 2008. MiR-15a and miR-16-1 cluster functions in human leukemia. Proceedings of the National Academy of Sciences of the United States of America 105: 5166-5171.

77. Bonci, D., V. Coppola, M. Musumeci, A. Addario, R. Giuffrida, L. Memeo, L. D'Urso, A. Pagliuca, M. Biffoni, C. Labbaye, M. Bartucci, G. Muto, C. Peschle, and R. De Maria. 2008. The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nature medicine 14: 1271-1277.

78. Bandi, N., S. Zbinden, M. Gugger, M. Arnold, V. Kocher, L. Hasan, A. Kappeler, T. Brunner, and E. Vassella. 2009. miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer. Cancer research 69: 5553-5559.

79. Palamarchuk, A., A. Efanov, N. Nazaryan, U. Santanam, H. Alder, L. Rassenti, T. Kipps, C. M. Croce, and Y. Pekarsky. 2010. 13q14 deletions in CLL involve cooperating tumor suppressors. Blood 115: 3916-3922.

219

80. Bojarska-Junak, A., I. Hus, S. Chocholska, E. Wasik-Szczepanek, M. Sieklucka, A. Dmoszynska, and J. Rolinski. 2009. BAFF and APRIL expression in B-cell chronic lymphocytic leukemia: correlation with biological and clinical features. Leukemia research 33: 1319-1327.

81. Stilgenbauer, S., L. Bullinger, P. Lichter, and H. Dohner. 2002. Genetics of chronic lymphocytic leukemia: genomic aberrations and V(H) gene mutation status in pathogenesis and clinical course. Leukemia 16: 993-1007.

82. Hallek, M., K. Fischer, G. Fingerle-Rowson, A. M. Fink, R. Busch, J. Mayer, M. Hensel, G. Hopfinger, G. Hess, U. von Grunhagen, M. Bergmann, J. Catalano, P. L. Zinzani, F. Caligaris-Cappio, J. F. Seymour, A. Berrebi, U. Jager, B. Cazin, M. Trneny, A. Westermann, C. M. Wendtner, B. F. Eichhorst, P. Staib, A. Buhler, D. Winkler, T. Zenz, S. Bottcher, M. Ritgen, M. Mendila, M. Kneba, H. Dohner, and S. Stilgenbauer. 2010. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet 376: 1164-1174.

83. Tam, C. S., J. Otero-Palacios, L. V. Abruzzo, J. L. Jorgensen, A. Ferrajoli, W. G. Wierda, S. Lerner, S. O'Brien, and M. J. Keating. 2008. Chronic lymphocytic leukaemia CD20 expression is dependent on the genetic subtype: a study of quantitative flow cytometry and fluorescent in-situ hybridization in 510 patients. British journal of haematology 141: 36-40.

84. Que, T. H., J. G. Marco, J. Ellis, E. Matutes, V. B. Babapulle, S. Boyle, and D. Catovsky. 1993. Trisomy 12 in chronic lymphocytic leukemia detected by fluorescence in situ hybridization: analysis by stage, immunophenotype, and morphology. Blood 82: 571-575.

85. Winkler, D., C. Schneider, A. Krober, L. Pasqualucci, P. Lichter, H. Dohner, and S. Stilgenbauer. 2005. Protein expression analysis of chromosome 12 candidate genes in chronic lymphocytic leukemia (CLL). Leukemia 19: 1211-1215.

86. Decker, S., K. Zirlik, L. Djebatchie, D. Hartmann, G. Ihorst, A. Schmitt-Graeff, D. Herchenbach, H. Jumaa, M. Warmuth, H. Veelken, and C. Dierks. 2012. Trisomy 12 and elevated GLI1 and PTCH1 transcript levels are biomarkers for Hedgehog-inhibitor responsiveness in CLL. Blood 119: 997-1007.

87. Dohner, H., S. Stilgenbauer, M. R. James, A. Benner, T. Weilguni, M. Bentz, K. Fischer, W. Hunstein, and P. Lichter. 1997. 11q deletions identify a new subset of B-cell chronic lymphocytic leukemia characterized by extensive nodal involvement and inferior prognosis. Blood 89: 2516-2522.

88. Stilgenbauer, S., T. Zenz, D. Winkler, A. Buhler, R. F. Schlenk, S. Groner, R. Busch, M. Hensel, U. Duhrsen, J. Finke, P. Dreger, U. Jager, E. Lengfelder, K. Hohloch, U. Soling, R. Schlag, M. Kneba, M. Hallek, and H. Dohner. 2009. Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocytic leukemia: clinical results and prognostic marker analyses from the CLL2H study of

220

the German Chronic Lymphocytic Leukemia Study Group. J Clin Oncol 27: 3994-4001.

89. Zenz, T., J. Mohr, J. Edelmann, A. Sarno, P. Hoth, M. Heuberger, H. Helfrich, D. Mertens, H. Dohner, and S. Stilgenbauer. 2009. Treatment resistance in chronic lymphocytic leukemia: the role of the p53 pathway. Leukemia & lymphoma 50: 510-513.

90. Chevallier, P., D. Penther, H. Avet-Loiseau, N. Robillard, N. Ifrah, B. Mahe, M. Hamidou, H. Maisonneuve, P. Moreau, H. Jardel, J. L. Harousseau, R. Bataille, and R. Garand. 2002. CD38 expression and secondary 17p deletion are important prognostic factors in chronic lymphocytic leukaemia. British journal of haematology 116: 142-150.

91. Delgado, J., B. Espinet, A. C. Oliveira, P. Abrisqueta, J. de la Serna, R. Collado, J. Loscertales, M. Lopez, J. A. Hernandez-Rivas, C. Ferra, A. Ramirez, J. M. Roncero, C. Lopez, A. Aventin, A. Puiggros, E. Abella, F. Carbonell, D. Costa, A. Carrio, and M. Gonzalez. 2012. Chronic lymphocytic leukaemia with 17p deletion: a retrospective analysis of prognostic factors and therapy results. British journal of haematology.

92. Starostik, P., T. Manshouri, S. O'Brien, E. Freireich, H. Kantarjian, M. Haidar, S. Lerner, M. Keating, and M. Albitar. 1998. Deficiency of the ATM protein expression defines an aggressive subgroup of B-cell chronic lymphocytic leukemia. Cancer research 58: 4552-4557.

93. Guo, Z., S. Kozlov, M. F. Lavin, M. D. Person, and T. T. Paull. 2010. ATM activation by oxidative stress. Science (New York, N.Y 330: 517-521.

94. Corney, D. C., A. Flesken-Nikitin, A. K. Godwin, W. Wang, and A. Y. Nikitin. 2007. MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. Cancer research 67: 8433-8438.

95. Fabbri, M., A. Bottoni, M. Shimizu, R. Spizzo, M. S. Nicoloso, S. Rossi, E. Barbarotto, A. Cimmino, B. Adair, S. E. Wojcik, N. Valeri, F. Calore, D. Sampath, F. Fanini, I. Vannini, G. Musuraca, M. Dell'Aquila, H. Alder, R. V. Davuluri, L. Z. Rassenti, M. Negrini, T. Nakamura, D. Amadori, N. E. Kay, K. R. Rai, M. J. Keating, T. J. Kipps, G. A. Calin, and C. M. Croce. 2011. Association of a microRNA/TP53 feedback circuitry with pathogenesis and outcome of B-cell chronic lymphocytic leukemia. Jama 305: 59-67.

96. Dohner, H., K. Fischer, M. Bentz, K. Hansen, A. Benner, G. Cabot, D. Diehl, R. Schlenk, J. Coy, S. Stilgenbauer, and et al. 1995. p53 gene deletion predicts for poor survival and non-response to therapy with purine analogs in chronic B-cell leukemias. Blood 85: 1580-1589.

221

97. Brosh, R., and V. Rotter. 2009. When mutants gain new powers: news from the mutant p53 field. Nature reviews. Cancer 9: 701-713.

98. Austen, B., J. E. Powell, A. Alvi, I. Edwards, L. Hooper, J. Starczynski, A. M. Taylor, C. Fegan, P. Moss, and T. Stankovic. 2005. Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL. Blood 106: 3175-3182.

99. Zenz, T., B. Eichhorst, R. Busch, T. Denzel, S. Habe, D. Winkler, A. Buhler, J. Edelmann, M. Bergmann, G. Hopfinger, M. Hensel, M. Hallek, H. Dohner, and S. Stilgenbauer. 2010. TP53 mutation and survival in chronic lymphocytic leukemia. J Clin Oncol 28: 4473-4479.

100. Gonzalez, D., P. Martinez, R. Wade, S. Hockley, D. Oscier, E. Matutes, C. E. Dearden, S. M. Richards, D. Catovsky, and G. J. Morgan. 2011. Mutational status of the TP53 gene as a predictor of response and survival in patients with chronic lymphocytic leukemia: results from the LRF CLL4 trial. J Clin Oncol 29: 2223-2229.

101. Byrd, J. C., J. G. Gribben, B. L. Peterson, M. R. Grever, G. Lozanski, D. M. Lucas, B. Lampson, R. A. Larson, M. A. Caligiuri, and N. A. Heerema. 2006. Select high-risk genetic features predict earlier progression following chemoimmunotherapy with fludarabine and rituximab in chronic lymphocytic leukemia: justification for risk-adapted therapy. J Clin Oncol 24: 437-443.

102. Woyach, J. A., A. S. Ruppert, N. A. Heerema, B. L. Peterson, J. G. Gribben, V. A. Morrison, K. R. Rai, R. A. Larson, and J. C. Byrd. 2011. Chemoimmunotherapy with fludarabine and rituximab produces extended overall survival and progression-free survival in chronic lymphocytic leukemia: long-term follow-up of CALGB study 9712. J Clin Oncol 29: 1349-1355.

103. Bosch, F., A. Ferrer, N. Villamor, M. Gonzalez, J. Briones, E. Gonzalez-Barca, E. Abella, S. Gardella, L. Escoda, E. Perez-Ceballos, A. Asensi, M. J. Sayas, L. Font, A. Altes, A. Muntanola, P. Bertazzoni, M. Rozman, M. Aymerich, E. Gine, and E. Montserrat. 2008. Fludarabine, cyclophosphamide, and mitoxantrone as initial therapy of chronic lymphocytic leukemia: high response rate and disease eradication. Clin Cancer Res 14: 155-161.

104. Bosch, F., P. Abrisqueta, N. Villamor, M. J. Terol, E. Gonzalez-Barca, C. Ferra, M. Gonzalez Diaz, E. Abella, J. Delgado, F. Carbonell, J. A. Garcia Marco, L. Escoda, S. Ferrer, E. Monzo, Y. Gonzalez, C. Estany, I. Jarque, O. Salamero, A. Muntanola, and E. Montserrat. 2009. Rituximab, fludarabine, cyclophosphamide, and mitoxantrone: a new, highly active chemoimmunotherapy regimen for chronic lymphocytic leukemia. J Clin Oncol 27: 4578-4584.

105. Rosenwald, A., E. Y. Chuang, R. E. Davis, A. Wiestner, A. A. Alizadeh, D. C. Arthur, J. B. Mitchell, G. E. Marti, D. H. Fowler, W. H. Wilson, and L. M. Staudt.

222

2004. Fludarabine treatment of patients with chronic lymphocytic leukemia induces a p53-dependent gene expression response. Blood 104: 1428-1434.

106. Zenz, T., S. Habe, T. Denzel, J. Mohr, D. Winkler, A. Buhler, A. Sarno, S. Groner, D. Mertens, R. Busch, M. Hallek, H. Dohner, and S. Stilgenbauer. 2009. Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial. Blood 114: 2589-2597.

107. Zenz, T., J. Mohr, E. Eldering, A. P. Kater, A. Buhler, D. Kienle, D. Winkler, J. Durig, M. H. van Oers, D. Mertens, H. Dohner, and S. Stilgenbauer. 2009. miR-34a as part of the resistance network in chronic lymphocytic leukemia. Blood 113: 3801-3808.

108. Neefjes, J., M. L. Jongsma, P. Paul, and O. Bakke. 2011. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nature reviews 11: 823-836.

109. Simonsson, B., A. Danersund, T. H. Totterman, K. Nilsson, and L. Wibell. 1986. Production of beta 2-microglobulin by chronic lymphocytic leukaemia cells in vitro. Scandinavian journal of haematology 36: 424-429.

110. Vilpo, J., L. Vilpo, M. Hurme, and P. Vuorinen. 1999. Induction of beta-2-microglobulin release in vitro by chronic lymphocytic leukaemia cells: relation to total protein synthesis. Leukemia research 23: 913-920.

111. Totterman, T. H., K. Nilsson, and B. Simonsson. 1986. Phorbol ester-induced production of beta-2-microglobulin in B-CLL cells: relation to IgM secretory response and disease activity. British journal of haematology 62: 95-103.

112. Berrebi, A., L. Bassous, M. Haran, M. Shtalrid, and L. Shvidel. 2010. The significance of elevated beta 2-microglobulin (b2-m) in chronic lymphocytic leukemia (CLL): Evidence of in vitro secretion following activation of CLL cells. Leukemia research 34: e248-249.

113. Bulian, P., M. Tarnani, D. Rossi, F. Forconi, G. Del Poeta, F. Bertoni, E. Zucca, M. Montillo, G. Pozzato, S. Deaglio, G. D'Arena, D. Efremov, R. Marasca, F. Lauria, V. Gattei, G. Gaidano, and L. Laurenti. 2011. Multicentre validation of a prognostic index for overall survival in chronic lymphocytic leukaemia. Hematological oncology 29: 91-99.

114. Antic, D., B. Mihaljevic, V. Cokic, M. D. Fekete, T. K. Djurasevic, S. Pavlovic, N. Milic, and I. Elezovic. 2011. Patients with early stage chronic lymphocytic leukemia: new risk stratification based on molecular profiling. Leukemia & lymphoma 52: 1394-1397.

115. Delgado, J., G. Pratt, N. Phillips, J. Briones, C. Fegan, J. Nomdedeu, C. Pepper, A. Aventin, R. Ayats, S. Brunet, R. Martino, D. Valcarcel, D. Milligan, and J. Sierra.

