uric acid as a growth factor for activated b … · uric acid as a growth factor for activated b...

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URIC ACID AS A GROWTH FACTOR FOR ACTIVATED B CELLS

by

Hajera Khaja

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Medical Biophysics

University of Toronto

© Copyright by Hajera Khaja 2009

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ABSTRACT

Uric acid as a growth factor for activated B cells

Hajera Khaja

Master of Science 2009

Graduate Department of Medical Biophysics

University of Toronto

Cancer vaccines targeted against tumour cells seek to mimic immune responses against

viral infections. Given the unique properties of B cells that allow them to present antigens

proficiently, activated B cells can be used in a vaccine setting to launch effective anti-tumour T

cell responses. Activation induced cell death, however, presents a major hurdle in the

generation of large numbers of B cells. Since apoptosis is mediated by oxidative stress, uric

acid was used as an antioxidant, enabling increased growth of normal B cells. Further

investigation in TK6 cells revealed that uric acid was mediating its effects extracellularly and

initiating signaling pathways that culminated in the activation of ERK and JNK proteins and

production of IL10, which was necessary, but not sufficient, in mediating the growth effects of

uric acid. The results outlined in this study implicate uric acid as a novel growth factor for

activated B cells.

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ACKNOWLEDGEMENTS

First and foremost, my sincerest gratitude goes out to Dr. David Spaner, my research

supervisor, for allowing me to pursue my graduate studies in his laboratory. David is an

exceptional educator and I would have faltered many times over were it not for his constant

guidance and support. Moreover, David‟s enthusiasm for scientific inquiry enabled me to vastly

broaden my areas of research and I greatly appreciate his advice and expertise. Many thanks are

also due to my committee members, Dr. Yaacov Ben-David, Dr. James Booth, and Dr. Richard

Wells for their advice and assistance.

It has been a pleasure to have been a member of the Spaner lab. To my labmates, Jelena,

Yonghong, Caitlin, Dave, and Suchi, your help and guidance is much appreciated. To Jelena,

Yonghong and Caitlin in particular, thank you kindly for your advice and friendship – I could

not have made it through grad school without you.

To my parents, Fareed and Wajeda, I cannot thank you enough for your unconditional

support and love. Your encouragement has always been a blessing and I strive to excel for you.

To my sisters, Javeria and Aisha, thank you for your enthusiasm and your prayers. I also owe

immense gratitude to my parents-in-law, Shabir and Sabitun, for their support and

encouragement. You are truly a blessing. To friends who have cheered from the sidelines:

Aaida, Asmaa, Asma Ali and Safiyyah, I am blessed to have your friendship and am overjoyed

to have been able to celebrate this milestone with you. To Aaida especially, you are an

unrelenting source of encouragement and I value your friendship dearly.

Last, but certainly not the least, I owe an immeasurable amount of gratitude to my

husband, Idrees, for his unwavering support and untiring optimism. Thank you for always being

there when I have needed you and for making this journey worthwhile. This thesis could not

have transpired without you and is in fact, more yours than it is mine.

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TABLE OF CONTENTS

Abstract ......................................................................................................................................... ii

Acknowledgements ...................................................................................................................... iii

Table of contents .......................................................................................................................... iv

List of figures................................................................................................................................ vi

List of abbreviations ................................................................................................................... vii

Chapter 1: Introduction and research hypotheses .................................................................... 1

1.1 Introduction........................................................................................................................... 2

1.1.1 Cancer Immunosurveillance .......................................................................................... 2

1.1.2 Immune Evasion: The Seventh Hallmark of Cancer .................................................... 5

1.1.3 Cancer Immunotherapy ................................................................................................. 7

1.1.4 Dendritic Cells and Cancer Vaccines ............................................................................ 9

1.1.5 B Cells as Alternative Antigen Presenting Cells ......................................................... 11

1.1.6 B Cell Expansion Protocol .......................................................................................... 14

1.1.6.1 Toll-like Receptor 7 ............................................................................................. 15

1.1.6.2 Interleukin 2 ......................................................................................................... 18

1.1.6.3 IL2 and TLR7 Stimulated B Cells ....................................................................... 20

1.1.7 Activation-Induced Cell Death in B Lymphocytes ..................................................... 21

1.1.7.1 Reactive Oxygen Species and AICD ................................................................... 22

1.1.8 Oxidative Stress and Uric Acid ................................................................................... 23

1.1.9 Epstein-Barr Virus Cell Lines as a Model System for Studies in Activated B Cells .. 27

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1.2 Rationale and research hypotheses ..................................................................................... 30

Chapter 2: Materials and methods ........................................................................................... 31

Chapter 3: Results ...................................................................................................................... 39

Chapter 4: Discussion and future directions ............................................................................ 56

4.1 Discussion ........................................................................................................................... 57

4.2 Future directions ................................................................................................................. 63

List of references .......................................................................................................................... 68

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LIST OF FIGURES

Figure 1. TLR7 signaling in B cells.

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Figure 2. Murine B cell cultures stimulated with IL2 and a TLR7 agonist

undergo proliferation and experience AICD which is partially

reversed by uric acid.

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Figure 3. Growth promoting effects of uric acid in activated human B cell

cultures.

44

Figure 4. Uric acid rescues TK6 cells from death associated with serum

withdrawal and displays functions beyond an antioxidant role.

47

Figure 5. Uric acid does not function as a metabolite as determined by

inhibition of uric acid transporters and G protein-coupled receptors.

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Figure 6. Activation of signaling pathways and cytokine production by uric

acid-treated TK6 cells.

52

Figure 7. IL10 inhibition leads to loss of uric acid‟s proliferative potential in

TK6 cells.

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Figure 8. Mechanism of uric acid‟s growth effects on activated B cells.

55

vii

LIST OF ABBREVIATIONS

∆ψm Mitochondrial membrane potential

2‟,5‟-dd-Ado 2‟,5‟-Dideoxyadenosine

2-ME 2-mercaptoethanol

7AAD 7-aminoactinomycin D

AC Adenylate cyclase

Ad D/F Advanced Dulbecco‟s modified Eagle‟s medium: Nutrient Mixture F12

AICD Activation-induced cell death

AIF Apoptosis-inducing factor

AP-1 Activator protein-1

APC Antigen presenting cell

BCR B cell receptor

Ca2+

Calcium

CLL Chronic lymphocytic leukemia

COX Cyclooxygenase

DC Dendritic cell

DPA Dipyridamole

EBV Epstein-Barr Virus

ERK Extracellular signal-regulated kinase

FBS Fetal bovine serum

GM-CSF Granulocyte-macrophage colony stimulating factor

GPCR G protein-coupled receptor

H-89 H-89, Dihydrochloride

HIV Human immunodeficiency virus

HLA Human leukocyte antigen

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HPRT Hypoxanthine-guanine phosphoribosyltransferase

IBMX 3-isobutyl-1-methylxanthine

IDO Indoleamine 2,3-dioxygenase

IFN Interferon

Ig Immunoglobulin

IκB Inhibitor of NF-κB

IKK IκB kinase complex

IL Interleukin

IRAK IL1 receptor-associated kinase

ITAG Insulin, Transferrin, Albumin, Glutamine

ITAM Immunoreceptor tyrosine-based activation motifs

JAK Janus kinase

JNK Jun N-terminal kinase

LMP Latent membrane protein

LRR Leucine-rich repeat

mAb Monoclonal antibody

MAPK Mitogen-activated protein kinase

MCP Monocyte chemoattractant protein

M-CSF Macrophage colony-stimulating factor

MDL-12 MDL-12,330A, Hydrochloride

MHC Major histocompatibility complex

MIIC MHC II rich compartment

MSU Monosodium urate

MyD88 Myeloid differentiation primary-response protein

NF-κB Nuclear factor- κB

NK Natural killer

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NOS Nitric oxide synthase

OAT Organic anion transporter

PBS Phosphate buffered saline

PDE Phosphodiesterase

PI Phosphatidylinositol

PKA Protein Kinase A

PKC Protein kinase C

PT Permeability transition

RAG Recombination-activating gene

redox Reduction/oxidation

ROS Reactive oxygen species

RPMI Roswell Park Memorial Institute medium

RT-PCR Reverse transcriptase polymerase chain reaction

S2 B cells S28690- and IL2-treated B cells

SAPK Stress-activated protein kinase

STAT Signal transducer and activator of transcription

TAB TAK1-binding protein

TAK TGFβ-activated kinase

TAP Transporter associated with antigen processing

TGF Transforming growth factor

TH T helper cell

TIR Toll/IL1 receptor

TLR Toll-like receptor

TNF tumour necrosis factor

TRAF TNF receptor associated factor

TRAIL TNF-related apoptosis-inducing ligand

x

TReg Regulatory T cell

URAT Uric acid transporter

VEGF Vascular endothelial growth factor

VSMC Vascular smooth muscle cell

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Chapter 1:

INTRODUCTION AND RESEARCH HYPOTHESES

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1.1 INTRODUCTION

1.1.1 Cancer Immunosurveillance

The hypothesis of cancer immunosurveillance argues that the immune system can

recognize cancer precursors as foreign entities and eliminate them from the body [1,2]. After

nearly a century of debate, it is now clear that effector cells, from both the innate and adaptive

immune systems, are involved in the recognition and destruction of tumours prior to the clinical

manifestation of cancer.

Several lines of evidence point to the veracity of immunosurveillance. Both occasional

spontaneous regression of tumours in immunocompetent hosts and increased prevalence of

cancer in immunocompromised patients suggest an involvement of the immune system in

keeping tumour progression in check. In a recent study, an examination of the rate of cancer

incidence in heart and/or lung transplant patients revealed that suppression of the immune

system in order to prevent graft rejection led to a dramatic increase in newly diagnosed cancers

in these recipients, and was 7.1 times greater than the rate for the general population [3].

Leukemias and lymphomas were the most common type of cancer detected with a rate that was

26.2 times as many as in the general population. While it is possible that the

immunosuppressive drugs used are mutagenic, another study revealed that the degree of natural

cytotoxicity of blood impacted cancer risk – a study conducted in Japan followed more than

3000 healthy people for 11 years and showed that subjects whose blood lymphocytes had a low

degree of natural cytotoxicity at the start of the investigation had a significantly higher risk of

developing cancer than did subjects whose lymphocytes had a medium or high degree of

cytotoxicity [4].

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Experimental animal models have also confirmed the existence of anti-tumour

immunity. Studies in mice have shown that when essential parts of the innate or adaptive

immune system are lacking, a corresponding increase in susceptibility to the development of

spontaneous or chemically induced tumours follows. This includes animals in which knockouts

and other experimental manipulations have resulted in the elimination of certain cells of the

immune system, such as T cells, B cells and natural killer T (NKT) cells brought about through

lack of the recombination-activating gene 2 (RAG2), as well as animals missing important

effector genes and molecules, such as interferon (IFN) receptor 1; the transcription factor,

signal transducer and activator of transcription 1 (STAT1), required for IFN-induced signaling;

perforin; or tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) [5].

Conversely, a reduction in the development of malignant disease in mouse models can be

brought about by the administration of an immunostimulatory regimen designed to increase the

number of immune effector cells [6].

Another line of evidence indicating that the immune system appears cognizant of

tumours in the body arises from the observation that an accumulation of tumour-infiltrating T

lymphocytes at tumour sites often correlates with improved prognosis [7]. A study of three

multicenter cohorts of colon cancer patients showed that rather than tumour stage and nodal

status, the current gold standards, a more accurate predictor of clinical outcome was the

presence or absence of T cells in the resected tumour [8]. Tumour samples from patients with

cervical cancer [9], breast cancer [10], urothelial carcinoma [11], and follicular lymphoma [12],

revealed similar observations. Likewise, results reported from a colorectal carcinoma study

revealed that when patients were found to express messenger transcripts encoding molecules

made by effector T helper 1 (TH1) and effector memory T cells, a reduced rate of metastasis and

increased survival time was observed compared to patients in which such molecules were absent

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[13]. On the other hand, an infiltration of regulatory T (TRegs) cells in the tumour predicts

diminished patient survival in the cases of ovarian carcinoma or melanoma [14,15].

As is apparent from the studies cited above, several diverse processes are employed by

the immune system to keep tumour growth in check upon detection. As a consequence of the

genotoxic stress of transformation, tumour growth can stir changes in the surrounding

microenvironment, resulting in the release of “danger” signals. Ideally, cells of the innate

immune system recognize these signals and induce inflammation alongside activating innate

effector cells that possess anti-tumour activity. Further action is taken by professional antigen-

presenting cells (APCs) which engulf tumour-derived antigens and in the context of major

histocompatibility (MHC) proteins, present them to B and T cells in the draining lymph nodes,

thereby triggering the adaptive immune system [16]. Effectors of the adaptive response include

T cells that express clonally distributed antigen receptors. When allied with MHC proteins, the

T cell receptors can recognize either unique tumour antigens, such as those resulting from

mutant or viral-derived proteins, or self-antigens, such as those derived from overexpressed

oncogenic proteins or aberrantly expressed proteins that are normally developmentally or tissue-

restricted [17].

Certain conditions must be met before effector T cells can mediate effective anti-tumour

responses – T cells must be activated by bone marrow-derived APCs that present tumour

antigens and also provide the necessary costimulatory signals; they must be able to migrate with

efficiency and access the tumour microenvironment; and they should be able to overcome

obstacles posed by the tumour and launch an effective attack that will not be abruptly withheld

and result in the induction of tolerance. When effective, T cell activation can lead to cessation

of tumour growth via production of inflammatory cytokines such as IFN and TNF. T cells

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can also cause direct lysis of malignant cells mediated by perforin and/or Fas and lead to

inhibition of angiogenesis [17].

