BAFF Regulation of Peripheral T Cell Responses

BAFF regulation of peripheral T cell responses

Andrew Peter Robert Sutherland

A submission to the University of New South Wales in candidature for the degree of Doctor of Philosophy

Arthritis and Inflammation Research Program Garvan Institute of Medical Research Darlinghurst, Sydney, Australia December 2005

ACKNOWLEDGEMENTS

Thanks go to my supervisors, Charles and Fabienne Mackay, for their financial and

intellectual support during the course of this project. I have enjoyed working on a project that fused both your areas of expertise and enabled us to explore new areas of BAFF biology. I have learnt a great deal under your tutelage. During this time I have also watched level 10 grow from a small nucleus of individuals to a substantial research department. It has been a pleasure to be part of such a vibrant, dynamic environment. I wish you all the best in your future endeavours.

A special thanks to Shane Grey for his constant and infectious enthusiasm. I have

greatly appreciated our interactions over the second half of my PhD and thank you for the

many helpful suggestions you have had along the way. You have played a significant

part in getting this thesis to completion.

Thanks to the members of the BAFF lab, both past and present. Lai Guan for his

tireless work during the initial stages of these projects, Rebecca for her guidance and

training, Carrie for her help with animal models, Blanche for providing a steady stream of

genotyped mice, Jo and Julie for suggestions and lab prowess, Cynthia and Jenny for

assistance with the production of monoclonal antibodies. Your help was invaluable.

To my colleagues and friends at the Garvan Institute, thank you for your kind words,

support and encouragement. There have been countless hours of enjoyment during my 6

year stint at the Garvan and I have made many strong friendships that I’m sure will last

me a lifetime.

Finally a special thanks to my family for their constant love, belief and reassurance.

Thank you for supporting me during the 21 years that I have been a student, and for

giving me this opportunity to quench my thirst for knowledge. Such a gift is afforded to very few people, and is one for which I am eternally grateful.

Experiments that were not the sole work of the author

All experiments were performed by the author at the Garvan Institute, with the exception of:

Fig. 3.6C, left panel – experiment performed by Ian Sutton

Fig. 3.8A – experiment performed by Rebecca Newton

Fig. 3.9A-D – experiments performed by Lai Guan Ng

Fig. 4.3B and C – experiments performed by Lai Guan Ng

Fig. 4.6B – analysis performed by Fabienne Mackay

iii

ABBREVIATIONS

AEEC Australian Experimentation Ethics Commitee ALPS autoimmune lymphoproliferative syndrome AP-1 activating protein-1 APC antigen presenting cell APRIL a proliferation inducing ligand BAFF B cell activation factor from the TNF family BAFF-R BAFF receptor BAL bronchoalveolar lavage BCMA B cell maturation antigen BCR B cell receptor BSA bovine serum albumin BTF Biological Testing Facility CDK cyclin dependent kinase CFA complete Freund’s adjuvant CFSE carboxyfluorescein diacetate succinimidyl ester CIA collagen induced arthritis CIITA class II transactivator CMV cytomegalovirus CRD cysteine rich domain CTLA cytotoxic T-cell-associated DC dendritic cell DD death domain DMEM Dulbecco’s modified Eagle’s medium DMSO dimethylsulfoxide DTH delayed type hypersensitity EAE experimental allergic encephlomyelitis EGFP enhanced green fluorescent protein ELISA enzyme linked immunosorbent assay ERK extracellular regulated kinase FACS fluorescence activated cell sorting FADD Fas associated death domain protein FO follicular GC germinal centre GFP green fluorescent protein H&E hematoxylin and eosin HEL hen egg lysozyme hIg polyclonal human immunoglobulin HIV human immunodeficiency virus ICAM intercellular adhesion molecule ICOS inducible costimulator IFN interferon IKK IκB kinase ITIM immunoreceptor tyrosine-based inhibitory motifs JNK c-Jun NH2-teminal kinases

KO knockout LCMV lymphocytic choriomeningitis virus LPS lipopolysaccharide mAb monoclonal antibody mBSA methylated bovine serum albumin MHC major histocompatibility complex MLR mixed lymphocyte reaction MZ marginal zone NFAT nuclear factor of activated T cells NFκB nuclear factor κB NIK NFκB inducing kinase OVA ovalbumin PAMP pathogen associated molecular pattern PBMC peripheral blood mononuclear cells PBS phosphate buffered saline PKC protein kinase C RA rheumatoid arthritis RBL rat basophilic leukemia RF rheumatoid factor SLE systemic lupus erythematosus SS Sjogren’s syndrome T1 transitional type 1 T2 transitional type 2 TACI transmembrane activator and calcium-modulator and cyclophilin interactor TCM central memory T cells TCR T cell receptor TEM effector memory T cells TFH follicular B helper T cells Tg transgenic Th helper T cells THD TNF homology domain TLR Toll like receptor TNF tumour necrosis factor TNFRSF tumour necrosis factor receptor superfamily TNFSF tumour necrosis factor superfamily TRADD TNF receptor associated death domain protein TRAF TNF receptor associated factor

ABSTRACT

The activation and effector function of CD4+ T cells are critical points of regulation

during an antigen specific T cell response. Dysregulation of these processes can lead to

the development of human diseases, encompassing both immunodeficiency and autoimmunity. Members of the TNF superfamily have recently emerged as important regulators of T cell responses, with their overexpression causing autoimmune inflammation in animal models. As overproduction of the novel TNF superfamily ligand

BAFF is associated with several autoimmune conditions, we sought to examine the potential role of BAFF as a regulator of T cell activation and effector function.

We initially demonstrated BAFF costimulation of T cell activation in vitro.

Generation of specific monoclonal antibodies identified BAFF-R as the only BAFF receptor present on T cells, and showed that it was expressed in an activation-dependent and subset-specific manner. Impaired BAFF costimulation in BAFF-R deficient mice indicated that BAFF-R was crucial for mediating BAFF effects in T cells. Analysis of T cell responses in vivo revealed that BAFF transgenic mice have increased T cell priming and recall responses to protein antigens, and showed a corresponding increase in the DTH model of Th1 cell-dependent inflammation. In addition, Th2-dependent allergic airway responses are suppressed in BAFF transgenic mice. Crossing to a B cell deficient background revealed that the proinflammatory effects of BAFF on T cell priming and

DTH rely on the presence of B cells, while the suppressive effects during allergic airway inflammation are B cell independent. These data demonstrated that BAFF regulated the outcome of T cell responses in vivo and identified BAFF dependent crosstalk between T and B cells. Stimulation of B cells with BAFF induced the upregulation of MHC class II

and ICOS-L both in vitro and in vivo. Induction of these cell surface molecules was associated with an increased capacity to induce T cell proliferation, however this effect was independent of ICOS-L expression. Thus it was demonstrated that BAFF regulated

T cell activation and effector function both directly, via stimulation of BAFF-R, and indirectly, by altering the function of B cells. These data suggest that BAFF dependent alterations in T cell function may be an additional causative factor in the association between elevated BAFF levels and the generation of autoimmunity.

vii

MANUSCRIPTS AND PRESENTATIONS ARISING

FROM THIS THESIS

Referred Journals

1. Sutherland AP*, Ng LG*, Fletcher CA, Shum B, Newton R, Grey ST, Rolph MS, Mackay F and Mackay CR. BAFF augments certain Th1-associated inflammatory responses. The Journal of Immunology, 2004, 174: 5537-44 *These authors contributed equally to the work

2. Ng LG*, Sutherland AP*, Newton R, Qian F, Cachero TG, Scott ML, Thompson JS, Wheway J, Chtanova T, Groom J, Sutton IJ, Xin C, Tangye SG, Kalled SL, Mackay F, Mackay CR. B cell- activating factor belonging to the TNF family (BAFF)-R is the principal BAFF receptor facilitating BAFF costimulation of circulating T and B cells. The Journal of Immunology, 2004, 173: 807-817 *These authors contributed equally to the work

Reviews

1. Sutherland AP, Mackay CR and Mackay F. Therapeutic targeting of BAFF in autoimmune and inflammatory disorders. Pharmacology and Therapeutics, manuscript in preparation

Presentations

1. Sutherland AP, Ng LG, Mackay F and Mackay CR. BAFF regulates Th1 and Th2 responses. Symposium session, Australasian Society for Immunology annual conference, Adelaide Australia, 13-16 December 2004

Posters

1. Sutherland AP, Ng LG, Mackay F and Mackay CR. BAFF regulates Th1 and Th2 responses. Poster session, Keystone conference, Dendritic Cells at the Center of Innate and Adaptive Immunity: Eradication of Pathogens and Cancer and Control of Immunopathology, Vancouver Canada, 1-7 February 2005

2. Sutherland AP, Ng LG, Mackay F and Mackay CR. BAFF stimulates dendritic cell function and regulates the Th1/Th2 balance. Poster session, Cytokines, Signalling and Diseases, International Society for Interferon and Cytokine Research (ISICR) annual meeting, Cairns Australia, 26-30 October 2003

3. Sutherland AP, Ng LG, Mackay F and Mackay CR. BAFF costimulates T cells, induces dendritic cell maturation and promotes Th1 type responses. Poster session, St Vincent’s Symposium, Sydney Australia, 11 September 2003

viii

Table of contents

Acknowledgements...... ii Abbreviations...... iv Abstract...... vi Manuscripts and presentations arising from this thesis ...... viii Chapter 1: Introduction...... 1 The adaptive immune system, tolerance and autoimmunity...... 1 Initiation and regulation of CD4+ T cell responses...... 2 T cell activation...... 2 Costimulation...... 3 CD28 and CTLA-4 ...... 5 ICOS and PD-1 ...... 7 Th1/Th2 differentiation and effector function ...... 11 TNF and TNF receptor superfamilies...... 16 Immune function...... 16 Structure...... 17 Signalling pathways...... 19 Control of T cell responses ...... 21 BAFF...... 27 Expression and regulation...... 27 Receptors...... 29 Function ...... 30 Signalling pathways...... 34 Autoimmunity...... 37 Therapeutic targetting ...... 41 Hypothesis and project aims ...... 42 Chapter 2: Materials and Methods...... 44 Buffers...... 44 Reagents...... 44 Animals...... 45 Lymphocyte isolation and culture...... 46 Cell stimulations and proliferation assays ...... 48 cDNA synthesis and LightCycler quantitative RT-PCR ...... 50 PCR and primer sequences ...... 51 Agarose gel electrophoresis ...... 52 GeneChip microarray analysis...... 52 Transfectant construction and DNA sequencing ...... 53 Production and specificity of mAbs to human BAFF receptors...... 55 Biotinylation ...... 56 ELISA ...... 57 Flow cytometry ...... 58 Histology, immunohistochemistry and immunofluorescence...... 59 Immunisations and antigen restimulations...... 61 DTH ...... 62 OVA-induced allergic airway inflammation ...... 62

Statistical analysis...... 63 Chapter 3: BAFF costimulates T cell activation via BAFF-R...... 64 Introduction...... 64 Results...... 65 Co-stimulatory effects of BAFF on human T-cell proliferation...... 65 BAFF has additional repressive effects on T cell proliferation via secondary cell types and factors ...... 67 BAFF-R but not TACI or BCMA transcripts are expressed by various T cell populations...... 69 Construction of transfectants for monoclonal antibody generation...... 71 Generation and validation of monoclonal antibodies to human BAFF-R and TACI ...... 74 BAFF-R, TACI and BCMA have unique expression patterns on human B cells. 74 Expression of BAFF-R by human T cells...... 78 BAFF-R is differentially expressed by T cell subsets and modulated during activation...... 80 BAFF-R is expressed on mouse T cells and mediates BAFF costimulation ...... 82 Discussion...... 85 Chapter 4: BAFF modulates T cell responses in vivo...... 90 Results...... 91 BAFF transgenic mice display overtly normal responses to mitogens in vitro .... 91 Increased antigen specific recall responses in BAFF transgenic mice ...... 91 Enhanced DTH responses in BAFF transgenic mice...... 95 BAFF levels determine the magnitude of the DTH response...... 97 BAFF transgenic mice have compromised allergic airway responses...... 99 Numbers of effector memory T cells are increased in BAFF transgenic mice, but not in μMT-/- x BAFF transgenic mice ...... 101 BAFF mediated enhancement of DTH is B cell dependent...... 103 BAFF mediated suppression of allergic airway inflammation is B cell independent ...... 105 Discussion...... 107 Chapter 5: BAFF regulation of T-B cell collaboration...... 114 Introduction...... 114 Results...... 116 BAFF regulates the expression of antigen presentation molecules via BAFF-R 116 BAFF stimulated B cells show a modest enhancement in allostimulatory capacity ...... 118 BAFF transgenic mice display altered expression of MHC class II and ICOS-L ...... 120 B cells from BAFF transgenic mice have enhanced allostimulatory capacity ... 122 Increased expression of ICOS-L is not responsible for the increased B cell stimulatory activity mediated by BAFF...... 124 B cell expansion alone cannot augment DTH responses: TACI-/- mice have normal DTH responses ...... 127 B cell expansion alone cannot augment DTH responses: Traf2 lox/lox X Mx-1 Cre mice have normal DTH responses ...... 129

Discussion...... 131 Chapter 6: General Discussion and Conclusions...... 137 References...... 148 Reprints of primary author publications ...... 189

xi CHAPTER 1 INTRODUCTION

CHAPTER 1: INTRODUCTION

THE ADAPTIVE IMMUNE SYSTEM, TOLERANCE AND AUTOIMMUNITY

The ability of the adaptive immune system to protect the host from pathogenic challenge lies in its unparalleled capacity to generate antigen receptor diversity. Random

junction of the VDJ gene families and unique mechanisms of somatic mutation accord

the receptors of the T and B lymphocytes the potential to bind almost any chemical

structure. While this represents a powerful form of pathogen recognition and clearance,

it poses the host with new dilemmas; how to discriminate the molecules that constitute

ones’ self from those of pathogenic organisms. The consistent ability of the adaptive

immune system to respond appropriately to foreign antigen is a central and enigmatic

issue in immunology.

In order to minimise the damaging effects of autoreactive lymphocytes, the immune

system has evolved multiple, integrated control mechanisms [1]. Functional censoring of

the lymphocyte repertoire occurs during maturation in the central immune tissues of the thymus and bone marrow. In these tissues, lymphocytes bearing functional antigen receptors are selected for (positive selection), while those with potentially autoreactive specificities are deleted (negative selection). This process balances functional ability against potential harm to the host and generates a restricted lymphocyte repertoire that then migrates to the peripheral lymphoid tissues. Despite these mechanisms of selection, a level of self-reactivity still exists in the normal peripheral lymphocyte repertoire [2], however, this is usually effectively controlled by additional peripheral mechanisms.

1 CHAPTER 1 INTRODUCTION

Disturbances in central or peripheral control mechanisms can result in the escape and

activation of self reactive clones. The result is a diverse spectrum of autoimmune

diseases, stemming from the generation of immune responses against the body’s own

tissues. While the incidence of specific disorders is relatively rare, together all of the

autoimmune diseases affect about 5% people in Western countries [3]. The incidence of autoimmune disease in developed nations continues to increase, particularly for diseases such as type I diabetes and systemic lupus erythematosus (SLE) [4]. While each autoimmune disease possesses an intrinsic etiology, it is likely that common mechanisms are involved in the genesis of the various diseases, as there is familial clustering for susceptibility to autoimmunity [3]. Our understanding of these common mechanisms and pathways is far from complete and, given the looming health burden of autoimmune syndromes, presents an important area of research.

INITIATION AND REGULATION OF CD4+ T CELL RESPONSES

T cell activation

Regulation of naïve CD4+ T cell activation is an essential component of peripheral

control. Naïve CD4+ T cells primarily recirculate through secondary lymphoid organs [5-

7], where their access to antigen is regulated. Here specialised antigen presenting cells

(APC) such as dendritic cells, B cell and macrophages, present captured antigen to CD4+

T cells, in the context of MHC class II molecules. Activation of naïve CD4+ T cells

proceeds in the T cell-rich areas of lymphoid tissue in response to APCs. While B cells

and macrophages can function as efficient APCs, they are mostly excluded from the T

cell rich areas of lymphoid tissue and thus are unlikely to be crucial in naïve CD4+ T cell

2 CHAPTER 1 INTRODUCTION

activation. Most T cell responses are seemingly normal in mice that lack either of these

cell types [8, 9]. These data indicate that the dendritic cell is the crucial APC for eliciting

activation of naïve CD4+ T cells.

Immature dendritic cells demonstrate little ability to induce CD4+ T cell activation,

instead inducing T cell tolerance [10]. The activating potential of dendritic cells is

modulated in response to maturation stimuli. In keeping with the “infectious non-self”

theory [11, 12] pathogen derived products e.g. DNA, RNA, lipopolysaccarides activate

dendritic cell maturation via Toll like receptors (TLR) [13, 14]. Additionally, molecules

that are released upon cell damage or infection e.g. heat-shock proteins and type I

interferons stimulate dendritic cell maturation, with the action of these endogenous

factors forming the basis of the “danger” hypothesis of immune regulation [15-17]. After

encountering maturation stimuli, dendritic cells acquire a mature phenotype characterized

by enhanced MHC class II and costimulatory molecule expression and increased proinflammatory cytokine production [18]. It is the induction of these cell surface and

soluble factors that imparts the mature dendritic cell with its potential to activate naïve T

cells.

Costimulation

The expression of costimulatory molecules on mature APCs is a critical component of

their activating potential. This is due to the requirement for two distinct signals before

CD4+ T cells can be effectively activated [19-21] (Fig. 1.1). Signal one constitutes the

interactions between T cell receptors (TCR) and peptide loaded MHC class II molecules.

Signal two constitutes the interactions of costimulatory molecules expressed on APCs

3 CHAPTER 1 INTRODUCTION

Figure 1.1: Costimulatory interactions control CD4+ T cell activation. Interactions with immature and mature DCs result in the opposing fates of tolerance and activation. Immature DCs present antigen to naïve T cells but lack the expression of B7 molecules. In the absence of this second signal naïve T cells are not activated efficiently. Mature dendritic cells express B7 molecules after exposure to maturation stimuli. Resultant engagement of CD28 induces the expression of IL-2 and efficient activation of naïve T cells.

4 CHAPTER 1 INTRODUCTION

with costimulatory receptors on CD4+ T cells. These interactions are critical for the initiation of T cell responses, with the presence or absence of costimulatory molecules controlling alternate fates for the interacting T cell. In the presence of signal one but absence of signal two, CD4+ T cells undergo an abortive activation program that is

characterized terminally by clonal deletion or unresponsiveness, and resultant immune

tolerance [22-26]. Alternatively, when CD4+ T cells encounter signal one and signal two

together, they are effectively activated and clonal proliferation and survival occurs. Thus

the appropriate provision of costimulation is an important mechanism that controls the

generation of effector T cell responses and the maintenance of self tolerance.

CD28 and CTLA-4

The prototypical costimulatory interactions are between the B7.1 (CD80) and B7.2

(CD86) molecules, which are induced after APC maturation, and the CD28 receptor

expressed constitutively by most naïve peripheral T cells (Fig. 1.2). Much experimental

evidence indicates that this interaction is a critical costimulatory event in the priming and

activation of naïve CD4+ T cells. In vitro addition of CD28 agonists in the presence of

TCR stimuli increases T cell proliferation [27]. Conversely, proliferation is reduced in

wildtype T cells activated in the presence of CD28 antagonists, and T cells from CD28

knockout mice [28, 29]. This reduction in proliferation is associated with the induction of an anergic state [23]. Enhanced IL-2 production due to CD28 activation is a key mediator of T cell activation [30] (Fig. 1.3), while the induction of T cell anergy in the absence of CD28 signalling is related to impaired production of IL-2 [27, 31-33].

The molecular basis for CD28 induced T cell costimulation is relatively well defined.

While signals through the TCR result in activation of the NFAT and ERK pathways, this

5 CHAPTER 1 INTRODUCTION is insufficient for T cell activation [34]. Engagement of CD28 leads to the activation of signalling intermediates such as IKK and JNK, which activate the NFκB and AP-1 transcription factors [35-38]. The activation of these transcription factors leads to their cooperative recruitment at regulatory regions controlling the transcription of key molecules such as IL-2 [39]. In addition they induce transcription of anti-apoptotic molecules (such as those of Bcl-2 family), cytokine production, entry into the cell cycle, proliferation and the generation of a T cell response [40].

CD28 dependent control of T cell responses has been defined in vivo via the generation of CD28 deficient mice. These mice have reduced T cell responses to viral challenge and altered cytokine profiles [41]. T cell help to B cells is retarded, resulting in reduced levels of T-dependent antibody production class switching [41]. Additionally, these mice possess defects in the generation of T cell dependent inflammation, having reduced responses in models of experimental autoimmune encephalomyelitis (EAE) and allergic airway inflammation [42, 43]. Thus CD28 plays a key role in regulating T cell activation in vitro and in vivo and its absence severely impairs the generation of effector

T cell responses.

The scope of the B7/CD28 interactions was extended with the discovery of CTLA-4.

This receptor is structurally related to CD28 and shares similar binding affinity for B7 molecules. Whilst CD28 is expressed by unactivated T cells, CTLA-4 is only expressed in response to activation [44, 45]. Treatment with CTLA4-Ig results in defective T cell responses, leading to prolonged allograft survival [46], severe disruption of T-dependent humoral responses [47, 48] and prevention of autoreactive T cell activation in the NOD and NZB/W F1 autoimmune models [49, 50]. These data suggested that CTLA-4 may

6 CHAPTER 1 INTRODUCTION

have similar functions to CD28. However, a definitive role for CTLA-4 as a negative

regulator of T cell activation was demonstrated in CTLA-4 KO mice, which develop

spontaneous autoimmunity [51-53]. Thus the impairment of T cell responses upon

CTLA4-Ig treatment was most likely due to blockade of CD28 engagement by B7

molecules. In its normal context CTLA-4 elicits important negative signals, with the

appropriate balance of CD28 and CTLA-4 signalling being essential for maintaining self tolerance.

ICOS and PD-1

Sequence homology searches of the human and murine genomes revealed the

existence of other B7 and CD28 like molecules. ICOS/ICOS-L and PD-1/PD-L1 and 2

were isolated based on their structural similarity to the CD28 and B7 families.

Functional analysis revealed that interactions between these sets of receptors and ligands provide additional levels of regulation during a developing T cell response. ICOS-L is constitutively expressed by a subset of APC, particularly B cells [54]. Like CTLA-4,

ICOS is expressed on the surface of T cells only after activation [55-58] indicating that the majority of its functions are directed at activated T cells after the primary CD28 signals have been integrated [59]. ICOS lacks the PYAP consensus SH-3 binding domain that is crucial for IL-2 production in CD28 [60], suggesting that this receptor does not regulate IL-2 dependent proliferation. Instead ICOS regulates important T cell effector functions such as secretion of cytokines and provision of B cell help [56] (Fig. 1.3).

ICOS-Ig treated or ICOS knockout mice display reduced cytokine production and

7 CHAPTER 1 INTRODUCTION

Figure 1.2: The B7 and CD28 family members. The B7 family of ligands are expressed on APCs and their cognate receptors are expressed on T cells. The B7.1 and B7.2 molecules interact with CD28 and CTLA-4. ICOS-L is the monogamous ligand for ICOS. PD-L1 and PD-L2 bind to PD-1, and may share receptor affinity for an unknown receptor. B7-H3 is a recently identified member whose receptor affinity is undefined. Reproduced from [59].

8 CHAPTER 1 INTRODUCTION

effector function in models of Th1 and Th2 mediated inflammation and are protected

from the induction of spontaneous autoimmunity [61-66]. ICOS interactions are

particularly important for enhancing T cell dependent B cell help. ICOS-L transgenic mice exhibit high levels of serum IgG [67], while ICOS deficient mice have profoundly reduced germinal centre formation [57, 68], antibody responses [55] and class switching

[57, 58]. The specialized B follicular helper T cells (TFH), which are located in germinal

centres and promote antibody responses, express particularly high amounts of ICOS [69,

70].

PD-1 is also expressed by activated T cells, B cells and monocytes. Its ligands, PD-L1

and PD-L2, are expressed by APCs and peripheral tissues such as the endothelium [59,

71-73]. PD-1 differs from CD28, CTLA-4 and ICOS, in that it contains intracellular

ITIM and ITSM domains, and can recruit the phosphatase SHP-2 [74]. These features indicate a probable function as a negative regulator. This was confirmed in vitro, as PD-1

engagement results in reduced TCR mediated proliferation and cytokine production [74-

76] (Fig. 1.3). Conversely, PD-1 knockout mice display enhanced proliferation of antigen specific T cells, and develop various autoimmune pathologies [77, 78]. Thus PD-

1 provides important negative signals to activated T cells that serve to limit the extent of

T cell responses and control of autoimmunity.

Expression of ICOS-L, PD-L1 and PD-L2 is not confined solely to APCs. PD-L1,

PD-L2 and ICOS-L are expressed on tissues such as the endothelium in response to pro-

inflammatory stimuli [73, 79-81]. In combination with the sequential expression of

9 CHAPTER 1 INTRODUCTION

Figure 1.3: Distinct functions of CD28 family members. The members of the CD28 family are differentially expressed by subsets of antigen presenting cells and elicit unique responses from resting and activated precursor T helper cells (THP). B7.1 (CD80) and B7.2 (CD86) interact with CD28 to induce T cell proliferation and IL-2 production, whilst interaction with CTLA-4 negatively regulates proliferation. Similar reductions in proliferation are observed after interaction of PD-L1 and PD-L2 with PD-1. Interactions between ICOS-L and ICOS are associated with the development of helper T cell effector functions such as cytokine secretion and are necessary for T dependent B cell responses. Reproduced from [82].

10 CHAPTER 1 INTRODUCTION

CD28, ICOS and PD-1 on the surface of activated T cells, these features suggest that the

molecules of the B7 and CD28 family control T cell responses in a temporal and spatial

manner [82] (Fig. 1.4). While signalling through CD28 is essential for the effective activation of T cells, signals from ICOS/ICOSL and PD-1/PD-L interactions may be utilized to direct the appropriate effector response in different locations of the body, controlled in part by local inflammation at the site of infection. These observations extend the concept of costimulation, from interactions that regulate early events of T cell activation in lymphoid tissue, to include interactions over the entire T cell response occurring at diverse locations.

Thus members of the CD28 family are essential regulators of T cell responses. CD28 interactions are crucial for the initiation of T cell proliferation, IL-2 production and survival after interactions with antigen loaded APCs. CTLA-4 is induced after activation and mediates negative feedback signals which oppose those of CD28. ICOS is responsible for the induction of B cell helper activity, via the induction of IL-10 and

CD40L, and is essential for the activation of T-dependent humoral responses. PD-1 transduces negative signals from a range of APCs and tissues via intracellular ITIM domains and SHP-1 recruitment. The coordinated action of these molecules is required for generation of competent and controlled T cell responses, as mice deficient in these molecules suffer alternatively from immune deficiencies or autoimmune activation.

Th1/Th2 differentiation and effector function

Activation of T cells is the first step in mounting a response to an invading pathogen.

However, to effectively control and eliminate the pathogen, T cells must also acquire the

11 CHAPTER 1 INTRODUCTION

Figure 1.4: Coordinated function of CD28 family members regulates developing immune responses. A, Naïve T cells recirculate in the blood and migrate to the T cell zone of peripheral lymphoid tissue. B, Here T cells form long lasting contacts with dendritic cells presenting cognate antigen. B7/CD28 interactions between naïve T cells and APCs constitute the primary costimulatory signal necessary for optimal priming of the T cell response. C, Activated T cells move to the border of the B cell zone where they encounter activated B cells of the same antigen specificity. ICOS-L:ICOS interactions between activated B and T cells are a critical secondary costimulatory interaction that elicit T cell help, and are necessary formation of D, germinal centres, high affinity antibody responses and effector T cells. E, and F, Negative signals provided by B7:CTLA-4 and PD-L:PD-1 interactions provide important counter regulation at multiple points during the developing T cells response. Reproduced from [82].

12 CHAPTER 1 INTRODUCTION

appropriate effector function. CD4+ T cells develop specialised effector functions that

primarily involve the activation of other cell types. These effector subsets are classified

as Th1 and Th2 cells and are broadly involved in the activation of macrophages tomediate intracellular killing or B cells to produce antibody. The capacity of Th1 and

Th2 cells to activate the cellular or humoral arms of the immune response respectively, lies in their alternate cytokine secretion profiles and cloned CD4+ T cell lines can be

classified into two distinct subsets based on their cytokine secretion profiles [83]. The

Th1 cells produce IFNγ, LT-α and IL-2, while Th2 cells produce IL-4, IL-5, IL-6, IL-9,

IL-10 and IL-13. These two subsets potently cross-regulate each other during their differentiation [84-86] and subsequently control the production of alternative antibody

subclasses [87] and recruitment of innate immune effector cells.

Th1 or Th2 cells can be derived from identical naïve, precursor T helper cells (THP) after culture with IL-12 or IL-4 respectively [88-91]. This demonstrates that environmental signals are responsible for directing the development of both the Th1 and

Th2 lineages. In addition to cytokines, Th1/Th2 differentiation is affected by other factors including antigen dose [92], affinity of antigens [93-96], signalling pathways [97-

99], costimulatory molecules [59, 100, 101] and specialized dendritic cell subsets [102-

104] (Fig. 1.5). The regulation of Th1/Th2 differentiation involves the interpretation of a myriad of signals, many of which are yet to be identified.

Although our understanding of the factors that regulate Th1/Th2 differentiation is not complete, in vitro differentiation systems using IL-4 and IL-12 have allowed molecular mechanisms of Th1/Th2 differentiation to be elucidated. During Th1 differentiation, IL-

13 CHAPTER 1 INTRODUCTION

Figure 1.5: Differentiation pathways of Th1 and Th2 effector T cells. Naïve precursor CD4+ T cells can be driven to differentiate into the alternate helper T cell subsets (Th1 or Th2) under the influence of environment factors such as dendritic cells subsets and cytokines. After differentiation, Th1 and Th2 cells express mutually exclusive panels of cytokines and are involved in the orchestration of alternate immune responses, characterized as the cellular and humoral arms of the immune system. Adapted from [105].

14 CHAPTER 1 INTRODUCTION

12 signaling via STAT4 [106, 107], collaborates with IFNγ [108] to induce the expression of T-bet, a T-box transcription factor [109]. Alternatively, IL-4 activation of

STAT6 [110-112] induces Th2 development via the activation of GATA-3 [113] and c-

Maf [114-116]. Forced expression of T-bet [109] or GATA-3 [113] in CD4+ T cells is sufficient to drive Th1 and Th2 differentiation, while knockout mice of either transcription factor fail to develop the appropriate effector lineage [117, 118]. The regulation of T-bet and GATA-3 activity is so precise that they antagonize each other by direct intermolecular interactions [119]. Thus Th1 and Th2 cells represent separate effector T cell lineages with distinct transcriptional profiles [69, 120], controlled by the master regulators T-bet and GATA-3, respectively.

The generation of the appropriate effector response is critical for immunity. The development of a predominantly Th1 response against the human pathogen

Mycobacterium leprae leads to efficient pathogen clearance, while the development of a

Th2 response results in unchecked pathogen growth and massive tissue destruction [121].

Dysregulation of these responses is implicated in the pathogenesis of several human diseases. Effector T cell responses against innocuous environmental antigens are associated with the development of allergic disorders such as asthma [122-127] and hayfever, and are essentially Th2 in nature. Th1 T cells are implicated in the pathology of certain autoimmune diseases such as type 1 diabetes [128] and multiple sclerosis [129,

130]. A more complete understanding of the factors that affect the generation of Th1 and

Th2 responses may provide avenues for new strategies for immunization and more effective treatments of infections, allergies and autoimmunity.

15 CHAPTER 1 INTRODUCTION

TNF AND TNF RECEPTOR SUPERFAMILIES

Immune function

The TNF superfamily (TNFSF) comprises a group of structurally related molecules named after the canonical family member, tumour necrosis factor (TNFα). Ligands from this family interact exclusively with members of the TNF receptor superfamily

(TNFRSF). 19 TNFSF and 29 TNFRSF members have presently been identified [131]

(Fig. 1.6). Their interactions mediate communication between a wide range of cell types and are essential for the development of numerous multicellular structures and tissues, such as lymphoid tissue, hair follicles, bone, and mammary gland [132-134].

Sequence alignment of TNF and TNFR family genes from the genomes of higher metazoans demonstrates that the majority of family members have diverged since the evolution of the adaptive immune system (~450 million years ago) [134]. Indeed many of these molecules are fundamental for the effective function of the adaptive immune system. TNFSF molecules are critical for control of immune processes of lymphocyte apoptosis (FasL/Fas) [135, 136], lymphoid organogenesis (LTβ/LTβR) [132], germinal centre formation (TNF/TNFRI) [137, 138] and antibody responses (CD40L/CD40) [139].

Loss of function mutations in CD40L or Fas/FasL lead to the development of Hyper IgM syndrome or type I autoimmune lymphoproliferative syndrome (ALPS) [134] respectively, demonstrating important roles for TNFSF molecules in human disease.

The potent immune modulatory functions of many TNFSF molecules mean that they are attractive therapeutic targets for the treatment of immune disorders. The proinflammatory effects of TNFα have been associated with the development of autoimmune syndromes such as rheumatoid arthritis (RA) [140]. In response TNFα

16 CHAPTER 1 INTRODUCTION neutralising agents such as Infliximab and Enbrel have been developed and demonstrate significant utility in the treatment of Crohn’s disease [141], RA [142] and various cancers

[143]. With the success of anti-TNFα therapies it is likely that agents targeting other

TNFSF members, in particular BAFF, will provide new avenues for intervention in a range of autoimmune conditions (see below).

Structure

The conserved structural domains common to TNFSF and TNFRSF members are essential for their receptor-ligand binding interactions. TNFSF ligands contain a TNF homology domain (THD), a 150 amino acid sequence that shares ~25-30% sequence homology between family members [144]. TNFRSF receptors contain a conserved cysteine-rich domain (CRD) and interactions between THD and CRD are involved in the trimeric assembly of ligands and receptors and adaptor molecules [134, 144]. As

TNFRSF receptors lack intrinsic enzymatic activity, the recruitment of these intracellular adaptor molecules is necessary to elicit signal transduction.

TNFSF ligands are able to form homo- or hetero-trimeric molecules [145, 146], and engage in monogamous or polygamous receptor interactions with TNFRSF members.

Most TNFSF members are active as both membrane-bound and soluble molecules, with the activity of specific proteases regulating their cleavage from the cell surface [144].

The membrane-bound and soluble forms often demonstrate differential receptor specificity and thus elicit alternate responses [147], indicating that cell surface cleavage is an important point of regulation for TNFSF activity [148, 149]. Some TNFRSF members share the ability to be cleaved from the cell surface with their ligands [150],

17 CHAPTER 1 INTRODUCTION

Figure 1.6: The TNF receptor superfamily and their ligands. The receptor:ligand interactions of these large families are demonstrated by arrows. The modular structure of the receptors is demonstrated on the left. Receptors display unique patterns of cysteine-rich domain (CRD) and death domain (DD) structure, and differential interactions with the TNF receptor-associated factors (TRAFs) adaptor proteins. The number of cytoplasmic amino acids is shown for each receptor. All ligands (right) are type II transmembrane proteins, with the exception of LTα (TNF-β) and VEGI. Most can be released from the cell surface after cleavage with specific proteases (triangles). Reproduced from [131].

18 CHAPTER 1 INTRODUCTION while others have also lost their membrane anchoring domains and function purely as decoy receptors [144]. Thus interactions between TNFSF ligands and their receptors are regulated at multiple levels.

Signalling pathways

Ligand engagement of TNFRSF receptors can result in distinct cellular outcomes; apoptosis, survival, proliferation or differentiation [151]. The outcome of any particular interaction is determined by the identities of the ligand and receptor and the cellular context in which they meet. The intracellular portion of each receptor contains structural motifs that enable the recruitment of specialised adaptor proteins. These adaptor proteins belong to either the death domain (DD) or TNF receptor-associated factors (TRAFs) families and activate distinct signalling pathways (Fig. 1.7). The DD proteins interact with the subset of TNFRSF members termed the “death receptors”. Fas and TNFRI are the most well characterised members of this family, while newer members such as DR3,

DR4 and DR5 have recently been identified [152]. Recruitment of DD proteins to “death receptors” leads to the engagement of pro-apoptotic machinery and results in cellular apoptosis. A family of DD proteins have evolved and show specific patterns of receptor interaction. FasL engagement of Fas leads to the recruitment of the DD protein Fas associated death domain (FADD). FADD then induces cellular apoptosis via the recruitment of pro-caspase 8 and initiation of the caspase cascade. TNFR associated death domain (TRADD) mediates a similar function after TNF engagement of TNFRI

[153-155].

