T Follicular Regulatory Cell-Derived Neuritin in the Regulation of Antibody Responses

T Follicular Regulatory Cell-derived Neuritin in the Regulation of Antibody Responses

A thesis submitted for the degree of Doctor of Philosophy of The Australian National University

The John Curtin School of Medical Research Department of Immunology and Infectious Disease ANU College of Medicine, Biology & Environment

Ms. Paula González-Figueroa

The work of this thesis has been conducted from March 2014 to October 2018 at the Department of Immunology and Infectious Disease, The John Curtin School of Medical Research (JCSMR), The Australian National University (ANU), Canberra, ACT, Australia. Unless otherwise referenced, the results and analyses in this document represent only my original work performed under the supervision of Professor Carola Vinuesa. This document has not been submitted for qualiﬁcations at any other academic institution.

Paula Gonzalez-Figueroa Prof. Carola Vinuesa

iii

Acknowledgements

Thanks to my supervisor Carola. I have learned so much from you. It has been an amazing experience and adventure. Thanks for your support, especially through tough times. To Lorena, my adoptive sister. Thank you for the laughs, the movies, the support (and the food). Nos vemos en Ecuador. To Pablo and Ilenia, my partners in science. Thanks for sharing the pain, the uncertainties, and the wonders of doing a PhD. To Julia, Sophie and Vicki for sharing your knowledge with me. To the rest of the awesome Humoral Immunity and Autoimmunity lab members I have had the privilege to work with. To David, for your friendship, ﬁlm festivals and apples. To Mick, Harpreet, Anne, and Cathy, for running the most outstanding facility in the JCSMR. A mi familia. Tóbal, I hope you pursue whatever interests you no matter where and how challenging. Mamá y papá, gracias por apoyar mis decisiones siempre, aún cuando me han llevado lejos de Uds. To my best friend, Jon. Thank you for supporting this thesis experimentally and conceptually but, above all, thanks for always being there, early mornings and weekends in the Animal Facility, late nights running FACS, and back home when all is done.

Abbreviations

Ab Antibody AID Activation-induced cytidine deaminase ANA Anti-nuclear antibodies APC Antigen presenting cell BAFF Bŋ-cell activating factor BCR B-cell receptor BDNF Brain-derived neurotrophic factor cDNA Complementary DNA CIA Collagen-induced arthritis CNS Central nervous system Cpg15 Candidate plasticity gene 15 CPM Counts per million CSR Class-switch recombination cTfh circulating T follicular helper cell CXCR5 Chemokine (C-X-C motif) receptor 5 DA Dopamine DAPI 4’,6-diamino-2-phenylindole DC Dendritic cell DNA Deoxyribonucleic acid EBI2 Epstein-Barr Virus Induced Gene 2 ELISA Enzime-linked immunosorbent assay

vii ENA Extractable nuclear antigens FACS Fluorescence-activated cell sorting FDC Follicular dendritic cell FGF Fibroblast growth factor FOXO1 Forkhead Box O1 FOXP3 Forkhead Box P3 GC Germinal centre GCPC Germinal centre-derived plasma cells GFP Green fluorescent protein GPCA Gastric parietal cell autoantibodies GPI Glycosyl-phosphatidyl-inositol Hep-2 Human epitelial type 2 H&E Haematoxylin and eosin ICAM-1 Intercellular Adhesion Molecule 1 ICOS Inducible T-Cell Costimulator Ig Immunoglobulin IF Immunofluorescence IIF Indirect immunofluorescence IFN Interferon INSR Insulin receptor IRF4 Interferon regulatory factor 4 LPS Lipopolysaccharide MACS Magnetic-activated cell sorting gMFI Geometric mean fluorescence intensity MHC-II Major histocompatibility complex class II mRNA Messenger ribonucleic acid NF-κB Nuclear factor κ-lightŋ-chain-enhancer of activated B-cells

viii NGF Nerve growth factor NMDA N-methyl-D-aspartate NRN1 Neuritin NT3 Neurotrophin 3 OVA Ovalbumin PC Plasma cell PCR Polymerase chain reaction PDŋ-1 Programmed cell death-1 PI3K Phosphatidylinositolŋ-4,5-Bisphosphate 3-kinase RA Rheumatoid arthritis RNA Ribonucleic acid RT-qPCR Reverse transcription quantitative realŋ-time PCR SLAM Signalling lymphocytic activation molecule SLE Systemic lupus erythematosus SRBC Sheep red blood cells STAT Signal Transducer And Activator Of Transcription TCR T-cell receptor TD Thymusŋ-dependent Tfh T follicular helper cell Tfr T follicular regulatory cell Th T helper cell TI Thymus-independent Treg T regulatory cell TSA Tissue-speciﬁc antigens WT Wild-type YFP Yellow ﬂuorescent protein

Abstract

T regulatory cells (Tregs) prevent the emergence of autoantibodies and excessive IgE production but the precise mechanisms are unclear. Here we show that BCL6-expressing Tregs, known as T follicular regulatory (Tfr) cells produce the neurotrophic factor neuritin that binds B cells. Mice lacking Tfr cells or neuritin in Tregs developed autoantibodies against systemic and tissue- expressed antigens and accumulated self-reactive early plasma cells in germinal centres (GCs). Upon immunisation, these mice also produced increased serum IgE. Addition of neuritin to activated human GC B cells dampened plasma cell differentiation and IgE production. Neuritin signals lowered ICOSL and augmented BCL6 in GC B cells in vitro, which respectively limit Tfh-cell help and plasma cell differentiation. Administration of neuritin to Tfr-deficient mice prevented the appearance of early plasma cells in GCs. Production of neuritin by Tfr cells emerges as a central mechanism to suppress B cell-driven autoimmunity.

Contents

Declaration iii

Acknowledgementsv

Abbreviations vii

Abstract xi

1 Introduction1

1.1 Preamble...... 1

1.1.1 Lymphatic tissue...... 3

1.2 B cells and Humoral Immune Responses...... 4

1.2.1 Regulation of Antibody Production...... 7

1.2.2 Germinal Centres...... 9

1.2.3 Tfh cells...... 12

1.2.4 Regulation of autoimmunity in the Germinal Centre... 13

1.2.5 ICOS:ICOSL Signalling in B cell Responses...... 15

1.3 T Regulatory cells...... 18

1.3.1 Foxp3...... 19

1.3.2 Treg diversity...... 21

1.3.3 Treg mechanisms of suppression...... 21

1.3.4 Tregs in health and disease...... 22

xiii 1.3.5 Tfr Cells...... 24

1.4 Pathogenic Antibody Production...... 27

1.4.1 Antibody-mediated autoimmunity...... 27

1.5 Neuroimmunology...... 29

1.6 Neuritin...... 31

1.7 Hypotheses and Aims...... 34

2 Materials and methods 37

2.1 Mice and human samples...... 37

2.2 Immunisations...... 38

2.3 Neuritin treatment in vivo ...... 38

2.4 Cell culture...... 39

2.5 Nojima cell coculture...... 39

2.6 Nucleosome extraction...... 39

2.7 Fluorescently labelled neuritin...... 40

2.8 TriCEPS...... 40

2.9 ELISA...... 41

2.9.1 Immunoglobulin ELISA...... 41

2.9.2 Antigen-speciﬁc ELISA...... 42

2.10 qPCR...... 42

2.11 Flow cytometry...... 43

2.11.1 Cytometric bead array...... 43

2.11.2 Mouse samples...... 44

2.11.3 Human samples...... 44

2.12 Immunoﬂuorescence...... 45

2.12.1 Tissue-speciﬁc autoantibody IF...... 46

2.13 IIF-ANA...... 46

2.13.1 Cytospin...... 47

xiv 2.14 Treg suppression assay...... 47

2.15 Western Blot...... 48

2.16 Statistical analysis...... 48

3 Results 49

3.1 T follicular regulatory cells...... 49

3.1.1 Generation of Tfr-deﬁcient mice...... 49

3.1.2 Tfr-deﬁcient mice lack Tfr cells but Tregs are normal in number and function...... 52

3.1.3 Young Tfr-deﬁcient mice have spontaneous GC accumulation that disappears with age...... 56

3.1.4 Tfr-deﬁcient mice have intact Tfh and GC responses after immunisation...... 59

3.1.5 Tfr-deﬁcient mice have elevated immunoglobulins.... 63

3.1.6 GCs in Tfr-deﬁcient mice are enriched in non-antigen- speciﬁc B cells...... 65

3.1.7 Tfr cells repress GC B cell-derived plasma cell formation 66

3.1.8 Tfr cells repress the emergence of autoantibodies to nuclear proteins...... 67

3.1.9 Tfr cells repress the emergence of tissue-speciﬁc antibodies 71

3.1.10 Tfr cells repress the increase in IgE titres upon immunization...... 74

3.2 Neuritin: a key eﬀector of B cell regulation by Tfr cells..... 77

3.2.1 Tfr cells express neuritin mRNA...... 77

3.2.2 Neuritin: a CNS neuropeptide...... 80

3.2.3 Neuritin protein is expressed by Tfr cells...... 81

3.2.4 Neuritin-expressing cells localise inside or in the vicinity of GCs...... 83

xv 3.2.5 Neuritin targets GC B cells...... 85 3.2.6 Neuritin represses plasma cell formation and increases BCL6...... 92 3.2.7 Neuritin represses IgE production in vitro ...... 96 3.2.8 Neuritin represses ICOSL expression...... 98 3.2.9 Neuritin limits Tfr and plasma cells in vivo ...... 100 3.2.10 Systemic cytokines in Tfr- and neuritin-deficient mice.. 103 3.2.11 Neuritin represses nucleosome-binding B cells and tissue- specific autoantibodies...... 110 3.2.12 Self-reactive GC B cells in neuritin-deficient mice display an "early-plasma cell" phenotype...... 114 3.2.13 Neuritin treatment suppresses abnormal pre-plasma cells in Tfr deficient mice...... 116

4 Discussion 121 4.1 Summary...... 121 4.2 Tfr cells and autoimmunity...... 121 4.3 Tfr-mediated IgE regulation...... 123 4.4 Tfr-derived neuritin in autoimmunity...... 125 4.5 Limitations and Future directions...... 132 4.5.1 Autoimmune mouse models...... 132 4.5.2 Neuritin overexpression...... 133 4.5.3 Neuritin-GFP mouse...... 133 4.5.4 Eﬀect of neuritin in Tfh:B cell interactions...... 133 4.5.5 Neuritin receptor in B cells...... 134 4.5.6 Disentanglement of the functions of Treg- from Tfr- derived neuritin...... 134

xvi List of Tables

1.1 Sequence similarity of neuritin protein across diﬀerent species.. 33

2.1 Primers list for qPCR analysis...... 43

xvii

List of Figures

3.1 Erythrocyte phenotype...... 51

3.2 Splenic and thymic Tregs...... 53

3.3 Functional Tregs in Tfr- mice...... 55

3.4 Spontaneous accumulation of GC B cells in Tfr- mice...... 57

3.5 Basal Tfh compartment in Tfr- mice...... 58

3.6 GC lymphocytes in Tfr-suﬃcient and Tfr-deﬁcient mice..... 60

3.7 Eﬀect of Tfr deﬁciency at early and late time points...... 62

3.8 Basal circulating Immunoglobulins...... 64

3.9 Antigen-speciﬁcity of GC B cells...... 66

3.10 Abnormal early-plasma cell phenotype in GC B cells from unimmunised Tfr-deﬁcient mice...... 67

3.11 Tfr- mice develop spontaneous autoantibodies against nuclear proteins...... 70

3.12 Tfr- mice develop spontaneous tissue-speciﬁc autoantibodies.. 73

3.13 Increased immunisation-induced IgE in Tfr-deﬁcient mice.... 75

3.14 Tfr-deﬁcient mice develop anaphylactic symptoms in response to a high dose re-challenge...... 77

3.15 Nrn1 expression in Tfr cells...... 79

3.16 Neuritin protein expression in lymphocytes...... 82

3.17 Neuritin-expressing cells localise nearby and within GCs.... 84

3.18 Mouse GC B cells bind neuritin in vitro ...... 86

xix 3.19 Human GC B cells bind neuritin in vitro ...... 87 3.20 Human B cells bind neuritin (TriCEPS assay)...... 89 3.21 GC B cells contain neuritin protein in the absence of Nrn1 mRNA 91 3.22 Neuritin suppresses plasma cell differentiation from GC B cells in vitro ...... 93 3.23 Neuritin induces upregulation of Bcl6 in cultured mouse B cells 95 3.24 Neuritin represses IgE production in vitro ...... 97 3.25 Human GC B cells bind neuritin in vitro ...... 99 3.26 Neuritin-deficient mice have normal Treg proportion and suppressive capacity...... 101 3.27 Antigen-specificity of GC B cells in neuritin-deficient mice... 102 3.28 Systemic cytokines in Tfr- and neuritin-deficient mice...... 104 3.29 Follicular lymphocyte populations in neuritin-deficient mice... 106 3.30 Neuritin-deficient mice baseline immunoglobulins...... 108 3.31 Neuritin-deficient mice immunisation-induced immunoglobulins. 109 3.32 Neuritin-deficient mice develop spontaneous autoantibodies... 111 3.33 Neuritin-deficient mice develop spontaneous tissue-specific autoantibodies...... 113 3.34 Self-reactive GC B cells in neuritin-deficient mice display a “early-plasma cell” phenotype...... 115 3.35 Neuritin treatment in vivo suppresses early plasma cells from GC B cells...... 117 3.36 Effect of neuritin treatment in autoantibody production..... 119

xx Chapter 1

Introduction

1.1 Preamble

We share and compete for space and resources with many different species and classes of organisms and have been constantly adapting to our environment and neighbours in order to thrive and survive. We have established intimate symbiotic associations with some organisms, while others can be potentially threatening. Physical, chemical and biological tools have evolved to protect us from pathogens and harmful substances and, at the same time, recognise favour- able or innocuous ones. This protection apparatus, the immune system, rests on three pillars that are not always in alignment: i) distinguishing self from foreign, ii) defining when to respond to self or foreign substances and when to tolerate them, and iii) being able to mount an immune response to defend the host when needed. These features hardly adhere to a strict dichotomy and will continue to mature as the individual interacts with different antigens in different contexts.

Higher vertebrates, specially mammals, have evolved sophisticated mechanisms that involve specialised cells, receptors, soluble factors and enzymatic machineries capable of editing and thus diversifying their receptor repertoires

1 CHAPTER 1. INTRODUCTION

and effector functions through highly controlled recombination events, i.e., somatic recombination, somatic hypermutation (SHM) and class switch recombination (CSR), to recognise a virtually infinite array of antigens. These refinements gave rise to the adaptive immune system, where immune responses are exquisitely tailored to each antigen and have the ability to remember previous assaults through immunological memory. Organisms where no longer limited to protection against old enemies, but could now anticipate for future ones.

Adaptive immunity is characterised by the recognition of molecules by antigen-receptors on the surface of B and T lymphocytes. B cells can recognise antigens in their native conformation and are responsible for antibody production by secreting their membrane B cell antigen-receptor (BCR), as opposed to the T cell receptor (TCR) that recognises intracellularly processed antigens coupled to major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs). T cells can help activate B cells or have cytotoxic properties.

The development of a system recognising an antigen as foreigner and dele- terious needed to be met with a canon or check-points capable to determine what is self or innocuous in order to ensure that such antigen can be tolerated. As in many other physiological processes, maintenance of tolerance against self is a multi-layered task that actively seeks to achieve homeostasis. Specialised adaptive immune cells known as T regulatory cells (Tregs), have emerged as one of the main peripheral tolerance gate-keepers. The absence or malfunction of these cells or key proteins can lead to various autoimmune disorders.

Human and animal genetic studies have shed light into some of the mech-

2 CHAPTER 1. INTRODUCTION anisms involved in the pathogenesis of diﬀerent cell- and antibody-driven autoimmune diseases. Still, the molecular and cellular players controlling pathogenic antibody production are not completely understood.

1.1.1 Lymphatic tissue

Most hematopoietic stem cells that give rise to immune cells develop in the bone marrow. Immune cell progenitors continue their diﬀerentiation and selection either in the bone marrow (B cells) or in the thymus (T cells) which are considered central or primary lymphoid organs. Immature cells then migrate from primary to secondary lymphoid organs such as lymph nodes, spleen, and gut-associated lymphoid tissue (GALT), that help centralise immune responses facilitating the encounter of circulating antigens and leukocytes.

Similar structure and cell distribution are observed in secondary lymphoid organs. The spleen in particular combines innate and adaptive cells and is anatomically divided into red pulp and white pulp. In the red pulp, blood is ﬁltered in a specialised vein system and macrophages, that reside in a privilege position, phagocyte and recycle iron from ageing erythrocytes. Macrophages can also phagocyte blood-borne antigens recognised by their pattern recognition receptors (PRRs). The white pulp, on the other hand, resembles the structure of lymph nodes. Is composed of lymphoid tissue that has no contact with the bloodstream and is enriched in T cells within the periarteriolar lymphoid sheath (PALS) also known as T cell zone, and B cells that localise in the B cell follicles due to their high expression of the CXC chemokine receptor 5 (CXCR5). The ligand for CXCR5 is CXCL13, which is expressed by follicular stromal cells(Cyster 2005). These lymphoid compartments are surrounded by the marginal zone (MZ), where innate-like MZ B cells and MZ macrophages

3 CHAPTER 1. INTRODUCTION

reside(Mebius and Kraal 2005). This compartmentalisation is achieved by adhesion molecules and chemokine gradients that attract speciﬁc lymphocytes to their corresponding microdomain(Okada and Cyster 2006).

Activated dendritic cells (DCs) can migrate to lymphatic tissues and enter the T cell zone to activate T cells. Primed T cells can migrate closer to the B cell follicles to provide cognate help to antigen-activated B cells, initiating an adaptive immune response.

1.2 B cells and Humoral Immune Responses

B cells and their ability to produce antigen-specific antibodies are essential for host protection against harmful microorganisms and substances. Individu- als that lack B cells or have serious deficiencies in antibody production are prone to life threatening infections. The mechanism by which B cells recognise antigens puzzled immunologist for decades. It soon became clear that there were not enough genes in the genome to account for the diversity of antibody specificities observed. Immunologists began to theorise about possible explan- ations, deliberately disregarding the limitations of the genome, for the time being. Over the first half of the 20th century, the predominant belief was the “instructionist theory”, whereby and antigen instructs a cell and acts as a template to reshape the immunoglobulin molecule to recognise the insulting antigen, meaning that a single cell could potentially produce an array of different antibodies(Breinl and Haurowitz 1930; Pauling 1940; Burnet and Fenner 1953).

The contender theory of “selectionism” was sparked by the speculations of Jerne’s natural selection theory and reﬁned by Burnet’s clonal-selection theory, and was experimentally supported in the following years(Jerne 1955;

4 CHAPTER 1. INTRODUCTION

Burnet 1957). According to selectionism, a cell bearing receptors with a unique speciﬁcity is selected for its intrinsic ability to recognise and bind to a given antigen. Burnet also introduced the concepts of “clonal commitment” and “clonal expansion” and, with surprising accuracy, he predicted that a randomisation of the genetic code of immunoglobulin genes was needed to generate diversity and it would inevitably produce self-reactive speciﬁcities that he suggested had to be eliminated by “in vivo absorption” to properly develop immune tolerance.

Today we have a better understanding of the central and peripheral mechanisms of tolerance that function to prevent self-reactive B cells from progressing during development in the bone marrow or to hinder their responsiveness afterwards. It is estimated that around 50-75% of immature B cells are polyreactive in the bone marrow, i.e., they recognise nuclear components or protein self-antigens such as insulin. This number is reduced to around 20-40% in immature and transitional B cells and further reduced to 20% in mature cells in the periphery by central mechanisms of counter-selection such as clonal deletion or secondary receptor editing by rearrangement of immunoglobulin light chain genes(Nemazee 2006). Peripheral mechanisms involve the antigen receptor de- sensitisation, anergy, and the induction of tolerance to antigens that co-engage inhibitory receptors, such as the sialic acid-binding immunoglobulin-like lectin (Siglec) and CD22(Duong et al. 2010; Sabouri et al. 2014; Macauley et al. 2013).

B cells are key to maintain antibody-driven immunity against the myriad pathogens that we are exposed to. B cells arise from hematopoietic stem cell progenitors in the bone marrow, where stromal cells promote B cell diﬀeren- tiation by providing critical growth factors and cytokines. Development of B cells can be divided in early stages that are antigen-independent, i.e., pro-B, pre-B, transitional B, and mature B1 and B2 cells; and late antigen-dependent

5 CHAPTER 1. INTRODUCTION

diﬀerentiation, that occurs in the periphery upon activation.

Immature B cells with functional BCRs that do not strongly react with self-antigens are positively selected and migrate from the bone marrow to secondary lymphoid organs as transitional B cells that will further diﬀerentiate into mature innate-like marginal zone (MZ) B cells or follicular B cells, the latter being the most abundant B cell compartment that strategically localises in B cell follicles next to the T cell zone.

Immune responses can be classified as T cell-dependent (TD) or T cell- independent (TI), depending on the nature of the immunising antigen. In the context of a TI immune response, MZ or B1 cells combine BCR with toll-like receptor (TLR) signals to rapidly differentiate into plasma cells and produce a wave of mostly germline low-affinity antibodies in response to mainly blood-borne antigens(Vinuesa and Chang 2013). Depending on the type of activation that the B cell undergoes, TI immune responses are further divided into type I (TI-I) that is BCR-independent, and type II (TI-II) or BCR-dependent responses. A common TI-I response is the activation of TLR4 by bacterial lipo- polysaccharides (LPS). In TI-II responses the BCR is crosslinked by antigens such as polysaccharides, glycolipids, and self-antigens derived from apoptotic cells(Kearney 2005). The second signals in TI-II responses are given by cells different than T cells, such as NKT cells, DCs or neutrophils(Cerutti et al. 2012).

On the other hand, in T cell-dependent responses, the BCR of follicular B cells recognise protein antigens on the surface of follicular dendritic cells (FDCs). The antigen is then internalised, processed in endosomal compartments and coupled to MHC-II molecules that are presented in the cell surface and can be

6 CHAPTER 1. INTRODUCTION recognised by cognate antigen-experienced CD4+ T cells. T cells give second signals or costimulatory signals to B cells in the form of CD40L, CD28 and ICOS, and third signals such as cytokines IL-4 and IL-21. These signals allow B cells to proliferate, class switch, enter the germinal centre (GC) reaction, acquire somatic mutations in their Ig genes, and diﬀerentiate into high-aﬃnity long-lived antibody-secreting plasma cells or memory B cells, ensuring long- term protection to the host.

1.2.1 Regulation of Antibody Production

Antibodies are members of the immunoglobulin superfamily (IgSF) of proteins. These divalent molecules are composed of two pairs of light and heavy chains, each with a constant and a variable region that determines its isotype or effector function, i.e., IgM, IgD, IgG, IgA and IgE, and its idiotype or antigen-specificity, respectively. Antibody specificity varies from one BCR to another in the sequence of amino acids found in their variable regions called complementarity-determining regions (CDRs).