223

2009. Beta2-microglobulin is a better predictor of treatment-free survival in patients with chronic lymphocytic leukaemia if adjusted according to glomerular filtration rate. British journal of haematology 145: 801-805.

116. Chiorazzi, N., and M. Ferrarini. 2011. Cellular origin(s) of chronic lymphocytic leukemia: cautionary notes and additional considerations and possibilities. Blood 117: 1781-1791.

117. Klein, U., Y. Tu, G. A. Stolovitzky, M. Mattioli, G. Cattoretti, H. Husson, A. Freedman, G. Inghirami, L. Cro, L. Baldini, A. Neri, A. Califano, and R. Dalla-Favera. 2001. Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. The Journal of experimental medicine 194: 1625-1638.

118. Ramachandra, S., R. A. Metcalf, T. Fredrickson, G. E. Marti, and E. Raveche. 1996. Requirement for increased IL-10 in the development of B-1 lymphoproliferative disease in a murine model of CLL. The Journal of clinical investigation 98: 1788-1793.

119. Planelles, L., C. E. Carvalho-Pinto, G. Hardenberg, S. Smaniotto, W. Savino, R. Gomez-Caro, M. Alvarez-Mon, J. de Jong, E. Eldering, A. C. Martinez, J. P. Medema, and M. Hahne. 2004. APRIL promotes B-1 cell-associated neoplasm. Cancer cell 6: 399-408.

120. Salerno, E., Y. Yuan, B. J. Scaglione, G. Marti, A. Jankovic, F. Mazzella, M. F. Laurindo, D. Despres, S. Baskar, C. Rader, and E. Raveche. 2010. The New Zealand black mouse as a model for the development and progression of chronic lymphocytic leukemia. Cytometry 78 Suppl 1: S98-109.

121. Kikushige, Y., F. Ishikawa, T. Miyamoto, T. Shima, S. Urata, G. Yoshimoto, Y. Mori, T. Iino, T. Yamauchi, T. Eto, H. Niiro, H. Iwasaki, K. Takenaka, and K. Akashi. 2011. Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer cell 20: 246-259.

122. Fais, F., F. Ghiotto, S. Hashimoto, B. Sellars, A. Valetto, S. L. Allen, P. Schulman, V. P. Vinciguerra, K. Rai, L. Z. Rassenti, T. J. Kipps, G. Dighiero, H. W. Schroeder, Jr., M. Ferrarini, and N. Chiorazzi. 1998. Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. The Journal of clinical investigation 102: 1515-1525.

123. Moreno, C., and E. Montserrat. 2008. New prognostic markers in chronic lymphocytic leukemia. Blood reviews 22: 211-219.

124. Stevenson, F. K., and F. Caligaris-Cappio. 2004. Chronic lymphocytic leukemia: revelations from the B-cell receptor. Blood 103: 4389-4395.

125. Lanasa, M. C., S. D. Allgood, S. L. Slager, S. S. Dave, C. Love, G. E. Marti, N. E. Kay, C. A. Hanson, K. G. Rabe, S. J. Achenbach, L. R. Goldin, N. J. Camp, B. K.

224

Goodman, C. M. Vachon, L. G. Spector, L. Z. Rassenti, J. F. Leis, J. P. Gockerman, S. S. Strom, T. G. Call, M. Glenn, J. R. Cerhan, M. C. Levesque, J. B. Weinberg, and N. E. Caporaso. 2011. Immunophenotypic and gene expression analysis of monoclonal B-cell lymphocytosis shows biologic characteristics associated with good prognosis CLL. Leukemia 25: 1459-1466.

126. Davis, R. E., V. N. Ngo, G. Lenz, P. Tolar, R. M. Young, P. B. Romesser, H. Kohlhammer, L. Lamy, H. Zhao, Y. Yang, W. Xu, A. L. Shaffer, G. Wright, W. Xiao, J. Powell, J. K. Jiang, C. J. Thomas, A. Rosenwald, G. Ott, H. K. Muller-Hermelink, R. D. Gascoyne, J. M. Connors, N. A. Johnson, L. M. Rimsza, E. Campo, E. S. Jaffe, W. H. Wilson, J. Delabie, E. B. Smeland, R. I. Fisher, R. M. Braziel, R. R. Tubbs, J. R. Cook, D. D. Weisenburger, W. C. Chan, S. K. Pierce, and L. M. Staudt. 2010. Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature 463: 88-92.

127. Tobin, G., U. Thunberg, A. Johnson, I. Eriksson, O. Soderberg, K. Karlsson, M. Merup, G. Juliusson, J. Vilpo, G. Enblad, C. Sundstrom, G. Roos, and R. Rosenquist. 2003. Chronic lymphocytic leukemias utilizing the VH3-21 gene display highly restricted Vlambda2-14 gene use and homologous CDR3s: implicating recognition of a common antigen epitope. Blood 101: 4952-4957.

128. Widhopf, G. F., 2nd, L. Z. Rassenti, T. L. Toy, J. G. Gribben, W. G. Wierda, and T. J. Kipps. 2004. Chronic lymphocytic leukemia B cells of more than 1% of patients express virtually identical immunoglobulins. Blood 104: 2499-2504.

129. Ghiotto, F., F. Fais, A. Valetto, E. Albesiano, S. Hashimoto, M. Dono, H. Ikematsu, S. L. Allen, J. Kolitz, K. R. Rai, M. Nardini, A. Tramontano, M. Ferrarini, and N. Chiorazzi. 2004. Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia. The Journal of clinical investigation 113: 1008-1016.

130. Messmer, B. T., E. Albesiano, D. G. Efremov, F. Ghiotto, S. L. Allen, J. Kolitz, R. Foa, R. N. Damle, F. Fais, D. Messmer, K. R. Rai, M. Ferrarini, and N. Chiorazzi. 2004. Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia. The Journal of experimental medicine 200: 519-525.

131. Tobin, G., U. Thunberg, K. Karlsson, F. Murray, A. Laurell, K. Willander, G. Enblad, M. Merup, J. Vilpo, G. Juliusson, C. Sundstrom, O. Soderberg, G. Roos, and R. Rosenquist. 2004. Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia. Blood 104: 2879-2885.

132. Steininger, C., G. F. Widhopf, 2nd, E. M. Ghia, C. S. Morello, K. Vanura, R. Sanders, D. Spector, D. Guiney, U. Jager, and T. J. Kipps. 2012. Recombinant antibodies encoded by IGHV1-69 react with pUL32, a phosphoprotein of cytomegalovirus and B-cell superantigen. Blood 119: 2293-2301.

225

133. Stamatopoulos, K., C. Belessi, C. Moreno, M. Boudjograh, G. Guida, T. Smilevska, L. Belhoul, S. Stella, N. Stavroyianni, M. Crespo, A. Hadzidimitriou, L. Sutton, F. Bosch, N. Laoutaris, A. Anagnostopoulos, E. Montserrat, A. Fassas, G. Dighiero, F. Caligaris-Cappio, H. Merle-Beral, P. Ghia, and F. Davi. 2007. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: Pathogenetic implications and clinical correlations. Blood 109: 259-270.

134. Murray, F., N. Darzentas, A. Hadzidimitriou, G. Tobin, M. Boudjogra, C. Scielzo, N. Laoutaris, K. Karlsson, F. Baran-Marzsak, A. Tsaftaris, C. Moreno, A. Anagnostopoulos, F. Caligaris-Cappio, D. Vaur, C. Ouzounis, C. Belessi, P. Ghia, F. Davi, R. Rosenquist, and K. Stamatopoulos. 2008. Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis. Blood 111: 1524-1533.

135. Kostareli, E., A. Hadzidimitriou, N. Stavroyianni, N. Darzentas, A. Athanasiadou, M. Gounari, V. Bikos, A. Agathagelidis, T. Touloumenidou, I. Zorbas, A. Kouvatsi, N. Laoutaris, A. Fassas, A. Anagnostopoulos, C. Belessi, and K. Stamatopoulos. 2009. Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors. Leukemia 23: 919-924.

136. Bomben, R., M. Dal Bo, D. Capello, D. Benedetti, D. Marconi, A. Zucchetto, F. Forconi, R. Maffei, E. M. Ghia, L. Laurenti, P. Bulian, M. I. Del Principe, G. Palermo, M. Thorselius, M. Degan, R. Campanini, A. Guarini, G. Del Poeta, R. Rosenquist, D. G. Efremov, R. Marasca, R. Foa, G. Gaidano, and V. Gattei. 2007. Comprehensive characterization of IGHV3-21-expressing B-cell chronic lymphocytic leukemia: an Italian multicenter study. Blood 109: 2989-2998.

137. Sutton, L. A., E. Kostareli, A. Hadzidimitriou, N. Darzentas, A. Tsaftaris, A. Anagnostopoulos, R. Rosenquist, and K. Stamatopoulos. 2009. Extensive intraclonal diversification in a subgroup of chronic lymphocytic leukemia patients with stereotyped IGHV4-34 receptors: implications for ongoing interactions with antigen. Blood 114: 4460-4468.

138. Gordon, M. S., R. M. Kato, F. Lansigan, A. A. Thompson, R. Wall, and D. J. Rawlings. 2000. Aberrant B cell receptor signaling from B29 (Igbeta, CD79b) gene mutations of chronic lymphocytic leukemia B cells. Proceedings of the National Academy of Sciences of the United States of America 97: 5504-5509.

139. Petlickovski, A., L. Laurenti, X. Li, S. Marietti, P. Chiusolo, S. Sica, G. Leone, and D. G. Efremov. 2005. Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells. Blood 105: 4820-4827.

140. Longo, P. G., L. Laurenti, S. Gobessi, S. Sica, G. Leone, and D. G. Efremov. 2008. The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals

226

downstream of the B-cell receptor in chronic lymphocytic leukemia B cells. Blood 111: 846-855.

141. Guarini, A., S. Chiaretti, S. Tavolaro, R. Maggio, N. Peragine, F. Citarella, M. R. Ricciardi, S. Santangelo, M. Marinelli, M. S. De Propris, M. Messina, F. R. Mauro, I. Del Giudice, and R. Foa. 2008. BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgV H unmutated chronic lymphocytic leukemia (CLL) cells. Blood 112: 782-792.

142. Uckun, F. M., S. Qazi, H. Ma, L. Tuel-Ahlgren, and Z. Ozer. 2010. STAT3 is a substrate of SYK tyrosine kinase in B-lineage leukemia/lymphoma cells exposed to oxidative stress. Proceedings of the National Academy of Sciences of the United States of America 107: 2902-2907.

143. Buchner, M., S. Fuchs, G. Prinz, D. Pfeifer, K. Bartholome, M. Burger, N. Chevalier, L. Vallat, J. Timmer, J. G. Gribben, H. Jumaa, H. Veelken, C. Dierks, and K. Zirlik. 2009. Spleen tyrosine kinase is overexpressed and represents a potential therapeutic target in chronic lymphocytic leukemia. Cancer research 69: 5424-5432.

144. Gobessi, S., L. Laurenti, P. G. Longo, L. Carsetti, V. Berno, S. Sica, G. Leone, and D. G. Efremov. 2009. Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells. Leukemia 23: 686-697.

145. Chen, L., S. Monti, P. Juszczynski, J. Daley, W. Chen, T. E. Witzig, T. M. Habermann, J. L. Kutok, and M. A. Shipp. 2008. SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma. Blood 111: 2230-2237.

146. Chen, L., J. Apgar, L. Huynh, F. Dicker, T. Giago-McGahan, L. Rassenti, A. Weiss, and T. J. Kipps. 2005. ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia. Blood 105: 2036-2041.

147. Perrot, A., C. Pionneau, S. Nadaud, F. Davi, V. Leblond, F. Jacob, H. Merle-Beral, R. Herbrecht, M. C. Bene, J. G. Gribben, S. Bahram, and L. Vallat. 2011. A unique proteomic profile on surface IgM ligation in unmutated chronic lymphocytic leukemia. Blood 118: e1-15.

148. Stadanlick, J. E., M. Kaileh, F. G. Karnell, J. L. Scholz, J. P. Miller, W. J. Quinn, 3rd, R. J. Brezski, L. S. Treml, K. A. Jordan, J. G. Monroe, R. Sen, and M. P. Cancro. 2008. Tonic B cell antigen receptor signals supply an NF-kappaB substrate for prosurvival BLyS signaling. Nature immunology 9: 1379-1387.

149. Rowland, S. L., K. F. Leahy, R. Halverson, R. M. Torres, and R. Pelanda. 2010. BAFF receptor signaling aids the differentiation of immature B cells into transitional B cells following tonic BCR signaling. J Immunol 185: 4570-4581.

227

150. Quiroga, M. P., K. Balakrishnan, A. V. Kurtova, M. Sivina, M. J. Keating, W. G. Wierda, V. Gandhi, and J. A. Burger. 2009. B-cell antigen receptor signaling enhances chronic lymphocytic leukemia cell migration and survival: specific targeting with a novel spleen tyrosine kinase inhibitor, R406. Blood 114: 1029-1037.

151. Vlad, A., P. A. Deglesne, R. Letestu, S. Saint-Georges, N. Chevallier, F. Baran-Marszak, N. Varin-Blank, F. Ajchenbaum-Cymbalista, and D. Ledoux. 2009. Down-regulation of CXCR4 and CD62L in chronic lymphocytic leukemia cells is triggered by B-cell receptor ligation and associated with progressive disease. Cancer research 69: 6387-6395.

152. Delmer, A., F. Ajchenbaum-Cymbalista, R. Tang, S. Ramond, A. M. Faussat, J. P. Marie, and R. Zittoun. 1995. Overexpression of cyclin D2 in chronic B-cell malignancies. Blood 85: 2870-2876.

153. Damle, R. N., F. M. Batliwalla, F. Ghiotto, A. Valetto, E. Albesiano, C. Sison, S. L. Allen, J. Kolitz, V. P. Vinciguerra, P. Kudalkar, T. Wasil, K. R. Rai, M. Ferrarini, P. K. Gregersen, and N. Chiorazzi. 2004. Telomere length and telomerase activity delineate distinctive replicative features of the B-CLL subgroups defined by immunoglobulin V gene mutations. Blood 103: 375-382.