Those TNFRSF receptors lacking a DD elicit signal transduction via the recruitment

19 CHAPTER 1 INTRODUCTION

Figure 1.7: Signalling pathways from DD and TRAF adaptor proteins. Engagement of TNFRSF induces the recruitment of DD or TRAF adaptor proteins. A, Fas engagement results in the recruitment of FADD to the intracellular DD of this receptor. Downstream activation of caspase-8 and effector caspases induces cellular apoptosis. B, TRAF proteins interact with TNFRSF members that lack DD. Activation of downstream signalling pathways is mediated by distinct protein domains and generally lead to cell survival and activation. Zinc finger domains are responsible for the activation of IKK, NIK and JNK kinases and resultant NFκB and AP-1 transcription. Adpated from [154, 156].

20 CHAPTER 1 INTRODUCTION of TRAF proteins. Each non DD containing TNFRSF receptor shows a complex pattern of interaction with TRAF proteins [131], and signalling from these receptors is generally associated with cell survival and activation rather than apoptosis [134, 151]. At present six TRAF proteins have been identified in mammals (named TRAF 1-6). All interact with TNFRSF receptors, with the exception of TRAF4, via a short amino acid sequence known as the TRAF binding motif [151, 157]. The individual TRAF family members differ in their ability to activate downstream signaling pathways. TRAF2, TRAF5 and

TRAF6 mediate the activation of protein kinases such as IKK and JNK, leading to NFκB and AP-1 activation, whilst TRAF1 and TRAF3 do not [151]. Many of the anti-apoptotic effects mediated by TRAF interacting TNFRSF members can be ascribed to the activation of these pathways [158]. However, the inability of TRAF1 and TRAF3 to activate these pathways implies that the full spectrum of signal transduction pathways activated by TRAF proteins is yet to be elucidated. Thus the intracellular domains of

TNFRSF receptors determine the alternate recruitment of DD or TRAF proteins. These adaptors can activate the caspase cascade, or NFκB and AP-1 transcription factors, and determine whether a given ligand/receptor interaction will result primarily in apoptosis or survival.

Control of T cell responses

Members of the TNFSF and TNFRSF have emerged as novel costimulators of T cells

[101, 159] and appear to play complementary roles to those of the B7 and CD28 families in regulating developing T cell activation and effector responses. T cell costimulatory interactions have been identified between five TNFSF members and their receptors,

21 CHAPTER 1 INTRODUCTION

Figure 1.8: Temporal expression patterns of the costimulatory TNF receptors on T cells. Multiple members of the TNF receptor superfamily are able to transmit costimulatory signals to T cells. One mechanism that directs the specificity of these signals is differential receptors expression during the course of T cell responses. OX40, 4-1BB, CD30 and CD27 show a similar pattern of expression that peaks during the effector phase, whilst being lower or absent at the naïve or memory stages. In contrast, HVEM is expressed highly by naïve and memory T cells but is potently down modulated during the effector phase. Reproduced from [100].

22 CHAPTER 1 INTRODUCTION namely: LIGHT/HVEM, CD70/CD27, OX40L/OX40, 4-1BBL/4-1BB and CD30L/CD30

[100]. These will herein be referred to as the costimulatory TNFSF molecules. Mice with targeted gene deletions of these ligands or receptors are compromised in their ability to mount a variety of T cell responses, whilst transgenic overexpression of these ligands is associated with excessive T cell activation and the development of autoimmunity [160-

163].

The costimulatory TNFRSF members are generally expressed by T cells and activated

APCs (DCs, macrophages, B cells). Their relevant ligands are predominantly expressed by activated APCs and T cells [100, 101]. LIGHT, being constitutively expressed by

APCs and downregulated in response to activation, is the exception [164]. Receptor expression on T cells is regulated in response to TCR and CD28 engagement, with dynamic cell surface expression of all five receptors occurring after activation [100] (Fig.

1.8). Regulation of receptor expression and ligand availability results in activated T cells receiving changing combinations of costimulatory TNFSF signals at different points of a

T cell response. Experimental data indicate that HVEM engagement is critical during initial activation, CD27 during clonal expansion and OX40, 4-1BB and CD30 for survival and memory formation [100].

Costimulatory TNFRSF receptors interact with TRAF molecules, but not DD proteins.

All five interact with TRAF2, while a subset also interacts with TRAF1, TRAF3 and

TRAF5 [131]. TRAF2 deficient mice, or mice transgenic for a dominant negative form of TRAF2, have impaired cytokine production in response to costimulatory TNFSF stimulation [165, 166] indicating that interaction with TRAF2 is important for mediating

23 CHAPTER 1 INTRODUCTION

Figure 1.9: Signalling pathways activated by the costimulatory TNF receptors. Engagement of the costimulatory TNFRSF by the ligands leads to the recruitment of TRAF adaptor proteins to the receptor, particularly TRAF2. TRAF2 can subsequently transmit activation signals to PI3K, NFκB and JNK. The combined actions of these signalling pathways leads to the upregulation of molecules important for T cell survival (Bcl-XL, Bcl-2), division (cyclins/CDKs) and effector functions (IL-2, IL-4, IFNγ). Reproduced from [100].

24 CHAPTER 1 INTRODUCTION

signalling from these receptors. The costimulatory TNFRSF receptors can all activate

NFκB and AP-1, with the exception of CD30 [100] (Fig. 1.9). Activation of these

signalling pathways leads to induction of Bcl-2 family anti-apoptotic proteins and

increased T cell survival [167, 168]. OX40 is also able to activate the PI3K/AKT

pathway which enhances T cell proliferation [169], however, it is unclear whether this pathway is downstream of the other TNFRSF costimulatory receptors.

Extensive functional analysis using agonistic/antagonistic antibodies and knockout

mice has elucidated the roles of each TNFRSF costimulatory receptor in regulating T cell

responses. OX40L/OX40 and 4-1BBL/4-1BB have been studied the most extensively.

Soluble OX40L and agonistic Abs enhance the proliferation and cytokine production of T

cells, primarily the CD4+ subset [170, 171]. OX40 and OX40L knockout mice have

reduced primary CD4+ T cell responses against model antigens and viral pathogens [172-

176]. Initial activation events appear intact in OX40 knockout mice, however, impaired

responses are observed 4-5 days post immunisation. At this time OX40 knockout CD4+

T cells have reduced expression of the anti-apoptotic molecules Bcl-XL, Bcl-2 and

survivin [168, 177], reduced proliferation and decreased specific, CD4+ effector T cells

survival [168]. This reduced frequency of effector CD4+ T cells is translated into

impaired development of memory CD4+ T cells [178], indicating an important role for

OX40 during memory formation. While some data suggest that OX40L can direct Th2

differentiation [179], it is clear that both Th1 and Th2 responses are suppressed by OX40

blockade or deficiency [180-186]. This suggests that OX40 does not control Th1 and

Th2 responses at the level of Th1/Th2 differentiation, but controls the size of the Th1 or

Th2 effector pool and generation of memory cells.

25 CHAPTER 1 INTRODUCTION

4-1BB and 4-1BBL knockout mice exhibit defects in CD8+ responses against a

number of viruses [187-189], without displaying defects in the CD4+ or antibody

compartments. Initial proliferative responses of CD8+ T cells appear to be intact in 4-

1BB knockout mice but survival of effector CTLs is impaired at the peak of the primary

response [190], most likely due to the defective induction of anti-apoptotic molecules

[167, 191, 192]. Thus OX40 and 4-1BB appear to play similar roles in sustaining

effector T cell survival and memory formation, with their action primarily directed at

CD4+ and CD8+T cells, respectively.

A connection has also been established between the action of the costimulatory

TNFSF molecules and the regulation of peripheral tolerance. Mice that transgenically

express OX40L, CD70 and LIGHT under the control of T cell or DC specific promoters

develop autoimmune inflammation [160, 161, 163]. In addition the administration of

soluble OX40L prevents induction of tolerance in response to high doses of peptide

antigens [193]. These data suggest that excessive stimulation via costimulatory TNFRSF

molecules leads to the activation of previously latent autoreactive clones in the periphery

and the induction autoimmune inflammation.

Thus the TNFSF molecules are an important new family of T cell regulators, which

show distinct and overlapping functions with molecules of the B7 family. They are

expressed by a variety of APCs, induce proliferation, cytokine production and the expression of anti-apoptotic molecules. Their activities regulate CD4+ and CD8+ T cell

responses at the levels of activation, effector generation and memory formation. The

overexpression of these molecules is associated with the generation of autoimmunity, implying that the action of these molecules play an important role for maintaining self

26 CHAPTER 1 INTRODUCTION

tolerance in the periphery. Whether other newly recognised members of the TNFSF and

TNFRSF similarly regulate the actions of T cells remains to be determined.

BAFF

Expression and regulation

BAFF (also called BLyS, TALL-1, THANK, zTNF-4, TNFSF13B and TNFSF20) is a

recently identified TNFSF member. It contains the characteristic TNF homology domain

(THD) and adopts the typical trimeric structure that is common to members of this

protein family [144]. It is most closely related in structure to another TNFSF molecule

called APRIL with which it shares ~50% sequence homology [144]. BAFF and APRIL share receptor affinity and mediate both unique and overlapping functions in the control of immune responses.

The active BAFF molecule exists as a homotrimer, which can be released as a soluble cytokine following proteolysis of the membrane-bound form by members of the subtilisin-like furin family of proteases. This process is regulated at both a stimulus and cell type level [194-197]. The differential regulation of the soluble and membrane bound forms suggests the possibility of distinct functions, however, their relative activities and functions are yet to be determined. In addition to control of expression, the biological activity of BAFF may also be regulated by other mechanisms such as heterotrimer formation, alternative splicing and protein localization. Heterotrimers incorporating a truncated splice variant called ΔBAFF constitute a non-cleavable form which is localised to the cell surface and lacks the normal biological activities of BAFF [198]. ΔBAFF transgenic mice have suppressed BAFF activity indicating that ΔBAFF downregulates

BAFF activity in vivo [199]. Additionally BAFF forms heterotrimers with APRIL.

27 CHAPTER 1 INTRODUCTION

These have been identified in vitro and in vivo, specifically in the context of some

autoimmune patients [145]. They have specific receptor affinity and thus may play a role

in regulating BAFF activity. Whether these particular molecules are activating or

suppressing in the context of autoimmune disease is unclear.

BAFF is expressed in a variety of cells types, predominantly leukocytes of the

peripheral blood, lymph nodes and spleen [200, 201], while low levels of expression are

observed in thymus and lung [195, 202]. BAFF production by leukocytes is induced by a

variety of proinflammatory stimuli. Type I and II interferons, IL-10 and LPS are potent inducers of BAFF in dendritic cells, macrophages and monocytes [196, 197, 203-205] while neutrophils stimulated with G-CSF- and IFNγ- express BAFF at very high levels

[194, 206]. Lower levels are produced by T cells and germinal centre B cells after TCR

[202, 204] and CD40L stimulation, respectively [207, 208].

There is growing evidence that non-hematopoietic cell types are also important

producers of BAFF. Both astrocytes and fibroblast like synoviocytes produce BAFF in

response to TNFα and IFNγ, indicating that stromal cells may be an important source of

BAFF during inflammation [209, 210]. Wildtype bone marrow transferred into BAFF knockout host animals is unable to restore normal serum levels of BAFF and reconstitute the peripheral B cell pool [211], indicating that the non-hematopeutic compartment is the major site of constitutive BAFF production and regulates the size of the peripheral B cell pool. Thus, there appears to be two distinct modes of BAFF expression: constitutive expression by stromal cells that controls B cell homeostasis, and inducible expression in response to inflammatory stimuli, which is presumably important for pathogen clearance

[212].

28 CHAPTER 1 INTRODUCTION

Receptors

BAFF interacts with 3 TNFRSF receptors, BAFF receptor (BAFF-R or BR3),

transmembrane activator and calcium-modulator and cyclophilin ligand interactor

(TACI), and B cell maturation antigen (BCMA). All three are primarily expressed by B

cells. BAFF is the sole ligand for BAFF-R, [213] while it shares receptor specificity for

TACI and BCMA with APRIL [214] (Fig. 1.10). All 3 receptors lack the ability to recruit DD proteins and instead interact with TRAF proteins [215-217]. This suggests the involvement of these receptors in cell survival and differentiation pathways.

BAFF-R, TACI and BCMA display unique but overlapping expression patterns, and functional analysis has revealed distinct roles for these three receptors. Cell surface expression of all three is modulated during B cell development. BAFF binding and receptor expression is minimal on early immature cells in the bone marrow [218].

Significant amounts of BCMA mRNA and lower levels of BAFF-R and TACI are detected in the later stages of B cell development [219] before migration to the peripheral lymphoid tissue. At this time BAFF-R and TACI are upregulated in immature B cells, coincident with the acquisition of BAFF responsiveness at the transitional type 1 (T1) stage of development [220]. BAFF-R is subsequently expressed at high levels on all mature peripheral B1 and B2 cell subsets in the peritoneum, lymphoid organs and peripheral blood [221, 222]. TACI is highly expressed in the T2, marginal zone and B-1

B cells, while being downregulated on follicular and germinal centre B cells [220, 223].

The expression pattern of BCMA is the most restricted of the BAFF receptors with cell surface expression being confined to germinal centre B cells [221], plasmablasts [221] and plasma cells [223].

29 CHAPTER 1 INTRODUCTION

Function

The discovery of BAFF has provided numerous insights into the regulation of the B cell repertoire and its activities link B cell homeostasis and the development of autoimmune syndromes [224, 225]. Functional data demonstrates that BAFF modulates key events in the lifetime of a B cell, particularly survival and maturation, germinal centre formation, antibody production and class switching [224, 225].

Recombinant BAFF augments B cell proliferation, antibody production and class- switching after anti-B cell receptor (BCR) stimulation in vitro [197, 202, 226] and results in expansion of peripheral B cell numbers when injected into mice [203]. In the absence of BCR stimulation, BAFF upregulated the expression of both Bcl-XL and Bfl-1 [218] and significantly increased splenic B cell survival in vitro [227]. Analysis of the surviving B cell subsets revealed that BAFF specifically enhances survival of the immature transitional type 2 (T2) B cell compartment and leads to the differentiation of the T2 subset to mature B cells when combined with a BCR stimulus [227]. Thus BAFF regulates the proliferation, survival and maturation of peripheral B cells.

BAFF transgenic mice display significant perturbations of the B cell compartment and develop symptoms of systemic autoimmunity with age. These mice have enlarged spleens and lymph nodes due to increased numbers of the T2, follicular and marginal zone B cell subsets [227, 228]. Splenic B cells from BAFF transgenic mice show significantly prolonged survival when cultured ex vivo [227, 229], which is consistent with their increased expression of Bcl-2 [228]. BAFF transgenic mice also display increased spontaneous germinal centre, formation [228] and increased levels of all antibody subclasses [229], indicating an increase in both antibody production and class

30 CHAPTER 1 INTRODUCTION switching. Increased numbers of effector/memory type T cells develop in BAFF transgenic mice as they age, demonstrating that T cell homeostasis is also disrupted

[228].

Conversely, BAFF deficient mice and those treated with BAFF blocking fusion proteins display profound defects in peripheral B cell numbers and responses [230-232].

BAFF knockout mice have severely impaired B cell maturation beyond the immature transitional T1 stage. This is associated with significantly reduced numbers of peripheral

B cells [230, 233] and a failure to upregulate B cell maturation markers, such as CD21 and CD23 [234]. Germinal centre reactions and antibody responses to T-dependent and

T-independent antigens are severely impaired [235]. Thus BAFF is an essential maturation factor for peripheral B cells.

Phenotypic analysis of gene deficient and mutant strains of mice has enabled the functions of the three BAFF receptors to be defined. BAFF-R knockout mice and the

A/WySnJ strain, which carries a mutation in the baff-r gene, have significantly reduced peripheral B cell numbers [236-238], mirroring the phenotype of BAFF knockout mice

[233]. Overexpression of Bcl-2 in B cells with defective BAFF-R signalling leads to partial restoration of peripheral B cell numbers [237, 239]. However, Bcl-2 overexpression does not complement the additional deficiencies in germinal centre formation, IgG responses and marginal zone B cell numbers in the absence of BAFF.

This indicates that these defects are not caused by impaired survival and suggests the existence of separate BAFF-R dependent maturation and functional pathways. Thus

BAFF-R is critical for survival, maturation and function of the mature B cell pool.

31 CHAPTER 1 INTRODUCTION

Figure 1.10: BAFF and APRIL receptor specificity and function. BAFF and APRIL have unique and overlapping receptor specificity for BAFF-R, BCMA and TACI. APRIL also specifically binds to proteoglycans. Functional analysis of the BAFF receptors has revealed distinct functions for these receptors. BAFF-R mediated effects are shown in yellow, BCMA in pink and TACI in light blue. The differential binding affinity of BAFF and APRIL, coupled with the distinct functions of the 3 receptors, elucidates the basis for the differences in functions for BAFF and APRIL. Reproduced from [240].

32 CHAPTER 1 INTRODUCTION

A number of TACI deficient mouse strains have been generated which display variable phenotypes [241-243]. Expanded numbers of mature B cells were found in peripheral lymphoid organs of all lines [241-243] and one line developed SLE like autoimmunity similar to BAFF transgenic mice [243]. Agonistic antibodies to TACI reduced wildtype B cell proliferation in response to activation [243]. Conversely, TACI knockout B cells were hyperproliferative in vitro and in vivo [241, 242]. TACI deficient mice also display markedly decreased antibody production and class switching in response to T-independent antigens, while their antibody responses to T-dependent antigens are normal [241, 242]. These results correlate with in vitro studies demonstrating that BAFF and APRIL can directly stimulate CD40L independent class switching [197, 244].

Analysis of two separate human cohorts identified a series of mutations in taci that are associated with the development of combined variable immunodeficiency (CVID) [245,

246]. These mutations were associated with reduced levels of serum IgM, IgG and IgA, and severe defects in APRIL induced B cell proliferation and antibody class switching

[245, 246]. Many patients with taci mutations also displayed lymphoproliferative disorders, with the incidence of autoimmune disease in CVID patients with taci mutations higher than the general CVID cohort [246]. Thus data from TACI knockout mice and human cohorts have defined a combination of roles for TACI, as both a positive regulator of antibody responses and a negative regulator of B cell proliferation.

Initial studies using BCMA knockout mice suggested that the functions of BCMA were redundant for normal immune regulation [247], however, recent advances have provided further insight in the function of this receptor. Analysis of BCMA deficient

33 CHAPTER 1 INTRODUCTION mice revealed a defect in the long term survival of plasma cells in the bone marrow, which is consist with their exclusive expression of BCMA [248]. In vitro stimulation of plasma cells with BAFF induces expression of the anti-apoptotic proteins Bcl-XL and

Mcl-1, but not Bcl-2, suggesting a qualitative difference in BAFF signals mediated by

BCMA and BAFF-R [248]. In addition, activation of BCMA enhances B cell antigen presentation. Crosslinking of BCMA leads to increased expression of MHC class II,

CD86, CD80, CD40 and ICAM-1 and results in significantly increased capacity to stimulate T cell proliferation and IL-2 production [249]. As BCMA is expressed primarily by germinal centre B cells, plasmablasts and plasma cells [221, 250, 251], these results suggest that BCMA may play an important role in regulating collaboration between these B cell subsets and T cells.

Signalling pathways

Definition of the signalling pathways activated by the 3 BAFF receptors has identified a molecular basis for their distinct functions (Fig. 1.11). Each receptor interacts with a different spectrum of TRAF proteins, BAFF-R recruiting TRAF3 alone, TACI recruiting

TRAF2, -5, -6 and BCMA recruiting TRAF1, -2, -3, -5, -6 [131]. These differential interactions are associated with clear distinctions in the ability of these receptors to activate the MAP kinases and NFκB. Both TACI and BCMA activate the canonical

NFκB1 pathway via the activation of IKK [131]. BCMA activates the MAP kinases,

JNK and p38, and Elk-1 [217, 249], while TACI activates AP-1 and NFAT [252]. In contrast, BAFF-R does not appear to interact with the MAP kinases, NFAT or NFκB1 pathways, instead activating the non-canonical NFκB2 pathway [253-255]. This

34 CHAPTER 1 INTRODUCTION

Figure 1.11: Differential activation of NFκB pathways by the BAFF receptors. The BAFF receptors show different patterns of TRAF protein interactions and subsequently activate alternative NFκB pathways. TACI and BCMA regulate the activation of the canonical (NFκB1) pathway which involves the formation of a heterodimeric transcriptional complex from the p50 and p65 subunits. In contrast, BAFF-R activates the upstream MAP kinase NIK and leads to the formation of p52 and RelB heterodimers. This pathway is known as the non-canonical (NFκB2) and is responsible for mediating the prosurvival effects of BAFF during B cell development. Reproduced from [214].

35 CHAPTER 1 INTRODUCTION alternative pathway is dependent upon NFκB inducing kinase (NIK) induced proteolytic cleavage of the p100 precursor to the p52 form. Transcription is initiated upon dimerisation of p52 with Rel-B [256, 257]. NFκB2 knockout mice and the aly/aly strain, which carries inactivating mutations in NIK, demonstrate impaired BAFF responsiveness in the B cell compartment [254], indicating that NFκB2 activation is critical for the mediation of BAFF survival signals.

Functional studies have eluciated critical interactions between BAFF-R, TRAF3 and

NIK that are essential for the BAFF induced activation of NFκB2. In the latent state prior to BAFF-R activation, TRAF3 forms stable associations with NIK, targeting newly translated NIK molecules for ubiquitinylation and constitutive degradation by the proteosome [258]. Engagement of BAFF at the cell surface results in rapid TRAF3 recruitment to BAFF-R via a unique sequence motif in the intracellular domain of the receptor [216, 259]. TRAF3 is subsequently degraded and the concentration of NIK protein increases, leading to induction of p100 processing [260] and production of functional p52 subunits [261]. Thus TRAF3 functions to link BAFF-R engagement to

NIK activation. The recruitment of TRAF3 to other TNFRSF members that activate

NFκB2, such as CD40 [262] and LTβR [263], indicates that TRAF3 is likely to be a critical positive regulator of the non-canonical NFκB pathway in general.

NFκB independent signalling pathways, such as those mediated by the protein kinase

C (PKC) family member PKCδ, are also important for signal transduction from BAFF-R.

BAFF stimulation of B cells induces a rapid reduction in the levels of nuclear PKCδ, while PKCδ deficient mice have increased B cell numbers and autoantibody production similar to BAFF transgenic mice [264, 265]. Thus PKCδ is a negative regulator of BAFF

36 CHAPTER 1 INTRODUCTION

survival signals in B cells, possibly mediating its function via repression of a target

molecule such as a nuclear transcription factor. Defects in BAFF induced survival are

also observed in B cells with specific deletion of the transcription factor c-Myb [266]. In

these cells, BAFF stimulation results in NFκB2 activation but does not lead to the

downregulation of nuclear PKCδ levels. Thus c-Myb may represent an important nuclear

target of PKCδ action. Together these data suggest that activation of NFκB2 alone is

insufficient to transmit fully competent BAFF survival signals and that PKCδ plays an

additional important role in this process.

The phenotype of TRAF2 conditional knockout mice suggests that TRAF2 may also

play a role in regulating downstream signalling from BAFF receptors. B cells that are

selectively deficient in TRAF2 show increased basal activation of NFκB2, increased Bcl-

XL expression and enhanced survival ex vivo. A particular expansion of marginal zone B

cells in secondary lymphoid organs is also observed [267]. Interestingly, while these

mice have many characteristics of increased BAFF stimulation, they do not develop

autoimmunity, possibly as the result of coincidental defects in CD40 signalling [267].

TRAF2 interacts directly with both TACI and BCMA [131], but is unable to interact with

BAFF-R [216]. Thus the enhanced B cell numbers in TRAF2 deficient mice may be the

result of impaired negative signals from TACI, or may indicate that TRAF2 negatively

regulates the interactions of TRAF3 and BAFF-R.

Autoimmunity

Multiple lines of BAFF transgenic mice have been generated, all of which display

significant disruptions of the B cell compartment and develop spontaneous autoimmunity. This is characterized by profound B cell hyperplasia, hyperglobulinaemia

37 CHAPTER 1 INTRODUCTION and production of circulating autoantibodies and immune complexes [228, 229, 268]. As they age, BAFF transgenic mice develop proteinuria and severe nephritis leading to kidney failure, reminiscent of human systemic lupus erythematosus (SLE). Mice also develop a secondary condition similar to human Sjögren’s syndrome (SS), characterized by leukocyte infiltration in the salivary glands, destruction of acinar cells and reduced saliva production [269]. Thus sustained overproduction of BAFF is sufficient to drive the generation of systemic autoimmunity. However, there is evidence to suggest that development of BAFF driven autoimmunity is affected by additional genetic modifiers.

Expression of a BAFF transgene in the presence of the Sle1 or Nba1 lupus susceptibility regions significantly increased disease onset [270]. One study has also noted that a small proportion of normal control subjects display elevated BAFF levels in the absence of infection or inflammation [271]. These data suggest that BAFF overexpression may only induce autoimmunity in the context of permissive genetic predisposition.

The enhanced production of auto antibodies in BAFF transgenic mice indicates that overproduction of BAFF allows self reactive B cells to elude normal tolerance checkpoints. To test this hypothesis Thien et al. used BCR transgenic B cell systems to study the effects of the BAFF transgene on B cell tolerance mechanisms [272]. Hen egg lysozyme (HEL) specific B cells that encounter HEL as neo-self antigen in vivo are normally functionally inactivated or deleted from the B cell repertoire in the bone marrow or peripheral lymphoid tissue [273-275]. While BAFF overexpression had no effect on B cell selection in the bone marrow, abnormal deletion of self reactive cells was observed during T1-T2 maturation in the spleen. These self reactive B cells eluded clonal deletion when BAFF was overexpressed and were subsequently selected to

38 CHAPTER 1 INTRODUCTION follicular and marginal zone niches, areas where they are normally excluded. HEL stimulation of these B cells in vitro indicated that these cells were not anergised and were fully capable of secreting autoantibody.

Survival of self reactive B cells was highly dependent on BCR affinity and the presence of competitor B cells. While B cells of intermediate BCR affinity were able to escape deletion in the presence of competitor cells in a BAFF transgenic host, those with high BCR affinity were not [272]. These findings imply that high affinity self reactive B cells possess an increased requirement for BAFF to support their survival. This conclusion was supported by another study using HEL specific BCR transgenic mice.

Lesley et al. demonstrated that high affinity self-reactive B cells can only survive where

BAFF availability is increased on a per cell basis, eg during B cell lymphopenia [201].

These studies suggest that the autoreactive cells that are supported by increased BAFF expression are unlikely to be of a high BCR affinity. Instead it may be the cells of intermediate BCR affinity, selected into the marginal zone, that are responsible for disease pathology.

The development of systemic autoimmunity in BAFF transgenic mice indicates that dysregulation of BAFF may also be relevant to the generation of human autoimmune syndromes. Cumulative evidence from mouse models of autoimmunity and autoimmune patient cohorts has strengthened this putative association. Spontaneous models of lupus, such as the NZB/W F1 and the MRL.lpr/lpr strains, and a model of chemically induced autoimmunity, have increased serum levels of BAFF [268, 276]. Systemic elevation in

BAFF is detected in a range of human autoimmune disease cohorts such as SLE [277-

279], SS [269, 279, 280], RA [278, 279] and Wegner’s granulomatosis [271].

39 CHAPTER 1 INTRODUCTION

Autoimmune patients also demonstrate local elevations of BAFF in affected tissues such as SS salivary glands [269, 281, 282], RA synovial fluid [283] and multiple sclerotic lesions [209]. Thus local and systemic increases in BAFF are associated with a range of autoimmune conditions, suggesting an important role for this molecule in stimulating lymphocytes during autoimmune inflammation.

The mechanisms responsible for BAFF overproduction during the development of autoimmune disease are unclear. There is some evidence to suggest that natural genetic variation regulates amounts of systemic BAFF, as polymorphisms in the baff gene are associated with increased BAFF production and the development of SLE in humans

[284]. More extensive cohort studies will be needed to further elucidate this in other autoimmune diseases. Alternatively initial increases in BAFF levels may be driven by inflammation or infection. The potent induction of BAFF in response to proinflammatory stimuli suggests that BAFF is part of the normal response to infectious agents, perhaps serving to increase B cell numbers during clonal expansion. A causative role for infection in BAFF production is suggested by the elevated BAFF levels in patients with HIV infection [285]. These elevations may be responsible for the high levels of autoantibodies and increased incidence of autoimmune diseases such as SLE in some HIV patients [285]. Further analysis of BAFF production during a range of infections is needed to elucidate the link between infection and BAFF production.

Additionally immune complexes bound to chromatin induce BAFF production from dendritic cells [286]. As autoantibodies to nuclear components such as chromatin are a consistent feature of SLE, this suggests that BAFF production in some patients may be a consequence of disease rather than causative. Regardless, BAFF production in response

40 CHAPTER 1 INTRODUCTION to chromatin:immune complex would initiate a powerful positive feedback loop, further compromising B cell tolerance mechanisms and providing a self sustaining driver of disease.

Therapeutic targetting

In keeping with the overproduction of BAFF during a range of autoimmune disease,

BAFF blocking agents have demonstrated efficacy in the treatment of animal models of

SLE and RA. Treatment of lupus prone NZB/WF1 and MRL.lpr/lpr mice with BAFF blocking fusion proteins successfully reduces many of their autoimmune symptoms.

Repeated administration of TACI-Ig and BAFF-R-Ig in NZB/WF1 mice substantially decreased total B cell numbers in peripheral blood, reduced proteinuria and increased mean survival [233, 253]. TACI-Ig treatment of MRL.lpr/lpr mice led to a long term reduction in splenic B cell and plasma cell numbers, reduction in total immunoglobulin levels, anti-dsDNA autoantibodies and rheumatoid factors (RF). This was sufficient to give long lasting reductions in proteinuria and improved survival 6 months after treatment [287]. This treatment was effective when given to mice with established autoimmunity as well as prior to disease onset, demonstrating potential utility for the treatment of established disease in humans. While TACI-Ig treatment was particularly effective in reducing disease parameters and autoantibody production in MRL.lpr/lpr mice, it produced only transient reductions of IgG in normal C57/B6 control animals

[287]. This suggests that TACI-Ig treatment may represent a novel treatment for human

SLE, enabling selective depletion of autoreactive B cells whilst leaving some normal humoral responses intact.

41 CHAPTER 1 INTRODUCTION

BAFF antagonists have also proven effective in preventing disease progression in

models of collagen induced arthritis (CIA) [233, 288]. Increased systemic BAFF is

observed during the course of this disease model and is coincident with the appearance of

anti collagen antibodies [289]. Treatment with TACI-Ig resulted in reduced levels of

anti-collagen antibodies and T cell responses, and an overall reduction in paw swelling

and joint destruction. This suggests that BAFF blockade has potential utility in treatment

of RA, however, further experiments testing the efficacy of TACI-Ig following disease

onset may be beneficial.

At present, several companies are developing BAFF blocking reagents for human

therapy. GlaxoSmithKlein/Human Genome Sciences have developed the human anti-

BAFF antibody LymphoStat-B [290] which has recently completed phase II trials for

efficacy in SLE and RA. Genentech/BiogenIdec and Zymogenetics/Serono are both

currently in phase I clinical trials with BAFF-R-Ig and TACI-Ig respectively, for the

treatment of SLE, RA and B cell malignancies. Thus it is likely that some form of BAFF

and APRIL blocking reagent will become part of the physician’s toolbox in the near

future.

HYPOTHESIS AND PROJECT AIMS

A large amount of data demonstrates the potent effects of BAFF on B cells. However, there is mounting evidence that BAFF may regulate aspects of T cell function as well.

BAFF is expressed by APCs in response to maturation signals [196, 197], placing it in

the relevant cellular context to regulate T cell activation. In vitro experiments

demonstrate that recombinant BAFF can enhance T cell proliferation and cytokine

production in response to suboptimal TCR stimulation [291], while blockade of

42 CHAPTER 1 INTRODUCTION

endogenous BAFF leads to reduced T cell responses [204, 288]. TACI is reported to be

expressed by a subset of activated T cells and is known to activate NFAT [252], a

transcription factor that is critical for T cell activation. Analysis of the peripheral T cell

compartment in BAFF transgenic mice reveals an increase in the number of CD4+ and

CD8+ effector T cells [228]. Significant levels of infiltrating T cells are observed in the tissues of BAFF transgenic mice (e.g. salivary glands) [269], suggesting that high levels of BAFF can drive effector T cell differentiation in vivo, and possibly the emergence of autoreactive specificities. These data suggest that BAFF may have an as yet unappreciated role in regulating T cell activation and effector function, which is in keeping with the emerging importance of TNFSF members as T cell costimulators and modulators of effector T cell responses [100]. Additionally, as anti-BAFF therapies move closer to the clinic, deciphering the contribution of BAFF to normal T cell responses is warranted.

Thus the experimental aims of this thesis are as follows:

¾ To determine a role of BAFF as a T cell costimulator

¾ To define BAFF receptor expression and usage by different T cell subsets

¾ To examine the ability of BAFF to modulate the course of T cell responses in

vivo

43 CHAPTER 2 MATERIALS AND METHODS

CHAPTER 2: MATERIALS AND METHODS

BUFFERS

BUFFER COMPONENTS MANUFACTURER 10 X PBS 3.6% Na2HPO4 Merck 0.2% KCl Merck 0.24% KH2PO4 Merck 8% Sodium chloride (NaCl) Ajax Finechem FACS Buffer 1 X PBS 0.5% BSA Gibco BRL MACS Buffer 1 x PBS 0.5% Fetal calf serum Gibco BRL 0.02 mM Na2EDTA Sigma ELISA coating buffer 50 mM NaHCO3 pH 9.6 Sigma ELISA wash buffer 1 x PBS 0.1% Tween 20 Pharmacia Biotech ELISA assay diluent 1 x ELISA wash buffer 1% BSA Gibco BRL Red Cell Lysis buffer 0.15 M NH4Cl Merck 0.01 M KHCO3 Sigma 0.15 M Na2EDTA Sigma 10 X TBE 89 mM Tris Base Sigma 89 mM Boric Acid Sigma 2 mM Na2EDTA Sigma

REAGENTS

Soluble forms of human BAFF and BAFFR-Fc, TACI-Fc and BCMA-Fc fusion proteins were supplied by Apotech (Epalinges, Switzerland). Human BAFF-Fc fusion protein and the plasmid contruct (ps805) containing cDNA for human TACI was a gift from Pascal Schneider (Lausanne, Switzerland). Anti human BAFF-R (clone #9-1) and

BCMA (clone #C4E2.2) were kindly provided Biogen-Idec (Cambridge, MA) and have been previously described [221]. Human BCMA-Ig was also provided by Biogen-Idec

44 CHAPTER 2 MATERIALS AND METHODS

and has been described [292]. Human BAFF, BAFF-R, IL-2 and IL-15 were supplied by

Peprotech (Rocky Hill, NJ). Denatured BAFF controls were prepared by incubation at

95oC for 2 h. LPS (from E. coli, serotype O111:B4), carbenicillin, methylated bovine

serum albumin (mBSA) and ovalbumin (OVA) was supplied by Sigma (St Louis, MO).

TM TM Imject CFA and Imject alum were purchased from Pierce (Rockford, IL). OVA323-

339 peptide was purchased from Auspep (Parkville, Victoria, Australia). Polyclonal

human immunoglobulin (hIg) was obtained from Novartis and used as a control for

receptor fusion proteins containing a human IgG Fc fragment. Murine ICOS-Fc and rat

anti mouse ICOS mAbs (clone #12A8 and 1C10) were kindly provided by Millenium

Pharmaceuticals (Cambridge, MA) and have been described [63]. Goat F(ab’)2 anti-

human μ chain Ab was purchased from Jackson ImmunoResearch Laboratories (West

Grove, PA). RPMI 1640, penicillin/streptomycin and bovine serum albumin (BSA) were

purchased from Gibco (Invitrogen, Mt. Waverley, Australia).

ANIMALS

Animals were housed under conventional barrier protection and handled in accordance with the Animal Experimentation Ethics Committee (AEEC), which complies with the

Australian code of practice for the care and use of animals for scientific purposes. AJ,

Balb/c and C57/B6 mice were bred in the BTF at the Garvan Institute of Medical

Research. The A/WySnJ mouse strain [238] was obtained from Jackson Laboratories

(Bar Harbor, Maine). BAFF transgenic mice [228], BAFF-/- mice [230] and TACI-/- [236]

were kindly supplied by Biogen-Idec on a mixed B6/129 background, and were backcrossed and maintained on a C57BL/6 background in Garvan BTF. BAFF transgenic mice were maintained homozygous for the BAFF transgene and wildtype mice

45 CHAPTER 2 MATERIALS AND METHODS

were used as controls, as previously described [293]. Two lines of BAFF transgenic mice

were used for experimentation, referred to as line 1 and 2, which differ slightly in BAFF

levels and immune responses (see chapter 4). μMT-/- mice [294] were purchased from

Jackson Laboratories (Bar Harbour, Maine). μMT-/- x BAFF transgenic mice were

established by breeding homozygous μMT-/- and BAFF transgenic line 1 mice and

subsequent interbreeding of the F1 generation. Homozygous μMT-/- x BAFF transgenic

mice and wildtype controls were derived from the F2 generation and breed as separate

lines for experimentation. To establish a Balb/c x BAFF transgenic line, line 1 BAFF

transgenic mice were backcrossed onto the Balb/c background for 10 generations. These

mice were bred from wildtype Balb/c x BAFF transgenic+/- crosses to yield wildtype or

BAFF transgenic+/- offspring. TACI-/- knockout mice were generated on a mixed C57/B6

x 129 background and subsequent backcrossed to C57/B6 for 6 generations. Separate wildtype and TACI-/- knockout lines were then bred from homozygous parents. A subline of TACI-/- knockout mice was subsequently generated by backcrossing TACI-/-

animals to the C57/B6 background for a further 2 generations. TACI+/- parents were then

mated to yield TACI-/- animals, and TACI+/- heterozygous and wildtype control littermates.