1.1.2 Immune Evasion: The Seventh Hallmark of Cancer

Despite the presence of a complex surveillance programme, the existence of a tumour in

the body is indicative of its ability to thwart detection and/or evade the immune response

launched against it. In fact, it has been postulated that apart from the six known hallmarks of

cancer (self-sufficiency in growth signals; insensitivity to anti-growth signals; evasion of

apoptosis; limitless replicative potential; sustained angiogenesis; and tissue invasion and

metastasis) [18], evasion of immunosurveillance could be the seventh hallmark of cancer

[19,20]. Due to the selective pressure exerted by the immune system (immunoselection),

tumours have developed a sophisticated array of strategies that allow them to escape the various

devices involved in immunosurveillance [2]. Developing tumours can initiate processes that

encourage evasion from immune recognition [16], suppress the triggering of an inflammatory

response via secretion of immunosuppressive factors [21], or escape death by blocking the

production of pro-inflammatory molecules by effector cells [22]. Even if evasion proves to be

unsuccessful, a cancerous growth can escape elimination via various mechanisms such as

deletion of targeted antigens to prevent immune attack, inducing growth and promotion of

regulatory T cells, and inducing anergy in, or causing deletion of tumour-reactive T cells

[23,24].

Downregulation or loss of expression of MHC class I molecules and disruption of

antigen processing and presentation is a frequent strategy designed to specifically dodge a T-cell

mediated adaptive response. Loss of MHC I expression is often observed in lung cancer and has

also been recorded in several epithelial cell cancers and melanomas. In fact, it has been

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proposed that the only tumour cells that progress into malignancy are those that have undergone

immunoselection owing to their loss of MHC class I expression, allowing them to escape

immune attack and develop into advanced cancer [25]. In the same vein, tumours frequently

downregulate molecules that partake in the processing and presentation of antigen by MHC I

molecules; for example, during the development of colorectal carcinoma, a progressive loss in

the expression of proteins involved in antigen processing, such as transporter associated with

antigen processing 1 (TAP1), low-molecular-mass protein 2 and 7, and tapasin has been

reported [26].

Immunosubversion, or the active suppression of the immune response, is yet another

tactic whereby tumour cell products can engage in direct repression of immune machinery [2].

For instance, increased arginase-1 activity and overproduction of nitric oxide by some tumours

can lead to inhibition of T cell function [27]. Constitutive overexpression of indoleamine 2,3-

dioxygenase (IDO) in many tumours, in particular prostate, colon and pancreatic carcinomas

[28], can not only block proliferation of CD8+ T cells at the local tumour site [28], but can also

induce apoptosis of CD4+ T cells [29]. The tumour microenvironment also evolves to aid in the

evasion of immunosurveillance and suppression of immune attack [30]. Various

immunosuppressive factors that can inhibit the differentiation, maturation, and function of

dendritic cells (DCs) can be found in tumour beds. These include the vascular endothelial

growth factor (VEGF), Interleukin (IL)6, IL10, transforming growth factor (TGF),

macrophage colony-stimulating factor (M-CSF), nitric oxide synthase (NOS)2, arginase-1, IDO,

and others [30]. Such factors can jeopardize anticancer immunity, which often relies on TH1-

cell responses, by polarizing the T-cell response from a TH1 response to a TH2 response, thus

rendering the immune response against the tumour ineffective [31]. Furthermore, it has been

demonstrated in mouse models that advanced cancer invariably undermines the anti-tumour

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immune response. At the initial stages of tumour growth, tumour-specific CD8+ T cells are

found to be activated, but eventually lose their cytotoxicity as the tumour expands [32].

Tumour-specific CD4+ T cells also sustain a progressive loss in their anti-tumour activity [33],

coinciding with a rise in the number of regulatory T cells [34]. Taken together, it appears that

induction of tolerance towards the tumour may be a prerequisite for the initial steps of

tumorigenesis [35].

There is hence a ceaseless cycle involved in the advent of cancer with the immune

system recognizing the tumour as a foreign entity and subsequently attacking it, and the tumour

evading and suppressing this response [19]. The progressive selection of weakly immunogenic

and/or immune-resistant malignant cells forms the eventual tumour that can spread in an

uninhibited fashion throughout the body [2]. It is starkly apparent that to win the fight against

cancer, it is necessary not only to develop chemotherapeutic strategies to efficiently kill all

cancer cells, but also to attempt to re-stimulate an immune response so that residual tumour cells

can be kept in check [36].

1.1.3 Cancer Immunotherapy

Despite the versatility of cancer, it is possible to reactivate a defunct immune system into

an anti-tumour state. Studies in both experimental models and cancer patients have shown that

augmenting natural anti-tumour responses can lead to therapeutic benefits [17]. Passive

antibody transfer into cancer patients was one of the first immunotherapeutic strategies

employed to control tumour growth. The initial testing of mouse monoclonal antibodies (mAb)

targeting the IL2 receptor expressed by many T cell leukemias and lymphomas in humans

provided proof of concept for the efficacy of this form of therapy [37]. Various mAbs are now

administered as part of standard treatment regimens. Rituximab, one of the most extensively

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used mAb, binds CD20 and can induce high remission rates in patients with B cell lymphomas

[38]. Clinical effectiveness has been achieved in other cancers also, such as the administration

of mAbs against tumour antigens in HER2-positive breast cancer (trastuzumab) [39], and head

and neck, lung, and colorectal cancers that express the epidermal growth factor receptor

(cetuximab) [40-42]. A challenge that still needs to be addressed is improving anti-tumour

activity without causing widespread toxicity to normal tissues; selective targeting of the mAb to

tumour sites is one plausible solution that needs to be further explored [43].

Another type of immunotherapy involves the intravenous administration of tumour-

specific autologous T cells that have been grown and expanded in vitro [44]. In a phase 1 study,

patients with metastatic melanoma were treated with infusions of CD8+ T cells specific for the

melanoma antigens MART-1, Melan-A, and glycoprotein 100 (gp100). T cell migration to the

tumour sites and metastatic regression was observed in 8 out of 10 patients [45]. Such studies

reveal that it is possible to harness the body‟s natural defences against cancer by devising

effective immunotherapeutic strategies based on the re-stimulation of an anti-tumour immune

response. As simple as it may sound, exploring this avenue of therapy is challenging for the

reason that most tumour antigens are unable to trigger a strong immune response, mainly due to

tolerance resulting from the fact that the vast majority of tumour-derived antigens are normal

proteins which are aberrantly expressed by cancer cells [43]. While some antigens may be

strongly immunogenic, the lack of a correspondingly strong immune attack reflects the

inconducive environment in which these antigens are presented.

Although administration of pre-formed tumour-specific antibodies or autologous T cells

is beneficial in initiating an immune response, such passive routes to immunotherapy will likely

not lead to the induction of long-lived memory T cells that have the ability to control tumour

outgrowth in the event of a relapse. A more attractive alternative is active immunotherapy,

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involving the use of cancer vaccines derived from professional APCs that can display tumour

antigens effectively, thereby allowing the vaccine to directly elicit or boost similar anti-tumour

antibodies and T cells in the patient [43]. Such an active approach is crucial to the induction of

productive T cell immunity involving both tumour-specific effector T cells and memory T cells

[46].

1.1.4 Dendritic Cells and Cancer Vaccines

Dendritic cells (DCs), activated macrophages, and activated B cells all possess the

capability to present peptides. DCs however, are considered to be the most efficient APCs as

they are not only exceedingly proficient at antigen capture and processing, but can also migrate

efficiently [47]. Encouraging data from animal models led to promising results in the first

report on DC vaccination in 1996 [48]. Since then, autologous DCs generated ex vivo and

loaded with antigen have been used as vaccines in various trials with melanoma, lymphoma, and

carcinoma patients, and have proven to be safe and effective [48-52]. Many different

approaches to the creation of DC vaccines have culminated in >100 clinical trials to date [53].

The generation of DC vaccines involves ex vivo isolation of DC subtypes from

peripheral blood and subsequent loading of tumour-derived or tumour-specific antigen followed

by reinjection. Clinically applicable protocols for DC differentiation and maturation have been

established for successfully generating immunostimulatory DCs from both proliferating CD34+

precursors and non-proliferating CD14+ monocytes [54,55]. Several tumour-derived and

tumour-associated products have been used as antigenic stimulants, including whole tumour

lysates, apoptotic or necrotic tumour debris, recombinant proteins, synthetic peptides, plasmid

DNA, RNA, and viral vectors [56]. Such mature and differentiated DCs can also successfully

migrate to secondary lymphoid organs [57]. Examples of cancer vaccines designed under such

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protocols are those generated against breast cancer (the HER2 antigen) [58], melanoma (DCs

loaded with tumour peptides or a mixture of necrotic and apoptotic tumour cells) [59,60], and

prostate cancer (DCs loaded with prostatic acid phosphatase) [61]. Evidence of an immune

response to the vaccines was demonstrated in each of these trials, and clinical responses with

minimal or adverse effects were also observed in a few cases.

Many issues regarding the use of DC vaccines, however, such as optimal method of

generation, delivery and dosage, remain to be resolved [56,62,63]. Furthermore, despite their

ability to induce T cell immunity with high efficiency, the use of DC-based cancer vaccines is

fraught with difficulties. Firstly, DCs are relatively scarce in peripheral blood and comprise

only 0.1 – 0.5% of human peripheral blood mononuclear cells, necessitating isolation via

apheresis [48] or from marrow sources [64]. Consequently, it is considerably difficult and

expensive to obtain adequate numbers of purified mature DCs [65]. Secondly, the use of DCs as

APCs presents a special problem in that the DC population is not homogenous but consists of

several subsets of functionally distinct cell types [66], including tolerogenic DCs [67].

Furthermore, the generation of sufficient numbers of DCs from non-stem cell sources requires

considerable expansion with cytokines like granulocyte M-CSF and IL4 [68]. Such expansion

protocols yield functional DCs, but with limited growth potential – within 2-3 weeks, DCs cease

to proliferate and also become less efficient in their antigen presenting ability [69]. Multiple

phlebotomies are thus needed to obtain fresh DCs with which to repetitively stimulate

autologous T cells.

Although DC-based cancer vaccines can be generated in sufficient numbers and tested in

clinical trials, the complexity and cost of preparing DC precursors and generating functional

DCs makes it difficult to harness their full potential [65]. Especially important is the

requirement for repeated high dose vaccinations to generate sustained T cell responses [63,70].

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Repetitive generation of DCs either from cryopreserved precursors or from fresh DC sources

will hence be required [69], not only rendering the process laborious and expensive, but also

limiting the clinic applicability of DC-based cancer vaccines [71].

1.1.5 B Cells as Alternative Antigen Presenting Cells

Owing to the disadvantages associated with DC-based cancer vaccines, effective

alternatives for active immunotherapy need to be explored. A possible approach is to use other

APCs that are available in large numbers, have the potential to be highly immunogenic, and may

have other unique advantages that enable them to elicit strong and effective vaccine responses.

The generation and expansion of such APCs should preferably be relatively straightforward and

should not require large precursor populations that are difficult to obtain or require prior

treatment of donors or patients with chemotherapeutic agents or other drugs. Generation from

small amounts of non-stem cell sources would be ideal, and once ready for vaccination, such

APCs should be capable of priming T cells and generating strong effector responses [72,73].

Furthermore, the production of such APCs should involve the use of recombinant growth factors

without the need to resort to usage of xenogeneic cells [74] or gene-transfer technology [75].

Based on these criteria, activated B cells appear to be a prime candidate for serving as surrogate

DC-based cancer vaccines. Several characteristics confer on B cells the ability to present

antigens efficiently: shortly after antigen encounter, activated B cells can quickly home to T

cells in secondary lymphoid organs; B cell receptor (BCR)-mediated endocytosis allows for

maximal concentration of minimal amounts of specific antigen; and signaling pathways can

guide the antigen processing machinery to give preference to the presentation of antigens

internalized through the BCR [76].

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After encounter, APCs can internalize antigen via phagocytosis, fluid-phase pinocytosis,

or receptor-mediated endocytosis. In B cells, endocytosis occurs through the BCR, a

membrane-bound immunoglobulin that has high affinity for its given antigen, allowing efficient

antigen presentation through the concentration of very small quantities of the specific antigen.

In fact, BCR affinity has been shown to be directly proportional to the degree to which B cells

are capable of presenting antigen to CD4 T cells. A progressive need for more antigen arises as

the affinity of the BCR-antigen interaction decreases. B cells with a very high affinity BCR (Ka

of 5 x 1010

M-1

) require very low concentrations of soluble antigen (as low as 0.05nM in one

study) to be able to induce CD4 T cell proliferation, whereas cells bearing a BCR of lower

affinity (Ka of 3 x 108 M

-1) require ten times more antigen. In contrast, about 5000 times more

antigen is needed when presentation occurs after uptake via fluid phase pinocytosis [77].

Upon antigen recognition, ligation of the BCR induces its internalization, which leads to

the activation of various signaling cascades essential for activation of the B cell [78-80]. Some

of these signals are directed towards antigen processing and facilitate the traffic of antigen and

MHC class II molecules. Once internalized, the BCR-antigen complex is directed through the

endocytic pathway towards MHC II rich compartments (MIIC) where the formation of peptide-

MHC II complexes occurs [81-84]. Alongside endocytosis, BCR signaling results in the

upregulation of MHC II expression and causes MHC II molecules to be channelled through the

same compartments used by the BCR-antigen complex to travel to the MIIC [85,86]. Moreover,

signals generated from BCR crosslinking also induce the formation of multivesicular bodies and

acidification of MIIC compartments in an effort to create a favourable environment for antigen

processing and peptide loading [87]. The degradation and loss of regions of the antigen bound

to the BCR is also blocked as a result of the strong affinity of the BCR-antigen interaction,

allowing for its preferential presentation [88,89]. B cells are thus able to link antigen

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recognition with the mechanisms responsible for antigen processing, thereby allowing the

presentation of BCR-internalized antigens preferentially over antigens taken up via other means

such as pinocytosis [76].

