Novel function for BAFFR signalling in the regulation of Germinal Centre and memory

Angelica Wing Yin Lau

A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy

St Vincent’s Clinical School, Faculty of Medicine, UNSW Sydney

Garvan Institute of Medical Research

Supervisor: Professor Robert Brink Co-supervisor: Dr. Tyani D. Chan May 2018 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Lau

First name: Wing Yin Angelica Other name/s:

Abbreviation for degree as given in the University calendar: PhD

School: St Vincent's Clinical School Faculty: Faculty of Medicine

Title: Novel function for BAFFR signalling in the. regulation of Germinal Centre and B cell memory

Abstract 350 words maximum: (PLEASE TYPE) BAFFR receptor (TNFRSF13C) and its ligand BAFF (TNFSF138) are imperative for the normal maturation and survival of naive B cells. BAFF:BAFFR signalling is also thought to be essential for the maintenance of T-dependent B cell responses. The immunisation of BAFFR-deficient or BAFF-deficient mice generated small germinal centres (GCs) that could not be sustained and a complete absence of memory B cells (MBCs). However, these deficient mice have abnormalities in their secondary lymphoid tissues and markedly reduced mature B cells; hence it remains unclear the extent to which BAFF:BAFFR signalling regulates B cell responses to T-dependent . To investigate this, this thesis utilised the high resolution in vivo mouse model (SWHEL), in which HEL-specific B cells were challenged and examined in various adoptive transfer approaches in order to dissect the cell-intrinsic function(s) of BAFF:BAFFR signalling in regulating GC and MSC responses. Results here demonstrated that the loss of BAFFR on responding B cells had no impact on GC B cell survivalbut the immaturity associated with BAFF or BAFFR deficiency led to the establishment of smaller GC responses. In addition, our results showed that BAFF:BAFFR signalling was dispensable for controlling the maturation and selection of GC B cells. BAFFR-deficient B cells were found to acquire normal somatic mutations in their immunoglobulin variable region that conferred high affinity binding for as well as the production of high affinity antibodies. Importantly, GC-independent MBCs were discovered to be particularly sensitive to the survival signals delivered through BAFFR. The loss of BAFFR expression led to a specific depletion in class-switched unmutated MBCs but had no impact on somatically mutated, GC-derived MBCs. Enforced signalling through BAFFR also preferentially prolonged the survival of these early MBCs. Data presented in this thesis indicate that the maintenance ofT-dependent MBC subsets are differentially regulated - that is the survival requirement for BAFF:BAFFR signalling is unique to GC-independent, early MBCs and not shared by BAFFR-independent MBCs that develop from GC precursors.

Declaration relating to disposition of project thesis/dissertation

I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such·'as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only) .

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FOR OFFICE USE ONLY Date of completion of requirements for Award: Table of Contents COPYRIGHT STATEMENT ...... i

AUTHENTICITY STATEMENT ...... i

ORIGINALITY STATEMENT ...... ii

Acknowledgments & Thank You ...... iii

Abstract ...... vi

Abbreviations, Conventions & Definitions ...... vii

Amino acid abbreviations ...... x

List of Diagrams & Tables ...... xi

List of Figures ...... xii

Chapter 1 ...... 1

1.1 Preamble 1 1.2 B cell origins and selection into the primary B cell repertoire ...... 1 1.2.1 B cell lymphopoiesis ...... 1 1.2.2 BCR rearrangement and selection ...... 2 1.2.3 B cell homeostasis in peripheral lymphoid tissue ...... 2

1.3 TNF ligand BAFF and its receptors ...... 5 1.3.1 Expression of BAFF and BAFF receptors in B cell homeostasis ...... 5 1.3.2 Activation of alternative NF-κB pathway by BAFF:BAFFR signalling in primary B cells ...... 6 1.3.3 BAFFR-mediated NF-κB2 activation and the induction of Bcl2-family in B cell survival ...... 9

1.4 T-dependent B cell responses ...... 10 1.4.1 B cell activation and early differentiation by T-dependent antigens ...... 10 1.4.2 Ig class switching and functionality ...... 12 1.4.3 The Germinal Centre Reaction ...... 13 1.4.4 Establishing immunological memory to T-dependent antigens ...... 18

1.5 BAFF:BAFFR signalling in B cell responses, health and diseases ...... 22

1.6 SWHEL system to study antigen-specific B cell responses in vivo ...... 25 1.7 Project aims and hypothesis ...... 27 Chapter 2 ...... 28

2.1 Mice and Adoptive Transfer ...... 28 2.1.1 Mice ...... 28 2.1.2 Recombinant HEL proteins ...... 28 2.1.3 Conjugation of HEL proteins to SRBC ...... 29 2.1.4 Recipe: Conjugation Buffer ...... 30 2.1.5 Preparation of Spleen, Bone Marrow and Blood ...... 31

2.1.6 Preparation of SWHEL splenocytes prior to adoptive transfer ...... 31 2.1.7 Recipe: 2% FBC in RPMI (2FR) Medium ...... 32 2.1.8 Recipe: RBC Lysis Buffer ...... 32 2.1.9 Adoptive Transfer ...... 32

2.2 Antibodies and reagents for data analyses ...... 33 2.2.1 Monoclonal antibodies (mAbs) ...... 33 2.2.2 Other reagents ...... 33

2.3 MACS Cell Separation ...... 35 2.3.1 MACS depletion of mature B cells ...... 35 2.3.2 Recipe: MACS Separation buffer ...... 36 2.3.3 Recipe: 2% FBS in DMEM (2FD) Medium ...... 36

2.4 Flow cytometry ...... 37 2.4.1 Surface staining procedure ...... 37 2.4.2 Data acquisition, processing and analysis ...... 39 2.4.3 Recipe: PBA (FACS) Buffer ...... 39

2.5 SHM analysis ...... 40 2.5.1 Standard protocol for single cell FACS ...... 40 2.5.2 Plate set up for single cell sort ...... 41 2.5.3 Primary PCR ...... 42 2.5.4 Secondary PCR ...... 44 2.5.5 PCR product screening ...... 46

2.5.6 Sequencing amplified VH10 PCR products ...... 46 2.5.7 Analysis of SHM ...... 47 2.5.8 Recipe: TBE Buffer (5X) ...... 47

2.6 ELISA 48 2.6.1 Staining procedure to detect HEL-specific antibodies ...... 48 2.6.2 Recipe: NPP Buffer ...... 49 2.6.3 Recipe: ELISA Wash Buffer ...... 49

2.7 Retroviral Transduction and Overexpression on primary B cells ...... 50 2.7.1 Cloning and Transformation ...... 50 2.7.2 Generation of viruses for Retroviral Transduction of primary B cell ...... 50

2.7.3 Retroviral Spin Transduction of SWHEL B cells ...... 51 2.7.4 Recipe: Super Optimal Broth ...... 52 2.7.5 Recipe: 10% FBS in DMEM (10FD) Medium ...... 52 2.7.6 Recipe: B Cell Medium (BCM) ...... 53

2.8 Immunofluorescence histology ...... 54 2.9 Generation of Mixed Bone Marrow Chimeras ...... 54 2.10 Statistical analysis ...... 55 Chapter 3 ...... 56