LYMPHOCYTE ISOLATION AND CULTURE

All human and mouse experiments were performed with approval of St. Vincent’s

campus human or animal ethics committees. Human PBMCs were isolated from human

blood by Ficoll-PaqueTM gradient centrifugation. Blood was diluted 1:1 with 1 X PBS,

then gently layered over 15ml of Ficoll-PaqueTM in 50ml Falcon tube. Gradient centrifugation was performed at 850 x g for 20 min at room temperature without brake.

46 CHAPTER 2 MATERIALS AND METHODS

The PBMC layer was collected and washed twice with PBS to remove platelets and

Ficoll-PaqueTM. Tonsils were obtained from routine tonsillectomies with informed consent from the patients and in accordance with Institutional Ethics Approval. Tonsillar

mononuclear cells were isolated by mechanical disruption, followed by Ficoll-PaqueTM density gradient centrifugation. Human CD3+ T cells were isolated from PBMC preparations using RosetteSep human T cell enrichment cocktail (StemCell Technologies,

USA) to >95% purity by flow cytometric analysis.

Splenocytes and lymph node cells were prepared by mechanical disruption with

frosted glass slides (Menzel-Glaser, Braunschweig, Germany) in 1 X PBS. Single cell

suspensions were obtained by filtering cells through 70µm nylon cell strainer (Falcon,

Becton Dickinson, Franklin Lakes, NJ) to remove connective tissue and debris.

Erythrocytes were removed by osmotic lysis with sterile red blood cell (RBC) lysis

solution, usually 5ml of lysis per spleen, incubated on ice for 1-2 min, then centrifuged at

300 x g for 5 min at 4°C.

Mouse CD3+ or CD4+ T cells were isolated from spleen via magnetic separation using

Pan T cell isolation kit or CD4+ T cell isolation kit respectively (Miltenyi Biotec, Auburn,

CA). Mouse B cells were isolated from spleen via magnetic separation using B cell

isolation kit (Miltenyi Biotec, Auburn, CA). Magnetic separations were performed using

LS columns and a VarioMACS magnet according to manufacturers instructions, to a

purity >95% by flow cytometric analysis. Cells were cultured in RPMI 1640,

supplemented with 10% fetal calf serum and penicillin/streptomycin (referred to as

lymphocyte culture media), at a concentration of 2x106 cells/ml unless otherwise noted.

47 CHAPTER 2 MATERIALS AND METHODS

Tissue culture flasks were purchased from Nunc (Nalge Nunc International, Rochester,

NY, USA).

CELL STIMULATIONS AND PROLIFERATION ASSAYS

Anti-CD3 induced T-cell proliferation assays were performed with human or mouse

PBMCs, splenocytes or purified T cells, activated in 96 well or 24 well plates at a concentration of 2x106 cells/ml. Anti-CD3 mAb and human BAFF (4 μg/ml) were coated onto plates overnight in PBS at 4oC, either separately or in combination, followed by two

1 X PBS washes before well seeding. Anti human CD3 mAb (mAb TR66) and mouse

CD3 mAb (mAb 145-2C11, BD Pharmingen) were used at a range of concentrations.

Anti human-CD28 Ab (mAb CD28.2, BD Pharmingen) and anti-mouse CD28 Ab (mAb

37.51, BD Pharmingen) was used in some assays at a concentration of 2-5 μg/ml and 2

μg/ml respectively. Anti-human IL-10 blocking Ab (mAb JES3-19F1, BD Pharmingen)

was used at 10 μg/ml. All assays were incubated for 72 hrs before harvesting. For long

term culture and expansion of activated T cells, cells were harvested at 72 hrs and

subsequently seeded into media supplemented human IL-2 and IL-15 (10 ng/ml) at 2x106

cells/ml, which was changed every 2-3 days for the duration of culture. Human BAFFR-

Fc, TACI-Fc, BCMA-Fc and hIg were used at 30 μg/ml.

B cell stimulations were performed in 24 well plates, in lymphocyte culture media with the addition of sterile β-mercaptoethanol (ICN Biomedical, Aurora, OH) to a final concentration of 5x10-5 M. Stimulations were performed for 24 or 48 hrs with human

BAFF (0.25 – 2μg/ml), human BAFF-R (20μg/ml), goat F(ab’)2 anti-human μ chain Ab

(5 μg/ml) and LPS (1 μg/ml). Cells were harvested and washed twice in media before use as stimulator cells other assays.

48 CHAPTER 2 MATERIALS AND METHODS

Human and mouse mixed lymphocyte reactions (MLR) were performed at a standard

concentration of 2 x 105 stimulator and responder cells per well, in 96 well plates.

Stimulator cells were inactivated before being seeded into culture using either mitomycin

C (Sigma) for human assays or γ-irradiation (250 rads) for mouse assays. Optimal assay duration corresponding to maximal proliferation was judged to be 6-7 days for human

MLR and 4-5 days for mouse MLR as the result of pilot studies. Mouse ICOS-Fc

(Millenium Pharmaceutics, Cambridge, MA) and hIg were used at 50 μg/ml during

ICOS-L blocking studies.

Antigen specific proliferation assays were performed using mice expressing the

DO11.10 αβ TCR transgene specific for the OVA323-339 peptide of chicken egg

ovalbumin and restricted to I-Ad [295, 296]. Stimulator cells were seeded at 2 x 105 cells per well, with 5 x 104 purified CD4+ D011.10 T cells used as the responder population.

Stimulator cells were inactivated using γ-irradiation (250 rads) and cultured with OVA323-

339 peptide at 0.03 – 3 μM. Optimal assay duration corresponding to maximal

proliferation was judged to be 4-5 days as the result of pilot studies.

For all mouse and human proliferation measurements involving incorporation of

radiolabel, cultures were pulsed with 1µCi/well [3H]-thymidine (Amersham,

Buckinghamshire, England) in 50 μl of media for 18 hrs prior to harvesting, and quantified using a β-scintillation counter. Alternatively proliferation was measured using dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes,

USA) by cell division. CFSE stocks were initially dissolved in dimethyl sulfoxide

(DMSO, BDH Merck, Victoria, Australia) at a concentration of 5 mM. Cell labelling was performed by resuspending cells at 1x107 cells/ml in RPMI 1640 without fetal calf

49 CHAPTER 2 MATERIALS AND METHODS

serum, CFSE was added to the cell suspension to make a final concentration of 10 μM.

The cell suspension was gently mixed immediately and incubated at 37°C for 10 min.

After labelling, cells were washed twice in lymphocyte culture media and cultured as described above. After the duration of the stimulation period, analysis of cell division

was performed using flow cytometry.

CDNA SYNTHESIS AND LIGHTCYCLER QUANTITATIVE RT-PCR

Total RNA was isolated from harvested cells using RNeasy Total RNA Isolation kit

(Qiagen) incorporating DNase treatment as per manufacturer’s instructions. Total RNA

(2 μg) was then used for cDNA synthesis via reverse transcription, with 4.5 units AMV

Reverse Transcriptase and MgCl2-containing buffer (Promega, Madison, WI), 20 nmol

dNTPs (Promega), and 0.02 nmol Oligo-p(dT)15 primer (Roche Molecular Biochemicals)

added in a total volume of 20 μl and incubated at 42°C for 90 min. cDNA was used for

LightCycler PCR (Roche Molecular Biochemicals) with the LightCycler FastStart Master

SYBR Green I kit (Roche Molecular Biochemicals) using 3mM MgCl2 and 0.5 μM

individual primers with the following specific protocol: 10 min @ 95°C activation of

FastStart Taq DNA polymerase; 45 cycles of 15 sec @ 95°C, 5 sec @ 63°C, 21 sec @

72°C; 5 min temperature ramp followed by melting point analysis. The primers used

were a combination of original sequences designed using Primer3 (http://www-

genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). LightCycler analyses used the

crossing point data for each gene during the logarithmic amplification program. The crossing point for each gene in each sample was normalised to the crossing point of

50 CHAPTER 2 MATERIALS AND METHODS

GAPDH. Respective genes were then compared between two samples and expressed as a

fold change.

PCR AND PRIMER SEQUENCES

Polymerase chain reaction (PCR) amplification of DNA fragments was performed

using the following standard conditions: 2 min 95°C denaturation; 45 cycles of 30sec @

95°C, 30sec @ 63°C, 1 min @ 72°C; 5 min @ 72°C final extension; 4°C hold.

Annealing temperatures were optimised for each primer set as determined by specific

product amplification. For determination of transcript expression, each reaction mixture

contained 1 μl of cDNA preparations, 1 mM MgCl2 (Promega, Madison, WI), 0.4 μM dNTPs (Promega, Madison, WI), 0.2 μM individual primers and 2.5 U of Taq

polymerase in a total volume of 50 μl. For amplification of DNA fragments for transfectant construction, the high fidelity DNA polymerase Pfu was used instead of Taq.

The reaction mix contained 1 μl of cDNA preparations, 0.4 μM dNTPs (Promega,

Madison, WI), 0.2 μM individual primers and 1.5 U of Taq polymerase in a total volume of 50μl. To improve the yield of DNA fragment from these reactions, DMSO was used at a concentreation range between 1-5% of final reaction volume in some reactions. All reactions were performed on the GeneAmp PCR System 2700 (Applied Biosystems).

All primers were purchased as sequencing/PCR grade from Geneworks (Hindmarsh,

South Australia). The nucleotide sequence for each primer is listed below:

Gene Product Primer name Sequence Human IL-10 IL-10-1 GCT GAG AAC CAA GAC CCA GAC A IL-10-2 CAT GGC TTT GTA GAT GCC TTT Human TGF-β TGFbeta-1 GGC TAC CAT GCC AAC TTC TG TGFbeta-2 GGA CAG CTG CTC CAC CTT GG Human BAFF-R BAFF-R1 GGC CCT GAG CAA CAA TAG CA

51 CHAPTER 2 MATERIALS AND METHODS

BAFF-R2 AGG CAA GCA CAC CAA ACT CC Human BCMA BCMA-1 CAC GGT GGA AGA ATG CAC CT BCMA-2 GGC ACT GCT CGA GTC GAA AT Human TACI TACI-5 CAA GTC TTC CCA GGA TCA CG TACI-6 GCA CAC ACA CAA TGC CAA G Human BAFF-R BAFF-R clonF2 GAA CAC GAA TTC ACC ATG AGG CGA GGG CCC CGG AGC CTG CGG (full length) BAFF-R clonR2 GAA TGT TCT AGA CTA CTA TTG TTG CTC AGG GCC GGC CGT CTT GGT GG Human GAPDH GADPH-1 GAC ATC AAG AAG GTG GTG AA GAPDH-2 TGT CAT ACC AGG AAA TGA GC

AGAROSE GEL ELECTROPHORESIS

Analysis of DNA fragements was performed using agarose gel electrophoresis.

Agarose (Progen BioSciences, Arderfield, Australia) was dissolved by heating in 1X

TBE, to a final concentration of 1% or 2%, and ethidium bromide (Sigma) was added to a final concentration of 0.5 ng/ml. DNA separation was achieved under 100V for 45 mins.

100bp ladder and 1kB ladder (Promega, Madison, WI) were used as molecular weight reference controls.

GENECHIP MICROARRAY ANALYSIS

For Genechip assessment of transcript expression, cRNA was prepared using the methods as described [297]. Hybridisation to the Affymetrix U133A and B Genechips and subsequent scanning and analysis was performed according to Affymetrix protocols.

Genechip analyses included human Th1 and Th2, purified eosinophils, cultured mast cells, IgE activated mast cells, purified neutrophils, immature DCs, mature DCs, macrophages, and splenic memory B cells. A full description of the preparation of these cells, and access to the full genechip results, is available at http://www.garvan.unsw.edu.au/public/microarrays.

52 CHAPTER 2 MATERIALS AND METHODS

TRANSFECTANT CONSTRUCTION AND DNA SEQUENCING

An expression DNA construct containing the human TACI cDNA sequence, named

ps805, was kindly provided by Pascal Schneider and was used to create tranfectants in

the rat basophilic leukaemia cell line, RBL. Briefly, 10 μg of purified ps805 plasmid

7 DNA in 30 μl of sterile H2O was added to 550 μl of RBL cells in RPMI 1640 at 1x10

cells/ml. Cells/DNA mixture was placed in electroporation cuvettes, incubated on ice for

10 mins, electroporated at 960 mF, 310 V in a Gene Pulser II system (Bio-Rad), and then

reincubated on ice for 30 mins. Cells were diluted in 50 ml of lymphocyte culture media

and seeded at 100 μl/well into 96 well plates. After overnight incubation, media was

removed from wells and replaced with lymphocyte culture media containing 350 μg/ml

G418 (Gibco, Invitrogen, Mt. Waverley, Australia) for selection of transfected clones. A

number of positive clones were isolated and expanded in G418 containing media, then

selected for cell surface expression of TACI protein using hBAFF-Fc binding assay

(described below).

To construct a human BAFF-R cell surface transfectant, the full length human BAFF-

R sequence was amplified from the human B cell line BJAB using BAFF-R clon-F2 and

R2 primer set. After PCR amplification, DNA fragments were gel purified using

QiaQuick gel elution kit (Qiagen). Prior to ligation into pGEM T-Easy vector (Promega,

Madison, WI) purified DNA fragments were A-tailed using 350 ng of DNA, 0.2 μM

dATP, 1 mM MgCl2 and 5 U of Taq polymerase in a total volume of 10 μl, and incubated for 30 mins @ 70°C. Ligations were performed overnight @ 4°C as per manufacturer’s instructions using 2 μl of ligation mix. Ligated mixtures were used to transform

53 CHAPTER 2 MATERIALS AND METHODS

chemically competent Top10 E. coli (Invitrogen, Mt. Waverley, Australia) as per

manufacturer’s instructions. Colonies were picked and grown overnight in 5 ml of LB

broth (prepared by Garvan media prep) with 100 μg/ml carbenicillin. Plasmid DNA was

purified using Qiaprep spin Miniprep kit (Qiagen) and checked for DNA insert by

restriction enzymes EcoRI and XbaI and subsequently sequenced to validate the integrity

of cDNA. BAFF-R insert DNA of the correct sequence was excised from pGEM construct with the restriction enzymes EcoRI and XbaI and directionally cloned into the pTracer CMV vector (Invitrogen, Mt. Waverley, Australia) as per manufacturer’s instructions. An appropriate BAFF-R/pTracer clone was identified after DNA sequencing, purified using Qiagen Plasmid midi kit (Qiagen) and used to transfect the mouse B cell line, L1.2 using Lipofectamine 2000 (Invitrogen, Mt. Waverley, Australia) as per manufacturer’s instructions. Cells were rested for 2 days post transfection then transferred to lymphocyte culture media containing 10 μg/ml blasticidin (Gibco,

Invitrogen, Mt. Waverley, Australia) and transferred to 96 well plates for selection of positive clones. Positive clones were isolated on the basis of GFP expression and binding of hBAFF-Fc to the cell surface as detected by flow cytometry (described below). To enhance the levels of BAFF-R expressed on the cell surface prior to immunisation for mAb generation, L1.2 transfectants were exposed to 5 mM butyric acid in lymphocyte culture medium overnight.

DNA sequencing was performed by the Supamac sequencing facility at the University of Sydney using ABI PRISM 3700 sequencing platform. 1 – 3 μg of purified DNA was prepared with 5 – 10 pmol of either T7 or SP6 sequencing primers for analysis, with reactions, cleanup and analysis performed by Supamac.

54 CHAPTER 2 MATERIALS AND METHODS

PRODUCTION AND SPECIFICITY OF MABS TO HUMAN BAFF RECEPTORS

Monoclonal antibodies to both human BAFF-R and human TACI were generated by

immunising C57/B6 mice and Wistar rats respectively, using the following immunisation

protocol: 2x107 irradiated transfectants were immunised intraperitoneally 6 times at 2

week intervals. This was followed 2 weeks later by a final intravenous injection of the

same quantity of transfectants. 3 days after intravenous injection spleen and lymph nodes

were harvested for hybridoma production as described previously [298]. Briefly spleen

and lymph node cells were mixed with myeloma cell line SP20. Cells were then washed

twice in warm DMEM (Gibco, Invitrogen, Mt. Waverley, Australia) and centrifuged for 5

min at 300 x g. After residual supernatant was removed polyethylene glycol (PEG,

Sigma) was added to the cell pellet gradually with constant mixing, at a rate of 1.5 – 2 ml

over 2 min. 2 ml of DMEM was added over 2 min followed by another 8 ml of DMEM

in a similar fashion. The tube was filled up to 50 ml with DMEM containing

hypoxanthine-aminopterin-thymidine (HAT, Sigma) and incubated for 1 h at 37°C

(fusions were performed by Cynthia Xin).

After incubation, hybridoma cells were then aliquoted (60 μl/well) into 10 x 384 well plates (Nalge Nunc International, Rochester, NY, USA). Three weeks after the fusion, supernatants from hybridomas surviving in HAT medium were tested individually for the presence of specific antibodies by ELISA, from which positive cultures were selected and expanded. After 3 – 5 days, supernatants were aspirated from each well and assayed for

reactivity against transfectants by flow cytometry. The hybridoma cultures with the

strongest transfectant affinity (as determined by FACS analysis) were subcloned.

55 CHAPTER 2 MATERIALS AND METHODS

Subcloning was performed three times to ensure the antibodies produced were monoclonal. After cloning, wells containing colonies derived from single cells were tested for antigen binding activity by ELISA and flow cytometry with transfectants.

Subsequently, positive clones (monoclonal) were grown in serum-free medium

(Thermotrace, Australia).

Monoclonal antibodies were isotyped using MonoAB ID/SP kits (Zymed Laboratories

Inc., CA, USA). Antibodies were purified from culture supernatants with Protein G

Sepharose (Amersham, UK). Supernatants from hybridoma cultures were passed through the packed column by gravitational force, washed with with 20 X bed volumes of 1 X

PBS and eluted with 2 ml of 0.1 M glycine buffer (pH 3.0-2.5). 1 M Tris-HCl, pH 9.0 was added to neutralise the eluted fractions at 1:10 dilution. Purified antibodies were then dialysed against 1 X PBS to remove contaminants in a 10KMWKO Slide-A-Lyzer™ cassette (Pierce, Rockford, IL) overnight at 4°C. The concentration of dialysed antibody was estimated by Bradford’s protein assay (Bio-Rad, Sydney, Australia).

BIOTINYLATION

Recombinant protein and purified monoclonal antibodies were labelled with biotin for ease of detection. Antibodies were first concentrated to 2 mg/ml using Centricon Plus 20

YM-10 columns (Millipore), recombinant proteins were resuspended at 2 mg/ml. A 20 fold molar excess of EZ-Link sulfo-NHC-LC biotin (Pierce, Rockford, IL) was added and samples incubated at room temperature for 30 min. Samples were then dialysed overnight in a 10KMWKO Slide-A-Lyzer™ cassette against 1 X PBS to remove unbound biotin reagent.

56 CHAPTER 2 MATERIALS AND METHODS

ELISA

Murine IL-4, IL-5, IFN-γ, and IL-10 ELISA kits were purchased from Becton

Dickinson (San Jose, CA) and assays were performed as per manufacturer’s instructions.

Goat anti-mouse AP conjugated antibodies were purchased from Southern Biotech

Laboratories (Birmingham, AL). Mouse BAFF ELISA was performed as previously described [293].

For the detection of positive hybridomas and determination of monoclonal antibody specificity, 384 well MaxiSorp ELISA plates (Nalge Nunc International, Rochester, NY,

USA) were coated with human BAFFR-Fc and TACI-Fc in ELISA coating buffer overnight at 4°C. Non-specific binding was then blocked with ELISA buffer for 1 hr at

37°C. After blocking, 20 μl of supernatant obtained from different hybridoma cultures or purified monoclonal antibody was added, and incubated for 1 hr at 37°C. HRP conjugated goat anti mouse IgG + IgM (H+L) or goat anti-rat IgG + IgM (H+L) were used at 1:10 000 dilution for the detection of mouse and rat monoclonal antibodies respectively by addition to each well and incubated for 1 hr at 37°C. Following additional washes detection was performed by the addition of TMB substrate reagent set and stopped by the addition of 2 M H2SO4.

mBSA-specific Ab responses was analyzed as follows: 384-well MaxiSorb plates

(Nalge Nunc International, Rochester, NY, USA) were coated with 50 μg/ml mBSA

diluted in 0.1 M sodium bicarbonate buffer (pH 9.6) at 4oC, overnight. Plates were

washed 3 times with ELISA wash buffer. Serial dilutions of mouse serum in ELISA

buffer (1% BSA in PBS) were added to the plate. Anti-mBSA antibodies were detected

using 0.125 μg/ml alkaline phosphatase (AP)-conjugated goat anti-mouse IgM, anti-

57 CHAPTER 2 MATERIALS AND METHODS

IgG1, anti-IgG2a, anti-IgG2b, anti-IgG3 or anti-IgA (Southern Biotechnology Associates

Inc., Birmingham, AL) at 1:3000 dilution. p-Nitrophenyl phosphate tablets (Sigma) were

used for detection and plates were read at an OD of 405 nm. The titre (log base 2) is

defined as the serum dilution giving an OD four times higher than that of background

(where 1=1/50 dilution).

FLOW CYTOMETRY

All anti mouse unconjugated, FITC-, PE-, CyChr-, PerCP-, PE-Cy7-, APC-Cy7-,

APC- and biotin-conjugated mAbs to various cell surface markers were from BD

Biosciences (San Diego, CA), with the exception of CCR7-FITC (R&D Systems,

Minneapolis). Immunofluorescent labelling of cells was performed by addition of 1x105

cells/well in V-bottomed 96 well plates. Primary antibody staining was performed in 100

μl of FACS buffer and incubated on ice for 25 mins. All anti human antibodies were

used at a concentration of 1 in 50 with the exception of CD45RO APC which was used at

1 in 200. All anti mouse FITC-conjugated antibodies were used at 1 in 100, while all PE-

, PerCP-, APC- and biotin-conjugated antibodies were used at 1 in 200. After primary antibody staining cells were washed twice with 150 μl of FACS buffer. Secondary

staining was performed using fluorochrome-conjugated streptavidin (BD Biosciences,

San Diego, CA) or appropriate secondary antibody staining reagents (Jackson

ImmunoResearch Laboratories, Inc., PA) at 1 in 500 in 50 μl of FACS buffer and incubated on ice for 15 mins. Cells were then washed once in 150μl of FACS buffer, resuspended in 200 μl of FACS buffer and analysed using BD FACSCaliburTM or LSRII flow cytometers (BD Biosciences, San Jose, CA). Six-color flow cytometric analysis to assess BAFF-R expression on naïve and memory subsets of CD4+ and CD8+ cells

58 CHAPTER 2 MATERIALS AND METHODS

employed anti-BAFF-R biotin (mAb 9-1) and streptavidin-PE; anti-CCR7-FITC; anti-

CD3-PerCP, anti-CD45RO-APC, anti-CD4-PE-Cy7 and anti-CD8-APC-Cy7. A

biotinylated mIgG1 (BD Pharmingen) was used as an isotype control for the BAFF-R

antibody.

Staining of transfectants and lymphocyte populations with hBAFF-Fc and biotinylated

BAFF to detect cell surface BAFF binding was performed as for antibody staining, with

both reagents being used at concentrations between 1 in 50 and 1 in 1000. Biotinylated

BAFF was detected as per biotinylated mAbs, while hBAFF-Fc was detected using PE-

conjugated F(ab')2 fragment goat anti human IgG + IgM (H+L) (Jackson

ImmunoResearch Laboratories, Inc., PA) at 1 in 500.

HISTOLOGY, IMMUNOHISTOCHEMISTRY AND IMMUNOFLUORESCENCE

Human and mouse tissues were either frozen in Tissue-Tek OCT compound (Sakura

Interational) or fixed in 10% buffered formalin and embedded in paraffin by the

Histology Dept. at the Univerity of New South Wales. Mouse paws was subject to a

further decalcification step prior to embedding in paraffin, which entailed 10% formalin

in PBS was supplemented with formic acid (Sigma) at a 9:1 ratio. 4-6 µm sections were

cut using a Cryostat (Leica, Wetzlar, Germany) from frozen tissue or a Microtome

(Leica) for paraffin embedded tissue.

Slides were stained with hematoxylin and eosin (H&E) for histologic examination.

Paraffin embedded tissue sections were de-waxed and re-hydrated by successive 10 min

baths in xylene (twice; Merck), 100% ethanol (twice), 70% ethanol then water. The rehydrated tissue was stained with hematoxylin (Merck) for 30 sec, washed for 2 min

59 CHAPTER 2 MATERIALS AND METHODS

under running deionised water, then emersed in Scott’s blueing solution for a further 2

min. Slides were then placed under running water for 2 min and then rinsed in 70%

ethanol for 1 min. Sections were then stained with eosin (Sigma) diluted to 30% in 70%

ethanol for 30 sec, followed by washes in 70% and 100% ethanol before rapid

dehydration in followed by xylene. Slides were mounted using Eukitt mounting solution

(Calibrated Instruments Inc., Hawthorne, NY).

For immunohistochemical visualisation of BAFF-R expression, antigen retrieval was

performed by immersing 4 μM thick paraffin sections of palatine tonsil in an EDTA-

based retrieval solution (pH 9.0) and heating for 20 min at 95-99oC in a water bath. After

cooling, sections were immunostained using a DakoCytomation Autostainer

(DakoCytomation Ca. Inc, Carpinteria, USA); following 5 min incubation with 3%

hydrogen peroxide, sections were incubated sequentially for 30 min with anti-BAFF-R

mAb (11C1) and Mouse EnvisionTM+ HRP (DakoCytomation Ca. Inc, Carpinteria,

USA). Anti-BAFF-R binding was visualized using Liquid DAB+ (DakoCytomation Ca.

Inc, Carpinteria, USA).

For immunofluorescent visualisation of BAFF-R expression 6 mM sections of human

palatine tonsil were cut from frozen in Tissue-Tek OCT compound. Sections were air dried for 30 mins, fixed in 1% paraformaldehyde in 0.1 M sodium phosphate buffer (pH

7.3) for 20 mins at room temperature. Sections were then wash twice in 1 X PBS for 5 min, permeabilised in 70% ethanol for 30 min at -20oC and washed again in 1 X PBS for

5 min. Sections were then blocked with 10% normal goat serum (Jackson

ImmunoResearch Laboratories, Inc., PA) in 1% BSA/PBS for 20 mins at room

temperature in a humidified chamber. Goat serum was aspirated and 1o Ab diluted in 1%

60 CHAPTER 2 MATERIALS AND METHODS

BSA/PBS incubated overnight at 4oC. Sections were washed 3 times with 1 X PBS for 5

min then incubated with 2o Ab for 1hr at room temperature in a humidified chamber,

followed by three 1 X PBS washes for 5 min. Excess liquid was then removed from the

slides, 1 drop of VectashieldTM mounting medium (Vector laboratories Inc., Burlingame,

CA, USA) added and coverslipped. The images were obtained using Zeiss fluorescent

microscope (AxioVision Software) or Leica TCS SP2 RS confocal microscope (Leica).

Anti human BAFF-R (clone #9-1) was used at 1 in 100, while anti human Bcl-6 (Ab #N-

3, polyclonal rabbit IgG) from Santa Cruz Biotechnology (Santa Cruz, CA), was used at

1 in 20.

IMMUNISATIONS AND ANTIGEN RESTIMULATIONS

Six to eight-week old mice were injected subcutaneously at the tail base with 200 μl of

1.25 mg/ml methylated bovine serum albumin (mBSA) in CFA (1:1 mix) or 1 mg/ml

OVA solution with alum (1:1 mix) on day 1. On day 7, inguinal lymph nodes were

collected and single cell suspensions prepared. Cultures were normalised to 2x106 T

cells/ml, seeded in 100 μl into 96 well plates and restimulated with the appropriate antigen for 72 hrs. Proliferation was measured by addition of [3H]-thymidine 18 hrs prior to harvesting, and subsequent β-scintillation counting and supernatants collected for measurement of cytokine production by ELISA. Lymph node cultures from mBSA/CFA immunized animals were maintained in X-Vivo 15 serum free medium (BioWhitakker,

Walkersville, MD) supplemented with penicillin/streptomycin, and restimulated with 40

μg/ml mBSA. Alternatively lymph node cultures from OVA/alum immunized animals

were cultured in lymphocyte culture medium and restimulated with 500 μg/ml OVA.

61 CHAPTER 2 MATERIALS AND METHODS

DTH

Six to eight-week old mice were injected subcutaneously at the tail base with 200 μl of

1.25 mg/ml methylated bovine serum albumin (mBSA) in CFA (1:1 mix) on day 1, as

described previously [299]. On day 7, mice were rechallenged in one hind footpad with

injection of 20 μl of a 10 mg/ml mBSA solution, while the opposite hind footpad

received 20 μl of 1X PBS. Paw swelling was measured 8-72 hrs after re-challenge with a

dial thickness gauge. For paw tissue section analysis, mice were sacrificed at 48 hrs post

challenge. The paws of mice were dissected, then fixed and decalcified as described

above. Paws were then embedded in paraffin, sectioned, and stained with H&E. For

analysis of mBSA specific Abs, mice were sacrificed 7 days post footpad challenge,

serum collected and mBSA specific Abs levels measured by ELISA.

Receptor fusion proteins or monoclonal antibody treatment regimens were as follows:

150 μg of BCMA-Ig or hIg per mouse at days -7, -3, 2 and 5

30 μg of anti ICOS (clone # 12A8 or 1C10) per mouse at days 1, 3, 5 and 7

In some DTH models additional B cells were transferred into recipient animals on day

0, 24 h before immunization. B cells were purified from whole spleens of donor animals

using magnetic separation and resuspended in PBS at 5x107 cells/ml. 1x107 cells in a

total volume of 200 μl were transferred by intravenous injection into the tail vein.

OVA-INDUCED ALLERGIC AIRWAY INFLAMMATION

Six to eight week old mice were injected i.p. with 200 μl of a 1 mg/ml OVA solution

with alum (1:1 mix) on day 1 and day 15. Mice were given a 20 min aerosol on days 28,

30, 32, 34 consisting of either PBS or 1% OVA in PBS. Mice were sacrificed on day 35, and bronchoalveolar lavage (BAL) fluid and organs were collected. Total BAL fluid cell

62 CHAPTER 2 MATERIALS AND METHODS

numbers were enumerated and differential cell counts were performed after cytospin and

Giemsa staining to establish the number and identity of infiltrating cells. Lung tissues were fixed in 10% formalin/PBS for 7 days and embedded in paraffin, then sectioned and stained with H&E. At the completion of the aerosol regimen peri-bronchial lymph nodes were collected, pooled into groups and cultured in lymphocyte culture medium. Cultures were normalised to 2x106 T cells/ml and restimulated for 72 hrs with 100 μg/ml OVA,

and proliferation and cytokine production measured as per inguinal lymph node cultures.

STATISTICAL ANALYSIS

Statistical significance was determined using a paired Student t-test, and significance

with relation to comparison data indicated as follows: p < 0.05 (*), p < 0.01 (**) and p <

0.005 (***).

63 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

CHAPTER 3: BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

INTRODUCTION

The appropriate control of T cell activation is essential for an effective response against pathogens and maintenance of self tolerance. Costimulatory molecules are critical for the control of naïve T cell activation and determine whether activation of tolerance ensues after interaction with APCs. In addition to the well characterised role of the B7 family molecules, certain TNFSF molecules e.g. LIGHT, OX40L and 4-1BBL have emerged as important costimulators of T cells [100, 101, 159]. These molecules provide important survival and proliferative signals during the processes of naïve T cell activation, effector generation and memory formation [100]. Overexpression of these molecules in transgenic mice results in activation of autoreactive peripheral lymphocytes and the development of autoimmune inflammation [160-163]. Thus novel members of the TNFSF also have the potential to be important regulators of T cell function.

Intense study of the TNFSF member BAFF indicates that it plays crucial roles in peripheral B cell survival, maturation and homeostasis [214, 225, 300] and that its overproduction is associated with autoimmune disease [228, 229, 233, 269, 278, 301,

302]. More recently it has been recognised that BAFF may also regulate T cell responses, in a similar fashion to other TNFSF ligands. Therefore the aims of this study were two-fold; first to further investigate the ability of BAFF to stimulate human T cells and secondly to thoroughly define BAFF receptor usage, expression and regulation on human T cells, via the production and characterisation of specific monoclonal antibodies.

64 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

RESULTS

Co-stimulatory effects of BAFF on human T-cell proliferation

A previous study demonstrated that BAFF was able to costimulate human T cell activation in the presence of suboptimal concentrations of anti-CD3, resulting in increased proliferation and cytokine production [291]. Initially we activated purified human CD3+ T cells at a range of anti-CD3 concentrations (1 μg/ml and 5 μg/ml), in the

presence or absence of recombinant BAFF (4 μg/ml). BAFF stimulation increased T cell

proliferation (Fig. 3.1A and B). This was most striking at the highest anti-CD3

concentration (5 μg/ml), where proliferation in the presence of BAFF was to anti-CD28

induced levels. These results confirmed those obtained previously [291]. To extend

these studies in an additional system of T cell activation, we examined the effects of

BAFF in an allogenic mixed lymphocyte reaction. Consistent with the effects observed

with anti-CD3 activation, BAFF increased proliferation of purified CD3+ T cells in MLR

(Fig. 3.1C). Similar to the previous study [291], we found that anti-CD3 stimulated T

cells responded to BAFF only when it was immobilised to plastic (Fig. 3.1A and B).

However, soluble BAFF was able to costimulate T cells in the MLR in the presence of

APCs (Fig. 3.1C).

We next determined the role of endogenous BAFF in regulating T cell activation in a

mixed cell environment. We assayed T cell proliferation in response to anti-CD3

activation in the presence or absence of Fc fusion proteins of the 3 BAFF receptors

(BAFFR-Fc, BCMA-Fc and TACI-Fc), allowing blockade of endogenous BAFF. All three fusion proteins inhibited T cell proliferation in response to anti-CD3 alone (1 μg/ml

65 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

Figure 3.1: BAFF costimulates T cell activation. Human CD3+ T cells were purified from blood by magnetic separation and activated with A, 1 μg/ml and B, 5 μg/ml anti-CD3 for 72 hrs in the presence or absence of BAFF. BAFF was used either as plate-bound (B), soluble (S) or denatured (DN). C, Human CD3+ T cells were purified from blood and inactivated PBMC stimulator cells used in a MLR for 7 days in the presence or absence of BAFF. PBMCs were activated with D, 1 μg/ml and E, 5 μg/ml anti-CD3 for 72 hrs, together with fusion proteins of BAFFR-Fc, BCMA-Fc, TACI-Fc or a hIgG control (30 μg/ml). Anti-CD28 was also added to some cultures. Proliferation was measured by thymidine incorporation. Statistical significance was determined using a paired Student T-test p < 0.05 (*) and p < 0.005 (***).

66 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R and 5 μg/ml) (Fig. 3.1D and E). However these fusion proteins demonstrated little effect on T cell proliferation in the presence of anti-CD28 costimulation (Fig 3.1E). These data demonstrate that endogenous BAFF regulates T cell proliferation in a mixed cell environment, however it effects are minimal in the presence of strong anti-CD28 costimulatory signals. Neutralisation of BAFF rather than APRIL appeared to be responsible for the observed effect, since BAFFR-Fc (BAFF- specific) reduced proliferation to the same extent as TACI-Fc or BCMA-Fc. Although it has been reported that BCMA-Fc has a lower affinity for BAFF [292], we found all three fusion proteins showed a similar ability to block BAFF activity, presumably because these fusion proteins were used at high concentrations (30 μg/ml).

BAFF has additional repressive effects on T cell proliferation via secondary cell types and factors

We next examined the costimulatory effects of BAFF in vitro, this time using anti-

CD3 stimulation to activate T cells in two distinct environments. These were in the context of total PBMC, or purified T cells alone. As previously shown (Fig. 3.1A),

BAFF increased proliferation of suboptimally stimulated purified CD3+ T cells (Fig.