154. Messmer, B. T., D. Messmer, S. L. Allen, J. E. Kolitz, P. Kudalkar, D. Cesar, E. J. Murphy, P. Koduru, M. Ferrarini, S. Zupo, G. Cutrona, R. N. Damle, T. Wasil, K. R. Rai, M. K. Hellerstein, and N. Chiorazzi. 2005. In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. The Journal of clinical investigation 115: 755-764.

155. van Gent, R., A. P. Kater, S. A. Otto, A. Jaspers, J. A. Borghans, N. Vrisekoop, M. A. Ackermans, A. F. Ruiter, S. Wittebol, E. Eldering, M. H. van Oers, K. Tesselaar, M. J. Kersten, and F. Miedema. 2008. In vivo dynamics of stable chronic lymphocytic leukemia inversely correlate with somatic hypermutation levels and suggest no major leukemic turnover in bone marrow. Cancer research 68: 10137-10144.

156. Calissano, C., R. N. Damle, G. Hayes, E. J. Murphy, M. K. Hellerstein, C. Moreno, C. Sison, M. S. Kaufman, J. E. Kolitz, S. L. Allen, K. R. Rai, and N. Chiorazzi. 2009. In vivo intraclonal and interclonal kinetic heterogeneity in B-cell chronic lymphocytic leukemia. Blood 114: 4832-4842.

157. Collins, R. J., L. A. Verschuer, B. V. Harmon, R. L. Prentice, J. H. Pope, and J. F. Kerr. 1989. Spontaneous programmed death (apoptosis) of B-chronic lymphocytic leukaemia cells following their culture in vitro. British journal of haematology 71: 343-350.

158. Levesque, M. C., C. W. O'Loughlin, and J. B. Weinberg. 2001. Use of serum-free media to minimize apoptosis of chronic lymphocytic leukemia cells during in vitro culture. Leukemia 15: 1305-1307.

228

159. Burger, J. A., P. Ghia, A. Rosenwald, and F. Caligaris-Cappio. 2009. The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood 114: 3367-3375.

160. Deaglio, S., and F. Malavasi. 2009. Chronic lymphocytic leukemia microenvironment: shifting the balance from apoptosis to proliferation. Haematologica 94: 752-756.

161. Soma, L. A., F. E. Craig, and S. H. Swerdlow. 2006. The proliferation center microenvironment and prognostic markers in chronic lymphocytic leukemia/small lymphocytic lymphoma. Human pathology 37: 152-159.

162. Herishanu, Y., P. Perez-Galan, D. Liu, A. Biancotto, S. Pittaluga, B. Vire, F. Gibellini, N. Njuguna, E. Lee, L. Stennett, N. Raghavachari, P. Liu, J. P. McCoy, M. Raffeld, M. Stetler-Stevenson, C. Yuan, R. Sherry, D. C. Arthur, I. Maric, T. White, G. E. Marti, P. Munson, W. H. Wilson, and A. Wiestner. 2011. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 117: 563-574.

163. Gururajan, M., A. Simmons, T. Dasu, B. T. Spear, C. Calulot, D. A. Robertson, D. L. Wiest, J. G. Monroe, and S. Bondada. 2008. Early growth response genes regulate B cell development, proliferation, and immune response. J Immunol 181: 4590-4602.

164. Paterson, A., C. I. Mockridge, J. E. Adams, S. Krysov, K. N. Potter, A. S. Duncombe, S. J. Cook, F. K. Stevenson, and G. Packham. 2012. Mechanisms and clinical significance of BIM phosphorylation in chronic lymphocytic leukemia. Blood 119: 1726-1736.

165. O'Neill, L. A., and A. G. Bowie. 2007. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nature reviews 7: 353-364.

166. Muzio, M., C. Scielzo, M. T. Bertilaccio, M. Frenquelli, P. Ghia, and F. Caligaris-Cappio. 2009. Expression and function of toll like receptors in chronic lymphocytic leukaemia cells. British journal of haematology 144: 507-516.

167. Rozkova, D., L. Novotna, R. Pytlik, I. Hochova, T. Kozak, J. Bartunkova, and R. Spisek. 2010. Toll-like receptors on B-CLL cells: expression and functional consequences of their stimulation. International journal of cancer 126: 1132-1143.

168. Puente, X. S., M. Pinyol, V. Quesada, L. Conde, G. R. Ordonez, N. Villamor, G. Escaramis, P. Jares, S. Bea, M. Gonzalez-Diaz, L. Bassaganyas, T. Baumann, M. Juan, M. Lopez-Guerra, D. Colomer, J. M. Tubio, C. Lopez, A. Navarro, C. Tornador, M. Aymerich, M. Rozman, J. M. Hernandez, D. A. Puente, J. M. Freije, G. Velasco, A. Gutierrez-Fernandez, D. Costa, A. Carrio, S. Guijarro, A. Enjuanes, L. Hernandez, J. Yague, P. Nicolas, C. M. Romeo-Casabona, H. Himmelbauer, E. Castillo, J. C. Dohm, S. de Sanjose, M. A. Piris, E. de Alava, J. San Miguel, R. Royo, J. L. Gelpi, D. Torrents, M. Orozco, D. G. Pisano, A. Valencia, R. Guigo, M.

229

Bayes, S. Heath, M. Gut, P. Klatt, J. Marshall, K. Raine, L. A. Stebbings, P. A. Futreal, M. R. Stratton, P. J. Campbell, I. Gut, A. Lopez-Guillermo, X. Estivill, E. Montserrat, C. Lopez-Otin, and E. Campo. 2011. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature 475: 101-105.

169. Bertilaccio, M. T., G. Simonetti, A. Dagklis, M. Rocchi, T. V. Rodriguez, B. Apollonio, A. Mantovani, M. Ponzoni, P. Ghia, C. Garlanda, F. Caligaris-Cappio, and M. Muzio. 2011. Lack of TIR8/SIGIRR triggers progression of chronic lymphocytic leukemia in mouse models. Blood 118: 660-669.

170. Cancro, M. P. 2009. Signalling crosstalk in B cells: managing worth and need. Nature reviews 9: 657-661.

171. Mackay, F., and P. Schneider. 2009. Cracking the BAFF code. Nature reviews 9: 491-502.

172. Kern, C., J. F. Cornuel, C. Billard, R. Tang, D. Rouillard, V. Stenou, T. Defrance, F. Ajchenbaum-Cymbalista, P. Y. Simonin, S. Feldblum, and J. P. Kolb. 2004. Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway. Blood 103: 679-688.

173. Endo, T., M. Nishio, T. Enzler, H. B. Cottam, T. Fukuda, D. F. James, M. Karin, and T. J. Kipps. 2007. BAFF and APRIL support chronic lymphocytic leukemia B-cell survival through activation of the canonical NF-kappaB pathway. Blood 109: 703-710.

174. Burger, J. A., N. Tsukada, M. Burger, N. J. Zvaifler, M. Dell'Aquila, and T. J. Kipps. 2000. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood 96: 2655-2663.

175. Tsukada, N., J. A. Burger, N. J. Zvaifler, and T. J. Kipps. 2002. Distinctive features of "nurselike" cells that differentiate in the context of chronic lymphocytic leukemia. Blood 99: 1030-1037.

176. Enzler, T., A. P. Kater, W. Zhang, G. F. Widhopf, 2nd, H. Y. Chuang, J. Lee, E. Avery, C. M. Croce, M. Karin, and T. J. Kipps. 2009. Chronic lymphocytic leukemia of Emu-TCL1 transgenic mice undergoes rapid cell turnover that can be offset by extrinsic CD257 to accelerate disease progression. Blood 114: 4469-4476.

177. Christopoulos, P., D. Pfeifer, K. Bartholome, M. Follo, J. Timmer, P. Fisch, and H. Veelken. 2011. Definition and characterization of the systemic T-cell dysregulation in untreated indolent B-cell lymphoma and very early CLL. Blood 117: 3836-3846.

178. Frydecka, I., A. Kosmaczewska, D. Bocko, L. Ciszak, D. Wolowiec, K. Kuliczkowski, and I. Kochanowska. 2004. Alterations of the expression of T-cell-related costimulatory CD28 and downregulatory CD152 (CTLA-4) molecules in

230

patients with B-cell chronic lymphocytic leukaemia. British journal of cancer 90: 2042-2048.

179. Motta, M., L. Rassenti, B. J. Shelvin, S. Lerner, T. J. Kipps, M. J. Keating, and W. G. Wierda. 2005. Increased expression of CD152 (CTLA-4) by normal T lymphocytes in untreated patients with B-cell chronic lymphocytic leukemia. Leukemia 19: 1788-1793.

180. Beyer, M., M. Kochanek, K. Darabi, A. Popov, M. Jensen, E. Endl, P. A. Knolle, R. K. Thomas, M. von Bergwelt-Baildon, S. Debey, M. Hallek, and J. L. Schultze. 2005. Reduced frequencies and suppressive function of CD4+CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine. Blood 106: 2018-2025.

181. D'Arena, G., L. Laurenti, M. M. Minervini, S. Deaglio, L. Bonello, L. De Martino, L. De Padua, L. Savino, M. Tarnani, V. De Feo, and N. Cascavilla. 2011. Regulatory T-cell number is increased in chronic lymphocytic leukemia patients and correlates with progressive disease. Leukemia research 35: 363-368.

182. Scrivener, S., E. R. Kaminski, A. Demaine, and A. G. Prentice. 2001. Analysis of the expression of critical activation/interaction markers on peripheral blood T cells in B-cell chronic lymphocytic leukaemia: evidence of immune dysregulation. British journal of haematology 112: 959-964.

183. Granziero, L., P. Ghia, P. Circosta, D. Gottardi, G. Strola, M. Geuna, L. Montagna, P. Piccoli, M. Chilosi, and F. Caligaris-Cappio. 2001. Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood 97: 2777-2783.

184. Tromp, J. M., S. H. Tonino, J. A. Elias, A. Jaspers, D. M. Luijks, A. P. Kater, R. A. van Lier, M. H. van Oers, and E. Eldering. 2010. Dichotomy in NF-kappaB signaling and chemoresistance in immunoglobulin variable heavy-chain-mutated versus unmutated CLL cells upon CD40/TLR9 triggering. Oncogene 29: 5071-5082.

185. Gricks, C. S., D. Zahrieh, A. J. Zauls, G. Gorgun, D. Drandi, K. Mauerer, D. Neuberg, and J. G. Gribben. 2004. Differential regulation of gene expression following CD40 activation of leukemic compared to healthy B cells. Blood 104: 4002-4009.

186. Binsky, I., M. Haran, D. Starlets, Y. Gore, F. Lantner, N. Harpaz, L. Leng, D. M. Goldenberg, L. Shvidel, A. Berrebi, R. Bucala, and I. Shachar. 2007. IL-8 secreted in a macrophage migration-inhibitory factor- and CD74-dependent manner regulates B cell chronic lymphocytic leukemia survival. Proceedings of the National Academy of Sciences of the United States of America 104: 13408-13413.

187. Luqman, M., S. Klabunde, K. Lin, G. V. Georgakis, A. Cherukuri, J. Holash, C. Goldbeck, X. Xu, E. E. Kadel, 3rd, S. H. Lee, S. L. Aukerman, B. Jallal, N. Aziz,

231

W. K. Weng, W. Wierda, S. O'Brien, and A. Younes. 2008. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells. Blood 112: 711-720.

188. Reittie, J. E., K. L. Yong, P. Panayiotidis, and A. V. Hoffbrand. 1996. Interleukin-6 inhibits apoptosis and tumour necrosis factor induced proliferation of B-chronic lymphocytic leukaemia. Leukemia & lymphoma 22: 83-90, follow 186, color plate VI.

189. Palacios, F., P. Moreno, P. Morande, C. Abreu, A. Correa, V. Porro, A. I. Landoni, R. Gabus, M. Giordano, G. Dighiero, O. Pritsch, and P. Oppezzo. 2010. High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease. Blood 115: 4488-4496.

190. Mainou-Fowler, T., S. Miller, S. J. Proctor, and A. M. Dickinson. 2001. The levels of TNF alpha, IL4 and IL10 production by T-cells in B-cell chronic lymphocytic leukaemia (B-CLL). Leukemia research 25: 157-163.

191. Mainou-Fowler, T., S. J. Proctor, S. Miller, and A. M. Dickinson. 2001. Expression and production of interleukin 4 in B-cell chronic lymphocytic leukaemia. Leukemia & lymphoma 42: 689-698.

192. Dancescu, M., M. Rubio-Trujillo, G. Biron, D. Bron, G. Delespesse, and M. Sarfati. 1992. Interleukin 4 protects chronic lymphocytic leukemic B cells from death by apoptosis and upregulates Bcl-2 expression. The Journal of experimental medicine 176: 1319-1326.

193. Steele, A. J., A. G. Prentice, K. Cwynarski, A. V. Hoffbrand, S. M. Hart, M. W. Lowdell, E. R. Samuel, and R. G. Wickremasinghe. 2010. The JAK3-selective inhibitor PF-956980 reverses the resistance to cytotoxic agents induced by interleukin-4 treatment of chronic lymphocytic leukemia cells: potential for reversal of cytoprotection by the microenvironment. Blood 116: 4569-4577.

194. Willimott, S., M. Baou, S. Huf, S. Deaglio, and S. D. Wagner. 2007. Regulation of CD38 in proliferating chronic lymphocytic leukemia cells stimulated with CD154 and interleukin-4. Haematologica 92: 1359-1366.

195. Ramsay, A. G., A. J. Johnson, A. M. Lee, G. Gorgun, R. Le Dieu, W. Blum, J. C. Byrd, and J. G. Gribben. 2008. Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug. The Journal of clinical investigation 118: 2427-2437.

196. Gorgun, G., T. A. Holderried, D. Zahrieh, D. Neuberg, and J. G. Gribben. 2005. Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells. The Journal of clinical investigation 115: 1797-1805.