3.1 Preamble 56 3.2 GC B cell differentiation leads to increased BAFFR expression ...... 57

3.3 Gating strategy to characterise BAFFR dynamics in antigen-specific SWHEL B cell responses ...... 59 3.4 Mapping the BAFFR expression during GC affinity maturation ...... 62 3.5 -derived BAFF is dispensable for GC and memory B cell survival ...... 66 3.6 Affinity-based selection in the GC does not require T cell-derived BAFF ...... 71 3.7 Wild type GC B cell responses show similar SHM patterns in chimeras with wild type versus BAFF-deficient T cells ...... 75 3.8 Discussion ...... 78 Chapter 4 ...... 81

4.1 Preamble 81 4.2 Loss of mature B cells in BAFF-deficient and BAFFR-deficient mice leads to abnormal lymphoid organ architecture ...... 82 4.3 Strategy to examine T-dependent responses of wild type B cells in BAFF- deficient and BAFFR-deficient mice ...... 86 4.4 Normal induction but attenuated endogenous GC and memory B cell responses in BAFF-deficient and BAFFR-deficient mice ...... 88 4.5 Cell-extrinsic factors compromise wild type B cell responses in BAFF- deficient and BAFFR-deficient mice ...... 91 4.6 GC affinity maturation is intact but cell-extrinsic absence of BAFF impairs memory B cell survival and absence of BAFFR accumulates low affinity memory B cells ...... 96 4.7 Absence of BAFFR extrinsic to responding wild type B cells promotes the persistence of low affinity memory B cells ...... 99 4.8 Wild type B cell responses accumulate unmutated memory B cells in BAFFR-deficient recipient mice ...... 104 4.9 Discussion ...... 108 Chapter 5 ...... 111

5.1 Preamble 111 5.2 Strategy to examine B cell-intrinsic function of BAFF:BAFFR signalling ..... 111 5.3 Immature BAFFR-deficient B cells show poor expansion but normal differentiation in response to T-dependent antigens ...... 113 5.4 Strategy to examine the impact of B cell maturity in BAFFR-deficient B cell responses ...... 116 5.5 Immature B cells form sustainable GC responses but memory B cells are significantly reduced in BAFFR-deficient responses ...... 119 5.6 Delayed affinity maturation but normal selection in immature BAFF- deficient and BAFFR-deficient GC B cell responses ...... 122 5.7 Low affinity but not high affinity memory B cells are depleted in BAFFR- deficient B cell responses ...... 125 5.8 Strategy to examine the cell-intrinsic function(s) of BAFFR signalling using immature wild type versus BAFFR-deficient B cells ...... 128 5.9 BAFF:BAFFR signalling is dispensable for GC maintenance but is critical for survival of memory B cells ...... 131 5.10 Persistence of low affinity memory B cells is dependent on BAFFR signalling but independent of cell maturity ...... 134 5.11 SHM analysis indicates normal affinity-based selection in BAFFR- deficient GC B cell responses ...... 137 5.12 SHM analysis reveals the absence of unmutated early memory B cells in BAFFR-deficient responses ...... 141 5.13 Discussion ...... 143 Chapter 6 ...... 148

6.1 Preamble 148 6.2 Strategy to examine the impact of enhanced BAFFR signalling on normal B cells during T-dependent responses ...... 149 6.3 BAFFR overexpression on responding B cells increases memory B cell output ...... 152 6.4 Normal GC affinity maturation but accumulation of low affinity memory B cells in responses of BAFFR-overexpressing B cells ...... 155 6.5 SHM analysis reveals the persistence of unmutated memory B cells in BAFFR-overexpressing B cell responses ...... 158 6.6 Strategy to examine constitutive activation of BAFFR signalling via TRAF3- inactivation ...... 161 6.7 TRAF3-inactivation on responding B cells increases memory B cell output but has no impact on the GC response ...... 163 6.8 TRAF3-deficient B cells undergo normal affinity maturation and selection in the GC but selectively perpetuate low affinity memory B cell responses ...... 166 6.9 Strategy to examine survival regulators in T-dependent memory B cell responses through the overexpression of pro-survival Bcl2-family proteins ...... 170 6.10 BCL-2 or BCL-xL overexpression on responding B cells generate abnormally expanded GC and memory B cell responses ...... 173 6.11 Excess BCL-2 or BCL-xL levels disturbs affinity-based selection in IgG1+ GC and memory B cell compartments ...... 177 6.12 Strategy to examine survival regulators in T-dependent memory B cell responses through the deletion of pro-apoptotic protein BIM ...... 183 6.13 BIM-deletion on responding B cells generates abnormally expanded GC and memory B cell responses ...... 185 6.14 Loss of BIM disturbs affinity-based selection in IgG1+ GC and memory B cell compartments ...... 188 6.15 Discussion ...... 194 Chapter 7 ...... 201

7.1 Research outcomes ...... 201 7.2 Future Directions ...... 204 Citations ...... 206 COPYRIGHT STATEMENT

'I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or partof this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only).

I have either used no substantial portions of copyright material in my thesis or l have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

Signed:

Dated:

AUTHENTICITY STATEMENT

'I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to the digital format.'

Signed:

Dated: ORIGINALITY STATEMENT

"I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.'

Signed:

Dated:

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Acknowledgments & Thank You

It almost brings me to tears reflecting on how much I have achieved and grown in the past couple of years. I have to admit I enjoyed the every bit of failure, success and excitement that arose throughout my PhD, but none of this would have been made possible without the past and current members of the wonderful Brink lab and the unending support of my family and friends.

To Prof. Rob Brink – my longest and biggest thank you goes to you. Thank you for being such a “calm and collected, reasonable but overly excitable over new data” supervisor. Your enthusiasm, passion and positivity towards my work had truly been infectious and I would not have come this far without your guidance. I could not have asked for a better mentor who is as patient and gentle as you had been. You respected me as the reserved, quiet achiever that I am, you’re always listening and respecting my ideas, and you would seriously consider my outrageous theories even though these might have mostly been wrong. You really are a great boss, teacher and supervisor deserving of the successes in your career. I had made the right decision to join your lab and have truly grown to love and enjoy B cell biology so much that even seeing acronyms like “B” or “GC” would excite me, so thank you! To Dr. Tyani Chan – thank you for having been a wonderful mentor, comforter and friend throughout these years. You have taught and helped me so much when I joined the lab that I might not be where I am now without the good foundation from the start. Thank you for the lovely coffee catch-ups and life discussions too because these are precious moments that really helped me refocus on my goal and tasks at hand.

Thank you also to Prof. Tri Phan – you’ve been a great mentor and friend. Thank you for your sincerity, honesty, support and encouragments, and for leading interesting scientific discussions that widen my views. I am really thankful and have learnt a lot from you! I especially need to thank Dr. Danyal Butt – my time in the lab overlapping with yours was perhaps the most fun and was also perhaps the reason that completely ruined my social life. I loved

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our permanent late night FACs, crazy sorts, late night dinners, sleepiness and weekend rendezvous in the lab. I would not have survived my PhD without your genuine and honest support and advice as a dear friend and former PhD in the Brink lab. I am truly blessed to have you as my friend and my support during the difficult times throughout my PhD, so thank you.