3.2A). However, when total PBMC were suboptimally stimulated by anti-CD3 in the presence of BAFF, proliferation was significantly reduced compared to a denatured

BAFF control (Fig. 3.2A). CFSE division profiling indicated that this reduction in proliferation occurred in both CD4+ and CD8+ T cell compartments (Fig. 3.2B). These results show that the presence of additional cell types has a profound

67 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

Figure 3.2: BAFF has indirect suppressive effects on T cell proliferation. A, Whole PBMC (white bars) or CD3+ T cells (black bars) were purified from blood and activated with 1 μg/ml anti-CD3 for 72 hrs in the presence or absence of BAFF. B, PBMC were labelled with CFSE and activated with 1 μg/ml anti- CD3 for 72 hrs in the presence of BAFF (green line) or a denatured control (purple). C, PBMC were activated with 1 μg/ml anti-CD3 for 4 hrs in the presence or absence of BAFF. BAFF was used either as plate-bound (B), soluble (S) or denatured (DN). Relative mRNA levels were determined for IL-10 and TGFβ using GAPDH as a reference control. D, PBMC were activated with 1 μg/ml anti-CD3 for 72 hrs in the presence or absence of BAFF. BAFF was used either as denatured (DN) or plate-bound (B), anti IL-10 Ab was used at 10 μg/ml. A, and D, proliferation was measured by thymidine incorporation. Statistical significance was determined using a paired Student T-test p < 0.05 (*) and p < 0.005 (***).

68 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

effect on the outcome of BAFF stimulation, and implies that the local environment in

which T cells encounter BAFF in vivo may be critical in determining whether BAFF

positively or negatively regulates T cell responses.

A candidate gene approach was used to identify molecules that may have been responsible for the repressive effects. Both IL-10 and TGFβ exert potent suppressive

effects on T cells and were therefore chosen for further study [303, 304]. Moreover IL-

10 was also reported to be significantly upregulated following BAFF stimulation of a B

lymphoma cell [305]. Stimulation of PBMC cultures with anti-CD3 (1 μg/ml) and BAFF

for 4 hrs resulted in a significant upregulation of IL-10 in response to BAFF (Fig. 3.2C).

TGFβ was not induced under identical conditions. Similar results were observed at 8 hrs

stimulation (data not shown). Fold induction of IL-10 varied with donor and a

representative plot is shown (Fig. 3.2C). Having determined that IL-10 was highly

regulated by BAFF at the mRNA level, we examined whether mAb blockade of IL-10

could reverse the observed proliferative defects in BAFF stimulated PBMC cultures.

Upon blockade of IL-10, proliferation was restored to levels similar to that observed in

the denatured BAFF control. These experiments implicate IL-10 from B cells as being

predominantly responsible for the indirect suppression of T cell proliferation by BAFF.

BAFF-R but not TACI or BCMA transcripts are expressed by various

T cell populations

To establish which BAFF receptors were expressed on T cells, and which of these was

mediating the direct costimulatory effects of BAFF, we performed RT-PCR on in vitro

69 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

Figure 3.3: T cells express detectable mRNA transcripts for BAFF-R but not TACI or BCMA. A, RT-PCR was performed on cDNA prepared from Th1/Th2 T cells, and Jurkat, BJAB and Raji cell lines using primers for BAFF receptors. B, A range of immune cell types were isolated and their transcript profile determined using Affymetrix U133A and B chips, containing probes for BAFF, APRIL, BCMA and TACI, (but not BAFF-R). Semi-quantitative expression values, expressed as a heat map, are shown. Affymetrix algorithms made calls of presence or absence for each gene, absence is indicated by an “A” in each square.

cultured T cells and the Jurkat T cell line with primer sets for the 3 BAFF receptors.

Results demonstrated that BAFF-R, but not TACI or BCMA, was present at the mRNA

level in several T cell RNA samples (Th1, Th2 and Jurkat) (Fig. 3.3A). The B cell lines

BJAB and Raji were used as positive controls in these experiments and showed

expression of all three BAFF receptors. To confirm these results at the RNA level, we

used the Affymetrix data mining tool to assess the expression of BCMA and TACI in numerous T cell subsets that we had previously profiled using Affymetrix GeneChip arrays. Unfortunately BAFF-R was not represented on these versions of the human chips

(version U95A). Fig. 3.3B shows that TACI was absent from all human T cell subsets assessed, including in vitro derived Th1 and Th2 cells, specialized subsets such as effector memory and central memory T cells, skin homing, gut homing, and T follicular

70 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

homing subsets. BCMA was also absent from all T cell samples analysed. All of the T

cell subsets analysed showed expression of BAFF. Transcripts for BAFF were also

expressed strongly in dendritic cells, mast cells, eosinophils and, particularly, neutrophils

which is consistent with reports from other groups of abundant BAFF production by

neutrophils [194, 206]. Transcripts for APRIL were also detected in mast cells,

eosinophils and neutrophils but at much lower levels than for BAFF (Fig. 3.3B);

transcripts of APRIL were absent from all T-cell subsets.

Construction of transfectants for monoclonal antibody generation

Expression of BAFF-R mRNA in a variety of T cell samples indicated that this was

the BAFF receptor most likely to be mediating BAFF effects on T cells. However,

despite the lack of TACI transcripts by RT-PCR or transcript profiling experiments there

was evidence from the literature suggesting that TACI was expressed on activated T cells

[252]. To clarify the nature of the BAFF receptors expressed by T cells, we resolved to

generate monoclonal antibodies to human BAFF-R and TACI. In the absence of large

quantities of purified BAFF-R and TACI protein we developed cell surface transfectant

cell lines with which to immunise mice or rats. The full length cDNA of human BAFF-R was amplified from the BJAB cell line, which is known to express high levels of BAFF-R

[213], using the BAFF-R clonF primer set. Efficient amplification was only achieved upon addition of 3 or 4% DMSO to the PCR (Fig. 3.4A, lanes 5 and 7). Amplification of a TACI PCR fragment from the ps805 plasmid vector was used as a positive control, using TACI-5 and -6 primer set (Fig. 3.4A, lane 2, 4, 6 and 8). The BAFF-R cDNA

71 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

Figure 3.4: Construction of transfectants expressing BAFF-R and TACI. A, cDNA from the coding region of BAFF-R mRNA was amplified via PCR. Pairs of reactions were performed at increasing DMSO concentrations, BAFF-R amplification (lanes 1, 3, 5 and 7) and positive control reactions (lanes 2, 4, 6 and 8). Reaction pairs contained 1% (lanes 1 and 2), 2% (lanes 3 and 4), 3% (lanes 5 and 6) and 4% (lanes 7 and 8) DMSO respectively. BAFF-R band marked with black arrow B, EcoRI/XbaI digests of 4 pGEM constructs containing the BAFF-R insert. C, EcoRI/XbaI digests of 5 pTracer constructs containing the BAFF-R insert. D, GFP expression by transfected L1.2 cells. Untransfected (thin grey line), empty pTracer vector (dotted grey line) and pTBAFF-R transfected (black line) are shown. E, BAFF-Fc binding of pTBAFF-R transfected L1.2 cells. Unstained (thin grey line), anti Fc PE alone (dotted grey line) and hBAFF-Fc + anti Fc PE (black line) are shown. F, BAFF-Fc binding of TACI transfected RBL cells. Unstained (thin grey line), anti Fc PE alone (dotted grey line) and hBAFF-Fc + anti Fc PE (black line) are shown.

72 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

fragment was subsequently cloned into the pGEM T-Easy vector. Plasmid clones were selected for inserts of the correct size (580bps) after digestion with the restriction enzymes EcoRI and XbaI (Fig 4.4B) and their sequence verified by nucleotide sequencing. Agarose gel purified fragments were directionally cloned into the pTracer expression vector, followed by validation of clones by EcoRI and XbaI restriction digest and nucleotide sequencing (Fig 4.4C). pTBAFFR vector was then transfected into the mouse B cell line L1.2. Stable transfectants were selected by blasticidin resistance, and

then cloned by limiting dilution. Stable integration was confirmed by GFP expression

(Fig 3.4D). Levels of human BAFF-R expression were determined by human BAFF-Fc

binding, which demonstrated that the L1.2/pTBAFF-R transfectant had increased cell

surface binding of human BAFF-Fc compared to untransfected and empty vector tranfected L1.2 cells (Fig. 3.4E, top panel). It has been noted previously that high levels

of cell surface expression by transfectant cell lines is necessary for the efficient

production of high affinity monoclonal antibodies [306], thus we attempted to increase

BAFF-R expression by L1.2/pTBAFFR transfectants before proceeding to immunisation

of animals. It was determined that incubation of transfectants with 5 mM butyric acid

overnight significantly increased expression of human BAFF-R (Fig. 3.4E, bottom

panel), thus this procedure was employed before each immunisation of C57/B6 mice.

TACI transfectants were generated in the rat basophilic leukaemia cell line, RBL

using a construct containing the full length cDNA for human TACI (ps805), which was

kindly provided by Dr. Pascal Schneider (Lausanne, Switzerland). ps805 was transfected

into RBL cells, positive clones selected by resistance to G-418, and optimal clones

selected for maximal binding of human BAFF-Fc to the cell surface. Clone 5H5

73 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

demonstrated strong BAFF binding (Fig. 3.4F), indicating high expression of TACI at the

cell surface, and was therefore used for immunisation of Wistar rats.

Generation and validation of monoclonal antibodies to human BAFF-R

and TACI

Following screening of fusions by ELISA and FACS staining, and cloning of the

candidate hybridomas, positive anti-human BAFF-R and anti-human TACI monoclonal

antibodies were isolated, named 11C1 and 1A1 respectively. These 2 clones bound

specifically to the appropriate transfectants (Fig. 3.5A). During the period of monoclonal

antibody generation we obtained additional monoclonal antibodies from collaborators at

Biogen-Idec. These monoclonals against human BAFF-R and BCMA were named 9-1

and C4E2.2, respectively. 9-1 also bound to our human BAFF-R transfectant line, at slightly higher levels than 11C1 (Fig. 3.5A). Binding of 9-1 and C4E2.2 to their appropriate transfectants has also been demonstrated previously. [221] The specifity of

9-1, 1A1 and C4E2.2 were determined by ELISA against recombinant fusion proteins of the 3 BAFF receptors (Fig. 3.5B), which demonstrated strong binding to their relevant antigen and minimal cross reactivity of these 3 antibodies.

BAFF-R, TACI and BCMA have unique expression patterns on human

B cells

To assess the staining of our antibodies on relevant cell types we assessed the

expression pattern of BAFF-R, BCMA and TACI on subsets of blood and tonsil B cells

in humans, which should express all three receptors. CD19+ blood B cells expressed

74 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

Figure 3.5: Validation of monoclonal antibody specificity. A, BAFF-R transfectants were stained with anti BAFF-R antibodies 11C1 and 9-1, a mouse IgG1 antibody was used as an isotype control (grey plots). TACI transfectants were stained with TACI antibody 1A1, a rat IgG2a was used as an isotype control. B, ELISA plates were coated with TACI-Fc, BCMA-Fc and BAFFR-Fc fusion proteins. The antibodies 9-1 (anti-BAFF-R), 1A1 (anti-TACI) and C4E2.2 (anti-BCMA) were used as detection antibodies and binding visualised with HRP conjugated secondary antibodies.

75 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

BAFF-R at a high level, with both 9-1 and 11C1 showing a similar pattern of staining

consistent with levels of BAFF binding (Fig. 3.6A). In contrast, no positively staining

population was observed with anti BCMA C4E2.2 indicating that BCMA was absent from B cells in blood (Fig. 3.6A). Staining with anti TACI 1A1 revealed TACI expression, but only on a proportion of blood B cells, and at a much lower level than

BAFF-R (Fig. 3.6A).

The staining of human tonsil cells with mAbs to BAFF-R, BCMA and TACI revealed that BAFF-R was also the predominant receptor expressed on tonsil B cells. Nevertheless

BAFF-R did show a variation in staining intensity between different B cell subsets (Fig.

3.6B). B cells with a germinal centre (GC) phenotype (CD38+, CD27+, CD39-, CD24-, and IgM-) expressed lower levels of BAFF-R, and this was clearly evident through

immunohistochemical and immunofluorescent staining of B cell follicles in tonsil (Fig.

3.6C). In contrast to the blood, where no B cells expressed BCMA, a proportion of tonsil

B cells did express low levels, and multi-colour flow cytometry revealed that these

BCMA+ B cells displayed a phenotype consistent with GC B cells (Fig. 3.6B).

Strikingly, TACI and BCMA were expressed on different subsets of the CD19+ B-cell

population, with the TACI+ subset being CD38-, CD27-, CD39+, CD24+ and IgM+ (ie a

non-GC phenotype) (Fig. 3.6B). The distinct difference between the TACI+ and BCMA+

subsets was further illustrated by a direct two-colour analysis (Fig. 3.6D), which showed

that the vast majority of BCMA+ B cells were TACI-; this was particularly evident when

B-cell blasts were gated and analysed (Fig. 3.6D). Thus BAFF-R, TACI and BCMA

expression was detected on blood and tonsillar B cells, validating the use of these

76 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

Figure 3.6: BAFF-R, TACI and BCMA have unique expression profiles on human blood and tonsillar B cells. A, FACS analysis of BAFF receptor expression on PBMC. Biotinylated BAFF and antibodies were detected with streptavidin PE. B, Three-colour FACS analysis of B cells (performed by gating on CD19+ B cells from tonsil) showing expression of BAFF-R (9-1), BCMA (C4E2.2) and TACI (1A1) in relation to the B-cell phenotypic markers CD38, CD39, CD24, CD27 and IgM. BAFF-R expression was down-regulated on B cells with a phenotype of GC B cells (CD38+, CD27+, CD39- and IgM-), whereas BCMA was expressed on these cells. C, Immunohistochemical staining of human tonsil with anti-BAFF-R mAb 11C1, showing intense staining of B cell follicles, and weaker staining of GC (left panel, X200). Immunfluorescent staining of human tonsil with anti-BAFF-R mAb 9-1 (green) and Bcl-6 (red) (right panel, X400). D, Two colour analysis of TACI (1A1) and BCMA (C4E2.2) on gated CD19+ B cells from tonsil. The left hand panel shows the entire CD19+ B-cell gate, and right hand panel shows CD19+ B-cell blasts, gated according to high forward and side scatter.

77 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

monoclonal antibodies for identification of BAFF receptors on T cells.

Expression of BAFF-R by human T cells

Reports from the literature and our own experiments demonstrated that BAFF co-

stimulated T cell responses in humans (Fig. 3.1A, B and C and [291]). Staining with

biotinylated BAFF demonstrated that a population of CD3+ T cells from human blood

were able to bind BAFF (Fig 3.7A). This was demonstrated to be a specific, reversible

interaction as biotinylated BAFF binding was prevented in the presence of 100 fold

excess of unlabelled BAFF (Fig 3.7A, right panel). B cells in the upper left quadrant (Fig

3.7A, middle panel) provide an internal positive control for this experiment. Staining

with antibodies to the BAFF receptors demonstrated expression of BAFF-R at low levels

on unactivated T cells from human blood (Fig. 3.7B, left panel), and this was consistent

with the levels of BAFF binding (Fig 3.7A, middle panel). BAFF-R was also detected on

T cells after anti-CD3 activation (1 μg/ml) for 72 hrs (Fig. 7B, left panel). Analysis of

BAFF-R expression on these cells in relation to well characterised T cell markers

revealed little segregation of BAFF-R expression with CD25, CD27, CD45RO or CCR7

(Fig 3.7C).

All human T cell populations examined, including blood T cells, in vitro anti-CD3 activated T cells (24 hrs and 48 hrs, data not shown), and tonsillar T cells were consistently negative for BCMA and TACI (Fig. 3.7B, middle and right panels, and data not shown). Thus, in agreement with the PCR and transcript profiling experiments presented in Fig. 3.3, we concluded that BAFF-R was the only BAFF receptor expressed by human T cells. Comparison of the staining profiles generated by the 2 BAFF-R

78 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

Figure 3.7: Human T cells express BAFF-R but not TACI or BCMA. A, A population of T cells binds BAFF. Human PBMC were incubated with biotinylated BAFF at 2 μg/ml, BAFF binding was detected with streptavidin PE. Specificity of binding was demonstrated by incubation with 160 μg/ml unlabelled BAFF. B, BAFF-R, TACI and BCMA expression on resting PBMC and 72 hrs post anti-CD3 activation. The x axis shows CD69 expression. C, PBMC were activated for 72 hrs with anti-CD3, and BAFF-R expression determined with respect to CD25, CD27, CD45RO and CCR7. D, BAFF-R staining with 9-1 and 11C1 anti BAFF-R antibodies was compared on two sets of donor PBMCs, showing that 9-1 gives superior staining of T cells.

79 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

clones, 9-1 and 11C1, indicated that 9-1 was a superior antibody giving consistently

stronger staining with a number of different donor PBMCs (Fig. 3.7D). This was

especially evident in the T cell compartment, with 11C1 only achieving staining

fractionally above background levels, while 9-1 was consistently able to resolve a BAFF-

R+ T cell subset. Thus 9-1 was used for all further studies investigating the expression of

BAFF-R on T cells.

BAFF-R is differentially expressed by T cell subsets and modulated

during activation

Our analysis of resting T cells from human blood indicated that there was a subset of

T cells that were positive for BAFF-R. To further define the nature of this subset(s) a

sensitive 6 colour flow cytometric analysis of BAFF-R expression on human peripheral

blood T cells was performed using a BD LSRII flow cytometer. BAFF-R expression was

+ + determined for naïve, central-memory (TCM) and effector-memory (TEM) CD4 and CD8 cells, as defined using the markers CCR7 and CD45RO [307]. Expression of BAFF-R was determined for the various populations and is represented by different shaded profiles, as indicated (Fig 3.8A). This analysis indicated that BAFF-R is expressed by both CD4+ and CD8+ T cells and by the naïve, central and effector memory subsets.

Higher expression of BAFF-R was consistently observed on central and effector memory

cells, suggesting that BAFF effects may be differentially regulated between naïve and

antigen experienced cells.

Due to the differential expression of BAFF-R by T cell subsets, we postulated that

BAFF-R would also be regulated during cellular events in response to antigen such as

80 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

Figure 3.8: BAFF-R is differentially expressed on T cell subsets and modulated during activation. A, + + Expression of BAFF-R on naïve, central-memory (TCM) and effector-memory (TEM) CD4 and CD8 cells. PBMC were gated first on CD3+ lymphocytes, and then CD4+ cells or CD8+ T cells, and assessed for CCR7 + - + + versus CD45RO to define naïve (CCR7 RO ), central-memory (TCM, CCR7 RO ), and effector-memory - + (TEM, CCR7 RO ) T cells (top panels). Expression of BAFF-R (lower panels) was determined for the various populations and is represented by different shaded profiles, as indicated. Isotype control staining was used to determine background staining. B, PBMC were activated for 72 hrs with various concentrations of anti-CD3 and anti-CD28. CD3+ cells were gated and BAFF-R expression (black line) displayed as a histogram. An isotype control is shown in grey. C, PBMC were activated for 72 hrs with 1 μg/ml anti-CD3 and then cultured for 20 days in media alone or supplemented with 10 ng/ml IL-2 or IL-15. CD3+ cells were gated and BAFF-R expression (black line) displayed as a histogram. An isotype control is shown in grey.

81 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

activation and effector differentiation. Human PBMC were activated with various combinations of anti-CD3 and anti-CD28 antibodies for 72 hrs and BAFF-R expression on T cells (dark lines) determined by gating on the CD3+ T cell population. An isotype

control antibody is shown in grey. These experiments demonstrated a downregulation of

cell surface BAFF-R in response to activation, compared to unstimulated controls (Fig.

3.8B). This downregulation was proportional to the strength of activation, as cells activated with 5 μg/ml anti-CD3 showed proportionally less cell surface BAFF-R than those activated with 1 μg/ml. Cells from the 1 μg/ml activation were then cultured in medium alone or with either IL-2 or IL-15 at 10 ng/ml for up to 3 weeks. Cells cultured in IL-2 or IL-15 were observed to downregulate BAFF-R further compared to medium alone for periods of up to 7 days of culture (Fig. 3.8B). Analysis at 20 days revealed reexpression of BAFF-R on the cell surface to levels similar to those observed in the original unstimulated controls. Thus, BAFF-R is differentially expressed on T cells subsets, showing higher expression on antigen experienced cells and is modulated during the course of T cell activation and effector expansion.

BAFF-R is expressed on mouse T cells and mediates BAFF

costimulation

Parallel studies examining the effects of BAFF on T cells and identification of the T

cell BAFF receptors in mice were performed in our laboratory by Lai Guan Ng.

Monoclonal antibodies were generated to mouse BAFF-R and TACI. Staining of splenocytes with anti-mouse BAFF-R antibodies revealed strong BAFF-R expression by

B cells and weaker expression on a subset of CD4+ T cells (Fig. 3.9A, right panel, gate

82 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

Figure 3.9: BAFF-R and not TACI is expressed on murine T cells and mediates the costimulatory effects of BAFF. A, Mouse splenocytes were stained with an anti BAFF-R antibody, showing strong staining on B cells and weaker staining on T cells (right panel). Little staining was observed with an isotype control antibody (left panel). B, Wildtype mouse splenocytes were stained with an anti TACI antibody (top panel). A population of B cells stained positively with this antibody, while T cells were negative. TACI knockout mice were stained to demonstrate the specificity of this antibody (bottom panel). CD3+ T cells were purified from mouse spleens and activated with 1 μg/ml anti-CD3 in the presence or absence of BAFF for 72 hrs. BAFF was used at 4 μg/ml in a plate bound form, denatured BAFF was used as a control (dBAFF). C, BAFF co stimulation is abolished in A/WySnJ mice (white bars) while D, TACI deficient T cells (white bars) are fully responsive to BAFF co stimulation. Relevant wildtype controls for both experiments are shown in black bars.

R2). An isotype control antibody showed minimal staining (Fig. 3.9A, left panel). Anti

TACI staining of splenocytes revealed a subset of TACI+ splenic B cells, however no staining was observed on T cells. Consistent lack of TACI expression was observed on both resting and activated T cells, stained 48 hrs after anti-CD3 activation (1 μg/ml) (Fig

3.9B, representative of all other time points tested, data not shown). Thus the staining

83 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

patterns of BAFF-R and TACI correlates strongly between mice and humans and

revealed that BAFF-R, but not TACI is expressed by T cells.

To functionally confirm that BAFF-R mediated BAFF effects on T cells, we performed BAFF costimulation assays on purified CD3+ T cells derived from two mutant strains of mice and their representative controls. These were the A/WySnJ strain which possess a natural mutation in BAFF-R resulting in impaired function [213, 232] and

TACI-deficient mice generated by gene targetting. The co-stimulatory effects of BAFF on purified T cells were abolished in A/WySnJ mice (Fig. 3.9C), in contrast to the A/J strain which served as the appropriate genetic control. Importantly, T cells from A/J and

A/WySnJ mice responded identically to anti-CD28 stimulation (Fig. 3.9C) indicating the defect in A/WySnJ T cells is BAFF specific and not related to a more general impairment of T cell function. In addition, others have reported that APC function and T cell proliferation in A/WySnJ mice is normal [308]. In keeping with our expression studies showing a lack of TACI expression on T cells, T cells from TACI-deficient mice were unaffected in their response to BAFF costimulation (Fig. 3.9D), confirming that BAFF does not costimulate T cells through this receptor.

84 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

DISCUSSION

Identifying the cell types that can respond to a particular ligand is key to

understanding its actions in a complex, multicellular environment. Thus, discerning the

precise expression of the various BAFF receptors is important to understanding how

BAFF can influence immune responses, and how and why over-production of BAFF

causes autoimmune disease. In this study we demonstrate differential expression of the 3

BAFF receptors by subsets of B cells, and demonstrate that BCMA and TACI have more

restricted expression patterns suggestive of specialized roles. In addition we establish

BAFF-R as the sole BAFF receptor mediating T cell costimulatory effects.

Our experiments clearly define important functions of direct BAFF stimulation of T cells. Addition of BAFF to purified T cell cultures led to significant increases in proliferation during activation, while blocking endogenous BAFF in mixed cell cultures

reduced proliferation. One notable caveat of these experiments was that only plate

immobilised BAFF, but not soluble BAFF, was able to provide costimulatory signals to

purified T cells (Fig 3.1A and B, and [291]). This suggests that perhaps BAFF signalling

through BAFF-R on T cells requires BAFF to be expressed in a membrane bound form

by APCs. It is also possible that APCs capture soluble BAFF from the environment, then

immobilize and present it to T cells in an active form, a conclusion supported by the

activity of soluble BAFF in a MLR system. These data would indicate a direct

relationship between BAFF levels and T cell proliferation in response to activation.

However, this simple interpretation is complicated by experiments that reveal crosstalk in

mixed cell systems in the presence of excess quantities of BAFF. Anti-CD3 stimulation of PBMCs in the presence of exogenous BAFF led to a potent suppression of T cell

85 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

proliferation, which occurred in an IL-10 dependent manner. Given the expression pattern of BAFF receptors, it would appear very likely that this effect is mediated by

BAFF stimulation of B cells and resultant IL-10 production, which is in keeping with previous reports [216, 305]. Extrapolating these in vitro results to an in vivo setting would suggest that cellular context would strongly influence the response of T cells to

BAFF. Membrane bound BAFF associated with APCs (e.g. dendritic cells) should provide a potent activating signal, while in a B cell rich environment BAFF would suppress T cells. This suggests differing outcomes of BAFF stimulation in the T cell and

B cell zones of lymphoid tissue.

The generation of high quality monoclonal antibodies to three BAFF receptors enabled a rigorous analysis of BAFF receptor expression on T cells. Our data show that

BAFF-R is the sole BAFF receptor expressed by T cells, and mediates the direct costimulatory effects of BAFF on T cells. This is in contrast with the prevailing literature which identified TACI as the T cell expressed BAFF receptor. A closer analysis of this previous study reveals that their conclusion was based on the use of a polyclonal antibody to TACI [252]. Thus the demonstration of TACI+ T cells may be an artefact due to non-

specific binding by this polyclonal antibody. All evidence that we have generated at both

the mRNA and protein levels, using both our monoclonal antibodies and another

commercial polyclonal antibody (data not shown) demonstrates that TACI is not

expressed on peripheral T cells.

Examination of BAFF-R expression on T cells showed that, in the resting state,

BAFF-R is more highly expressed by central memory and effector memory T cells

compared to their naïve counterparts. This implies that BAFF may have preferential

86 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

proliferative effects on memory cells compared to naïve cells, or that memory cells are

more dependent on BAFF for important signals such as survival. BAFF induces Bcl-2

expression in T cells (Lai Guan Ng, personal communication) similar to its effects on B

cells [309], which suggests that BAFF may enhance T cell survival, particularly by T

cells with increased BAFF-R expression such as effector/memory subsets. The observation that effector/memory T cells are expanded in BAFF transgenic mice along with mature B cells, splenic T2 and MZ B cells [227, 228] supports this view.

Monoclonal antibody staining revealed that cell surface levels of BAFF-R were actively downmodulated during the course of in vitro activation. This downregulation was in

response to TCR receptor signalling and exposure to the proliferative cytokines IL-2 and

IL-15, which regulate survival and effector functions in activated T cells [310-312].

These data suggest that T cells might only be responsive to BAFF during defined periods

of the T cell response, most likely during the early stages of activation.

The most striking finding in regard to BAFF receptor expression on B cells was the

restricted patterns of TACI and BCMA expression, and the unique receptor expression

pattern after B cell differentiation to GC cells. The most obvious expression of BCMA

was by tonsillar B cells with a GC B cell phenotype i.e. CD38+, CD27+, CD39- and IgM-

[313, 314]. BAFF is important for the GC reaction, since blocking BAFF in vivo

attenuates the GC reaction [315], and those GCs that do form in BAFF deficient mice

have impaired maturation and function [316]. BAFF-R was also expressed on GC B cells,

but at lower levels compared to mature B cells. TACI was largely absent from GC B

cells suggesting that its proposed role as a negative regulator of B-cell activation [241,

243] does not extend to the GC reaction. Its loss from GC B cells would be consistent

87 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R with the down-regulation by GC B cells of another inhibitory receptor, LAIR-1 [317] and the phosphatase SHP-1 [318]. This would serve to limit the function of inhibitory receptors, and thus reduce the threshold for activation and proliferation in the course of

B-cell selection and differentiation within GCs. TACI mAb stained subsets of mature naïve B cells, memory B cells and activated B cells, suggesting that TACI regulates the responses of each these B cell subsets, but not GC B cells. The acquisition of BCMA by

GC B cells (and plasmablasts [221]) presumably plays a role in regulating their survival, although the high expression of BAFF-R, albeit at lower levels compared to circulating B cells, implies a role for this receptor as well. The reason for BCMA up-regulation on GC cells is uncertain, although newly discovered functions for BCMA in activating antigen presentation pathways [249] correlates with the importance of B cell antigen presentation functions during antibody affinity maturation and class switching in the GC [319].

Certainly one consequence might be the acquired ability to signal in response to APRIL as well as BAFF, therefore enabling the BCMA+ B cell pool to use additional survival resources and regulate their numbers somewhat independently of the normal B cell pool.

In conclusion, we provide further evidence that BAFF can function as a costimulator of T cells. We demonstrate that the cellular context determines the effect of BAFF on T cells. We identify BAFF-R as the only BAFF receptor present on T cells in either a resting or activated state, and demonstrate activation-dependent and subset-specific regulation of this receptor. We show that BAFF-R is critical for mediating the costimulatory effects of BAFF to T cells, while TACI is not. Finally, we define specific expression patterns for the three BAFF receptors on B cell subsets and show that while

88 CHAPTER 3 BAFF COSTIMULATES T CELL ACTIVATION VIA BAFF-R

BAFF-R is expressed globally, TACI and BCMA have more restricted expression patterns suggesting specialised roles for these receptors.

89 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

CHAPTER 4: BAFF MODULATES T CELL RESPONSES IN VIVO

INTRODUCTION

In Chapter 3 we demonstrated that BAFF influences T cell responses in vitro,

signalling directly via BAFF-R expressed on T cells. Our next aim was to test the

relevance of the in vitro effects during the regulation of T cell responses in vivo. At the

time we commenced this study, there was evidence in the literature demonstrating a role

for BAFF in the regulation of particular T cell responses in vivo. TACI-Fc treatment of T

cell-dependent CIA substantially reduced inflammation and inhibited bone and cartilage

destruction [288]. Additionally, BAFF knockout and BAFF-R deficient (A/WySnJ) mice

showed impaired T cell-mediated allograft rejection [320]. Given the results of our in

vitro studies and the data from animal models we resolved to study the role of BAFF in

the generation of T cell responses in vivo. As the generation and regulation of Th1 and

Th2 responses is a critical component of CD4+ effector T cell function, we aimed to

understand how BAFF regulates these two types of responses, by investigating this issue

in classical models of Th1- and Th2-mediated inflammation.

90 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

RESULTS

BAFF transgenic mice display overtly normal responses to mitogens in

vitro

Previously, it was noted that BAFF transgenic mice have altered composition of the peripheral T cell compartment, primarily by increases in the numbers of effector/memory type T cells [228]. To determine whether this was of functional significance we cultured

splenocytes or purified CD3+ T cells from wildtype and BAFF transgenic mice in the presence of various activating stimuli. Activation of splenocytes with a range of PHA concentrations for 72 hrs revealed no significant difference between wildtype and BAFF

transgenic cells (Fig. 4.1A). Similar activation of splenocytes with anti-CD3 and anti-

CD28 antibodies for 72 hrs again revealed no significant difference between cells from

wildtype and BAFF transgenics (Fig. 4.1B, left and right panels). When CD3+ T cells

were purified from spleen and activated with anti-CD3 and anti-CD28 in combination for

72 hrs, slightly but significantly increased proliferation was observed in BAFF transgenic

T cells at higher concentrations of anti-CD3 (Fig 4.1C, right panel). This trend was not

observed with anti-CD3 activation alone (Fig 4.1C, left panel). Thus the response of

BAFF transgenic T cells to activating stimuli in vitro appeared mostly normal, with slightly increased proliferation under some experimental conditions.

Increased antigen specific recall responses in BAFF transgenic mice

To determine whether elevated levels of BAFF were able to alter T cell responses towards antigen in vivo, we performed immunisation and in vitro restimulation

91 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

Figure 4.1: BAFF transgenic T cells display normal responses to mitogens in vitro. A, Splenocytes from wildtype and BAFF transgenic mice were cultured for 72 hrs with a range of PHA concentrations (n=4). B, Splenocytes from wildtype and BAFF transgenic mice were cultured for 72 hrs with a range of anti-CD3 concentrations, in the absence (left) or presence (right) of anti-CD28 (n=4). C, Splenic CD3+ T cells from wildtype and BAFF transgenic mice were purified via magnetic separation and cultured for 72 hrs at a range of anti-CD3 concentrations, in the absence (left) or presence (right) of anti-CD28 (n=4). Wildtype mice are shown in white bars, BAFF transgenic mice in black. Proliferation was measured by thymidine incorporation. Statistical significance was determined using a paired Student T-test p < 0.05 (*).

92 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO experiments. We used 2 different sets of antigens and adjuvants, methylated bovine serum albumin (mBSA) in complete Freund’s adjuvant (CFA) and ovalbumin (OVA) in alum, to generate specific Th1 and Th2 T cell responses, respectively. Six to eight week old wildtype and BAFF transgenic mice were immunised at the tailbase with mBSA/CFA

(as described in Materials and Methods), and inguinal lymph nodes were collected 7 days later. Lymph node cells were restimulated in vitro with mBSA for 72 hrs. As expected, wildtype lymph node cells showed increased proliferation upon stimulation with mBSA, indicative of a T cell response against this antigen. T cells from BAFF transgenic mice gave a significantly stronger recall response compared to wildtype mice, displaying a 3- fold increase in proliferation (Fig. 4.2A), and a 10-fold higher IFN-γ production in culture supernatants (Fig. 4.2B). IL-4 and IL-5 levels were below the level of detection in both wildtype and BAFF transgenic mice (data not shown). Alternatively, mice were immunised at the tailbase with OVA/alum mix, and inguinal lymph nodes were removed after 7 days and restimulated in vitro for 72 hrs. In concordance with the mBSA/CFA experiments, BAFF transgenic lymph node cells showed an approximately 3-fold increase in proliferation over wildtype mice (Fig. 4.2C). In addition, cytokine ELISA of supernatants from restimulated cultures from BAFF transgenic mice demonstrated significantly increased levels of IL-5 and IFN-γ compared to wildtype mice (Fig. 4.2D).

Thus in the presence of high levels of BAFF, T cell responses to antigen were augmented in the presence of both Th1 and Th2 priming adjuvants. This might be due to qualitative changes in the T cells involved, or an increased frequency of Ag-specific memory or

93 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

Figure 4.2: Increased in vitro recall responses in BAFF transgenic mice. A, Mice were injected at the tailbase with 250 μg of mBSA in CFA, and inguinal lymph nodes were collected 7 days later. Cultures were normalised to 2x105 T cells/well and cultured in triplicate with medium alone or 40 μg/ml mBSA for 72 hrs. B, IFN-γ levels from culture supernatants were measured by ELISA (n=3). C, Mice were injected at the tailbase with 100 μg of OVA in alum and inguinal lymph nodes collected 7 days later (n=4). Cultures were normalised to 2x105 T cells/well and were cultured in triplicate with medium alone or 100 μg/ml OVA for 72 hrs. D, Cytokine levels from culture supernatants were measured by ELISA (n=4). Proliferation was measured by thymidine incorporation in A and C. Statistical significance was determined using a paired Student T-test p < 0.05 (*) and p < 0.01 (**).

94 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

effector T cells.

Enhanced DTH responses in BAFF transgenic mice

Having shown that BAFF transgenic mice mount enhanced antigen specific Th1

responses, we determined whether this could affect the outcome of a delayed type

hypersensitity (DTH) model, which constitutes a typical model of Th1 driven

inflammation. Six to eight week old mice were immunised with a subcutaneous injection of mBSA in CFA at the base of the tail and challenged 7 days later with an intra-dermal injection of mBSA into the footpad. BAFF transgenic mice from 2 distinct transgenic lines (line 1 and line 2) displayed significantly increased paw swelling compared to wildtype mice (Fig.4.3A and B). Paw swelling was comparable in wildtype and BAFF transgenic mice at early time points post-challenge (Fig. 4.3A), but was significantly higher in BAFF transgenic mice 48 hrs and 72 hrs later. DTH responses in BAFF transgenic mice at 48 hrs were also characterised by increased erythema (Fig. 4.3B) and histological analysis of paw tissue sections revealed an increased cellular infiltrate into the footpads (Fig. 4.3C). Isotypic serum anti-mBSA Ab compostitions were measured 7 days post-footpad challenge and showed BAFF transgenic mice to have significantly higher titres of mBSA specific IgG1, IgG2a and IgG2b compared to wildtype mice (Fig.