The diversity in the set of germline speciﬁcities that an individual bears, or its repertoire, is produced by somatic recombination of variable (V), diversity (D) and joining (J) segments in the genome (V, D and J segments in heavy chain genes, and V and J segments in light chain genes) by recombinases in the RAG complex. Pairing of heavy and light chains is a stochastic process, adding more diversity to the repertoire. Because DNA rearrangements occur in a random fashion, probability dictates that self-reactive lymphocytes can and will be generated(Hozumi and Tonegawa 1976; Brack et al. 1978; Davidson et al. 1987). Appropriate mechanisms to suppress autoreactive cells

7 CHAPTER 1. INTRODUCTION

are required to obviate diseases arising from the immune system generating pathogenic antibodies and mounting responses against tissues from its own host.

Antibody production is the result of a complex series of events that involve the interaction of different cell types as well as endogenous and exogenous stimuli. Initially, DCs orchestrate immune responses by presenting antigens to T cells and polarising their differentiation to distinct T cell subsets (e.g., Th1, Th2, Th17) depending on the cytokine milieu and costimulatory signals. Each T cell subset is characterised by a master transcription factor and cytokine profile tailored to its specific effector function. After priming, T cells migrate close to the B cell follicle, to a region designated as the T:B border within secondary lymphoid organs. On the other hand, B cell receptors (BCR) on B cells recognise antigens in circulation or on the surface of follicular DCs (FDCs) localised in the B cell follicle(Heesters et al. 2014). Follicles are B cell niches that, in the absence of an active immune response, are composed mostly of IgD+/IgM+ naive B cells.

If during the course of a primary immune response, an antigen-activated naive B cell is deemed competent and safe, it can differentiate through: i) the extrafollicular (EF) pathway(Lee et al. 2011), ii) the germinal centre (GC) pathway(MacLennan 1994), or a GC-independent pathway leading to low- affinity memory B cells(Takemori et al. 2014). Through the EF pathway, naive B cells can rapidly differentiate to become short-lived plasma cells that produce low-affinity antibodies as a first line of defence. Through the GC pathway, naive B cells first differentiate to GC B cells and accumulate somatic mutations in their immunoglobulin light and heavy chain variable regions aiming to increase their affinity for the antigen to become high-affinity long-lived plasma cells or GC-derived memory B cells(Ho et al. 1986; Jacob et al. 1991; Inamine et al.

8 CHAPTER 1. INTRODUCTION

2005). It is still unclear what is the precise mechanism that determines the fate of a given clone. Recent studies have shown that a single naive B cell can potentially give rise to all subsets, and that it is the ability to avoid apoptosis and the aﬃnity of the individual B cell what will determine its fate(Taylor et al. 2015). Other theories propose a stochastic model where B cells diﬀerentiate according to the number of cell divisions they have undergone or depending on signals derived from Tfh cells(Hasbold et al. 2004; Tarlinton 2012).

1.2.2 Germinal Centres

Antigen-experienced B cells upregulate chemokine receptor 7 (CCR7) that responds to gradients of the chemokine ligand 21 (CCL21) produced in the T cell zone, allowing them to migrate to the T:B border. This initial B cell activation induces the upregulation of the activation-induced cytidine deaminase (AID) which, in association with other enzymes (APE1, APE2, UNG), is central for immunoglobulin (Ig)-gene somatic hypermutation (SHM) and class switch recombination (CSR)(Pavri and Nussenzweig 2011). The rate of SHM in GCs has been calculated to be increased 106 fold compared to the normal somatic mutation rate(Berek and Milstein 1987). Activation at the T:B border also induces the formation of extrafollicular foci or a GC reaction.

Germinal centres (GCs) are transient microenvironments that form in follicles within secondary lymphoid organs and are characterised by antigen- speciﬁc B cell clonal expansion in the context of T cell-dependent (TD) immune responses(MacLennan 1994; Chan and Brink 2012). Depending on the nature of the antigen, GCs begin to form a few days after exposure and continue to expand in the following days to form a mature GC that pushes non-responder

9 CHAPTER 1. INTRODUCTION

naive B cells to the periphery to form the B cell mantle.(Victora and Nussenz- weig 2012).

GC B cells have an activated phenotype that differentiates them from naive B cells in the follicle. They downregulate surface IgD, upregulate the ligand of GL-7 antibody, n-glycolylneuraminic acid, bind to peanut agglutinin (PNA), increase their size, and develop uropods. Some phenotypic changes differ in human and mice, such as the expression of CD38 that is increased in human GC B cells and decreased in mice. Additionally, upregulation of S1P2 and downregulation of Ebi2 maintain GC B cells confined in the centre of B cell follicles. Finally, GC B cells are prone to apoptosis due to, in part, their high expression of Fas receptor and B cell lymphoma 6 (Bcl6)-mediated downregulation of anti-apoptotic Bcl2(Saito et al. 2009). At the same time, GC B cells are highly resistant to DNA-damage cause by SHM, a characteristic that is also mediated by Bcl6(Phan and Dalla-Favera 2004; Ranuncolo et al. 2007).

The transcriptional repressor Bcl6 controls GC B cell program(Kitano et al. 2011). Mice lacking Bcl6 are unable to form GCs and produce antibodies of lower quality(Dent et al. 1997).

GCs are highly polarised structures. Histological studies using cat lymph nodes and light microscopy conducted almost 90 years ago, clearly showed that GCs were compartmentalised into dark and light regions(Röhlich 1930). The Dark zone (DZ) was shown to be packed with proliferating B cells closer to the T cell zone, while the light zone (LZ), closer to the MZ in the spleen, is more diverse in their cell composition. In addition to GC B cells, the LZ contains FDCs that act as antigen deposits(Mandels et al. 1980), T follicular cells, and naive IgD+ B cells or newly activated B cells that transit through the

10 CHAPTER 1. INTRODUCTION

GC(Schwickert et al. 2009). Additionally, a specialised type of macrophages called tingible bodies phagocyte dying GC B cells throughout the GC(Fliedner 1967).

The dynamic nature of GCs and the migration patterns of B cells within them was visually conﬁrmed by intravital-imaging using multiphoton laser scanning(Hauser et al. 2007; Allen et al. 2007b; Schwickert et al. 2007). These and other studies were also fundamental to better comprehend the diﬀerences between DZ and LZ B cells and to validate new surface markers to clearly demarcate GC B cells in both mouse and human.

GC B cells in the DZ or centroblasts are highly mitotic cells with cell cycles as short as 7 hours(Liu et al. 1991). In fact, ∼2/3 of the GC B cell population correspond to proliferative cells(Victora and Nussenzweig 2012). Centroblasts express the DZ marker CXC chemokine receptor 4 (CXCR4), important for cell migration from the LZ to the DZ(Bannard et al. 2013). Centroblasts also express higher amounts of AID enzyme than their LZ counterpart. This expression pattern is associated to their division rate to facilitate SHM and aﬃnity maturation, as AID can only execute its function in dividing cells.

In situ photoactivation of intact GC structures coupled to transcriptional analysis of DZ and LZ B cells provided a new definition for DZ and LZ B cells. Today, the consensus is that DZ B cells can be identified by flow cytometry as CD83lo CD86lo CXCR4hi, and LZ B cells as CD83hi CD86hi CXCR4lo (Victora et al. 2010). Additionally, LZ B cells or centrocytes have an activated phenotype evidenced by CD69 upregulation and expression of genes relevant for T:B cell interaction, supporting the model in which B cells positioned in the LZ area undergo affinity-based selection through competition for limiting Tfh cell-

11 CHAPTER 1. INTRODUCTION

derived help(Victora et al. 2010; Allen et al. 2007a; Victora and Nussenzweig 2012).

1.2.3 Tfh cells

T follicular helper (Tfh) cells are a specialised subset of CD4+ cells that give essential survival and differentiation signals to cognate B cells within B cell follicles. Antigen presenting cells (APCs) can direct CD4+ T helper cell differentiation into Tfh cells in the presence of IL-6, IL-12, and IL-21(Eto et al. 2011; Karnowski et al. 2012; Ma et al. 2009). Under physiological conditions, Tfh cells are necessary for GC formation and maintenance, and act as a limiting factor in B cell selection and differentiation to memory or antibody-secreting plasma cells(Victora et al. 2010).

The first differentiation stages of Tfh cells depend on the expression of Achaete-Scute Family bHLH Transcription Factor 2 (Ascl2) that controls initial upregulation of CXCR5 to promote their migration to the T:B interface. After this initial stage, ICOS-stimulation mediated by DCs induces the upregulation of the transcription factor Bcl6 that takes control as the master regulator responsible for the follicular program in Tfh cells(Choi et al. 2011; Yu et al. 2009). Bcl6 sustains CXCR5 expression and upregulates PD-1 (CD279) important to prevent uncontrolled proliferation and localisation(Sage et al. 2013; Shi et al. 2018), and ICOS while downregulating CCR7, S1PR1, and EBI-2, also important for migration and the correct positioning of Tfh cells closer to the B cell follicle(Liu et al. 2014). Bcl6 can also act as a transcriptional repressor inhibiting the expression of antagonistic transcription factors, such as Blimp-1 (Johnston et al. 2009), Rorγt, T-bet and Foxo1(Kerdiles et al. 2010); thus, blocking differentiation of Bcl6-expressing T cells to other non-Tfh effector

12 CHAPTER 1. INTRODUCTION subsets(Crotty 2014).

T helper cells govern the fate of responding naive B cells in a T cell- dependent (TD)-immune response. Cognate interactions between B and T cells are initiated at the T:B border with the recognition of the MHC class II-peptide complex (pMHCII) on B cells by the TCR. Upon activation, T cells express CD40-L, ICOS, and LFA-1 that can interact with CD40, ICOSL, and ICAM-1/ICAM-2 on the surface of B cells(Tafuri et al. 2001; Carrasco et al. 2004). Other costimulatory molecules, such as B7.1 and B7.2 (CD80 and CD86, respectively) on B cells, interact with CD28 on the surface of T cells(Walker et al. 2000). In consequence, Tfh cells prime cognate B cells to proliferate and subsequently decide whether to initiate an extrafollicular(Lee et al. 2011) or a germinal centre response. Further diﬀerentiation of Tfh cells into GC-Tfh cells relies on SLAM-associated protein (SAP)-dependent interactions that help to stabilise the expression of CXCR5 and ICOS, and the downregulation of EBI-2, which allows the migration into the follicle to support the formation of GCs (Crotty et al. 2003; Qi et al. 2008).

T helper cells can also give help to antigen-speciﬁc B cells in the form of soluble factors, such as cytokines IL-21(Linterman et al. 2010; Zotos et al. 2010), IL-4, and IFN-γ, that pair with receptors on B cells to promote B cell survival, selection, and diﬀerentiation(Mills and Cambier 2003; Ozaki et al. 2002; Kim et al. 2004; Takemori et al. 2014).

1.2.4 Regulation of autoimmunity in the Germinal Centre

Germinal centres are privilege sites for somatic hypermutation (SHM)- mediated aﬃnity maturation(Berek et al. 1991). The stochastic nature of

13 CHAPTER 1. INTRODUCTION

this process constitutes an advantage and, at the same time, a risk. A low affinity clone can become a high-affinity plasma cell after undergoing affinity maturation and acquiring the right mutations. This means that there are no strict criteria to establish the usefulness, danger, or irrelevance of a given clone a priori. This is also the reason why our repertoire, while vast, has no need to be infinite. Nevertheless, the generation of autoreactive clones after SHM is always a possibility(Davidson et al. 1987).

Besides central tolerance mechanisms in the bone marrow that select against lymphocytes that show strong antigen-receptor signalling in response to self- antigens, different checkpoints at different levels are put in place to avoid selection of self-reactive B cells. In the periphery, the limiting number of Tfh cells acts as a barrier ensuring that only the fittest B cell clones can access T cell help and are selected to produce antibodies. However, because cognate T and B cells might recognise completely different epitopes from the same antigenic complex, it is possible that tolerised T cells give help to self-reactive B cells that have processed antigen complexes containing self- and non-self-antigens.

There is a building body of evidence showing different mechanisms that the immune system has evolved to control for the potential surge of autoimmunity at different stages of immune cell development and immune responses, like the maintenance of a pro-apoptotic state in GC B cells that ensures the survival of antigen-specific clones only. This is achieved by upregulation of pro-apoptotic molecules such as EAF2 and Fas, and downregulation of anti- apoptotic molecules, such as Bcl2,(Li et al. 2016).

14 CHAPTER 1. INTRODUCTION

1.2.5 ICOS:ICOSL Signalling in B cell Responses

Other mechanisms involve B cell-extrinsic signals delivered to them that modulate B cell responses. Among these, CD28:B7.1/B7.2, PD-1:PD-1L, and ICOS:ICOSL signalling have been shown to have critical roles in the regulation of TD-immune humoral responses(Esensten et al. 2016; Francisco et al. 2010; Frey et al. 2010).

ICOS is expressed mainly in APCs and binds exclusively to ICOSL(Wiken- heiser and Stumhofer 2016). ICOS:ICOSL signalling has been implicated in the differentiation of T cells to Th1 and Th2 effector programs(Wilson et al. 2006) and to IL-21 production in Tfh cells(Vogelzang et al. 2008). Naive B cells express low levels of ICOS-L constitutively, whereas ICOS expression on T cells is triggered after TCR stimulation(Liang et al. 2002). Within GCs, ICOS expression appears to be restricted to the LZ(Hutloff et al. 1999), an area of active antigen-presentation to Tfh cells, suggesting a role in the GC reaction. ICOS signalling in T cells at the T:B border is critical for the initial BCL6 upregulation and acquisition of a follicular phenotype(Wikenheiser et al. 2016).

Mice lacking Icos signalling (Icos-/- or Icosl-/-) have no apparent defect in the production of IgM antibodies in response to TD antigens; however, IgG antibody production was significantly impaired. Additionally, Icos signalling appeared to be essential for the size of primary GCs and the formation of secondary GCs in response to boost immunisations with TD antigens(Wong et al. 2003; Dong et al. 2001; Mak et al. 2003). Besides quantitative differences, the quality of the humoral response is also affected. Icos-/- mice immunised with NP-OVA produced less antigen-specific IgG1 and presented impaired affinity

15 CHAPTER 1. INTRODUCTION

maturation, as shown by a reduction in the NP4/NP23 IgG1 ratio 6 weeks after immunisation(Mak et al. 2003). This phenotype was lost 10 weeks post immunisation.

Other approaches using blocking antibodies against Icos showed a reduction in CXCR5 expression in CD4+ cells from mice immunised with TD antigens during primary and secondary responses(Hu et al. 2009; Akiba et al. 2005). In the context of a viral immunisation, Icos-/- T cells were unable to properly upregulate Bcl6 and CXCR5 compared to wild type T cells at early time points (day 3 and 4)(Choi et al. 2011). This effect was shown to be B cell-independent, suggesting that the early differentiation of pre-Tfh cells is dependent on other APCs, such as DCs, while B cells have a more relevant role after GC onset. Consistent with this idea, Liu et al. showed that ICOS is essential for Tfh:B cell engagement within GCs, probably by stabilising and increasing the length of the interaction(Liu et al. 2015). In mice where MHC-II is restricted to DCs, pre-Tfh cells form normally but are unable to further differentiate into GC Tfh cells, highlighting the importance of interactions with B cells to support the GC reaction(Goenka et al. 2011).

Diﬀerences in ICOS expression can have an eﬀect on the development of autoimmunity. Hypomorphic, or null mutations in ICOS can translate in Tfh cell dysfunction and can impact germinal centre-mediated antibody production. Similarly, blockade of Icos using monoclonal antibodies prevents the progression of lupus-like disease in NZB/W F1 mice(Iwai et al. 2003). Icos signalling has also been associated to increased morbidity and mortality in murine models of lupus such as BXSB-Yaa, MRL-Faslpr, and Roquinsan/san, where Icos overexpression leads to Tfh accumulation and systemic autoimmunity characterised by hypergammaglobulinemia and excessive IL-21 production(Vinuesa et al.

16 CHAPTER 1. INTRODUCTION

2005; Bubier et al. 2009; Teichmann et al. 2015).

Patients with homozygous null mutations in ICOS develop common variable immunodeﬁciency (CVID) characterised by hypogammaglobulinemia and opportunistic infections(Grimbacher et al. 2003). Circulating Tfh cells from patients with active SLE show enhanced responsiveness to ICOS stimulation in vitro, producing more IFN-γ than Tfh cells from healthy controls. ICOS ligation also induced the production of polyreactive antibodies in autologous T:B cell cocultures from SLE patients(Kawamoto et al. 2006). Additionally, a higher frequency of ICOS+ Tfh cells was found in the blood of SLE patients (Kawamoto et al. 2006; Simpson et al. 2010).

ICOS-signalling also has a role in Tfh migration across the follicle in a costimulation-independent manner. Engagement with follicular bystander B cells expressing ICOSL promotes the persistence and motility of Tfh cells at the T:B border. This interaction further improves the responsiveness of Tfh cells towards CXCL13 in a PI3K-dependent manner, and thus the migration into the follicle(Xu et al. 2013). More recently, this migration has been shown to be ﬁne-tuned in GCs by PD-1. Activation of PD-1 by its ligand, PD-L1, also expressed by follicular bystander B cells suppresses the activity of the axis CXCR5-PI3K, preventing the migration of Tfh cells within GCs(Shi et al. 2018).

More recently, our group has described the role of Tfh-derived dopamine in the rapid upregulation of surface ICOSL in human B cells(Papa et al. 2017). ICOSL upregulation enhanced CD40L accumulation at the immunological synapse, increasing its contact area and thus, accelerating the GC output.

In summary, these studies support the role of ICOS:ICOSL interaction

17 CHAPTER 1. INTRODUCTION

in the establishment, diﬀerentiation, maintenance, and outcome of the GC reaction, where dysregulated signalling can result in immunodeﬁciencies or autoimmunity.

1.3 T Regulatory cells

The main goal of immune tolerance is to achieve homeostasis by preserving the integrity of the host without compromising its defence. After the association between helper T cells and B cells was established in the 1960s, Gershon postulated that thymic-derived cells were also important to induce tolerance (Gershon and Kondo 1970). Even 20 years later, the existence of a specialised suppressor T cell subset was still considered a bold overinterpretation of the available evidence, “bordering on the mystical”(Green and Webb 1993).

Although the existence of rogue self-reactive T cells clones in the periphery that escaped thymic-deletion was undeniable at the time, the mechanisms in place that controlled them to prevent the onset of autoimmunity were vague. With the discovery of Th1 and Th2 cells, the general belief was that suppression was achieved by cytokines with opposing functions. Soon after, Sakaguchi et al. showed that around 10% of the T cell pool coexpressed the IL-2Rα subunit (CD25), and in the absence of these cells, mice showed histological and serolo- gical signs of systemic autoimmunity(Sakaguchi et al. 1995) ﬁnally pinpointing a marker for the elusive suppressive subset. Further characterisations showed CD25+ T cells also express several other activation markers, such as CTLA-4, OX40, CD62L, and glucocorticoid-induced TNFR-related protein (GITR).

Naturally occurring thymic-educated Tregs suppress inﬂammatory responses against self- and foreign-antigens to prevent tissue damage by repressing spe-

18 CHAPTER 1. INTRODUCTION cialised effector lymphocyte subsets. During their differentiation and selection in the thymus, medullary thymic epithelial cells (mTECs) negatively select potentially pathogenic self-reactive T cells. This selection constitutes the core of T cell central tolerance. A subset of mTECs express the genesAIRE and FEZF2 that act as transcriptional regulators allowing the promiscuous expression of ectopic tissue-specific antigens (TSAs)(Mathis and Benoist 2009). Besides mTECs, thymic DCs and B cells can also present antigens to T cells (Takahama et al. 2017). T cells that show high affinity for TSAs have, besides undergoing apoptosis, an alternative path: becoming Tregs(Klein et al. 2014).

Mutations in the AIRE gene can result in diﬀerent autoimmune disorders. In humans, loss-of-function mutations leads to a rare autosomal recessive disorder called Autoimmune Polyglandular Syndrome Type-I (APS-1, also known as APECED) that is characterised by susceptibility to chronic mucocutaneous candidiasis (CMC), development of autoantibodies against cytokines IL-17F and IL-22, and multiorgan autoimmunity(Cheng and Anderson 2012). A similar phenotype was found in Aire-deﬁcient mice(Anderson et al. 2002); although, the pattern of autoantibodies produced did not seem to be the same(Pöntynen et al. 2006). Many of the manifestations of APS-1 have been associated to the escape of self-reactive T cells and defects in Treg selection.

1.3.1 Foxp3

The Scurfy mouse model was key for the identiﬁcation of the master regulator of suppressive CD25+ T cells. Genetic studies by Brunkow et al. characterised the gene responsible for the Scurfy phenotype as a member of the forkhead/winged-helix family of transcriptional regulators, and called it forkhead box protein 3 (Foxp3 )(Brunkow et al. 2001). The Foxp3 gene encoded

19 CHAPTER 1. INTRODUCTION

for a protein that, besides a forkhead domain, had a leucine-zipper and a zinc-ﬁnger DNA-binding domain.

Foxp3 mRNA expression is restricted to CD4+ T cells and because of its specificity it is the gold standard for Treg identification, unlike other ubiquit- ous activation markers previously associated with Tregs. Foxp3 is a master transcription factor that regulates the expression of key genes that aid in the acquisition of a suppressive phenotype. This was further confirmed by in vitro studies using retroviral transduction or transgenic T cells where Foxp3 overexpression was sufficient to gain regulatory capacity in CD25− CD4+ cells that originally lacked suppressive activity(Khattri et al. 2003; Hori et al. 2003; Fontenot et al. 2003). Moreover, Foxp3 was shown to not only be important for the onset of a regulatory phenotype but for its maintenance that required sustained expression(Gavin et al. 2007; Lin et al. 2007; Williams and Rudensky 2007).

Mutations in the human FOXP3 gene that result in a complete or partial loss of function, or interfere with its DNA-binding specificities can lead to splenomegaly, excessive organ-specific inflammation (including pancreas, liver, and lungs), lymphadenopathy, skin and gastrointestinal pathologies, and to immune dysregulation poly-endocrinopathy enteropathy X-linked syndrome (IPEX), the human counterpart of Scurfy mice. This syndrome is characterised by severe allergy associated to hyper IgE, multiorgan autoimmune conditions, thyroiditis, type 1 diabetes mellitus, and inflammatory bowel disease(Chatila et al. 2000; Wildin et al. 2001; Bennett et al. 2001; Wildin and Freitas 2005).