A special thank you to all of Brink lab and Phan lab. All of you have contributed to this supportive environment for me to achieve what I have today. I feel very loved by all the generous giving of advice, stories, food, coffee and good laughs – thank you so much! I especially want to acknowledge Katherine Bourne, Jana Hermes and Clara Young for the genotyping and general housekeeping in the lab, making it an easy and smooth environment to work in. I’d also like to thank Imogen Moran, Dr. Chris Sundling and Dr. Dan Suan for some interesting scientific discussions over the years. Thank you also to the wonderful lab mates: Julia, Bethany, Geetha, Etienne, Debbie, Emily, Danielle, Simon – you’ve all been especially supportive and wonderful friends to share the work environment with during my time at Garvan and I am truly grateful for this. My thanks also extend to the all my other colleagues who make this a wonderful place to work in.

Thank you to my most dependable and best friends Lauren, Eric, Jason, Steffi, Marcello, Anna, Nikita, Mayura – these are all brilliant and important people who journeyed with me throughout these important years of my career and are the foundations of my successes today. We share and shoulder each others’ troubles, worries, successes, achievements and failures all the time and I cannot express how much your genuine friendships mean to me. Most importantly, I’d like to thank my dearest parents and my brother who are the most patient and loving people in the universe. These people have greatly influenced who I am and where I am today. Their encouragements and unconditional love for me are the real driving force for me to pursue my dreams and interests. Words cannot express my gratitude enough, thank you and I love you all.

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Thank you especially to my best friend and my love, Eric Lee. There are so many things that I must thank you for. You have been there for me this entire journey, including the many sleepless 4am FACS, patiently waiting for me and making sure I have a way to go home and rest. Thank you for coming out to have dinner dates with me in the lab. Thank you for always being there for me whenever things don’t go the way it should. This draws a new chapter in my life and no words can express how much I love you and how grateful I am to have your support during all these years. You deserve this PhD as much as I do. Thank you so much for all the time and effort you have sacrificed for me, I will forever remember.

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Abstract

BAFFR receptor (TNFRSF13C) and its ligand BAFF (TNFSF13B) are imperative for the normal maturation and survival of naïve B cells. BAFF:BAFFR signalling is also thought to be essential for the maintenance of T-dependent B cell responses. The immunisation of BAFFR-deficient or BAFF- deficient mice generated small germinal centres (GCs) that could not be sustained and a complete absence of memory B cells (MBCs). However, these deficient mice have abnormalities in their secondary lymphoid tissues and markedly reduced mature B cells; hence it remains unclear the extent to which BAFF:BAFFR signalling regulates B cell responses to T-dependent antigens. To investigate this, this thesis utilised the high resolution in vivo mouse model (SWHEL), in which HEL-specific B cells were challenged and examined in various adoptive transfer approaches in order to dissect the cell- intrinsic function(s) of BAFF:BAFFR signalling in regulating GC and MBC responses. Results here demonstrated that the loss of BAFFR on responding B cells had no impact on GC B cell survival but the immaturity associated with BAFF or BAFFR deficiency led to the establishment of smaller GC responses. In addition, our results showed that BAFF:BAFFR signalling was dispensable for controlling the maturation and selection of GC B cells. BAFFR-deficient B cells were found to acquire normal somatic mutations in their immunoglobulin variable region genes that conferred high affinity binding for antigen as well as the production of high affinity antibodies. Importantly, GC-independent MBCs were discovered to be particularly sensitive to the survival signals delivered through BAFFR. The loss of BAFFR expression led to a specific depletion in class-switched unmutated MBCs but had no impact on somatically mutated, GC-derived MBCs. Enforced signalling through BAFFR also preferentially prolonged the survival of these early MBCs. Data presented in this thesis indicate that the maintenance of T-dependent MBC subsets are differentially regulated – that is the survival requirement for BAFF:BAFFR signalling is unique to GC-independent, early MBCs and not shared by BAFFR- independent MBCs that develop from GC precursors.

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Abbreviations, Conventions & Definitions

AID Activation-Induced cytidine Deaminase APRIL A PRoliferation Inducing Ligand BAFF B cell Activating Factor BAFFR B cell activating Factor Receptor Bcl-2 B-cell lymphoma 2 Bcl-xL B cell lymphoma extra-large BCM B Cell Media BCMA B Cell Maturation Antigen BCR B Cell Receptor Bim Bcl-2 Interacting Mediator of cell death BSA Bovine Serum Albumin CH Ig Heavy Chain Constant region cIAP1/2 Cellular Inhibitor of Protein 1 and 2 CCL Chemokine (C-C motif) Ligand CCR Chemokine (C-C motif) Receptor CD Cluster of Differentiation CLP Common Lymphoid Progenitor CSR Class Switch Recombination CXCL Chemokine (C-X-C motif) Ligand CXCR Chemokine Receptor Concatenated Pooled, combined (FACS data) DAPI 2- (4-Aminopenyl)-1H-indole-6-carboxamidine DMEM Dulbecco’s Modified Eagle Medium DPBS Dulbecco’s Phosphate Buffered Saline Eptstein-Barr Virus-Induced G-protein coupled EBI2 Receptor 2 (N-(3-Dimethylaminoapropyl)-N’-ethylcarbodiimide EDCI hydrochloride) eGFP Enhanced Green Fluorescent Protein ELISA -linked Immunosorbent Assay FACS Fluorescence-Activated Cell Sorting FBS Fetal Bovine Serum FDC Follicular Dendritic cell Fo (B cell) Follicular B cell FSC-A Forward Light Scatter-Area (cell size) GC Germinal Centre GC-independent Also referred to as early memory B cells memory B cells GC-dependent Also referred to as GC-derived or late memory B cells memory B cells GFP Green Fluorescent Protein hr Hour

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Hapten A small molecule conjugated to an antigenic carrier HEL Hen Egg Lysozyme Hen Egg Lysozyme with 3 mutations at R21Q, R73E, HEL3X and D101R. HSC HyHEL10 anti-Hen Egg Lysozyme antibody i.v. Intravenous tail vein injections IFN Interferon Ig Immunoglobulin IgH Immunoglobulin Heavy Chain IL Interleukin () IRES Internal Ribosome Entry Site LTα1β1 Lymphotoxin alpha-1-beta-1 mAb Monoclonal Antibody MACS Magnetic-Activated Cell Sorting MBC Memory B Cell MCS Multiple Cloning Site MFI Mean Fluorescence Intensity MHC Major Histocompatibility Complex min Minute MS Magnetic Separation MSCV Murine Stem Cell Virus Retrovirus System MZ (B cell) Marginal Zone B cell Nuclear Factor of Kappa like polypeptide enhancer NF-κB2 in B cells NIK NF-κB2 Inducing Kinase NP 4-Hydroxy-3-nitrophenylacetyl NP-CGG 4-Hydroxy-3-nitrophenylacetyl-Chicken Gamma Globulin 4-Hydroxy-3-nitrophenylacetyl-Keyhole Limpet NP-KLH Hemocyanin NPP p-nitrophenyl phosphate P (statistics) P-value, indication of statistical significance PBA 0.1% BSA with 0.1% Azide in 1x PBS (FACS buffer) PB Plasmablast PBS 1x Phosphate buffered saline pCL-Eco Retrovirus Packaging Vector PCR Polymerase Chain Reaction PC PNA Peanut Agglutinin Polybrene Hexadimethrine Bromide RAG Recombination-Activating Gene RBC RPMI Roswell Park Memorial Institute medium S31R Serine (Ser) to Arginine (Arg) at amino acid position 31