4.3D), while mBSA-specific IgA levels showed a non significant trend towards an increase (data not shown). Levels of mBSA specific IgM and IgG3 were similar in wildtype and BAFF transgenic mice (data not shown). Thus elevated levels of BAFF

95 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

Figure 4.3: BAFF transgenic mice show increased DTH responses. A, Increased paw swelling over time in 2 lines of BAFF transgenic mice (n=10 line 1; n=6, line 2). B, Increased erythema in paws of BAFF transgenic mice at 48 hrs, compared to WT mice. C, H&E staining of paw sections at 48 hrs reveals increased leukocyte infiltration in BAFF transgenic mice compared to WT controls. D, Increased serum levels of mBSA specific antibodies in BAFF transgenic mice 7 days after footpad challenge (n=11 WT, n=10 BAFF transgenic). Statistical significance was determined using a paired Student T-test p < 0.05 (*), p < 0.01 (**) and p < 0.005 (***).

96 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

were sufficient to enhance Th1 associated inflammation in the DTH reaction.

BAFF levels determine the magnitude of the DTH response

The concentration of BAFF in human blood varies considerably; most individuals

express low levels however some autoimmune patients express over 100 ng/ml [269].

BAFF transgenic mice also show variable levels of BAFF in blood, due to secondary

endogenous BAFF production in response to autoimmune activation [293]. To assess the

relationship between serum BAFF levels and paw swelling, serum from both wildtype

and BAFF transgenic mice was collected 48 hrs after challenge and the level of BAFF in

serum was determined by ELISA. Both lines of BAFF transgenic mice displayed high

levels of BAFF in serum (line 1 average concentration 215.03 ng/ml ± 50.69, line 2

average concentration 1128.2 ng/ml ± 315.8), whereas lower levels of BAFF were

detected in wildtype mice (5.27 ng/ml ± 1.47), similar to levels in unimmunised mice

[293]. When the level of BAFF in the serum was correlated with paw swelling for each

mouse (Fig. 4.4A), a strong correlation was observed (r2 = 0.593). Thus modulation of

systemic BAFF levels may regulate the magnitude of certain T cell responses.

We next examined DTH responses in BAFF deficient mice. These mice were competent in mounting a normal DTH response and showed no differences compared to wildtype strains (Fig. 4.4B). Also, wildtype mice treated with BCMA-Ig showed no consistent reduction in DTH responses compared to untreated mice (Fig. 4.4C). Thus

BAFF is not essential for basal DTH responses, but in circumstances of BAFF over-

97 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

Figure 4.4: DTH responses correlate with serum BAFF levels, although BAFF deficiency does not eliminate DTH responses. A, Serum levels of BAFF in BAFF transgenic mice were measured using ELISA and plotted against paw swelling at 48 hrs following footpad rechallenge. B, DTH responses in BAFF-/- mice, and mice heterozygous for the knockout allele (n=6). C, C57/B6 mice treated with BCMA- Ig showed no reduction in DTH severity compared to control (PBS) treated mice (n=6).

98 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO production, the magnitude of DTH responses correlates with levels of BAFF.

BAFF transgenic mice have compromised allergic airway responses

We next asked whether elevated levels of BAFF were able to augment a Th2 response in a similar fashion to the enhanced DTH response. To answer this question we used the

OVA induced allergic airway inflammation model of Th2 T cell function and examined the responses of wildtype and BAFF transgenic mice. Measurement of cell numbers in the BAL fluid showed that exposure of wildtype mice to an OVA aerosol resulted in substantial eosinophil infiltration (Fig. 4.5A), which is characteristic for this model [321].

In contrast, BAFF transgenic mice showed a significant reduction in eosinophil infiltration. In accordance with BAL fluid cell numbers, histochemical staining of lung tissue sections revealed greatly reduced numbers of peribronchial and perivascular leukocytes in BAFF transgenic mice compared to wildtype controls (Fig. 4.5B). There was no significant difference in the amount of OVA specific IgE between wildtype and

BAFF transgenic mice (data not shown).

Lymphocytes from peri-bronchial lymph nodes were collected after the final aerosol exposure, and restimulated in vitro with OVA for 72 hrs. Wildtype lymphocytes showed a strong increase in proliferation after restimulation with OVA, in contrast to lymphocytes from BAFF transgenic mice (Fig. 4.5C). Cytokine measurements showed that wildtype lymph node cells produced high levels of IL-5 after restimulation with

OVA, while cells from BAFF transgenic mice showed 10-fold reduced levels (Fig. 4.5D).

In addition, small but demonstrable levels of IFN-γ were detected in some BAFF transgenic cultures, although this result was not always reproducible. Of note, there was

99 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

Figure 4.5: Suppression of allergic airway inflammation in BAFF transgenic mice. A, BAL fluid was recovered from BAFF transgenic mice and wildtype controls after an 8 day aerosol exposure regimen, and constituent cell types determined by cytospin and Giemsa staining (n=5). B, Lung sections stained with H&E revealed greatly reduced leukocyte infiltration in BAFF transgenic mice. C, Peri-bronchial lymph nodes were collected and pooled. Cultures were normalised to 2x105 T cells/well and were cultured in triplicate with media alone or 100 μg/ml OVA for 72 hrs, and proliferation was measured by thymidine incorporation. D, IL-5 and IFN-γ levels from culture supernatants were measured by ELISA. Statistical significance was determined using a paired Student T-test p < 0.005 (***).

100 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

no compensatory increase in IL-10 production in BAFF transgenic cultures (data not

shown). Thus BAFF transgenic mice show a suppression of Th2-mediated allergic

airway inflammation, and an associated reduction in lymph node T cell proliferation and

IL-5 production in response to OVA challenge.

Numbers of effector memory T cells are increased in BAFF transgenic

mice, but not in μMT-/- x BAFF transgenic mice

We also examined changes in the make-up of the T cell immune system that result

from long-term exposure to high levels of BAFF, and effects that expanded subsets of B

cells could have on T cell responses in BAFF transgenic mice. Previous studies of BAFF

transgenic mice revealed altered CD4+ T cell subset ratios [228]. Splenocytes from

wildtype and BAFF transgenic mice were isolated and stained with antibodies to CD4,

CD44 and CD62L. We observed a substantial increase in the proportion of CD44hi

CD62Llo effector memory CD4+ cells, and a decrease in CD44lo CD62Lhi naïve CD4+ T cells, in BAFF transgenic mice (Fig. 4.6A, top panels). Functionally, effector memory T cells are closely related to effector T cells and have the capacity to migrate to peripheral tissues [307, 322]. To investigate whether the expansion of mature B cell subsets seen in

BAFF transgenic mice [228] might in some way connect to the increased numbers of effector memory CD4+ T cells, we generated B cell deficient BAFF transgenic (μMT-/- x

BAFF transgenic) mice and these showed no increase in effector memory T cell numbers

(Fig. 4.6A, bottom panels). The ratio of effector/naïve T cells in line 2 BAFF transgenic

mice was increased ~4-fold (Fig. 4.6B, left panel), and line 1 BAFF transgenic mice also

showed a statistically significant (although smaller) increase (Fig. 4.6B, right panel).

101 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

Figure 4.6: Altered T cell phenotype and increased DTH severity in BAFF transgenic mice is B cell dependent. A, Splenocytes were stained with anti-CD4, anti-CD44 and anti-CD62L (CD4+ gated cells are shown, n=3 for all genotypes with a representative plot for each genotype shown). B, Cell ratios for CD4+ T cells calculated based on the following subsets: naïve = CD62Lhi CD44lo; effector = CD62Llo CD44hi. Left panel shows the ratios for line 2 BAFF transgenic mice and wildtype controls, right panel shows ratios for the μMT-/- x BAFF transgenic line 1 F2 generation littermates. Statistical significance was determined using a paired Student T-test p < 0.01 (**) and p < 0.005 (***).

102 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

Analysis of μMT-/- mice showed a reduced ratio, indicating an overall reduction in effector cells, which was not increased in μMT-/- x BAFF transgenic mice (line 1). Thus

the altered makeup of the T cell compartment in BAFF transgenic mice is dependent on

B cells.

BAFF mediated enhancement of DTH is B cell dependent

We examined whether the B cell dependency of expanded memory populations in

μMT-/- x BAFF transgenic mice could affect Th1 T cell generation and the course of the

DTH reaction. Wildtype, μMT-/- and μMT-/- x BAFF transgenic mice were immunised

with mBSA/CFA, lymph nodes collected after 7 days and lymph node restimulations

performed. Restimulated cells showed comparable proliferation (Fig 4.7A) and IFNγ production (Fig 4.7B) between wildtype, μMT-/- and μMT-/- x BAFF transgenic mice. In

addition all three strains of mice displayed similar levels of footpad swelling over the

course of DTH (Fig. 4.7C). Paw swelling at 48 hrs was plotted against serum BAFF

levels for each animal, and linear regression analysis performed (Fig. 4.7D). The

correlation coefficient (r2) of <0.01 indicated a lack of correlation between BAFF levels

and the DTH response in the absence of B cells. The lack of increased proliferation and

IFNγ production in the μMT-/- x BAFF transgenic strain suggests that the lack of

augmented DTH responses in this strain were at the level of T cell priming, expansion or

function in the local lymph node. Thus BAFF associated increases in Th1 T cell numbers

103 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

Figure 4.7: Enhanced DTH responses in BAFF transgenic mice are B cell dependent. A, Mice were injected at the tailbase with 250 μg of mBSA in CFA, and inguinal lymph nodes were collected 7 days later. Cultures were normalised to 2x105 T cells/well and cultured in triplicate with medium alone or 40 μg/ml mBSA for 72 hrs. Proliferation was measured by thymidine incorporation. B, IFN-γ levels from culture supernatants were measured by ELISA (n=3). C, μMT-/- x BAFF transgenic mice have similar levels of paw swelling to WT and μMT-/- control mice (n =6). D, Serum levels of BAFF were measured using ELISA and plotted against paw swelling at 48 hrs.

104 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

and function and DTH responses require the presence of B cells.

BAFF mediated suppression of allergic airway inflammation is B cell

independent

Our results demonstrating that μMT-/- x BAFF transgenic mice were protected from

the augmented DTH responses led to the question of whether BAFF-mediated

suppression of airway inflammation would be relieved in μMT-/- x BAFF transgenic

mice. Differential cell counts from BAL fluid showed no significant differences in cell

numbers between wildtype and μMT-/- mice in response to OVA aerosol, with

characteristic eosinophil infiltration observed in either case (Fig. 4.8A). As for BAFF

transgenic mice, μMT-/- x BAFF transgenic mice showed a significant reduction in the

eosinophil infiltration in response to OVA challenge. Restimulation with OVA of peri-

bronchial lymph node T cells from wildtype and μMT-/- mice showed strong induction of

proliferation (Fig. 4.8B) and IL-5 production (Fig. 4.8C), whereas there was strong

suppression of proliferation and IL-5 production in μMT-/- x BAFF transgenic mice (Fig.

4.8B and C), as we observed in BAFF transgenic mice. There were no detectable levels

of IFN-γ or IL-10 in any of the cultures (data not shown). These results demonstrate that

BAFF-mediated suppression of Th2-dependent allergic airway inflammation occurs by mechanisms that are B cell-independent.

105 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

Figure 4.8: Suppression of allergic airway inflammation in BAFF transgenic mice is B cell independent. A, BAL fluid was recovered from WT, μMT-/- and μMT-/- x BAFF transgenic mice after aerosol exposure, and constituent cell types determined by cytospin and Giemsa staining (n=5). B, Peri- bronchial lymph nodes were collected and pooled into groups. Cultures were normalised to 2x105 T cells/well and restimulated with medium alone or 100 μg/ml OVA for 72 hrs, with proliferation measured by thymidine incorporation. C, IL-5 and IFN-γ levels from culture supernatants were measure by ELISA. Statistical significance was determined using a paired Student T-test p < 0.05 (*) and p < 0.005 (***).

106 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

DISCUSSION

The data presented in Chapter 3 demonstrates that T cells express BAFF-R and are responsive to BAFF during T cell activation in vitro, suggesting that BAFF may be important in the regulation of T cell responses in vivo. The results of this chapter demonstrate that overexpression of BAFF modulates antigen specific T cell responses and influences the outcome of two models of T cell dependent inflammation in vivo.

These data indicate that BAFF controls both B cell and T cell responses in vivo, and that overproduction of BAFF has profound effects on both the B and T cell systems.

Initial analysis of in vitro proliferative responses of CD3+ T cells purified from wildtype and BAFF transgenic mice suggested that long term exposure to BAFF does not result in any gross alterations to mitogenic responses. The small increase in reactivity of

BAFF transgenic T cells to anti-CD3 and anti-CD28 stimulation is probably a reflection of increased numbers of memory T cells, which are known to be more responsive to activation [323]. Immunisation with antigen and subsequent restimulation of draining lymph nodes revealed a significant enhancement in proliferation and cytokine production in BAFF transgenic mice. These data correlate well with our previous finding from in vitro systems which demonstrated that BAFF can directly costimulate T cell activation, and indicate that the BAFF transgenic environment strongly enhances T cell priming.

To examine whether the increased function of Th1 T cells in BAFF transgenic mice translated to increased Th1 induced inflammation, we utilised a model of DTH. DTH is a classical Th1-mediated response involving Ag-specific T cell activation and production of Th1 cytokines including TNFα and IFN-γ [324]. BAFF transgenic mice developed significantly prolonged DTH responses, with an accompanying increase in class switched

107 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

antibody production. Since BAFF increased antigen specific proliferation and cytokine production in vivo, we suggest that this augmented DTH response is due to increased effector T cell expansion, survival and effector function. Of particular note was the

correlation between BAFF levels and the degree of footpad swelling in BAFF transgenic

mice. BAFF augmented the DTH response in a concentration dependent manner through

a range of serum concentrations that are in line with those observed in some patients with

autoimmune diseases [269]. However, whilst BAFF did augment DTH responses, it was

not necessary for basal responses. These data would indicate that BAFF may play little role during the course of many T cell responses that occur in a normal inflammatory environment in vivo. Our own in vitro studies suggested that BAFF blockade could only suppress T cell responses in the absence of strong T cell costimulation with anti-CD28

(Chapter 3, Fig. 3.1 E). A strong inflammatory environment such as that induced in responses to CFA (with accompanying activation of various TLRs and other PAMP receptors) is likely to result in the potent induction of CD86 and CD80 expression on

APCs and thus supersede the requirement for BAFF in the T cell response. This model however, would not discount enhancement of T cell responses under conditions where there are high serum levels of BAFF. Possible examples of this situation are ongoing autoimmune diseases, or persistent viral or bacterial infections, all of which can be associated with high levels of BAFF in serum [269, 271, 277-280, 285]. These circumstances of high BAFF levels may lead to increased Th1 T cell activity and effector

T cell responses.

Our data with μMT-/- x BAFF transgenic mice demonstrate that B cells are important

for regulating the composition of the peripheral T cell pool. μMT-/- x BAFF transgenic

108 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO mice lack the specific expansion of effector type T cells that are observed in BAFF transgenic mice, suggesting that BAFF alters T cell homeostasis indirectly via the action of B cells. Interestingly this type of B-cell dependent effector T cell expansion is observed in other models of SLE [323, 325]. The most straightforward explanation for our results is that high levels of BAFF affect the numbers and phenotype of peripheral B cells [269]. Subsequent interactions between T and B cells of altered frequency or function would therefore affect the makeup and responses of the T cell immune system.

This has important functional consequences as the presence of B cells is fundamental in the enhanced lymph node responses and the dose-dependent effects of BAFF on cutaneous DTH responses.

This model suggests two possible mechanisms to explain the B cell augmentation of T cell responses. It is possible that the expansion of effector memory T cells would result in a more BAFF responsive T cell pool, as effector memory T cells expressed BAFF-R at the highest levels (Chapter 3, Fig. 3.8A). This subset of T cells responds more vigorously to activation than other T cell subsets [323], which could explain the observed increases in proliferation and cytokine production (Fig. 4.2). Therefore direct BAFF stimulation of T cells would be responsible for the effects, but would rely on B cell- dependent changes in the frequency of particular T cell subsets prior to immunisation.

Alternatively, BAFF stimulation of B cells may affect specific functions e.g. increased antigen presentation and/or co stimulation and thus allowing B cells to enhance T cell activation and effector generation during antigen specific interactions in local lymph nodes. The role of these mechanisms will be explored further in Chapter 5. Curiously the B cell involvement in this model appears to enhance T cell function in response to

109 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

BAFF, while BAFF stimulated B cells were inhibitory to T cell activation in our previous study (Fig. 3.2). Clearly in vitro systems do not always provide adequate approximations of the situation in vivo, where multicellular, inflammatory responses operate at multiple locations. We would suggest this discrepancy might arise from exposure to inflammatory factors in vivo and differential B cell composition in peripheral blood and lymphoid organs, with associated differential BAFF receptor expression.

The potentiation of Th1 responses by BAFF suggests that this cytokine plays a role in immune responses to viral or bacterial infections. BAFF is produced at sites of inflammatory reactions, particularly by leukocyte types associated with type 1 responses such as neutrophils [194, 206] and macrophages [196]. To date most studies have been confined to autoimmune diseases, with few addressing BAFF levels following viral or bacterial infection, due partly to lack of commercial reagents. In HIV-1 infection, BAFF levels were elevated compared to controls, particularly during later stages of infection when CD4 counts had declined [285]. In addition, HIV-1 infected patients show alterations in their B cell immune system consistent with high BAFF levels, notably hypergammaglobulinemia and altered B cell differentiation [222]. BAFF produced at an inflammatory site by activated neutrophils, activated T cells, or macrophages, would be transported via the lymph to local lymph nodes, and augment T cell activation, and probably provide survival signals to the central and effector memory T cells that express

BAFF-R [250]. In BAFFhi individuals, increased proportions of effector memory T cells, possibly predisposed to become Th1-type effector T cells, would promote clearance of viral or bacterial agents.

110 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

BAFF transgenic mice had suppressed allergic airway responses, with a marked

reduction in eosinophils in BAL fluid and infiltrating leukocytes around airways and

pulmonary blood vessels. Lung-draining lymph node cells from BAFF transgenic mice

showed markedly reduced T cell proliferation and IL-5 production after OVA re-

stimulation. This would account for the reduced eosinophils in the airways, since IL-5 is

the key cytokine regulating eosinophil production and recruitment in this model [326-

328]. It is possible that this suppression of Th2 effector function was the result of a

skewing of the OVA-specific T cell response towards that of a more Th1-like profile.

There was some evidence for this, since small amounts of IFN-γ were produced by T

cells in lung-draining lymph nodes from BAFF transgenic mice, and this was not

observed in wildtype mice. Since re-stimulation of non-lung-draining (inguinal) lymph

nodes from primed BAFF transgenic mice revealed significantly increased proliferation

and cytokine production these mice were effectively primed to OVA and displayed

increased production of IFNγ from inguinal nodes after immunisation (Fig. 2D). It is

therefore possible that the increase in IFNγ production during T cell priming may lead to

antagonism of Th2 differentiation, since IFN-γ induces the expression of the Th1

transcription factor T-bet which can suppress IL-5 production [329].

The localised suppression of T cell responses in the lung-associated lymph nodes in

BAFF transgenic mice, but not other lymph nodes, might also favour the notion of a pulmonary Th-1-like regulatory T cell, as has been described in a recent study [330, 331].

These T-bet and Foxp3 expressing regulatory T cells suppressed the development of airway hyper-reactivity, possibly through the actions of IL-10 [331]. In addition other investigators have demonstrated that a large proportion of CD4+CD25+ regulatory T cells

111 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

in mice express BAFF-R [320]. These data suggest that regulatory cells may be

responsible for the suppressed airway inflammation in BAFF transgenic mice. However,

we have found no strong evidence for a suppressive IL-10-mediated T cell response in

BAFF transgenic lung-associated lymph nodes and no changes in CD4+CD25+ regulatory

T cells or increased levels of IL-10 (data not shown). Whether any role for increased regulatory T cell function exists in BAFF transgenic mice remains to be determined.

At present T and B cells are the only cell types with strong evidence of BAFF receptor expression, so the mechanism presumably relates to BAFF signalling to either a T cell or

B cell subset. However, the inhibition by BAFF of allergic airway responses in μMT-/-x

BAFF transgenic mice appears to exclude B cell involvement, suggesting that this is a T

cell-dependent phenomenon. Further experimentation could be performed in this system

to more fully elucidate the nature of the defect in BAFF transgenic mice. OVA specific

OT-II cells [332] could be activated in vitro, then transferred into mice, followed by an aerosolisation regimen. These experiments would enable the priming stage of the response to be differentiated from the effector phase, and determine whether fully functional effector T cells are suppressed in the BAFF transgenic environment. One important qualification that must be noted is that the enhanced DTH response in BAFF transgenic mice was in skin, whereas the suppressed response in the allergic model was in mucosal tissues. It is conceivable that BAFF effects somehow relate to the nature of cutaneous or mucosal responses, perhaps due to differential regulation of T cell chemotaxis and homing patterns. Thus experiments which track chemokine receptor expression, homing patterns and effector functions of antigen specific cells may provide further insight.

112 CHAPTER 4 BAFF MODULATES T CELL RESPONSES IN VIVO

In conclusion, we show that BAFF over-expression modulates the outcome of T cell- dependent responses in vivo. We demonstrate an increase in T cell priming and recall responses to multiple antigens and show a corresponding increase in the DTH model of

Th1 cell-dependent inflammation. In addition we show that BAFF transgenic mice have suppressed Th2-dependent allergic airway responses. Finally, we demonstrate that the proinflammatory effects of BAFF in T cell priming and DTH rely on the presence of B cells, while the suppressive effects during allergic airway inflammation are B cell independent. This elucidates mechanisms of crosstalk between these two BAFF responsive cell types. Since overproduction of BAFF has been associated with various autoimmune diseases [228, 229, 233, 269, 278, 302] determining whether these mechanisms contribute to the generation of autoimmunity in BAFF transgenic mice will be pursued in future studies.

113 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

CHAPTER 5: BAFF REGULATION OF T-B CELL COLLABORATION

INTRODUCTION

T and B cells are unique in their ability to express a large diversity of antigen

receptors after recombination of VDJ genes. This results in the generation of rare T and

B cells that can respond to the same antigen and results in antigen specific pairs whose

functions are intimately linked. During an immune response, T and B cells that recognise

the same antigen form intercellular contacts in lymphoid tissue and engage in a crosstalk

of signals that direct both the cellular and humoral immune responses. T cell help to B cells is required for antibody production in response to T-dependent antigens. Follicular

B helper T cells (TFH) orchestrate the production of high affinity, class switched

antibodies in the germinal centre and are critical for the evolution of improved antibody

specificities [333-336]. Conversely B cells are likely to play important roles in the

regulation of peripheral T cell responses and tolerance [20]. For example B cells can

become competent antigen presenting cells via their expression of MHC class II and

costimulatory molecules [337-339] that potently stimulate T cell proliferation, while B cells are required for the generation of many antiviral CD8+ T cell responses [340-342].

B and T cells are the only cells (to date) found to express receptors specific for BAFF,

and exhibit BAFF responsiveness. This suggests that BAFF regulates important

functions of these two cell types in tandem. In our previous studies we demonstrated that

BAFF alters T cell responses by direct stimulation of T cells via BAFF-R (Chapter 3). In

addition, we highlighted significant roles for indirect effects, via BAFF stimulation of B

114 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION cells, in modulating the outcome of these T cell responses (Chapters 3 and 4). Therefore the aim of this study was to explore further the behaviour of B cells in response to BAFF, with specific focus on mechanisms that would enable B cells to interact with and affect the function of T cells.

115 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

RESULTS

BAFF regulates the expression of antigen presentation molecules via

BAFF-R

Purified splenic B cells were cultured with/without 250 ng/ml of recombinant BAFF for 24 hrs and the expression of a panel of antigen presentation and costimulation molecules monitored by flow cytometry. The expression of both MHC class II and

ICOS-L was significantly upregulated on the cell surface in response to BAFF treatment

(Fig. 5.1A), while no change was observed in MHC class I, CD40 or CD86 (Fig. 5.1A).

Additionally no change was observed in the expression of CD80 and ICAM-1 (data not shown). A similar pattern of regulation was observed with 500 ng/ml of recombinant

BAFF and up to 48 hrs of culture (data not shown). The specificity of this effect was demonstrated by the lack of MHC class II and ICOS-L regulation in the presence of an 80 fold excess of recombinant BAFF-R protein (data not shown).

To determine which of the known BAFF receptors was mediating the upregulation of these molecules we performed similar stimulation of purified B cells from receptor mutant or knockout strains. 24 hrs stimulation of purified B cells from the AJ strain resulted in a similar upregulation of MHC class II and ICOS-L, although to a somewhat lower level than C57/B6 mice (Fig 5.1B, top panel). In contrast, BAFF stimulation of B cells from the A/WySnJ strain resulted in no significant upregulation of MHC class II or

ICOS-L (Fig. 5.1B, bottom panel), indicating that BAFF-R was responsible for mediating these effects. To test the role of TACI in this response we performed BAFF stimulation of B cells from TACI deficient mice and their relevent wildtype control strain. BAFF

116 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

Figure 5.1: BAFF regulates MHC class II and ICOS-L expression on B cells. Splenic B cells from a variety of mouse strains were purified by magnetic separation and stimulated for 24 hrs with 250 ng/ml BAFF. Cell surface expression was measured by flow cytometry with antibodies against MHC class II, MHC class I, CD40, CD86 and ICOS-L. A, C57/B6 mice. B, Control (A/J) and BAFF-R mutant (A/WySnJ) mice. C, Control (TACI+/+) and TACI-/- mice.

117 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

induced the upregulation of both MHC class II and ICOS-L in both wildtype and TACI

deficient mice (Fig 5.1C), indicating that TACI was not responsible for mediating these

effects. BCMA deficient mice were not tested, although we have not detected BCMA

expression on peripheral B cells, other than germinal centre B cells (Chapter 3),

plasmablasts [221] and plasma cells [248].

BAFF stimulated B cells show a modest enhancement in allostimulatory

capacity

We next asked whether the BAFF stimulation of B cells was capable of increasing

their ability to prime a T cell response, given the importance of both MHC class II and

ICOS-L in this process [67, 81, 343-345]. Purified splenic B cells from C57/B6 mice

were stimulated for 24 hrs with BAFF (2 μg/ml), with LPS (1 μg/ml) and anti-IgM (5

μg/ml). LPS and anti-IgM were used as positive controls as they enhance T cell priming

by B cells [337, 346, 347]. Flow cytometric analysis of cell surface markers revealed a

significant upregulation of MHC class II in response to BAFF, LPS and anti-IgM (Fig.

5.2A, left panel). CD86 was upregulated in response to LPS and anti-IgM but not by

BAFF (Fig. 5.2A, middle panel) while ICOS-L was upregulated by BAFF, slightly downregulated by LPS and extinguished by anti-IgM (Fig 5.2A, right panel). After stimulation, B cells were harvested, inactivated and used as stimulator cells in an allogeneic activation of CD4+ T cells purified from Balb/c mice. LPS and anti-IgM

stimulated B cells induced significantly increased proliferation in responding CD4+ T cells (Fig. 5.2B) while BAFF stimulated B cells induced only slightly increased proliferation.

To confirm these results in another assay system we used an OVA specific DO11.10

118 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

Figure 5.2: BAFF stimulation of splenic B cells leads to increased T cell stimulatory capacity. A, Purified C57/B6 splenic B cells were stimulated with BAFF (pink), LPS (blue) or anti-IgM (orange) for 24 hrs. MHC class II, CD86 and ICOS-L expression were measured by flow cytometry. Unstimulated (purple) and denatured BAFF (green) controls are shown (n=3). B, B cells stimulated with BAFF, LPS and anti-IgM were used as stimulators in a MLR assay. Balb/c CD4+ T cells were used as the responder population. C, Balb/c B cells were stimulated for 24 hrs with BAFF and anti-IgM were used as presenting cells in an OVA specific system. Responses of DO11.10 CD4+ T cells were measured in the presence of range of OVA323-339 peptide concentrations (n=1). Figure 5.2C shows proliferation in response to unstimulated control (white bars) and anti-IgM (grey bars) stimulated B cells. D, Proliferation in response to denatured BAFF (white bars) and BAFF (grey bars) stimulated B cells. Proliferation was measured by thymidine incorporation.

119 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

TCR transgenic system, where it is possible to manipulate the amount of cognate antigen.

As the DO11.10 is restricted to I-Ad all experiments were performed on a Balb/c background. Purified Balb/c B cells were stimulated for 24 hrs with BAFF (2 μg/ml) and anti-IgM (5 μg/ml), harvested, inactivated and used as stimulator cells in the presence of various concentrations of the OVA323-339 peptide. Anti-IgM stimulated B cells significantly increased proliferation of purified CD4+ DO11.10 T cells at the lower concentrations of OVA323-339 peptide (Fig 5.2C), acting as a positive control, and validating this system for further study. BAFF stimulated B cells induced a modest but significant increase in proliferation at a range of OVA323-339 peptide concentrations (Fig

5.2D). These results correlated with those observed in the allogeneic MLR system and indicate that BAFF stimulation of B cells increases their ability, albeit modestly, to prime

T cell responses.

BAFF transgenic mice display altered expression of MHC class II and

ICOS-L

To determine whether the in vitro regulation of MHC class II and ICOS-L by BAFF was relevant in vivo, we analysed wildtype and BAFF transgenic B cells for their expression of MHC class II and ICOS-L. Splenocytes were stained with B220, IgM and

IgD, and follicular, transitional type 2 and marginal zone B cells gates using these markers. Expansion of the B cell compartments was observed in BAFF transgenic mice, especially the marginal zone compartment (Fig 5.3A) consistent with previous observations [228]. Flow cytometric analysis revealed increased levels of MHC class II and ICOS-L expression on BAFF transgenic B cells compared to wildtype (Fig. 5.3B),

120 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

Figure 5.3: B cells from BAFF transgenic mice display increased levels of MHC class II and ICOS-L. A, The subset composition of splenic B cells from wildtype and BAFF transgenic mice was determined by flow cytometry after staining with antibodies against B220, IgD and IgM. Populations were gated on B220+ cells IgMlo IgDhi = follicular (FO), IgMhi IgDhi = transitional type 2 (T2), IgMhi IgDlo = marginal zone (MZ). B, Cell surface expression of MHC class II, MHC class I, CD40, CD86 and ICOS-L by wildtype (purple) and BAFF transgenic (green) B220+ gated B cells was measured by flow cytometry (n=5). C, B220+ gated B cells were gated on follicular (FO) and marginal zone (MZ) B cell subsets. MHC class II and ICOS-L expression by wildtype (purple) and BAFF transgenic (green) mice was measured by flow cytometry.

121 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

with no differential regulation of MHC class I, CD40, or CD86 (Fig. 5.3B). BAFF

transgenic mice have altered B cell subset distribution, and B cells from the follicular and marginal zone subsets express MHC class II and ICOS-L at different levels (Fig. 5.3C), therefore it was possible that the increased levels of these molecules in the BAFF transgenic mice may have been due simply to altered B cell subset distribution and numbers. To determined whether MHC class II and ICOS-L were upregulated on a per cell basis we specifically gated on the follicular and marginal zone B cell populations and determined MHC class II and ICOS-L expression for each subset. This analysis revealed that both follicular and marginal zone B cells from BAFF transgenic mice have significantly increased levels of MHC class II and ICOS-L (Fig. 5.3C), suggesting a true upregulation on the cell surface.

B cells from BAFF transgenic mice have enhanced allostimulatory

capacity

We subsequently performed allogeneic and antigen specific T cell activation assays to

determine whether BAFF transgenic B cells were better stimulators of T cell proliferation

than wildtype B cells. Purified wildtype and BAFF transgenic B cells were inactivated

and used as the stimulator population in an allogeneic MLR, with Balb/c CD4+ T cells as

responders. BAFF transgenic B cells induced significantly increased proliferation in the

responder population compared to wildtype B cells (Fig. 5.4A). Additional studies were

performed using antigen specific DO11.10 TCR transgenic T cells. For these studies we

made use of BAFF transgenic mice that had been backcrossed to a Balb/c background for

10 generations. Balb/c x BAFF transgenic B cells elicited an increased proliferative

122 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

Figure 5.4: BAFF transgenic splenic B cells display increased T cell stimulatory capacity. A, Wildtype (white bars) and BAFF transgenic (grey bars) purified splenic B cells were used as stimulator cells in a MLR assay with Balb/c CD4+ responder T cells (n=4). B, Purified Balb/c (white bars) and Balb/c x BAFF transgenic (grey bars) B cells were used as presenting cells in the presence of OVA323-339 peptide and DO11.10 CD4+ responder T cells (n=3). Proliferation was measured by thymidine incorporation.

123 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

response compared to Balb/c controls, at a range of OVA323-339 peptide concentrations.

Statistical significance was reached at 0.3 μM and 1 μM OVA323-339 while demonstrating a consistent trend towards an increase at other concentrations (Fig. 5.4B). Thus we concluded that BAFF transgenic B cells were superior stimulators of T cell responses compared to wildtype controls.

Increased expression of ICOS-L is not responsible for the increased B cell stimulatory activity mediated by BAFF

As ICOS-L regulation appeared to be a consistent response of B cells to BAFF stimulation, we postulated that increased levels of ICOS-L were responsible for the increased stimulatory nature of BAFF stimulated B cells. To test this hypothesis, mouse

ICOS-Fc was used to block ICOS-L on wildtype and BAFF transgenic B cells in an allogeneic MLR. ICOS-Fc addition to B cells significantly reduced the available ICOS-L on the cell surface, determined by inhibition of ICOS-L antibody binding by flow cytometry (Fig. 5.5A). Levels of ICOS-L on BAFF transgenic B cells were blocked by

ICOS-Fc to approximately the same level as wildtype mice, suggesting that these reagents were suitable for this study.

Increased proliferation was observed in response to BAFF transgenic B cells in allogeneic MLRs (Fig. 5.5B). In the presence of ICOS-Fc, the response to wildtype B cells was significantly increased, reaching levels similar to those seen for BAFF transgenic B cells (Fig. 5.5B). In contrast, ICOS-Fc addition to BAFF transgenic B cells had no effect on proliferation. These results were of an unexpected nature and are not

124 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

Figure 5.5: Blockade of ICOS:ICOS-L interactions does not normalise B cell dependent T cell responses in BAFF transgenic mice. A, Purified splenic B cells from wildtype and BAFF transgenic mice were incubated with ICOS-Fc (green), hIg (orange) or alone (purple). ICOS-L levels were then detected using ICOS-L specific antibody. B, Wildtype (white bars) and BAFF transgenic (grey bars) B cells were used as stimulators in a MLR with Balb/c CD4+ responder T cells. Cells were cultured with ICOS-Fc, hIg or without additive (n=4). C, Wildtype and BAFF transgenic mice were treated with an ICOS blocking antibody (12A8), an ICOS non blocking antibody (1C10) or PBS as a control. DTH responses were then measured (n=4). D, Typical DTH responses for wildtype and BAFF transgenic strains.

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easily rationalised given the known biology of ICOS-L. Regardless, they probably

indicate that signals from ICOS-L are not responsible for the increased allogeneic

proliferation in response to BAFF transgenic B cells.

In parallel studies, we examined the role of ICOS:ICOS-L interactions in regulating

DTH responses in wildtype and BAFF transgenic mice. We reasoned that upregulation

of ICOS-L on B cells may be regulating the augmented DTH responses that were

observed in BAFF transgenic mice (Chapter 4, Fig. 4.4A), as this augmentation was

known to be B cell-dependent (Chapter 4, Fig. 4.8A). To test this we used a pair of rat

anti-mouse ICOS monoclonal antibodies: clone #12A8, blocking anti-ICOS antibody, and clone #1C10, a non blocking anti ICOS isotype control antibody [63]. Six to eight week old mice were treated on days 1, 3, 5, and 7 with 30 μg of the relevant antibody or a

PBS control and their DTH responses were measured. Wildtype mice mounted a typical

DTH response which was not altered by treatment with either clone #12A8 or #1C10

(Fig. 5.5C). The control PBS treated, BAFF transgenic group failed to elicit an enhanced

DTH response as expected for this strain, however we judged this to be the result of outliers in a small sample size (n=3), as the response of this group did not conform to the normal kinetics illustrated in Fig. 5.5D. BAFF transgenic mice treated with either clone

#12A8 or #1C10 displayed normal DTH responses, with no difference observed between the two antibodies (Fig. 5.5C). These data indicate that blockade of ICOS has no effect on DTH responses in wildtype and BAFF transgenic mice.

126 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

B cell expansion alone cannot augment DTH responses: TACI-/- mice have normal DTH responses

To further examine the B cell dependence of DTH responses in BAFF transgenic

mice further, we measured DTH responses in other mouse strains that exhibit increases in

B cells numbers. Both the TACI-/- and Traf2 lox/lox X Mx-1 Cre strains exhibit

expanded B cell compartments in the absence of elevated BAFF levels. The phenotypes

of these mice have been described in detail previously [241-243, 267]. Flow cytometry

revealed expanded B cell subsets in TACI-/- mice (Fig. 5.6A), particularly the follicular

population, increases in MHC class II expression and reduced ICOS-L expression (Fig.