20 CHAPTER 1. INTRODUCTION

1.3.2 Treg diversity

Over the last 15 years, a variety of regulatory T cell subpopulations have been described, from IL-10-producing Tr1 cells(Roncarolo et al. 2006), TGF- β-producing T helper type 3 cells(Faria and Weiner 2005), CD8 + suppressor T cells(Chang et al. 2002), natural killer T (NKT) cells(Kronenberg 2005), CD4−CD8− antigen-speciﬁc regulatory T cells(Zhang et al. 2000), and γδ T cells(Hayday and Tigelaar 2003).

Tregs have been shown to co-opt specialised eﬀector functions in order to best modulate their target cells. For example, Foxp3+ Tregs can coexpress T- bet, IRF4, Stat-3, or Bcl6 to suppress Th1(Koch et al. 2009), Th2(Zheng et al. 2009), Th17(Chaudhry et al. 2009), or B cells(Chung et al. 2011; Linterman et al. 2011; Wollenberg et al. 2011), respectively. Additionally, Tregs have been shown to regulate cytotoxic CD8+ T cells(Maeda et al. 2014) and other innate immune cells(Gasteiger and Rudensky 2014).

Besides conventional thymic-derived Foxp3-expressing natural Tregs (nTregs), there are a plethora of peripherally induced suppressive T cells (iTregs)(Cassis et al. 2005) that can be generated by polarisation of T cells with inhibitory cytokines such as IL-10, TGF-β, or IL-35(Collison and Vignali 2008; Collison et al. 2010). Of note, not all iTregs express Foxp3.

1.3.3 Treg mechanisms of suppression

How do Tregs exert their diverse suppressive functions? Although still a developing area of research, we now have a better understanding of some of the mechanisms that Tregs use to support tolerance to innocuous and self-antigens

21 CHAPTER 1. INTRODUCTION

(Miyara and Sakaguchi 2007; Rivas and Chatila 2016). These mechanisms include: i) inhibitory receptors like LAG-3 and CTLA-4, the latter important for its ability to transendocytose the ligands for CD28, i.e., B7.1 (CD80) and B7.2 (CD86), in antigen presenting cells (APCs), such as dendritic cells (DCs), limiting the priming of self-reactive T cells(Walker and Sansom 2015). CTLA-4 on Tregs has also been associated to enhanced indoleamine 2,3-dioxygenase (IDO) activity in APCs(Puccetti and Grohmann 2007); ii) contact-independent mechanisms that involve anti-inflammatory cytokines IL-10(Kearley et al. 2005; Rubtsov et al. 2008), IL-35, and TGF-β (Strauss et al. 2007); and cytotoxic molecules such as granzyme and perforin(Cao et al. 2007; Zhao et al. 2006); iii) metabolic disruption by cAMP(Kobie et al. 2006) and CD25 which can act as a “sponge” for IL-2 (cytokine-sink-model) thus limiting the survival and differentiation to other effector T helper cells(Pandiyan et al. 2007; Kastenmuller et al. 2011). Considering that T effector:Treg interactions are not particularly stable (Tang et al. 2006) contact-independent mechanisms are of special relevance.

Treg activation depends on TCR stimulation by APCs, in an antigen-specific manner. However, after activation Tregs can amplify their scope as bystander suppressors regulating T cells with other specificities in a non-specific manner.

1.3.4 Tregs in health and disease

T regulatory cells (Tregs) are essential players in the induction of tolerance to food nutrients(Kim et al. 2016), self-antigens(Sakaguchi 2004; Fujio et al. 2012), and microsymbionts(Russler-Germain et al. 2017), and to curtail innate and adaptive immune responses leading to safe resolution(Shevach et al. 2001). Tregs can also limit fetal rejection(Aluvihare et al. 2004; Samstein et al. 2012)

22 CHAPTER 1. INTRODUCTION and in the context of transplantation, limit graft versus host disease (GVHD) (Di Ianni et al. 2011). Specialised Treg subsets play multiple and varied regulatory roles within non-lymphoid tissues, such as muscle, fat, the nervous system in the context of multiple sclerosis(DiSpirito et al. 2018; Vasanthakumar et al. 2015; Viglietta et al. 2004), and within solid tumours(Shang et al. 2015; Tanaka and Sakaguchi 2017).

Tregs have also been shown to prevent the emergence of pathogenic antibodies including autoantibodies to systemic and tissue-specific antigens and excessive IgE in mice and humans(Fujio et al. 2012; Krogulska et al. 2015; Ramsdell and Ziegler 2014; Wing et al. 2008) and this effect can be directly (Lim et al. 2005; Zhao et al. 2006; Iikuni et al. 2009) or indirectly (through the suppression of effector T cell subsets) targeted to B cells. Indeed, Scurfy mice lacking the Treg transcriptional regulator Foxp3 develop high titres of organ-specific antibodies and hyper-IgE(Lahl et al. 2007; Kim et al. 2007). These autoimmune manifestations are also present in mice lacking Aire, required for ectopic expression of tissue-specific antigens in the thymus and known to drive the development of self-peptide-specific Tregs(Mathis and Benoist 2009).

The manipulation of Tregs has been pursued for its relevance in clinical settings either dampening their function to enhance an immune response against tumour cells or chronic pathogen infections, or strengthening them to alleviate autoimmune conditions(Miyara et al. 2014). The use of monoclonal antibodies to deplete CTLA-4+ or CCR4+ cells is being discussed as a potential therapeutic approach to deplete eﬀector Tregs in cancer patients(Tanaka and Sakaguchi 2017).

More recently, Tregs have been associated to additional roles in tissue

23 CHAPTER 1. INTRODUCTION

regeneration and stem cell proliferation in muscle and lung by secreting the growth factor Amphiregulin, an epidermal growth factor receptor (EGFR) ligand(Burzyn et al. 2013; Arpaia et al. 2015). Hui et al. have shown that Foxp3+ Tregs in zebrafish can migrate to injured tissue and secrete different organ-specific growth factors in heart, spinal cord and retina(Hui et al. 2017). Of interest, two out of these three repair factors are neuron-derived products, i.e., Neurotrophin-3 (NT3) and Neuregulin-1 (Nrg1).

Tregs express a variety of neuron-associated genes and it was recently reported that neurons can support Treg diﬀerentiation in vitro(Szklany et al. 2016).

1.3.5 Tfr Cells

Bcl6 expressing Tregs, known as T follicular regulatory (Tfr) cells, were first described as repressors of GC B cell reactions, particularly limiting the magnitude of GCs, enhancing affinity maturation, and repressing the emergence of non-antigen-specific B cells(Chung et al. 2011; Linterman et al. 2011; Wollenberg et al. 2011; Aloulou et al. 2016; Sage et al. 2014b; Wing et al. 2014).

Tfr cell differentiation appears to be comparable to the Tfh cell differentiation path that is dependent on BCL6, CD28, SAP, and ICOS expression (Chung et al. 2011; Linterman et al. 2011). Initial differentiation of Tfr cells has been shown to be dependent on IL-2 responsiveness that can be dampened by IL-21(Jandl et al. 2017). IL-4 also seems to promote Tfr differentiation(Crotty 2014). In contrast to Tregs, Tfr cells downregulate IL-2Rα as they mature into fully differentiated GC-Tfr cells(Botta et al. 2017; Ritvo et al. 2017; Wing et al. 2017). Besides Bcl6, Tfr cells also express Blimp-1, the transcription

24 CHAPTER 1. INTRODUCTION factor important for the expression of Treg eﬀector molecules, such as IL-10 (Bankoti et al. 2017; Cretney et al. 2018).

Several studies using different immunisation strategies and different approaches to remove Tfr cells have shown varying effects of these cells on Tfh cell expansion at the peak of the GC reaction, affinity maturation and on the magnitude of antigen-specific antibody responses, with reports ranging from very significant to weak inhibitory effects(Chung et al. 2011; Linterman et al. 2011; Wollenberg et al. 2011; Sage et al. 2014b; Wing et al. 2014; Sage et al. 2014a). Intriguingly, in mixed bone marrow chimera models constructed to lack Tfr cells, GCs accumulated B cell specificities not directed against the immunising antigen(Linterman et al. 2011), suggesting that Tfr cells may prevent the emergence of unwanted antibody specificities. Given the known selection of thymic Tregs on the basis of recognition of tissue-specific self-antigens (TSAs) in the thymus, it is conceivable that some of those Tregs will become Tfr cells to access follicles during immune responses and repress B cells that present cognate self-peptides.

In most studies, the functional consequences of Tfr cell activity have either been shown in vitro, or with mouse models in which Tfr depletion was either transient, or achieved by adoptive transfers of Tregs lacking CXCR5 into TCR-α and β KO mice, or through generation of mixed bone marrow chimeras. None of these approaches allowed accurate evaluation of autoantibody responses. Recently, mice genetically engineered to naturally lack Tfr cells have been generated and reported to develop anti-dsDNA antibodies in a pristane-induced lupus model(Wu et al. 2016). A role for Tfr cells in the prevention of spontaneous anti-nuclear and Sjogren’s Syndrome-associated antibodies has also been recently described(Fu et al. 2018). In this study, Fu et al. showed that

25 CHAPTER 1. INTRODUCTION

Tfr-deficiency leads to late onset of autoimmunity characterised by lymphocyte infiltration in lung, pancreas and salivary gland and antibody deposition in kidney and salivary gland. However, it is unclear if these antibodies are specific for tissue antigens, as polyreactive antibody deposition in kidneys can occur when chromatin fragments from necrotic cells bind with high affinity to glomerular basement membrane structures in the absence of proper Dnase1 expression (Mortensen and Rekvig 2009).

In the last decades there has been a sustained rise in the prevalence of allergic disorders that has urged researchers to elucidate the mechanisms responsible for this break in tolerance and to propose means of restoring the balance. The generation of antigen-speciﬁc Tregs is pivotal to maintain tolerance to innocuous antigens, such as food nutrients.

Tissues that have direct contact with the environment, such as digestive (Josefowicz et al. 2012a) and respiratory tract(Duan and Croft 2014; Jaffar et al. 2009) are known to be important niches of induction of antigen-specific Tregs. APCs in these tissues, such as gut CD103+CD11c+ dendritic cells or alveolar macrophages, are not efficient T effector (Teff) cell activators, but they are well suited to drive the differentiation of naive T cells into inducible Treg (iTreg) allergen-specific cells in the presence of TGF-β1 and retinoic acid (Soroosh et al. 2013; Coleman et al. 2013).

The role of Tfr cells in preventing allergic responses also remains unclear. Excessive IgE responses to environmental and innocuous food antigens are associated with allergic reactions and anaphylaxis(Galli et al. 2008). Recently, Tfh cells have been shown to induce IgE responses against airborne-antigens (Kobayashi et al. 2017). This may not be surprising, since Tfh cells are known

26 CHAPTER 1. INTRODUCTION to be important for the induction of both follicular and extrafollicular antibody responses to thymus-dependent antigens. Indeed, with some exceptions, the appearance of both IgM and IgG antibody responses to protein antigens is dependent on Bcl6-expressing Tfh cells(Lee et al. 2011). IgE is strongly regulated, with evidence of selection against IgE+ B cells in both the GC and post-GC response through several B cell-intrinsic and extrinsic mechanisms (Harris et al. 1999; He et al. 2013; Yang et al. 2012; Butt et al. 2015; Haniuda et al. 2016), but whether Tfr cells play a role in limiting IgE is not yet known. Given the known role of Foxp3+ Tregs in the repression of IgE responses and allergic reactions at mucosal sites(Khattri et al. 2003; Lin et al. 2005; Kim et al. 2010; Wing and Sakaguchi 2013), and that most allergy patients do not have decreased Treg numbers, it is plausible that Tfr cells repress IgE responses within secondary lymphoid organs.

A number of molecules and mechanisms have been postulated to participate in Treg and Tfr-mediated regulation of B cell responses, including changes in B cell metabolism and expression of CTLA-4, consistent with what has been found in Tregs(Sage and Sharpe 2016). However, how Tregs/Tfr cells prevent the emergence of autoantibodies and excessive IgE remains unknown.

1.4 Pathogenic Antibody Production

1.4.1 Antibody-mediated autoimmunity

Autoimmunity can arise from unfavourable environment and habits combined with hormonal and genetic predisposition that together can disrupt immune self-tolerance. Some of the basic symptoms are well characterised

27 CHAPTER 1. INTRODUCTION

and shared among diﬀerent autoimmune disorders, but the patient-speciﬁc cellular, molecular and genetic causes are extremely heterogeneous and still poorly understood.

In later years, the development of biotherapeutics such as recombinant proteins, monoclonal antibodies or new small molecule drugs targeting complete cell lineages, multiple pathways, or speciﬁc receptors and lymphokines, has boosted the array of possibilities for clinical immunologists to treat patients. Still, only a fraction of the patients will respond to any given therapy while clinicians continue to relied on symptomatology to allocate individual treat- ments. Current therapies including corticoid steroids are based on the unspeciﬁc silencing of the immune system and can have detrimental consequences on the patient’s health and quality of life. For example, making them more susceptible to opportunistic infections that take advantage of the depressed immune system.

There are more than 80 autoimmune diseases described in the literature that can be broadly divided into tissue-specific disorders affecting particular organs (e.g., type 1 diabetes (T1D), inflammatory bowel disease (IBD), multiple sclerosis (MS), Hashimoto’s thyroiditis, and Grave’s disease) and systemic autoimmunity, that generates immune responses directed to common cellular components (e.g., rheumatoid arthritis, RA; systemic lupus erythematosus, SLE; and Sjogren’s syndrome, SS). Additionally, depending on the effector mechanism that underpins the pathology, autoimmune disorders can be cell- mediated, when immune cells, such as innate cells or cytotoxic T lymphocytes infiltrate tissues and promote tissue damage; or antibody-mediated, when the production of autoantibodies is directed to self-antigens, which can lead to fatal end organ failure.

28 CHAPTER 1. INTRODUCTION

Antibody-mediated autoimmune diseases can arise from molecular mimicry, where the immune system recognises pathogen antigens that resemble structures found in self-tissues but are diﬀerent enough to mount an immune response that will cross-react with the host’s own tissues(Albert and Inman 1999), or from a break in tolerance after peptide modiﬁcation (e.g., peptide citrullination) that can be triggered by environmental factors (air pollution, ozone, diet, medication, etc.)(Klareskog et al. 2009; Suzuki et al. 2003). There is a strong genetic component in the prevalence of autoimmunity, with around 25% to 75% of homozygotic twins sharing the same phenotype. Still, the aetiology of many autoimmune disorders remains a mystery.

Autoantibodies found in patients with autoimmune disorders have been shown to be of high-aﬃnity, suggesting that the B cells that produce them have gone through a process of aﬃnity-maturation, most likely within GCs, although somatic hypermutation has been shown to occur in extrafollicular foci in autoimmunity(Herlands et al. 2007). The role of T follicular helper (Tfh) cells in autoimmunity has been actively studied since their discovery(Zhu et al. 2015).

More recently, Tfr cells have been implicated in the production of systemic (Wu et al. 2016) and Sjogren’s syndrome-speciﬁc-autoantibodies(Fu et al. 2018).

1.5 Neuroimmunology

The idea of a close relationship between the immune and nervous system was already being discussed in the early 1960s(Silverstein 1963; Jerne 1967; Cohn 1970; Edelman and Mountcastle 1978). This association was also established

29 CHAPTER 1. INTRODUCTION

on the origin of the thymus, where its constitutive components were thought to be recycled from cellular and molecular neuroendocrine precursors, and was further supported by the observation that stress and inﬂammation can have comparable physiological outcomes. The concept of “immunological synapse”, introduced by Norcross in the early 1980s, only started to gain attraction more than a decade later, with the work of Grakoui and subsequent characterisations by Dustin and Coleman, and Bromeley.(Grakoui et al. 1999; Bromley et al. 2001; Dustin and Colman 2002). The immunological synapse between B and T cells characterised by a loss in T cell locomotion, junction formation, and cell polarisation(Choudhuri et al. 2014).

Besides clear similarities in the way lymphocytes and neurons interact with neighbouring cells through stable junctions or synapses, the discovery of the immunoglobulin superfamily-related molecules (e.g., ICAM-5 (Telenchephalin), axonal cell adhesion molecules (e.g., AxCAM and DSCAM) in the nervous system, only strengthened the theory of an evolutionary association with the immune system(Rutishauser and Jessell 1988; Yoshihara et al. 1994; Wojtowicz et al. 2004). It has become evident that nervous and immune systems join forces in previously unappreciated ways to protect the host after infection (Pavlov et al. 2018). Even more, both systems share signalling and sensory pathways(Chiu et al. 2013).

The ﬁeld of immunology can no longer be understood in isolation. By embracing other areas of biomedical research, such as oncology, genetics, molecular biology and neuroscience, we are now integrating our knowledge of diﬀerent systems interactions to make sense out of observations we could not previously explain. This interdisciplinary approach has led to the successful use of antibodies in the treatment of B cell malignancies, autoimmune and

30 CHAPTER 1. INTRODUCTION cytokine-mediated diseases, as well as a variety of immunotherapies to treat diﬀerent types of cancer. This highlights the importance of studying basic and applied immunology for the advancement of human medicine.

1.6 Neuritin

Neuritin is encoded by the Nrn1 gene, also known as Cpg15 (candidate plasticity gene 15), and was discovered in the early 90s in a screening searching for activity-dependent genes in the rat hippocampus dentate gyrus after stimulation with the glutamate agonist, kainic acid(Nedivi et al. 1993; Naeve et al. 1997). Since then, it has been associated to many activity-driven roles, such as dendrite arborisation and rearrangements, axonal growth(Nedivi et al. 1998), migration(Zito et al. 2014), and survival of embryonic cortical progenitors (Putz et al. 2005).

Structurally, human NRN1 localised in chromosome 6 and consists of a 2072 bp long transcripts with 2 introns and 3 exons. In mice, Nrn1 is found in chromosome 13. The mouse and human gene sequence share a 97% of similarity. The open reading frame (ORF) has 426 bp that encode for a 142 aminoacid (aac) protein composed of a 27 aac signal peptide, an 88 aac mature peptide, and a 27 aac GPI-anchor. Position 116 marks a lipidation site for post-transcriptional modiﬁcation in the GPI-anchor(Naeve et al. 1997).

In humans, neuritin has three different transcript variants. Variants 1 and 2 differ only in their 5’ UTR regions and encode for the same isoform 1, whereas variant 3 differs in the 5’ UTR and 5’ coding region, rendering a protein with a longer N-terminus that is distinctive of isoform 2. As a GPI-anchored protein, neuritin has a consensus cleavage sequence that can be targeted by

31 CHAPTER 1. INTRODUCTION

phosphatidylinositol-speciﬁc phospholipase C (PI-PLC) in a catalytic process that is dependent on the phosphorylation of this enzyme. Neuritin lacks an intracellular domain and thus, it is likely to signal extracellularly.

Cpg15-2 (neuritin-2), the only paralog of neuritin in mouse and human, has a similar function in vitro but has a different temporal and spatial expression pattern in different tissues. Additionally, neuritin seems to be more efficiently secreted than neuritin-2. Both neuritin and neuritin-2 form homodimers and heterodimers that are highly stable even after boiling them to induce denatura- tion. Dimeric forms can also occur in membrane-bound forms, suggesting that they can dimerised intracellularly rather than in solution. This implies that homophilic interactions of neuritin or neuritin-2 between molecules in opposing cells are unlikely to happen(Fujino et al. 2008; Fujino et al. 2008). Because neuritin and neuritin-2 are not coexpressed in the same tissue, heterodimers may not occur in vivo.

Neuritin has been studied in this context of the nervous system for the last 25 years. Neuritin can act as a neurotrophic factor and its transcription can be activated by Ca2+, N-methyl-D-aspartate (NMDA) receptors, L-type voltage-gated calcium channels (VGCC) and other neuromodulators, such as nerve growth factor (NGF), neurotrophin-3 (NT3), and brain derived neurotrophic factor (BDNF). The expression and activity of neuritin in post-mitotic presynaptic neurons correlates with periods of active synaptogenesis and maturation of synapse(Nedivi et al. 1998). Postnatally, neuritin expression is mainly evident in high plasticity zones, including the olfactory bulb and the auditory and visual cortex(Corriveau et al. 1999). Recent studies have found that recombinant neuritin administration can promote nerve regeneration after injury and has a critical role in learning and memory processes in vivo (Fargo

32 CHAPTER 1. INTRODUCTION

Specie Length (aac) Protein Identity (%) H. sapiens 142 – P. troglodytes 142 100 M. mulatta 142 100 C. lupus 142 100 R. norvegicus 142 100 M. musculus 142 99 B. taurus 142 99 G. gallus 144 76 D. rerio 142 72 X. tropicalis 144 71

Table 1.1. Sequence similarity of neuritin protein across diﬀerent species. et al. 2008; Karamoysoyli et al. 2008; Fujino et al. 2011; Zhao et al. 2015; Wang et al. 2016). More recently, Zhang et al. described the interaction of neuritin and the E3 ligase NEURL1 in inhibiting endocytosis of Notch-ligand as a possible mechanism to explain neuritinâĂŹs ability to promote neurite extension(Zhang et al. 2017b).

Nrn1 encodes for a highly conserved protein, with orthologs in 208 diﬀer- ent species from Xenopus tropicalis to Homo sapiens and there is only one aminoacid change at the C-terminus of the neuritin protein between human and mouse, while mature peptides are identical (Table 1.1).

Neuritin complete knockout (KO) mice were recently generated using a Cre/loxP system. Fujino et al. studied the lack of neuritin with a focus on their CNS development and behavioural abnormalities, highlighting a role for synapse stabilisation and learning processes(Fujino et al. 2011). No other gross abnormalities have been reported in these mice, probably due to neuritin-2 redundant functions in neurons.

33 CHAPTER 1. INTRODUCTION

Elucidating the mechanisms of action of neuritin is still an active and challenging area of research due, in part, to its receptor is still unknown. Some studies have pointed towards receptor tyrosine kinases in neurons(Yao et al. 2012; Shimada et al. 2016).

Neuritin expression is mostly conﬁned to the central and peripheral nervous system. Signiﬁcant expression is also found in fat and placenta, as well as lung and spleen to a lesser extent(Fagerberg et al. 2014). Open source data-bases also report neuritin expression in some leukocytes, such as mast cells and Foxp3+CD4+ T lymphocytes in humans and mice, where its role has not been studied so far. Mast cells and helper T cells are specialised subsets that can interact and instruct B cells as both of them express B cell helper molecules, such as CD40L and the cytokine IL-4(Merluzzi et al. 2010; Merluzzi et al. 2015).

1.7 Hypotheses and Aims

Previous work from our lab identiﬁed Tfr cell markers in mice by comparing the transcriptome of Tfr and Tfh cells(Linterman et al. 2011). Among more than a thousand diﬀerentially expressed genes, Nrn1 was the most highly upregulated gene in Tfr cells. Surprisingly, this gene encodes for a neurotrophin that has been extensively studied in the nervous system, but not in the immune system.