Yet another sophisticated aspect of antigen processing in B cells that is absent in other

APCs is the expression of human leukocyte antigen (HLA)-DO, a non-classical MHC II

molecule [90]. Antigen-derived peptide loading occurs in acidic compartments and is mediated

through the activity of another non-classical MHCII molecule, HLA-DM. HLA-DO is an

inhibitor of HLA-DM – HLA-DO activity predominates in compartments with high pH, limiting

HLA-DM activity to low pH compartments [91,92]. This pH-dependent inhibition by HLA-DO

serves to restrict peptide-loading to MHC II in the MIIC, where the acidic conditions are a

requirement for the processing of the stable BCR-antigen complex [93,94]. In this manner, the

processing of other non-BCR derived epitopes is inhibited, thus influencing the immune

response against BCR-internalized antigens.

B cells are thus capable of acquiring characteristics reminiscent of highly proficient

antigen presenting cells: after encountering antigen in secondary lymphoid organs, they can

efficiently internalize antigens specific for their BCR and preferentially process and present

those antigens to T cells. The ensuing response to BCR crosslinking, the first signal for B cell

activation, is proliferation and survival of the B cell. BCR crosslinking also induces expression

of costimulatory molecules required for naïve CD4 T cell activation [95,96]. In addition, B cells

can also present cytoplasmic antigens in the context of MHC class I molecules, thereby leading

to direct activation of CD8 positive T lymphocytes [76,97]. As a result, B cells can be strongly

immunogenic and possess the capability of presenting tumour-derived foreign antigens to other

components of the immune surveillance machinery, allowing for an immediate and speedy

response to cancer cells.

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Since activated B cells are capable of functioning as potent APCs, cancer vaccines on a

B cell platform may be a more feasible alternative to DC-based vaccines. Unlike their DC

counterparts, B cells possess a greater proliferative capacity and can be easily obtained from

peripheral blood [98]. Consequently, the most apparent and immediate advantage of B cell-

derived cancer vaccines is the potential of generating unlimited cell numbers for repeated

vaccinations required for the induction of a sufficient immune response. Although it unclear

how many activated B cells can replace one activated DC, titration experiments comparing the T

cell stimulatory capability of the two APCs suggest that a T cell:APC ratio of 4:1 is optimal for

activated B cells while a ratio of 20:1 or less is optimal for DCs [99]. However, to generate

about 108 DCs requires roughly 1 litre of blood (or 10

9 peripheral blood mononuclear cells),

while the same number of activated B cells can be generated from only 4-8cc of peripheral

blood [100]. In other words, large numbers of B cells can be obtained and expanded easily from

a small and manageable amount of blood, yielding a pure and homogenous population of

activated B cells [98]. Like DCs, in vitro activation of B cells can be accomplished by treatment

with inflammatory cytokines and toll-like receptor (TLR) ligands [101], leading to enhanced

MHC and costimulatory molecule expression, as well as an increase in cytoskeletal activity and

capacity to stimulate T cells [69,102-104]. Moreover, owing to the deficiencies often

encountered in DCs of patients with advanced cancers [105], activated B cells could substitute

as alternative APCs either by replacing DCs, or boosting T cell responses that have been already

primed by DCs.

1.1.6 B Cell Expansion Protocol

Previous studies in the lab involving the in vitro manipulation of B cells from patients

with chronic lymphocytic leukemia (CLL) had set a precedent for an expansion protocol for

15

normal activated B cells. During the course of investigations on the molecular basis of tumour

immunogenicity, it was found that upon treatment with a TLR7 agonist, S28690 [106,107], and

the inflammatory cytokine IL2 [108], leukemic B cells could be induced to undergo

proliferation in vitro. Subsequent treatment with a Protein Kinase C (PKC) activator caused the

cells to acquire a phenotype resembling that of mature DCs [109]. Although TLR7-stimulated

and IL2-treated cells are less able to elicit an immune response than their PKC-activated

counterparts, normal B cells treated with S28690 and IL2 should induce proliferation in vitro

and generate sufficient quantities of activated B cells to be used as APCs for repeated

administrations in clinical trials of cancer vaccines.

1.1.6.1 Toll-like Receptor 7

Toll-like receptors recognize molecular patterns on infectious agents and play an

important role in the defence initiated against invading pathogens [110]. Stimulation of the toll-

like receptors is an efficient means for the induction of an immune response as TLRs regulate

both innate and adaptive immunity. In fact, activation of the innate immune system is a

prerequisite for the instigation of the adaptive immune response, especially with regards to a

TH1 response, which is central to cancer immunity [36].

The eleven members of the TLR family are type I integral membrane glycoproteins, and

are a subset of a larger superfamily that includes the interleukin 1 receptors (IL1Rs) based on

significant homology in their cytoplasmic regions. The extracellular region of TLRs contain

leucine-rich repeat (LRR) motifs and the cytoplasmic tail has a conserved region of ~200 amino

acids known as the Toll/IL1R (TIR) domain [111]. Although B cells express many members of

the TLR family, they are stimulated most potently by TLR7 and TLR9 ligands [112]. Unlike

other TLRs such as TLR1, TLR2 and TLR4, which are located on the cell surface, TLR3, TLR7

16

and TLR9 are endosomal receptors and are involved in the recognition of nucleic-acid-like

structures [113-115]. TLR7 is primarily expressed by B cells and monocytes and is involved in

the recognition of single-stranded viral RNA in endosomes [116,117]. Oxidized guanosines

[118] and imidazoquinolines [119] are synthetic human TLR7 agonists. Subsequent to ligand

binding, TLRs dimerize and undergo conformational changes necessary for the recruitment of

proteins involved in downstream signaling pathways (Figure 1), which include the adaptor

molecule myeloid differentiation primary-response protein 88 (MyD88), IL1R-associated

kinases (IRAKs), TGF-activated kinase (TAK1), TAK1-binding protein 1 (TAB1), TAB2 and

TNF-receptor-associated factor 6 (TRAF6) [120,121].

Upon receptor engagement, the TIR domain in the intracellular region of TLR7

associates with the adaptor protein, MyD88 [111]. The TLR7-MyD88 complex then recruits

IRAK1 and IRAK4, members of the IRAK family of kinases. IRAK4 is activated first via

MyD88 binding, and then proceeds to activate IRAK1 by phosphorylating specific residues in

its kinase-activation loop. Activation of the intrinsic kinase activity of IRAK1 induces

autophosphorylation of residues in its N-terminus, enabling the recruitment of TRAF6 to the

complex. IRAK1 and TRAF6 then disengage from TLR7 and interact with a plasma

membrane-associated pre-formed complex consisting of TAK1 and two TAK-1 binding

proteins, TAB1 and TAB2. This interaction leads to the phosphorylation of TAB2 and TAK1,

which translocate to the cytoplasm along with TRAF6 and TAB1, leaving IRAK1 to be

degraded at the membrane. TAK1 kinase activity is then triggered in the cytosol upon

ubiquitination of TRAF6, and activated TAK1 phosphorylates components of the inhibitor of

nuclear factor (NF)-B (IB) kinase complex (IKK). Activated IKK then phosphorylates IB,

causing its degradation and permitting release of NF-B dimers, allowing them to enter the

nucleus and function as transcription factors. TAK1 also functions to activate stress-activated

17

Figure 1. TLR7 signaling in B cells. Activation of TLR7 leads to association with MyD88, which in turn allows

for the recruitment of IRAK4 and IRAK1. Activation of IRAK1 allows TRAF6 to associate with the complex,

leading to the activation of TAK1 and TAB2. TAK1 is responsible for the activation of MAPKs, as well as NF-B.

18

protein kinase (SAPK) pathways via activation of mitogen-activated protein kinase kinases such

as MKK4 or MKK3 and MKK6, which lead to phosphorylation and activation of c-Jun-NH2-

kinases (JNK) or p38, respectively. Upon activation, JNK and p38 translocate to the nucleus

and activate c-Jun and c-Fos, components of activator protein-1 (AP-1), resulting in the

transcription of genes encoding molecules such as TNF [111]. TLR engagement also leads to

the activation of transcription factors that modulate the expression of costimulatory molecules

such as CD80 and CD86, and cytokines such as IFN and IL12 that skew the immune response

towards type 1 immunity [122].

Consistent with its capability to induce an immunogenic phenotype, one

immunotherapeutic route may be to manipulate TLR signaling in order to exact an anti-tumour

response. For instance, a recent study demonstrated that treatment with a TLR7 agonist caused

the disappearance of leukemic skin infiltrates in a patient with CLL [106]. In yet another study,

the sensitivity of CLL cells to chemotherapeutic agents was increased with exposure to a TLR7

agonist, Loxoribine [123]. Similarly, if TLR7 stimulation allows for the proliferation of normal

B cells and renders them capable of activating an immune response against tumours, such

activated B cells may have the potential of replacing DC-based vaccines.

1.1.6.2 Interleukin 2

The inflammatory cytokine IL2 is a member of the common cytokine-receptor -chain

(c; also known CD132)-binding family of cytokines, which includes IL4, 7, 9, 15, and 21 [124].

Apart from the common c receptor subunit shared by all members of this family, the IL2

receptor (IL2R) is a heterotrimeric receptor and consists of the IL2 receptor -chain (IL2R)

and the IL2 receptor alpha-chain (IL2R), associated with the high-affinity form of the receptor

[125,126]. IL2R is expressed primarily by activated T and B cells; exposure to IL2 induces

19

expression of IL2R, assisting in further activation [127]. IL2 is a mediator of adaptive

immunity [108] and signaling through the IL2R triggers the activation of multiple pathways,

including stimulation of the phosphatidylinositol-3-kinase (PI3)-AKT pathway, and stimulation

of the RAS-RAF-mitogen-activated-protein-kinase pathway. It also involves the common Janus

kinase (JAK) and STAT signaling molecules [124]. IL2 activation can thus lead to the

expression of various genes mediated by the STAT molecules, and FOS- and JUN-containing

transcription-factor complexes [128]. As a result, exposure to IL2 can lead to a variety of

responses, such as proliferation of T cells, proliferation of and synthesis of immunoglobulin by

activated B cells, and generation of cytotoxic T lymphocytes [129].

Owing to its immunogenic properties, IL2 is being tested as a therapeutic agent for the

treatment of various cancers and has been approved for use in patients with metastatic renal-cell

carcinoma and malignant melanoma [130]. Its anti-tumour effect likely arises from its

proliferation-inducing properties as IL2 stimulation not only leads to expansion of lymphocyte

populations in vivo, including anti-tumour T cells, but also strengthens their effector functions

[130,131]. Rather than have a direct effect on cancer cells, IL2 thus mediates its anti-tumour

activity by altering immune reactions to the tumour.

The growth-stimulating property of IL2 is also exploited by malignant cells of various T

and B cell leukemias. In such situations, one therapeutic strategy aims to develop antibodies

specific for IL2R so as to block the IL2-IL2R interaction, leading to cytokine starvation and

permitting death of IL2 dependent cancer cells directly. The unique IL2R subunit is an

especially attractive component to target as it is constitutively expressed by cancerous T and B

lymphocytes, and is not expressed by any resting cells except TReg cells [125]. Two such

antibodies, basiliximab (Simulect; Novartis AG), a chimeric antibody, and daclizumab (also

known as anti-Tac; Zenapax; F.Hoffman-LaRoche Ltd), the first humanized antibody, have both

20

been approved by the US Food and Drug Administration [132-134]. IL2 thus has a proven

track-record of serving as an immunotherapeutic agent in some forms of cancer, leading to the

hypothesis that its use could allow for proliferation of normal B cells whilst enabling them to

acquire an immunogenic phenotype via simultaneous TLR stimulation.

1.1.6.3 IL2 and TLR7 Stimulated B Cells

As mentioned above, the data from leukemic B cells from CLL patients treated in vitro

with IL2 and the TLR7 agonist S28690 showed that such treatment results in rapid proliferation,

accompanied by IL10 secretion and expression of the costimulatory molecules, CD80 and CD86

[109]. S28690 treatment activated the NF-B, p38 and SAPK pathways, while IL2 induced

activation of extracellular signal-regulated kinase (ERK) family members. STAT signaling was

also apparent 24 hours after treatment. In addition, it was noted that S28690 treatment

sensitized CLL cells to IL2, in part via the increased expression of IL2R. Initially, signaling

from the two stimulants was observed to be merely additive, perhaps due to the difference in

receptor locations with the IL2R on the plasma membrane and TLR7 residing in the endosomal

compartment [114]. Over time however, the signaling patterns became more synergized as

TLR7 activation led to an increase in IL2R expression, allowing for stronger IL2 signaling.

This interaction amplified growth and proliferation, increased costimulatory molecule

expression, and also led to STAT1 and STAT3 activation, as well as cytokine production.

Further stimulation by a PKC agonist allowed for the expression of CD83, a maturation marker,

and led to an increase in the production of TNF and , thus enabling the activated CLL cells to

become strongly immunogenic [109]. Based on these studies, it was hypothesized that normal B

cells procured from healthy individuals would become activated and proliferate in response to

TLR7 stimulation and IL2 treatment. Subsequent activation with a PKC agonist should enable

21

these cells to acquire characteristics reminiscent of potent APCs, allowing for further

investigation of their potential use for cancer vaccines.

1.1.7 Activation-Induced Cell Death in B Lymphocytes

The in vitro activation and proliferation of lymphocytes can prove to be problematic due

to the potential complication of activation-induced cell death (AICD). As the term implies,

AICD refers to apoptosis as an ensuing response to lymphocyte activation. Indeed, after a few

days of robust proliferation, normal B cells stimulated with the TLR7 agonist, S28690 and IL2

seemed to reach the end of their growth potential (elaborated upon in Results below), presenting

a major hurdle in the attempt to generate large numbers of activated B cells.

When B cells encounter their specific antigen, the response that follows can either be

activation and proliferation, or anergy and AICD [135]. In other words, BCR crosslinking and

internalization does not necessarily culminate in an immune response, but can result in tolerance

when other survival signals are lacking. AICD is mechanistically achieved by the regulation of

signaling pathways that promote apoptosis, such as the activation of effector caspases,

expression of pro-apoptotic genes, and inhibition of pro-survival genes. Regulation of the

immune response in mature lymphocytes is thus controlled by AICD, which is an essential

property of lymphocytes [135].