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SA-AP Streptavidin conjugated Alkaline Phosphatase SHM Somatic Hypermutation SRBC Sheep Red Blood Cells TACI Transmembrane activator and CAML Interactor Tfh (cell) T Follicular Helper cell Tg Transgenic TBE Tris/Borate/EDTA (buffer) TNF Tumor Necrosis Factor TNFSF/TNFSFR Tumor Necrosis Factor Superfamily Ligand/Receptor TRAF TNF Receptor Associated Factor VH10 HyHEL10 Heavy Chain Variable Region vs. versus WT Wild Type Tyrosine (Tyr) to Aspartic Acid (Asp) at amino acid Y53D position 53 Tyrosine (Tyr) to Phenylalanine (Phe) at amino acid Y58F position 58 2FD 2% FBS in DMEM medium 2FR 2% FBS in RPMI medium 10FD 10% FBS in DMEM medium Δ (Delta) Gene deletion

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Amino acid abbreviations

Arginine Arg R Histidine His H Lysine Lys K Aspartic acid Asp D Glutamic acid Glu E Serine Ser S Threonine Thr T Asparagine Asn N Glutamine Gln Q Cysteine Cys C Selenocysteine Sec U Glycine Gly G Proline Pro P Alanine Ala A Valine Val V Isoleucine Ile I Leucine Leu L Methionine Met M Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W

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List of Diagrams & Tables

Diagram 1. Mechanisms of BAFF:BAFFR signalling in NF-κB2 activation...... 8 Table 2.2. List of monoclonal Abs, clones, conjugates and sources used for tissue culture, flow cytometry, immunofluorescence histology, MACS separation and ELISA analysis...... 34 Table 2.4. Staining procedure for surface marker analysis by flow cytometry...... 38 Table 2.5.1. Staining strategy for sorting total donor IgG1+ GC and IgG1+ memory B cells ...... 40 Table 2.5.2. Reaction mix for FACS sorted single cell digest ...... 41 Table 2.5.3. Single cell digest reaction condition ...... 41 Table 2.5.4. Reagents for single cell primary PCR ...... 42 Table 2.5.5. Primary PCR reaction condition ...... 43 Table 2.5.6. Reagents for single cell secondary PCR ...... 44 Table 2.5.7. Secondary PCR reaction condition ...... 45

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List of Figures

Figure 3.1. BAFFR is differentially expressed on activated B cell blasts upon activation by T-dependent antigen SRBC ...... 58 Figure 3.2. Gating strategy to identify donor-derived high vs. low affinity GC B cells, MBCs and early PBs using multicolour cell surface markers...... 60

Figure 3.3. BAFFR is differentially expressed on activated SWHEL B cells responding T-dependent antigen HEL...... 63 Figure 3.4. BAFFR expression does not correlate with high and low affinity compartments in the GC...... 65 Figure 3.5. Generation of mixed bone marrow chimera to investigate the role of T cell-derived BAFF in regulating GC and MBC responses. .. 68 Figure 3.6. T cell-derived BAFF is dispensable for the survival and maintenance of donor-derived GC and MBC responses...... 69 Figure 3.7. T cell-derived BAFF is dispensable for GC B cell affinity maturation and selection into the MBC compartment...... 72 Figure 3.8. T cell-derived BAFF is not required for the generation and selection of high affinity antibodies...... 74 Figure 3.9. GC B cells undergo normal SHM and selection for affinity- enhancing mutations in the absence of T cell-derived BAFF...... 76 Figure 4.1. BAFF-deficient and BAFFR-deficient mice are lymphopenic and lack mature B cells in their secondary lymphoid organs ...... 83 Figure 4.2. Abnormal lymphoid organ architecture in BAFF-deficient and BAFFR-deficient mice...... 85 Figure 4.3. Adoptive transfer strategy to investigate the impact of BAFF- deficient and BAFFR-deficient conditions on wild type B cell responses...... 87 Figure 4.4 BAFF-deficient and BAFFR-deficient mice fail to sustain endogenous GC and MBC responses to SRBC...... 89 Figure 4.5. BAFF-deficient and BAFFR-deficient environments inhibit the progression of wild type B cell responses...... 93 Figure 4.6. Attenuated wild type GC responses but intact MBC responses in BAFFR-deficient not BAFF-deficient recipient mice...... 94 Figure 4.7. Wild type responses accumulate low affinity MBCs despite normal GC affinity maturation in BAFFR-deficient recipient mice...... 97 Figure 4.8. BAFFR-deficient environment attenuates GC but not MBC responses of wild type B cells...... 100 Figure 4.9. Wild type B cell responses accumulate low affinity MBCs but not low affinity GC B cells in BAFFR-deficient condition...... 102 Figure 4.10. Single cell SHM analysis reveals wild type B cell responses accumulate unmutated, early MBCs in BAFFR-deficient environment...... 106

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Figure 5.1. Adoptive transfer strategy to elucidate B cell-intrinsic functions of BAFFR in T-dependent responses...... 112 Figure 5.2. Poor clonal expansion but normal differentiation of activated BAFFR-deficient B cells...... 114 Figure 5.3. Adoptive transfer strategy to investigate B cell-intrinsic BAFFR signalling and cell maturity in regulating T-dependent responses...... 117 Figure 5.4. Loss of BAFFR signalling but not cell immaturity impairs BAFFR- deficient IgG1+ MBC survival...... 120 Figure 5.5. Cell immaturity delays affinity maturation but has no impact on affinity-based selection in BAFF or BAFFR deficient GC responses...... 123 Figure 5.6. BAFFR-deficient low affinity MBCs fail to persist but high affinity MBCs remain intact during T-dependent responses...... 126 Figure 5.7. Adoptive transfer strategy to compare immature B cell responses using wild type and BAFFR-deficient mice...... 129 Figure 5.8. BAFFR-deficient B cells form normal GC responses but diminished MBC responses compared to immature wild type B cells...... 132 Figure 5.9. Normal affinity maturation in the GC but an absence of low affinity MBCs in BAFFR-deficient donor responses...... 135 Figure 5.10. Single cell SHM analysis reveals normal selection for high affinity somatic mutations in BAFFR-deficient GC B cells...... 139 Figure 5.11. BAFFR-deficient B cells produce high affinity antibody responses comparable to immature wild type B cells...... 140 Figure 5.12. Single cell SHM analysis shows unmutated, early MBCs are absent in BAFFR-deficient donor responses...... 142 Figure 6.1. Adoptive transfer strategy to investigate enforced BAFFR signalling via retroviral overexpression ...... 150 Figure 6.2. BAFFR-overexpression on B cells significantlly increases IgG1+ MBC numbers during T-dependent responses...... 153 Figure 6.3. BAFFR-overexpressing B cell responses show normal affinity maturation in the GC but accumulate low affinity MBCs...... 156 Figure 6.4. Single cell SHM analysis reveals the accumulation of unmutated, early MBCs in BAFFR-overexpressing donor responses...... 160 Figure 6.5. Adoptive transfer strategy to examine the impact of hyperactivated BAFFR signalling in T-dependent responses...... 162 Figure 6.6. TRAF3 inactivation has no impact on GC size but significantly increases MBC numbers...... 164 Figure 6.7. TRAF3-deficient GC B cells undergo normal affinity maturation and affinity-based selection...... 167 Figure 6.8. TRAF3-deficient responses accumulate low affinity MBCs but generate comparable numbers of high affinity MBCs...... 168 Figure 6.9. Adoptive transfer strategy to examine BCL-2 family proteins in regulating B cell responses...... 171