The association between the nervous and the immune system has been well characterised and documented during the last decades. Today, accumulating evidence has proven that the interaction between the two systems is bidirectional and this dialogue is fundamental to maintain immune homeostasis in the host(Eskandari and Sternberg 2002). Constant crosstalk between immune

34 CHAPTER 1. INTRODUCTION and nervous systems is supported by common molecules and receptors that constitute a lingua franca. Examples of these interactions are: i) the cytokine interleukin-1 (IL-1), essential for inflammatory processes, that was discovered for its role in eliciting fever by immunoreactive innervation of the hypothalamus in humans(Breder et al. 1988); ii) nitric oxide, a molecule produced by immune cells that is involved in immunomodulation and inflammation(Scholz and Woolf 2007); and iii) neuropilin-1 in Tregs, a glycoprotein transmembrane receptor that is important in the nervous system for axon guidance, angiogenesis, and remyelination but is also relevant in the immune system, specifically in stabilising Tregs(Delgoffe et al. 2013). The main aim of this thesis is to investigate the role of Tfr cells in the regulation of germinal centre responses, especially in the context of autoimmunity. In particular, the work here presented seeks to investigate a novel neuroimmune Tfr-derived factor and elucidate its role in regulating antibody production.

Chapter 2

Materials and methods

2.1 Mice and human samples

Bcl6fl/fl mice were generated by Alexander Dent (Idiana University, US) (Hollister et al. 2013). Neuritinfl/fl mice were generated by Elly Nedivi (Mas- sachusetts Institute of Technology, US)(Fujino et al. 2011). Cre Foxp3-YFP (CreFoxp3) mouse strain was a kind gift from Alexander Rudensky(Rubtsov et al. 2008). All mice were bred and maintained in pathogen-free conditions at the Australian Phenomics Facility (APF), ANU, Australia. When comparing the effect of two genotypes we used age-matched littermate controls. Only males were used for Tfr-deficient mice (BCL6fl/fl.CreFoxp3) and neuritin-deficient mice (Neuritinfl/fl.CreFoxp3) experiments. All mouse procedures carried out were approved by the Australian National University’s Animal Experimentation Ethics Committees.

Mouse lymphocyte single-cell suspensions were prepared by mechanically disrupting the tissue through 70 µm nylon mesh ﬁlters (BD Bioscience) in complete RPMI 1640 media (Sigma). and depleted of RBCs in ammonium chloride lysis buﬀer.

37 CHAPTER 2. MATERIALS AND METHODS

Human lymphocytes were obtained from consenting donors undergoing routine tonsillectomy at Calvary John James Hospital and The Canberra Hospital (Canberra, Australia). Tonsillar tissue was mechanically disrupted and leukocytes were isolated by the Ficoll Hypaque gradient centrifugation method (GE Healthcare Life Sciences). Experiments involving human samples were approved by the Human Experimentation Ethics Committee at The Australian National University and the University Hospitals Institutional Review Board.

2.2 Immunisations

Mice were immunised intraperitoneally with either 200 µl containing 100 µg of Ovalbumin (OVA) precipitated in 9% alum or intravenously with 200 µl of sheep red blood cells (SRBCs). For food allergy experiments mice were sensitised on day zero and day 7 with 1 mg of peanut extract and 10 µg of cholera toxin in PBS 1x by oral gavage. Animals were boosted on day 21 with

5 mg peanut extract. Blood was collected by cardiac puncture right after CO2 asphyxia on day 28.

2.3 Neuritin treatment in vivo

6 week old control (Bcl6+/+.CreFoxp3) and Tfr-deficient (Bcl6fl/fl.CreFoxp3) mice were treated intravenouslly with 200 µg of recombinant neuritin (ab- 69755, Abcam) twice a week for 4 weeks. Serum was collected by retro-orbital

bleeding before treatment and after treatment by cardiac puncture after CO2 asphyxiation.

38 CHAPTER 2. MATERIALS AND METHODS

2.4 Cell culture

Human B cells isolated from tonsils were puriﬁed by either FACS sort or MACS sort and cultured in complete RPMI 1640 media (Sigm-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), 2 mM L-Glutamine (Gibco), 100 mM Hepes (Gibco), 0.1 mM nonessential amino acids (Gibco), and 55 mM β-mercaptoethanol (Gibco). Cells were cultured in an incubator with 5%

CO2 at 37°C. For ICOSL downregulation in vitro experiments, B cells were isolated using MACS column separation system (#130-090-862, Miltenyi Biotec) according to the manufacturer’s instructions. B cells were then stimulated for 30 min with neuritin 2 µg/ml. For plasma cell suppression experiments, cells were cultured with anti-CD40 5 µg/ml, IL-4 40 ng/ml, IL-21 10 ng/ml, and recombinant neuritin 250 ng/ml, 500 ng/ml, 1 µg/ml, or 2 µg/ml.

2.5 Nojima cell coculture

Mouse naive B cells were isolated from total splenocytes by magnetic colum sort (#130-090-862, Miltenyi Biotec) and cocultured with Nojima cells(Nojima et al. 2011) per well seeded the day before in a 6 well plate. Cells were supplemented with murine IL-4 (10ng/ml, PeproTech). B cells were then recovered, washed, stained and analysed by FACS.

2.6 Nucleosome extraction

Native nucleosome were isolated following an extraction protocol described elsewhere(Gies et al. 2017). In summary, HEK293 cells were cultured in Dulbecco’s Modiﬁed Eagle’s Medium supplemented with 10% (v/v) FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Buﬀers A (0.015 M Tris-HCl

39 CHAPTER 2. MATERIALS AND METHODS

pH 7.4, 0.015 M NaCl, 0.060 M KCl, 0.005 M MgCl2), B (0.01 M Tris-HCl pH 7.4, 0.001 M KCl; 0,0015 M MgCl2), C (0.015 M Tris-HCl pH 7.4, 0.015 M NaCl, KCl 0.060 M, 0.005 M MgCl2, 0.001 M CaCl2), and D (0.001 M Tris-HCl pH 7.4, 0.001 M EDTA pH 7.4) were sequentially used to purify nucleosomes from 1x109 HEK293 cells. Micrococcal nuclease (0.1 unit per absorbance unit) (Sigma-Aldrich) was added to lyse remaining free DNA.

All buﬀers were supplemented with 100 µL of protease inhibitor cocktail (Sigma-Aldrich) before use. Nucleosome samples were stored at −20°C.

2.7 Fluorescently labelled neuritin

Commercial recombinanat human neuritin (ab-69775, Abcam) was conjugated to Alexa Fluor 647 fluorophore using the microscale proptein labeling kit (#A3009, Invitrogen) and following the manufacturer’s instructions. Following surface staining, total splenocytes from a SRBC-immunised mouse or total tonsillar cells were incubated with neuritin-AF 647 conjugate for 20 minutes at room temperature. Cells were washed and fixed using the Foxp3/Transcription Factor Staining Buffer Set (#00-5523-00, eBioscience) and analysed by FACS.

2.8 TriCEPS

We used a TriCEPS-based ligand-receptor capture approach (LRC-TriCEPS; Dualsystems Biotech AG) to conﬁrm neuritin binding to B cells, following the manufacturer’s instructions. In summary, human neuritin (Abcam), glycine (Sigma-Aldrich) or human transferrin (Sigma-Aldrich) were coupled to biotinylated TriCEPS trivalent molecule. Coupled ligands were then incubated either with naive or GC B cells freshly sorted from human tonsils. Ligand binding to the target cells was asses by staining cells with SA-PE and analysing

40 CHAPTER 2. MATERIALS AND METHODS by FACS.

2.9 ELISA

Antibodies against ds-DNA (#5110, Alpha Diagnostics), extractable nuclear antigens (#5210, Alpha Diagnostics), gastric parietal cell (#MBS9364474, My- Biosource) were measured in the serum of unimmunised mice using commercial kits following the manufacturer’s instructions. Absorbance was measured at 450 nm using a Inﬁnite® 200 PRO plate reader (Tecan) using the software i-controlTM version 1.9.

2.9.1 Immunoglobulin ELISA

Sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure baseline and immunisation-induced immunoglobulins in mouse serum. Immun- isation induced-IgE and IgG were detected in plasma taken before, at the endpoint, and other time points as indicated for each immunisation experi- ment. 96-well ﬂat bottom ELISA plates (#3855, Thermo Fisher) were coated with rat anti-mouse IgE (R35-72, BD Bioscience) or goat anti-mouse kappa- UNLB (#1050-01, Southern Biotech). Plates were washed and blocked with 1% bovine serum albumin (BSA) in PBS 1x for 1.5h at 37°C. Serum was diluted 1:20 (IgE), 1:500 (IgA), 1:5000 (IgM), 1:10000 (IgG, IgG1). Target antibodies were detected using rat anti-mouse IgE-biotin (R35-118, BD) followed by AP-conjugated Streptavidin, AP-conjugated goat anti-mouse IgG (#1030-04, SouthernBiotech), AP-conjugated rat anti-mouse IgG1 (#1144-04, SouthernBiotech), AP-conjugated rat anti-mouse IgA (#1165-04, SouthernBi- otech), or AP-conjugated goat anti-mouse IgM (#1020-04, SouthernBiotech). Plates were developed using phosphatase substrate tablets 1 mg/ml (#S0942, Sigma-Aldrich) and optical density was read at 405 nm and 650 nm with a

41 CHAPTER 2. MATERIALS AND METHODS

Inﬁnite® 200 PRO plate reader (Tecan) using the software i-controlTM version 1.9. Puriﬁed mouse IgE κ-isotype control (#557079, BD Biosciences), mouse IgA (#0106-01, SouthernBiotech), mouse IgG1 isotype control (#ab27479, Abcam), and mouse IgM (#02-6800, Thermo Fisher) were used as standards. Immunoglobulin titers were calculated using second order polynomial standard curves.

2.9.2 Antigen-speciﬁc ELISA

Autoantibodies against native nucleosomes in circulation were measured by ELISA. Plates (#9018, Costar, Corning®) were coated overnight with 3 µg/ml of purified nucleosomes, purified core histones, recombinant histone H1, or 5 µg/ml of OVA or peanut extract. Plates were washed and blocked with 1% bovine serum albumin (BSA) in PBS 1x for 1.5h at 37°C. Mouse serum was diluted 1:50 and incubated overnight at 4°C. Plates were then washed, incubated with biotinylated anti-kappa antibody (#407204, BioLegend) 1h at 37°C, washed again and incubated with anti-biotin antibody conjugated to horseradish peroxidase (HRP) (#ab19221, Abcam) 1 hr at 37°C. Plates were developed using tetramethylbenzidine (TMB) (#T0440, Sigma-ALdrich) and absorbance was measured at 370 nm every 5 min until samples reached a value of 0.8 – 1. At this point, the developing reaction was stopped using sulfuric acid 2 N. Absorbance was measured at 450 nm using a Infinite® 200 PRO plate reader (Tecan) using the software i-controlTM version 1.9.

2.10 qPCR

Total RNA was extracted from mouse samples using Trizol reagent (# 15596026, Thermo Fisher). RNA quality and concentration were determined with an Agilent 2100 Bioanalyzer instrument (Agilent Technologies). Samples

42 CHAPTER 2. MATERIALS AND METHODS

Target Sense Sequence Exon human neuritin F 5’-TGTTTGCTCAAGCTGGGCGACA-3’ 2 " R 5’-TGGCAATCCGTAAGGGCTGTGA-3’ 3 human RPL13A F 5’-GCCATCGTGGCTAAACAGGTA-3’ 2 " R 5’-GTTGGTGTTCATCCGCTTGC-3’ 4 mouse neuritin F 5’-TGTTTGCTCAAGCTGGGCGACA-3’ 2 " R 5’-CTTCCTGGCAATCCGTAAGAGC-3’ 3 mouse Ubc F 5’-AGCCCAGTGTTACCACCAAG-3’ 1 " R 5’-ACCCAAGAACAAGCACAAGG-3’ 1

Table 2.1. qPCR primer sets for the indicated target genes. F = forward primer, R = reverse primer. Exon column indicates the exon in which each primer aligns. with a RIN score ≥ 8 were selected for downstream analysis. Digestion of gen- omic DNA was performed with RQ1 RNase–Free DNase (#M6101, Promega). 1 µg of total RNA was transcribed into cDNA using SuperScript III Reverse Transcriptase (# 18080093, Thermo Fisher) according to the manufacturer’s instructions. qPCR reactions for each target gene were performed using 5 µl of Power SYBR Green Master Mix (# 4367659, Thermo Fisher) with 250 nM of forward (F) and reverse (R) primers (see Table 2.1). Samples were measured in triplicate using an Applied Biosystems 7900HT Fast Real-Time machine (Thermo Fisher). qPCR thermal proﬁle: 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 30s at 95°C followed by 1 min at 60°C, and melting curve analysis from 60°C to 95°C. Data is expressed as a fold-change using the ∆∆CT method.

2.11 Flow cytometry

2.11.1 Cytometric bead array

For IgE and IgG measurement in culture supernatants, we used the BD Cytometric Bead Array (CBA) Human IgE Flex Set (Bead C9) (#558682, BD Biosciences) and the Human IgG Flex Set (Bead C6) (#558679) respectively,

43 CHAPTER 2. MATERIALS AND METHODS

according to the manufacturer’s instructions.

2.11.2 Mouse samples

Single cell suspensions were prepared by mechanically disrupting the tissue through 70 µm nylon mesh filters (BD Bioscience) in complete RPMI 1640 media (Sigma). Surface staining was performed using anti–CD4 FITC or APC–Cy7 (RM4–5, BioLegend), or PE–Cy7 (RM4–5, BD Pharmingen), or PerCP (RM4–5, iCyt), anti–B220 PerCP or APC (RA3–6B2, BD), or PE Cy7 or APC–Cy7 (RA3–6B2, BioLegend), anti-CXCR5 biotin (2G8, BD Pharmin- gen), anti–PD–1 PE (J43, eBioscience), or Brilliant Violet 421 (29F.1A12, BioLegend), anti–CD44 Alexa Fluor 700 or Pacific Blue (IM7, BioLegend), anti-Foxp3 FITC or Alexa Fluor 700 (FJK–16s, eBioscience), anti–GL–7 Alexa Fluor 647 (GL7, BioLegend), anti-Fas PE or PE Cy7 (Jo2, BD Pharmin- gen), anti-CD21/35 Brilliant Violet 605 (7G6, BD Pharmingen), anti–T-bet PerCP Cy5.5 (eBio4B10, eBioscience), anti–GATA–3 Alexa Fluor 647 (L50– 823, BD Pharmingen), streptavidin-Brilliant Violet 605 (#405229, BioLegend), streptavidin-PE Cy7 (#557598, BD Pharmingen) and Zombie Aqua fixable viability kit (#423102, BioLegend). Intracellular staining was performed using the Foxp3/Transcription Factor Staining Buffer Set (#00-5523-00, eBioscience) according to the manufacturer’s instructions. Samples were acquired on a LSRII or Fortessa cytometer (BD) and analysed using FlowJo software v10.3 (Tree Star).

2.11.3 Human samples

Tonsillar cell suspensions were prepared by mechanically disrupting the tissue through 70 µm nylon mesh ﬁlters (BD Bioscience) in complete RPMI 1640

44 CHAPTER 2. MATERIALS AND METHODS media (Sigma-Aldrich). Lymphocytes were isolated using the Ficoll Hypaque gradient centrifugation method (GE Healthcare Life Sciences). Cells were stained with the following anti-human antibodies: CD4 APCCy7 (RPA-T4, BD Biosciences), CXCR5 Alexa 488 or Alexa 647 (RF8B2, BD Biosciences), PD-1 PE (MIH4, eBioscience) or BV605 or BV421 (EH12.2H7, BioLegend), CD127 FITC (11-1278, eBioscience) or BV 421 (A019D5, BioLegend), CD25 biotin (BC96, eBioscience or BioLegend) or PE-Cy7 (BC96, BD Biosciences or BioLegend), BCL6 Alexa 647 or PE-Cy7 (K112-91, BD Biosciences), CD3 APC (HIT3a, BD Biosciences) or Alexa 700 (UCHT1, BD Biosciences), CD27 FITC or APC (M-T271, BD Biosciences), CD38 FITC (HIT2, BD Biosciences) or PE (HB7, BD Biosciences), ICOSL APC (2D3, BioLegend), FAS PE-CF594 (DX2, BD Bioscience), CD40 APCCy7 (5C3, BioLegend), BAFFR PECy7 (11C1, BioLegend), CD19 PECy7 or BV605 (SJ25C1, BD Bioscience), IL21R BV421 (17A12, BioLegend), CD86 BV421 (2331/FUN-1, BD Bioscience). All surface stains were performed in the presence of Human TruStain FcX (cat. 422302, BD Bioscience). Intracellular staining was performed using the FOXP3/Transcription Factor Staining Buﬀer Set (eBioscience) according to the manufacturer’s instructions. Cells were stained with primary antibodies followed by secondary reagents for 30 min at 4°C. Data were collected on a LSRII or Fortessa cytometer (BD) and analysed with FlowJo software (TreeStar). 7-AAD (Invitrogen) or Zombie Aqua (BioLegend) staining was used to exclude dead cells from analysis.

2.12 Immunoﬂuorescence

Frozen spleen sections were ﬁxed in cold 4% PFA for 20 min and blocked using 3% BSA in PBS. Sections were then stained using and anti-IgD FITC (Biolegend), anti-neuritin (Cat #PA5-20368, Invitrogen) overnight at 4°C in

45 CHAPTER 2. MATERIALS AND METHODS

the dark, followed by anti-rabbit Alexa Fluor 594 (#A-11058, Invitrogen) for 30 min at RT in the dark. Stained sections were mounted using Vectashield with DAPI mounting media (#H-1200, Vector Laboratories). Images were collected with an Olympus IX71 microscope with DP Controller software (Olympus) using a 40x objective and compiled by using Adobe Illustrator software version 22.1.0.

2.12.1 Tissue-speciﬁc autoantibody IF

For the detection of autoantibodies against tissue-specific antigens by immunofluorescence in mouse serum, we harvested organs from RAG1−/− mice. Organs were embedded in O.C.T (Tissue-Tek) before frezzing them in dry ice. Cryosections were obtained using a Bright cryostat. Samples were left to air dry for at least 2 hrs and were subsequently fixed with PFA 4%. Samples were blocked with 5% horse serum in PBS 10 minutes at room temperature, washed and incubated with mouse serum diluted 1:30 for 1 h at room temperature. Slides were then washed and stained with Alexa Fluor-488-conjugated donkey anti-mouse IgG (#A-21202, Invitrogen) diluted 1:400 for 30 minutes at room temperature. Slides were then washed and prepared for microscopy using Vectashield with DAPI (#H-1200, Vector Laboratories). Images were collected with an Zeiss Axiophot fluorescence microscope with Zen software (Zeiss) using a 10x objective and compiled by using Adobe Illustrator software version 22.1.0.

2.13 IIF-ANA

Indirect immunoﬂuorescence using commercial Hep-2 (human laryngeal squamous cell carcinoma cells) preﬁxed slides (Innova) were used to detect anti-nuclear antigens (ANAs) by microscopy in the serum of mice. Mouse serum was diluted 1:40 and incubated on Hep-2 slides for 45 min at room temperature.

46 CHAPTER 2. MATERIALS AND METHODS

Slides were then washed and incubated with Alexa Fluor-488-conjugated donkey anti-mouse IgG (#A-21202, Invitrogen) diluted 1:400 for 30 minutes at room temperature. Slides were then washed and prepared for microscopy using Vectashield (#H-1000, Vector Laboratories). Images were collected with an Olympus IX71 microscope with DP Controller software (Olympus) using a 40x objective and compiled by using Adobe Illustrator software version 22.1.0.

2.13.1 Cytospin

Human T naive (CD4+CD25-CD45RA+PD-1−CXCR5−), T effector/memory (CD4+CD25−CD45RA−PD-1−CXCR5−), Tregs (CD4+CD25−PD-1−CXCR5−), Tfh (CD4+CD25−CD45RA−PD-1+CXCR5+), Tfr (CD4+CD25+CD45RA−PD- 1+CXCR5+), and GC B (CD19+CD38+CD27int) cells were FACS sorted on a BD FACS Aria I. Cells were then fixed in paraformaldehyde 4% for 20 min at room temperature, washed and resuspended in PBS at 106 cells/ml. 100 µl of cells were placed in cytospin chambers and spun down 5 minutes at 300 g using a Cellspin cytocentrifuge (Tharmac). Cells were air dried and subsequently stained with polyclonal anti-neuritin (Cat #PA5-20368, Invitrogen) and slides were prepared for microscopy using Vectashield with DAPI (#H-1200, Vector Laboratories). Images were collected with an Zeiss Axiophot fluorescence microscope with Zen software (Zeiss) using a 10x objective and compiled by using Adobe Illustrator software version 22.1.0.

2.14 Treg suppression assay

CTV labeled FACS puriﬁed T cells (CD4+CD3+CD25−) were co–cultured in the presence or absence of serially diluted non-follicular (PD-1−CXCR5−) T regulatory (CreFoxp3-YFP+) cells starting with 10,000 cells. Cells were stimulated with CD3 and CD28 microbeads (Miltenyi) at a 1:2 bead to cell

47 CHAPTER 2. MATERIALS AND METHODS

ratio. After 3 days, cells were stained with 7-AAD and proliferation was subsequently analysed by ﬂow cytometry.

2.15 Western Blot

Total protein extraction from lymphocyte subsets was performed using RIPA buﬀer (Thermo Fisher) supplemented with protease and phosphatase inhibitor (Roche). 10 µg of whole-cell extracts from each cell subset were separated by SDS-PAGE (12% w/v), blotted onto nitro-cellulose membranes and incubated with polyclonal anti-neuritin antibody (Cat #PA5-20368, Invitrogen). β-actin was used as a loading control (#A5441, Sigma- Aldrich). Membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) and then Enhanced chemiluminescence (ECL) development was performed using Pierce ECL Western Blotting Substrate reagent (Thermo Fisher). Images were acquired on an Image Quant LAS 4000 machine (GE Healthcare Life Sciences). Band quantiﬁcation was performed by densitometry analysis using Image Studio software version 5.2.5 (LI-COR Biosciences).

2.16 Statistical analysis

All data were analysed with non-parametric Mann-Whitney test (U test). All statistical analyses were performed with Prism software (version 7, GraphPad Software). Statistically significant differences are indicated as ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001 and ns = not significant.