In B cells, AICD was first thought to be primarily restricted to the assembling of the

immune repertoire where elimination of self-reactive immature cells occurs upon crosslinking of

surface IgM receptors [136]. Later studies revealed that mature B cells can also undergo

apoptosis upon stimulation through extensive surface IgM ligation with immobilizing antibodies

[137,138] and MHC class II crosslinking [139]. Also, the finding that antigen-specific B cells

apoptose when they encounter soluble antigen in germinal centres [140], suggests that

22

lymphocytes are affected by AICD throughout their ontogeny, serving as a means for

maintaining peripheral tolerance [141]. Nonetheless, the vast array of signaling processes

involved in the AICD of mature B cells is not entirely understood. IL2 is a likely candidate as it

is known to play a crucial role in AICD in T cells, resulting in the clearance of self-reactive T

lymphocytes [142]. It is possible that cell mortality due to oxidative stress can also lead to

AICD.

1.1.7.1 Reactive Oxygen Species and AICD

The apoptotic process is mediated by intracellular reactive oxygen species (ROS) in

several systems, including excitotoxic neural cell death [143], glucocorticoid-mediated cell

death of thymocytes [144], HIV-induced death of T cells [145], and also in the case of the

immature B cell-line WEHI-231 treated with anti-IgM [146]. In fact, cytotoxicity induced by

increased levels of intracellular ROS results in alterations very similar to those observed in cells

undergoing apoptosis, such as plasma and nuclear membrane blebbing, elevated intracellular

Ca2+

levels, and protease activation [147]. Shortly after the induction of apoptosis,

mitochondrial permeability transition (PT), referring to an increase in the permeability of the

mitochondrial membrane, and loss of mitochondrial membrane potential (m) occur. These

changes allow for the release of the pro-apoptotic proteins, cytochrome C and apoptosis-

inducing factor (AIF), from the mitochondrial intermembrane space into the cytosol, leading to

the activation of effector caspases and thus committing the cell to apoptosis [148,149]. ROS

production is one of the means via which disruption of m occurs [141], and is an early event

upstream of caspase activation [150,151]. Activation of the pro-apoptotic members of the Bcl-2

family, such as Bax, Bad, Bak, Bid, Bik and Bim also leads to loss of m [152].

23

If apoptosis is mediated by ROS, it is worth investigating whether B cell activation

creates an intracellular environment that encourages ROS production, leading to AICD. This

hypothesis is quite plausible since cellular activation necessitates increased oxidative

phosphorylation. The increased rate of respiration in the mitochondria of activated cells could

lead to ROS generation, which is also a by-product of the mitochondrial electron transport

chain. In fact, a recent study demonstrated that activation of B cells with soluble antigen leads

to the generation of intracellular ROS, resulting in the disturbance of the reduction/oxidation

(redox) potential [141], which controls mitochondrial PT [153]. The intracellular redox

potential regulates the proclivity of cells to undergo apoptosis [154,155]; an oxidative shift in

the cellular redox state can hence modify the nature of the stimulatory signal received, resulting

in death instead of proliferation [156]. Moreover, it has been reported that BCR engagement on

activated B cells induces m breakdown, leading to caspase 9 activation, followed by further

activation of downstream effector caspases [157]. Elevated intracellular ROS levels could result

in the activation of genes known to mediate apoptosis, perhaps through an oxidative stress-

responsive nuclear transcription factor such as NF-B [141]. A strong case can hence be made

for the role of intracellular ROS generation in serving as the missing link in the mechanism

behind the induction of AICD in mature B cells. In light of this, it was postulated that the

introduction of strong antioxidants in a cell culture system may allow for decreased ROS

generation, thereby enabling prolonged growth.

1.1.8 Oxidative Stress and Uric Acid

Oxidative damage to basic cellular constituents – DNA and RNA, protein, lipids, and

carbohydrates – is an inevitable consequence of fuel-efficient aerobic metabolism [158]. While

low levels of ROS are a typical occurrence under physiological conditions, their effect becomes

24

detrimental once their rate of production rises enough to overpower the protective measures

cells have in place to combat the harmful consequences of oxidative stress. Notably, a role for

oxidative stress has been implicated in more than a hundred physiological disorders, including

ageing, cancer, and cardiovascular disease [159]. In response, evolution in aerobic organisms

has led to the amassing of a variety of defence mechanisms for inhibiting oxidant formation and

lipid peroxidation, as well as for counteracting and repairing oxidative damage. These

protective measures include enzymes, such as superoxide dismutase [160] and the selenium-

containing glutathione peroxidase, as well as antioxidants and radical scavengers, such as -

tocopherol (vitamin E) and -carotene confined to the lipid portion of the cell, and glutathione

and ascorbic acid restricted to the aqueous phase [161]. Another defence mechanism against

ROS is increased levels of the antioxidant uric acid in the plasma [161].

Uric acid is derived from adenine- and guanine-based compounds and is a product of

purine catabolism. Xanthine oxidoreductase is the sole enzyme responsible for its production

[162]. Whereas most mammals express the enzyme uricase (urate oxidase), which converts uric

acid to allantoin and urea, it is not expressed in humans and higher primates due to a non-

transcribed defective gene as a result of a nonsense mutation, hence making uric acid the final

product of purine degradation in these species [163-165]. As a result, the plasma level of uric

acid in humans is more than 10 times the amount found in most mammals [162]. Uric acid is

also found within cells and in all body fluids at lower concentrations than in plasma, where it

exists mostly in its ionized form, urate, at physiological pH [159].

Prior to the knowledge of its antioxidant and radical scavenging properties [166-168],

uric acid was traditionally viewed as being biologically dispensable, perhaps owing to the

harmful consequences arising from its enhanced retention or abnormally high production,

manifesting in crystal formation (due to its limited solubility in water) and leading to conditions

25

such as gout, where urate accumulates in joints and causes arthritis. This presumption was not

an unfounded one given that with regards to the human purine balance, uric acid is truly a waste

product. Unlike other purines, no further utilization of xanthine or uric acid is possible [169].

Whereas compounds such as adenosine, inosine, hypoxanthine, adenine, and guanine can be

salvaged from blood and added back to the cellular purine pool, any purine component that is

degraded to uric acid must be resynthesized anew with considerable energy expenditure, or

replaced by salvaging the appropriate precursors [169]. Upon ingestion, however, purines are

converted to uric acid via mucosal xanthine dehydrogenase, and instead of being reabsorbed in

the gastro-intestinal tract, uric acid is the only purine derivative that appears in the systemic

circulation [170]. Moreover, although it is filtered in the kidneys and then secreted, about 90%

of uric acid is typically reabsorbed and returned to the blood [161].

If the physiological handling of uric acid is any indication, it seems unlikely that it is an

expendable waste product. In fact, the idea that uric acid is one of the most powerful

antioxidants in the body was first put forth nearly 30 years ago, when in vitro studies

demonstrated uric acid to be a powerful scavenger of peroxyl and hydroxyl radicals, as well as

singlet oxygen [161]. About 30-65% of the peroxyl radical-scavenging capacity of plasma is

ascribed to uric acid [171,172], and in severe preeclampsia, a spike in uric acid levels accounts

for more than 90% of the increase in the radical-scavenging capabilities of the plasma [162]. It

was also shown that uric acid can serve as an electron donor, in particular, for heme

protein/H2O2 systems [159]. Beyond its role as a radical scavenger, uric acid also has the ability

to chelate metal ions such as iron and copper, rendering them incapable of catalyzing free

radical reactions [173-175]. More recently, it was discovered that uric acid acts as a specific

inhibitor of radicals resulting from the breakdown of peroxynitrite (ONOO-) [176,177], a

powerful oxidant that can inflict a variety of injuries by damaging essential cellular components

26

[178,179]. Peroxynitrite can also erode the structure and function of proteins by causing

nitration of their tyrosine residues [179,180]. In fact, given that uric acid is the strongest

antioxidant in blood plasma [161], it may prove to be useful in an in vitro setting for curtailing

ROS generation and relieving oxidative stress.

The initial studies on uric acid starting in the early 80s led a few investigators to

speculate that the antioxidant properties of uric acid may have allowed it to be a contributor to

the increased life span in humans [161,162]. An interspecies comparison of uric acid levels in

primates and other mammals reveals that there is a positive association between plasma uric

acid concentration and species longevity [162]. The majority of mammals have less than 0.5mg

uric acid/100ml of blood, while the levels in humans are close to its maximum solubility limit,

about 5mg/100ml of blood [161]. This stark difference arose over the course of about 60

million years of evolution and correlated with a substantial increase in life span as well as brain

size. Increased uric acid levels may also correlate with superior health. In patients diagnosed

with multiple sclerosis, for instance, serum uric acid levels are generally lower than those of

healthy individuals. Given that oxidative stress plays an important role in multiple sclerosis, it

is interesting to note that very seldom do patients with hyperuricaemia develop the disease

[181]. In addition, in patients with acute stroke, uric acid concentration in the plasma correlates

inversely with neurological impairments at the onset of stroke and size of the infarct upon

follow-up [182]. Similar studies reporting confirmatory results have led to the suggestion that

uric acid administration may be beneficial to patients suffering from conditions of heightened

oxidative stress, such as acute ischemic stroke [183].

Given the antioxidant properties of uric acid and the possible involvement of ROS in

AICD, the role of uric acid in the minimization of AICD in B cells was investigated. To this

27

end, a model system in which to study the effects of uric acid was required, and an Epstein-Barr

Virus (EBV) cell line was used.

1.1.9 Epstein-Barr Virus Cell Lines as a Model System for Studies in Activated B Cells

EBV is a human herpes virus that infects B cells primarily, but can infect other cell types

as well, especially epithelial cells. Given its ability to remain in a transcriptionally repressed

state indefinitely, almost all individuals (>95%) test positive for the virus and are infected for

life [184]. In certain circumstances however, at times involving even a single mutated protein,

this seemingly innocuous virus can prove to be deadly [185,186]. In fact, EBV was first

detected in Burkitt‟s lymphoma and is now known to be associated with various lymphomas,

carcinomas and other neoplastic disorders [187]. Of more significance is the fact that EBV can

infect resting B cells in vitro and compel them into activation, thereby causing their

transformation into proliferating lymphoblasts [188,189].

It is possible for EBV to achieve this dual status of benign and malignant infection by

altering its programme of gene expression based on the site and differentiation state of infected

cells. In vivo, EBV-infected B cells express one of four different gene expression programmes,

three of which are associated with latent infection and one of which is used for the manufacture

of infectious virus. The three latency transcription programmes are: the growth programme,

which demands expression of all nine latent proteins; the default programme, which prescribes

expression of only three latent proteins; and the latency programme, which is associated with

the expression of few, if any, latent genes [184]. Infected resting memory B cells circulating in

the peripheral blood express the latency programme, thereby allowing the virus to persist in the

infected individual for life [190-192]. Latent infection however, does not involve a state of

simple passivity but is associated with an immune response against the virus – a stable number

28

of infected B cells constantly circulate through the blood [193], along with stable levels of

cytotoxic T cells and serum antibodies to lytic and latent-stage proteins, leading to consistent

shedding of infectious EBV into the saliva [194,195]. The virus uses the growth gene

expression programme to drive resting B cells into an activated state, the same programme that

enables development of lymphoblastoid cell lines in vitro, such as TK6 [196]. Two important

proteins expressed in the growth programme enable this transformation – Latent membrane

protein 1 (LMP1) and LMP2A [197,198].

LMP1 and LMP2A are both multiple membrane-spanning proteins that lack functional

extracellular domains but signal in a constitutive fashion by acting as ligand-independent

receptors [199,200]. LMP1 is a functional homologue of CD40 [201,202], a member of the

TNF receptor (TNFR) family of receptors, and interacts with a host of proteins known as

TRAFs [203]. CD40 is an essential receptor on germinal centre B cells and interacts with

CD154 on T helper cells to deliver a costimulatory survival signal [204]. The signaling domains

of LMP1 and CD40 are comparable [200]; both proteins interact with JAK3 and STAT

molecules and lead to AP-1 activation via the JNK signaling pathways [205-207]. Signaling

through LMP1 thus serves to propel B cells into a proliferative state by mimicking signals

induced by the CD40 ligand via activation of the NF-B, MAPK, PI3K, and JAK3/STAT

pathways [207-211]. In this fashion, LMP1 is indispensable for the immortalization of B cells

in vitro [212]. LMP2A on the other hand, does not cause B cells to grow in vivo, but delivers an

important survival signal [213]. The amino-terminal domain of LMP2A contains the same

immunoreceptor tyrosine-based activation motifs (ITAMs) found in the - and -chains of the B

cell receptor [214], and associates with Lyn, a member of the Src family of tyrosine kinases

[215,216]. LMP2A mimics the non-proliferative BCR-initiated signal that is delivered in the

absence of antigen and is vital for the survival of all B cells [217]. Signaling via LMP2A

29

culminates in Protein Kinase C activation, leading to the resultant changes in gene expression

[199].

Between them, LMP1 and LMP2A bear the signaling capabilities necessary to mimic

signals from the BCR and T-helper cells – signals that are essential for the growth and survival

of activated B cells [218,219]. It was recently recognized that TLR signaling may serve as a

third signal for optimal B-cell activation [220]. There is evidence to indicate that this third

proliferative signal is also induced by EBV infection – EBV infection results in enhanced

expression of TLR7 and MyD88, an adaptor molecule that is an important constituent in TLR

signal transduction, which also plays a role in the initial B cell growth response [221].

EBV transformed cell lines thus mimic the pro-proliferative effects of TLR7 activation

as well, thereby rendering the collective signaling processes in the EBV-transformed line, TK6

analogous to those of normal activated B cells resulting from S28690 and IL2 treatment. TK6

cells can thus be used as a model in which to study the role that uric acid plays in overcoming

AICD of activated B cells.