5.6B). The expression of MHC class I, CD40 and CD86 were similar to wildtype

controls (Fig. 5.6B). DTH responses were measured in 2 lines of TACI deficient mice, a

description of which can be found in Chapter 2. Initial experiments were performed on wildtype and TACI deficient mice that were bred as separate lines from homozygous parents. Experiments from these mice consistently indicated that TACI-/- mice mounted a

strongly enhanced DTH response (Fig. 5.6C). However, the kinetics of the response

were slightly unusual, exhibiting a particularly strong response at early time points. To

confirm these results we measured DTH responses in a second line of TACI-/- mice, bred

from heterozygous parents, allowing the use of true littermate controls. Results from

these experiments demonstrated that TACI-/- mice did not exhibit increased responses

compared to TACI+/- and wildtype controls (Fig. 5.6D). In addition, restimulation of

inguinal lymph nodes after immunisation revealed no differences in proliferation (Fig.

5.6E) or IFNγ production (Fig. 5.6F) between wildtype and TACI-/- mice. From these

data

127 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

Figure 5.6: TACI deficient mice do not display increased Th1 T cell function or increased DTH responses. A, The subset composition of splenic B cells from wildtype and TACI deficient mice was determined by flow cytometry after staining with antibodies against B220, IgD and IgM. Populations were gated on B220+ cells IgMlo IgDhi = follicular (FO), IgMhi IgDhi = transitional type 2 (T2), IgMhi IgDlo = marginal zone (MZ). B, Cell surface expression of MHC class II, MHC class I, CD40, CD86 and ICOS-L by wildtype (purple) and TACI deficient (green) B220+ gated B cells was measured by flow cytometry (n=5). C, DTH responses were measured in wildtype (white bars) and TACI deficient (grey bars) mice bred as established lines (n=5). D, DTH responses were measured in wildtype (white bars), TACI+/- (grey bars) and TACI-/- (black bars) mice bred from heterozygous parents (n=4). E, Wildtype (white bars) and TACI deficient (black bars) mice were immunised at the tailbase with 250 μg of mBSA in CFA, and inguinal lymph nodes were collected 7 days later. Cultures were normalised to 2x105 T cells/well and cultured in triplicate with medium alone or 40 μg/ml mBSA for 72 hrs. Proliferation was measured by thymidine incorporation (left). IFN-γ levels from culture supernatants were measured by ELISA (right), (n=3).

128 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

we conclude that TACI-/- mice do not exhibit increased DTH or Th1 T cell responses.

B cell expansion alone cannot augment DTH responses: Traf2 lox/lox X

Mx-1 Cre mice have normal DTH responses

Traf2 lox/lox X Mx-1 Cre mice were identified as another strain that exhibited increased B cell numbers in the absence of elevated serum BAFF levels. Analysis of splenic B cell subsets revealed a particular expansion of marginal zone B cells in the

Traf2 lox/lox X Mx-1 Cre mice compared to Traf2 lox/lox controls (Fig. 5.7A), which correlates with the published phenotype [267]. This was accompanied by increases in

MHC class II and ICOS-L expression in the Traf2 lox/lox X Mx-1 Cre strain in a similar

fashion to BAFF transgenic mice (Fig. 5.7B), while levels of MHC class I, CD40 and

CD86 were unchanged. Measurement of DTH responses in Traf2 lox/lox X Mx-1 Cre

mouse strains revealed a completely normal DTH response compared to their Traf2

lox/lox littermate controls (Fig. 5.7C). Finally we examined DTH responses in C57/B6

mice after B cell transfer. 1x107 purified B cells from wildtype, BAFF transgenic and

TACI-/- animals were transferred by intravenous injection into the tail vein of recipient

C57/B6 mice 24 hrs before immunisation, and DTH responses measured. These data show that transfer of B cells from any of the 3 genotypes could not transfer increases in the DTH responses. Thus these experiments imply that while augmented DTH in BAFF transgenic mice is a B cell-dependent phenomenon, it is integrally tied to the elevated levels of BAFF that exist in these mice, and is not replicated by B cell expansion alone.

129 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

Figure 5.7: Traf2 lox/lox X Mx-1 Cre mice do not display increased DTH responses. A, The subset composition of splenic B cells from Traf2 lox/lox and Traf2 lox/lox X Mx-1 Cre mice was determined by flow cytometry after staining with antibodies against B220, IgD and IgM. Populations were gated on B220+ cells IgMlo IgDhi = follicular (FO), IgMhi IgDhi = transitional type 2 (T2), IgMhi IgDlo = marginal zone (MZ). B, Cell surface expression of MHC class II, MHC class I, CD40, CD86 and ICOS-L by Traf2 lox/lox (purple) and Traf2 lox/lox X Mx-1 Cre (green) B220+ gated B cells was measured by flow cytometry (n=5). C, DTH responses were measured in Traf2 lox/lox (white bars) and Traf2 lox/lox X Mx- 1 Cre (grey bars) mice (n=6). D, 1x107 purified splenic B cells from wildtype (dark grey bars), BAFF transgenic (light grey bars), and TACI deficient mice (black bars) were transferred into C57/B6 recipients and DTH responses measured. C57/B6 mice without B cell transfer were used as a control (white bars) (n=7).

130 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

DISCUSSION

The appropriate regulation of T cell responses is essential for effective pathogen clearance and maintenance of self tolerance. The provision of cognate antigen, costimulatory molecules and cytokines is critical to activation, and is controlled by a range of antigen presenting cells (APCs) such as dendritic cells, macrophages and B

cells. B cells are uniquely positioned amongst the stable of antigen presenting cells in

their ability to capture and present molecules in an antigen specific manner [337, 339,

346, 348]. Soluble molecules that regulate the expression of antigen presentation and

costimulatory molecules on APCs, such as type I IFNs, can profoundly affect the

outcome of a developing T cell response [349, 350]. In turn overproduction of type I

IFNs is associated with autoimmune diseases such as SLE [351, 352]. In this study we

demonstrated that BAFF regulates the expression of antigen presentation and

costimulatory molecules on B cells, and in so doing, increases the capacity of B cells to

prime T cell responses. This may constitute an important mechanism for the induction of

autoimmunity in BAFF transgenic mice.

BAFF stimulation of purified splenic B cells resulted in the potent upregulation of

both MHC class II and ICOS-L on the cell surface. This effect was seen to be dependent

on signalling via BAFF-R and independent of TACI. The role of BCMA was not tested

due to lack of availability of knockout mice. It would seem unlikely that BCMA would

mediate this effect, as all B cells stimulated with BAFF displayed increased class II and

ICOS-L, while the expression pattern of BCMA is restricted to specific subsets of B cells

(Chapter 3, Fig. 3.6B).

131 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

B cells constitutively express both MHC class II [353, 354] and ICOS-L [54, 81],

which are important for normal B cell function. Both MHC class II and ICOS-L

expression is significantly reduced on the small number of peripheral B cells present in

BAFF deficient mice (data not shown), suggesting that BAFF may drive the constitutive

expression of these molecules [201], similarly to CD21 and CD23 [234]. The increased

expression of MHC class II after BAFF stimulation may result from increased production

or altered subcellular relocalisation of class II molecules. MHC class II expression in B cells is mainly regulated at the level of transcription [353, 354], primarily under the control of the class II transactivator (CIITA) [319, 355]. Thus BAFF may enhance MHC

class II transcription via increased CIITA recruitment or activity. Alternatively, BAFF

may induce changes in class II subcellular localisation, such as occur during dendritic cell

maturation [356, 357], although this would seem less likely. Both options could be

explored in further studies.

The regulatory mechanisms controlling ICOS-L expression are still being defined.

Thus far CD40 is the main inducer of ICOS-L expression on B cells [54]. As both

BAFF-R and CD40 signal via the NFκB2 pathway, it seems likely that NFκB2 activation

is involved in the regulation of ICOS-L expression in B cells. Elevated expression of

ICOS-L in Traf2 lox/lox X Mx-1 Cre mice, which exhibit constitutive NFκB2 activation

[267], adds further weight to this hypothesis. BAFF stimulation of NFκB2 deficient or

aly/aly NIK mutant B cells [254] would define the role of NFκB2 in regulating ICOS-L

expression further.

Induction of antigen presentation and costimulatory molecules in response to BAFF

implied that BAFF might regulate T cell priming by B cells. B cells stimulated with

132 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

BAFF in vitro increased T cell proliferative responses, although this increase was

relatively small in magnitude compared with classical activators such as BCR stimulation

or LPS. Interestingly, B cells purified from BAFF transgenic mice showed greater

effects on T cell proliferation. It is possible that additional factors increase the effects of

BAFF stimulation in vivo. Additionally, differing receptor usage and expression or

changes in B cell subset distribution between wildtype and BAFF transgenic mice may be responsible. Marginal zone B cells are better activators of naïve T cells than follicular B

cells [358] and their numbers are increased in BAFF transgenic mice [272] thus

expansion of marginal zone B cells seems a likely mechanism. We attempted to prove

this by sorting follicular and marginal zone B cells from wildtype and BAFF transgenic

mice and using these cells as stimulators in MLR assays. Unfortunately robust proliferative responses could not be obtained from these experiments, most likely as the

result of cell stress and damage during the cell sorting procedure. In future Balb/c x

BAFF transgenic mice and DO11.10 TCR transgenic T cells will be used for similar sorting experiments, as this system produced more robust data than the MLR system.

Interactions between ICOS and ICOS-L regulate Th1 and Th2 T cell driven inflammation [62-64], pathogen clearance [61], germinal centre formation [68] and CD40 mediated antibody class switching [55, 57, 58]. Contact hypersensitivity responses are altered in the presence ICOS stimulation [67] and Th1 responses in models of EAE are altered upon ICOS blockade or in ICOS deficient mice [55, 62]. Given the B cell- dependent nature of the increased DTH response in BAFF transgenic mice, we judged

ICOS-L to be a likely molecular mediator of this phenomenon. However, blocking the function of ICOS-L proved to be ineffective in normalising the increased T cell

133 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

proliferative responses in response to BAFF transgenic B cells in MLRs. In addition

blocking antibodies were unable to normalise the DTH response in BAFF transgenic

mice. These data suggest that enhanced ICOS-L expression was not mediating the observed increases in T cell responses. Increased ICOS-L may however be responsible for the increased size and frequency of spontaneous germinal centres [228] and increased

T-dependent antibody responses [293] that are observed in BAFF transgenic mice.

Assays directed at T-B interactions during antibody responses in BAFF transgenic mice may be a worthwhile area for future studies.

The effect of increased B cell numbers in DTH responses was further examined using mice strains that exhibit B cell expansion, in the absence of elevated BAFF. Both TACI deficient and Traf2 lox/lox X Mx-1 Cre mice display increased numbers of B cells and inappropriate localisation of particular subsets e.g. marginal zone B cells in peripheral lymph nodes (data not shown and [267]). Preliminary studies in TACI deficient mice indicated an increased DTH response in this strain, however experiments performed with littermate controls indicated otherwise. A possible explanation for these discrepancies is incomplete backcrossing of TACI knockout lines to the C57/B6 background, resulting in cosegregation of 129 alleles that potentiate inflammation independently of the TACI locus. These results highlight the importance of effective breeding strategies and maintenance of knockout lines. Traf2 lox/lox X Mx-1 Cre mice also displayed no increases in DTH responses, while transfer of B cells also failed to increase DTH. In summary, this series of experiments suggests that increased DTH responses in BAFF transgenic mice are unlikely to be the result of increased B cell numbers or inappropriate cellular localization, and likely result from elevated BAFF levels.

134 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

Recent evidence suggests that increased systemic BAFF concentrations may increase

the rate at which antigen specific B cells proliferate, and thus the numbers of antigen specific B cells during clonal expansion [359]. This would result in increased numbers of antigen-specific B cells and an increased probability of interaction with antigen-specific

T cells. Thus an argument could be made that increases in antigen-specific B cell numbers are responsible for augmented DTH in BAFF transgenic mice. Alternatively there may be distinct alterations in B cell function. Recently a clearer functional distinction between the 3 BAFF receptors has emerged with the observation that activation of BCMA leads to the upregulation of MHC class II, CD86, CD80, CD40 and

ICAM-1. This resulted in enhanced presentation of OVA to DO11.10 T cells and increased proliferative responses [249]. Therefore, it is possible that the increased DTH responses in BAFF transgenic mice may result from increased numbers and activity of

BCMA+ B cells during T cell priming.

BAFF increases the numbers of BCMA+ plasmablasts in human in vitro systems

[221], and our own data showed that BCMA is highly expressed by germinal centre B

cells (Chapter 3, Fig. 3.3.6B, and [250]), an important site of B and T cell interaction.

BCMA was also reported to be regulated by mouse splenic B cells in response to IL-4 and IL-6 [249] indicating that this receptor may be induced by interactions with T cells or during inflammation. Thus activation in an inflammatory environment may increase generation of antigen-specific BCMA+ plasmablasts or germinal centre B cells. These

cells would then respond to elevated BAFF levels by increasing expression of MHC class

II and costimulatory molecules and increase their capacity to stimulate T cells. Thus

during B cell:T cell interactions in the local lymph nodes, antigen-specific T cells would

135 CHAPTER 5 BAFF REGULATION OF T-B CELL COLLABORATION

receive increased activation signals, leading to increased T cell activation, clonal expansion and effector function. This model would account for the increased proliferation and IFNγ production observed in BAFF transgenic mice (Chapter 4, Fig.

4.3A and B).

Given further time this hypothesis would have been tested in a series of experiments.

Anti HEL specific transgenic B cells would be used to study antigen specific B cell responses in wildtype and BAFF transgenic mice. These cells would first be labeled with

CFSE, then transferred into hosts. These animals would then be immunized with

HEL/CFA and their proliferation kinetics, BAFF receptor expression and costimulatory molecule induction monitored by flow cytometry. A resultant correlation between

BCMA expression and increased expression of costimulatory molecules could be tested functionally by reconstituting wildtype and BAFF transgenic animals with BCMA knockout bone marrow, followed by measurement of DTH responses.

In conclusion, we have examined the effects of BAFF on the antigen presenting and costimulatory molecule expression of B cells. We highlight BAFF-induced regulation of

MHC class II and ICOS-L both in vitro and in vivo and demonstrate that BAFF stimulated B cells possess improved activating potential for alloreactive and antigen- specific T cells. Blockade of ICOS-L did not alter the course of in vitro assays (such as

MLR) using B cells as stimulators, or the outcome of the B cell dependent DTH response in BAFF transgenic mice. Finally, we demonstrated that B cell expansion is not responsible for the enhanced DTH in BAFF transgenic mice, and conclude that the B cell augmentation of this response is likely to be of a site- and antigen-specific nature, possibly via the stimulation of BCMA.

136 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

CHAPTER 6: GENERAL DISCUSSION AND CONCLUSIONS

In this thesis we provide evidence that strongly support a role of the TNFSF molecule

BAFF as a novel regulator of peripheral T cell function. We clearly demonstrate that

BAFF stimulation of T cells regulates proliferative responses in vitro and that this occurs

via the expression of BAFF-R on the cell surface. Furthermore, we show that the overexpression of BAFF alters the course of two models of T cell-dependent inflammation, indicating that BAFF is a regulator of T cell responses in vivo. These experiments extend the biological effects of BAFF to another important immune cell type and provide a solid basis for further study of the mechanisms by which BAFF regulates T cell responses.

Cellular proliferation during T cell activation is a highly regulated process balancing the competing objectives of rapid responses to pathogens and prevention of self reactivity. Naïve T cells respond to extracellular signals via the activation of intracellular signalling pathways, de novo transcription, entry into the cell cycle and cellular proliferation, all of which are under fine molecular control. Thus defining the ways in which BAFF regulates important molecular events during T cell activation would improve our understanding of BAFF co-stimulation at the molecular level. Many families of signalling and transcription factor molecules have well established, critical roles during T cell activation e.g. NFAT, ERK, JNK and NFκB. These molecules are differentially regulated by TCR and costimulatory signals and are activated by both the

CD28 and TNFRSF costimulatory families. NFAT and ERK are activated in response to

137 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

TCR signals [34], while activation of NFκB and JNK are important events in mediating costimulatory signals from CD28, especially IL-2 production [35, 37]. Thus targeted study of these molecules in response to BAFF co stimulation would provide an effective strategy for coupling the observed cellular events to molecular events.

BAFF-R is uncommon amongst the costimulatory TNFRSF in that it is unable to interact with TRAF2, [216] a known activator of the JNK and NFκB pathways [100] and a central mediator of other TNFRSF costimulatory responses [165, 166]. This implies that BAFF-R may mediate qualitatively distinct signals to those of the other TNFRSF members, especially at the level of Map kinase and NFκB activation. Instead BAFF-R interacts with TRAF3 only [216], which is unable to activate the Map kinases JNK, p38 or ERK, or the canonical NFκB1 pathway. Experiments in B cells indicate that engagement of BAFF-R is coupled to the activation of the NFκB2 pathway [254], the prosurvival serine/threonine kinase Pim-2 [201, 305] and suppression of PKCδ [264].

The ability to activate particular signalling pathways is determined in part by the modular structure of the receptor, thus it seems likely that similar pathways would be activated by BAFF-R in T cells. There is evidence demonstrating that Pim-2 [360] and particularly NFκB2 are important for the regulation of T cell survival and proliferation.

Experimental evidence shows that T cell lines express NFκB2 p100 and that overexpression of NIK (the kinase associated with NFκB2 processing and activation) results in increased levels of p100 processing [256, 361]. In addition overexpression or constitutive processing of p100 is associated with cutaneous T cell lymphomas [362] and lymphoma derived cell lines [256]. These data suggest that the NFκB2 pathway is likely to be active in T cells and that transcriptional targets of p52 may be important regulators

138 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

of T cell survival. Thus the NFκB2 pathway may have an important role in inducing cell survival during peripheral T cell responses. The identification of the NFκB1 dependent, but NFκB2 independent [363], nature of CD28 co-stimulation suggests that BAFF activation of NFκB2 may provide an important complementary signal to that induced by

TCR/CD28. Experiments could be designed to address this hypothesis. The activation of

NFκB 1 and 2 pathways in BAFF stimulated T cells could be analysed, paying particular attention to p100 processing and p52 nuclear localisation. Similar analyses of unstimulated T cells from BAFF transgenic mice may also be revealing. Additionally experiments could be designed to probe the downstream effects of NFκB2 activation in T cells. Specifically, over-expression of NIK and/or p100 in T cell lines, combined with transcript profiling may provide a good system in which to determine the downstream effects of NFκB2 activation in T cells.

Regulation of cell survival/apoptosis and cytokine production as a result of BAFF co stimulation are two additional areas that would warrant further exploration. The Bcl-2 family proteins are key regulators of cell survival in lymphocytes [364], and their induction in response to appropriate external stimuli is important for T cell survival during activation and clonal expansion. Bcl-2 is regulated by BAFF in B cells [228, 365] and our experiments show that BAFF induces Bcl-2 in T cells [250]. In addition Bcl-XL induction by CD28 and TNFRSF co stimulation [167, 168, 366-370] is associated with T cell survival during activation. Thus Bcl-2 and Bcl-XL may be important molecular

mediators of BAFF costimulation. Analysis of BAFF induced cytokine production could

be addressed by direct measurement of cytokine gene transcription at the mRNA level

and use of reporter constructs containing promoter regions of genes such as IL-2. In

139 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

addition, more detailed dissection of division kinetics by CFSE as previously described

[371] and analysis of cell cycle entry and progression may provide further insight. A more global understanding of the transcriptional programs induced by BAFF in T cells would be provided by transcript profiling experiments. This would provide additional clarity and possibly provide novel targets of BAFF that may be important for regulating T cell responses aside from the well-studied molecules already described.

The series of experiments presented in Chapter 4 provide the first demonstration that modulation of the level of BAFF, such as in BAFF transgenic mice, can lead to altered outcomes of T cell driven inflammation. Parallel studies performed in our laboratory demonstrate that BAFF transgenic mice display altered T cell responses in models of

EAE and allograft rejection (I. Sutton and S. Grey, unpublished data). These experiments further strengthen our conclusion that BAFF levels can modulate T cell responses in vivo.

The BAFF transgenic mice used in these studies were generated by placing murine BAFF

under the transcriptional control of the α1-antitrypsin promoter/ApoE enhancer element

[228]. This causes significant production of soluble BAFF in the liver and elevated

serum levels of BAFF compared to normal mice [293]. This overabundance of BAFF is

therefore freely available to all responding cell types and is not specifically targeted to T

cells. The relevance of this becomes clear with the demonstration of important T and B

cell interactions in BAFF transgenic mice, which result in the modulation of DTH

responses (Chapter 4 and 5). Thus while our experiments demonstrate significant

modulation of T cell responses in a high BAFF environment, the interpretation of the

direct role of BAFF stimulation on T cells in this process remains unclear.

140 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

Cell intrinsic tools, which would enable the cell type specificity of these responses to

be determined, provide the best way to further refine these studies. The tool of choice is

clearly BAFF-R deficient animals. Since BAFF-R is critical for maintaining numbers,

survival and function of the B cell pool and regulating T and B cell interactions, targeted

deletion of BAFF-R in T cells alone would be desirable. Construction of conditional

BAFF-R knockout mice via incorporation of loxP sites and subsequent crossing to T cell

specific Lck-Cre mice would enable T cell specific deletion. Alternatively, generating

mixed chimeras using bone marrow from germline BAFF-R knockout and TCRβ

knockout mice would also achieve T cell specific deletion. Additionally, crossing of

BAFF-R knockout mice to TCR transgenic strains such as the OVA specific OT-I and

OT-II strains would provide important tools for transfer experiments and enable antigen

specific CD8+ and CD4+ responses to be examined.

Once these systems are established, it would be possible to probe the role of

physiological levels of BAFF in various T-dependent responses and clarify the role of

BAFF-R signalling in T cell development, survival, function and tolerance. In vitro

activation would be performed using anti-CD3 and anti-CD28 or in vitro derived

dendritic cells. Proliferative and cytokine responses would subsequently be measured.

BAFF-R deficient OT-I and OT-II TCR transgenic T cells could also be CFSE labelled

and transferred into recipient hosts. Immunisation with OVA protein or the appropriate

MHC restricted OVA peptides would allow in vivo proliferation, Th1/Th2 differentiation and cytotoxic CD8+ responses to be measured. This system would also allow the

contributions of BAFF to clonal expansion and memory formation to be determined,

processes in which other TNFRSF members have been shown to play important roles

141 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

[100]. Responses to a range of pathogenic organisms, such as viruses (LCMV), Listeria

or schistosome eggs, and tumour cell lines could be measured. Additionally important

models of human diseases such as allergic airway inflammation, EAE, colitis and

allograft rejection could be performed. Expression of BAFF-R in the thymus [213]

suggests that elements of thymic T cell development may be regulated by BAFF; as such

use of transgenic TCR and antigen combinations would allow contribution of BAFF to

thymic selection to be determined. CD4+CD25+ regulatory T cells have been shown to

express high levels of BAFF-R [320], thus experiments probing regulatory T cell

function in the absence of BAFF-R may also be revealing. Assays measuring classical

suppressor function in vitro and cotransfer with CD45RBhi cells in vivo would determine

any contribution of BAFF to regulatory T cell function.

Given the obvious advantages of using a receptor knockout approach to decipher cell specific functions, we attempted to use the A/WySnJ strain as a model of BAFF-R deficiency. Unfortunately these experiments served to demonstrate many problems

associated with using this strain as a model for BAFF-R deficiency. The baff-r allele

present in the A/WySnJ mice codes for a truncated protein, which probably mediates

some signalling due to the discrepancy between the phenotypes of the BAFF knockout,

BAFF-R knockout and A/WySnJ mice [213, 230, 236, 237]. Consultation with the

original literature also indicates that this strain has additional genetic lesions which reside

in separate loci to baff-r and result in mastocytosis [372]. Thus we determined that in

vivo experiments in A/WySnJ mice would not be interpretable. Instead we reasoned that

transfer of A/WySnJ T cells into A/J or Balb/c mice would allow some analysis of

BAFF-R function in T cells. However, baseline proliferation and cytokine responses to

142 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

anti-CD3 stimulation revealed that A/WySnJ T cells had abnormally enhanced

production of Th2 cytokines, particularly IL-5 and IL-10 (A Sutherland, unpublished

data). We could not demonstrate similar responses upon activation of T cells from BAFF

knockout mice, or wildtype T cells activated in the presence of BAFF blockers (A.

Sutherland, unpublished data). Thus we determined that rigorous analysis of BAFF-R deficiency using A/WySnJ mice was not possible. Indeed our data would question the validity of another study which examines the role of BAFF in the context of allograft rejection using the A/WySnJ strain [320]. Ye et al. demonstrated enhanced allograft survival in A/WySnJ mice, leading to the interpretation that deficiencies in BAFF-R signalling in T cells prevents normal graft rejection [320]. However we would offer an alternative interpretation. Instead other genetic lesions in A/WySnJ mice, which are independent of the baff-r locus, lead to enhanced Th2 responses in these mice and result in predominantly Th2-type responses at the site of the graft [320]. This would result in the suppression of the characteristic Th1-type response and lead to enhanced graft survival that was independent of deficiencies in BAFF-R signalling. As this study [320] provides one of the few pieces of evidence that BAFF can regulate T cell responses in vivo (besides our own), experiments using mice with targeted deletion of BAFF-R in the

T cell compartment are all the more necessary.

The biological functions of BAFF are likely to be controlled in a cell type- and stimulus specific- manner at the level of expression and regulation of activity. The significance of any of these mechanisms in the broader context of homeostasis, pathogen clearance or disease has not been established. For example, B cell homoestasis is dependent upon BAFF production by an unidentified stromal cell population in the spleen

143 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

[211] while induction by proinflammatory mediators in leukocytes may function to

support clonal expansion during immune responses. Associations have been established

between elevated BAFF levels and pathogen infection [285] or autoimmune disease [268,

269, 277, 278], however the cellular basis for this is unknown. A better understanding of

BAFF expression may provide better opportunities to target this molecule in humans.

One way to establish this would be the creation of BAFF reporter mice by knock-in of a

BAFF allele containing EGFP adjacent to an internal ribosome entry site (IRES) site, an approach that has been used successfully employed for other cytokine molecules such as

IL-4 [373]. This would allow co-expression of EGFP with BAFF under its normal regulatory elements and allow cell type-specific expression of BAFF to be easily measured at the whole body level. This would be particularly useful for defining important BAFF producing cell types during pathogen invasion or progression of autoimmune disease.

Expression studies alone will not be sufficient to understand the biology of BAFF. In addition to regulation of mRNA expression, other important mechanisms exist such as alternative splicing [198], heterodimer formation [145] and posttranslational modification

[202]. Many TNFSF members are regulated by the alternate production of membrane bound and soluble forms, which can have significantly altered activities [149].

Differential regulation of the membrane bound and soluble forms also occurs for BAFF implying that differential functions may exist between these two forms of BAFF. Our in vitro studies demonstrated that binding of recombinant BAFF to cell culture plates was necessary before its costimulatory activities could be observed, while soluble BAFF was capable of mediating its effects on B cells. This suggests that BAFF conformation is

144 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

important for mediating cell type-specific effects. Thus we hypothesise that soluble

BAFF would function to maintain B cell homeostasis, but would not regulate T cell

activation. It would seem more likely that membrane bound BAFF expressed by leukocytes such as dendritic cells, would be responsible for regulating T cell activation.

To test this hypothesis, non cleavable mutants of BAFF could be created by mutagenesis of the furin protease site. These mutants could then be used to probe the differential functions of membrane and soluble BAFF by creating transgenic or knockin mice.

Targetting of this transgenic construct to myeloid cells, similarly to Gavin et al [199], and subsequent analysis of T cell priming in vitro or in vivo would determine if overexpression by myeloid APCs could alter T cell function. Additionally, monitoring the ability of this non-cleavable molecule to generate B cell expansion and autoimmunity would be of interest. Alternatively, the non cleavable mutant form of BAFF could be placed under the control of its normal regulatory elements thus resulting in more physiologically relevent levels of expression. Analysis of B cell homeostasis and T cell responses during inflammation in these mice would give clearer insight into the normal function of membrane versus soluble BAFF.

The experiments presented in Chapter 5 reveal interesting mechanisms of crosstalk between T and B cells, especially in the context of BAFF overexpression. Given the ability of B cells to potently enhance antigen specific T cell responses and modulate effector T cell numbers in BAFF transgenic mice, we would postulate that interactions between B and T cells are important for the generation of autoimmunity in these mice.

Data from BAFF transgenic mice suggest that B cells acquire an enhanced ability to stimulate T cells after exposure to BAFF. As exposure to high levels of BAFF also

145 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

enables the survival of B cells with significant affinity for self antigens [201, 272], it

would seem likely that significant quantities of self antigen would be captured and

presented on the surface of B cells in BAFF transgenic mice. This would lead to the

activation of self reactive T cells in the periphery, which may augment the function of

autoreactive B cells by providing T cell help. An important function for B cells as

antigen presenting cells has been demonstrated in other models of lupus. Crossing of

MRL-lpr/lpr mice to a B cell deficient background prevented the spontaneous activation

of T cells in inflamed tissues, expansion of CD4+ CD44hi CD62Llo effector/memory

populations [323] and the development of autoimmunity [374]. Interestingly crossing of

BAFF transgenic mice to the μMT-/- B cell deficient background resulted in similar

outcomes (F. Mackay, unpublished data). Generation of mice that are unable to secrete

antibody revealed that the B cell-dependent autoimmunity in the MRL-lpr/lpr mice was

antibody independent [375], and was probably related to enhanced antigen presentation,

as T cell activation was maintained. Thus we propose a similar role for B cell antigen

presentation in the development of autoimmunity in BAFF transgenic mice. This

hypothesis is currently being tested in two ways. First we have specifically targetted antigen presentation functions in B cells using MHC class II knockout mice. We have constructed mixed bone marrow chimeras in BAFF transgenic mice using μMT-/-, I-Aβ-/- and wildtype bone marrow, which results in the absence of MHC class II expression specifically from the B cell compartment, while leaving it intact in other cell types [376,

377]. These mice are being left to age and symptoms of autoimmunity will be monitored over time to determine disease progression in the absence of B cell antigen presentation.

Secondly, we have crossed BAFF transgenic mice to immunoglobulin non-secretor mice

146 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSIONS

developed by Chan et al. [375] and will monitor disease progression. This system will enable us to define whether secreted antibody is necessary for disease progression, which

will provide clues to the relative role of antigen presentation in the generation of

autoimmune disease.

In conclusion, we show that BAFF functions as a regulator of T cell responses in vitro,

demonstrating direct costimulatory effects on T cells via BAFF-R. We demonstrate that

modulating BAFF levels influences the outcome of T cell dependent models of

inflammation, showing that BAFF regulates T cell function in vivo. We also provide evidence that BAFF regulates important antigen presentation and costimulatory functions in B cells, which can play additional roles in regulating T cell responses. The definition of BAFF mediated signalling pathways in T cells, and studies with targeted deletion of

BAFF-R in T cells, would significantly improve our understanding of the action of this

molecule in T cell responses. In addition, attempts to understand the regulation and

function of the membrane and soluble forms of BAFF during infection and autoimmunity

will provide important new insights. Finally, we suggest that enhanced B cell antigen

presentation of self antigen may be an important mechanism in the generation of autoimmunity in BAFF transgenic mice.

147

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188

REPRINTS OF PRIMARY AUTHOR PUBLICATIONS

189 The Journal of Immunology

BAFF Augments Certain Th1-Associated Inﬂammatory Responses1

Andrew P. R. Sutherland,2* Lai Guan Ng,2* Carrie A. Fletcher,* Bennett Shum,*† Rebecca A. Newton,* Shane T. Grey,* Michael S. Rolph,*† Fabienne Mackay,*† and Charles R. Mackay3*†

B cell-activating factor belonging to the TNF family (BAFF; BLyS) is a critical regulator of B cell maturation and survival, and its overexpression in BAFF transgenic (Tg) mice results in the development of autoimmune disorders. BAFF also affects T cell function through binding to one of the BAFF receptors, BAFF-R. Using BAFF Tg mice, we examined a typical Th1-mediated response, the cutaneous delayed-type hypersensitivity reaction, and found a much greater degree of paw swelling and inflammation than in control mice. Importantly, delayed-type hypersensitivity scores correlated directly with BAFF levels in serum. Con- versely, in a Th2-mediated model of allergic airway inflammation, BAFF Tg mice were largely protected and showed markedly reduced Ag-specific T cell proliferation and eosinophil infiltration associated with the airways. Thus, local and/or systemically distributed BAFF affects Th1 and Th2 responses and impacts on the course of some T cell-mediated inflammatory reactions. Our results are consistent with the idea that BAFF augments T cell as well as B cell responses, particularly Th1-type responses. Results in BAFF Tg mice may reflect the situation in certain autoimmune patients or virally infected individuals, because BAFF levels in blood are comparable. The Journal of Immunology, 2005, 174: 5537Ð5544.

cell-activating factor belonging to the TNF family leted (12, 14). BAFF also enhances the survival of plasmablasts (BAFF;4 BLyS, TALL-1, zTNF-4, THANK) is a TNF (15, 16) and stimulates peripheral B cell proliferation, Ab produc- B superfamily member that plays an important role in im- tion, and class switching (17–19). mune responses (1, 2). Excessive production of BAFF is associ- BAFF binds three receptors: BAFF-R, transmembrane activator ated with the development of autoimmune diseases, because trans- and calcium modulator cyclophilin ligand interactor (TACI), and B genic (Tg) mice that overproduce BAFF develop severe cell maturation Ag (BCMA) (1). BAFF-R is the predominant autoimmune disorders that resemble systemic lupus erythematosus BAFF receptor on circulating B cells (18) and is also important for and Sjo¨gren’s syndrome (3–6). Also, high levels of BAFF have the regulation of B cell maturation in the spleen, because BAFF- been found in the blood of patients with autoimmune diseases, R-deficient mice show disrupted B cell maturation, similar to that particularly systemic lupus erythematosus and Sjo¨gren’s syndrome seen in BAFF-deficient mice (13, 20). BAFF-induced survival is (5, 7–9). Thus, excess BAFF may cause a breakdown in immune mediated by up-regulation of the antiapoptotic molecule Bcl-2 (3), tolerance through inappropriate survival signals to splenic and pe- and Bcl-2 overexpression restores B cell survival in the absence of ripheral B cells (10–12). In the spleen, BAFF promotes B cell BAFF (21). The other receptors, TACI and BCMA, have restricted maturation, particularly at the critical T1-T2 immune tolerance patterns of expression on specific B cell subsets (18); BCMA ap- checkpoint (10), and splenic B cells in BAFF-deficient mice fail to pears to be particularly important for survival or stimulation of mature past the T1 stage (13). Autoreactive B cells are expanded plasmablasts and germinal center cells (15, 18), whereas TACI in lymphoid tissues of BAFF Tg mice (3, 14), possibly as a result most likely serves as a negative regulator of B cell activity, par- of BAFF rescue of autoreactive cells that would normally be de- ticularly on activated B cells (22). More recently, BAFF has emerged as a regulator of T cell function. BAFF provides costimulatory signals to T cells in the *Arthritis and Asthma Research Program, Garvan Institute of Medical Research, Syd- presence of suboptimal TCR stimulation and enhances T cell pro- † ney, Australia; and Cooperative Research Center for Asthma, Sydney, Australia liferation and cytokine production (18, 23, 24). TACI-Ig and Received for publication December 13, 2004. Accepted for publication February BAFF-R-Ig decoy receptors inhibit T cell activation in vitro (18, 14, 2005. 23) and in vivo (25), and BAFF-deficient mice show impaired T The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cell-mediated allograft rejection (26). Thus, physiological levels of with 18 U.S.C. Section 1734 solely to indicate this fact. BAFF appear necessary for the generation of optimal T cell re- 1 This work was supported by the Australian National Health and Medical Research sponses. These effects occur through BAFF-R, because T cells Council, the Wellcome Trust, and the Cooperative Research Center for Asthma. from BAFF-R mutant mice fail to respond to BAFF, and BAFF-R 2 A.P.R.S. and L.G.N. contributed equally. expression (but not that of TACI or BCMA) is present on the 3 Address correspondence and reprint requests to Dr. Charles R. Mackay, Arthritis surface of subsets of central and effector memory T cells (18). and Inflammation Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia. E-mail address: These findings suggest that BAFF may play a role in stimulating T [email protected] cell function in vivo, particularly during T cell-mediated pathogenic 4 Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the reactions. Interestingly, BAFF is highly up-regulated in astrocytes in TNF family; BAFF-R, BAFF receptor; BAL, bronchoalveolar lavage; DTH, delayed- multiple sclerosis plaques (27) and is also produced by fibroblast-like type hypersensitivity; mBSA, methylated BSA; Tg, transgenic; WT, wild type; TACI, transmembrane activator and calcium modulator cyclophilin ligand interactor; synoviocytes from rheumatoid arthritis patients (28). Both of these BCMA, B cell maturation Ag. autoimmune diseases have a strong T cell association.