232

197. Burger, J. A., M. P. Quiroga, E. Hartmann, A. Burkle, W. G. Wierda, M. J. Keating, and A. Rosenwald. 2009. High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation. Blood 113: 3050-3058.

198. Panayiotidis, P., D. Jones, K. Ganeshaguru, L. Foroni, and A. V. Hoffbrand. 1996. Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro. British journal of haematology 92: 97-103.

199. Shehata, M., S. Schnabl, D. Demirtas, M. Hilgarth, R. Hubmann, E. Ponath, S. Badrnya, C. Lehner, A. Hoelbl, M. Duechler, A. Gaiger, C. Zielinski, J. D. Schwarzmeier, and U. Jaeger. 2010. Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia. Blood 116: 2513-2521.

200. Balakrishnan, K., J. A. Burger, M. P. Quiroga, M. Henneberg, M. L. Ayres, W. G. Wierda, and V. Gandhi. 2010. Influence of bone marrow stromal microenvironment on forodesine-induced responses in CLL primary cells. Blood 116: 1083-1091.

201. Balakrishnan, K., J. A. Burger, W. G. Wierda, and V. Gandhi. 2009. AT-101 induces apoptosis in CLL B cells and overcomes stromal cell-mediated Mcl-1 induction and drug resistance. Blood 113: 149-153.

202. Pedersen, I. M., S. Kitada, L. M. Leoni, J. M. Zapata, J. G. Karras, N. Tsukada, T. J. Kipps, Y. S. Choi, F. Bennett, and J. C. Reed. 2002. Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1. Blood 100: 1795-1801.

203. Granziero, L., P. Circosta, C. Scielzo, E. Frisaldi, S. Stella, M. Geuna, S. Giordano, P. Ghia, and F. Caligaris-Cappio. 2003. CD100/Plexin-B1 interactions sustain proliferation and survival of normal and leukemic CD5+ B lymphocytes. Blood 101: 1962-1969.

204. Bhattacharya, N., S. Diener, I. S. Idler, J. Rauen, S. Habe, H. Busch, A. Habermann, T. Zenz, H. Dohner, S. Stilgenbauer, and D. Mertens. 2011. Nurse-like cells show deregulated expression of genes involved in immunocompetence. British journal of haematology 154: 349-356.

205. Ding, W., T. R. Knox, R. C. Tschumper, W. Wu, S. M. Schwager, J. C. Boysen, D. F. Jelinek, and N. E. Kay. 2010. Platelet-derived growth factor (PDGF)-PDGF receptor interaction activates bone marrow-derived mesenchymal stromal cells derived from chronic lymphocytic leukemia: implications for an angiogenic switch. Blood 116: 2984-2993.

206. Ding, W., G. S. Nowakowski, T. R. Knox, J. C. Boysen, M. L. Maas, S. M. Schwager, W. Wu, L. E. Wellik, A. B. Dietz, A. K. Ghosh, C. R. Secreto, K. L.

233

Medina, T. D. Shanafelt, C. S. Zent, T. G. Call, and N. E. Kay. 2009. Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression. British journal of haematology 147: 471-483.

207. Abrams, S. T., B. R. Brown, M. Zuzel, and J. R. Slupsky. 2010. Vascular endothelial growth factor stimulates protein kinase CbetaII expression in chronic lymphocytic leukemia cells. Blood 115: 4447-4454.

208. Burger, J. A., M. Burger, and T. J. Kipps. 1999. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood 94: 3658-3667.

209. Niedermeier, M., B. T. Hennessy, Z. A. Knight, M. Henneberg, J. Hu, A. V. Kurtova, W. G. Wierda, M. J. Keating, K. M. Shokat, and J. A. Burger. 2009. Isoform-selective phosphoinositide 3'-kinase inhibitors inhibit CXCR4 signaling and overcome stromal cell-mediated drug resistance in chronic lymphocytic leukemia: a novel therapeutic approach. Blood 113: 5549-5557.

210. Vaisitti, T., S. Aydin, D. Rossi, F. Cottino, L. Bergui, G. D'Arena, L. Bonello, A. L. Horenstein, P. Brennan, C. Pepper, G. Gaidano, F. Malavasi, and S. Deaglio. 2010. CD38 increases CXCL12-mediated signals and homing of chronic lymphocytic leukemia cells. Leukemia 24: 958-969.

211. Till, K. J., K. Lin, M. Zuzel, and J. C. Cawley. 2002. The chemokine receptor CCR7 and alpha4 integrin are important for migration of chronic lymphocytic leukemia cells into lymph nodes. Blood 99: 2977-2984.

212. Redondo-Munoz, J., M. Jose Terol, J. A. Garcia-Marco, and A. Garcia-Pardo. 2008. Matrix metalloproteinase-9 is up-regulated by CCL21/CCR7 interaction via extracellular signal-regulated kinase-1/2 signaling and is involved in CCL21-driven B-cell chronic lymphocytic leukemia cell invasion and migration. Blood 111: 383-386.

213. Burkle, A., M. Niedermeier, A. Schmitt-Graff, W. G. Wierda, M. J. Keating, and J. A. Burger. 2007. Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia. Blood 110: 3316-3325.

214. Wang, X., H. Yuling, J. Yanping, T. Xinti, Y. Yaofang, Y. Feng, X. Ruijin, W. Li, C. Lang, L. Jingyi, T. Zhiqing, O. Jingping, X. Bing, Q. Li, A. E. Chang, Z. Sun, J. Youxin, and T. Jinquan. 2007. CCL19 and CXCL13 synergistically regulate interaction between B cell acute lymphocytic leukemia CD23+CD5+ B Cells and CD8+ T cells. J Immunol 179: 2880-2888.

215. Gattei, V., P. Bulian, M. I. Del Principe, A. Zucchetto, L. Maurillo, F. Buccisano, R. Bomben, M. Dal-Bo, F. Luciano, F. M. Rossi, M. Degan, S. Amadori, and G. Del Poeta. 2008. Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia. Blood 111: 865-873.

234

216. Zucchetto, A., D. Benedetti, C. Tripodo, R. Bomben, M. Dal Bo, D. Marconi, F. Bossi, D. Lorenzon, M. Degan, F. M. Rossi, D. Rossi, P. Bulian, V. Franco, G. Del Poeta, S. Deaglio, G. Gaidano, F. Tedesco, F. Malavasi, and V. Gattei. 2009. CD38/CD31, the CCL3 and CCL4 chemokines, and CD49d/vascular cell adhesion molecule-1 are interchained by sequential events sustaining chronic lymphocytic leukemia cell survival. Cancer research 69: 4001-4009.

217. Binsky, I., F. Lantner, V. Grabovsky, N. Harpaz, L. Shvidel, A. Berrebi, D. M. Goldenberg, L. Leng, R. Bucala, R. Alon, M. Haran, and I. Shachar. 2010. TAp63 regulates VLA-4 expression and chronic lymphocytic leukemia cell migration to the bone marrow in a CD74-dependent manner. J Immunol 184: 4761-4769.

218. Redondo-Munoz, J., E. Escobar-Diaz, R. Samaniego, M. J. Terol, J. A. Garcia-Marco, and A. Garcia-Pardo. 2006. MMP-9 in B-cell chronic lymphocytic leukemia is up-regulated by alpha4beta1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration. Blood 108: 3143-3151.

219. Redondo-Munoz, J., E. Ugarte-Berzal, J. A. Garcia-Marco, M. H. del Cerro, P. E. Van den Steen, G. Opdenakker, M. J. Terol, and A. Garcia-Pardo. 2008. Alpha4beta1 integrin and 190-kDa CD44v constitute a cell surface docking complex for gelatinase B/MMP-9 in chronic leukemic but not in normal B cells. Blood 112: 169-178.

220. Redondo-Munoz, J., E. Ugarte-Berzal, M. J. Terol, P. E. Van den Steen, M. Hernandez del Cerro, M. Roderfeld, E. Roeb, G. Opdenakker, J. A. Garcia-Marco, and A. Garcia-Pardo. 2010. Matrix metalloproteinase-9 promotes chronic lymphocytic leukemia b cell survival through its hemopexin domain. Cancer cell 17: 160-172.

221. Santanam, U., N. Zanesi, A. Efanov, S. Costinean, A. Palamarchuk, J. P. Hagan, S. Volinia, H. Alder, L. Rassenti, T. Kipps, C. M. Croce, and Y. Pekarsky. 2010. Chronic lymphocytic leukemia modeled in mouse by targeted miR-29 expression. Proceedings of the National Academy of Sciences of the United States of America 107: 12210-12215.

222. Dearden, C. E. 2006. T-cell prolymphocytic leukemia. Medical oncology (Northwood, London, England) 23: 17-22.

223. Pekarsky, Y., C. Hallas, and C. M. Croce. 2001. The role of TCL1 in human T-cell leukemia. Oncogene 20: 5638-5643.

224. Bichi, R., S. A. Shinton, E. S. Martin, A. Koval, G. A. Calin, R. Cesari, G. Russo, R. R. Hardy, and C. M. Croce. 2002. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proceedings of the National Academy of Sciences of the United States of America 99: 6955-6960.

235

225. Yan, X. J., E. Albesiano, N. Zanesi, S. Yancopoulos, A. Sawyer, E. Romano, A. Petlickovski, D. G. Efremov, C. M. Croce, and N. Chiorazzi. 2006. B cell receptors in TCL1 transgenic mice resemble those of aggressive, treatment-resistant human chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America 103: 11713-11718.

226. Johnson, A. J., D. M. Lucas, N. Muthusamy, L. L. Smith, R. B. Edwards, M. D. De Lay, C. M. Croce, M. R. Grever, and J. C. Byrd. 2006. Characterization of the TCL-1 transgenic mouse as a preclinical drug development tool for human chronic lymphocytic leukemia. Blood 108: 1334-1338.

227. Gorgun, G., A. G. Ramsay, T. A. Holderried, D. Zahrieh, R. Le Dieu, F. Liu, J. Quackenbush, C. M. Croce, and J. G. Gribben. 2009. E(mu)-TCL1 mice represent a model for immunotherapeutic reversal of chronic lymphocytic leukemia-induced T-cell dysfunction. Proceedings of the National Academy of Sciences of the United States of America 106: 6250-6255.

228. Hofbauer, J. P., C. Heyder, U. Denk, T. Kocher, C. Holler, D. Trapin, D. Asslaber, I. Tinhofer, R. Greil, and A. Egle. 2011. Development of CLL in the TCL1 transgenic mouse model is associated with severe skewing of the T-cell compartment homologous to human CLL. Leukemia 25: 1452-1458.

229. Kobayashi, R., G. Picchio, M. Kirven, G. Meisenholder, S. Baird, D. A. Carson, D. E. Mosier, and T. J. Kipps. 1992. Transfer of human chronic lymphocytic leukemia to mice with severe combined immune deficiency. Leukemia research 16: 1013-1023.

230. Hummel, J. L., B. D. Lichty, M. Reis, I. Dube, and S. Kamel-Reid. 1996. Engraftment of human chronic lymphocytic leukemia cells in SCID mice: in vivo and in vitro studies. Leukemia 10: 1370-1376.

231. Marcus, H., A. Shimoni, D. Ergas, A. Canaan, B. Dekel, D. Ben-David, M. David, E. Sigler, Y. Reisner, and A. Berrebi. 1997. Human/mouse radiation chimera generated from PBMC of B chronic lymphocytic leukemia patients with autoimmune hemolytic anemia produce anti-human red cell antibodies. Leukemia 11: 687-693.

232. Shimoni, A., H. Marcus, A. Canaan, D. Ergas, M. David, A. Berrebi, and Y. Reisner. 1997. A model for human B-chronic lymphocytic leukemia in human/mouse radiation chimera: evidence for tumor-mediated suppression of antibody production in low-stage disease. Blood 89: 2210-2218.

233. Shimoni, A., H. Marcus, B. Dekel, R. Shkarchi, F. Arditti, L. Shvidel, M. Shtalrid, W. Bucher, A. Canaan, D. Ergas, A. Berrebi, and Y. Reisner. 1999. Autologous T cells control B-chronic lymphocytic leukemia tumor progression in human-->mouse radiation chimera. Cancer research 59: 5968-5974.

236

234. Durig, J., P. Ebeling, F. Grabellus, U. R. Sorg, M. Mollmann, P. Schutt, J. Gothert, L. Sellmann, S. Seeber, M. Flasshove, U. Duhrsen, and T. Moritz. 2007. A novel nonobese diabetic/severe combined immunodeficient xenograft model for chronic lymphocytic leukemia reflects important clinical characteristics of the disease. Cancer research 67: 8653-8661.

235. McDermott, S. P., K. Eppert, E. R. Lechman, M. Doedens, and J. E. Dick. 2010. Comparison of human cord blood engraftment between immunocompromised mouse strains. Blood 116: 193-200.

236. Bagnara, D., M. S. Kaufman, C. Calissano, S. Marsilio, P. E. Patten, R. Simone, P. Chum, X. J. Yan, S. L. Allen, J. E. Kolitz, S. Baskar, C. Rader, H. Mellstedt, H. Rabbani, A. Lee, P. K. Gregersen, K. R. Rai, and N. Chiorazzi. 2011. A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease. Blood 117: 5463-5472.

237. Loisel, S., K. L. Ster, I. Quintin-Roue, J. O. Pers, A. Bordron, P. Youinou, and C. Berthou. 2005. Establishment of a novel human B-CLL-like xenograft model in nude mouse. Leukemia research 29: 1347-1352.

238. Bertilaccio, M. T., C. Scielzo, G. Simonetti, M. Ponzoni, B. Apollonio, C. Fazi, L. Scarfo, M. Rocchi, M. Muzio, F. Caligaris-Cappio, and P. Ghia. 2010. A novel Rag2-/-gammac-/--xenograft model of human CLL. Blood 115: 1605-1609.

239. Nabhan, C., S. Coutre, and P. Hillmen. 2007. Minimal residual disease in chronic lymphocytic leukaemia: is it ready for primetime? British journal of haematology 136: 379-392.