30

1.2 RATIONALE AND RESEARCH HYPOTHESES

Although dendritic cell-based cancer vaccines have demonstrated potential as a useful

form of cancer immunotherapy, their production is fraught with many difficulties. One of the

main challenges that needs to be addressed is the difficulty involved in generating large numbers

of DCs for repeated vaccinations, required for sustained T cell responses against tumours. A

promising alternative is the use of other professional antigen presenting cells such as B cells,

which possess unique qualities that can make them potent APCs upon activation. Healthy

activated B cells can hypothetically be generated in large numbers via IL2 and TLR7

stimulation; however, AICD presents a major obstacle. Since oxidative stress is a mediator of

apoptosis, it appeared quite conceivable that the activated B cells were undergoing apoptosis

due to ROS-induced oxidative stress resulting from consistent stimulation over a prolonged

period of time. If true, the introduction of strong antioxidants into the culture media could tip

the redox balance and reduce ROS generation. Given that uric acid is the most powerful

antioxidant in plasma, its addition to the growth media should curtail oxidative stress and allow

for a prolonged proliferative capacity. To study in more detail the role of uric acid, and in

particular, to explore its possible function as a growth factor for activated B cells, the EBV-

transformed cell line, TK6, was used as a model system.

31

Chapter 2:

MATERIALS AND METHODS

32

Antibodies and Reagents

Clinical grade IL2 (Chiron Corp., San Francisco, CA) was purchased from the

Sunnybrook Health Sciences Centre hospital pharmacy. The TLR7 agonist S28690 was

obtained from 3M Pharmaceuticals (St. Paul, MN). The powder was dissolved in a mixture of

AIM-V medium (Invitrogen Life Technologies, Carlsbad, CA) with 33% DMSO at 1.3 mg/ml

and stored at 4oC in the dark. Phycoerythrin- or FITC-labelled CD19 and CD3 mouse

antibodies were purchased from BD Pharmingen (Mississauga, ON). Advanced D-MEM/F12,

Knockout Serum Replacement and Trypan blue were purchased from Invitrogen Life

Technologies. Thy1.2 antibody was purchased from the Sunnybrook Research Institute

Antibody Core Facility and Rabbit Complement was from Cedarlane Laboratories Ltd.

(Burlington, ON). Transferrin, 2-mercaptoethanol (2-ME), 7-aminoactinomycin D (7AAD),

dipyridamole (DPA), uric acid, Probenecid and Benzbromarone were from Sigma-Aldrich

Canada Ltd. (Oakville, ON). 2‟,5‟-Dideoxyadenosine (2‟,5‟-dd-Ado), MDL-12,330A,

Hydrochloride (MDL-12), Pentoxyfylline, 3-isobutyl-1-methylxanthine (IBMX), 14-22 Amide,

and H-89, Dihydrochloride (H-89) were from Calbiochem (San Diego, CA). Insulin (Humulin

RTM

, Eli Lilly Canada Inc., Toronto, ON) and 25% Human Albumin (Talecris Biotherapeutics

Ltd., Mississauga, ON) were obtained from the hospital pharmacy and blood bank. Phosphate

buffered saline (PBS) and Glutamine was purchased from Wisent Bioproducts (St. Bruno, QC).

Abs against phosphorylated JNK, phosphorylated ERK, and phosphorylated Akt were obtained

from Cell Signaling Technology (Beverly, MA). -Actin Ab was obtained from Sigma-Aldrich.

Recombinant human IL10 was obtained from R&D Systems, Inc. (Minneapolis, MN) and anti-

human IL10 antibody was purchased from eBioscience (San Diego, CA), along with IL15 and

IL7. TK6 cells were a generous gift from Dr. H. Liber (Colorado State University, Fort Collins,

CO).

33

Human blood samples and cell purification

Heparinized blood (30 ml) was collected from consenting healthy volunteers with

approval from the Research Ethics Board, Sunnybrook Health Sciences Centre. B cells were

isolated immediately after collection from each sample through negative selection using the

RosetteSep Human B Cell Enrichment Cocktail (StemCell Technologies, Vancouver, BC)

according to the manufacturer‟s instructions.

Isolation of murine B Cells

Spleens and lymph nodes were obtained from C57BL/6J wild type mice and

lymphocytes were obtained via ficoll gradient density centrifugation (Lympholyte-M, Cedarlane

Laboratories Ltd.). The isolated cells were T cell depleted with Thy1.2 antibody and Rabbit

complement. Briefly, cells were suspended at 107cells/ml in RPMI 1640 medium with 10%

FBS. 10g/ml of Thy1.2 antibody was added to the lymphocyte suspension and incubated for

30 minutes on ice. After two washes in PBS at 1000 rmp, the supernatant was discarded and

pelleted cells were resuspended in rabbit complement (diluted to 1:7 with media) for 30 minutes

at 37oC. Complement-treated cells were washed with PBS twice and depletion of T cells was

confirmed via flow cytometric analysis of the B and T cell marker, CD19 and CD3,

respectively.

Cell culture

Murine T cell-depleted lymphocytes were cultured in RPMI 1640 (Invitrogen Life

Technologies) with 5% Fetal Bovine Serum (FBS) (Lot No. 115615, Wisent) in flat bottom 96

well plates (BD Labware, Franklin Lakes, NJ) at 37oC in 5% CO2. 100 U/ml IL2 and 0.1 g/ml

S28690, the TLR7 agonist were added to 5 x 105 cells/ml at the start of the culture period. Cells

34

were re-stimulated with the two growth factors, along with addition of fresh media every two

days for the duration of the culture period. When uric acid was included in the media, the

appropriate amount was weighed (so as to obtain 0.1 mg/ml) and added to RPMI + 5% FBS.

The mixture was warmed at 37oC for about 15 minutes, or until completely dissolved, and then

filter sterilized via sterile 0.20m filters. The process was repeated every time cells were re-fed.

Human B cells were also plated at 5 x 105 cells/ml and cultured in 24 well plates (BD

Labware) in the indicated media. The same concentrations of S28690 and IL2 were used as for

mouse cells, and cells were maintained in the same manner as the murine B cell cultures.

TK6 cells were harvested every 3 days and re-cultured at approximately 1 x 105 cells/ml

in RPMI + 5% FBS. After a 1-2 month culture period, cells were discarded and a freshly

thawed aliquot from the original TK6 stock was used to set up a new culture. For experiments

assessing proliferation and/or growth inhibition, TK6 cells were plated at 5 x 104 cells/ml in 24

well plates in serum-free media consisting of RPMI plus 1 g/ml Insulin, 5 g/ml Transferrin,

25% Human Albumin, and 10mM Glutamine (ITAG).

Determination of live and dead cell populations

When simple cell counts were employed, live cells were determined by Trypan Blue

exclusion and counted in a hemocytometer. To determine percentages of live vs. dead cells,

approximately 1 x 106 cells were collected into a 100-200 l volume and 4 l of 7AAD solution

was added to the cell suspension. The cells were then analyzed for 7AAD staining on a

FACScan flow cytometer (Beckton Dickinson, San Jose, CA) and at least 10 000 events were

collected in each experiment.

When utilizing a colorimetric assay for proliferation, 20 l of Alamar blue (Biosource

International, Camarillo, CA) was added to cells cultured in 96-well round-bottomed plates (in

35

200 l volumes) and incubated at 37oC for 6-8 hours [222]. An absorbance colorimeter

microplate reader (Molecular Devices, Menlo Park, CA) was used to determine absorbance at

wavelengths of 540 nm (reduced state) and 595 nm (oxidized state).

Flow Cytometry

Flow cytometric analyses of surface CD19 and CD3 expression on murine lymphocytes

was performed after T cell depletion. Isotype-matched irrelevant antibodies were used as

negative controls (Pharmingen, Franklin Lakes, NJ). At least 10 000 events were collected in

each experiment and viable nucleated cells were determined by gating on 7AAD negative cells.

Before each experiment, SpheroParticles (Spherotech Inc., Chicago, IL) were used to

standardize measurements obtained from a FACScan flow cytometer. Data was analyzed using

CELLQUEST software (Becton Dickinson).

Cytokine Measurements

Cytokine levels in culture supernatants from TK6 were measured via a multi-analyte

fluorescent bead assay with a Luminex-100 system (Luminex Corp, Austin, TX). A kit

allowing measurements for IL5, IL6, IL10, Granulocyte-macrophage colony stimulating factor

(GM-CSF) and TNF was used according to the manufacturer‟s instructions (R&D Systems,

Inc.). Individual cytokine concentrations were ascertained from standard curves using Bio-Plex

2.0 software (BioRad, Mississauga, ON). For each cytokine, assays were linear between 3 and

10 000 pg/ml.

36

Western Blots

TK6 cells growing in RPMI and 5% FBS were washed twice in PBS and cultured in 6

well plates in 4ml volumes at 5 x 105 cells/ml. Cells were collected at the indicated time points,

and after an initial wash in cold PBS, they were lysed on ice for 30 minutes in lysis buffer (0.5%

Triton X-100, 25mM MES, 150mM NaCl, 1mM Na3VO4, 2mM EDTA, 1mM PMSF, 1 g/ml

aprotinin), followed by 10 minutes of high speed centrifugation. Protein extracts were

quantified [223] after collection and prepared for Western blot analysis by 1:4 dilution in 5X

sample buffer (8% [wt/vol] SDS, 8% [vol/vol] 2-ME, 250mM Tris, 40% glycerol, 2%

bromophenol blue in dd-H2O) and denaturation at 95oC for 5 minutes. Protein samples were

then loaded on a discontinuous polyacrylamide gel consisting of 7.5% resolving and 4%

stacking gels. PVDF membranes were then preactivated with 100% methanol and separated

proteins from the resolving gel were transferred onto the membranes. Phosphorylation status of

ERK and JNK proteins were determined through the use of antibodies listed in Antibodies and

Reagents. After overnight incubation at 4oC with the primary antibodies, blots were incubated

with anti-rabbit antibody conjugated with horseradish peroxidase for 1 hour, according to the

manufacturer‟s instructions. Chemiluminescence signals were detected with Supersignal West

Pico Luminol Enhancer and Stable Peroxide Solution (Pierce, Rockford, IL). -actin was used

as a loading control.

Isolation of total RNA and cDNA synthesis

Total RNA was extracted from 4 ml volumes of TK6 cells plated at 5 x 105 cells/ml at

the indicated time points. RNA was isolated using the RNeasy kit (Qiagen) according to the

manufacturer‟s instructions and concentration was measured in a spectrophotometer at 260nm.

37

cDNA was synthesized using the Superscript First Strand Synthesis System (Invitrogen

Life Technologies) in a 20 l reaction volume comprising 3 g of total collected RNA, 20 mM

Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 10 mM DTT, 0.5 g oligo dT18, 0.5 mM

dATP, dGTP, dCTP, dTTP, and 200 U of Superscript II Reverse Transcriptase. The mixture

was incubated at 70oC for 5 minutes and cooled to 4

oC to anneal the priming oligonucleotide to

the total RNA. Reverse transcription was performed at 42oC for 2 hours and the cDNA obtained

from the reaction was stored at -20oC.

Real-time polymerase chain reaction

cDNA was amplified using the following primers: IL-10 forward, 5'-

CATCGATTTCTTCCCTGTGA-3', IL-10 reverse, 5'-CGTATCTTCATTGTCATGTAGGC-3';

hypoxanthine-guanine phosphoribosyltransferase (HPRT) forward, 5'-

GAGGATTTGGAAAGGGTGTT-3' HPRT reverse, 5'-ACAATAGCTCTTCAGTCTGA -3'.

PCR was performed on a DNA engine Opticon System (MJ Research Inc, Waltham, MA) using

SYBR Green I as a double-strand DNA-specific binding dye. PCR reactions were cycled 34

times after initial denaturation (95°C, 15 minutes) with the following parameters: denaturation

at 95°C for 15 seconds, primer annealing at 54°C and 52°C for IL10 and HPRT, respectively,

for 20 seconds, and extension at 72°C for 20 seconds. Fluorescent data were acquired during

each extension phase. After each PCR reaction, a melting curve analysis of the amplification

products was performed. Rapid fluorescence loss was uniquely observed at the

denaturing/melting temperature of the amplified DNA fragment. Standard curves were

generated with serial 10-fold dilutions of cDNAs obtained using the above primers. Expression

values of IL10 were normalized to those of HPRT.

38

Statistical analysis

Data are presented as mean standard error, unless stated otherwise. P values for

differences between sample means were determined using the Student t test, and P values for

assessing the significance of differences between two measurements were determined using the

paired t test. Statistical significance was set at P 0.05.

39

Chapter 3:

RESULTS

40

Murine B cells activated with IL2 and S28690 proliferate and then die

Mouse cells were first used as a model system to study B cell expansion with IL2 and

TLR stimulation. Cells isolated from the spleen and lymph nodes of B6 mice were T cell-

depleted using Thy 1.2 antibody, which binds T cells, and complement lysis of the antibody-

coated cells to obtain a predominantly B cell population. The negligible heterogeneity of the

population was confirmed by high CD19 staining, as determined via flow cytometry, and

staining for the T cell marker CD3, which decreased dramatically upon T cell depletion (Figure

2a). At the start of the culture period, and every two days thereafter, the isolated cells were

stimulated with 0.1 g/ml of the TLR7 agonist, S28690, and 100 U/ml IL2 (subsequently

referred to as S2 B cells), and re-fed with fresh media to prevent death associated with nutrient

exhaustion. Upon doubling, the culture was split and allowed to re-grow starting at the initial

cell concentration. In this manner, a roughly 5X expansion was achieved in one week from

mouse S2 B cells before the culture disintegrated as a result of large amounts of cell death

(Figure 2b).