48 Chapter 3

Results

3.1 T follicular regulatory cells

3.1.1 Generation of Tfr-deﬁcient mice

In order to understand how T follicular regulatory cells (Tfr) cells maintain B cell tolerance, we generated mice lacking Tfr cells. For this, we first obtained Bcl6 floxed (Bcl6fl/fl) mice, provided by Alex Dent (Idiana University, US) (Hollister et al. 2013). In these mice, exons 7 to 9 of the Bcl6 gene encoding the zinc finger domain are flanked by loxP sites, allowing the excision of the DNA-binding site of BCL6 when crossed to a Cre-expressing strain. This results in the expression of a non-functional protein. Since Bcl6fl/fl mice were generated using embryonic stem (ES) cells from a 129Sv background, and it was unclear the number of times the strain had been backcrossed, we first ran a panel of microsatellite markers across the genome to determine the content of 129Sv genetic background. We established that the mice contained predominantly C57BL/6 genome with the exception of the area surrounding the Bcl6 gene. We proceeded to backcross the Bcl6fl/fl strain to C57BL/6 mice for 5 generations before crossing them to CreFoxp3 mice.

49 CHAPTER 3. RESULTS

The CreFoxp3 strain was kindly provided by Alexander Rudensky (Sloan Kettering Institute, US). In these mice the Cre recombinase fused to yellow fluorescent protein (YFP) has been knocked into the Foxp3 gene locus. Forma- tion of Cre-YFP occurs via an internal ribosomal entry site (IRES)(Rubtsov et al. 2008). The F1 generation was intercrossed to produce F2 intercross (F2IC) Bcl6fl/fl.CreFoxp3 mice that lack functional BCL6 protein in Foxp3-expressing cells. Bcl6fl/fl.CreFoxp3 mice bred well and appeared healthy. Previous communications from Takaharu Okada (Riken, Japan) had suggested a similar cross generated in Japan, albeit with different origin of the F1 strains, died prematurely due to anaemia, at least in part due to erythrocyte-intrinsic defects. Bcl6fl/fl.CreFoxp3 mice used in this project had normal survival and displayed normal Ter119+ red blood cell (RBC) maturation in all peripheral stages of development, i.e., orthochromatic erythroblasts (IV), reticulocytes (V) and mature red blood cells (VI), as determined by flow cytometric analysis using forward scatter area (FSC-A) and CD44 expression (Fig. 3.1).

50 CHAPTER 3. RESULTS

a Control Tfr- IV Orthochromatic Erythroblasts IV Orthochromatic Erythroblasts 2.95 2.64

V Reticulocytes V Reticulocytes 75.2 76.6

VI mature RBC VI mature RBC 16.6 15.6 CD44

FSC-A

b Orthochromatic Erythrocytes erythroblasts (IV) 10 5

8 in t 4 cells ) 6 + 3 19 FSC- A i h er 1

4 T 2 (% of Live cells ) + 19 2 CD4 4 1 (% of er 1 T 0 0 Control Tfr- Control Tfr-

Reticulocytes (V) Mature RBC (VI) 100 30

o l o 80 l 24 cells ) cells ) + +

60 SC- A 18 19 19 FSC- A in t er 1 er 1 T 40 T 12 CD44- F CD4 4 20 6 (% of (% of 0 0 Control Tfr- Control Tfr- Figure 3.1. Erythrocyte phenotype. a) Representative flow cytometric plots and b) quantification of different peripheral erythrocyte developmental stages in control (Bcl6+/+.CreFoxp3, n=4) and Tfr- (Bcl6fl/fl.CreFoxp3, n=4) mice. Bars indicate median values. Each dot represents data obtained from one mouse.

51 CHAPTER 3. RESULTS

The Foxp3 locus is in the X chromosome, and thus, it is susceptible to random lyonisation and mosaicism when using heterozygous female mice, or to double dosage of the Cre transgene when using homozygous females if inclomplete silencing of the Foxp3 locus occurs(Wildin and Freitas 2005). Previous work with these mice performed by Alexander Rudensky restricted all experimental work to male mice(Rubtsov et al. 2008). To avoid potentially confounding eﬀects, we decided to use the same approach throughout this thesis, using only male mice.

3.1.2 Tfr-deﬁcient mice lack Tfr cells but Tregs are nor-

mal in number and function

Spleens from Tfr-deficient (Bcl6fl/fl.CreFoxp3) and sufficient (Bcl6+/+.CreFoxp3) mice were examined by flow cytometry to determine the efficiency of the deletion of Bcl6 in Tregs, and to confirm that Cre expression in the Foxp3 locus or Bcl6 deletion in Tregs did not alter total Treg numbers or Treg suppressive function.

Conventional Tregs gated as Foxp3+ CD4+ T cells were present in comparable proportions and numbers with a median of 15% and 16% of all CD4+ cells expressing Foxp3 in Tfr-deficient and sufficient mice respectively (Fig. 3.2). Foxp3+ populations in the thymus of Tfr-deficient mice also appeared normal when compared to Tfr-sufficient mice.

52 CHAPTER 3. RESULTS

a b Control Tfr- 25

reg (% of CD4) 5 T

FoxP3 0 CD44 Control Tfr-

c Control Tfr- d 6 cells ) + 4

2 (% of CD4 + FoxP3

Foxp3 0 CD4 Control Tfr- Control Tfr- 0.10 cells ) + 0.08

0.06

0.04 (% of CD8 + 0.02

FoxP3 0.00 Foxp3 CD8 Control Tfr-

Figure 3.2. Splenic and thymic Tregs. a) Representative flow cytometric plots and b) quantification of splenic Foxp3+ Treg cells (gated on B220− CD4+ T cells). c) Representative flow cytometric plots and d) quantification of Foxp3+ CD4+ (upper panels) and Foxp3+ CD8+ (lower panels) cells in the thymus of control (Bcl6+/+.CreFoxp3, n=4) and Tfr-deficient (Tfr-: Bcl6fl/fl.CreFoxp3, n=4) mice. Bars indicate median values. Each dot represents data obtained from one mouse.

53 CHAPTER 3. RESULTS

We also tested the ability of Tregs from mice lacking Tfr cells to suppress naive T cell proliferation in vitro. Using a conventional suppression assay, where CD4+ T cells are cultured with Tregs in the presence of antigen presenting cells (APCs), Tregs from Tfr-deficient mice were capable of suppressing T cell proliferation to the same extent as Tregs from Tfr-sufficient mice over a range of Treg : T naive ratios (Fig. 3.3). These data suggest that the introduction of the transgene into the Foxp3 locus and the deletion of Bcl6 have no effect on the suppressive function of Tregs.

54 CHAPTER 3. RESULTS

a Control Treg Tfr- Treg No Treg

9.05 7.55 67.4 Normalised To Mode Normalised To CTV b 80 Control

io n Tfr- 60 era t 40 proli f f o

20 % 0 0:1 1:16 1:8 1:4 1:2 1:1 Treg : T naive ratio Figure 3.3. Functional Tregs in Tfr- mice. a) Representative flow cytometric histograms showing proliferation profiles and b) quantification of proliferating naive T cells (B220−CD4+Foxp3−CD44−CD62L+) cocultured with Tregs (B220−CD4+Foxp3+CD44lowPD1−) from control (Bcl6+/+.CreFoxp3, in green) or Tfr- (Bcl6fl/fl.CreFoxp3, in light blue) mice in the presence of antigen presenting cells (APCs, CD4−) from 3 mice from each genotype and two independent experiments. Vertical bars and dots show range and median values, respectively.

55 CHAPTER 3. RESULTS

3.1.3 Young Tfr-deﬁcient mice have spontaneous GC ac-

cumulation that disappears with age

Since their discovery in 2011, Tfr cells have been thought to have a major role in controlling the size of the GC B cell and Tfh cell compartments(Chung et al. 2011; Linterman et al. 2011; Wollenberg et al. 2011). Our lab previously reported a spontaneous accumulation of Tfh cells and GC B cells in bone marrow chimeric mice lacking Tfr cells(Linterman et al. 2011). To investigate whether this was also a feature of Tfr-deficient mice, we first examined young unimmunised Bcl6fl/fl.CreFoxp3 mice. At 8 weeks of age there was a statistically significant accumulation of spontaneous GC B cells in Tfr-deficient mice (Fig. 3.6). Contrary to what was observed in chimeric mice, there was no spontaneous accumulation of Tfh cells in Bcl6fl/fl.CreFoxp3 mice compared to control littermates at 8 weeks of age.

56 CHAPTER 3. RESULTS

a b Control Tfr- 0.8 P = 0.0022 ** 0.6

0.4 (% of B cells) +

FAS 0.2 + 7 - 8 week old mice L

G 0.0 Control Tfr-

2.0 P = 0.1604

1.5

1.0 (% of B cells) +

FAS 0.5 + 7 GL-7 - 26 week old mice L

G 0.0 FAS Control Tfr- Figure 3.4. Spontaneous accumulation of GC B cells in Tfr- mice. a) Flow cytometric plots and b) quantification of GL-7+FAS+ GC B cells as a percentage of B220+ cells from 8- and 26-week-old mice of the indicated genotype (Control: Bcl6+/+.CreFoxp3, n=7; Tfr-: Bcl6fl/fl.CreFoxp3, n=5).

57 CHAPTER 3. RESULTS

a b Control Tfr- 20 P = 0.6970 hi 15 PD-1 hi 10

5 CXCR5 (% of Teff cells) 8 week old mice 0 Control Tfr-

15 P = 0.1667 hi

10 PD-1 hi

5 CXCR5 (% of Teff cells) PD-1 26 week old mice

0 CXCR5 Control Tfr- Figure 3.5. Basal Tfh compartment in Tfr- mice. a) Flow cytometric plots and b) quantification of CXCR5hi PD-1hi Tfh cells as a percentage of T effector/memory cells (CD4+ CD44+ Foxp3−) from 8 and 26 week old mice of the indicated genotype (Control: Bcl6+/+.CreFoxp3, n=7; Tfr-: Bcl6fl/fl.CreFoxp3, n=5).

58 CHAPTER 3. RESULTS

As mice aged, the differences in GC B cell numbers and proportions between Tfr-sufficient and deficient mice disappeared. Indeed, mice sacrificed at 6-7 months of age did not display the accumulation of GC B cells seen at earlier ages. These data suggest that repression of spontaneous GC formation is not the most relevant function of Tfr cells in vivo.

3.1.4 Tfr-deﬁcient mice have intact Tfh and GC responses

after immunisation

Next, we immunised mice with sheep red blood cells (SRBC) to determine whether Tfr deficiency changed Tfh and GC B cell proportions at the peak of the GC response (day 6). As expected, Bcl6fl/fl.CreFoxp3 mice were specifically deficient in Tfr cells (p = 0.0238) whilst the proportions of Tfh cells (p = 0.3095) and GC B cells (p = 0.4206) were similar to those of Tfr-sufficient Bcl6+/+.CreFoxp3 mice (Fig. 3.6a, b). Analysis by Immunofluorescence staining of GC formation in frozen spleen sections from Tfr-deficient mice also appeared morphologically normal (Fig. 3.6c).

59 CHAPTER 3. RESULTS

a b Control Tfr- 30 30 ) 3

20 20 s (x10 fh cells el l c T 10 10 % Tfh

# PD-1 0 0 CXCR5 Control Tfr- Control Tfr- 8 8 ) 5 0 s 1

6 x 6 (

cel l

B 4 4 cell s

C B G

2 C 2 % CD38 G

# FAS 0 0 Control Tfr- Control Tfr- 10 5 )

** 3 ** 8 4 s el l s (x10

c 6 3

el l c Tf r

4 2 % Tf r

PD-1

2 # 1

CXCR5 0 0 Control Tfr- Control Tfr- c Control Tfr- BCL6 IgD

Figure 3.6. GC lymphocytes in Tfr-sufficient and Tfr-deficient mice. a) Representative flow cytometric plots and b) quantification of PD-1hi CXCR5hi Tfh cells (pre-gated on CD4+ Foxp3− CD44+ cells), CD38− FAS+ GC B cells (pre-gated on B220+ cells), and PD-1hi CXCR5hi Tfr cells (pre-gated on CD4+ Foxp3+ cells). Plots are representative of two independent experiments. c) Immunofluorescence of frozen sections from the spleen of SRBC-immunised mice of the indicated genotype (Control: Bcl6+/+.CreFoxp3, n=5; Tfr-: Bcl6fl/fl.CreFoxp3, n=5). Bars represent medians and dots represent individual mice.

60 CHAPTER 3. RESULTS

The kinetics of Tfr expansion upon immunization in which their peak coin- cides with the decline in Tfh cells suggested that Tfr cells may be important to curtail Tfh cells at the peak of the GC response. We have shown above that Tfh cells did not expand at the peak of the response in immunised Tfr-deﬁcient mice (Fig. 3.6a-b).

We sought to analyse GC B cells and Tfh cells in the early and late stages of the GC response to a foreign TD antigen. For this, mice were immunised with ovalbumin (OVA) precipitated in alum and sacrificed at days 6 and 28 after challenge. GC B cell numbers were reduced in Tfr-deficient mice at day 6 after immunisation, a difference that was not seen at later time points, while Tfh cells were comparable in Tfr-deficient and sufficient mice at all time points (Fig. 3.7). Together, these results suggest that the absence of Tfr cells do not influence the proportions of Tfh or GC B cells at the peak or late stages of the GC reaction, but it might support expansion of GC B cells in the initial stages of an immune response.

61 CHAPTER 3. RESULTS

Day 6 Day 28 3 0.8

0.6 2

0.4

1 0.2 Tfh (% of T cells) T Tfh (% of Tfh (% of T cells) T Tfh (% of

0 0.0 Control Tfr- Control Tfr-

1.5 0.4 *** ** 0.3 1.0

0.2

0.5 0.1 Tfr (% of T cells) T Tfr (% of Tfr (% of T cells) T Tfr (% of

0.0 0.0 Control Tfr- Control Tfr- ) ) 1.5 3 + + *

1.0 2

0.5 1 GC B cells (% of B220 GC B cells (% of B220 0.0 0 Control Tfr- Control Tfr- Figure 3.7. Effect of Tfr deficiency at early and late time points. Quanti- fication of PD-1hi CXCR5hi Tfh cells (pre-gated on CD4+ Foxp3− CD44+), PD-1hi CXCR5hi Tfr cells (pre-gated on CD4+ Foxp3+ cells), and CD38− FAS+ GC B cells (pre-gated on B220+ cells) in OVA-immunised mice of the indicated genotype at day 6 and day 28 after challenge (Control: Bcl6+/+.CreFoxp3, n=7 for day-6 and n=9 for day-28; Tfr-: Bcl6fl/fl.CreFoxp3, n=9 for day-6 and n=6 for day-28). Bars indicate median values. Each dot represents an individual mouse.

62 CHAPTER 3. RESULTS

3.1.5 Tfr-deﬁcient mice have elevated immunoglobulins

We next sought to compare the titres of circulating IgM and total IgG in Tfr-sufficient and -deficient mice. Evaluation of antibody titres in unimmunised Tfr-deficient and sufficient mice revealed normal total IgM and IgG in circulation. Nevertheless, assessment of IgG subclasses revealed a 2.93-fold selective elevation in IgG1 (Fig. 3.8).

A median 3.7-fold increase in IgA was also seen in Tfr-deficient mice (p < 0.05). Basal IgE was also significantly elevated in mice lacking Tfr cells, who had 5.3-fold increase total IgE than Tfr-sufficient mice (p < 0.0001).

63 CHAPTER 3. RESULTS

104 104 103 ***

103 103 g/ ml

102 g/ml 102 102 IgG1

IgM μ g/ml 1 1 10 IgG μ 10

0 0 10 10 101 Control Tfr- Control Tfr- Control Tfr-

102 5 * 10 **** 4

1 l

ml 10 m 103 ng / ng /

A E 102 g 0 g I 10 I 101

10-1 100 Control Tfr- Control Tfr- Figure 3.8. Basal circulating Immunoglobulins. Quantification of immunoglobulins IgM, IgG, IgG1, IgA, and IgE in the serum of unimmunised mice of the indicated genotype by ELISA. Control: Bcl6+/+.CreFoxp3; Tfr-: Bcl6fl/fl.CreFoxp3. Bars indicate median values. Each dot represents data obtained from one mouse. Plots are representative of three independent experiments.

64 CHAPTER 3. RESULTS

3.1.6 GCs in Tfr-deﬁcient mice are enriched in non-antigen-

speciﬁc B cells

Our group previously reported that mixed bone marrow chimeric mice that cannot form Tfr cells have GCs that are enriched in non-antigen-speciﬁc B cells(Linterman et al. 2011). We tested whether this was also the case in the current model of Tfr deﬁciency.

Mice were primed with OVA in alum, and then challenged 3 weeks later with soluble OVA intravenously. Five days after the challenge spleens were harvested and GC B cells and OVA-specific B cells were enumerated. We confirmed a paucity of OVA-specific GC B cells amongst total GC B cells in mice lacking Tfr cells compared with littermate controls (Fig. 3.9). This suggests that Tfr cells may repress the recruitment of non-antigen-specific B cells or B cells with low avidity for the immunising antigen.

65 CHAPTER 3. RESULTS

a b

Control 25

s ** 20 cel l

AS B F 15 C G

GC B A 10 V O 5 Tfr- % 0 Control Tfr- AS F

GC B CD38 FAS OVA Figure 3.9. Antigen-specificity of GC B cells. a) Representative flow cytometric plots and b) quantification of OVA-specific cells pre-gated on GC B cells in mice of the indicated genotype (Control: Bcl6+/+.CreFoxp3; Tfr-: Bcl6fl/fl.CreFoxp3). Mice were immunised intraperitoneally with OVA in alum on day 0, boosted with soluble OVA intravenously on day 21, and sacrificed on day 5 after boost. Bars indicate median values. Each dot represents an individual mouse.

3.1.7 Tfr cells repress GC B cell-derived plasma cell form-

ation

In our attempt to investigate in more depth the GC B cell compartment in Tfr-deficient mice, we observed an intriguing population of CD138+ cells within Fas+CD38−Bcl6+ GC B cells that has not been previously observed in wild type umimmunised mice. Pre-plasmablasts arising in the GC have been identified on the basis of IRF4 expression but these cells do not express CD138 (Ise et al. 2018). We speculate this CD138+ GC B cells represents a population of cells that have recently differentiated into plasma cells, and we designate them hereafter as “early GC-derived plasma cells” (GCPC). Enumeration of CD138+ cells amongst GC B cells revealed that this population was practically non-existent in WT mice but present in almost all Tfr-deficient littermate mice

66 CHAPTER 3. RESULTS

a :: B220 B220 Comp-BUV 395- A 0.99 8.47

CD138

b c

) 20 * Non-GC B 15

B c ell s GC B Mod e

o GCPC

f G C 10 o ed t s (%

C 5 P C Normali G 0 Bcl6 Control Tfr- Figure 3.10. Abnormal early-plasma cell phenotype in GC B cells from unimmunised Tfr-deficient mice. a) Representative flow cytometric plots and b) quantification of CD138+ GC B cells in mice of the indicated genotype (Control: Bcl6+/+.CreFoxp3; Tfr-: Bcl6fl/fl.CreFoxp3). c) Flow cytometric histogram showing Bcl6 expression in non-GC B cells (CD38+FAS−), GC B cells (CD38−FAS+), and GC plasma cells (GCPC, CD138+B220lo GC B cells) Data is representative of two independent experiments.

(Fig. 3.10a-b). They still express intermediate levels of Bcl6 further suggesting a GC origin and lack of terminal diﬀerentiation into BCL6− plasma cells (Fig. 3.10c). Together these data suggest that Tfr cells repress plasma cell formation from GCs, and given the role of regulatory cells in the control of autoimmunity, this repression might be speciﬁcally targeted to self-reactive B cells.

3.1.8 Tfr cells repress the emergence of autoantibodies

to nuclear proteins

Given the enrichment in non-antigen-speciﬁc B cells in GCs from Tfr- deﬁcient mice and the propensity of these cells to commence plasma cell

67 CHAPTER 3. RESULTS

diﬀerentiation, we considered the possibility that these cells may include self- reactive B cells that can produce autoantibodies.

We first analysed the serum from Tfr-deficient and sufficient mice for the presence of lupus-associated antibodies against antinuclear antibodies (ANAs) and extractable nuclear antigens (ENAs). We did not see obvious differences in the proportion of ANA+ mice between Tfr-sufficient and Tfr-deficient mice (Fig. 3.11b).

Intriguingly, and despite the absence of elevated ANAs, all Tfr-deﬁcient mice tested had detectable ENAs (Fig. 3.11a). As opposed to ANAs, that are predominantly directed against nuclear DNA, the ENA panel detects the presence of autoantibodies in the blood that react with proteins in the cell nucleus, including Ro/SS-A (Sjogren’s Syndrome-related antigen A), La/SS-B (Sjogren’s Syndrome-related antigen B), Sm (Smith antigen), RNP (Ribonuclear protein), Scl-70 (Topoisomerase 1), and Jo-1 (histidyl tRNA synthetase). We considered this interesting given that Tfr cells are likely to recognise peptides presented on MHC class II rather than DNA, and it is likely that the peptide recognised is a self-antigen.

We developed a FACS-based assay to detect nucleosome-binding B cells, adapted from Gies et al. (Gies et al. 2017). For this, nucleosomes were extracted from the human HEK293 cell line by salt extraction and conjugated to the fluorochrome Alexa 488. Total mouse splenocytes were incubated with fluorochrome-conjugated nucleosomes for 30 minutes, and the proportion of nucleosome-binding cells were enumerated by flow cytometry. Very low numbers of nucleosome-binding B cells were detected in Tfr-sufficient mice, which did not change significantly in mice lacking Tfr cells (Fig. 3.11c-d).

68 CHAPTER 3. RESULTS

Given the reactivity of Tfr-deficient mouse serum with ENAs, we also tested reactivity against purified nucleosomes by ELISA. Nucleosomes consist of a octamer core of histones H2A, H2B, H3, and H4 tightly wrapped by 147 base pairs (bp) of DNA. Cores are then compacted into a higher structure in a manner dependent on the linker H1 histone(Schalch et al. 2005). Serum from Tfr-deficient mice did not display obvious reactivity against salt-extracted nucleosomes (Fig. 3.11a).

Antibodies against native or post-transcriptionally modified histones are typically found in B cell-mediated autoimmune diseases including SLE, lupus- nephritis, drug-induced lupus, juvenile rheumatoid arthritis and rheumatoid arthritis patients(Catrina et al. 2017). Histones are presented in the thymus in an AIRE-dependent manner(Adamopoulou et al. 2013). It is therefore likely that Tregs recognising histones are produced in the thymus, and may later differentiate into Tfr cells that could regulate B cell responses against nucleosome components. We considered the possibility that the DNA wrapping the histones may not be allowing detection of anti-histone antibodies. To test whether Tfr deficient mice developed anti-histone antibodies, we purchased human purified core histones as well as H1 histone, and performed ELISA using serum from unimmunised mice (Fig. 3.11a).