ϩ CD4 effector T cells often polarize to either Th1 or Th2 phe- were killed at 48 h after challenge. The paws of mice were dissected and notypes (29). Th1 responses control intracellular pathogens such as fixed in 10% formalin (Sigma-Aldrich) in PBS for 5 days. Paws were viruses and are associated with the production of IFN-␥, IL-2, and decalcified for 10% formalin in PBS supplemented with formic acid (Sig- ␤ ma-Aldrich) at a 9:1 ratio before being embedded in paraffin, sectioned, TNF- and recruitment of phagocytic leukocytes. In contrast, Th2 and stained with H&E. responses control infections by large extracellular parasites, in part For analysis of mBSA-specific Abs, mice were killed 7 days after foot- through production of IL-4, IL-5, and IL-13, and recruitment of pad challenge, serum was collected, and mBSA-specific Abs levels were eosinophils. Dysregulation of Th1 or Th2 responses may contrib- measured by ELISA. In vitro recall responses were measured by collecting inguinal lymph nodes 7 days after immunization. Cultures were normalized ute to the pathogenesis of autoimmune diseases as well as allergic to 2 ϫ 106 T cells/ml and were restimulated for 72 h with 40 ␮g/ml mBSA diseases such as asthma. The mechanisms that initiate and main- in X-Vivo 15 serum-free medium (BioWhittaker) supplemented with pen- tain polarization of T cell responses to Th1 or Th2 are incom- icillin/streptomycin. Supernatants were collected for measurement of cy- pletely understood, but instructive signals in the form of cytokines tokine production by ELISA. Proliferation was measured by addition of 1 ␮ 3 and cell surface molecules expressed by APC are important (30– Ci of [ H]thymidine (Amersham Biosciences)/well 18 h before harvesting and subsequent beta scintillation counting. 33). Th1 and Th2 responses counteract each other, in that strong polarization to a Th1 response suppresses Th2 responses and vice OVA-induced allergic airway inflammation versa (34). Given the ability of BAFF to regulate T cell activation Six- to 8-wk-old mice were injected i.p. with 200 ␮l of a 1 mg/ml OVA and its association with autoimmune diseases, we investigated solution with alum (1/1 mix) on days 1 and 15. Mice were given a 20-min whether BAFF affects the outcome of Th1- and Th2-mediated T aerosol on days 28, 30, 32, and 34 consisting of either PBS or 1% OVA in cell responses in vivo. PBS. Mice were killed on day 35, and bronchoalveolar lavage (BAL) fluid We show that systemic overexpression of BAFF in BAFF Tg and organs were collected. Total BAL fluid cell numbers were enumerated, and differential cell counts were performed after cytospin and Giemsa mice exacerbates the severity of Th1-mediated delayed-type hy- staining to establish the number and identity of infiltrating cells. Lung persensitivity (DTH) responses by enhanced T cell proliferation tissues were fixed in 10% Formalin/PBS for 7 days and embedded in par- and IFN-␥ production in local lymph nodes. Enhanced DTH re- affin. Standard protocols for H&E staining were used to stain tissue sec- sponses in BAFF Tg mice correlated directly with levels of BAFF tions. At the completion of the aerosol regimen, peribronchial lymph nodes were collected, pooled into groups, and cultured in RPMI 1640 supple- in serum, suggesting that the varying levels of BAFF seen in the mented with 10% heat-inactivated FCS and penicillin/streptomycin. Cul- blood of human subjects might also relate to varying capacities for tures were normalized to 2 ϫ 106 T cells/ml and restimulated for 72 h with DTH Th1 responses. High levels of BAFF in BAFF Tg mice also 100 ␮g/ml OVA, and proliferation and cytokine production were measured resulted in subdued Th2-mediated responses in an allergic airways as per DTH. In vitro recall responses were measured by immunizing mice ␮ disease model. The effects of BAFF on Th1/Th2 responses oper- at the tail base with 200 l of 1 mg/ml OVA in alum (1/1 mix) and collecting inguinal lymph nodes 7 days later. Cells were restimulated in vitro as described ated at multiple levels, because enhanced Th1 responses depended above for peribronchial lymph nodes, using 500 ␮g/ml OVA. on B cells, whereas BAFF inhibition of Th2 responses was B cell independent. These findings demonstrate that BAFF augments cer- ELISA tain Th1 responses in vivo, but not Th2 responses, and illustrate The mouse BAFF ELISA was performed as previously described (36). The the importance of BAFF for T cell as well as B cell responses. Ab response to mBSA was analyzed as follows. MaxiSorb plates (Nalge Nunc International; 384-well) were coated with 50 ␮g/ml mBSA diluted in Materials and Methods 0.1 M sodium bicarbonate buffer (pH 9.6) at 4°C overnight. Plates were washed three times with PBS-0.05% Tween 20. Serial dilutions of mouse Reagents and Abs serum in ELISA buffer (1% BSA in PBS) were added to the plate. Anti- ␮ Methylated BSA (mBSA) and OVA were purchased from Sigma-Aldrich. mBSA Abs were detected using 0.125 g/ml alkaline phosphatase-conju- Imject CFA and Imject alum were purchased from Pierce. All anti-mouse gated goat anti-mouse IgM, anti-IgG1, anti-IgG2a, anti-IgG2b, anti-IgG3, FITC-, PE-, CyChrome-, PerCP-, and allophycocyanin-conjugated mAbs or anti-IgA (Southern Biotechnology Associates). p-Nitrophenyl phosphate were obtained from BD Pharmingen. Murine IL-4, IL-5, IFN-␥, and IL-10 tablets (Sigma-Aldrich) were used for detection, and plates were read at an ELISA kits were purchased from BD Biosciences. Goat anti-mouse alka- OD of 405 nm. The titer (log base 2) is defined as the serum dilution giving ϭ line phosphatase-conjugated Abs were purchased from Southern Biotech- an OD four times higher than that of background (where 1 1/50 dilution). nology Associates. Human BCMA-Ig was provided by Biogen-Idec and has been described previously (35). Statistical analysis Mice Statistical significance was determined using t test, and significance with relation to comparison data is indicated by p values in the figure legends. Animals were housed under conventional barrier protection and handled in accordance with the animal experimentation ethics committee, which com- Results plies with the Australian code of practice for the care and use of animals Enhanced DTH responses in BAFF Tg mice for scientific purposes. BAFF Tg mice (3) and BAFFϪ/Ϫ mice (13) were supplied by Biogen-Idec and were bred in our animal facility. BAFF Tg Six- to 8-wk-old mice were immunized intradermally at the base of mice were maintained as mice homozygous for the BAFF transgene, and the tail with mBSA in CFA and challenged 7 days later with an s.c. wild-type (WT) mice were used as controls, as previously described (36). injection of mBSA into the footpad. BAFF Tg mice from two Two lines of BAFF Tg mice were used for experimentation, referred to as lines 1 and 2, which differ slightly in BAFF levels and immune responses distinct transgenic lines (lines 1 and 2) displayed significantly in- (see below). ␮MTϪ/Ϫ mice (37) were purchased from The Jackson Lab- creased paw swelling compared with WT mice (Fig. 1, A and B). oratory. ␮MTϪ/Ϫ ϫ BAFF Tg mice were established by breeding homozy- Paw swelling was comparable in WT and BAFF Tg mice at early ␮ Ϫ/Ϫ gous MT and BAFF Tg line 1 mice and subsequent breeding of the F1 time points after challenge (Fig. 1A), but was significantly higher generation. Homozygous ␮MTϪ/Ϫ ϫ BAFF Tg mice and WT controls in BAFF Tg mice 48 and 72 h after challenge. DTH responses in were derived from the F2 generation and bred as separate lines for experimentation. All mice were maintained on a C57BL/6 background BAFF Tg mice at 48 h were also characterized by increased erythema (Fig. 1B), and histological analysis of paw tissue sections DTH responses revealed an increased cellular infiltrate into the footpads (Fig. 1C). Six- to 8-wk-old mice were injected s.c. at the tail base with 200 ␮l of 1.25 Serum anti-mBSA Ab isotypes measured 7 days after footpad mg/ml mBSA in CFA (1/1 mix) on day 1, as described previously (38). On challenge showed BAFF Tg mice to have significantly higher titers day 7, mice were rechallenged in one hind footpad with an injection of 20 ␮l of a 10 mg/ml mBSA solution, whereas the opposite hind footpad re- of mBSA-specific IgG1, IgG2a, and IgG2b compared with WT ceived 20 ␮lof1ϫ PBS. Paw swelling was measured 8–72 h after rechal- mice (Fig. 1D), whereas mBSA-specific IgA levels showed a non- lenge with a dial thickness gauge. For paw tissue section analysis, mice significant trend toward an increase (data not shown). Levels of The Journal of Immunology 5539

FIGURE 1. BAFF Tg mice show increased DTH responses. A, Increased paw swelling over time in two lines of BAFF Tg mice (n ϭ 10, line 1; n ϭ 6, line 2). B, Increased erythema in paws of BAFF Tg mice at 48 h compared with WT mice. C, H&E staining of paw sections at 48 h reveals increased leukocyte inﬁltration in BAFF Tg mice compared with WT controls. D, In- creased serum levels of mBSA-speciﬁc Abs in BAFF Tg mice 7 days after footpad challenge (n ϭ 11, WT; n ϭ 10, .p Ͻ 0.005 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .(BAFF Tg

mBSA-speciﬁc IgM and IgG3 were similar in WT and BAFF Tg mice (data not shown).

BAFF levels determine the magnitude of the DTH response The concentration of BAFF in human blood varies considerably; most individuals express low levels, but some autoimmune patients express levels Ͼ100 ng/ml (5). BAFF Tg mice also show variable levels of BAFF in blood due to secondary endogenous BAFF production in response to autoimmune activation (36). To assess the relationship between serum BAFF levels and paw swelling, serum from both WT and BAFF Tg mice was collected 48 h after challenge, and the level of BAFF in serum was determined by ELISA. Both lines of BAFF Tg mice displayed high levels of BAFF in serum (line 1 average concentration, 215.03 Ϯ 50.69 ng/ml; line 2 average concentration, 1128.2 Ϯ 315.8 ng/ml), whereas lower levels of BAFF were detected in WT mice (5.27 Ϯ 1.47 ng/ml), similar to levels in unimmunized mice (36). When the level of BAFF in serum was correlated with paw swelling for each mouse (Fig. 2A), a strong correlation was observed (r2 ϭ 0.593). Thus systemic BAFF levels may regulate the magnitude of certain T cell responses. FIGURE 2. DTH responses correlate with serum BAFF levels, although We next examined DTH responses in BAFF-deﬁcient mice. BAFF deﬁciency does not eliminate DTH responses. A, Serum levels of BAFF in BAFF Tg mice were measured using ELISA and plotted against These mice were competent in mounting a normal DTH response paw swelling 48 h after footpad rechallenge. B, DTH responses in (Fig. 2B). Also, WT mice treated with BCMA-Ig showed no con- BAFFϪ/Ϫ mice, and mice heterozygous for the knockout allele (n ϭ 6). C, sistent reduction in DTH responses compared with untreated mice C57/B6 mice treated with BCMA-Ig showed no reduction in DTH severity (Fig. 2C). Thus, BAFF is not essential for basal DTH responses, compared with control (PBS-treated) mice (n ϭ 6). 5540 BAFF AND Th1 RESPONSES but in circumstances of BAFF overproduction, the magnitude of DTH responses correlates with the level of BAFF.

Increased mBSA-specific T cell proliferation and IFN-␥ production in BAFF Tg mice The mechanisms underlying the enhanced DTH response in BAFF Tg mice were investigated, first by examining T cell responses to mBSA in vitro. WT and BAFF Tg mice were immunized at the tail base as described above, and inguinal lymph nodes were collected 7 days later. Lymph node cells were restimulated in vitro with mBSA for 72 h. T cells from BAFF Tg mice gave a significantly stronger recall response compared with WT mice, displaying a 3-fold increase in proliferation (Fig. 3A) and a 10-fold higher IFN-␥ production in culture supernatants (Fig. 3B). IL-4 and IL-5 levels were below the level of detection in both WT and BAFF Tg mice (data not shown). Thus, in an environment of high BAFF levels, T cell responses to Ag are augmented, and this might be due to qualitative changes in the T cells involved or an increased frequency of Ag-specific memory or effector T cells.

Numbers of effector memory T cells are increased in BAFF Tg mice, but not in ␮MTϪ/Ϫ ϫ BAFF Tg mice We also examined changes in the make-up of the T cell immune system that result from long term exposure to high levels of BAFF and effects that expanded subsets of B cells could have on T cell and DTH responses in BAFF Tg mice. Previous studies of BAFF Tg mice revealed altered CD4ϩ T cell subset ratios (3). Spleno- cytes from WT and BAFF Tg mice were isolated and stained with Abs to CD4, CD44, and CD62L. We observed a substantial increase in the proportion of CD44highCD62Llow effector memory CD4ϩ cells and a decrease in CD44lowCD62Lhigh naive CD4ϩ T cells in BAFF Tg mice (Fig. 4A, top panels). Functionally, effector memory T cells are closely related to effector T cells and have the capacity to migrate to peripheral tissues (39, 40). To investigate FIGURE 4. Altered T cell phenotype and increased DTH severity in whether the expansion of mature B cell subsets seen in BAFF Tg BAFF Tg mice are B cell dependent. A, Splenocytes were stained with mice (3) might in some way connect to the increased numbers of anti-CD4, anti-CD44, and anti-CD62L (CD4ϩ gated cells are shown; n ϭ ϩ effector memory CD4 T cells, we generated B cell-deficient 3 for all genotypes with a representative plot for each genotype shown). B, BAFF Tg (␮MTϪ/Ϫ ϫ BAFF Tg) mice, and these showed no Cell ratios for CD4ϩ T cells calculated based on the following subsets: increase in effector memory T cell numbers (Fig. 4A, bottom pan- naive, CD62LhighCD44low; and effector, CD62LlowCD44high. Left panel, els). The ratio of effector/naive T cells in line 2 BAFF Tg mice was Ratios for line 2 BAFF Tg mice and WT controls; right panel, ratios for the ␮MTϪ/Ϫ ϫ BAFF Tg line 1 F generation littermates. C, ␮MTϪ/Ϫ ϫ increased ϳ4-fold (Fig. 4B, left panel), and line 1 BAFF Tg mice 2 BAFF Tg mice have similar levels of paw swelling as WT and ␮MTϪ/Ϫ also showed a statistically significant (although smaller) increase control mice (n ϭ 6). D, Serum levels of BAFF were measured using ␮ Ϫ/Ϫ p Ͻ ,ءءء ;p Ͻ 0.01 ,ءء .Fig. 4B, right panel). Analysis of MT mice showed a re- ELISA and plotted against paw swelling at 48 h) duced ratio, indicating an overall reduction in effector cells, which 0.005. was not increased in ␮MTϪ/Ϫ ϫ BAFF Tg mice (line 1). Thus, the altered makeup of the T cell compartment in BAFF Tg mice is ␮ Ϫ/Ϫ dependent on B cells. We examined whether disrupted T cell subset ratios in MT ϫ BAFF Tg mice could affect the course of the DTH reaction. WT, ␮MTϪ/Ϫ, and ␮MTϪ/Ϫ ϫ BAFF Tg mice displayed similar levels of footpad swelling over the course of DTH (Fig. 4C). Paw swelling at 48 h was plotted against serum BAFF levels for each animal, and linear regression analysis was performed (Fig. 4D). The correlation coefficient (r2)ofϽ0.01 indicated a lack of correlation between BAFF levels and DTH response in the absence of B cells. Thus, BAFF-mediated changes in B cell numbers or function affect T cell subset composition and responses in the DTH reaction.

BAFF Tg mice have compromised allergic airway responses FIGURE 3. Increased mBSA-specific in vitro recall responses by T We next asked whether the enhanced DTH response in BAFF Tg cells in BAFF Tg mice. A, Mice were injected at the tail base with 250 ␮g of mBSA in CFA, and inguinal lymph nodes were collected 7 days later. mice was the result of a general increase in T cell responsiveness, Cultures were normalized to 2 ϫ 105 T cells/well and were cultured in particularly by effector memory T cells, or was related more to a triplicate with medium alone or 40 ␮g/ml mBSA for 72 h. Proliferation was Th1-specific enhancement. The responses of WT and BAFF Tg measured by thymidine uptake. B, IFN-␥ levels from culture supernatants mice were examined in a Th2-driven OVA model of allergic air- p Ͻ 0.05. way inflammation. Measurement of cell numbers in the BAL fluid ,ء .(were measured by ELISA (n ϭ 3 The Journal of Immunology 5541 showed that exposure of WT mice to OVA aerosol resulted in tween WT and BAFF Tg mice. However, in our experience, BAL substantial eosinophil infiltration (Fig. 5A), which is characteristic fluid measurements for cytokines have not been particularly reli- for this model (41). In contrast, BAFF Tg mice showed a signif- able. In summary, BAFF Tg mice show a suppression of Th2- icant reduction in eosinophil infiltration. In accordance with BAL mediated allergic airway inflammation and an associated reduction fluid cell numbers, histochemical staining of lung tissue sections in lymph node T cell proliferation and IL-5 production in response revealed greatly reduced numbers of peribronchial and perivascu- to OVA challenge. lar leukocytes in BAFF Tg mice compared with WT controls (Fig. 5B). Levels of anti-OVA specific IgE Abs in serum were roughly Increased OVA-specific recall responses from lymph nodes of equivalent in WT and BAFF Tg mice (not shown), suggesting that BAFF Tg mice the suppressed airway inflammation in BAFF Tg mice may relate To determine whether initial priming defects were responsible for to BAFF effects on T cell function rather than those on B cells or the observed reduction in airway eosinophilia in BAFF Tg mice, Ab production. we performed immunization and in vitro recall experiments. Mice Lymphocytes from peribronchial lymph nodes were collected were immunized at the tail base with an OVA/alum mix, and in- after the final aerosol exposure and restimulated in vitro with OVA guinal lymph nodes were removed after 7 days and restimulated in for 72 h. WT lymphocytes showed a strong increase in prolifera- vitro for 72 h. As expected, WT lymph node cells showed in- tion in response to restimulation with OVA, in contrast to lym- creased proliferation upon stimulation with OVA. BAFF Tg lymph phocytes from BAFF Tg mice (Fig. 5C). Cytokine measurements node cells showed an ϳ3-fold increase in proliferation over WT showed that after restimulation with OVA, WT lymph nodes cells mice (Fig. 6A). In addition, cytokine ELISA from supernatants of produced high levels of IL-5, whereas cells from BAFF Tg mice restimulated cultures from BAFF Tg mice demonstrated signifi- showed 10-fold reduced levels (Fig. 5D). In addition, small, but cantly increased levels of IL-5 and IFN-␥ compared with WT mice demonstrable, levels of IFN-␥ were detected in some BAFF Tg (Fig. 6B). Thus, BAFF Tg mice have increased OVA-specific re- cultures, although this result was not always reproducible. Of note, call responses, and the defect in lung eosinophilia observed in there was no compensatory increase in IL-10 production in BAFF BAFF Tg mice was not due to impaired priming to OVA. Tg cultures (data not shown). We also examined cytokine levels in BAL fluid, but observed no differences in IL-4 or IL-5 levels be- BAFF-mediated suppression of allergic airway inflammation is B cell independent Our results demonstrating that ␮MTϪ/Ϫ ϫ BAFF Tg mice were protected from the augmented DTH responses seen in BAFF Tg mice led to the question of whether BAFF-mediated suppression of airway inflammation would be relieved in ␮MTϪ/Ϫ ϫ BAFF Tg mice. Differential cell counts from BAL fluid showed no significant differences in cell numbers between WT and ␮MTϪ/Ϫ mice in response to OVA aerosol, with characteristic eosinophil infiltration observed in either case (Fig. 7A). Similar to BAFF Tg mice, ␮MTϪ/Ϫ ϫ BAFF Tg mice showed a significant reduction in the eosinophil infiltration in response to OVA challenge. Restimula- tion with OVA of peribronchial lymph node T cells from WT and ␮MTϪ/Ϫ mice showed strong induction of proliferation (Fig. 7B) and IL-5 production (Fig. 7C), whereas there was strong suppression of proliferation and IL-5 production in ␮MTϪ/Ϫ ϫ BAFF Tg mice (Fig. 7, B and C), as seen in BAFF Tg mice. There were no detectable levels of IFN-␥ or IL-10 in any of the cultures (data not shown). These results demonstrate that BAFF-mediated suppression of Th2-dependent allergic airway inflammation occurs by mechanisms that are B cell independent.

FIGURE 5. Suppression of allergic airway inflammation in BAFF Tg mice. A, BAL fluid was recovered from BAFF Tg mice and WT controls after an 8-day aerosol exposure regimen, and constituent cell types were determined by cytospin and Giemsa staining (n ϭ 5). B, Lung sections FIGURE 6. Increased OVA-specific in vitro recall responses in BAFF stained with H&E reveal greatly reduced leukocyte infiltration in BAFF Tg Tg. A, Mice were injected at the tail base with 100 ␮g of OVA in alum, and mice. C, Peribronchial lymph nodes were collected and pooled. Cultures inguinal lymph nodes were collected 7 days later (n ϭ 4). Cultures were were normalized to 2 ϫ 105 T cells/well and were cultured in triplicate with normalized to 2 ϫ 105 T cells/well and were cultured in triplicate with medium alone or 100 ␮g/ml OVA for 72 h, and proliferation was measured medium alone or 100 ␮g/ml OVA for 72 h. Proliferation was measured by by thymidine uptake. D, IL-5 and IFN-␥ levels from culture supernatants thymidine uptake. B, Cytokine levels from culture supernatants were mea- .p Ͻ 0.01 ,ءء .p Ͻ 0.005. sured by ELISA ,ءءء .were measured by ELISA 5542 BAFF AND Th1 RESPONSES

bacterial infection, there could be increased Th1 T cell activity and increased effector T cell responses. Our data for ␮MTϪ/Ϫ ϫ BAFF Tg mice showed that B cells were necessary for the dose-dependent effect of BAFF on cutaneous DTH responses. The most straightforward explanation for our results is that high levels of BAFF affect the numbers and phenotype of peripheral B cells (5), which, in turn, affect the makeup of the T cell immune system, particularly the proportion of effector memory T cells. These effector memory T cells, many of which are most probably commit- ted to a Th1-type response, might also receive additional survival or costimulatory signals from the high levels of BAFF present in serum or on the surface of APC. BAFF augmented the DTH response, a typical Th1 response, suggesting that this cytokine may play a role in immune responses to viral or bacterial infections. BAFF is produced at sites of inflammatory reactions, particularly by leukocyte types associated with type 1 responses, such as neutrophils (43, 44) and macrophages (45). To date, most studies have been confined to autoimmune diseases, with few addressing BAFF levels after viral or bacterial infection due partly to the lack of commercial reagents. In HIV-1 infection, BAFF levels were elevated compared with con- FIGURE 7. Suppression of allergic airway inflammation in BAFF Tg trol values, particularly during later stages of infection when CD4 mice is B cell independent. A, BAL fluid was recovered from WT, counts had declined (46). In addition, HIV-1-infected patients ␮MTϪ/Ϫ, and ␮MTϪ/Ϫ ϫ BAFF Tg mice after aerosol exposure, and show alterations in their B cell immune system consistent with constituent cell types were determined by cytospin and Giemsa staining high BAFF levels, notably hypergammaglobulinemia and altered (n ϭ 5). B, Peribronchial lymph nodes were collected and pooled into B cell differentiation (47). BAFF produced at an inflammatory site groups. Cultures were normalized to 2 ϫ 105 T cells/well and restimulated by activated neutrophils, activated T cells, or macrophages would with medium alone or 100 ␮g/ml OVA for 72 h, with proliferation mea- be transported via the lymph to local lymph nodes, augment T cell ␥ sured by thymidine uptake. C, IL-5 and IFN- levels from culture super- activation, and probably provide survival signals to the central and Ͻ ءءء Ͻ ء natants were measured by ELISA. , p 0.05; , p 0.005. effector memory T cells that express BAFF-R (18). In BAFFhigh individuals, increased proportions of effector memory T cells, possibly predisposed to become Th1-type effector T cells, would pro- Discussion mote the clearance of viral or bacterial agents. Overproduction of BAFF has been associated with various auto- BAFF Tg mice had suppressed allergic airway responses, with immune diseases (3–6, 8, 9). This association was originally a marked reduction in eosinophils in BAL fluid and infiltrating thought to be through BAFF effects on B cell maturation and sur- leukocytes around airways and pulmonary blood vessels. Restimu- vival. However, it is now clear that BAFF promotes T cell acti- lation of lung-draining lymph node cells with OVA showed mark- vation (18, 23, 24) and effector function (25, 26), which may con- edly reduced T cell proliferation and IL-5 production in BAFF Tg tribute to autoimmunity. Autoimmune diseases are often mice. This would account for the reduced eosinophils in the air- associated with Th1-type T cells and the cytokines they produce, ways, because IL-5 is the key cytokine regulating eosinophil pro- such as IFN-␥ and TNF-␣. Our results suggest that BAFF is an duction and recruitment in this model (48). Presumably this sup- important cytokine for both B and T cell responses in vivo, and pression of Th2 effector function was the result of skewing of the that overproduction of BAFF has profound effects on both the T OVA-specific T cell response toward that of a more Th1-like pro- cell and B cell systems. file. There was some evidence for this, because small amounts of Our findings with the DTH model highlight the role for BAFF IFN-␥ were produced by T cells in lung-draining lymph nodes in modulating T cell responses in vivo. DTH is a classical Th1- from BAFF Tg mice, and this was not observed in WT mice. mediated response involving Ag-specific T cell activation and pro- However, other explanations exist, including local regulatory T duction of Th1 cytokines, including TNF-␣ and IFN-␥ (42). BAFF cells that may be particularly BAFF responsive and associated Tg mice developed significantly prolonged DTH responses, and with airway tissue and its draining lymph nodes. Such regulatory we suggest that this augmented DTH response is due to increased T cells have been shown to inhibit Th2 airway responses in mice effector T cell expansion, survival, and effector function. Restimu- (49). However, at the whole animal level, BAFF Tg mice were lation of lymph node cells supported this conclusion, showing in- effectively primed to OVA, because restimulation of non-lung- creased T cell proliferation and a 10-fold increase in IFN-␥ pro- draining (inguinal) lymph node T cells from primed BAFF Tg duction by T cells isolated from BAFF Tg mice. Thus, in a high mice revealed significantly increased proliferation and IL-5 and BAFF environment, T cells receive additional costimulatory sig- IFN-␥ production, similar to the enhanced recall response seen in nals, expand, and provide augmented effector functions. mBSA-primed BAFF Tg mice. It is also possible that an increase Of particular note was the correlation between BAFF levels and in IFN-␥ production at the initial stages of priming may lead to the degree of swelling in BAFF Tg mice. BAFF augmented the antagonism of Th2 differentiation. IFN-␥ induces the Th1 tran- DTH response in a concentration-dependent manner. However, al- scription factor T-bet, which can suppress IL-5 production (50). though BAFF did augment DTH responses, it was not necessary However, the very localized suppression of T cell responses in for basal responses. Indeed, BAFF may play only a small role lung-associated lymph nodes, but not other lymph nodes, might during the course of many normal T cell responses. However, in favor the notion of a pulmonary Th1-like regulatory T cell, as has situations in which there are high serum levels of BAFF, such as been described in a recent study (51). These T-bet- and forkhead/ in an ongoing autoimmune disease or possibly persistent viral or winged helix transcription factor gene-expressing regulatory T The Journal of Immunology 5543 cells suppressed the development of airway hyper-reactivity, pos- 14. Thien, M., T. G. Phan, S. Gardam, M. Amesbury, A. Basten, F. Mackay, and sibly through the actions of IL-10 (51). However, we have found R. Brink. 2004. Excess BAFF rescues self-reactive B cells from peripheral deletion and allows them to enter forbidden follicular and marginal zone niches. no strong evidence for a suppressive IL-10-mediated T cell re- Immunity 20:785. sponse in BAFF Tg lung-associated lymph nodes and no changes 15. Avery, D. T., S. L. Kalled, J. I. Ellyard, C. Ambrose, S. A. Bixler, M. Thien, in CD4ϩCD25ϩ regulatory T cells or increased levels of IL-10 R. Brink, F. Mackay, P. D. Hodgkin, and S. G. Tangye. 2003. BAFF selectively ϩ ϩ enhances the survival of plasmablasts generated from human memory B cells. (data not shown). Interestingly, a large proportion of CD4 CD25 J. Clin. Invest. 112:286. regulatory T cells in the mouse express BAFF-R (26). At present, 16. Balazs, M., F. Martin, T. Zhou, and J. F. Kearney. 2002. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent Immune T and B cells are the only cell types with strong evidence of responses. Immunity 17:341. BAFF-R expression, so the mechanism presumably relates to 17. Schneider, P., F. Mackay, V. Steiner, K. Hofmann, J. L. Bodmer, N. Holler, BAFF signaling to eitheraTorBcell subset. However, the inhi- C. Ambrose, P. Lawton, S. Bixler, H. Acha-Orbea, et al. 1999. BAFF, a novel Ϫ/Ϫ ligand of the tumor necrosis factor (TNF) family, stimulates B-cell growth. bition by BAFF of allergic airway responses in ␮MT ϫ BAFF J. Exp. Med. 189:1747. Tg mice appears to exclude B cell involvement. One important 18. Ng, L. G., A. P. Sutherland, R. Newton, F. Qian, T. G. Cachero, M. L. Scott, J. S. Thompson, J. Wheway, T. Chtanova, J. Groom, et al. 2004. B cell-activating qualification that must be noted is that the enhanced DTH response factor belonging to the TNF family (BAFF)-R is the principal BAFF receptor in BAFF Tg mice was in skin, whereas the suppressed response in facilitating BAFF costimulation of circulating T and B cells. J. Immunol. the allergic model was in mucosal tissues. It is conceivable that 173:807. 19. Litinskiy, M., B. Nardelli, B. M. Hilbert, H. Bing, A. Schaffer, P. Casali, and BAFF effects somehow relate to the nature of cutaneous or mu- A. Cerutti. 2002. DCs induce CD40-independent immunoglobulin class switch- cosal responses. ing through BLyS and APRIL. Nat. Immunol. 3:822. In conclusion, we show that BAFF overexpression in vivo pro- 20. Thompson, J. S., S. A. Bixler, F. Qian, K. Vora, M. L. Scott, T. G. Cachero, C. Hession, P. Schneider, I. D. Sizing, C. Mullen, et al. 2001. BAFF-R, a novel motes the DTH reaction, which is a classical Th1 response, and TNF receptor that specifically interacts with BAFF. Science 293:2108. suppresses Th2-dependent allergic airway responses. The effects 21. Tardivel, A., A. Tinel, S. Lens, Q. G. Steiner, E. Sauberli, A. Wilson, F. Mackay, of BAFF on the T cell system were profound in terms of subset A. G. Rolink, F. Beermann, J. Tschopp, et al. 2004. The anti-apoptotic factor high Bcl-2 can functionally substitute for the B cell survival but not for the marginal ratios and Th1 or Th2 responses. Whether BAFF human sub- zone B cell differentiation activity of BAFF. Eur. J. Immunol. 34:509. jects also show augmented Th1-type responses or suppressed Th2 22. Seshasayee, D., P. Valdez, M. Yan, V. M. Dixit, D. Tumas, and I. S. Grewal. 2003. Loss of TACI causes fatal lymphoproliferation and autoimmunity, estab- responses will be an interesting topic for future studies. lishing TACI as an inhibitory BLyS receptor. 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B Cell-Activating Factor Belonging to the TNF Family (BAFF)-R Is the Principal BAFF Receptor Facilitating BAFF Costimulation of Circulating T and B Cells1

Lai Guan Ng,2* Andrew P. R. Sutherland,2*† Rebecca Newton,* Fang Qian,§ Teresa G. Cachero,§ Martin L. Scott,§ Jeffrey S. Thompson,§ Julie Wheway,* Tatyana Chtanova,*† Joanna Groom,* Ian J. Sutton,* Cynthia Xin,*† Stuart G. Tangye,‡ Susan L. Kalled,§ Fabienne Mackay,*† and Charles R. Mackay3*†

BAFF (B cell-activating factor belonging to the TNF family) is a cell survival and maturation factor for B cells, and overproduction of BAFF is associated with systemic autoimmune disease. BAFF binds to three receptors, BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B cell maturation Ag (BCMA). Using speciﬁc mAbs, BAFF-R was found to be the predominant BAFF receptor expressed on peripheral B cells, in both humans and mice, and antagonist mAbs to BAFF-R blocked BAFF-mediated costimulation of anti-␮ responses. The other BAFF receptors showed a much more restricted expression pattern, suggestive of specialized roles. BCMA was expressed by germinal center B cells, while TACI was expressed predominantly by splenic transitional type 2 and marginal zone B cells, as well as activated B cells, but was notably absent from germinal center B cells. BAFF was also an effective costimulator for T cells, and this costimulation occurs entirely through BAFF-R. BAFF-R, but not TACI or BCMA, was expressed on activated/memory subsets of T cells, and T cells from BAFF-R mutant A/WySnJ mice failed to respond to BAFF costimulation. Thus, BAFF-R is important not only for splenic B cell maturation, but is the major mediator of BAFF-dependent costimulatory responses in peripheral B and T cells. The Journal of Immunology, 2004, 173: 807–817.

he B cell-activating factor from the TNF family (BAFF)4 and salivary glands of Sjo¬gren’s syndrome patients (7). These el- (also known as BLyS, TALL-1, zTNF-4, THANK, evated levels of BAFF in blood and tissues in human autoimmune T TNFSF 13b), is emerging as an important regulator of B patients likely affect peripheral B cell survival as well as B cell cell and T cell responses. BAFF was originally identified as a maturation and activation in the spleen, similar to effects of BAFF factor responsible for B cell survival and maturation (reviewed in in BAFF transgenic mice. Although high levels of BAFF have Refs. 1Ð3). BAFF was subsequently associated with autoimmune been associated mostly with autoimmune diseases, BAFF is likely disease, because transgenic mice overproducing BAFF produce important for normal immune responses to microbial challenge, by autoantibodies and develop diseases akin to human systemic lupus priming or enhancing T and B cell activity to facilitate clearance erythematosus and Sjo¬gren’s syndrome (4Ð8). In humans, high (11). The baff gene, located in humans on chromosome 13, has levels of BAFF are detectable in the blood of a proportion of been identified as a likely determinant for susceptibility to Ascaris patients with autoimmune rheumatic diseases, particularly sys- infection (12). temic lupus erythematosus and Sjo¬gren’s syndrome (7, 9, 10), and BAFF binds to several receptors. These include transmembrane BAFF is present at high levels in rheumatoid synovial fluid (9), activator and calcium modulator and cyclophilin ligand interactor (TACI), BAFF-R (BR3), and B cell maturation Ag (BCMA) (13Ð 17). Another TNF family member termed a proliferation-inducing *Arthritis and Asthma Research Program, The Garvan Institute of Medical Research, Sydney, †Cooperative Research Center for Asthma, Sydney, ‡Centenary Institute of ligand (APRIL) also binds to TACI and BCMA (14, 18). BAFF-R Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia; and appears to be particularly important for the regulation of B cell ¤Department of Research, Biogen Idec Inc., Cambridge, MA 02142 survival and maturation in the spleen, because A/WySnJ mice ex- Received for publication March 15, 2004. Accepted for publication May 14, 2004. pressing a defective BAFF-R have disrupted B cell maturation, The costs of publication of this article were defrayed in part by the payment of page similar to that seen in BAFF-deficient mice (17, 19). The BCMA charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. receptor appears not to play a role in B cell maturation but is 1 This work was supported by the Wellcome Trust, the Cooperative Research Center involved in plasma cell survival (20). In TACI-deficient mice, for Asthma, and the National Heath and Medical Research Council of Australia. there are increased B cell numbers, marked splenomegaly, and 2 L.G.N. and A.S. contributed equally to this work. mice develop autoimmune disorders (21). Antigenic challenge in 3 Address correspondence and reprint requests to Dr. Charles R. Mackay, Arthritis these mice results in enhanced Ab production (21). Because an and Inflammation Research Program, The Garvan Institute of Medical Research, 384 agonistic mAb to TACI inhibited B cell proliferation, signaling Victoria Street, Sydney, New South Wales 2010, Australia. E-mail address: [email protected] through TACI may in fact serve to down-regulate B cell activity (21). 4 Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the TNF family; BCMA, B cell maturation Ag; h., human; hIgG, human IgG; GC, ger- BAFF and possibly APRIL also act on T cells. In vitro studies minal center; m., mouse; MZ, marginal zone; RBL, rat basophilic leukemia; TACI, of human T cells showed that BAFF provided a complete costimu- transmembrane activator and calcium modulator and cyclophilin ligand interactor; T1, transitional type 1; T2, transitional type 2; WT, wild type; APRIL, a proliferation- latory signal together with anti-TCR stimulation (22, 23). In mice, inducing ligand. a TACI-Fc fusion protein blocked activation of T cells in vitro, and