IL2 as a stimulator of AICD

AICD was observed to be a major limiting factor in the expansion of S2 B cells and

evidence of apoptosis from cell counts was apparent in the cell culture starting as early as two

days after initial stimulation. IL2 is known to cause AICD in T cells [142] and could be

functioning in the same manner in activated B cells. Using other cytokines with similar

proliferative effects may allow the cells to stay alive without causing AICD. To test this

possibility, IL2 was replaced with other members of the c binding family of cytokines, which

comprises IL2, IL15, IL4, IL7, IL9, and IL21 [124]. IL15 seemed likely to be the most obvious

41

(a) (d)

(b)

0

5

10

15

20

25

30

35

40

45

day 0 day 2 day 5 day 7 day 9

Cell

co

un

ts (

x10^

5)

untreated

s28 only

s28/IL2

s28/IL15

s28/IL7

(c)

0

10

20

30

40

50

60

day 0 day 2 day 5 day 7

Cell

co

un

ts (

x10^

5)

untreated

s28/IL2

s28/IL2 & UA

Figure 2. Murine B cell cultures stimulated with IL2 and a TLR7 agonist undergo proliferation and

experience AICD which is partially reversed by uric acid. (a) B cells were isolated from the spleens and lymph

nodes of healthy B6 mice. After T cell depletion, an aliquot from the remaining lymphocytes was stained with B

and T cell markers to assess the purity of the population. A representative example shows the percentages of CD19

and CD3 expressing cells immediately before (left panel) and after (right panel) T cell depletion, as determined by

flow cytometric analysis. (b) After undergoing T cell depletion, B cells were plated at 5 x 105 cells/ml in RPMI +

5% FBS. Cells were treated with 0.1 g/ml of the TLR7 agonist, S28690 (abbreviated s28), and either 100 U/ml

IL2, 1 ng/ml IL15, or 1 g/ml IL7. Live cells, as assessed by lack of trypan blue uptake, were counted in a

hemocytometer on the indicated days of the culture period. Cell counts are an average of at least two separate

experiments with error bars indicating standard error of the means. (c) In another set of experiments, B cells were

stimulated with either S28690 and IL2 alone, or with S28690, IL2 and 0.1 mg/ml uric acid (UA). Live cells were

determined and plotted as above. P < 0.05 for the difference in cell counts between s28/IL2 and s28/IL2 & UA on

day 5. (d) After seven days of culture, mouse B cells were harvested and stained with 7AAD and analyzed via flow

cytometry. Cell size (forward scatter) is shown on the x-axis and the percentage of live cells is determined by

7AAD negative staining (shown in the gated regions).

18% live cells 52% live cells

S28/IL2

S28/IL2 & UA

CD3

CD

19

CD19: 38%

CD3: 53%

CD19: 82%

CD3: 2%

42

replacement candidate as it is known to be functionally similar to IL2 owing to the unique

heterotrimeric construction of their receptors. Moreover, IL15 serves an anti-apoptotic factor in

many systems and was shown to inhibit IL2-induced AICD in some circumstances [224]. IL2

was replaced with 1ng/ml of IL15, and although there was an initial significantly large increase

in cell growth observed 5 days into the culture period, IL15 treatment was unable to overcome

AICD (Figure 2b). Similar to S2 B cells, cells treated with S28690 and IL15 all died after a

week in culture. IL15 was also used in conjunction with IL2 to determine if the dual cytokine

combination would affect cells differently, but identical results were obtained, and in fact, there

seemed to be no difference between the S2+IL15 cultures and the S2 B cell cultures (data not

shown). Similar observations were made when IL7, a pre-pro-B cell growth factor that plays a

role in the survival and expansion of lymphoid precursors [225], was used in place of, or in

addition to IL2 (Figure 2b; data not shown). As with IL15 treatment, despite increased

proliferation when S26890 was used with 1 g/ml IL7 instead of IL2, after 7 days there were

very few live cells left in the culture, and within 2 more days, all remaining cells died. Due to

lack of significant differences with the use of other cytokines, the IL2 and S28690 combination

was used for B cell stimulation in all subsequent experiments as it stimulated B cell proliferation

as best as seemed possible and allowed for the highest amount of sustained growth.

Oxidative stress as a mediator of AICD

Since cellular apoptosis is mediated by ROS [143], it is highly plausible that due to

repeated stimulation over a prolonged period of time, oxidative stress led to increased ROS

production in S2 B cells, resulting in AICD [135,141]. If true, the introduction of strong

antioxidants into the cell culture medium should curtail oxidative stress and reduce the amount

of cell death observed. Uric acid, a product of purine degradation, is known to be the most

43

potent antioxidant in plasma [161] and was added to the S2 B cell growth media. Indeed, it

appeared that treatment with 0.1 mg/ml uric acid not only reduced death, but also enabled

increased proliferation, acting as a growth supplement for activated B cells (Figure 2c). In a

representative experiment for instance, after about one week, only 18% of cells in the S2 culture

were alive (Figure 2d), as confirmed by 7AAD staining (a dye that penetrates cell membranes of

dying or dead cells and intercalates into double stranded nucleic acids). In contrast, 52% of S2

B cells growing in medium that contained uric acid were still alive at the end of 7 days,

demonstrating that uric acid could partially rescue S2 activated B cells from AICD.

Human B cell expansion cultures

Based on the positive preliminary results from mouse B lymphocytes, the expansion

protocol for murine B cells was extended to human B cells isolated from peripheral blood

collected from healthy volunteers. Human B cells were cultured in the same manner as were

murine B cells with the exception of the culture medium: serum-free AIM-V with the

antioxidant 2-ME (5 x 10-5

M) was observed to be a superior media than RPMI supplemented

with FBS (data not shown). Apart from increased amino acid and vitamin concentrations, this

difference in growth may be attributed to the inclusion of serum replacing components in AIM-

V, such as transferrin and lipids, as well as trace elements and antioxidants (Patent No.

WO/1988/002774) [226]. Initial experiments with IL2 and S28690 treated human B cells

yielded results that were almost identical to their murine counterparts. After several days in

culture, S2 B cells began undergoing apoptosis and AICD was reduced when 2-ME was

replaced by the more powerful antioxidant, uric acid (Figure 3). Replacement with uric acid

also allowed for the generation of larger numbers of S2 B cells. However, as with mouse

cultures, uric acid was unable to fully rescue cells from AICD and after about 12 days in culture,

44

0

5

10

15

20

25

day 0 day 4 day 8 day 12

Cell c

ou

nts

(x 1

0^

5)

AIM-V + 2-ME

AIM-V + UA

Ad D/F + ITAG

Ad D/F + ITAG + UA

Figure 3. Growth promoting effects of uric acid in activated human B cell cultures. Human B cells were

isolated via negative selection from the peripheral blood of healthy donors and plated at 5 x 105 cells/ml in one of

the following four growth media: AIM-V with 2-ME (5 x 10-5

M), AIM-V with 0.1 mg/ml uric acid, Advanced D-

MEM/F-12 (Ad D/F) with ITAG (1 g/ml Insulin, 5 g/ml Transferrin, 25% Albumin, and 10mM Glutamine)

alone, or Advanced D-MEM/F-12 with ITAG and uric acid. All cells were stimulated with 0.1 g/ml S28690 and

100 U/ml IL2. Cell counts of live cells in a hemocytometer based on trypan blue exclusion are an average of at

least two separate experiments with error bars indicating standard error.

45

most, if not all activated B cells succumbed to death, despite the use of a rich growth medium

supplemented with antioxidants to stabilize culture conditions.

Further examination of the AIM-V patent revealed that it contained Indomethacin, a

non-selective inhibitor of cyclooxygenase (COX)1 and COX2. COX1 is required for tissue

homeostasis and is constitutively expressed, whereas COX2 is expressed in response to various

stimuli ranging from cytokines and growth factors to stress inducers such as free radicals and

hypoxia [227]. COX2 is expressed at high levels in activated human B cells and possesses

growth-promoting properties that are exploited in many cancers including B cell malignancies

[228], while its inhibition leads to decreased proliferation and survival of CLL cells [229].

Moreover, it has been shown that inhibitors like Indomethacin severely restrict the ability of

human B cells to produce IgG and IgM in vitro and negatively affect proliferation as well [229].

In light of this information, it was apparent that the full proliferative potential of activated B

cells could not be realized in the AIM-V medium. Instead, Advanced D-MEM/F-12 (Ad D/F), a

medium rich in strong antioxidants such as ascorbic acid and glutathione seemed more likely to

provide an improved environment conducive to the continued growth of activated human B cells

and was used in subsequent experiments. In an effort to keep culture conditions free of animal-

derived components, it was also essential to replace the bovine-derived serum growth

requirement. More than three decades ago, Iscove and Melchers discovered that when growth

media for B cells is supplemented with additional amino acids and vitamins, the growth-

inducing properties of serum can be completely recovered with the use of albumin, transferrin

and lipids, thus rendering the requirement of FBS redundant [226]. Ad D/F was thus

supplemented with 1 g/ml Insulin, 5 g/ml Transferrin, 25% lipid-rich Albumin [230], and

10mM Glutamine (subsequently referred to as ITAG), and these components together were

46

found to be an adequate and desirable serum replacement given the lack of FBS-associated

xenogeneic components.

Cell counts of live cells revealed that S2 human B cells cultured in Ad D/F + ITAG were

able to stay alive longer than cells cultured in AIM-V medium (Figure 3). Addition of uric acid

to the new media enabled acquisition of the highest yield of cells with the least amount of death

compared to all other alternatives that were tested (Figure 3). Notably, despite the fact that Ad

D/F is rich in antioxidants, a significantly greater number of cells were attained with the

addition of uric acid (P < 0.05), indicating that perhaps uric acid was possibly playing a role

beyond that of an antioxidant.

Uric acid plays an additional role above that of an antioxidant in supporting proliferation of TK6

cells

To study the role that uric acid plays in the minimization of AICD in S28690 and IL2

treated B cells, the spontaneous EBV transformed lymphoblastoid B cell line, TK6, was used as

a model system for activated B cells. The cell line is derived from a paediatric patient with non-

malignant hereditary haemolytic anemia [231,232]. TK6 cells can serve as a model for S2 B

cells in particular, given that the transformation of these cells is achieved via signaling pathways

analogous to those activated upon IL2 stimulation and TLR7 activation [184,221].

While TK6 cells are typically grown in FBS supplemented media, serum starvation

causes these cells to alter their signaling and undergo a death process (Figure 4a) that is

morphologically similar to the AICD observed in S2 B cell cultures. When uric acid is added to

the serum-free TK6 growth media (RPMI + ITAG) however, cell death is inhibited and robust

47

(a)

(b)

Figure 4. Uric acid rescues TK6 cells from death associated with serum withdrawal and displays functions

beyond an antioxidant role. (a) TK6 cells growing in RPMI + 5% FBS were washed twice in PBS and plated at 5

x 104 cells/ml in RPMI plus ITAG alone, with 2-ME (5 x 10

-5 M), or with uric acid (0.1 mg/ml). Cells were

harvested after 3-4 days and viability was assessed by 7AAD exclusion on a flow cytometer. Average percentages

of the 7AAD negative populations are shown along with standard errors of the indicated number of experiments.

(b) TK6 cells were plated in RPMI plus ITAG with either uric acid, DPA (0.3 g/ml), or both, and percentage of

live cells was determined 3-4 days after start of the culture period via flow cytometric analysis of 7AAD staining.

The left panel depicts differences between the percentage of viable cells in uric acid-treated and DPA-treated TK6

cells and P value, the significance of the differences between the two treatments, was determined via a paired t test

for the indicated number of experiments. The right panel depicts differences between DPA- and uric acid plus

DPA-treated cells, and P value was also determined via a paired t test for the indicated number of experiments.

65

70

75

80

85

90

UA DPA

% L

ive

ce

lls

65

70

75

80

85

90

DPA UA + DPA

% L

ive c

ells

P < 0.05 P < 0.01

(n=4) (n=4) (n=4) (n=4)

0

10

20

30

40

50

60

70

80

90

ITAG 2-ME UA

% L

ive c

ell

sP < 0.05

(n=3) (n=2) (n=2)

48

growth is exhibited by these cells (Figure 4a), akin to the observation that uric acid partially

rescues normal activated B cells from AICD. TK6 is thus a valid and applicable system in

which to investigate the relieving effects of uric acid and to determine if it has a previously

underappreciated mechanism of action in activated B cells.

Other antioxidants besides uric acid such as dipyridamole (DPA) [233] and 2-ME were

also able to rescue TK6 cells in a serum-free setting from death, although uric acid delivered the

most amount of growth (Figure 4a and b). When DPA was added along with uric acid, an

additive effect was observed and an increased percentage of cells in the culture were viable,

implying that uric acid was perhaps acting as more than just an antioxidant (Figure 4b). Taken

together, the observations that uric acid had a consistent growth effect in activated mouse B

cells, in activated human B cells, and in TK6 cells; that uric acid enabled a more pronounced

growth effect than other antioxidants; and that an additive effect was observed when uric acid

was used in combination with DPA, led to the hypothesis that uric acid was acting as a growth

factor for activated B cells.

Uric acid does not function intracellularly as a metabolite

Since uric acid is a purine derivative, and given that the dose of uric acid which seemed

most effective in all three systems (activated murine B cell cultures, activated human B cell

cultures, and TK6 cells) was 0.1 mg/ml (600 μM), falling within the dose range in which purine

metabolites are known to exert their effects [234], it was possible that uric acid was functioning

as a metabolite, rather than a growth factor acting via a receptor. If this were to be the case,

uric acid would need entry into the cell to be functional, and exposure to transport inhibitors

would abrogate its survival effect. To test this hypothesis, two uric acid transport inhibitors

were used –Probenecid, an inhibitor of the organic anion transporter (OAT), which transports

49

uric acid from urine into plasma [235], and Benzbromarone, a uricosuric agent that inhibits the

reabsorption of uric acid regulated by the uric acid transporter, URAT1 [236]. Neither of these

two inhibitors caused a reduction in uric acid-mediated TK6 proliferation as assessed by a

colorimetric assay (Figure 5a), implying that uric acid was exerting its effects from within the

extracellular milieu and did not require entry into the cell, but was acting at the cell surface.