240. Tam, C. S., and M. J. Keating. 2010. Chemoimmunotherapy of chronic lymphocytic leukemia. Nature reviews. Clinical oncology 7: 521-532.

241. Jaglowski, S. M., L. Alinari, R. Lapalombella, N. Muthusamy, and J. C. Byrd. 2010. The clinical application of monoclonal antibodies in chronic lymphocytic leukemia. Blood 116: 3705-3714.

242. Weiner, L. M., R. Surana, and S. Wang. 2010. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nature reviews 10: 317-327.

243. Schlette, E. J., J. Admirand, W. Wierda, L. Abruzzo, K. I. Lin, S. O'Brien, S. Lerner, M. J. Keating, and C. Tam. 2009. p53 expression by immunohistochemistry is an important determinant of survival in patients with chronic lymphocytic leukemia receiving frontline chemo-immunotherapy. Leukemia & lymphoma 50: 1597-1605.

244. Ziegler, H. W., N. E. Kay, and J. M. Zarling. 1981. Deficiency of natural killer cell activity in patients with chronic lymphocytic leukemia. International journal of cancer 27: 321-327.

237

245. Chan, A. C., and P. J. Carter. 2010. Therapeutic antibodies for autoimmunity and inflammation. Nature reviews 10: 301-316.

246. Zitvogel, L., O. Kepp, and G. Kroemer. 2011. Immune parameters affecting the efficacy of chemotherapeutic regimens. Nature reviews. Clinical oncology 8: 151-160.

247. Obeid, M., A. Tesniere, F. Ghiringhelli, G. M. Fimia, L. Apetoh, J. L. Perfettini, M. Castedo, G. Mignot, T. Panaretakis, N. Casares, D. Metivier, N. Larochette, P. van Endert, F. Ciccosanti, M. Piacentini, L. Zitvogel, and G. Kroemer. 2007. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nature medicine 13: 54-61.

248. Green, D. R., T. Ferguson, L. Zitvogel, and G. Kroemer. 2009. Immunogenic and tolerogenic cell death. Nature reviews 9: 353-363.

249. Martins, I., O. Kepp, F. Schlemmer, S. Adjemian, M. Tailler, S. Shen, M. Michaud, L. Menger, A. Gdoura, N. Tajeddine, A. Tesniere, L. Zitvogel, and G. Kroemer. 2011. Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress. Oncogene 30: 1147-1158.

250. Apetoh, L., F. Ghiringhelli, A. Tesniere, M. Obeid, C. Ortiz, A. Criollo, G. Mignot, M. C. Maiuri, E. Ullrich, P. Saulnier, H. Yang, S. Amigorena, B. Ryffel, F. J. Barrat, P. Saftig, F. Levi, R. Lidereau, C. Nogues, J. P. Mira, A. Chompret, V. Joulin, F. Clavel-Chapelon, J. Bourhis, F. Andre, S. Delaloge, T. Tursz, G. Kroemer, and L. Zitvogel. 2007. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nature medicine 13: 1050-1059.

251. Ghiringhelli, F., L. Apetoh, A. Tesniere, L. Aymeric, Y. Ma, C. Ortiz, K. Vermaelen, T. Panaretakis, G. Mignot, E. Ullrich, J. L. Perfettini, F. Schlemmer, E. Tasdemir, M. Uhl, P. Genin, A. Civas, B. Ryffel, J. Kanellopoulos, J. Tschopp, F. Andre, R. Lidereau, N. M. McLaughlin, N. M. Haynes, M. J. Smyth, G. Kroemer, and L. Zitvogel. 2009. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors. Nature medicine 15: 1170-1178.

252. Piper, K. P., M. Karanth, A. McLarnon, E. Kalk, N. Khan, J. Murray, G. Pratt, and P. A. Moss. 2011. Chronic lymphocytic leukaemia cells drive the global CD4+ T cell repertoire towards a regulatory phenotype and leads to the accumulation of CD4+ forkhead box P3+ T cells. Clinical and experimental immunology 166: 154-163.

253. Zitvogel, L., L. Apetoh, F. Ghiringhelli, and G. Kroemer. 2008. Immunological aspects of cancer chemotherapy. Nature reviews 8: 59-73.

238

254. Carballido, E., M. Veliz, R. Komrokji, and J. Pinilla-Ibarz. 2012. Immunomodulatory drugs and active immunotherapy for chronic lymphocytic leukemia. Cancer Control 19: 54-67.

255. Wierda, W. G., M. J. Cantwell, S. J. Woods, L. Z. Rassenti, C. E. Prussak, and T. J. Kipps. 2000. CD40-ligand (CD154) gene therapy for chronic lymphocytic leukemia. Blood 96: 2917-2924.

256. Biagi, E., R. Rousseau, E. Yvon, M. Schwartz, G. Dotti, A. Foster, D. Havlik-Cooper, B. Grilley, A. Gee, K. Baker, G. Carrum, L. Rice, M. Andreeff, U. Popat, and M. Brenner. 2005. Responses to human CD40 ligand/human interleukin-2 autologous cell vaccine in patients with B-cell chronic lymphocytic leukemia. Clin Cancer Res 11: 6916-6923.

257. Foster, A. E., F. V. Okur, E. Biagi, A. Lu, G. Dotti, E. Yvon, B. Savoldo, G. Carrum, M. A. Goodell, H. E. Heslop, and M. K. Brenner. 2010. Selective elimination of a chemoresistant side population of B-CLL cells by cytotoxic T lymphocytes in subjects receiving an autologous hCD40L/IL-2 tumor vaccine. Leukemia 24: 563-572.

258. Spaner, D. E., Y. Shi, D. White, J. Mena, C. Hammond, J. Tomic, L. He, M. A. Tomai, R. L. Miller, J. Booth, and L. Radvanyi. 2006. Immunomodulatory effects of Toll-like receptor-7 activation on chronic lymphocytic leukemia cells. Leukemia 20: 286-295.

259. Tomic, J., D. White, Y. Shi, J. Mena, C. Hammond, L. He, R. L. Miller, and D. E. Spaner. 2006. Sensitization of IL-2 signaling through TLR-7 enhances B lymphoma cell immunogenicity. J Immunol 176: 3830-3839.

260. Spaner, D. E., Y. Shi, D. White, S. Shaha, L. He, A. Masellis, K. Wong, and R. Gorczynski. 2010. A phase I/II trial of TLR-7 agonist immunotherapy in chronic lymphocytic leukemia. Leukemia 24: 222-226.

261. Hollyman, D., J. Stefanski, M. Przybylowski, S. Bartido, O. Borquez-Ojeda, C. Taylor, R. Yeh, V. Capacio, M. Olszewska, J. Hosey, M. Sadelain, R. J. Brentjens, and I. Riviere. 2009. Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy. J Immunother 32: 169-180.

262. Brentjens, R. J., I. Riviere, J. H. Park, M. L. Davila, X. Wang, J. Stefanski, C. Taylor, R. Yeh, S. Bartido, O. Borquez-Ojeda, M. Olszewska, Y. Bernal, H. Pegram, M. Przybylowski, D. Hollyman, Y. Usachenko, D. Pirraglia, J. Hosey, E. Santos, E. Halton, P. Maslak, D. Scheinberg, J. Jurcic, M. Heaney, G. Heller, M. Frattini, and M. Sadelain. 2011. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 118: 4817-4828.

239

263. Giordano Attianese, G. M., V. Marin, V. Hoyos, B. Savoldo, I. Pizzitola, S. Tettamanti, V. Agostoni, M. Parma, M. Ponzoni, M. T. Bertilaccio, P. Ghia, A. Biondi, G. Dotti, and E. Biagi. 2011. In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor. Blood 117: 4736-4745.

264. Porter, D. L., B. L. Levine, M. Kalos, A. Bagg, and C. H. June. 2011. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. The New England journal of medicine 365: 725-733.

265. Hanahan, D., and R. A. Weinberg. 2011. Hallmarks of cancer: the next generation. Cell 144: 646-674.

266. Schreiber, R. D., L. J. Old, and M. J. Smyth. 2011. Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion. Science (New York, N.Y 331: 1565-1570.

267. Zou, W., and L. Chen. 2008. Inhibitory B7-family molecules in the tumour microenvironment. Nature reviews 8: 467-477.

268. Matsuzaki, J., S. Gnjatic, P. Mhawech-Fauceglia, A. Beck, A. Miller, T. Tsuji, C. Eppolito, F. Qian, S. Lele, P. Shrikant, L. J. Old, and K. Odunsi. 2010. Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer. Proceedings of the National Academy of Sciences of the United States of America 107: 7875-7880.

269. Derre, L., J. P. Rivals, C. Jandus, S. Pastor, D. Rimoldi, P. Romero, O. Michielin, D. Olive, and D. E. Speiser. 2010. BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination. The Journal of clinical investigation 120: 157-167.

270. Fourcade, J., Z. Sun, O. Pagliano, P. Guillaume, I. F. Luescher, C. Sander, J. M. Kirkwood, D. Olive, V. Kuchroo, and H. M. Zarour. 2012. CD8(+) T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1. Cancer research 72: 887-896.

271. Brahmer, J. R., C. G. Drake, I. Wollner, J. D. Powderly, J. Picus, W. H. Sharfman, E. Stankevich, A. Pons, T. M. Salay, T. L. McMiller, M. M. Gilson, C. Wang, M. Selby, J. M. Taube, R. Anders, L. Chen, A. J. Korman, D. M. Pardoll, I. Lowy, and S. L. Topalian. 2010. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J Clin Oncol 28: 3167-3175.

272. Robert, C., L. Thomas, I. Bondarenko, S. O'Day, D. J. M, C. Garbe, C. Lebbe, J. F. Baurain, A. Testori, J. J. Grob, N. Davidson, J. Richards, M. Maio, A. Hauschild, W. H. Miller, Jr., P. Gascon, M. Lotem, K. Harmankaya, R. Ibrahim, S. Francis, T. T. Chen, R. Humphrey, A. Hoos, and J. D. Wolchok. 2011. Ipilimumab plus

240

dacarbazine for previously untreated metastatic melanoma. The New England journal of medicine 364: 2517-2526.

273. Yuan, J., M. Adamow, B. A. Ginsberg, T. S. Rasalan, E. Ritter, H. F. Gallardo, Y. Xu, E. Pogoriler, S. L. Terzulli, D. Kuk, K. S. Panageas, G. Ritter, M. Sznol, R. Halaban, A. A. Jungbluth, J. P. Allison, L. J. Old, J. D. Wolchok, and S. Gnjatic. 2011. Integrated NY-ESO-1 antibody and CD8+ T-cell responses correlate with clinical benefit in advanced melanoma patients treated with ipilimumab. Proceedings of the National Academy of Sciences of the United States of America 108: 16723-16728.

274. Barclay, A. N., M. J. Clark, and G. W. McCaughan. 1986. Neuronal/lymphoid membrane glycoprotein MRC OX-2 is a member of the immunoglobulin superfamily with a light-chain-like structure. Biochemical Society symposium 51: 149-157.

275. Gorczynski, R. M. 2005. CD200 and its receptors as targets for immunoregulation. Curr Opin Investig Drugs 6: 483-488.

276. Wright, G. J., H. Cherwinski, M. Foster-Cuevas, G. Brooke, M. J. Puklavec, M. Bigler, Y. Song, M. Jenmalm, D. Gorman, T. McClanahan, M. R. Liu, M. H. Brown, J. D. Sedgwick, J. H. Phillips, and A. N. Barclay. 2003. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol 171: 3034-3046.

277. Gorczynski, R., Z. Chen, Y. Kai, L. Lee, S. Wong, and P. A. Marsden. 2004. CD200 is a ligand for all members of the CD200R family of immunoregulatory molecules. J Immunol 172: 7744-7749.

278. Rijkers, E. S., T. de Ruiter, A. Baridi, H. Veninga, R. M. Hoek, and L. Meyaard. 2008. The inhibitory CD200R is differentially expressed on human and mouse T and B lymphocytes. Molecular immunology 45: 1126-1135.

279. Gorczynski, R. M., J. Hu, Z. Chen, Y. Kai, and J. Lei. 2002. A CD200FC immunoadhesin prolongs rat islet xenograft survival in mice. Transplantation 73: 1948-1953.

280. Gorczynski, R. M., Z. Chen, K. Yu, and J. Hu. 2001. CD200 immunoadhesin suppresses collagen-induced arthritis in mice. Clinical immunology (Orlando, Fla 101: 328-334.

281. Gorczynski, R. M., Z. Chen, J. Hu, Y. Kai, and J. Lei. 2001. Evidence of a role for CD200 in regulation of immune rejection of leukaemic tumour cells in C57BL/6 mice. Clinical and experimental immunology 126: 220-229.

282. Hoek, R. M., S. R. Ruuls, C. A. Murphy, G. J. Wright, R. Goddard, S. M. Zurawski, B. Blom, M. E. Homola, W. J. Streit, M. H. Brown, A. N. Barclay, and J. D.

241

Sedgwick. 2000. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science (New York, N.Y 290: 1768-1771.

283. Gorczynski, R. M., Z. Chen, W. He, I. Khatri, Y. Sun, K. Yu, and I. Boudakov. 2009. Expression of a CD200 transgene is necessary for induction but not maintenance of tolerance to cardiac and skin allografts. J Immunol 183: 1560-1568.

284. Gorczynski, R. M., Z. Chen, I. Khatri, and K. Yu. 2011. Graft-infiltrating cells expressing a CD200 transgene prolong allogeneic skin graft survival in association with local increases in Foxp3(+)Treg and mast cells. Transplant immunology 25: 187-193.

285. Gorczynski, R., I. Khatri, L. Lee, and I. Boudakov. 2008. An interaction between CD200 and monoclonal antibody agonists to CD200R2 in development of dendritic cells that preferentially induce populations of CD4+CD25+ T regulatory cells. J Immunol 180: 5946-5955.

286. Costello, D. A., A. Lyons, S. Denieffe, T. C. Browne, F. F. Cox, and M. A. Lynch. 2011. Long term potentiation is impaired in membrane glycoprotein CD200-deficient mice: a role for Toll-like receptor activation. The Journal of biological chemistry 286: 34722-34732.