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Figure 6.10. Overexpression of BCL-2 or BCL-xL significantly expands both IgG1+ GC and IgG1+ MBC numbers...... 175 Figure 6.11. BCL-2 or BCL-xL overexpression prolongs the survival of low affinity cells during affinity-based selection in the GC...... 179 Figure 6.12. B cell responses overexpressing BCL-2 or BCL-xL accumulate significantly increased numbers and frequencies of low affinity MBCs...... 181 Figure 6.13. Adoptive transfer strategy to examine Bcl2-family protein BIM in regulating T-dependent B cell responses...... 184 Figure 6.14. BIM-deletion increases GC B cell and MBC numbers and prolongs their survival during T-dependent responses...... 186 Figure 6.15. Normal affinity maturation but impaired counter-selection of low affinity GC B cells in BIM-deficient donor responses...... 190 Figure 6.16. BIM-deletion increases high affinity MBC output but accumulates significantly increased numbers and frequencies of low affinity MBCs...... 192

xiv Chapter 1: Introduction

Chapter 1

Introduction

1.1 Preamble

A remarkable feature of B cells is their ability to establish lifelong memory and antibodies that are critical for maintaining long-lived immunity. This introductory chapter will provide a brief overview of the components crucial for B cell homeostasis, followed by a review on the existing knowledge on how germinal centre (GC) B cell responses and memory B cells (MBCs) are regulated, all of which are key to understanding the rationale of the topic presented in this thesis.

1.2 B cell origins and selection into the primary B cell repertoire

1.2.1 B cell lymphopoiesis

B and T cells are generated from pluripotent hematopoietic stem cells (HSC) residing in the bone marrow. HSCs lose their self-renewing ability upon downregulation of fms-like tyrosine kinase-3 (Flt3) controlled by transcription factor Pax5 (1-3) and become multipotent progenitor cells that give rise to both the common myeloid progenitor (CMP) whereby all myeloid cells are derived (4), and the early lymphoid progenitors (ELP) through which all lymphoid cells are derived (5). In mice, these lymphoid precursors are nurtured by the bone marrow niche, in which survival factors Flt3 ligand (6-9), interleukin 7 (IL-7) (10), and other stem cell growth factors are essential for their survival and subsequent differentiation into common lymphoid progenitors (CLP). CLPs commitment to the B cell lineage is regulated through a set of transcription factors (11, 12), namely PU.1 (13, 14), E2A (15), EBF1 (16) and Pax5 (3). These transcription factors orchestrate the ligand/receptors critical for the survival, maturation and progression from pro-B, pre-B to new immature-B cells inside the bone marrow (17) and finally transitional-B cells that egress to peripheral lymphoid organs to undergo further maturation and selection.

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1.2.2 BCR rearrangement and selection

The key determinant that governs B cell fate is the assembly of a functional B cell receptor (BCR) first initiated at the pro-B cell stage. During this developmental stage, the rearrangement of multiple V(D)J segments of the immunoglobulin (Ig) genes (18, 19) yield variable gene exons that encode the amino-terminal components of the Ig heavy and chains of the BCR unique to each B cell. Two critical , the products of Recombination Activating Genes 1 and 2 (RAG1 and RAG2) (20, 21), first mediate VDJ recombination to form the heavy chain variable region gene. During pre-B cell stage, intact µ- membrane (µm) heavy chain is synthesised and paired with surrogate light chain components (22, 23), as well as the Igα and Igβ transmembrane proteins to allow surface expression of the pre-BCR. Successful selection through pre-BCR signalling initiates the final transition, in which RAG1/RAG2 mediate VJ recombination to form a productive κ or λ light chain that subsequently pairs with µm to express a functional IgM BCR on immature B cells (24). Immature B cells expressing BCRs that interact strongly with self-antigen in the bone marrow are removed from the newly emerged primary B cell pool either by deletion or RAG-mediated receptor editing (24-26).

1.2.3 B cell homeostasis in peripheral lymphoid tissue

Immature B cells that have egressed from bone marrow primarily migrate to the spleen via mechanisms that are still not entirely understood. These transitional (T) B cells undergo further negative selection based on reactivity with self-antigen and can either be deleted or enter a state of anergy (27). Subsequent maturation into mature follicular (Fo) or marginal zone (MZ) B cells are distinguished from immature B cells by their reduced CD93 expression (AA4.1) (28, 29) and exhibit distinct functions and anatomical locations in peripheral lymphoid organs.

1.2.3.1 Transitional B cells

As immature B cells mature into transitional B cells, they undergo extended transcription and alternative splicing of their Ig heavy chain genes

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resulting in the co-expression of the IgM and IgD antigen receptors carrying identical variable region sequences but distinct constant region sequences (30). Within the spleen, three types of immature transitional B cells (T1, T2, T3) have been identified based on their surface expression of IgM, IgD, CD21 and CD23 (28, 29). T1 B cells (IgMhi, IgDlo, CD21lo and CD23lo) are recent immature emigrants from the bone marrow. T1 B cells undergo further maturation to become T2 B cells (IgMhi, IgDhi, CD21hi and CD23hi) before they start migrating to distal peripheral lymph nodes. T3 B cells (IgMlo, IgDhi, CD21hi and CD23hi) appear to be made up of self-reactive B cells that have entered a state of anergy (27, 31).

BCR expression and "tonic" signalling is essential for the survival of transitional B cells and their maturation into and survival as both MZ and Fo B cell subsets (32-35). Upon reaching the T2 stage, B cells also become absolutely dependent on the survival cytokine, BAFF (TNFSF13B) (36, 37) and its receptor BAFFR (TNFRSF13C) (38). BAFF:BAFFR signalling is also essential for the subsequent maturation of T2 B cells into mature B cells in the spleen (39, 40) and has been shown to regulate selection of self-tolerant B cells into the mature B cell compartment (41, 42).

1.2.3.2 Mature B cell subsets

Mature B cells are categorised into two types, B1 and B2 cells that display distinct definitions and functions and are distinguished from immature subset by loss of CD93 (AA4.1) expression (28, 29). B1 cells (IgMhi, CD19hi, CD1dint, IgDlo, CD23- and CD43+) reside in peritoneal and pleural cavities that function to secrete IgM+ “natural antibodies” against a wide range of self- antigens and antigens derived from pathogens such as LPS (43, 44). B2 cells refer to marginal zone B cells (CD21hi, CD23lo/int, IgMhi, IgDlo/int, CD1dhi) and follicular B cells (CD21lo/int, CD23hi, IgMlo/int, IgDhi, CD1dlo/int) that occupy distinct anatomical regions of secondary lymphoid organs (45).