Proliferative effects of uric acid are not mediated by a G protein-coupled receptor

Many metabolites are known to signal via G protein-coupled receptors (GPCRs), such as

amino acid receptors, glutamate receptors, purinergic receptors, etc. [237]. If uric acid was

acting as a metabolite at the cell surface, it would be possible that its effects were mediated

through a G-protein and subsequent Protein Kinase A (PKA) activation. To test this theory,

various inhibitors in the PKA activation pathway were employed and their effects on uric acid-

mediated proliferation were tested (a brief illustration of this pathway in relation to the sites of

inhibitor action can be found in Figure 5b). As a control, the inhibitors were also used on TK6

cells growing with the support of the antioxidant DPA to rule out nonspecific growth inhibitory

or toxicity effects. An examination of cell viability through flow cytometric analysis revealed

that the adenylate cyclase (AC) inhibitors, 2‟,5‟-Dideoxyadenosine (2‟,5‟-dd-Ado) and MDL-

12,330A Hydrochloride (MDL-12), decreased the viability of uric acid-treated TK6 cells, but

also decreased the viability of DPA-treated TK6 cells, precluding interpretation of its inhibitory

effects (Figure 5c). In accordance with these results, phosphodiesterase (PDE) inhibitors,

Pentoxyfylline and 3-isobutyl-1-methylxanthine (IBMX) did not increase viability in the uric

acid culture (DPA was not used as a control since it has been characterized as a PDE inhibitor as

well [238]; Figure 5c). Treatment with 14-22 Amide and H-89 Dihydrochloride (H-89), two

PKA inhibitors, also did not decrease the viability of these cells. Taken together, results from

50

(a) (b)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

UA Probenecid + UA Benzbromarone +

UA

Re

lati

ve

Gro

wth

(c)

Figure 5. Uric acid does not function as a metabolite as determined by inhibition of uric acid transporters

and G protein-coupled receptors. (a) TK6 cells were plated in a 96 well plate in RPMI plus ITAG with uric acid

alone, or with either Probenecid (1mM) or Benzbromarone (1 M). Doses were based on toxicity experiments on

cells growing in FBS and the highest non-toxic dose was used (data not shown). After 3-4 days in culture,

proliferation was assessed by Alamar blue reduction in a colorimetric assay as described in Materials and Methods.

Absorbance measures were normalized to the proliferation readout for uric acid-treated TK6 cells. The experiment

was repeated three times with similar results. (b) G protein-coupled receptor (GPCR) activation by ligand binding

induces the translocation of G proteins (Gs) associated with the receptor, leading to activation of adenylate cyclases

(ACs), which catalyze conversion of ATP to 3‟5‟-cyclic AMP (cAMP) and pyrophosphate. Phosphodiesterases

(PDEs) degrade cAMP to AMP, thereby inhibiting Protein Kinase A (PKA) activation. While AC and PKA

inhibitors should decrease PKA-dependent growth, PDE inhibitors should allow for enhanced growth of cells that

rely on PKA activity. (c) TK6 cells were plated at 5 x 104 cells/ml in RPMI plus ITAG alone, or with 2‟,5‟-

Dideoxyadenosine (2‟,5‟-dd-Ado) (1 M), MDL-12,330A Hydrochloride (MDL-12) (20 M), Pentoxyfylline (250

M), 3-isobutyl-1-methylxanthine (IBMX) (25 M), 14-22 Amide (1 M), or H-89 Dihydrochloride (H-89) (1

M). Cells were also plated in RPMI + ITAG plus uric acid with or without all the listed inhibitors, as well as in

RPMI + ITAG with DPA either with or without all the listed inhibitors except for pentoxyfylline and IBMX. Doses

for all inhibitors were based on toxicity experiments on cells growing in FBS and the highest non-toxic dose was

used (data not shown). After 3-4 days in culture, viability was assessed by flow cytometry using 7AAD exclusion.

The experiments have been repeated at least three times with similar results.

0

10

20

30

40

50

60

70

80

90

100

Controls 2'5'-dd-Ado MDL-12 Pentoxyfylline IBMX 14-22 Amide H-89

% L

ive

ce

lls

ITAG

UA

DPA

AC inhibitors PDE inhibitors PKA inhibitors

ligand

GPCR

Gs Gs AC

ATP cAMP AMP

PDE

inactive PKA

active PKA

2’5’-dd-Ado

MDL-12

Pentoxyfylline

IBMX

14-22 Amide

H-89

51

the GPCR pathway inhibitor studies led to the conclusion that uric acid does not act as a

metabolite and exerts its effect through a mechanism other than PKA activation.

Uric acid activates signaling pathways and leads to cytokine production

To determine if uric acid was acting via signal transduction pathway(s) other than PKA,

proteins from various signaling pathways were examined. Immunoblotting revealed that uric

acid treated TK6 cells had enhanced pERK and pJNK activation five minutes after stimulation,

compared to cells growing in non-supplemented media with ITAG alone (Figure 6a). Both

signals quickly diminished and were barely visible when examined ten minutes after

stimulation, suggestive of receptor activation and ensuing signaling. Notably, one hour after

stimulation, the state of both ERK and JNK activation in uric acid-treated cells was similar to

that of cells grown in media supplemented with serum, indicative of the stable state of the cells

in both cultures (Figure 6a). TK6 cells in serum-free ITAG supplemented media, on the other

hand, exhibit what seemed to be an aberrant signaling pattern with highly enhanced phospho-

ERK expression after one hour, likely a reflection of the stress-inducing conditions in the

culture. The activation statuses of other signaling molecules, namely, pAkt, pIB, pIRAK1,

pMARCKS, pVASP, pSTAT3, and pSTAT1, were also examined and did not reveal any

differences (data not shown).

In addition to protein activation, uric acid-treated TK6 cells also engaged in cytokine

production. While an increase in both IL6 and IL10 was observed, the level of IL10 in uric

acid-treated cells was more than 100X greater than those in cells growing in FBS or serum-free

media (ITAG alone), both of which had negligible IL10 production (Figure 6b). RT-PCR was

also performed to confirm increased IL10 message production in uric acid-treated cells. Indeed,

52

(a)

(b) (c)

Figure 6. Activation of signaling pathways and cytokine production by uric acid-treated TK6 cells. (a) TK6

cells growing in RPMI and 5% FBS were washed twice in PBS and cultured in 4ml volumes at 5 x 105 cells/ml in

RPMI + ITAG alone (Ø) or RPMI + ITAG with uric acid (UA), or RPMI + 5% FBS (FBS). Cells were collected at

the indicated time points (5, 10, 30, and 60 minutes for Ø and UA, and 60 minutes only for FBS) and

phosphorylation of JNK and p42/44 ERK were determined by immunoblotting with phosphospecific Abs as

described in the Materials and Methods section. -actin was also probed for as a loading control. Similar results

were obtained from three separate experiments. (b) Supernatants were collected from TK6 cells growing in RPMI

+ 5% FBS, RPMI + ITAG and RPMI + ITAG with uric acid 24 hours after start of the culture period, and cytokine

production from each treatment was measured as described in the Materials and Methods section. The averages

and standard errors of IL6 and IL10 production for the indicated number of experiments are shown. (c) mRNA

expression was measured from TK6 cells growing in ITAG with or without uric acid 1 hr and 4 hr after treatment as

determined via RT-PCR (see Materials and Methods). Expression values are normalized to mRNA amounts of the

housekeeping gene, HPRT. The averages and standard errors of IL10 expression for the indicated number of

experiments are shown. * indicates P < 0.05.

β-actin

pERK

Ø UA Ø UA Ø UA Ø UA FBS

5mins 10mins 30mins 1 hr _

β-actin

pJNK

Ø UA Ø UA Ø UA Ø UA FBS

5mins 10mins 30mins 1 hr _

53

1 and 4 hours after stimulation, a greater amount of IL10 expression at the mRNA level was

observed in these cells (Figure 6c).

IL10 inhibition partially blocks the proliferative effects of uric acid

In order to determine if increased IL10 production was the mechanism via which uric

acid was exerting its proliferative effects, recombinant human IL10 was added to serum-free

media to test if IL10 could mimic the growth effects observed in uric acid-treated TK6 cells. No

proliferation was observed in cells treated with IL10, and in fact, these cells behaved exactly

like those cultured in ITAG alone and died within 2-3 days (data not shown). Although

enhanced IL10 levels appeared insufficient in explaining its growth effects, it was possible that

IL10 was partly responsible for uric acid-induced proliferation. This did indeed prove to be the

case as inhibition of IL10 resulted in a significant decrease in the number of live uric acid-

treated TK6 cells (Figure 7, left panel). As assessed by cell counts based on trypan blue uptake,

both treatments, however, exhibited similar amounts of cell death, implying that in the absence

of IL10 production, uric acid still allowed TK6 cells to grow, but at a significantly slower pace

(Figure 7). These results indicate that uric acid, at least in part, exerts its proliferative effects via

production of the IL10 cytokine, and that IL10, although necessary, is insufficient to fully

recapitulate the growth effects of uric acid (Figure 8).

54

Figure 7. IL10 inhibition leads to loss of uric acid-dependent proliferative potential in TK6 cells. 5 x 104

TK6 cells were plated in RPMI + ITAG, RPMI + ITAG + uric acid, with or without anti-IL10 antibody (1 g/ml).

After 3-4 days of growth in culture, the number of live (left panel) and dead cells (right panel) were assessed by

trypan blue uptake and counted in a hemocytometer. Proliferation is depicted as differences in cell counts between

uric acid-treated and uric acid plus IL10-treated cells and differences in the number of dead cells in the two

treatments is also shown. P value was determined by a paired t test for the indicated number of experiments.

0

10

20

30

40

50

UA UA + anti-IL10

Liv

e c

ells

(x

10

^4

)

0

10

20

30

40

50

UA UA + anti-IL10D

ea

d c

ells

(x

10

^4

) (n=3) (n=3) (n=3) (n=3) *

P < 0.05

55

Figure 8. Mechanism of uric acid-dependent growth effects on activated B cells. The results from this study

implicate uric acid as a growth factor for activated B cells that likely functions at the cell surface via a receptor.

Upon receptor engagement, various signaling pathways appear to be activated, including those involving ERK and

JNK, as demonstrated by increased expressions of the phosphorylated forms of the two proteins. Cytokine

production, particularly that of IL10, and to a lesser extent, that of IL6 is also observed. Inhibition studies

involving anti-IL10 have revealed that uric acid does, in fact, exert some of its proliferative effects via enhanced

levels of IL10 production.

ERK JNK

IL6 IL10

Cell Growth

Uric acid

Cytosol

Nucleus

56

Chapter 4:

DISCUSSION AND FUTURE DIRECTIONS

57

4.1 DISCUSSION

The idea that activated B cells can serve as APCs and replace DC-based cancer vaccines

is not novel. In fact, many researchers in the last decade have been studying B cells in a quest to

find viable alternatives to generate autologous vaccines [69,71,98,99,239]. This search is

especially relevant to cancer patients in whom large volume leukapheresis is not practical, such

as pediatric patients or breast cancer patients that have had to undergo mastectomy and axillary

lymph node dissection. While clinical trials have yet to take place, most recently, activated B

cells generated from canine lymphoma patients with CD40 and IL4 stimulation and loaded with

antigen-specific mRNA showed T cell responses in vitro [239].

Earlier this year, Wiesner et al. also reported that they were successfully able to generate

long-term activated human B cell cultures that have the capacity to undergo proliferation

indefinitely via activation of the cell surface CD40 receptor in the presence of IL4 [240]. The

CD40/IL4 combination (CD40-B cells) mimics B cell activation by T helper cells, and these

cells were originally used as an in vitro model system to study B cell differentiation into plasma

cells [103,241]. CD40-B cells exhibited uninhibited CD40 ligand (CD40L)- and IL4-dependent

growth, with more than 370 population doublings, thereby justifying characterization as

„conditionally immortalized‟ [240,242]. Moreover, CD40-B cells could also activate and induce

expansion of antigen-specific CD8+ T cells, demonstrating their potential as effective APCs in a

vaccine setting.

Weisner‟s results provide evidence for the ability to generate long term activated B-cell

cultures. Such cells however, may not serve as adequate DC-based vaccine replacements due to

problematic generation protocols that rely on the use of xenogeneic components like FBS and

murine fibroblast feeder cells (NIH3T3) transfected with human CD40L as a source of CD40

58

stimulation. NIH3T3 cells in particular, cannot be used under good manufacturing practice

(GMP) conditions due to their xenogeneic origins. In addition, regulations concerning the

therapeutic use of such activated B cells for the purpose of creating cancer vaccines would be

similar to those for xenotransplantion and gene therapy, resulting in restricted clinical

applicability [243]. The use of a soluble recombinant human CD40L is preferable in that regard

[71]. Alternatively, Mason et al. described a novel method which involves transfection of a

human erythroleukemic cell line with a CD40L expression plasmid, resulting in the generation

of feeder cells that are able to provide stimulation to the CD40 receptor [239]. The main

concern in instances where the use of non-human components is employed is the potential

danger to patients as a result of contact with foreign entities, which has the possibility of causing

widespread inflammation in the body. Detrimental effects may result upon exposure to animal

proteins in the cells or infections caused by animal microbes. The concern is relevant to other

situations as well, most notably that of human embryonic stem cell generation and expansion,

where the search for xenogeneic-free culture components is a dominant topic of discussion in

the field [244-247].

The need to replace serum from bovine origins is also crucial. Not only is FBS

potentially harmful due its xenogeneic nature, but its use also entails the drawback of including

an unknown and uncontrolled set of substances in the expansion protocol, making it harder to

interpret growth requirements [226]. Although the use of autologous human serum is a

possibility, it may not be the best alternative given the added procedural requirements. In

addition, it makes for the generation of long-term activated B cell cultures more cumbersome

and potentially inefficient if several phlebotomies are needed for the purposes of serum

derivation. A better solution is to replace FBS with its crucial active ingredients and supplement

growth media with such components.