69 CHAPTER 3. RESULTS m

a 15 1.5 n 3 *** 450

10 D 450n m 1.0 2 O - D - RU O -

5 EN A 0.5 1

0 dsDN A 0.0 0 Control Tfr- Control Tfr- Nucleosomes Control Tfr-

2.5 4 ** 450n m 2.0 b D 3 Positive O

- 1.5 100 Borderline 2 Negative 1.0 ones 1

His t 0.5 50

0.0 Histone H1 - OD 450 nm 0 Core Control Tfr- Control Tfr-

0 Control Tfr- c Control Tfr- d 3 cells )

B 2 f G C o 1 (% CD38

Nuc+ 0 Control Tfr- Nucleosome Figure 3.11. Tfr- mice develop spontaneous autoantibodies against nuclear proteins. a) Quantification of autoantibodies against the indicated self-antigen by ELISA. b) Anti-nuclear antigen antibodies (ANAs) were measured in the serum of unimmunised mice by indirect immunofluorescence on Hep-2 slides. Data is representative of two independent experiments using at least 10 mice per genotype. c) Representative flow cytometric plot and d) quantification of the proportion of nucleosome-binding GC B cells in mice of the indicated genotype (Control: Bcl6+/+.CreFoxp3; Tfr-: Bcl6fl/fl.CreFoxp3). Bars indicate median values. Each dot represents data obtained from one mouse.

70 CHAPTER 3. RESULTS

Together these results suggest that Tfr mice develop autoantibodies to nuclear components other than DNA, including histones.

3.1.9 Tfr cells repress the emergence of tissue-speciﬁc an-

tibodies

Following our observation of autoantibodies against nuclear protein antigens in Tfr-deficient mice, we considered the possibility that Tfr cells may play a broader role in the repression of autoantibodies to tissue-specific antigens (TSAs) ectopically expressed in the thymus. Mice lacking Tregs or the AIRE protein that is required for thymic expression of a broad range of tissue antigens develop autoantibodies to eye(DeVoss et al. 2006) and stomach(Gavanescu et al. 2007). Thus, we evaluated the presence of the autoantibodies to pancreas, stomach and salivary gland (previously described in Aire-deficient mice) in addition to retina, colon and skin that have been described in other autoimmune diseases(Folwaczny et al. 1997; Witte et al. 2018; Adamus 2018). For this, we incubated serum from Tfr-deficient and sufficient mice on tissue sections from RAG1−/− mice, which lack B cells and thus, endogenous antibodies.

We were surprised to detect antibodies binding these three tissues in a large proportion of mice lacking Tfr cells. Specifically, 80%, 60% and 100% of Tfr-deficient mice had antibodies against exocrine pancreas, stomach and salivary gland respectively, compared to 20%, 0% and 20% of Tfr-sufficient mice (Fig. 3.12a, c). We also specifically tested autoantibodies to the main stomach autoantigen, the gastric parietal cell proton pump (GPCA) using a commercial ELISA kit and again found elevated anti-GPCA titres in Tfr-deficient mice (Fig. 3.12d). By contrast, and similar to what has been reported in Aire−/− mice, no autoantibodies were detected in liver, retina, colon, and brain (Fig.

71 CHAPTER 3. RESULTS

3.12b).

72 CHAPTER 3. RESULTS

a Pancreas Stomach Salivary Gland Control Tfr-

Liver Eye Colon Control Tfr-

Not affected b Affected c Pancreas Stomach Salivary G. 1.6 100 **

80 mic e 1.4 (RU )

ed 60 ec t f 40 1.2 a f o

20 anti-GPC A

% 1.0 0 Control Tfr- Tfr- Tfr- Tfr- Control Control Control Figure 3.12. Tfr- mice develop spontaneous tissue-specific autoantibodies. a) Representative images of indirect immunofluorescence using Rag1−/− mouse tissue frozen sections stained with serum from mice of the indicated genotype. b) Percentage of mice of the indicated genotype harbouring autoantibodies against the specified organ. c) Relative quantification of autoantibodies against gastric parietal cells (GPCA) in mice of the indicated genotype by ELISA. Control: Bcl6+/+.CreFoxp3; Tfr- : Bcl6fl/fl.CreFoxp3. Bars indicate median values. Each dot represents data obtained from one mouse.

73 CHAPTER 3. RESULTS

3.1.10 Tfr cells repress the increase in IgE titres upon

immunization

Having observed an increase in basal IgE in mice lacking Tfr cells, we next tested whether Tfr cells also repressed IgE responses to exogenous protein antigens. For this, mice were first immunised with OVA precipitated in alum intraperitoneally and boosted intravenously with soluble OVA on day 21. Mice were bled on day 19 and 34 after prime. While there was a clear IgG1 response after OVA-immunisation, total and OVA-specific IgG1 titres were comparable between Tfr-sufficient and Tfr-deficient mice (Fig. 3.13a). The differences in baseline IgE between the two groups of mice in basal IgE were maintained after immunisation, with a 2-fold increase in total and OVA-specific IgE seen in Tfr-deficient mice compared with Tfr-sufficient mice (Fig. 3.13b).

A similar result was observed when mice were immunised with peanut extract, a well-established food allergen known to induce IgE responses and anaphylaxis. Mice were orally sensitised with 1 mg of peanut extract and 10 µg of cholera toxin on days 0 and 7, boosted orally on day 21 with peanut extract in saline solution, and sacrificed on day 28. We observed a robust total IgG1 response but there was no statistically significant differences between Tfr-deficient and -sufficient mice. Tfr-deficient mice had greater IgE without significantly enhancing antigen-specific IgG production (Fig. 3.13a-b).

74 CHAPTER 3. RESULTS

a OVA Peanut Extract Control ns 0.6 ns 3 Tfr-

ns 2 0.4 ns 1 otal IgG1 (mg/ml) T 0 0 15 80

** 60 **** 10 40 5 * 20 * otal IgE ( μ g/ml) T 0 0 d19 d34 d0 d28 b OVA d34 Peanut extract d28 4 ns 3 ns

2 2

Ag-speciﬁc IgG1 (OD) 0 0

4 ns 2 * 3

1 2

1 0

Ag-specific IgE (OD) 0 Control Tfr- Control Tfr- Figure 3.13. Increased immunisation-induced IgE in Tfr-deficient mice. a) Quantification of total serum IgG1 and IgE after OVA in alum and peanut extract immunisation at the indicated time points. b) Quantification of antigen-specific IgG1 and IgE as for a). Control: Bcl6+/+.CreFoxp3; Tfr-: Bcl6fl/fl.CreFoxp3. Bars indicate median values. Each dot represents data obtained from one mouse.

75 CHAPTER 3. RESULTS

Strikingly, Tfr-deﬁcient mice did not survive an intravenous boost with OVA in alum 28 days later, with 100% mice succumbing from anaphylaxis within less than 10 minutes of the intravenous boost, and all Tfr-suﬃcient control littermates surviving (Fig. 3.14). By this time point, 28 days post-primary, IgE had increased further (Fig. 3.13a)

76 CHAPTER 3. RESULTS a b Control Tfr-

l 100 a v 100 μg OVA 50 μg Clinical i in alum (i.p.) OVA (i.v.) Scoring 50 en t s ur v er c P D0 D28 D28 0 Challenge Boost +40 min 0 10 20 30 40 Minutes after OVA boost Figure 3.14. Tfr-deficient mice survival after high dose re-challenge. a) OVA in alum immunisation strategy. b) Kaplan-Meier curve showing the survival of Tfr-deficient (n=4) and Tfr-sufficient (n=4) mice after prime-boost immunisation. Control: Bcl6+/+.CreFoxp3, blue dashed line; Tfr-: Bcl6fl/fl.CreFoxp3, red dotted line.

3.2 Neuritin: a key eﬀector of B cell regulation

by Tfr cells

3.2.1 Tfr cells express neuritin mRNA

In an effort to identify the key molecules that may mediate the ability of Tfr cells to regulate B cell responses, we analysed a previously published RNA expression microarray dataset, comparing T naive, Tfr, Tfh and Treg cells (Linterman et al. 2011). Out of 1283 differentially expressed genes, the neurotrophic factor neuritin (encoded by the Nrn1 gene) was the second most highly upregulated gene after IL2Ra (CD25) in Tfr cells (2.96 fold-change) compared to Tfh cells. Of note, Nrn1 was practically absent in T naive and Tfh cells (Fig. 3.15a). Compared to naive T cells, Tfh, T effector, Tregs and Tfr cells had an increase of 1.4, 1.45, 1.93, and 4.36 fold-change in Nrn1 expression, respectively.

We conﬁrmed selective expression of Nrn1 in mouse Tfr cells by sorting CD4+ CXCR5+ PD-1hi Foxp3+ cells from Foxp3-GFP reporter mice. Tfr cells had 5-fold higher expression of Nrn1 than Tfh cells and other T cell subsets

77 CHAPTER 3. RESULTS

including Tregs (Fig. 3.15b).

Next, we evaluated expression of NRN1 in human follicular T cell subsets by qPCR. As observed in mice, Nrn1 was predominantly expressed by human Tfr- like cells (CD4+CD25hiCD127loCXCR5hiPD-1hi) and absent in naive, memory, GC, and PC B cells (Fig. 3.15c).

78 CHAPTER 3. RESULTS

a Down-regulated in Tfr Upregulated in Tfr 0.05 Bcl6 1e−02 Myc IL21

1e−03 IL4

1e−04 p-value

1e−05 Nrn1 1e−06 Cd25 −2 0 2 log2 FC b Mouse c Human 25 5

20 4 expressio n expressio n 15 3

2 10 Nrn1 Nrn1 ive ive 5 1 Rela t

Rela t 0 0

Tfr Tfh reg Tfr Tfh reg PB T T GC B ff/mem naive ff/mem naive e T e T Mem BNaive B T T Figure 3.15. Nrn1 expression in Tfr cells. a) Volcano plot showing diﬀerentially expressed genes in murine Tfr cells compared to Tfh cells by microarray analysis. b) Nrn1 gene expression in mouse and b) human samples by qPCR. In dot plots, each dot represents data obtained from one mouse b) or donor c). Data is presented relative to Tfh values using the ∆∆Cq method. qPCR data is representative of three independent experiments.

79 CHAPTER 3. RESULTS

3.2.2 Neuritin: a CNS neuropeptide

As mentioned in Chapter 1, neuritin can act as a neurotrophic factor downstream of NMDA receptors and other neuromodulators such as NGF, NT3 and BDNF. The expression and activity of neuritin in post-mitotic presynaptic neurons correlates with periods of active synaptogenesis and maturation of synapse(Nedivi et al. 1998). Neuritin in vivo administration can promote nerve regeneration after injury and has a critical role in learning and memory formation(Fargo et al. 2008; Karamoysoyli et al. 2008; Fujino et al. 2011; Zhao et al. 2015; Wang et al. 2016). More recently, Zhang et al. described the interaction of neuritin and the E3 ligase NEURL1 in inhibiting endocytosis of Notch-ligand as a possible mechanism to explain neuritin’s ability to promote neurite extension(Zhang et al. 2017a).

Neuritin is a GPI-anchored protein that can be cleaved by the enzyme phosphatidylinositol-speciﬁc phospholipase C (PI-PLC). Neurtin has no intracellular domain, thus, it can signal extracellularly. Neuritin has been shown to be highly expressed in the nervous system and in some cancer cell lines but it has not been shown to be signiﬁcantly expressed or have a relevant role in the immune system so far.

To date, the receptor for neuritin remains unknown, however, it has been suggested that it might act in the signalling pathway of receptor tyrosine kinases possibly involving the insulin(Yao et al. 2012) or the ﬁbroblast growth factor (FGF)(Shimada et al. 2016) receptor pathway.

80 CHAPTER 3. RESULTS

3.2.3 Neuritin protein is expressed by Tfr cells

Having identified neuritin mRNA in Tfr cells, we next sought to evaluate Neuritin protein expression in human lymphocytes. Commercial antibodies to neuritin did not appear to work well by flow cytometric staining, but had been reported to work well by immunofluorescence and western blot. We analysed neuritin expression in sorted T cell subsets using these approaches.

FACS-purified T cell subsets were subjected to cytospin and immunofluores- cent staining using a rabbit polyclonal anti-neuritin antibody. Neuritin staining appeared specific and confirmed that most Tfr cells expressed neuritin protein in human samples (Fig. 3.16a). Neuritin protein was also found in a small proportion of Tregs and was absent in Tfh cells.

By western blot (using the same antibody), we observed a band of the expected size (approx. 30 kDa) for dimerised complete neuritin protein in Tfr cells. Tfr cells contained the highest expression of neuritin, with low amounts detected on Tregs and Tfh cells (Fig. 3.16b, c).

81 CHAPTER 3. RESULTS

a Tfr Tfh

Treg

b c Neuritin

l 15 35 kDa e le v 10 ein pro t

e v

i 5 β-Actin Rela t 0 40 kDa Tfr Tfh Treg naive f/memT ef Tfr Tfh Treg T eff/mem T naive T

Figure 3.16. Neuritin protein expression in lymphocytes. a). Cytospin images of FACS sorted human Tfr, Tfh, and Treg cells stained with DAPI (blue) and anti-neuritin (red) b) Western Blot of Neuritin in the indicated T cell subsets. β-actin was used as a loading control. c) Quantiﬁcation of Neuritin protein levels by densitometry as for blot in (b). Expression levels were normalised to those of T eﬀ/mem cells. Dotted grey lines connect samples derived from the same donor.

82 CHAPTER 3. RESULTS

3.2.4 Neuritin-expressing cells localise inside or in the

vicinity of GCs

We also determined the anatomical localisation of neuritin-expressing cells. Staining for neuritin in frozen sections from immunised mouse spleen and human tonsil revealed rare neuritin-positive cells within GCs and at the T:B border (Fig. 3.17), consistent with the described localisation of Tfr cells in mouse(Sage et al. 2013) and human GCs(Sayin et al. 2018).

Of note, neuritin-expressing cells were found to be adjacent to B cells staining positive for IgD in most of the analysed tissue samples. This was the case for both neuritin-expressing cells found within GCs and those at the T:B border. While it is possible that these IgD+ cells represent naive B cells, IgD+ cells have been described within GCs(Liu et al. 1996) and it has been suggested these may represent self-reactive B cells(Sabouri et al. 2016; Sabouri et al. 2014).

83 CHAPTER 3. RESULTS

Figure 3.17. Neuritin-expressing cells localise nearby and within GCs. Immunoﬂuorescence images of frozen a) SRBC-immunised mouse spleen and b) human tonsil. Cryosections were stained with anti-IgD (green), anti-neuritin (red), and DAPI (blue).

84 CHAPTER 3. RESULTS

3.2.5 Neuritin targets GC B cells

Neuritin is produced as both a secreted and a membrane-bound protein. We next aimed to identify the cells binding secreted neuritin. Given that the receptor for neuritin is still unknown, we conjugated recombinant active neuritin to a ﬂuorophore and incubated this conjugate with single lymphoid cell suspensions from mouse spleen and human tonsil. Flow cytometric analysis revealed that neuritin preferentially bound to mouse GC B cells (Fig. 3.18). In humans, neuritin bound to most human B cell subsets, with the highest amounts also found in GC B cells, followed closely by memory B cells, and slightly lower levels in naive B cells (Fig. 3.19).

85 CHAPTER 3. RESULTS

a B cells GCB

Th cells GL-7 B220

Non-GCB CD4 FAS

b c FMO I

F 2400 Th cells M Non-GCB 1800

647 GCB 1200

in-A F 600 Neuri t

Normalised To Mode Normalised To 0 Neuritin-AF 647 FMO cells GCB T Non-GCB

Figure 3.18. Mouse GC B cells bind neuritin in vitro. a) Flow cytometric plots showing gating strategy used to purified Th cells (B220− CD4+), non-GC B cells (B220+ GL-7− FAS−), and GC B cells (B220+ GL-7+ FAS+). b) Representative flow cytometric histogram and c) quantification of neuritin-Alexa Fluor 647 gMFI in different populations of lymphocytes. Each dot represents data obtained from one mouse from independent experiments. Dotted grey lines connect samples derived from the same tonsil donor.

86 CHAPTER 3. RESULTS

B cells a B mem

GCB CD27 CD19

Th cells B naive CD4 CD38 b c FMO

I Th cells F 250

M B naive 200 B mem 647 150 GCB 100 in-A F 50

Neuri t 0 Normalised To Mode Normalised To

Neuritin-AF 647 FMO cells GCB T B naiveB mem Figure 3.19. Human GC B cells bind neuritin in vitro. a) Flow cytometric plots showing gating strategy used to purified Th cells (CD19− CD4+), memory B cells (B mem: CD19+CD38− CD27+), naive B cells (B naive: CD19+CD38− CD27−), and GC B cell (GCB: CD19+CD38+CD27int) from human tonsils. b) Representative flow cytometric histogram and c) quantification of neuritin-Alexa Fluor 647 gMFI in different populations of lymphocytes. Dotted grey lines connect samples derived from the same tonsil donor. Data is representative of three independent experiments.

87 CHAPTER 3. RESULTS

To further confirm the presence of a receptor for neuritin in B cells, we took advantage of a chemoproteomic approach using the TriCEPS reagent (Frei et al. 2013). TriCEPs is a trivalent biotinylated molecule that can be coupled to an orphan ligand of interest and, at the same time, to oxidised glycosylated receptors on the surface or intracellular space of living cells. To establish the binding of an orphan ligand to a particular cell type potentially bearing a specific receptor, ligand-coupled TriCEPs is incubated with living cells to allow TriCEPs-mediated crosslinking of the putative receptor and the ligand of interest. Cells are then lysed and the biotin tag is used to purify the ligand-TriCEPs-receptor complexes to be analysed by quantitative mass spec- trometry (Fig. 3.20a). Freshly sorted GC and naive B cells were incubated with ligand-coupled TriCEPS and binding was assessed using fluorescently-labelled streptavidin (SA). TriCEPs coupled to transferrin and quenched with glycine were used as positive and negative controls respectively, because B cells like all lymphoid cells express transferring receptors but glycine on the other hand has no binding preference for a receptor. Flow cytometric analysis revealed that neuritin can indeed bind to receptors in human GC B cells and also to naive B cells to a lesser extent (Fig. 3.20b).

88 CHAPTER 3. RESULTS

a Attachment to N-terminus and lysines (NHS-ester) Ligand of the ligand of interest

Cross-linking to Visualisation oxidised glycans Biotin and purification using Streptavidin Receptor SA b Naive B cells GC B cells FMO (no SA) Glycine-TriCEPS Neuritin-TriCEPS Transferrin-TriCEPS Normalised To Mode Normalised To TriCEPS-SA Figure 3.20. Human B cells bind neuritin (TriCEPS assay). a) Schematic representation of TriCEPS trivalent molecule. b) Flow cytometric histogram showing the gMFI of TriCEP coupled to glycine (blue, negative control), neuritin (magenta, ligand of interest) or transferrin (green, positive control) in the indicated B cell subset.

89 CHAPTER 3. RESULTS

Intriguingly, and despite the absence of neuritin mRNA in human GC B cells by either qPCR (Fig. 3.15c) or RNA-seq (Fig. 3.21a), GC B cells appeared to contain neuritin protein as assessed by immunoﬂuorescence (Fig. 3.21b). It is therefore possible that GC B cells can uptake neuritin from the extracellular space. This hypothesis needs further experimental conﬁrmation.

90 CHAPTER 3. RESULTS

a human Nrn1 human CD19 4 800

3 600

2 400 RPK M RPK M 1 200

0 0

PB B cell onsil PC PB B cell onsil PC onsil naive onsil GCBT onsil naive onsil GCBT Cord naive BM plasmaT T Cord naive BM plasmaT T onsil B memory onsil B memory T T b

DAPI Nrn1

Figure 3.21. GC B cells contain neuritin protein in the absence of Nrn1 mRNA. a) RNA-seq data showing Nrn1 and CD19 expression in FACS-puriﬁed human B cell populations. b) Representative immunoﬂuorescence images of FACS sort human GC B cell cytospin. Cells were stained with DAPI (blue) and anti-neuritin (red).

91 CHAPTER 3. RESULTS

3.2.6 Neuritin represses plasma cell formation and in-

creases BCL6

We next investigated the eﬀect of soluble full-length human neuritin on sorted human GC B cells cultured for 5 days with anti-CD40 and the cytokine IL-4, which promote survival and plasma cell diﬀerentiation. We observed a reduction in the percentage of plasma cells (CD138+) in the presence of neuritin (Fig. 3.22a-b).

In parallel to, and consistent with, the decline in plasma cell formation, neuritin treatment augmented BCL6 protein formation on human GC B cells (Fig. 3.22c-d). BCL6 is the transcription factor that drives GC B cell diﬀer- entiation and antagonises the function of Blimp-1, the master transcription factor needed for plasma cell diﬀerentiation.

92 CHAPTER 3. RESULTS

a Gated on CD27+CD38+ Nil 250 ng/ml 1 μg/ml 2 μg/ml CD138

CD38 b c d 30 Human 1600

s *

Nil I el l c

F 1200 20 Neuritin o Mode a M

m 800 la s 10 CL 6 P B

400 % 0 0 Nil Neuritin Normalised BCL6 Nil Neuritin Figure 3.22. Neuritin suppresses plasma cell differentiation from GC B cells in vitro. a) Representative flow cytometric plots and b) quantification of the proportion of CD138+ cells from CD27int CD38+ GC B cells cultured for 4 days in the presence of anti-CD40, IL-4, and the indicated neuritin concentration. c) Representative flow cytometric histogram and d) quantification of BCL6 MFI from cultured CD27+ CD38+ GC B cells as for (a) stimulated with neuritin 2 µg/ml.

93 CHAPTER 3. RESULTS

Given that mouse GC B cells fail to survive in culture, we took advantage of the Nojima murine ﬁbroblast feeder cell line that stably expresses BAFF, IL-21 and CD40L to support survival and B cell diﬀerentiation from naive B cells through to GC B cells and plama cells. When naive B cells isolated from mouse spleen where cocultured with Nojima cells for 4 days, recombinant neuritin also increased Bcl6 protein formation (Fig. 3.23), as previously observed in human GC B cells (Fig. 3.22c-d).

94 CHAPTER 3. RESULTS

a Mouse Nil Neuritin Normalised To Mode Normalised To Bcl6 b Nil 500 ng/ml 2 μg/ml

14.9 17.2 31 FAS

Bcl6 c ) 50 )

6000 8 + 10 ) + cells )

B 40 8

f 6 4000 o 30

CD138 6 (% lo

+ 4 20 as 4

2000 F + 2 10 2 Blc6 PC (B220

pre-PC GC B (CD138 Bcl6 MFI (GC B cells

0 % 0 0 0 NIL Neuritin NIL Neuritin NIL Neuritin NIL Neuritin Figure 3.23. Neuritin induces upregulation of Bcl6 in cultured mouse B cells. a) Representative flow cytometric histogram showing Bcl6 gMFI and b) percentage of Bcl6+FAS+ from MACS-purified mouse naive B cells co-cultured for 3 days with Nojima feeder cells in the presence of IL-4 and neuritin at the indicated concentration. c) Quantification of the indicated cell subset or gMFI in cells cultured as per (a-b), stimulated with neuritin 2 µg/ml.