Marginal zone (MZ) B cells are located in the marginal zone of the spleen between the marginal sinus and follicle and normally constitute 5 to 10% of

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splenic B cells (46, 47). From the marginal sinus, bacterial derived T- independent antigens can be delivered by slow-flowing blood into the MZ and directly activate resident MZ B cells (47). Crosslinking of BCR on MZ B cells by T-independent antigens leads to rapid production of unswitched (IgM) antibodies that are typically of low affinity (46, 48) but are crucial for effective control of T-independent pathogens. However, recent studies have demonstrated MZ B cells can also participate in T-dependent responses to generate antibodies that are isotype-switched and have mutated BCRs, albeit at a slower kinetics compared to neighbouring follicular B cells (49, 50).

Follicular (Fo) B cells are functionally and phenotypically distinct from MZ B cells. Fo B cells dominate the peripheral B cell pool (70-80%) (45) and they are the main subset involved in response to T-dependent antigens. Fo B cells express chemokine receptor CCR7, thus they are conveniently located adjacent to the T cell zone (51). Upon activation by T-dependent antigens, activated Fo B cells subsequently migrate into GCs to undergo somatic hypermutation (SHM) and selection for enhanced BCR affinity for antigen so as to generate high affinity antibody and memory responses (50, 52).

B cells express a number of chemotactic receptors that direct their migration within and between secondary lymphoid tissues. These include CXCR5 (CXCL13 receptor), CCR7 (CCL19/21 receptor), EBI2 (7α,25- dihydroxycholestrol receptor) and the sphingosin-1 phosphate receptors S1PR1 and S1PR2 (53-56). Follicular dendritic cells (FDCs) in secondary lymphoid organ produce CXCL13 (57). In response to this chemokine gradient, Fo B cells migrate to populate the follicular region to form characteristic B cell follicles in spleens and lymph nodes (55, 58). FDCs are also critical for presentation of immune complexes that drive GC responses to T-dependent antigens (59-61). Mature Fo B cells are essential for the formation and maturation of FDC network through a positive feedback loop for LTα1β2-signalling that maintains the cellular integrity of stromal and B cell meshwork (57, 60, 62, 63). Mature FDCs in turn provide survival factors such as BAFF (64, 65) and other important chemokines and to assist the survival and aggregation of Fo B cells

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into well-define microstructures, in order to promote effective antigen-encounter during T-dependent B cell responses (60, 66, 67).

All mature B cells continue to require intact signalling through its BCR to survive (68, 69). A second indispensable signal is delivered to naïve B cells through BAFFR, which is essential for the normal maturation and establishment of peripheral B cell repertoire (37, 40, 70-72).

1.3 TNF ligand BAFF and its receptors

1.3.1 Expression of BAFF and BAFF receptors in B cell homeostasis

B cell activating factor (BAFF also known as TNFSF13B, Blys, THANK, TALL-1, zTNF4) is a survival cytokine crucial for B cell maturation and survival in vivo (34, 39, 73, 74). BAFF is expressed by myeloid cells, stromal cells and also by T cells (75, 76). However, mice reconstituted with BAFF-deficient bone marrow demonstrated that BAFF derived from resident radiation-resistant cells is sufficient to support naïve B cell maturation and survival (77, 78). Transgenic (Tg) mice over-expressing BAFF typically display mature B cell hyperplasia and autoimmune symptoms (79-81). Conversely, deficiency in BAFF leads to B cell lymphopenia and a developmental block at the transitional T2 stage (76), resulting in severely impaired B cell responses (82).

BAFF is expressed on cell surface and can also be cleaved into its soluble form. BAFF acts as a functional trimer or 60-mer protein that show different activity and binding (83, 84) to three TNF receptors (TNFR) restricted mainly to the B cell lineage: B Cell Maturation Antigen (TNFRSF17; BCMA) (73, 85), Transmembrane Activator and Calcium Modulator and Cyclophilin Ligand Interactor (TNFRSF13B; TACI) (86, 87) and BAFF Receptor (TNFRSF13C, BAFFR or BR3) (88, 89). A PRoliferation Inducing Ligand (TNRSF13; APRIL) is also closely related to BAFF, but shows specificity only to TACI and BCMA and has weak affinity towards BAFFR (73).

BCMA is mostly expressed on plasma cells (PCs) and has been shown to exhibit pro-survival effects on human plasmablasts (PBs) (90) and bone

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marrow PCs (91). TACI is expressed on transitional B cells but is not required for their survival (82, 92, 93). TACI potently interacts with APRIL and the multimeric BAFF 60-mer (94) and have been shown to be a negative regulator in B cell survival, as TACI-deficient animals have significantly expanded B cell compartment (95).

BAFFR is first expressed on T2 B cells and is maintained at high levels throughout maturation and B cell activation (40, 96). BAFFR is the only one of the three receptors for BAFF that bears high specificity for this ligand (73, 89). As mentioned previously, BAFF:BAFFR signalling is critical for naïve B cell survival (73). In humans and in mice, defective signalling in BAFFR (such as A/WySnJ mice) (97) or absent expression results in similar B cell defects as those associated with BAFF-deficiency (73, 76). Lack of BAFF:BAFFR signalling leads to the termination of B cell maturation at the T2 stage, impaired naïve B cell survival and significantly reduced numbers of mature B cells in peripheral lymphoid tissues (73, 76).

1.3.2 Activation of alternative NF-κB pathway by BAFF:BAFFR signalling in primary B cells

BAFFR (TNFRSF13C) is a type III transmembrane protein that contains a single binding site (PVPAT) for TNFR-Associated Factor 3 (TRAF3) but not other TRAF proteins (84, 98, 99). TRAF3 does not activate the canonical NF-κB pathway or c-jun N-terminal (JNK) pathway (98-101) but instead specifically suppresses the alternative NF-κB pathway (NF-κB2 activation) by assisting the degradation of NF-κB-inducing kinase (NIK) (102, 103); hence activation of NF-κB2 is a major regulator of the survival in primary B cells. Mice deficient in NF-κB2 or any other upstream components such as NIK or I-κB kinase α (IKKα), displayed a significant loss of peripheral B cells that phenotypically resembled BAFF-deficient or BAFFR-deficient mice (104, 105).

The molecular mechanisms that underlie BAFF:BAFFR signalling pathway in NF-κB2 activation is depicted in the following diagram (Diagram 1) (103). The inactive state of BAFFR maintains the stable interactions of

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cytoplasmic TRAF3, TRAF2 and the cellular Inducer of Apoptosis Proteins 1 or 2 (cIAP1/2). During steady state, TRAF3/TRAF2/cIAP1/2 form a complex that readily ubiquitinylates NIK and direct its degradation (106, 107). Conversely, the ligation of BAFFR results in the dissociation of the TRAF3/TRAF2/cIAP1/2 complex, from which TRAF3 is recruited directly to bind the cytoplasmic domain of BAFFR resulting in its proteasomal degradation (107). The degradation of TRAF3 releases NIK from targeted degradation, which in turn activates NF-κB2 signalling (103). Thus NIK triggers processing of p100 NF-κB2 protein to its active p52 component, which is then recruited into the nucleus to activate the NF-κB2-regulated gene transcription in primary B cells (89, 108). Since TRAF3 suppresses the alternative NF-κB pathway by binding NIK and directing its degradation (102), inactivation of TRAF3 in murine B cells results in increased NF-κB2 activation (109). TRAF3-inactivation constitutively activates the BAFFR/NF-κB2 signalling cascade and relieves B cells from dependence on BAFF (106, 107, 109, 110).