59

The above-mentioned considerations led to the use of an antioxidant rich medium

(Invitrogen‟s Advanced D-MEM/F-12) supplemented with insulin, transferrin, albumin, and

glutamine for the generation of human activated B cells. AICD was still a major concern and

uric acid demonstrated an enhanced ability to alleviate oxidative stress and allowed for

increased cell proliferation. The use of uric acid thus presents a new approach for the

cultivation of B cells as APCs. In fact, the data presented herein not only confirmed the

antioxidant properties of uric acid, but also pointed towards a novel function beyond its

ostensible role as an antioxidant, affording new insights into B cell growth. Apart from

addressing the challenge of AICD, uric acid activated ERK signaling in TK6 cells and increased

IL10 production, which was shown to be necessary for its proliferative effects. Taken together,

the results from this study led to the proposition that uric acid functions as a growth factor for

activated B cells, and also implicated the presence of a cell surface receptor that mediates uric

acid signaling.

Until recently, uric acid was for the most part viewed simply as an antioxidant and

interest in the molecule was marginally sustained due to its role in causing gout [248], and also

due to a longstanding correlation between hyperuricemia and cardiovascular disease [249,250].

Lately, however, uric acid has garnered renewed interest because of its recent identification as a

danger signal that is released by injured cells, enabling it to function as a stimulus for activating

immune responses [251]. The “danger model” [252,253] posits that APCs such as DCs can be

activated by endogenously produced molecules released by injured cells into the extracellular

environment. Such signals serve to promote DC activation and engender subsequent T cell

responses alongside antigen presentation. Employing a host of in vitro as well as in vivo studies,

Shi et al. identified uric acid to be a primary immunological danger signal [251]. A significant

increase in uric acid levels was observed when mouse lymphoma cells (EL4 cells) were forced

60

into an injurious state, and exposure to uric acid led to a strong increase in the expression of

costimulatory molecules in cultured DCs from healthy mice. In vivo significance of the

molecule was demonstrated by showing that mice injected with uric acid and antigen displayed

enhanced CD8+ T cell priming. Furthermore, when injured cells treated with allopurinol (a uric

acid analogue that reduces production of uric acid) and uricase were injected into mice that were

also exposed to the two inhibitors, significantly reduced plasma uric acid concentrations were

observed and priming of effector T cells was also reduced by almost 90% in the mice.

Subsequent investigations by other groups have further demystified this danger-alerting role of

uric acid. Increased levels of uric acid were observed in tumour cells undergoing death

processes, either due to responsiveness to chemotherapy, or as a result of immune rejection

[254]. Its withdrawal delayed the rejection process, whereas uric acid administration enhanced

it. Also, in the absence of T cell expansion, augmented antibody immunity was shown to be

induced by uric acid [255].

Given that uric acid is the end product of purine catabolism, it is likely that when cells

get injured and degrade their nucleic acids, the resultant purine build up allows for increased

accumulation of uric acid [251]. Dying cells are thus able to produce greater amounts of danger

signal without relying on protein synthesis [256]. With relation to immune surveillance, Shi et

al. propose a model which comprises dying cells simultaneously releasing antigens that are

picked up by DCs upon ingestion of cellular debris, as well as uric acid. The uric acid „danger

signal‟ allows DCs to mature and become immunostimulatory, thereby allowing the immune

system to respond even to tumours and infections that may not be associated with adjuvant

stimulation [251].

Although seemingly incongruent upon first glance, the newly identified role of uric acid

as a growth factor of activated B cells is not inconsistent with the recent reports describing uric

61

acid as an immunostimulatory danger signal. The key difference lies in the dose – the

concentration of uric acid at which DCs are stimulated corresponds to the point at which uric

acid precipitates into crystals. Unlike preformed monosodium urate (MSU) crystals, soluble

concentrations of uric acid (such as that used in the described experiments (0.1mg/ml)) cannot

activate DC responses [251]. Though uric acid is relatively insoluble in biological fluids, its

concentration in the cytosol is much higher (> 4 mg/ml) and increases dramatically in dying

cells as a result of DNA and RNA degradation. Upon lysis, the extracellular environment then

becomes flooded with high levels of uric acid, thus favouring MSU formation. These crystals

can also stimulate other immunological constituents like monocytes, leading to cytokine

production and further activation of the immune system [257]. In the same vein, along with

other endogenous danger signals, hyperuricemia may inspire autoimmunity in genetically

predisposed individuals [251]. In fact, it was recently reported that uric acid in the context of

gout can activate the NALP3 inflammasome [258], which is a known sensor of other

endogenous danger signals like extracellular ATP or hypotonic stress [259]. Soluble uric acid

on the other hand, seems to function as a separate molecule and possesses activity that appears

to be distinct from that of its crystalline form. The proliferative advantage of uric acid in

activated B cell cultures was observed only at the soluble dose of 0.1 mg/ml and this effect

could not be replicated at higher doses (data not shown). When used at 0.75 mg/ml, uric acid

precipitated out into the culture medium as crystals and was incapable of rescuing cells from

AICD.

That uric acid can possess growth factor activity at soluble levels has also been reported

in rat vascular smooth muscle cells (VSMCs) in an in vitro setting [260]. Uric acid was shown

to stimulate DNA synthesis, and as seems to be the case with activated B cells, this proliferative

capacity was independent of its antioxidant function. In addition, uric acid-induced growth in

62

VSMCs also implicated the involvement of signaling pathways as confirmed by the activation

of ERK1/2 [261], and an increase in the expression levels of platelet-derived growth factor

[260]. Further investigations led to the discovery that uric acid at the same soluble doses can

also directly exert proinflammatory effects on VSMCs [262]. The chemokine, monocyte

chemoattractant protein-1 (MCP-1), which is a major mediator of inflammation and is known to

play an active role in the initiation of atherosclerosis-associated lesions [263], was produced at

much higher levels upon exposure to uric acid. MCP-1 production was mediated via p38

MAPK, as well as activation of the nuclear transcription factors NF-B and AP-1. Uric acid

stimulation also resulted in the production of other inflammatory molecules such as IL1, IL6

and TNF [262]. Given that uric acid was found to stimulate IL10 production in TK6 cells, it

may be that it also possesses similar immunogenic capabilities in activated B cells as in VSMCs.

The results emerging from VSMC studies not only point to a potential mechanism

through which uric acid mediates vascular disease, but also highlight the many facets of uric

acid stimulation in different settings, as illustrated by the results presented here as well. In fact,

in light of the deleterious effects on vascular cells, and its role in cardiovascular disease,

atherosclerosis and kidney disease, many researchers are beginning to question the previously

unchallenged role of uric acid as an antioxidant [264-266]. How this information plays out with

regards to the role of uric acid in B cells and the distinction between its soluble and insoluble

crystalline form remains to be ascertained.

63

4.2 FUTURE DIRECTIONS

In summary, the results from this study suggest a novel role for soluble uric acid as a

growth stimulant for activated B cells. This conclusion was arrived at primarily from

experiments conducted in TK6 cells. In order to validate these results and ensure the soundness

of their implications, it is essential to perform these studies in normal human B cells, and

secondarily, it is also of interest to validate these results in murine B cells. In particular,

experiments involving uric acid transport inhibitors, confirmation of ERK and JNK activation,

confirmation of IL10 production, and IL10 inhibitor studies need to be repeated in normal B

cells so that it can be assessed if the results observed in TK6 cells are an aberrant response due

to the malignant nature of the cell line, or if they hold true for both normal and transformed B

cells. The applicability of the uric acid response to healthy B cells is especially crucial if the

results are to be interpreted in the context of normal B cell growth and function. Eventually, the

effects of uric acid on T cells, macrophages, and DCs can also be studied to determine if a

functional pattern can be ascribed to the activity of uric acid in immune cells.

Along with repeating the experiments in normal B cells, better characterization of the

response induced by uric acid in TK6 cells is needed. The experiments described here only

surveyed those proteins involved in major signaling pathways (as determined by their

phosphorylation status) and cytokine production was also only minimally examined. A more

large scale study involving gene arrays assessing activation of transcription factors and

production of messenger RNA needs to be undertaken in order to fully assess the signaling

pathways activated in TK6 cells by uric acid. A study of this magnitude would be particularly

beneficial in specifying the nature of the immunostimulatory (or not) response to uric acid.

Likewise, an examination of the changes in gene expression upon IL10 inhibition will help

64

further elucidate the role of this cytokine and shed light on the mechanism(s) via which IL10

mediates the proliferative effects of uric acid.

Although it seems likely that a receptor at the cell surface is activated upon exposure to

uric acid, further studies need to be conducted before the issue of receptor involvement can be

conclusively settled. A dose response study of uric acid as it relates to ERK activation, for

instance, would be helpful in this regard as the determination of the precise dose of uric acid that

leads to ERK activation would enable more fine-tuned studies to be performed, particularly as it

pertains to receptor identification. Blocking experiments involving ERK inhibitors and how the

uric acid response changes on a genome wide scale as observed in gene expression studies

would also be beneficial in elucidating the precise role of ERK and whether its activation is

sufficient and/or necessary in mediating the effects of uric acid. Studying the effects of JNK

inhibitors along with ERK inhibitor studies would also be helpful in determining if multiple

contradictory or confirmatory pathways are activated upon stimulation via uric acid.

If it becomes more apparent that uric acid functions extracellularly via engagement with

a cell surface receptor, a strategy to identify and clone the receptor needs to be determined.

Since there are no known receptors of uric acid, it is difficult to pinpoint likely candidates. In

terms of receptor class, given that uric acid is a purine derivative, it is possible that it activates a

purine receptor and mediates its effects through one of the known purinergic receptors. The

adenosine or P1 receptors, all of which couple to G proteins, and P2 receptors that primarily

engage ATP, ADP, UTP, and UDP are the two main families of purine receptors [267].

Although it would be worthwhile to examine P1 receptors, it is likely that uric acid does not act

via this family given that its members are GPCRs and lack of PKA activity (normally induced

upon engagement of GPCRs) did not abrogate the survival effects of uric acid in TK6 cells. P2

receptors, on the other hand, can be subdivided into two families, one of which consists of

65

GPCRs (P2Y receptors), and the other, the P2X receptors, which are ligand-gated ion channels

[267]. In fact, the P2X subclass of purine receptors are known to be expressed on VSMCs

[268], and it may be that uric acid exerts its effects on these cells via the activation of a P2X

receptor. The P2X7 receptor (P2X7R), in particular, is an attractive candidate given its

involvement in mediating immune responses in macrophages and leukemic lymphocytes [267].

In fact, it has been reported that IL1 secretion in murine macrophages as a response to

extracellular stimuli is not possible without the autocrine activation of P2X7R by ATP [269].

More recently, it has been reported that danger signals such as MSU crystals induce endogenous

ATP release and that antagonists of P2X7R can block the MSU-induced IL1 and IL18 secretion

in human monocytes [270]. While this information may prove to be irrelevant, it may be a

pragmatic starting point to determine if soluble non-crystalline uric acid can engage any of the

P2X receptors, in particular, P2X7, via use of receptor antagonists [267]. For the sake of

thoroughness, a quick screening of the results obtained by blocking P2Y receptors and P1

receptors in uric acid-treated TK6 cells should also be undertaken.

If uric acid is unable to act as a ligand for purine receptors, other strategies for

identification of its receptor will be required. Although it may be cumbersome, the activation of

ERK upon uric acid treatment can be taken advantage of for the development of a more direct

approach to receptor identification via functional screening of ERK activation and subsequent

cloning strategies. A reporter cell line that displays activation of the AP1 transcription factor,

which is a downstream activator of the ERK protein, can be used in an assay to monitor ERK

activity. Such a reporter line can be transfected with subsets of the TK6 gene library and ERK

activation can be assessed via luciferase or GFP activity upon treatment with uric acid. Isolation

of GFP/luciferase-positive clones in subsequent phases involving smaller and smaller gene

regions would eventually lead to the isolation of the uric acid receptor gene, which can then be

66

identified by cDNA cloning. Reporter cell lines are widely available and can be purchased

directly. Panomics Inc. (Fremont, CA) for instance, produces an AP1 reporter line derived from

human 293T cells that induces luciferase-tagged AP1 activity upon treatment with known

activators like phorbol myristate acetate. Such a reporter line, if easily transfectable, may be

ideal for a screening-based identification strategy.

Discovery of the uric acid receptor will shed further light on the intracellular

mechanisms via which uric acid exerts its effects, and perhaps reveal more information

pertaining to the disparity between the functions of soluble and crystalline uric acid, which

needs to be further explored. Moreover, based on the nature of the identified receptor, it can be

determined if the growth-inducing properties of uric acid are unique to certain cell types or not.

The implication arising from the antioxidant properties of uric acid and its apparent

function as a growth factor is that uric acid can be used in the generation of large numbers of

activated B cells that can serve as antigen presenting platforms in cancer vaccines. Further

studies characterizing the immunogenicity of uric acid treated human S2 B cells will be required

before investigations related to the development of vaccines can be initiated. Based on

previously reported results with CLL cells [109], it is plausible that normal activated B cells will

also require further exposure to PKC agonists before they are able to achieve maximal

upregulation of costimulatory molecules and produce inflammatory cytokines that can mediate

sufficiently strong CD8+ T cell responses. However, it may well be that soluble uric acid alone

can induce such immunostimulatory changes, as reported in VSMCs.

It is also worth investigating whether the two „danger signal‟ and growth factor roles of

uric acid can be married into an effective immunotherapeutic strategy for cancer treatment.

Perhaps soluble uric acid can be used to induce proliferation of S2 B cells and exposure to the

crystalline form can subsequently enable the acquisition of a DC-like phenotype. Such a

67

protocol will be more efficient and save time and money with regards to vaccine development.

Even if this is not the case, the implications for immunotherapy are still exciting given that uric

acid crystals could potentially be administered with B cell-based cancer vaccines for the

induction of potent DC activation, thereby dispensing the need for an initial T cell boost with

DC-based vaccines.

As further progress is made with regards to its mechanisms of action, a cohesive picture

of the seemingly disparate roles of uric acid in maintaining health and disease will hopefully

emerge. The data presented in this thesis have furthered this endeavour and have shed light on

the diverse nature of this molecule that is only now beginning to be appreciated.

68

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