287. Wang, X. J., S. Zhang, Z. Q. Yan, Y. X. Zhao, H. Y. Zhou, Y. Wang, G. Q. Lu, and J. D. Zhang. 2011. Impaired CD200-CD200R-mediated microglia silencing enhances midbrain dopaminergic neurodegeneration: roles of aging, superoxide, NADPH oxidase, and p38 MAPK. Free radical biology & medicine 50: 1094-1106.

288. Foster-Cuevas, M., G. J. Wright, M. J. Puklavec, M. H. Brown, and A. N. Barclay. 2004. Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor. Journal of virology 78: 7667-7676.

289. Foster-Cuevas, M., T. Westerholt, M. Ahmed, M. H. Brown, A. N. Barclay, and S. Voigt. 2011. Cytomegalovirus e127 protein interacts with the inhibitory CD200 receptor. Journal of virology 85: 6055-6059.

290. Moreaux, J., D. Hose, T. Reme, E. Jourdan, M. Hundemer, E. Legouffe, P. Moine, P. Bourin, M. Moos, J. Corre, T. Mohler, J. De Vos, J. F. Rossi, H. Goldschmidt, and B. Klein. 2006. CD200 is a new prognostic factor in multiple myeloma. Blood 108: 4194-4197.

291. Tonks, A., R. Hills, P. White, B. Rosie, K. I. Mills, A. K. Burnett, and R. L. Darley. 2007. CD200 as a prognostic factor in acute myeloid leukaemia. Leukemia 21: 566-568.

292. Coles, S. J., E. C. Wang, S. Man, R. K. Hills, A. K. Burnett, A. Tonks, and R. L. Darley. 2011. CD200 expression suppresses natural killer cell function and directly inhibits patient anti-tumor response in acute myeloid leukemia. Leukemia 25: 792-799.

242

293. Coustan-Smith, E., G. Song, C. Clark, L. Key, P. Liu, M. Mehrpooya, P. Stow, X. Su, S. Shurtleff, C. H. Pui, J. R. Downing, and D. Campana. 2011. New markers for minimal residual disease detection in acute lymphoblastic leukemia. Blood 117: 6267-6276.

294. Siva, A., H. Xin, F. Qin, D. Oltean, K. S. Bowdish, and A. Kretz-Rommel. 2008. Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200. Cancer Immunol Immunother 57: 987-996.

295. Petermann, K. B., G. I. Rozenberg, D. Zedek, P. Groben, K. McKinnon, C. Buehler, W. Y. Kim, J. M. Shields, S. Penland, J. E. Bear, N. E. Thomas, J. S. Serody, and N. E. Sharpless. 2007. CD200 is induced by ERK and is a potential therapeutic target in melanoma. The Journal of clinical investigation 117: 3922-3929.

296. Kawasaki, B. T., and W. L. Farrar. 2008. Cancer stem cells, CD200 and immunoevasion. Trends in immunology 29: 464-468.

297. Kawasaki, B. T., T. Mistree, E. M. Hurt, M. Kalathur, and W. L. Farrar. 2007. Co-expression of the toleragenic glycoprotein, CD200, with markers for cancer stem cells. Biochemical and biophysical research communications 364: 778-782.

298. Gorczynski, R. M., Z. Chen, J. Diao, I. Khatri, K. Wong, K. Yu, and J. Behnke. 2010. Breast cancer cell CD200 expression regulates immune response to EMT6 tumor cells in mice. Breast cancer research and treatment 123: 405-415.

299. Rygiel, T. P., G. Karnam, G. Goverse, A. P. van der Marel, M. J. Greuter, R. A. van Schaarenburg, W. F. Visser, A. B. Brenkman, R. Molenaar, R. M. Hoek, R. E. Mebius, and L. Meyaard. 2011. CD200-CD200R signaling suppresses anti-tumor responses independently of CD200 expression on the tumor. Oncogene.

300. Gorczynski, R. M., D. A. Clark, N. Erin, and I. Khatri. 2011. Role of CD200 expression in regulation of metastasis of EMT6 tumor cells in mice. Breast cancer research and treatment 130: 49-60.

301. Stumpfova, M., D. Ratner, E. B. Desciak, Y. D. Eliezri, and D. M. Owens. 2010. The immunosuppressive surface ligand CD200 augments the metastatic capacity of squamous cell carcinoma. Cancer research 70: 2962-2972.

302. Huovila, A. P., A. J. Turner, M. Pelto-Huikko, I. Karkkainen, and R. M. Ortiz. 2005. Shedding light on ADAM metalloproteinases. Trends in biochemical sciences 30: 413-422.

303. Fortini, M. E. 2002. Gamma-secretase-mediated proteolysis in cell-surface-receptor signalling. Nat Rev Mol Cell Biol 3: 673-684.

304. Murphy, G. 2008. The ADAMs: signalling scissors in the tumour microenvironment. Nature reviews. Cancer 8: 929-941.

243

305. Su, G., S. A. Blaine, D. Qiao, and A. Friedl. 2008. Membrane type 1 matrix metalloproteinase-mediated stromal syndecan-1 shedding stimulates breast carcinoma cell proliferation. Cancer research 68: 9558-9565.

306. Vaisar, T., S. Y. Kassim, I. G. Gomez, P. S. Green, S. Hargarten, P. J. Gough, W. C. Parks, C. L. Wilson, E. W. Raines, and J. W. Heinecke. 2009. MMP-9 sheds the beta2 integrin subunit (CD18) from macrophages. Mol Cell Proteomics 8: 1044-1060.

307. Chetty, C., S. K. Vanamala, C. S. Gondi, D. H. Dinh, M. Gujrati, and J. S. Rao. 2012. MMP-9 induces CD44 cleavage and CD44 mediated cell migration in glioblastoma xenograft cells. Cellular signalling 24: 549-559.

308. Richens, J., L. Fairclough, A. M. Ghaemmaghami, J. Mahdavi, F. Shakib, and H. F. Sewell. 2007. The detection of ADAM8 protein on cells of the human immune system and the demonstration of its expression on peripheral blood B cells, dendritic cells and monocyte subsets. Immunobiology 212: 29-38.

309. Chaimowitz, N. S., R. K. Martin, J. Cichy, D. R. Gibb, P. Patil, D. J. Kang, J. Farnsworth, E. C. Butcher, B. McCright, and D. H. Conrad. 2011. A disintegrin and metalloproteinase 10 regulates antibody production and maintenance of lymphoid architecture. J Immunol 187: 5114-5122.

310. Li, N., K. Boyd, P. J. Dempsey, and D. A. Vignali. 2007. Non-cell autonomous expression of TNF-alpha-converting enzyme ADAM17 is required for normal lymphocyte development. J Immunol 178: 4214-4221.

311. McGinn, O. J., W. R. English, S. Roberts, A. Ager, P. Newham, and G. Murphy. 2011. Modulation of integrin alpha4beta1 by ADAM28 promotes lymphocyte adhesion and transendothelial migration. Cell biology international 35: 1043-1053.

312. Gibb, D. R., M. El Shikh, D. J. Kang, W. J. Rowe, R. El Sayed, J. Cichy, H. Yagita, J. G. Tew, P. J. Dempsey, H. C. Crawford, and D. H. Conrad. 2010. ADAM10 is essential for Notch2-dependent marginal zone B cell development and CD23 cleavage in vivo. The Journal of experimental medicine 207: 623-635.

313. Bozkulak, E. C., and G. Weinmaster. 2009. Selective use of ADAM10 and ADAM17 in activation of Notch1 signaling. Molecular and cellular biology 29: 5679-5695.

314. Sulis, M. L., P. Saftig, and A. A. Ferrando. 2011. Redundancy and specificity of the metalloprotease system mediating oncogenic NOTCH1 activation in T-ALL. Leukemia 25: 1564-1569.

315. Shukla, C. J., C. J. Pennington, A. C. Riddick, K. K. Sethia, R. Y. Ball, and D. R. Edwards. 2008. Laser-capture microdissection in prostate cancer research: establishment and validation of a powerful tool for the assessment of tumour-stroma interactions. BJU international 101: 765-774.

244

316. Reiss, K., and P. Saftig. 2009. The "a disintegrin and metalloprotease" (ADAM) family of sheddases: physiological and cellular functions. Seminars in cell & developmental biology 20: 126-137.

317. Lee, S. B., A. Schramme, K. Doberstein, R. Dummer, M. S. Abdel-Bakky, S. Keller, P. Altevogt, S. T. Oh, J. Reichrath, D. Oxmann, J. Pfeilschifter, D. Mihic-Probst, and P. Gutwein. 2010. ADAM10 is upregulated in melanoma metastasis compared with primary melanoma. The Journal of investigative dermatology 130: 763-773.

318. Zubel, A., C. Flechtenmacher, L. Edler, and A. Alonso. 2009. Expression of ADAM9 in CIN3 lesions and squamous cell carcinomas of the cervix. Gynecologic oncology 114: 332-336.

319. Oh, S. T., A. Schramme, A. Stark, W. Tilgen, P. Gutwein, and J. Reichrath. 2009. The disintegrin-metalloproteinases ADAM 10, 12 and 17 are upregulated in invading peripheral tumor cells of basal cell carcinomas. Journal of cutaneous pathology 36: 395-401.

320. Gavert, N., M. Sheffer, S. Raveh, S. Spaderna, M. Shtutman, T. Brabletz, F. Barany, P. Paty, D. Notterman, E. Domany, and A. Ben-Ze'ev. 2007. Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis. Cancer research 67: 7703-7712.

321. Shintani, Y., S. Higashiyama, M. Ohta, H. Hirabayashi, S. Yamamoto, T. Yoshimasu, H. Matsuda, and N. Matsuura. 2004. Overexpression of ADAM9 in non-small cell lung cancer correlates with brain metastasis. Cancer research 64: 4190-4196.

322. Al-Fakhri, N., J. Wilhelm, M. Hahn, M. Heidt, F. W. Hehrlein, A. M. Endisch, T. Hupp, S. M. Cherian, Y. V. Bobryshev, R. S. Lord, and N. Katz. 2003. Increased expression of disintegrin-metalloproteinases ADAM-15 and ADAM-9 following upregulation of integrins alpha5beta1 and alphavbeta3 in atherosclerosis. Journal of cellular biochemistry 89: 808-823.

323. Buckley, C. A., F. N. Rouhani, M. Kaler, B. Adamik, F. I. Hawari, and S. J. Levine. 2005. Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch. American journal of physiology. Lung cellular and molecular physiology 288: L1132-1138.

324. Schlondorff, J., J. D. Becherer, and C. P. Blobel. 2000. Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). The Biochemical journal 347 Pt 1: 131-138.

325. Killock, D. J., and A. Ivetic. 2010. The cytoplasmic domains of TNFalpha-converting enzyme (TACE/ADAM17) and L-selectin are regulated differently by p38 MAPK and PKC to promote ectodomain shedding. The Biochemical journal 428: 293-304.

245

326. Abrams, S. T., T. Lakum, K. Lin, G. M. Jones, A. T. Treweeke, M. Farahani, M. Hughes, M. Zuzel, and J. R. Slupsky. 2007. B-cell receptor signaling in chronic lymphocytic leukemia cells is regulated by overexpressed active protein kinase CbetaII. Blood 109: 1193-1201.

327. Mackay, H. J., and C. J. Twelves. 2007. Targeting the protein kinase C family: are we there yet? Nature reviews. Cancer 7: 554-562.

328. Overall, C. M., and C. P. Blobel. 2007. In search of partners: linking extracellular proteases to substrates. Nat Rev Mol Cell Biol 8: 245-257.

329. Sanderson, M. P., S. N. Erickson, P. J. Gough, K. J. Garton, P. T. Wille, E. W. Raines, A. J. Dunbar, and P. J. Dempsey. 2005. ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx. The Journal of biological chemistry 280: 1826-1837.

330. Litterst, C., A. Georgakopoulos, J. Shioi, E. Ghersi, T. Wisniewski, R. Wang, A. Ludwig, and N. K. Robakis. 2007. Ligand binding and calcium influx induce distinct ectodomain/gamma-secretase-processing pathways of EphB2 receptor. The Journal of biological chemistry 282: 16155-16163.

331. Le Gall, S. M., P. Bobe, K. Reiss, K. Horiuchi, X. D. Niu, D. Lundell, D. R. Gibb, D. Conrad, P. Saftig, and C. P. Blobel. 2009. ADAMs 10 and 17 represent differentially regulated components of a general shedding machinery for membrane proteins such as transforming growth factor alpha, L-selectin, and tumor necrosis factor alpha. Molecular biology of the cell 20: 1785-1794.

332. Fournier, S., M. Rubio, G. Delespesse, and M. Sarfati. 1994. Role for low-affinity receptor for IgE (CD23) in normal and leukemic B-cell proliferation. Blood 84: 1881-1886.

333. Hubmann, R., J. D. Schwarzmeier, M. Shehata, M. Hilgarth, M. Duechler, M. Dettke, and R. Berger. 2002. Notch2 is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia. Blood 99: 3742-3747.

334. Duechler, M., M. Shehata, J. D. Schwarzmeier, A. Hoelbl, M. Hilgarth, and R. Hubmann. 2005. Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2. Leukemia 19: 260-267.

335. Knauf, W. U., B. Ehlers, B. Mohr, E. Thiel, I. Langenmayer, M. Hallek, B. Emmerich, D. Adorf, C. Nerl, and T. Zwingers. 1997. Prognostic impact of the serum levels of soluble CD23 in B-cell chronic lymphocytic leukemia. Blood 89: 4241-4242.

336. Meuleman, N., B. Stamatopoulos, M. Dejeneffe, H. El Housni, L. Lagneaux, and D. Bron. 2008. Doubling time of soluble CD23: a powerful prognostic factor for newly

246

diagnosed and untreated stage A chronic lymphocytic leukemia patients. Leukemia 22: 1882-1890.

337. Fourie, A. M., F. Coles, V. Moreno, and L. Karlsson. 2003. Catalytic activity of ADAM8, ADAM15, and MDC-L (ADAM28) on synthetic peptide substrates and in ectodomain cleavage of CD23. The Journal of biological chemistry 278: 30469-30477.