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A B C BAFF

BAFFR BAFFR BAFFR

c-IAP1 K48 Ub TRAF3 K48 Ub c-IAP1 c-IAP1 TRAF2 TRAF3 TRAF3 Degradation Inactivation TRAF2 NIK K63 Ub TRAF2 K63 Ub TRAF3

NIK NIK NIK P IKK Degradation NIK

K48 Ub P P P p100 RelB Activation

p100 RelB

p52 RelB

Nucleus

Bcl2, BclxL p52 RelB Bcl2a1

Cell Survival Figure adapted from Gardam & Brink, 2013, Front. Immu.

Diagram 1. Mechanisms of BAFF:BAFFR signalling in NF-B2 activation.

(A) Inactive BAFFR signalling pathway consists of TRAF3, TRAF2 and cIAP1/2 facilitating the degradation of NIK. This preserves p100 NF-B2 protein and inhibits NF-B2 activation by sequestering RelB in the cytoplasm. (B) BAFF:BAFFR ligation leads to TRAF3 recruitment to the receptor and subsequent degradation by the TRAF2/cIAP1/2 complex. TRAF3 degradation liberates NIK from degradation, allowing it to accumulate and degrade p100 via direct phosphorylation and the phosphorylation of IKK. p100 is partially degraded into p52 active subunit and dimerises with RelB to migrate to the nucleus and initiate NF-B2 specific gene transcription. (C) Inactivation of TRAF3 mimics BAFF:BAFFR ligation, similarly prevents NIK from degradation and activates the downstream signalling cascade to initiate NF-B2 specific gene transcription.

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1.3.3 BAFFR-mediated NF-κB2 activation and the induction of Bcl2-family proteins in B cell survival

The effects of BAFF:BAFFR signalling are associated with the induction of Bcl2-family proteins that are critical in regulating hematopoietic cell death and survival. Several studies have shown the effects of BAFF signals on B cells consequently led to the induction of NF-κB2-dependent pro-survival genes such as Bcl-2, Bcl-xL and Bcl2a1 (39, 81, 108, 111, 112) and the repression of pro- apoptotic genes such as Bim (Bcl2l11) (110, 113-116).

Studies on BCL-2-deficient and NF-κB2-deficient mice revealed global defects in immune cells and phenotypes similar to those observed in BAFF- deficient and BAFFR-mutant mice (104, 105, 117). The deficiency in BCL-2 and NF-κB2 resulted in markedly reduced peripheral B cell numbers and significant maturation defects in B cell development. Conversely, Bcl-2 Tg mice closely mimicked Baff Tg mice (81, 118), or those that lack the pro-apoptotic Bcl2- family protein BIM (119). These mice all presented with splenomegaly, increased B cell numbers and prolonged B cell survival. These phenotypic similarities suggest that Bcl2-family proteins play a key role in the BAFF- mediated survival of primary B cells.

Several studies have investigated the convergence of these pathways by crossing BAFFR-mutant (A/WySnJ) mice onto the Bcl-xL Tg or Bcl-2 Tg backgrounds (120, 121). Interestingly, transgenic expression of BCL-2 or BCL- xL overcame the survival defect in B cells with impaired BAFFR signalling and peripheral B cell numbers were restored in vivo (120, 122). These studies suggest that survival signals delivered through BAFF:BAFFR are mediated by the inhibition of mitochondrial-mediated cell death (intrinsic apoptotic pathway) by inducing Bcl2-family proteins in a NF-κB2-dependent manner. However, the transgenic expression of BCL-2 and BCL-xL that rescued B cell survival in BAFFR-mutant mice failed to restore the B cell maturation defect (121), hence indicating that additional pathways are activated by BAFF:BAFFR engagement to mediate the B cell maturation in vivo.

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1.4 T-dependent B cell responses

1.4.1 B cell activation and early differentiation by T-dependent antigens

Antibody responses to protein antigens require B cells to present antigen to cognate CD4+ T helper cells (123, 124). Protein antigen captured by the BCR is internalised and degraded within the lysosome before antigenic peptide are loaded onto the major histocompatibility complex class II (MHC II) for presentation to helper T cells (125, 126). Soon after antigen-encounter, activated B cells express chemokine receptor EBI2 and migrate in response to its ligand 7α,25-dihydroxycholestrol towards the inter-follicular and outer- follicular region of secondary lymphoid organs (127, 128). Here activated B cells increase their surface CCR7 expression and meander to the boundary between the B cell follicle and T cell zone towards chemokines CCL19/21 produced by fibroblastic reticular cells within the T cell-rich area (54, 55, 129, 130). Simultaneously, antigen-activated T cells also migrate to the T:B border to interact with cognate B cells. Here the lengthy interactions between antigen- specific activated T and B cells (131) drive the activated B cells to differentiate along one of three distinct fates: i) extrafollicular plasmablasts (PBs), ii) early memory B cells (MBCs) and iii) GC B cells. The formation of GCs is the hallmark of T-dependent B cell responses where SHM of B cell Ig genes occur that gives rise to affinity matured high affinity antibodies. Positive and negative selection of GC B cells take place within these microstructures to produce high affinity long-lived plasma cells (PCs) (52, 132) and GC-derived MBCs that provide long-lived immunity (133).

Extrafollicular PBs are GC-independent, short-lived antibody-producing cells with a half-life of only 3-5 days (134, 135). These effector cells are located in the bridging channel of the spleen or medullary chords of the lymph nodes, guided by the G protein-coupled receptor EBI2 (127). Inside the bridging channels, activated B cell blasts proliferate and actively produce low affinity antibodies of both IgM and IgG sub-classes during early stages in a T- dependent response (135, 136). However, some evidence suggests

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extrafollicular PBs can also enter the long-lived PC pool in the bone marrow (137, 138).

Our laboratory has shown that the initial strength of BCR engagement impacted upon by antigen affinity and epitope density impacts on the relative size of the PB versus the GC response (135, 136). High affinity interactions between the BCR and its cognate antigen promote the extrafollicular PB response and low affinity interactions preferentially promote differentiation into GC B cells for further affinity maturation (135, 136). In addition, the inhibition of intrinsic apoptosis was shown to liberate the differentiation decisions based on BCR affinity for the antigen (139). Together these demonstrated that BCR signalling strength controls B cell apoptosis and regulates B cell selection into the GC-independent extrafollicular PB or GC B cell responses. Inside the GC, influential decisions are made to determine the death and progression of antibodies with a spectrum of affinities, where antigen-accessibility and competition for T cell help are primary drivers of this highly dynamic selection process (52, 140, 141).

CD4+ T cells are the primary mediators for GC B cell commitment by providing the TNF ligand CD40L necessary for driving the proliferation of activated B cells through CD40 ligation (142-145). The full activation of antigen- activated T cells is mutually dependent on cognate B cells provision of ligands CD80 (B7-1) and CD86 (B7-2) that bind the stimulatory receptor CD28 on T cells. This second signal promotes T cell expansion and induction of key cytokines such as IL-2, IL-4 and IFN-γ (146-148), which are required for the induction of antibody isotype switching. CD80 and CD86 also bind to the inhibitory receptor CTLA-4 on T cells, which acts to negatively regulate B cell responses by competing for these ligands to dampen CD28 signalling (149).