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 808 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS inhibited T cell priming in vivo (24). Also, treatment with hIgG (Novartis Pharmaceuticals, East Hanover, NJ), h.BCMA-Fc, or anti- TACI-Fc substantially inhibited inflammation, as well as bone and mouse BAFF-R (m.BAFF-R) mAbs (29). For Ag-specific T cell assays, cartilage destruction, in a mouse model of rheumatoid arthritis splenic T cells from DO11.10 TCR transgenic mice were purified by magnetic separation, and 5 ϫ 104 cells/well were cultured with 1 ϫ 105 cells/ (24). T cells from transgenic mice that overexpressed human well of mitomycin C-inactivated APC, together with OVA peptide in 96- APRIL showed greatly enhanced survival in vitro and in vivo (25). well U-bottom plates. BAFF costimulation of T cells was performed by In autoimmune patients, inappropriate costimulation of T and B adding 4 ␮g/ml soluble BAFF or denatured BAFF to wells. For inhibition cells by BAFF may be an important component of disease patho- assays, cells were cultured with anti-m.BAFF-R mAbs (2 ␮g/ml), h.B- CMA-Fc, or hIgG (2 ␮g/ml). genesis. The receptor for BAFF on T cells is largely unknown, For anti-CD3 T cell proliferation assay, human T cells were activated although one study reported TACI expression on a subset of acti- using plate-bound anti-human CD3 mAb (mAb TR66), and mouse T cells vated T cells using a polyclonal Ab (26). Expression of BCMA and were activated with anti-mouse-CD3 mAb 145-2C11 (BD Pharmingen). BAFF-R is thought to be restricted to B cells (16, 17, 27, 28). Anti-CD3 mAb and BAFF were coated onto plates overnight in PBS at 4¡C either separately or in combination, followed by two PBS washes. A total The critical functions of BAFF for B and T cell biology are ϩ of 2 ϫ 106 PBMC (human) or CD3 (human or mouse) cells/ml were facilitated by the regulated expression of BAFF receptors. Accord- added to tissue culture plates containing immobilized anti-CD3 Ϯ plate- ingly, we have investigated the capacity of BAFF to stimulate B bound or soluble BAFF, and harvested after 72 h. For all mouse and human and T cells, and have used specific antagonistic mAbs as well as proliferation measurements, cultures were pulsed with [3H]thymidine (1 strains of mice with mutant BAFF receptors to ascertain precise ␮Ci/well) 18 h before harvesting and quantified using a beta-scintillation counter. expression patterns and functional roles for the three receptors. Materials and Methods Production and specificity of mAbs to human and mouse BAFF Reagents and flow cytometry receptors Soluble forms of human BAFF and BAFF receptors were supplied by An expression DNA construct containing the human TACI cDNA se- Apotech (Epalinges, Switzerland) and P. Schneider (Institute of Biochem- quence was kindly provided by P. Schneider, and a human BAFF-R ex- istry, BIL Biomedical Research Center, University of Lausanne, Epalinges, pression construct was developed using the p-Tracer vector, as per man- Switzerland). Denatured BAFF controls were prepared by incubation at ufacturer’s protocol (Invitrogen, Mt. Waverley, Australia). Cell surface 95¡C for 2 h. Unconjugated and FITC-, PE-, CyChrome-, PerCP-, PE- receptor-expressing transfectants were made using the rat basophilic leu- Cy7-, allophycocyanin-Cy7- and allophycocyanin-conjugated mAbs to kemia (RBL) mast cell line (human and mouse TACI, mouse BAFF-R), or various cell surface markers were from BD Biosciences (San Diego, CA), mouse B cell lymphoma L1.2 cells (human BAFF-R). Anti-h.TACI mAb ϫ 7 with the exception of CCR7-FITC (R&D Systems, Minneapolis, MN). Im- 1A1 (rat IgG1) was generated by immunizing Wistar rats with 2 10 munofluorescent staining was performed using standard procedures with irradiated TACI-transfected RBL cells, 6 times at 2-wk intervals. Cell fu- appropriate secondary staining reagents (Jackson ImmunoResearch Labo- sion was performed as described (31). Similarly, anti-h.BAFF-R mAb ratories, West Grove, PA), and cells were analyzed using BD FACSCalibur 11C1 (mouse IgG1) was generated using the same procedures except that or LSRII flow cytometers (BD Biosciences). Six-color flow cytometric BAFF-R-L1.2 transfectants were used to immunize C57BL/6 mice. In ad- analysis to assess BAFF-R expression on naive and memory subsets of dition, anti-h.BAFF-R (clone 9-1) and hamster anti-human BCMA (clone CD4ϩ and CD8ϩ cells used anti-human BAFF-R (h.BAFF-R) biotin (mAb C4E2.2) mAbs were used. These were generated by immunizing with re- 9-1) and streptavidin-PE, anti-CCR7-FITC, anti-CD3-PerCP, anti-CD45RO- ceptor-Ig fusion proteins, as detailed previously (28). The rat anti-mouse allophycocyanin, anti-CD4-PE-Cy7, and anti-CD8-allophycocyanin-Cy7. A TACI mAbs, 8F10 (IgG2a) and 5L7 (IgG1), were generated by immuniz- biotinylated mIgG1 (BD Pharmingen, San Diego, CA) was used as an isotype ing Wistar rats with soluble TACI extracellular domain protein, 6 times at control for the BAFF-R Ab. 2-wk intervals. Anti-m.BAFF-R mAbs B2G1 and P1B8 were produced by immunizing hamsters with murine BAFF-R-Fc protein. Rat anti- Animals, lymphocyte preparations, and T and B cell stimulations m.BAFF-R mAbs 7H22-E16 and 3I4 (both IgG1) were produced by immunizing Wistar rats with soluble m.BAFF-R-Fc protein, 5 times at 2-wk All human and mouse experiments were performed with approval of St. intervals. Vincent’s campus human or animal ethics committees. Human PBMCs For immunohistochemical visualization of BAFF-R expression, Ag re- were isolated from human blood by Ficoll gradient centrifugation. Human trieval was performed by immersing 4-␮M-thick paraffin sections of pal- splenocytes or tonsil cells were obtained from resected human spleen or ϩ atine tonsil in an EDTA-based retrieval solution (pH 9.0) and heating for tonsil, and were prepared by gentle teasing with forceps. Human CD3 T 20 min at 95Ð99¡C in a water bath. After cooling, sections were immuno- cells were isolated from PBMC preparations by magnetic separation stained using a DakoCytomation Autostainer (DakoCytomation, Carpinte- (MACS; Miltenyi Biotec, Sydney, Australia). CD3ϩ cells were then iso- Ͼ ria, CA); following 5 min incubation with 3% hydrogen peroxide, sections lated in a magnetic field, to 98% purity. For human B cell stimulation, were incubated sequentially for 30 min with anti-BAFF-R mAb (11C1) and 5 ␮ PBLs were incubated in 96-well plates (10 cells/well in 100 l RPMI Mouse EnVisionϩ HRP (DakoCytomation). Anti-BAFF-R binding was 1640 supplemented with 10% FBS) for 48 h with 75 ng/ml soluble BAFF ϩ ␮ Ј ␮ visualized using Liquid diaminobenzidine (DakoCytomation). in the presence of 5 g/ml goat F(ab )2 anti-human -chain Ab (Jackson ImmunoResearch Laboratories), and with different concentrations of anti- h.BAFF-R mAb, h.BAFF-R-Fc, or human IgG (hIgG) control (29). GeneChip microarray analysis (Affymetrix, Millenium Science, Homozygous TACIϪ/Ϫ mice were generated as previously described for Victoria, Australia), and real-time LightCycler PCR (Roche targeting the BAFF and BCMA loci (30). Briefly, a tailless human CD2 Molecular Biochemicals, Sydney, Australia) reporter cDNA was inserted in frame at the ATG that normally initiates TACI translation and just upstream of a phosphoglycerate kinase 1-pro- Total RNA was isolated from harvested cells using Qiagen RNeasy Total moted neomycin resistance cassette. The resulting deletion of the TACI RNA Isolation kit (Valencia, CA). Total RNA (2 ␮g) was then used for genomic locus spanned 6.12 kb of DNA immediately downstream of the cDNA synthesis, with 4.5 U AMV reverse transcriptase and MgCl2-con- initiating ATG. This mutation introduced premature stop codons in the taining buffer (Promega, Madison, WI), 20 nmol dNTPs (Promega), and three possible reading frames downstream of the CD2 reporter and elim- 0.02 nmol oligo-p(dT)15 primer (Roche Molecular Biochemicals) and in- inated all the nucleotides encoding aa 1Ð89 of normal mouse TACI (Gen- cubated at 42¡C for 90 min. cDNA was used for LightCycler PCR (Roche Bank accession number NM_021349). The A/WySnJ mouse strain (17) Molecular Biochemicals) with the LightCycler FastStart Master SYBR was obtained from The Jackson Laboratory (Bar Harbor, ME). Mouse Green I kit (Roche Molecular Biochemicals), using 3 mM MgCl2 and 0.5 CD3ϩ cells were isolated from spleen via magnetic separation using Pan T ␮M individual primers with the following specific protocol: 10 min 95¡C cell isolation kit (MACS; Miltenyi Biotec). For mouse B cell proliferation activation of FastStart TaqDNA polymerase (Roche Molecular Biochemi- assays, B cells were isolated from spleens of 2-mo old C57BL/6 mice using cals), and 40 cycles of 95¡C for 15 s, 63¡C for 5 s, and 72¡C for 21 s. The B cell recovery columns (Accurate Chemical & Scientific, Westbury, NY). primers used were a combination of original sequences designed using Prim- Mouse B cells were incubated in 96-well plates (105 cells/well in 50 ␮l er3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Light- RPMI 1640 supplemented with 10% FBS) for 72 h with 75 ng/ml BAFF Cycler analyses used the crossing point data for each gene during the loga- (Biogen Idec, Cambridge, MA), in the presence of 2 ␮g/ml goat anti-mouse rithmic amplification program. The crossing point for each gene in each ␮-chain Ab (Jackson ImmunoResearch Laboratories), and with the indi- sample was normalized to the crossing point of GAPDH. Respective genes cated concentrations of previously described reagents, including polyclonal were then compared between two samples and expressed as a fold change. The Journal of Immunology 809

For GeneChip assessment of transcript expression, cRNA was prepare- Results dusing the methods as described (32). Hybridization to the Affymetrix Production of mAbs to human and mouse BAFF receptors U133A and B GeneChips and subsequent scanning and analysis was con- ducted exactly according to Affymetrix protocols. GeneChip analyses in- mAbs were raised to human TACI, BAFF-R, and BCMA, and to cluded human Th1 and Th2, purified eosinophils, cultured mast cells, IgE- activated mast cells, purified neutrophils, and splenic memory B cells. A full mouse TACI and BAFF-R, to determine the functional signifi- description of the preparation of these cells, and access to the full GeneChip cance of these three receptors for B and T cells, and to determine results, is available at http://www.garvan.unsw.edu.au/public/microarrays. their precise expression pattern. Various immunization strategies

FIGURE 1. Production and specificity of mAbs to human and mouse BAFF receptors. A, Reactivity of various mAbs against transfectants of human TACI and BAFF-R. The different shaded profiles represent different BAFF-R transfectants; lighter profiles are human BAFF-R transfectants (in mouse L1.2 cells, a B lymphoma line), and darker profiles are human TACI transfectants (in rat RBL cells). Negative control staining used mAbs of irrelevant specificity, and anti-h.TACI or anti-h.BAFF-R staining on irrelevant transfectants resembled this staining (data not shown). B, Mouse TACI and BAFF-R transfectants (both in rat RBL cells); lighter profiles are m.BAFF-R transfectants and darker profiles are m.TACI transfectants. In all instances, Ab staining of nontransfected cells resembled negative control staining. C, ELISA of anti-human BAFF-R, TACI, and BCMA mAbs against human TACI-Fc, BAFF-R-Fc, or BCMA-Fc fusion proteins. The mAbs used were anti-h.TACI mAb 1A1, anti-h.BCMA mAb C4E2.2, and anti-h.BAFF-R mAb 9-1. Values are shown as relative absorbance after subtraction of background values. 810 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS were used, including receptor fusion proteins and transfectants ex- BAFF-R blocked proliferation to the same level as did the positive pressing high levels of these receptors. All of the ensuing mAb control BCMA-Fc (Fig. 2B), indicating that in mice, BAFF-R is raised against either human or mouse BAFF-R or TACI stained the principal costimulatory BAFF receptor for resting mature B their respective receptor transfectants (Fig. 1, A and B) or reacted cells. specifically against receptor-Ig fusion proteins by ELISA (Fig. TACI expression was detected on T cells using a polyclonal Ab 1C), without cross-reacting with the other BAFF receptors. The (26), however, a kinetic analysis of TACI expression on T cells specificity of a mAb raised against human BCMA-Fc, C4E2.2, has failed to detect TACI expression on 48-h-activated T cells (Fig. been reported previously (28). Anti-human BAFF-R mAb 9-1 2C), or on resting T cells or T cells at other activation time points blocked BAFF binding to BAFF-R expressing cells (data not (data not shown). However, TACI was expressed on a subset of shown). Similarly, anti-murine BAFF-R mAb B2G1 was blocking, ϩ peripheral B220 B cells, and, as expected, mAb to TACI was while mAb P1B8 was nonblocking (see below). Ϫ Ϫ unreactive with all B cell subsets from TACI / mice. The most BAFF-R is the predominant BAFF receptor expressed on mouse notable expression of TACI was by maturing subsets of splenic B T and B cells, whereas TACI marks maturing splenic B cell cells. Transitional type 1 (T1), transitional type 2 (T2), and mar- subsets ginal zone (MZ) B cells were distinguished using multicolor flow mAb to mouse BAFF-R stained the vast majority of mouse splenic cytometry using IgM, CD21, and CD23 (see Ref. 33). In wild-type B cells (CD4-negative cells, Fig. 2A) and lymph node B cells (data (WT) mice, T2 and MZ B cells expressed very high levels of TACI not shown). A few resting T cells were clearly BAFF-Rϩ (Fig. 2A) (Ͼ95% positive, Fig. 2D), whereas on other splenic B cell subsets and PCR analysis of mouse T cells and T cell lines revealed ex- such as follicular B cells and T1 B cells, TACI expression was low pression of BAFF-R and absence of TACI and BCMA (data not or absent, respectively. However, TACI expression by T2 or MZ shown). In experiments using BAFF costimulation of anti- B cells was not essential for B cell maturation in the spleen, be- ␮-mediated B cell proliferation, an antagonistic mAb to mouse cause all B cell subsets developed in TACIϪ/Ϫ mice, although

FIGURE 2. BAFF-R is the principal BAFF receptor for mouse B cells, and is expressed on a subset of T cells while TACI is expressed on T2 and MZ B cells. A, Expression of BAFF-R (mAb P1B8) by the vast majority of splenocytes (comprising mostly B cells), and also by a small subset of CD4ϩ T cells. B, Inhibition of BAFF costimulatory effects on anti-␮-mediated (2 ␮g/ml) B cell proliferation by anti-m.BAFF-R mAb B2G1. hIgG as well as the nonblocking mAb, P1B8, were used as negative controls, and BCMA-Fc was used as a positive BAFF neutralizing agent. Each point was done in triplicate and the experiment was performed three times with identical results. C, TACI is expressed on a small subset of B cells in the spleen, but is absent from T cells. The left panels shows TACI expression on a subset of B220ϩ splenocytes from WT but not TACIϪ/Ϫ mice. The right panels shows activated splenic CD4ϩ T cells, either from WT or TACIϪ/Ϫ mice, stained with anti-m.TACI mAb 8F10 (y-axis) and with anti-CD69 mAb (x-axis). Activated CD4ϩ T cells were produced following T cell stimulation in vitro with anti-CD3 (4 ␮g/ml, optimal concentration) and anti-CD28 (2 ␮g/ml) for 48 h. D, On murine splenic B cells, TACI is expressed highly by T2 and MZ B cells. Mature B cells, T1, T2, and MZ phenotypes were identified using multicolor flow cytometry by staining for IgM, CD21, and CD23, as described (33). Control mAb staining resembled TACIϪ/Ϫ mouse splenocyte staining (dotted line). The Journal of Immunology 811 these mice displayed splenomegaly and peripheral B cell hyper- least in part, through the up-regulation of the survival factor Bcl-2 plasia, as described for other strains of TACIϪ/Ϫ mice (21, 34, 35). (33, 37). Bound BAFF, but not denatured BAFF, also resulted in When splenic B cells were stimulated with anti-␮ for 24 h, TACI an up-regulation of Bcl-2 in purified mouse T cells suboptimally was strongly up-regulated (36) and the expression profile resem- stimulated for 72 h with anti-CD3 (Fig. 3C). bled that shown for MZ or T2 B cells. To confirm that BAFF-R was facilitating BAFF-mediated effects on T cells, we studied BAFF costimulation of anti- BAFF costimulation of T cells in mice is mediated by BAFF-R CD3-mediated T cell proliferation using two mutant strains of We next assessed BAFF responses by mouse T cells, using an mice, TACI-deficient mice, and A/WySnJ mice with a defective OVA peptide-specific proliferation by DO11.10 TCR transgenic BAFF-R (17, 19). Fig. 3D shows that the costimulatory effect of mouse T cells. Fig. 3A shows that an antagonistic mAb recogniz- BAFF on purified T cells was abolished in A/WySnJ mice, in ing murine BAFF-R (mAb B2G1) inhibited T cell proliferation by contrast to T cells from WT mice of the same genetic background ϳ20%, indicating that endogenous BAFF participates in T cell expressing a functional BAFF-R, which responded to BAFF co- costimulation, for an optimal response (Fig. 3A), as previously stimulation. T cells from A/WySnJ mice were costimulated by shown by others (23). As a control, BCMA-Fc inhibited prolifer- anti-CD28, similar to T cells from control A/J mice (Fig. 3D) ation to the same extent. Furthermore, addition of exogenous sol- indicating that T cells from A/WySnJ mice were competent and uble BAFF also increased T cell proliferation in the presence of responded normally. Therefore, the defect in the ability to respond APC (Fig. 3B). The effects of BAFF on B cells in mice occur, at to BAFF costimulation was due to a lack of functional BAFF-R

FIGURE 3. BAFF costimulates mouse T cells through BAFF-R. A, Inhibition of BAFF costimulation by h.BCMA-Fc and anti-m.BAFF-R blocking mAb B2G1. Purified DO11.10 T cells were stimulated with 0.05 ␮g/ml OVA peptide, together with 2 ␮g/ml anti-m.BAFF-R mAbs (P1B8, nonblocking; B2G1, blocking), or 2 ␮g/ml h.BCMA-Fc. B, Soluble BAFF (BAFF), but not denatured BAFF (DN BAFF), costimulates Ag-specific T cell proliferation at suboptimal concentrations of OVA peptide (0.01 ␮g/ml). BAFF was used at 4 ␮g/ml. C, Up-regulation of Bcl-2 in 72 h anti-CD3-stimulated, purified splenic T cells upon incubation with bound BAFF (B BAFF). Shaded and dotted profiles are similarly treated T cells, stimulated with denatured BAFF or no BAFF. D, T cell costimulation by BAFF is compromised in BAFF-R mutant mice (strain A/WySnJ). T cell costimulation by BAFF of purified T cells from the relevant A/J control background (f), and lack of costimulation of A/WySnJ T cells (Ⅺ). T cells obtained from A/WySnJ mice responded well to anti-CD28 (2 ␮g/ml) costimulation. These T cells were purified from spleens by magnetic sorting, and cultures were activated with 1 ␮g/ml plate-bound anti-CD3, Ϯ an optimal amount of plate-bound BAFF (4 ␮g/ml). E, T cells form TACIϪ/Ϫ mice, stimulated with anti-CD3 Ϯ BAFF (Ⅺ), compared with T cells from control littermates (f) (mixed B6/129 background). 812 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS expression, rather than an intrinsic defect in T cell function. In express low levels of TACI (Fig. 4A); however, TACI is known to addition, others have reported that APC function and T cell pro- repress B cell activation, and TACI is dispensable for BAFF stim- liferation in A/WySnJ mice is normal (38). It is noteworthy that ulation of B cell responses to anti-␮ (21). proliferation of T cells from A/J mice, without addition of exogenous BAFF, was consistently higher than for A/WySnJ mice, sug- B cells with a germinal center (GC) phenotype show altered gesting that activated T cells may be a source of BAFF (see mi- expression of BAFF receptors croarray data below). In contrast, T cells from TACI-deficient The staining of human tonsil cells with mAbs to BAFF-R, BCMA, mice were unaffected in their response to BAFF costimulation and TACI revealed that BAFF-R was also the predominant recep- (Fig. 3E), indicating that BAFF does not costimulate T cells tor expressed on tonsil B cells. Nevertheless, BAFF-R did show a through this receptor. Similar to the previous study (22), we also variation in staining intensity between different B cell subsets (Fig. found that anti-CD3-stimulated T cells responded to BAFF only 5A). B cells with a GC phenotype (CD38ϩ, CD27ϩ, CD39Ϫ, when it was immobilized to plastic, although soluble BAFF was CD24Ϫ, and IgMϪ) expressed lower levels of BAFF-R, and this able to costimulate T cells in the Ag-specific assay in the presence was clearly evident through immunohistochemical staining of B of APCs (Fig. 3B). cell follicles in tonsil (Fig. 5B). In contrast to the blood, where no B cells expressed BCMA, a proportion of tonsil B cells did express BAFF-R is the predominant BAFF receptor expressed on human low levels, and multicolor flow cytometry revealed that these blood B cells BCMAϩ B cells displayed a phenotype consistent with GC B cells The studies described above established BAFF-R as the predom- (Fig. 5A). Strikingly, TACI and BCMA were expressed on differ- inant BAFF receptor for mouse B and T cells. The expression ent subsets of the CD19ϩ B cell population, with the TACIϩ sub- pattern of BAFF-R, BCMA, and TACI was also assessed on subset being CD38Ϫ, CD27Ϫ, CD39ϩ, CD24ϩ, and IgMϩ (i.e., a sets of blood and tonsil B cells in humans. On blood B cells, non-GC phenotype). The distinct difference between the TACIϩ BAFF-R was expressed at a high level on all CD19ϩ B cells, and BCMAϩ subsets was further illustrated by a direct two-color whereas BCMA was absent (Fig. 4A). TACI was expressed, but analysis (Fig. 5C), which showed that the vast majority of only on a proportion of blood B cells, and at a much lower level BCMAϩ B cells were TACIϪ; this was particularly evident when than BAFF-R. We confirmed that BAFF-R was also the principal B cell blasts were gated and analyzed (Fig. 5C). stimulatory receptor for human blood B cells, by inhibition of BAFF costimulation of anti-␮-treated blood B cells using a block- Expression of BAFF-R by human T cells ing mAb to BAFF-R. Fig. 4B shows that anti-BAFF-R mAb 9-1, BAFF has been reported to costimulate T cell responses in humans which interferes with binding of BAFF to BAFF-R, inhibited (22), as well as in mice (see above). However, mAb staining sug- BAFF costimulation of B cells. A proportion of these B cells did gested that TACI and BCMA were absent from human blood T

FIGURE 4. BAFF-R is the predominant BAFF receptor expressed on human blood B cells, and facili- tates the vast majority of BAFF costimulation of anti-␮ proliferation of B cells. A, Two-color expression analysis of BAFF-R, TACI, and BCMA on CD19ϩ B cells from human blood. Cells were stained with CD19 Ab and respective biotinylated BAFF receptor mAbs followed by streptavidin-PE. B, Inhibition of BAFF costimulatory effects on anti- ␮-mediated (5 ␮g/ml) B cell proliferation by anti-h.BAFF-R mAb 9-1 (2 ␮g/ml) in the presence of 75 ng/ml BAFF. Anti-h.BAFF-R mAb (F) vs hIgG (E). h.BAFF-R Fc was used as a positive BAFF-neutralizing agent (Œ) The Journal of Immunology 813

FIGURE 5. B cells regulate BAFF receptors upon differentiation to GC phenotype. A, Three-color FACS analysis of B cells (performed by gating on CD19ϩ B cells from tonsil) showing expression of BAFF-R, BCMA, and TACI in relation to the B cell phenotypic markers CD38, CD39, CD24, CD27, and IgM. BAFF-R expression was down-regulated on B cells with a phenotype of GC B cells (CD38ϩ, CD27ϩ, CD39Ϫ, and IgMϪ), whereas BCMA was expressed on these cells. B, Immunohistochemical staining of human tonsil with anti-h.BAFF-R mAb 11C1, showing intense staining of B cell follicles, and weaker staining of GC (magnification, ϫ200). Anti-h.TACI and anti-h.BCMA mAbs failed to work in immunohistochemistry, presumably because epitopes were not retained by the fixation process. C, Two-color analysis of TACI and BCMA on gated CD19ϩ B cells from tonsil. The left panel shows the entire CD19ϩ B cell gate, and right panel shows CD19ϩ B cell blasts, gated according to high forward and side scatter. The mAbs used were 9-1 (anti-h.BAFF-R), 1A1 (anti-h.TACI), and C4E2.2 (anti-h.BCMA). cells, and that BAFF-R was expressed at very low levels (Fig. 6A). Affymetrix data-mining tool to assess the presence of BCMA and To further define the precise expression pattern of the receptors, TACI transcripts in numerous T cell subsets (BAFF-R was not particularly BAFF-R, a sensitive multicolor flow cytometric anal- represented on the Affymetrix human U133 chips). TACI and ysis was performed using a BD LSRII or FACSCalibur. All human BCMA were absent from all human T cell subsets assessed, in- T cell populations examined, including blood T cells, in vitro anti- cluding effector memory and central memory T cells, Th1 and Th2 CD3-activated T cells (24 and 48 h, data not shown), and tonsillar cells generated in vitro, and specialized subsets such as T follic- T cells were found to be consistently negative for BCMA and ular-homing cells (Fig. 6D). The GeneChip results also showed TACI (Fig. 6A, and data not shown). In contrast, a fraction of T that TACI and BCMA were undetectable in all nonlymphoid leu- cells expressed BAFF-R, especially T cells activated for 72 h with kocyte types, such as mast cells, neutrophils, and eosinophils, and anti-CD3 (Fig. 6A). This led us to examine expression of BAFF-R staining by flow cytometry with the specific mAbs confirmed these on naive, central memory, and effector memory CD4ϩ and CD8ϩ results (data not shown). BCMA and TACI were detectable in cells as defined using the markers CCR7 and CD45RO. Expression certain B cell populations, particularly memory cell subsets (Fig. of BAFF-R was determined for the various populations and is 6D). Interestingly, all of the T cell subsets analyzed did show represented by different shaded profiles, as indicated (Fig. 6B). expression of BAFF. Transcripts of BAFF were also expressed BAFF-R was expressed predominantly by central and effector strongly in mast cells, eosinophils, and particularly neutrophils, memory T cells, and not naive T cells, in keeping with the up- which is consistent with a recent report on production of BAFF by regulation of BAFF-R on activated T cells. neutrophils (39). Transcripts of APRIL were also detected in mast RT-PCR showed that mRNA for BAFF-R, but not TACI or cells, eosinophils, and neutrophils but at much lower levels than BCMA, was demonstrable in several T cell RNA samples, BJAB for BAFF (Fig. 6D); transcripts of APRIL were absent from all T and RAJI cells were used as control (Fig. 6C). We also used the cell subsets. 814 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS

FIGURE 6. BAFF-R but not TACI or BCMA is expressed on human T cells. A, TACI, BAFF-R, and BCMA expression on resting PBMC and 72 h anti-CD3-activated T cells. The x-axis shows CD69 expression. B, Expression of BAFF-R on naive, central memory (Tcm), and effector memory (Tem) CD4ϩ and CD8ϩ cells. PBMC were gated first on CD3ϩ lymphocytes, and then CD4ϩ cells or CD8ϩ T cells, and assessed for CCR7 vs CD45RO to define naive (CCR7ϩROϪ), central memory (Tcm, CCR7ϩROϩ), and effector memory (Tem, CCR7ϪROϩ) T cells (top panels). Expression of BAFF-R (middle panels) was determined for the various populations and are represented by different shaded profiles, as indicated. Isotype control staining was used to determine background staining (bottom panels). C, PCR for BAFF-R, BCMA, and TACI in pure cell populations/lines. GAPDH was used a positive control. D, Data mining was performed on a series of Affymetrix GeneChip experiments, performed on various purified leukocyte populations. Affymetrix U133A and B chips contain probes for BAFF, APRIL, BCMA, and TACI, (but not BAFF-R), and semiquantitative expression values, expressed as a heat map, are shown. Affymetrix algorithms made calls of presence or absence for each gene, absence is indicated by an “A” in each square.

Costimulatory effects of BAFF on human T cell proliferation unlike the mouse experiments described above, anti-h.BAFF-R ␮ Similar to the mouse results and previous study in humans (22), mAb (2Ð30 g/ml used) failed to inhibit T cell proliferation, sug- BAFF increased cell proliferation of suboptimally anti-CD3- gesting that the way BAFF-R signals in B and T cells is different treated human T cells, to a level similar to that observed with and possibly activated by a different interaction between BAFF anti-CD28 stimulation (Fig. 7A). However, we observed that the and BAFF-R. ability of BAFF to costimulate human T cell responses was variable, and was highly dependent on T cell purity. We next used Ig fusion proteins of TACI, BCMA, and BAFF-R to block BAFF Discussion (and APRIL) activity in a suboptimal anti-CD3 T cell activation The expression of BAFF receptors by subsets of B cells and T cells assay (Fig. 7B). All three fusion proteins inhibited T cell prolifer- regulates survival or costimulation at critical stages of maturation ation in response to anti-CD3, demonstrating the importance of or functional responses. Discerning the precise role of the various endogenous BAFF for normal T cell stimulation. Neutralization of BAFF receptors is important to understanding how BAFF and BAFF rather than APRIL appeared to be responsible for the ob- APRIL influence immune responses, and how and why overpro- served effect, because BAFF-R-Fc (BAFF specific) reduced production of BAFF causes autoimmune disease. This study estab- liferation to the same extent as TACI-Fc or BCMA-Fc. Although lishes BAFF-R as the important BAFF costimulatory receptor on it has been reported BCMA-Fc has a lower affinity for BAFF (29), circulating T and B cells, and shows that BCMA and TACI display we found all three fusion proteins showed a similar ability to block restricted expression patterns suggestive of specialized roles. BAFF activity, presumably because these fusion proteins were The role of BAFF-R in peripheral B cell survival and costimu- used at high concentrations (30 ␮g/ml). We did observe a degree lation has been difficult to gauge using BAFF-R mutant mice, be- of person to person variation, as TACI-Fc failed to inhibit T cell cause splenic B cell maturation is halted at the T1 stage (17). As proliferation in one individual of five (data not shown). In addition, BAFF-R was the only known BAFF receptor detectable on most The Journal of Immunology 815

FIGURE 7. BAFF costimulates human T cells. A, Human T cells were puriﬁed from blood by magnetic separation and activated with anti-CD3 (1 ␮g/ml) for 72 h in the presence or absence of BAFF. BAFF was used either as plate-bound (B), soluble (S), or denatured (DN) at 5 ␮g/ml. B, Inhibition of BAFF costimulation by BAFF receptor fusion proteins. PBMCs were activated with anti-CD3 for 72 h, together with fusion proteins of h.BAFF-R-Fc, h.BCMA- Fc, TACI-Fc, or a hIgG control (30 ␮g/ml). C, Anti-h.BAFF-R failed to inhibit T cell proliferation. PBMCs were activated with anti-CD3 in the presence of anti-h.BAFF-R, h.TACI-Fc, and h.IgG (30 ␮g/ml) were used as controls. All cultures were pulsed with [3H]thymidine for 18 h.

mature resting B cells, and anti-BAFF-R mAbs completely inhib- cells (43). The MZ has been suggested as a refuge for autoreactive ited BAFF-mediated costimulation of B cells, we conclude that B cells (44, 45), and increased TACI expression facilitating re- positive BAFF responses up to the stage of CD38ϩ plasmablasts pression of B cell proliferation might help contain autoreactive occur exclusively through this receptor. An assessment of BCMA cells within this population. and TACI deficient mice supports this conclusion. Early studies Although BAFF and its receptors have been associated mostly with BCMA-deficient mice found no unusual phenotype (30, 40, with B cell responses, we showed that BAFF also had profound 41), although recent studies showed impaired survival of long- effects on T cell costimulation, occurring through BAFF-R. How- lived bone marrow plasma cells (20). Our expression analysis also ever, immobilized BAFF rather than soluble BAFF provided these showed that BCMA is most likely relevant for later stages of B cell costimulatory signals to T cells (this report and Ref. 22) suggesting maturation or survival, i.e., for CD38ϩ plasmablasts (28) and GC that BAFF signaling through BAFF-R on T cells requires mem- B cells (this report). BCMA is also up-regulated on mouse plasma brane expression by APC. It is also conceivable that APCs immo- cells (20). Likewise, TACI was expressed only weakly on a small bilize and present soluble BAFF. BAFF-R has been identified as a subset of peripheral B cells. TACI-deficient mice showed in- survival receptor for B cells and it may serve a similar role for T creased B cell numbers, and splenomegaly (Refs. 21, 34, 35, and cells, rather than act as a classic costimulatory receptor. For in- this report). TACI has been proposed to play a role as a negative stance, signals through BAFF-R increase Bcl-2 expression in T regulator for B cells, rather than as an essential survival-related cells (Fig. 3) as they do for B cells (37), which may result in receptor (21). However, TACI may play an important role in some enhanced basal T cell survival, particularly by BAFF-Rϩ T cells aspects of B cell maturation or function within the spleen, because such as effector and central memory subsets. Enhanced T cell sur- T2 and MZ B cells expressed high levels of TACI. MZ B cells are vival would then augment the number of T cells capable of acti- essential for T-independent immune responses (42), and TACI vation. The activity of BAFF as an important component of co- knockout mice have impaired T-independent but normal T-depen- stimulation (or survival factor) for T cell proliferation is intriguing dent responses (34). TACI was clearly dispensable for splenic B because of the very high levels of BAFF in inflammatory lesions cell maturation, as shown by the production of mature B cells in and blood of certain patients with autoimmune diseases (7, 9, 10, TACIϪ/Ϫ mice. Nevertheless, BAFF (or APRIL) signaling through 46), although whether BAFF in such patients affects T cell biology TACI at the T2 or MZ stages may constrain B cell proliferation at is uncertain. Interestingly, the cell types that are expanded in critical stages of tolerance induction. The T1-T2 maturation step is BAFF transgenic mice include mature B cells, splenic T2 and MZ likely an important immune tolerance checkpoint for maturing B B cells, and also effector memory T cells (4, 33). Our in vivo data 816 BAFF-R IS THE PRINCIPAL BAFF RECEPTOR FOR B AND T CELLS has also shown that BAFF enhances the delayed-type hypersensi- tional relevance for T and B cell responses, represents an attractive tivity reaction, which is a classical T-dependent immune response target for intervention in autoimmune diseases. (our unpublished data). Taken together, these results clearly indicate that BAFF is not only an important B cell factor; it is also a Acknowledgments critical factor for T cell responses. The reason why only immobi- We gratefully thank Ian Mackay and Shane Grey for suggestions; lized BAFF costimulated T cells is unresolved; it is possible that Sabine Zimmer, Sue Liu, Mary Sisavanh, Michael Rolph, Kim Good, and immobilization of BAFF on the plastic plate reproduces elements Melinda Frost for Affymetrix expression data; Pascal Schneider for sharing of membrane expression by APCs. A final feature of BAFF-R DNA constructs; Eric Schmied and Carrie Fletcher for animal husbandry; worthy of comment is that BAFF-R binds BAFF but not APRIL, and Jenny Thatcher for assistance in Ab purification. and so the reported effects of APRIL on T cells (25) presumably occur through an unidentified receptor, or through indirect References mechanisms. 1. Mackay, F., P. Schneider, P. Rennert, and J. Browning. 2003. BAFF and APRIL: BAFF receptors were regulated during B cell differentiation to a tutorial on B cell survival. Annu. Rev. Immunol. 21:231. 2. Mackay, F., and C. Ambrose. 2003. The TNF family members BAFF and APRIL: GC cells. The most obvious expression of BCMA was by B cells the growing complexity. Cytokine Growth Factor Rev. 14:311. ϩ in tonsil with a phenotype consistent with GC B cells, i.e., CD38 , 3. Kalled, S. L., C. Ambrose, and Y. M. Hsu. 2003. BAFF: B cell survival factor and CD27ϩ, CD39Ϫ, and IgMϪ (47, 48). 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