338. Lemieux, G. A., F. Blumenkron, N. Yeung, P. Zhou, J. Williams, A. C. Grammer, R. Petrovich, P. E. Lipsky, M. L. Moss, and Z. Werb. 2007. The low affinity IgE receptor (CD23) is cleaved by the metalloproteinase ADAM10. The Journal of biological chemistry 282: 14836-14844.

339. Chen, J. R., B. J. Gu, L. P. Dao, C. J. Bradley, S. P. Mulligan, and J. S. Wiley. 1999. Transendothelial migration of lymphocytes in chronic lymphocytic leukaemia is impaired and involved down-regulation of both L-selectin and CD23. British journal of haematology 105: 181-189.

340. Li, Y., J. Brazzell, A. Herrera, and B. Walcheck. 2006. ADAM17 deficiency by mature neutrophils has differential effects on L-selectin shedding. Blood 108: 2275-2279.

341. Palumbo, G. A., N. Parrinello, G. Fargione, K. Cardillo, A. Chiarenza, S. Berretta, C. Conticello, L. Villari, and F. Di Raimondo. 2009. CD200 expression may help in differential diagnosis between mantle cell lymphoma and B-cell chronic lymphocytic leukemia. Leukemia research 33: 1212-1216.

342. El Desoukey, N. A., R. A. Afify, D. G. Amin, and R. F. Mohammed. 2012. CD200 expression in B-cell chronic lymphoproliferative disorders. J Investig Med 60: 56-61.

343. Spaner, D. E., and A. Masellis. 2007. Toll-like receptor agonists in the treatment of chronic lymphocytic leukemia. Leukemia 21: 53-60.

344. McWhirter, J. R., A. Kretz-Rommel, A. Saven, T. Maruyama, K. N. Potter, C. I. Mockridge, E. P. Ravey, F. Qin, and K. S. Bowdish. 2006. Antibodies selected from combinatorial libraries block a tumor antigen that plays a key role in immunomodulation. Proceedings of the National Academy of Sciences of the United States of America 103: 1041-1046.

345. Moreaux, J., J. L. Veyrune, T. Reme, J. De Vos, and B. Klein. 2008. CD200: a putative therapeutic target in cancer. Biochemical and biophysical research communications 366: 117-122.

346. Kretz-Rommel, A., F. Qin, N. Dakappagari, E. P. Ravey, J. McWhirter, D. Oltean, S. Frederickson, T. Maruyama, M. A. Wild, M. J. Nolan, D. Wu, J. Springhorn, and K. S. Bowdish. 2007. CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy. J Immunol 178: 5595-5605.

247

347. Pallasch, C. P., S. Ulbrich, R. Brinker, M. Hallek, R. A. Uger, and C. M. Wendtner. 2009. Disruption of T cell suppression in chronic lymphocytic leukemia by CD200 blockade. Leukemia research 33: 460-464.

348. Shi, Y., D. White, L. He, R. L. Miller, and D. E. Spaner. 2007. Toll-like receptor-7 tolerizes malignant B cells and enhances killing by cytotoxic agents. Cancer research 67: 1823-1831.

349. Tweeddale, M. E., B. Lim, N. Jamal, J. Robinson, J. Zalcberg, G. Lockwood, M. D. Minden, and H. A. Messner. 1987. The presence of clonogenic cells in high-grade malignant lymphoma: a prognostic factor. Blood 69: 1307-1314.

350. Hammond, C., Y. Shi, J. Mena, J. Tomic, D. Cervi, L. He, A. E. Millar, M. Debenedette, A. C. Schuh, J. L. Baryza, P. A. Wender, L. Radvanyi, and D. E. Spaner. 2005. Effect of serum and antioxidants on the immunogenicity of protein kinase C-activated chronic lymphocytic leukemia cells. J Immunother 28: 28-39.

351. Chen, D. X., and R. M. Gorczynski. 2005. Discrete monoclonal antibodies define functionally important epitopes in the CD200 molecule responsible for immunosuppression function. Transplantation 79: 282-288.

352. Flies, D. B., and L. Chen. 2007. The new B7s: playing a pivotal role in tumor immunity. J Immunother 30: 251-260.

353. Crispen, P. L., Y. Sheinin, T. J. Roth, C. M. Lohse, S. M. Kuntz, X. Frigola, R. H. Thompson, S. A. Boorjian, H. Dong, B. C. Leibovich, M. L. Blute, and E. D. Kwon. 2008. Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma. Clin Cancer Res 14: 5150-5157.

354. Roth, T. J., Y. Sheinin, C. M. Lohse, S. M. Kuntz, X. Frigola, B. A. Inman, A. E. Krambeck, M. E. McKenney, R. J. Karnes, M. L. Blute, J. C. Cheville, T. J. Sebo, and E. D. Kwon. 2007. B7-H3 ligand expression by prostate cancer: a novel marker of prognosis and potential target for therapy. Cancer research 67: 7893-7900.

355. Oikonomopoulou, K., L. Li, Y. Zheng, I. Simon, R. L. Wolfert, D. Valik, M. Nekulova, M. Simickova, T. Frgala, and E. P. Diamandis. 2008. Prediction of ovarian cancer prognosis and response to chemotherapy by a serum-based multiparametric biomarker panel. British journal of cancer 99: 1103-1113.

356. Comin-Anduix, B., Y. Lee, J. Jalil, A. Algazi, P. de la Rocha, L. H. Camacho, V. A. Bozon, C. A. Bulanhagui, E. Seja, A. Villanueva, B. R. Straatsma, A. Gualberto, J. S. Economou, J. A. Glaspy, J. Gomez-Navarro, and A. Ribas. 2008. Detailed analysis of immunologic effects of the cytotoxic T lymphocyte-associated antigen 4-blocking monoclonal antibody tremelimumab in peripheral blood of patients with melanoma. Journal of translational medicine 6: 22.

357. Rezvany, M. R., M. Jeddi-Tehrani, H. Rabbani, N. Lewin, J. Avila-Carino, A. Osterborg, H. Wigzell, and H. Mellstedt. 2000. Autologous T lymphocytes may

248

specifically recognize leukaemic B cells in patients with chronic lymphocytic leukaemia. British journal of haematology 111: 608-617.

358. Rezvany, M. R., M. Jeddi-Tehrani, H. Wigzell, A. Osterborg, and H. Mellstedt. 2003. Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia. Blood 101: 1063-1070.

359. Kofler, D. M., C. Mayr, and C. M. Wendtner. 2006. Current status of immunotherapy in B cell malignancies. Current drug targets 7: 1371-1374.

360. Mayr, C., D. M. Kofler, H. Buning, D. Bund, M. Hallek, and C. M. Wendtner. 2005. Transduction of CLL cells by CD40 ligand enhances an antigen-specific immune recognition by autologous T cells. Blood 106: 3223-3226.

361. Hus, I., M. Schmitt, J. Tabarkiewicz, S. Radej, K. Wojas, A. Bojarska-Junak, A. Schmitt, K. Giannopoulos, A. Dmoszynska, and J. Rolinski. 2008. Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response. Leukemia 22: 1007-1017.

362. Palma, M., L. Adamson, L. Hansson, P. Kokhaei, R. Rezvany, H. Mellstedt, A. Osterborg, and A. Choudhury. 2008. Development of a dendritic cell-based vaccine for chronic lymphocytic leukemia. Cancer Immunol Immunother 57: 1705-1710.

363. Dorfman, D. M., and A. Shahsafaei. 2011. CD200 (OX-2 membrane glycoprotein) expression in b cell-derived neoplasms. American journal of clinical pathology 134: 726-733.

364. Kretz-Rommel, A., F. Qin, N. Dakappagari, R. Cofiell, S. J. Faas, and K. S. Bowdish. 2008. Blockade of CD200 in the presence or absence of antibody effector function: implications for anti-CD200 therapy. J Immunol 180: 699-705.

365. Dunn, G. P., C. M. Koebel, and R. D. Schreiber. 2006. Interferons, immunity and cancer immunoediting. Nat Rev Immunol 6: 836-848.

366. Clark, D. A., K. Wong, D. Banwatt, Z. Chen, J. Liu, L. Lee, R. M. Gorczynski, and M. A. Blajchman. 2008. CD200-dependent and nonCD200-dependent pathways of NK cell suppression by human IVIG. Journal of assisted reproduction and genetics 25: 67-72.

367. Ramsay, A. G., and J. G. Gribben. 2009. Immune dysfunction in chronic lymphocytic leukemia T cells and lenalidomide as an immunomodulatory drug. Haematologica 94: 1198-1202.

368. Hegde, U., A. Chhabra, S. Chattopadhyay, R. Das, S. Ray, and N. G. Chakraborty. 2008. Presence of low dose of fludarabine in cultures blocks regulatory T cell expansion and maintains tumor-specific cytotoxic T lymphocyte activity generated with peripheral blood lymphocytes. Pathobiology 75: 200-208.

249

369. Mone, A. P., C. Cheney, A. L. Banks, S. Tridandapani, N. Mehter, S. Guster, T. Lin, C. F. Eisenbeis, D. C. Young, and J. C. Byrd. 2006. Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism. Leukemia 20: 272-279.

370. Gorczynski, R. M., Z. Chen, L. Lee, K. Yu, and J. Hu. 2002. Anti-CD200R ameliorates collagen-induced arthritis in mice. Clinical immunology (Orlando, Fla 104: 256-264.

371. Chitnis, T., J. Imitola, Y. Wang, W. Elyaman, P. Chawla, M. Sharuk, K. Raddassi, R. T. Bronson, and S. J. Khoury. 2007. Elevated neuronal expression of CD200 protects Wlds mice from inflammation-mediated neurodegeneration. The American journal of pathology 170: 1695-1712.

372. Bell, J. H., A. H. Herrera, Y. Li, and B. Walcheck. 2007. Role of ADAM17 in the ectodomain shedding of TNF-alpha and its receptors by neutrophils and macrophages. Journal of leukocyte biology 82: 173-176.

373. Matthews, V., B. Schuster, S. Schutze, I. Bussmeyer, A. Ludwig, C. Hundhausen, T. Sadowski, P. Saftig, D. Hartmann, K. J. Kallen, and S. Rose-John. 2003. Cellular cholesterol depletion triggers shedding of the human interleukin-6 receptor by ADAM10 and ADAM17 (TACE). The Journal of biological chemistry 278: 38829-38839.

374. Sanderson, M. P., S. N. Erickson, P. J. Gough, K. J. Garton, P. T. Wille, E. W. Raines, A. J. Dunbar, and P. J. Dempsey. 2005. ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx. J. Biol. Chem. 280: 1826-1837.

375. Wong K, S. S., Spaner DS, and Gorczynski R. 2009. Journal of Immunology 182: 15.

376. Wong, K. K., I. Khatri, S. Shaha, D. E. Spaner, and R. M. Gorczynski. 2010. The role of CD200 in immunity to B cell lymphoma. Journal of leukocyte biology 88: 361-372.

377. Seiffert, M., A. Schulz, S. Ohl, H. Dohner, S. Stilgenbauer, and P. Lichter. 2011

Soluble CD14 is a novel monocyte-derived survival factor for chronic lymphocytic leukemia cells, which is induced by CLL cells in vitro and present at abnormally high levels in vivo. Blood 116: 4223-4230.

378. Eisterer, W., O. Bechter, O. Soderberg, K. Nilsson, M. Terol, R. Greil, J. Thaler, M. Herold, L. Finke, U. Gunthert, E. Montserrat, and R. Stauder. 2004. Elevated levels of soluble CD44 are associated with advanced disease and in vitro proliferation of neoplastic lymphocytes in B-cell chronic lymphocytic leukaemia. Leukemia research 28: 1043-1051.

250

379. Brizard, A., F. Morel, J. C. Lecron, B. Dreyfus, F. Brizard, A. Barra, and J. L. Preud'homme. 1994. Proliferative response of B chronic lymphocytic leukemia lymphocytes stimulated with IL2 and soluble CD23. Leukemia & lymphoma 14: 311-318.

380. Ruchlemer, R., A. C. Wotherspoon, J. N. Thompson, J. G. Swansbury, E. Matutes, and D. Catovsky. 2002. Splenectomy in mantle cell lymphoma with leukaemia: a comparison with chronic lymphocytic leukaemia. British journal of haematology 118: 952-958.

381. Cheson, B. D. 2011

Monoclonal antibody therapy of chronic lymphocytic leukaemia. Best practice & research 23: 133-143.

382. Di Giovanni, S., G. Valentini, P. Carducci, and P. Giallonardo. 1989. Beta-2-microglobulin is a reliable tumor marker in chronic lymphocytic leukemia. Acta haematologica 81: 181-185.

383. Hallek, M., L. Wanders, M. Ostwald, R. Busch, R. Senekowitsch, S. Stern, H. D. Schick, I. Kuhn-Hallek, and B. Emmerich. 1996. Serum beta(2)-microglobulin and serum thymidine kinase are independent predictors of progression-free survival in chronic lymphocytic leukemia and immunocytoma. Leukemia & lymphoma 22: 439-447.

384. Haiat, S., C. Billard, C. Quiney, F. Ajchenbaum-Cymbalista, and J. P. Kolb. 2006. Role of BAFF and APRIL in human B-cell chronic lymphocytic leukaemia. Immunology 118: 281-292.

385. Sawicka-Powierza, J., E. Jablonska, J. Kloczko, J. Piszcz, M. Garley, and W. Ratajczk-Wrona. 2011

Evaluation of TNF superfamily molecules release by neutrophils and B leukemic cells of patients with chronic B - cell lymphocytic leukemia. Neoplasma 58: 45-50.

386. Mellstedt, H., and A. Choudhury. 2006. T and B cells in B-chronic lymphocytic leukaemia: Faust, Mephistopheles and the pact with the Devil. Cancer Immunol Immunother 55: 210-220.

387. Scrivener, S., R. V. Goddard, E. R. Kaminski, and A. G. Prentice. 2003. Abnormal T-cell function in B-cell chronic lymphocytic leukaemia. Leukemia & lymphoma 44: 383-389.