95 CHAPTER 3. RESULTS

3.2.7 Neuritin represses IgE production in vitro

We have previously shown that mice lacking Tfr cells have elevated IgE. To test whether neuritin can directly act on B cells to regulate IgE production, we cultured human GC B cells in the presence of IL-13, known to promote IgE production. We observed a significant reduction in the amount of IgE in the supernatant of cultured cells treated with neuritin compared to those untreated (Fig. 3.24a) and this effect was dose-dependent (Fig. 3.24b). No differences were observed in IgG production, GC B cell survival or proliferation in these cultures. Reduced IgE could be a consequence of reduced plasma cell differentiation and general reduction in immunoglobulin production. To know whether the reduced IgE was selective, we also measured IgG in these cultures and found no significant differences at any neuritin concentration tested (Fig. 3.24b).

96 CHAPTER 3. RESULTS

a 15000 **

FI 10000 E g M g

I 5000

0 Nil Neuritin b c 400 * 32000

300 24000 FI FI

200 16000 E g M E g M g g I I 100 8000

0 0

g/ml g/ml μg/mlμ Blank Blank 0 ng/ml 1 2 0ng/ml 1μ 2μg/ml 180 ng/ml500 ng/ml 250ng/ml500ng/ml Figure 3.24. Neuritin represses IgE production in vitro. a) Quantification of IgE gMFI in FACS-purified human GC B cells before (grey) and after (red) stimulation with neuritin (2 µg/ml) by cytometric bead array (CBA). Data representative of three independent experiments. Each dot represents one donor b) Quantification of IgE and IgG gMFI in FACS-sorted human GC B cells after stimulation with neuritin at the indicated concentration by CBA. Bars depict medians, and each dot represents data obtained from one donor.

97 CHAPTER 3. RESULTS

3.2.8 Neuritin represses ICOSL expression

Given that neuritin is a neurotrophic factor acting through synaptic interactions in the brain, we investigated the possibility of short-term effects of neuritin, as we described recently for dopamine(Papa et al. 2017). Opposite to the ability of Dopamine to cause translocation of ICOSL to the cell membrane within 30 minutes of culture, 30 minutes incubation with neuritin alone in the absence of second signals (anti-CD40) or third signals (cytokine stimulation), led to downregulation of surface ICOSL (Fig. 3.25). We have shown previously that ICOSL upregulation by GC B cells is important to increase the area of Tfh:B cell synaptic interaction and the focused delivery of CD40L to GC B cells. Our opposite results with neuritin – reducing ICOSL expression – suggest that this neurotrophic factor may increase the threshold for Tfh cell help delivery or facilitate dissociation of Tfh:GC B cell conjugates and, in doing so, decreases the chances of plasma cell differentiation. Of note, we observed no differences in CD25, CD86 or IL-21R expression in B cells after 30 minutes of incubation with neuritin alone.

98 CHAPTER 3. RESULTS

3000 ** 1500 FI FI

M 2000 1000 M g L b S O C 1000 CD8 6 g 500 I Nil Neuritin o Mode 0 T Nil neuritin Nil neuritin

500 2000

400 Normalised

FI 1500 FI

M ICOSL 300 M 1000 200 L-21R g CD2 5 g I 500 100

0 0 Nil neuritin Nil neuritin Figure 3.25. Neuritin induces downregulation of ICOSL on GC B cells in vitro. a) Representative ﬂow cytometric histogram and b) quantiﬁcation of ICOSL gMFI in human GC B cells before (grey) and after (red) stimulation with neuritin treatment (2 µg/ml). Data is representative of three independent experiments. Each dot represents data obtained from one donor.

99 CHAPTER 3. RESULTS

3.2.9 Neuritin limits Tfr and plasma cells in vivo

In order to test the role of neuritin in vivo, we generated mice that selectively lack neuritin in Foxp3-expressing T cells (Tregs and Tfr cells) by crossing a neuritin floxed mouse provided by Elly Nedivi (Massachusetts Institute of Technology, US)(Fujino et al. 2011) to the same Cre Foxp3 strain used to generate Tfr-deficient mice. Male neuritin-deficient mice (Neuritinfl/fl.CreFoxp3) showed normal number and proportion of Tregs compared to CreFoxp3 control mice after intraperitoneal immunisation of OVA in alum (Fig. 3.26a). The Treg suppressive capacity was not affected in the absence of neuritin (Fig. 3.26b-c).

100 CHAPTER 3. RESULTS

a Control Tfr- Nrn1- FoxP3 CD44 b 27 2400 Control Tfr- Nrn1- 18 1600 cell s reg T

9 800 Foxp3 MF I %

0 0

Tfr- Tfr- Nrn1- Nrn1- Control Control c d 80 Control Tregs Nrn1- Tregs No Tregs Control

io n Nrn1- 60 era t 40 proli f f o

20 %

Normalised To Mode Normalised To 0 CTV 0:1 1:16 1:8 1:4 1:2 1:1 Treg : T naive ratio

Figure 3.26. Neuritin-deficient mice have normal Treg proportion and suppressive capacity. a) Representative flow cytometric plots and b) quantification of Tregs (CD4+ Foxp3+). Bars indicate median values. Each dot represents data obtained from one mouse. c) Representative flow cytometric histograms showing proliferation profiles and d) quantification of proliferating naÃŕve T cells (B220− CD4+ Foxp3− CD44− CD62L+) cocultured with Tregs (B220− CD4+ Foxp3+ CD44lo PD-1−) from control (Neuritin+/+.CreFoxp3, in green) or Nrn1- (Neuritinfl/fl.CreFoxp3, in blue) mice in the presence of antigen presenting cells (APCs, CD4−) from 3 mice from each genotype. Vertical bars and dots show range and median values, respectively. Data is representative of two independent experiments.

101 CHAPTER 3. RESULTS

Interestingly, we did not observe the same effect seen in Tfr-deficient mice on GC B cell antigen specificity after OVA immunisation in neuritin-deficient mice (Fig. 3.27).

a b

Control 22

s 20 cel l

B 18 F

G 16

GC B A

V 14 O

Nrn1- % 12 10 Control Nrn1- AS F

GC B CD38

FAS OVA Figure 3.27. Antigen-specificity of GC B cells in neuritin-deficient mice. a) Representative flow cytometric plots and b) quantification of OVA-specific cells pre-gated on GC B cells in mice of the indicated genotype (Control: Bcl6+/+.CreFoxp3, n=6; Nrn1-: Neuritinfl/fl.CreFoxp3, n=5). Mice were immunised intraperitoneally with OVA in alum on day 0, boosted with soluble OVA intravenously on day 21, and sacrificed on day 5 after boost. Bars indicate median values. Each dot represents an individual mouse.

102 CHAPTER 3. RESULTS

3.2.10 Systemic cytokines in Tfr- and neuritin-deﬁcient

mice

We tested whether Tfr cell or neuritin deficiency caused any significant cytokine dysregulation. Evaluation of serum revealed a cytokine pattern that differed from sanroque mice (homozygous Roquinsan/san mutant) that have overactive Tfh cells(Vinuesa et al. 2005; Odegard et al. 2009). As opposed to sanroque mice, Tfr-deficient mice did not have elevated IL-6 or IL-21, both associated with Tfh responses in mice. By contrast, IL-17, which is normally not detected in the serum of sanroque mice, was modestly elevated in mice lacking Tfr cells. We did find augmented IFN-γ in unimmunized Tfr-deficient mice, although this cytokine is much higher in sanroque mice (Fig. 3.28). Excessive IFN-γ and/or IL-17 production, have individually been associated with the development of inflammatory and autoimmune reactions to both systemic and organ-specific antigens(Lee et al. 2012; Hsu et al. 2008). Only IL-6 appeared elevated in neuritin-deficient mice.

103 CHAPTER 3. RESULTS

5 100 * 4 80

3 60 pg/m l 2 40 IL-6 pg/m L IFN- γ 1 20

0 0 Control Tfr- Nrn1- Control Tfr- Nrn1-

40 50

l 40 30 30 20 pg/m l 20

IL-10 pg/m 10 IL-17 A 10

0 0 Control Tfr- Nrn1- Control Tfr- Nrn1- Figure 3.28. Systemic cytokines in Tfr- and neuritin-deficient mice. Quan- tification of cytokines IFN-γ, IL-6, IL-10, and IL-17 in the serum of unimmunised mice of the indicated genotype by meso-scale. Control: Bcl6+/+.CreFoxp3; Tfr-: Bcl6fl/fl.CreFoxp3. Bars indicate median values. Each dot represents data obtained from one mouse.

To investigate follicular populations in the absence of neuritin, we immunised mice with OVA precipitated in alum intraperitoneally and then sacriﬁced 12 days later. As seen in Tfr-deﬁcient mice, mice lacking neuritin in Foxp3+ cells had modestly elevated Tfh cells but comparable GC B cell and Ki-67+ GC B compartments. Notably, mice lacking neuritin in Foxp3+ cells had elevated Tfr cells, suggesting that neuritin may directly or indirectly control Tfr numbers (Fig. 3.29).

Interestingly, and consistent with the in vitro data, Treg-derived neuritin appeared to limit plasma cell (PC) formation, as neuritin-deficient mice had a 2-fold increase in Blimp1+CD138+ plasma cells and a modest but statistically- significant increase in the geometric mean fluorescence intensity (gMFI) of Blimp-1 within plasma cells (Fig. 3.29).

104 CHAPTER 3. RESULTS

Neuritin-deﬁcient mice showed normal GC formation after SRBC immunisation (day 6) as shown by immunoﬂuorescence on spleen sections.

105 CHAPTER 3. RESULTS

a b Control Tfr- Nrn1- 15 6 ** * s s

el l **

10 el l 4 c c

Tf h Tf r

5 2 PD-1 % % CXCR5 0 0 Control Tfr- Nrn1- Control Tfr- Nrn1-

3 100 s 80 el l PD-1 c B c ell s 2 60 B

CXCR5

C 40 1 G 20 % i-67 + G C

0 K 0 Control Tfr- Nrn1- Control Tfr- Nrn1- CD38 1.6 3200 ** FAS * I ell s 1.2 F 2400 M

ma c 0.8 1600 la s limp-1 P 0.4 800

B % Blimp-1 0.0 0 CD138 Control Tfr- Nrn1- Control Tfr- Nrn1- c Control Tfr- Nrn1-

BCL6 IgD Figure 3.29. Follicular lymphocyte populations in neuritin-deficient mice. a) Representative flow cytometric plots and b) quantification of Tfh (CD4+ Foxp3− PD-1+ CXCR5+), Tfr (CD4+ Foxp3+ PD-1+ CXCR5+), GC B (CD38− FAS+), and plasma cells (Blimp-1+ CD138+) in mice of the indicated genotype. c) Immunofluor- escence of frozen sections from the spleen of SRBC-immunised mice of the indicated genotype (Bcl6: green; IgD: red). Control: CreFoxp3; Tfr-: Bcl6fl/fl.CreFoxp3; Nrn1-: Neuritinfl/fl.CreFoxp3. Bars indicate median values. Each dot represents data obtained from one mouse.

106 CHAPTER 3. RESULTS

Tfr-deficient mice showed elevated IgE, IgG1 and IgA in the absense of immunisation (Fig. 3.8). To evaluate if neuritin has a role controlling directly or indirectly the production of baseline immunoglobulins we analysed the sera of neuritin-deficient mice. No significant differences were found between control and neuritin-deficient littermates, although some Tfr-deficient mice had elevated IgG1 (Fig. 3.30).

107 CHAPTER 3. RESULTS

a 25 100 20 10 15

g/ml μ

A Ig ml IgM mg/ IgM 0.1 5

0 0.01 Control Nrn1- Control Nrn1- 40 10000

30 1000

20 100

ml ng/ IgE

IgG1 mg/ml IgG1 10 10

0 1 Control Nrn1- Control Nrn1- Figure 3.30. Neuritin-deficient mice baseline immunoglobulins. a) Dot plots showing quantification of IgM, IgG1, IgA, and IgE measured by ELISA in serum of unimmunised mice from the indicated genotype. Bars indicate median values. Each dot represents data obtained from one mouse. Control: Bcl6+/+.CreFoxp3; Nrn1-: Neuritinfl/fl.CreFoxp3.

In order to evaluate antibody responses in vivo in the absence of neuritin- expressing Tfr/Treg cells, neuritin-deficient mice and control CreFoxp3 littermates were immunised intraperitoneally with OVA precipitated in alum. Both Tfr-deficient and neuritin-deficient mice had modestly elevated total IgG1 and IgE 16 days after OVA immunisation (Fig. 3.31).

108 CHAPTER 3. RESULTS

a 100 μg OVA in alum (i.p.) Bleed

D0 D16 Challenge Serum analysis

b 8 80 ** * 6 * 60 *

4 g/ml 40 IgE μ IgG1 mg/m l 2 20

0 0 Control Tfr- Nrn1- Control Tfr- Nrn1-

Figure 3.31. Neuritin-deficient mice immunisation-induced immunoglobulins. a) Immunisation regime. b) Dot plots showing quantification of IgG1 and IgE measured by ELISA in serum of OVA-immunised mice from the indicated genotype. Bars indicate median values. Each dot represents data obtained from one mouse. Control: Bcl6+/+.CreFoxp3; Nrn1-: Neuritinfl/fl.CreFoxp3. Data representative of three independent experiments.

109 CHAPTER 3. RESULTS

3.2.11 Neuritin represses nucleosome-binding B cells and

tissue-speciﬁc autoantibodies

Previous reports have suggested that self-reactive B cells may arise within GCs as a consequence of somatic hypermutation in the context of an immune response to pathogen related antigens that result in the production of antibodies that crossreact with self-antigens(Suurmond and Diamond 2015; Putterman et al. 1996; Di Zenzo et al. 2012).

We asked then whether neuritin could constitute a mechanism by which Tfr cells repress autoreactive B cells. In order to detect self-reactive GC B cells, we incubated splenocytes with ﬂuorescently labelled nucleosomes for 30 mins ex vivo. Nucleosome-binding GC B cells were increased in mice lacking neuritin in Treg/Tfr cells. These nucleosome-binding B cells were mainly found within the GC B cell gate (Fig. 3.32a-b). As shown in the previous section, nucleosome-binding B cells could not be detected in Tfr-deﬁcient mice (Fig. 3.11a, c, d).

Anti-nucleosome autoantibodies were also detected in the circulation of these mice (Fig. 3.32c). Finding more self-reactive B cells in mice lacking neuritin in Tregs compared to Tfr-deﬁcient mice suggests that neuritin derived from a small subset of neuritin-expressing Tregs may also play a role in the suppression of anti-nucleosome antibodies.

Although there was a trend for increased ENAs as shown in the previous chapter for Tfr-deficient mice, this was not statistically significant in neuritin- deficient mice compared to littermate controls (Fig. 3.32c).

110 CHAPTER 3. RESULTS

a Control Nrn1- b ) 4 * ell s c 3 B

C G

f 2 o

% ( 1 + CD38 Nu c 0 Nucleosome Control Nrn1- c 8 2.0 m 2.5 n * m

6 45 0 2.0 1.5 D U 450 n

O R

4 1.5 D - -

O 1.0

A -

N 2 1.0 A E N 0.5 0 D 0.5 s d 0.0 0.0 Nucleosome s Control Nrn1- Control Nrn1- Control Nrn1-

m 4 4 ** m n

450 n

3 45 0 3 D

D O

- O

2 2 1 H one s

t 1 1 Hi s on e

0 Hi s 0 Cor e Control Nrn1- Control Nrn1-

Figure 3.32. Neuritin-deficient mice develop spontaneous systemic autoantibodies. a) Representative flow cytometric plots showing the gating strategy used to identify nucleosome-binding B cells in Tfr-sufficient (control) and Tfr-deficient (Tfr-) mice. b) Dot plot showing quantification as for (a). c) Dot plots showing the quantification of autoantibodies to systemic self-antigens in unimmunised mice of the indicated genotype. Bar represent medians and each dot individual mice. Each dot represents data obtained from one mouse.

111 CHAPTER 3. RESULTS

Similar to what was seen in Tfr-deficient mice, neuritin-deficient mice also developed spontaneous anti-gastric parietal cell (GPC) autoantibodies (Fig. 3.33) and autoantibodies against other tissue antigens including exocrine pancreas (78%), pancreatic islets (44%), stomach (78%), eye (66%), skin (66%), and colon (33%) (Fig. 3.33). These data suggest that neuritin is an important mediator of autoantibody repression and both Treg- and Tfr-derived neuritin are likely to contribute to this effect.

112 CHAPTER 3. RESULTS a Colon Eye Pancreas Stomach Control Nrn1-

Liver Salivary Gland Control Nrn1- b Not affected c Pancreas Pancreas Affected 1.6 exocrine Islets Stomach Eye Skin Colon *** 100

80 (RU ) 1.4 mic e

ed 60

ec t 1.2 f 40 a f o

20 anti-GPC A

% 1.0 0 Control Nrn1-

ControlNrn1- ControlNrn1- ControlNrn1- ControlNrn1- ControlNrn1- ControlNrn1- Figure 3.33. Neuritin-deficient mice develop spontaneous tissue-specific autoantibodies. a) Representative images of indirect immunofluorescence using Rag1−/− mouse tissue frozen sections stained with serum from mice of the indicated genotype. b) Percentage of mice of the indicated genotype harbouring autoantibodies against the specified organ (Pancreas exocrine: Control n=8, Nrn1- n=9; Pancreas islets: Control n=8, Nrn1- n=9; Stomach: Control n=8, Nrn1- n=9; Eye: Control n=3, Nrn1- n=3; Skin: Control n=3, Nrn1- n=3; Colon: Control n=5, Nrn1- n=3. c) Relative quantification of autoantibodies against gastric parietal cells (GPCA) in mice of the indicated genotype by ELISA. Control: Neuritin+/+.CreFoxp3; Tfr-: Neuritinfl/fl.CreFoxp3. Bars indicate median values. Each dot represents data obtained from one mouse.

113 CHAPTER 3. RESULTS

3.2.12 Self-reactive GC B cells in neuritin-deﬁcient mice

display an "early-plasma cell" phenotype

We described in the previous chapter how Tfr-deficient mice have an abnormal population of plasma cells within the GC B cell compartment. We evaluated if this phenotype could also be found in neuritin-deficient mice. We found that, similar to Tfr-deficient mice, neuritin-deficient mice have a significantly increased B220loCD138+ population in GC B cells in the absence of immunisation (Fig. 3.34a-b).

We next attempted to assess whether nucleosome-binding GC B cells could be selected to become antibody-producing plasma cells. It seemed plausible to us that this abnormal pre-plasma cell population, that is normally repressed by Tfr cells, contains self-reactive B cells including nucleosome-binding B cells. For this, we gated on the nucleosome-binding GC B cell population and examined the appearance of the plasma cell marker CD138. Over 40% of nucleosome-binding GC B cells expressed CD138 in neuritin-deficient mice compared to around15% in littermate controls (Fig. 3.34b). This prehontype was not observed in Tfr-deficient mice, consistent with our data showing that lack of Tfr cells does not lead to nucleosome-specific GC B cells.

114 CHAPTER 3. RESULTS

a Control Tfr- Nrn1-

1.01 9.94 3.49 B220 CD138 b *

15 ) *

* B 80

G + B cells 60 10

C Nuc (% og GC B)

+ f f

o 40

5 +

CD138 20 lo pre-plasma G

0 CD138 0 B220 Control Tfr- Nrn1- Control Tfr- Nrn1- Figure 3.34. Self-reactive GC B cells in neuritin-deficient mice display a “early-plasma cell” phenotype. a) Representative flow cytometric plots showing the proportion of B220lo CD138+ GC B cells (early plasma cells). b) Quantification of early-plasma cells in mice as per (a) (left panel) and quantification of CD138+ pregated in nucleosome+ GC B cells (right panel). Control: Neuritin+/+.CreFoxp3; Tfr-: Neuritinfl/fl.CreFoxp3. Bars indicate median values. Each dot represents data obtained from one mouse.

115 CHAPTER 3. RESULTS

3.2.13 Neuritin treatment suppresses abnormal pre-plasma

cells in Tfr deﬁcient mice

To study the potential effect of neuritin treatment in the prevention of autoantibody-development, we treated Tfr-deficient mice, which develop autoantibodies to ENA, specific organs and histone H1 (Fig. 3.11a), with 200 µg of recombinant neuritin intravenously twice a week for 4 weeks starting at 6 weeks of age. Strikingly, treatment of Tfr-deficient mice with recombinant neuritin protein led to the disappearance of the abnormal GC-derived early plasma cell population (Fig. 3.35a). Neuritin treatment did not have an obvious effects on the percentages of Tfr cells, Tregs or in Foxp3 expression within Tregs, nor changed the proportions of Tfh cells but it had a mild effect increasing the proportion of GC B cells, consistent with in vitro Bcl6 upregulation after culturing B cells with neuritin in human (Fig. 3.22c-d) and mouse (Fig. 3.23). Neuritin treatment also had a noticeable effect in reducing the proportion of splenic B220loCD138+ plasma cells (Fig. 3.35).

116 CHAPTER 3. RESULTS a Control Tfr- Tfr- + neuritin

3.46 0.077 0.13 PD-1 CXCR5

2.15 7.95 2.11 B220 b CD138 5 20 12 s s 4 s el l 15 el l c

el l 8

3 c

re g 10 C T

Tf h

2 f

o 4

% 5

1 %

0 0 0 0.5 4000 4

t 0.4 3000 3 I s 0.3 F M cel l

2000 2 mabla s

0.2 Tf r

la s oxp3 P 1000 % F 1 0.1 % 0.0 0 0

s 25

el l Control c

B Tfr-

C 15 Tfr- + neuritin G

+ 10

5 CD13 8

% 0

Figure 3.35. Neuritin treatment in vivo suppresses early plasma cells from GC B cells. a) Representative flow cytometric plots showing the proportion of CD4+ Foxp3+ PD-1+ CXCR5+ Tfr cells (pre-gated in Tregs) and abnormal CD138+ early plasma cells from GC B cells in mice of the indicated genotype. b) Dot plots showing the quantification of lymphocyte populations and Foxp3 gMFI from Tregs in mice of the indicated genotype and treatment. Control: Bcl6+/+.CreFoxp3; Tfr-: Bcl6fl/fl.CreFoxp3. Bars indicate median values. Each dot represents data obtained from one mouse. 117 CHAPTER 3. RESULTS

We have shown that Tfr-deficient mice have increased anti-histone H1 autoantibodies. We looked at the effect of neuritin treatment in vivo in autoantibody production in the serum of treated and untreated Tfr-deficient mice. Consistently with our previous experiments, Tfr-deficient mice showed increased anti-histone H1 autoantibodies but they were not significantly reduced by neuritin treatment (p = 0.5962). Surprisingly, Tfr-deficient mice showed a modest reduction in anti-nucleosome autoantibodies after neuritin treatment (p = 0.0541) (Fig. 3.36).