388. Tam, C. S., and M. J. Keating. 2007. Chemoimmunotherapy of chronic lymphocytic leukemia. Best practice & research 20: 479-498.

389. Rodriguez, J., and A. Gutierrez. 2008. Pharmacokinetic properties of rituximab. Reviews on recent clinical trials 3: 22-30.

251

390. Oaks, M. K., and K. M. Hallett. 2000. Cutting edge: a soluble form of CTLA-4 in patients with autoimmune thyroid disease. J Immunol 164: 5015-5018.

391. Zhang, G., Y. Xu, X. Lu, H. Huang, Y. Zhou, B. Lu, and X. Zhang. 2009. Diagnosis value of serum B7-H3 expression in non-small cell lung cancer. Lung cancer (Amsterdam, Netherlands) 66: 245-249.

392. Frigola, X., B. A. Inman, C. M. Lohse, C. J. Krco, J. C. Cheville, R. H. Thompson, B. Leibovich, M. L. Blute, H. Dong, and E. D. Kwon. 2011. Identification of a soluble form of B7-H1 that retains immunosuppressive activity and is associated with aggressive renal cell carcinoma. Clin Cancer Res 17: 1915-1923.

393. Arribas, J., and A. Borroto. 2002. Protein ectodomain shedding. Chemical reviews 102: 4627-4638.

394. Oaks, M. K., K. M. Hallett, R. T. Penwell, E. C. Stauber, S. J. Warren, and A. J. Tector. 2000. A native soluble form of CTLA-4. Cellular immunology 201: 144-153.

395. Chalaris, A., N. Adam, C. Sina, P. Rosenstiel, J. Lehmann-Koch, P. Schirmacher, D. Hartmann, J. Cichy, O. Gavrilova, S. Schreiber, T. Jostock, V. Matthews, R. Hasler, C. Becker, M. F. Neurath, K. Reiss, P. Saftig, J. Scheller, and S. Rose-John. 2010. Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice. The Journal of experimental medicine 207: 1617-1624.

396. Garton, K. J., P. J. Gough, and E. W. Raines. 2006. Emerging roles for ectodomain shedding in the regulation of inflammatory responses. Journal of leukocyte biology 79: 1105-1116.

397. Gu, B., L. J. Bendall, and J. S. Wiley. 1998. Adenosine triphosphate-induced shedding of CD23 and L-selectin (CD62L) from lymphocytes is mediated by the same receptor but different metalloproteases. Blood 92: 946-951.

398. Smalley, D. M., and K. Ley. 2005. L-selectin: mechanisms and physiological significance of ectodomain cleavage. Journal of cellular and molecular medicine 9: 255-266.

399. Suenaga, N., H. Mori, Y. Itoh, and M. Seiki. 2005. CD44 binding through the hemopexin-like domain is critical for its shedding by membrane-type 1 matrix metalloproteinase. Oncogene 24: 859-868.

400. Ashiru, O., P. Boutet, L. Fernandez-Messina, S. Aguera-Gonzalez, J. N. Skepper, M. Vales-Gomez, and H. T. Reyburn. 2010. Natural killer cell cytotoxicity is suppressed by exposure to the human NKG2D ligand MICA*008 that is shed by tumor cells in exosomes. Cancer research 70: 481-489.

252

401. Nuckel, H., M. Switala, L. Sellmann, P. A. Horn, J. Durig, U. Duhrsen, R. Kuppers, H. Grosse-Wilde, and V. Rebmann. 2010. The prognostic significance of soluble NKG2D ligands in B-cell chronic lymphocytic leukemia. Leukemia 24: 1152-1159.

402. Lesesve, J. F., A. M. Florence, G. M. Maynadie, and P. Feugier. 2001. Prognostic relevance of soluble CD23 levels in CLL. Hematol J 2: 355.

403. Schwarzmeier, J. D., M. Shehata, M. Hilgarth, I. Marschitz, N. Louda, R. Hubmann, and R. Greil. 2002. The role of soluble CD23 in distinguishing stable and progressive forms of B-chronic lymphocytic leukemia. Leukemia & lymphoma 43: 549-554.

404. Seiffert, M., A. Schulz, S. Ohl, H. Dohner, S. Stilgenbauer, and P. Lichter. 2010. Soluble CD14 is a novel monocyte-derived survival factor for chronic lymphocytic leukemia cells, which is induced by CLL cells in vitro and present at abnormally high levels in vivo. Blood 116: 4223-4230.

405. Wong, K., Shaha, S., Brenneman F., Chesney A., Spaner D., and Gorczynski R. 2011. Soluble CD200 is an in vivo growth factor for Chronic Lymphocytic Leukemia. Submitted.

406. Bennett, T. A., E. B. Lynam, L. A. Sklar, and S. Rogelj. 1996. Hydroxamate-based metalloprotease inhibitor blocks shedding of L-selectin adhesion molecule from leukocytes: functional consequences for neutrophil aggregation. J Immunol 156: 3093-3097.

407. Ahrens, I., C. Ellwanger, B. K. Smith, N. Bassler, Y. C. Chen, I. Neudorfer, A. Ludwig, C. Bode, and K. Peter. 2008. Selenium supplementation induces metalloproteinase-dependent L-selectin shedding from monocytes. Journal of leukocyte biology 83: 1388-1395.

408. Lambert, E., E. Dasse, B. Haye, and E. Petitfrere. 2004. TIMPs as multifacial proteins. Critical reviews in oncology/hematology 49: 187-198.

409. Gomez-Pina, V., A. Soares-Schanoski, A. Rodriguez-Rojas, C. Del Fresno, F. Garcia, M. T. Vallejo-Cremades, I. Fernandez-Ruiz, F. Arnalich, P. Fuentes-Prior, and E. Lopez-Collazo. 2007. Metalloproteinases shed TREM-1 ectodomain from lipopolysaccharide-stimulated human monocytes. J Immunol 179: 4065-4073.

410. Prevost, J. M., J. L. Pelley, W. Zhu, G. E. D'Egidio, P. P. Beaudry, C. Pihl, G. G. Neely, E. Claret, J. Wijdenes, and C. B. Brown. 2002. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory stimuli up-regulate secretion of the soluble GM-CSF receptor in human monocytes: evidence for ectodomain shedding of the cell surface GM-CSF receptor alpha subunit. J Immunol 169: 5679-5688.

253

411. Kveiborg, M., R. Instrell, C. Rowlands, M. Howell, and P. J. Parker. 2011. PKCalpha and PKCdelta regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways. PloS one 6: e17168.

412. Horiuchi, K., S. Le Gall, M. Schulte, T. Yamaguchi, K. Reiss, G. Murphy, Y. Toyama, D. Hartmann, P. Saftig, and C. P. Blobel. 2007. Substrate selectivity of epidermal growth factor-receptor ligand sheddases and their regulation by phorbol esters and calcium influx. Molecular biology of the cell 18: 176-188.

413. Borland, G., G. Murphy, and A. Ager. 1999. Tissue inhibitor of metalloproteinases-3 inhibits shedding of L-selectin from leukocytes. The Journal of biological chemistry 274: 2810-2815.

414. Mohler, K. M., P. R. Sleath, J. N. Fitzner, D. P. Cerretti, M. Alderson, S. S. Kerwar, D. S. Torrance, C. Otten-Evans, T. Greenstreet, K. Weerawarna, and et al. 1994. Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature 370: 218-220.

415. Chen, Z., D. X. Chen, Y. Kai, I. Khatri, B. Lamptey, and R. M. Gorczynski. 2008. Identification of an expressed truncated form of CD200, CD200tr, which is a physiologic antagonist of CD200-induced suppression. Transplantation 86: 1116-1124.

416. Hargreaves, P. G., F. Wang, J. Antcliff, G. Murphy, J. Lawry, R. G. Russell, and P. I. Croucher. 1998. Human myeloma cells shed the interleukin-6 receptor: inhibition by tissue inhibitor of metalloproteinase-3 and a hydroxamate-based metalloproteinase inhibitor. British journal of haematology 101: 694-702.

417. Fitzgerald, M. L., Z. Wang, P. W. Park, G. Murphy, and M. Bernfield. 2000. Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase. The Journal of cell biology 148: 811-824.

418. Amour, A., C. G. Knight, A. Webster, P. M. Slocombe, P. E. Stephens, V. Knauper, A. J. Docherty, and G. Murphy. 2000. The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3. FEBS letters 473: 275-279.

419. Aichem, A., M. Masilamani, and H. Illges. 2006. Redox regulation of CD21 shedding involves signaling via PKC and indicates the formation of a juxtamembrane stalk. Journal of cell science 119: 2892-2902.

420. Nakamura, H., N. Suenaga, K. Taniwaki, H. Matsuki, K. Yonezawa, M. Fujii, Y. Okada, and M. Seiki. 2004. Constitutive and induced CD44 shedding by ADAM-like proteases and membrane-type 1 matrix metalloproteinase. Cancer research 64: 876-882.

421. Gooz, M. 2010. ADAM-17: the enzyme that does it all. Critical reviews in biochemistry and molecular biology 45: 146-169.

254

422. Feehan, C., K. Darlak, J. Kahn, B. Walcheck, A. F. Spatola, and T. K. Kishimoto. 1996. Shedding of the lymphocyte L-selectin adhesion molecule is inhibited by a hydroxamic acid-based protease inhibitor. Identification with an L-selectin-alkaline phosphatase reporter. The Journal of biological chemistry 271: 7019-7024.

423. Thorp, E., T. Vaisar, M. Subramanian, L. Mautner, C. Blobel, and I. Tabas. 2011. Shedding of the Mer tyrosine kinase receptor is mediated by ADAM17 protein through a pathway involving reactive oxygen species, protein kinase Cdelta, and p38 mitogen-activated protein kinase (MAPK). The Journal of biological chemistry 286: 33335-33344.

424. Ghia, P., N. Chiorazzi, and K. Stamatopoulos. 2008. Microenvironmental influences in chronic lymphocytic leukaemia: the role of antigen stimulation. Journal of internal medicine 264: 549-562.

425. Kim, J., J. Lin, R. M. Adam, C. Lamb, S. B. Shively, and M. R. Freeman. 2005. An oxidative stress mechanism mediates chelerythrine-induced heparin-binding EGF-like growth factor ectodomain shedding. Journal of cellular biochemistry 94: 39-49.

426. Timmermann, M., and P. Hogger. 2005. Oxidative stress and 8-iso-prostaglandin F(2alpha) induce ectodomain shedding of CD163 and release of tumor necrosis factor-alpha from human monocytes. Free radical biology & medicine 39: 98-107.

427. Chalaris, A., B. Rabe, K. Paliga, H. Lange, T. Laskay, C. A. Fielding, S. A. Jones, S. Rose-John, and J. Scheller. 2007. Apoptosis is a natural stimulus of IL6R shedding and contributes to the proinflammatory trans-signaling function of neutrophils. Blood 110: 1748-1755.

428. Kamiguti, A. S., E. S. Lee, K. J. Till, R. J. Harris, M. A. Glenn, K. Lin, H. J. Chen, M. Zuzel, and J. C. Cawley. 2004. The role of matrix metalloproteinase 9 in the pathogenesis of chronic lymphocytic leukaemia. British journal of haematology 125: 128-140.

429. Buggins, A. G., A. Levi, S. Gohil, K. Fishlock, P. E. Patten, Y. Calle, D. Yallop, and S. Devereux. 2011. Evidence for a macromolecular complex in poor prognosis CLL that contains CD38, CD49d, CD44 and MMP-9. British journal of haematology 154: 216-222.

430. Howard, L., R. A. Maciewicz, and C. P. Blobel. 2000. Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28. The Biochemical journal 348 Pt 1: 21-27.

431. Thery, C., M. Ostrowski, and E. Segura. 2009. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9: 581-593.

432. Cocucci, E., G. Racchetti, and J. Meldolesi. 2009. Shedding microvesicles: artefacts no more. Trends in cell biology 19: 43-51.

255

433. Ghosh, A. K., C. R. Secreto, T. R. Knox, W. Ding, D. Mukhopadhyay, and N. E. Kay. 2010. Circulating microvesicles in B-cell chronic lymphocytic leukemia can stimulate marrow stromal cells: implications for disease progression. Blood 115: 1755-1764.

434. Zhao, L., M. Shey, M. Farnsworth, and M. O. Dailey. 2001. Regulation of membrane metalloproteolytic cleavage of L-selectin (CD62l) by the epidermal growth factor domain. The Journal of biological chemistry 276: 30631-30640.

435. Gasbarri, A., F. Del Prete, L. Girnita, M. P. Martegani, P. G. Natali, and A. Bartolazzi. 2003. CD44s adhesive function spontaneous and PMA-inducible CD44 cleavage are regulated at post-translational level in cells of melanocytic lineage. Melanoma research 13: 325-337.

436. Schwager, S. L., A. J. Chubb, R. R. Scholle, W. F. Brandt, R. Mentele, J. F. Riordan, E. D. Sturrock, and M. R. Ehlers. 1999. Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease. Biochemistry 38: 10388-10397.

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

438. Rygiel, T. P., and L. Meyaard. 2012. CD200R signaling in tumor tolerance and inflammation: A tricky balance. Current opinion in immunology.

439. Gorczynski, R. M. 2001. Transplant tolerance modifying antibody to CD200 receptor, but not CD200, alters cytokine production profile from stimulated macrophages. European journal of immunology 31: 2331-2337.

440. Guo, B., T. T. Su, and D. J. Rawlings. 2004. Protein kinase C family functions in B-cell activation. Current opinion in immunology 16: 367-373.

441. Mahadevan, M., M. C. Lanasa, M. Whelden, S. J. Faas, T. L. Ulery, A. Kukreja, N. Li, C. L. Bedrosian, and L. T. Heffner. 2010. First-In-Human Phase I Dose Escalation Study of a Humanized Anti-CD200 Antibody (Samalizumab) In Patients with Advanced Stage B Cell Chronic Lymphocytic Leukemia (B-CLL) or Multiple Myeloma (MM). Blood (ASH Annual Meeting Abstracts) 116: Abstract 2645.

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