118 CHAPTER 3. RESULTS

0.5962 0.0541 3 4 * 0.1457 405nm

D 2 O 450n m -

D 2 O 1 -

H1 1 Nucleosome 0 0

Tfr- Tfr- Tfr- Tfr- Control Control

+ neuritin + neuritin Figure 3.36. Effect of neuritin treatment in autoantibody production. a) Dot plots showing the quantification of anti-nucleosome and anti-histone H1 autoantibodies in mice of the indicated genotype and treatment. Control: Bcl6+/+.CreFoxp3; Tfr-: Bcl6fl/fl.CreFoxp3. Bars indicate median values. Each dot represents data obtained from one mouse.

Collectively, these results suggest that intravenous administration of recombinant neuritin can target peripheral B cells to reduce potentially pathogenic antibody-producing plasma cells.

119

Chapter 4

Discussion

4.1 Summary

4.2 Tfr cells and autoimmunity

Here we have shown that Tfr cells are particularly important to control antibody responses against self-tissues and innocuous food antigens. There is evidence that Tfr cells can be induced against both self and foreign antigens (Aloulou et al. 2016), including those protein antigens that Tfh cells are responding to. Tfr cells that develop in response to the same antigens as Tfh cells, may serve as a natural homeostatic mechanism to curtail Tfh cell responses, as appears to be the case for specialised Tregs controlling Th17, Th2 and Th1 responses(Josefowicz et al. 2012b; Koch et al. 2009; Duhen et al. 2012).It is also possible that diﬀerent subsets of Tfr cells develop against foreign vs innocuous environmental or self-antigens.

Thymus-derived Tregs that stably express Foxp3 are selected on the basis of their ability to recognise self-antigen in the thymus(Hsieh et al. 2012). Given that Tregs give rise to Tfr cells in immunised mice(Chung et al. 2011;

121 CHAPTER 4. DISCUSSION

Linterman et al. 2011; Wollenberg et al. 2011; Aloulou et al. 2016; Sage et al. 2014b; Wing et al. 2014) these thymic-derived Tregs are ideal candidates to repress responses against self. Data presented in this thesis showing tissue- speciﬁc autoantibodies in Tfr-deﬁcient mice lend support to this proposition: A fraction of Tregs selected in the thymus on the basis of their ability to recognise tissue-expressed antigens (TSAs) co-opt the follicular pathway to become Tfr cells and act to prevent formation of B cells secreting antibody against these tissue antigens. Whether the Tfr cells that recognise food allergens are thymic derived or induced in the periphery is unknown. It has been proposed that Tregs in the airways and gut can emerge from Foxp3− precursors. Their follicular counterparts, Tfr cells, would be ideally positioned to regulate IgE production against environmental and dietary antigens, and it is thus not surprising that their follicular counterpart is particularly well positioned to exert this control.

The data shown here suggest that Tfr cells may act at different stages of the immune response, from the time of T:B cell priming at the T:B interface (i.e., neuritin+ cells interacting with IgD+ B cells in the periphery of GCs), during GC B cell selection (i.e., accumulation of early plasma cells within GCs of Tfr-deficient mice) and potentially in the reactivation of memory B cells (i.e., increased IgE response after secondary challenge). This would be in line with the known diverse functions of Tfh cells in the early activation of B cells required for differentiation towards GC B cells or extrafollicular plasma cells, their role in GC B cell selection and in reactivation of memory B cells(Vinuesa et al. 2016). It is therefore possible that Tfr cells engage with B cells at all these stages, and may form cognate interactions with those cells expressing self-peptides. Thus, Tfr cell, or at least a subset of them, may selectively control self-tissue-specific B cells at the different maturation stages.

122 CHAPTER 4. DISCUSSION

Intriguingly, although young Tfr-deficient mice had increased spontaneous GCs, we did not observe changes in the magnitude of GCs at the peak of the GC response, which contrasts with previous findings in Tfr-deficient mice made through different approaches, e.g., bone marrow chimeras, cell transfers to KO mice. Regardless of potential differences in the experimental models used, our results suggest that one of the main roles of Tfr cells may be the regulation of responses to self rather than to foreign antigens.

Thus, we speculate that Tfr cells may be predominantly important to influence B cells soon after migrating to the T:B border upon engaging antigen, prior to or during the process of priming by pre-Tfh cells. It is also possible that Tfr cells within GCs are selectively engaged in eliminating tissue specific B cells, and do not largely influence responses to foreign antigen; thus not changing dramatically the numbers of Tfh cells and GC B cells in established GCs.

4.3 Tfr-mediated IgE regulation

The work presented here supports the regulation of IgE responses to protein antigens and common food allergens by Tfr cells. This regulation appears to be particularly strong for IgE, and to a lesser extent for IgA and IgG1 responses. Elimination of Tfr cells exacerbates IgE responses despite the presence of intact Tregs. Tfr cells may exert this effect through multiple mechanisms, including direct repression of B cells and indirect repression by limiting the production of pro-inflammatory cytokines such as IFN-γ. The increase in IL-17 observed in Tfr-deficient mice is particularly interesting, especially in association with elevated IgA and autoantibodies to antigens against tissues in the digestive

123 CHAPTER 4. DISCUSSION

tract (salivary gland, stomach, and pancreas). IL-17 is known to promote gut inﬂammation, often in response to alterations in the composition of gut microbiota(Honda and Littman 2016). It will be interesting to explore the link between diet, microbiota, defective Tfr generation in gut mucosa and IgE/IgA autoantibodies to gut tissue antigens and food allergens.

Our work did not analyse the precise stage at which Tfr cells repress IgE production, whether pre-GC, GC, or post GC/memory stages. The substantial increase in IgE upon secondary challenge of Tfr-deficient mice suggests that Tfr regulation may occur during formation of allergen-reactive memory B cells or during reactivation of such memory cells. GCs have been shown to be sites where IgE-expressing B cells are regulated. IgE+ B cells are already at a survival disadvantage due to their lower BCR expression and signalling(He et al. 2013; Yang et al. 2012). Lower BCR density would decrease presentation of antigen to T cells, which together with lowered BCR-mediated survival signals would make IgE+ GC B cells more dependent on Tfh cell help for survival and differentiation, as shown recently(Yang et al. 2016). Also, if Tfr cells are particularly good at regulating memory B cells, there is strong evidence that sequential switching of IgG memory B cells is an important pathway for the production of antigen-specific IgE in the late phase of immune responses(Xiong et al. 2012).

In terms of possible direct eﬀects on B cells, Tfr cells may regulate their eﬀects on IgE production through several mechanisms including Tfr-mediated metabolic regulation of B cells, as recently reported to occur in mice, CTLA- 4-dependent mechanisms(Sage et al. 2016), or IL-10-mediated repression of IgE-switching(Jeannin et al. 1998). As shown here and discussed below, neuritin emerges as a new layer of Tfr-mediated regulation.

124 CHAPTER 4. DISCUSSION

4.4 Tfr-derived neuritin in autoimmunity

Tfr cells have emerged as Tregs specialised in regulating B cell responses but the precise mechanisms that specifically regulate antibody responses remain poorly understood. Tfr cells are derived from thymic Tregs and can also be induced in the periphery. Tregs have been shown to use various effector molecules and mechanisms to regulate antigen-presenting cells (APCs) and T cell responses, including transedocytosis of CD28 ligands via CTLA-4(Walker and Sansom 2011) and IL-10 secretion(Wing et al. 2018). While patients lacking functional Foxp3 or with perturbed Treg development, i.e., in the absence of AIRE, develop tissue-specific B cell autoreactivity, these features are less prominent in patients with selective defects in CTLA-4(Schubert et al. 2014), and are not a feature of IL-10-deficient mice. We considered the possibility that there may be additional molecules that make Tfr cells particularly effective at preventing autoantibody responses. Comparison of transcriptomic profiles between Tfh and Tfr cells identified neuritin as a putative candidate.

In this thesis we add a novel Tfr eﬀector molecule, neuritin, that is predominantly expressed by Tfr cells and appears to be involved in the repression of self-reactive GC B cell selection and diﬀerentiation to prevent the emergence of autoantibody-secreting plasma cells.

This thesis provides the ﬁrst evidence of Tfr-derived neuritin in the control of antibody responses. Although our online searches found communications of a putative role for neuritin in the promotion and maintenance of the Treg pool, no articles have yet been published(Barbi et al. 2016). Moreover, in the absence of neuritin speciﬁcally in Foxp3+ cells, we did not observe a reduction in the Treg pool nor in the amount of Foxp3 expressed per cell, and the Treg

125 CHAPTER 4. DISCUSSION

suppressive capacity was unaltered.

Here, we have shown neuritin-mediated effects on B cells both in vitro and in vivo and evidence that neuritin can bind to B cells. Intriguingly, our data suggests that Tfr-derived neuritin can be taken up by B cells. In deed, we were surprised to find sorted cytospinned GC B cells appeared to stain for cytoplasmic or membrane neuritin, as Tfr cells did. Given the lack of Nrn1 mRNA expression in B cells, we can only speculate that GC B cells take up neuritin, or there may be a receptor for this peptide that is internalised after ligand binding. Finding such neuritin protein in most GC B cells is at odds with our hypothesis of preferential interaction of Tfr cells with self-reactive B cells. It is plausible that Tfr cells screen recently activated B cells prior to follicular entry at the T:B border, and transfer neuritin during this interaction. Additional experiments analysing neuritin content in the different follicular T and B cell populations using different detection methods will be important to understand the significance of finding neuritin in GC B cells.

We have shown that B cells bind neuritin using two diﬀerent methods. Our experiments incubating ﬂuorescently labelled neuritin with total spleen or tonsil cells, or experiments cross-linking neuritin to oxidised receptors on B cells have shown strong and preferential binding of neurititin to human and mouse B cells. While in mouse, GC B cells bound more neuritin than other B cell subsets, most human B cells appeared to bind neuritin. These results suggest that in both humans and mice, Tfr cells may be capable of transmitting neuritin to interacting B cells.

To date, the receptor for neuritin, even in the nervous system, remains elusive. Experiments blocking INSR (insulin receptor) in nerve cells have proposed

126 CHAPTER 4. DISCUSSION that this might be its receptor, at least in neurons(Yao et al. 2012). Never- theless, the broad effects of restricting insulin signalling cannot be overlooked, especially in neurons. Neurotrophins can have different roles depending on their form (monomer, hetero, or homodimer) and on the receptors they might use in different tissues. A more recent report by Shimada et al. proposed that neuritin cooperates with the FGF (fibroblast growth factor) receptor to promote mossy fiber sprouting by enhancing the recruitment of FGF receptors to the cell surface, amplifying the effects observed after FGF treatment(Shimada et al. 2016). A similar scenario might explain the results observed by Yao et al., where neuritin might indirectly act on the insulin receptor without necessarily binding to it. Additional experiments are needed to definitively pinpoint the receptor for neuritin in lymphocytes.

Expression proﬁles and immunoﬂuorescent staining of neuritin in sorted lymphocyte subsets shows that Tfr cells are the main source of neuritin, although some Tregs can still produce it. At this stage, it is still unclear what the signals inducing Tfr cells to produce neuritin are.

Our tissue immunohistochemistry shows neuritin-expressing T cells can be found located within GCs and at the T:B border. This suggests that Tfr cells may interact first with recently activated B cells and later again with GC B cells as they compete for T cell help in GCs. At both of these stages, neuritin may exert its repressive effects. Finding a small amount of neuritin binding on T cells, if specific, may lead to an autocrine effect of neuritin in Tfr cells themselves, which would explain the ∼4-fold increase in Tfr cells in mice lacking neuritin in Foxp3-expressing cells after OVA-immunisation.

Surprisingly, most of the neuritin-expressing cells were found to be in contact

127 CHAPTER 4. DISCUSSION

with IgD+ B cells in the T:B interface as well as inside GCs. IgD+ cells within GCs might be recirculating naive B cells or a subset of GC B cells reported to be hypermutated and possibly autoreactive(Noviski and Zikherman 2018; Sabouri et al. 2014). IgD expression has been reported to be less sensitive to endogenous antigens than IgM. Additionally, autoreactive B cells downregulate IgM but not IgD. It is possible that neuritin+ cells interact with IgD+ naive B cells at the T:B border as a check point to control the access to the GC and limiting Tfh-mediated B cell activation. In GCs, neuritin+ Tfr cells might interact more stably with self-reactive IgD+ GC B cells to control their selection and diﬀerentiation.

Neuritin can be expressed as a membrane-associated molecule and in secreted form as a soluble intracellularly dimerised peptide. The latter has been shown to efficiently induce neurite outgrowth in vitro (Putz et al. 2005). While the defects seen in neuritin-deficient mice may be a consequence of the combined action of membrane and soluble forms, our in vitro cultures and in vivo administration of soluble neuritin indicate that several of the observed effects, particularly the repression of plasma cell differentiation from GC B cells and autoantibody formation, can be an effect of the secreted form.

Recently, mice engineered to naturally lack Tfr cells have been generated and reported to develop systemic anti-DNA antibodies in a pristane-induced autoimmune mouse model(Wu et al. 2016). A role for Tfr cells in the prevention of spontaneous anti-nuclear and Sjogren’s Syndrome-associated antibodies has also been recently reported(Fu et al. 2018). These studies showed that Tfr-deﬁciency leads to late onset of autoimmunity characterised by lymphocyte inﬁltration in lung, pancreas and salivary gland, and antibody deposition in kidney and salivary gland. However, detection of autoantibodies to self-peptides

128 CHAPTER 4. DISCUSSION was not assessed in detail. It is unclear if the antibody deposition observed in salivary gland and kidney from Tfr-deficient mice were specific for tissue- specific antigens or if they were polyreactive antibodies deposited in response to the presence of chromatin fragments from necrotic cells that can bind with high affinity to glomerular basement membrane structures, especially in the absence of proper Dnase1 expression(Mortensen and Rekvig 2009; Napirei et al. 2004). To avoid these confounding effects, we screened mouse serum using organ cryosections from RAG1+/+ mice that lack endogenous antibodies. Additionally, we used ELISAs to test for the presence of antibodies against purified proteins (gastric parietal cell antigens and histones).

Fu et al. also described the presence of spontaneous GCs in Tfr-deficient mice from 12 weeks of age but not at 8 weeks of age, anti-dsDNA antibody production in aged mice, and no differences in serum IFN-γ; however, our results showed otherwise. We only see statistical differences in GC B cells from young mice and we did not detect basal anti-dsDNA autoantibodies. We also found that IFN-γ was significantly increased in the serum of Tfr-deficient mice. These differences could be due to the origin of the founder strains used to generate Tfr-deficient mice, the sex of the experimental mice used, and to mouse housing conditions, including diet and microbiota composition.

Finding rapid neuritin-mediated downregulation of ICOSL is reminiscent of recent observations made by our group – albeit in the opposite polarity – of dopamine-mediated upregulation of ICOSL in human GC B cells, shown to occur within an immunological Tfh:B cell synapse(Papa et al. 2017). It is therefore likely that neuritin is also transmitted in a secreted form, or polarised in lipid rafts in its GPI-anchored form, during an immunological synapse between Tfr cells and GC B cells. This may counteract the eﬀects of Tfh cells,

129 CHAPTER 4. DISCUSSION

limiting Tfh:B cell contact. Neuritin in neurons stabilises synapses; thus, it is possible that Tfr:B cell interactions might be enhanced by neuritin, preventing Tfh:B cell interactions. Neuritin polarisation was observed ex vivo by cytospin immunoﬂuorescence, further supporting this idea. It would be interesting to measure Tfh:B cell entanglement and aﬃnity maturation in the presence and absence of neuritin.

Soluble neuritin has also been shown to have potent effects in vivo in a rat model of brain injury(Zhang et al. 2017a). Our findings in mice treated with soluble protein support the idea that secreted neuritin can target lymphoid cells. One of the most striking effects of neuritin administration in vivo was the ability to prevent the differentiation of B220loCD138+ plasma cells, particularly the abnormal appearance of early plasma cells from GC B cells.

Additionally, neuritin treatment appeared to have a positive effect preventing the production of nucleosome-specific autoantibodies but this did not reach statistical significance (p = 0.0541). As mentioned above, neuritin-deficiency in Treg/Tfr cells led to an increase in self-reactive (nucleosome-binding) GC B cells that possess a plasma cell phenotype. The mechanism underlying the repression of self-reactive B cell differentiation into plasma cells could be explained by our observation that neuritin augments BCL6 expression in GC B cells. This is likely to limit Blimp-1 upregulation and plasma cell differentiation in B cells that have bound neuritin. BCL6 upregulation could also sustain a GC phenotype to induce further cycling and somatic hypermutation in GC B cells that may remove self-reactivity(Tan et al. 2016; Reed et al. 2016).

The accumulation of early plasma cells in GCs has not been previously reported in Tfr-deﬁcient mice, and it could help explain the role of Tfr cells in

130 CHAPTER 4. DISCUSSION repressing autoimmunity. Both are likely to be linked if Tfr cells predominantly target self-reactive B cells, given that they derive from Tregs that have been selected in the thymus on the basis of self-antigen recognition. Tfr recognition of B cells presenting self-peptides would also explain another intriguing observation: Tfr and neuritin-deficient mice do not form typical anti-ssDNA or dsDNA antibodies, at least in the absence of immunisation, but do form autoantibodies to protein nuclear antigens, which are disease markers in systemic lupus erythematosus (SLE)(Suleiman et al. 2009). Protein nuclear antigens such as histones, as many other tissue-specific antigens, are expressed by an Aire-dependent mechanism in the thymus(Adamopoulou et al. 2013) and thus, are seen by histone-reactive Tregs selected in the thymus. It makes sense therefore that Tfr cells are specialised in recognising tissue-specific antigens (TSAs), as opposed to antigens not typically presented to T cells, such as DNA. Not surprisingly, mice lacking Tfr cells and neuritin did not present with pure antinuclear antibodies or anti-dsDNA antibodies, but instead developed abundant antibodies to other TSAs including autoantibodies to gastric parietal cells, goblet cells, retina, pancreas, and salivary gland.

We have shown that neuritin is important for the repression of human and mouse plasma cells in vitro and in vivo. Although some of the pathologic features of Tfr-deficient mice are shared with neuritin-deficient mice (i.e., tissue-specific autoantibodies, spontaneous generation of early plasma cells within GCs, increased IgG1 and IgE after immunisation) some of the others are surprisingly different. Tfr-deficient mice develop autoantibodies against ENAs and histone H1, but not to nucleosomes or core histones. This differs from neuritin-deficient mice that develop autoantibodies to nucleosomes and core histones. Some of these discrepancies might be explained by differences in their non-follicular Treg compartment. While Tfr-deficient mice have intact

131 CHAPTER 4. DISCUSSION

Tregs, neuritin-deficient mice have Tregs that lack neuritin. We found neuritin expression in a small fraction of Tregs. It is therefore possible these may be important in the control of nucleosome/core histone-reactive B cells. It would be interesting to further characterise neuritin-deficient vs neuritin-sufficient Tregs, as they might have subtle differences in cytokine expression or localisation. Additionally, neuritin derived from the earliest Tfr cells that may not yet be dependent on Bcl6 expression – present in Tfr-deficient mice – might play a role in controlling responses of self-reactive B cells.

4.5 Limitations and Future directions

4.5.1 Autoimmune mouse models

We have shown that neuritin-deficiency and neuritin treatment in vivo can have targeted effects on B cells. For this we used a mouse model that naturally lacks Tfr cells. Tfr-cell deficiency, as shown here, is associated to the development of humoral autoimmunity; however, none of these antibodies appeared to be pathogenic, similar to what is observed in Aire-deficiency(Mathis and Benoist 2009). In order to test if neuritin-deficiency or neuritin treatment can have a relevant role in the progression of antibody-mediated autoimmune disorders, where autoantibodies are known to play a role in pathogenesis, we will use collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis (RA), in control, tfr-deficient, and neuritin-deficient mice. The incidence of CIA in mice is dependent on their MHC haplotype. C57BL/6 mice are considered to be resistant to CIA and we speculate that Tfr- or neuritin-deficiency may render them more susceptible. CIA has also been linked to a pathogenic increase in IL- 17 and IFN-γ in serum. Thus, we hypothesise that Tfr-deficient mice would be more vulnerable to disease progression(Nakae et al. 2003). Similarly, it would

132 CHAPTER 4. DISCUSSION be interesting to test the signiﬁcance of neuritin- and Tfr-deﬁciency, as well as neuritin treatment in other autoimmune models, such as EAE (experimental autoimmune encephalomyelitis).

4.5.2 Neuritin overexpression

Neuritin can act as membrane bound GPI-anchored protein or as a soluble secreted peptide and both forms have been shown to be important for the development and maturation of neurons. Thus, it would be interesting to investigate if the same eﬀects are seen in B cells when they are cocultured with either a feeder cell line or T cells overexpressing the membrane-bound form of neuritin.

4.5.3 Neuritin-GFP mouse

Antibodies against neuritin are of poor quality and there are currently no antibodies for ﬂow cytometry. To further analyse the temporal and spatial expression pattern of neuritin in Tfr cells, we have generated a CRISPR mouse strain that expresses GFP under the control of the neuritin promoter. We are now performing the initial tests to validate this model that will allow us to phenotype neuritin+ cells by FACS and sort them to further characterise them by RNA-seq.

4.5.4 Eﬀect of neuritin in Tfh:B cell interactions

Given the known role of neuritin in synapse stabilisation, investigating the eﬀect of neuritin treatment in the way Tfh and B cells interact would shed light into its possible mechanism of action.

133 CHAPTER 4. DISCUSSION

4.5.5 Neuritin receptor in B cells

The receptor for neuritin is unknown. Most neurotrophins bind to tyrosine receptor kinases (Trk) that are then internalised in target cells. However, the evidence suggesting putative receptors for neuritin in neurons, or any other cell type, is far from deﬁnitive. One possible approach is the use of the recently developed ligand-receptor capture technology(Frei et al. 2012). We cannot ignore the possibility that neuritin might be transferred from Tfr cells in vesicles and endocytosed by B cells in the immunological synapse. This would explain why this receptor has been particularly elusive.

4.5.6 Disentanglement of the functions of Treg- from Tfr-

derived neuritin

Neuritin is expressed mainly by Tfr cells, although a small fraction of Tregs can also produce it. Our current model of neuritin-deficiency cannot distinguish between the effects mediated by Treg- or Tfr-derived neuritin. We have shown here that the phenotype of Tfr-deficient mice does not always overlap with that of neuritin-deficient mice, suggesting that Treg-derived neuritin might play an important role too. One approach to overcome this would be to transfer Tregs from Tfr-deficient mice, that are unable to differentiate into Tfr cells, into neuritin-deficient mice in order to generate a mouse that will have normal neuritin-suficient Tregs and will only lack neuritin in Tfr cells.

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