The generation of early MBCs requires CD40-signalling from T cells, but these are generated independently of the GC reaction and can be formed in the absence of GCs (150, 151). A fraction of activated B cells undergo rounds of proliferation and Ig class switch recombination (152) within the first few days post-immunisation. Some of these clonally expanded early B cell blasts obtain a

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memory phenotype to become functionally distinct IgM+ and IgG+ early MBCs (135, 150, 151, 153, 154). These early MBCs are generally short-lived in the spleen and exit the response readily by either migration to peripheral lymph nodes or into the circulation (133, 135, 150, 154). At present, unswitched early MBCs can be distinguished from class-switched GC-dependent MBCs by their surface phenotype of IgM+, CD73-, PD-L2-, CD80- (150, 154), but no definitive phenotypic surface markers have been identified to distinguish class-switched GC-independent MBCs from class-switched GC-derived MBCs (150, 154) and the factors that maintain these GC-independent early MBCs also remain poorly understood (155).

1.4.2 Ig class switching and functionality

B cells enhance their antibody effector function by class switch recombination (CSR) of its Ig heavy chain constant region (CH) (154, 156, 157). Under the influence of T cell help and cytokine signalling, activated B cells express the DNA editing enzyme activation-induced (cytidine) deaminase (AID) that specifically targets a repetitive sequences of DNA called switch regions that are located upstream of all but one (Cδ) of the Ig constant region genes (158, 159). AID induces DNA breaks at the Ig switch region sequences to initiate DNA repair (160) that ultimately leads to intrachromosomal recombination events

(161) and replaces the IgM-encoded Cµ gene with one of the downstream CH genes. This gives rise to IgG (Cγ), IgA (Cα) and IgE (Cε) antibody isotypes (162) that are functionally distinct in their effector capabilities and in eliminating site- specific pathogens (163-165). The induction of both IgG1 and IgE switching require IL-4 and CD40-signalling from T cell (166-169), while IgG2a/c switching is mediated by cytokines IL-2 and IFN-γ (169). Class switching of activated B cells are concomitant with cell division (170, 171).

Although class switching is thought to occur within the GC, our laboratory and others have shown that extensive switching from IgM to IgG can occur prior to GC formation (135). Thus, CSR of activated B cell blasts can occur prior to their differentiation into the GC, memory or extrafollicular PB fates (135, 172-

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174). However, the provision of T cell help and the availability to specific cytokines are essential to initiate this process (143, 144).

1.4.3 The Germinal Centre Reaction

1.4.3.1 Organisation and dynamics of the germinal centre

Germinal centres are distinct microstructures formed inside B cell follicles of secondary lymphoid organs in response to T-dependent antigens. Here, antigen-specific B cells undergo active clonal expansion and somatic hypermutation (SHM) of their Ig variable region genes by the aforementioned AID enzyme thereby generating a diverse BCR repertoire (159, 175, 176). The GC provides an optimal niche for the selection of BCR with high affinity for the antigen, a process known as affinity maturation (177). SHM generates a wide spectrum of BCR affinities for the foreign antigen, some of which might inadvertently acquire increased affinity for self-antigens while increasing affinity for the foreign antigen (178-181). Thus, the positive selection of high affinity GC B cells for the foreign antigen in conjunction with the negative selection and clonal deletion of self-reactive GC B cells, together regulate and maintain effective but non-pathogenic antibody output (52, 182-185).

GC B cells are B220+ (186) and can be identified by high levels of CD24 (186), Fas (187) and n-glycolyneuraminic acid (bound by GL-7 antibody) expression (186). GC B cells lose surface IgD and instead express only IgM or a class-switched BCR class (e.g. IgG1), bind peanut agglutinin (PNA) (186) and have dynamic expression of CD38 that is downregulated in mice, but upregulated in humans (186, 188). GC B cells also highly express MHC-II (186) that is important for antigen presentation and display regulated expression of chemokine receptors EBI2 (128), S1PR2 (77), CXCR4 and CXCR5 (189) that determines their anatomical location and identity. In this thesis, GC B cells and their class-switched progenies will be identified based on B220hi, CD38lo, Fashi and IgG1+ and will be confirmed based on antigen-specificity.

Clonally expanded GC B cells upregulate CXCR4 and CXCR5. CXCR4 is consequently differentially expressed to polarize the GC microenvironment

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into two functionally distinct compartments, the dark zone and light zone (189). The control of CXCR4 expression is mediated primarily through the transcription factor FOXO1 (190). GC B cells fluidly cycle between the two zones during the course of affinity maturation and affinity-based selection to develop BCR specificities of increased antigen affinities (191-193). Dark zone cells are phenotypically CXCR4hi, CD86lo and CD83lo (189). The dark zone develops proximal to the splenic T-cell zone or the medullary cord region in lymph nodes. Here B cells undergo intense proliferation, have increased AID expression and the error-prone DNA polymerase eta (Polη) responsible for introducing point mutations into DNA when repairing AID-induced lesions (193-195). In this way, amino acid substitutions are introduced into the complementary determining regions (CDR) of the Ig variable gene to generate diverse BCR affinities for the foreign antigen (196-198). GC B cells transition from the dark zone to the CXCL13-rich light zone via CXCR5 and the downregulation of CXCR4 (189).

The light zone of the GC is proximal to the splenic marginal zone or the lymph node capsule (199, 200). The light zone comprises of GC B cells that are lo hi hi phenotypically CXCR4 , CD86 and CD83 (189). It is in the light zone that GC B cells undergo affinity-based selection regulated by competition for antigen (201) and accessibility to T cell help (202). Inside the light zone, GC B cells are interspersed amongst a network of FDCs that present antigen in the form of immune complexes that are critical for GC B cell selection (60). Light zone B cells compete for antigens displayed on complement and Fc receptors of FDCs (59, 203), such that GC B cells with highest affinity for foreign antigen are positively selected to survive and proliferate to increase interactions with their cognate GC-resident T follicular helper (Tfh) cells (131, 141, 191, 204, 205). Here in the light zone, high affinity GC B cells are generally selected to become PCs or return to the dark zone for further SHM, while low affinity cells become MBCs or undergo apoptosis (141, 191, 193, 206, 207).

1.4.3.2 Germinal centre populations and their mutual dependence

A complex multicellular network constitutes a functional GC microenvironment and these components act in concert to control antibody

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production and provide long-lived immunity. Tfh cells are specialised CD4+ T cells regulated by transcription factor BCL-6, with a characteristic phenotype of CXCR5hi, CCR7lo (208, 209) that determines their residence in the light zone within the GC microenvironment (210). Although present at relatively small numbers, Tfh cells in the GC provide the CD40L stimulus essential for delivering cognate help to GC B cells (142, 202, 206). However, Tfh cells mutually require contact with cognate B cells to differentiate and maintain their specialised phenotype and functions during the GC reaction (211-213). Tfh cells also provide costimulation through CD28 (142, 214), ICOS (215) and PD-1 (210, 211, 216, 217), as well as cytokines such as IL-4 and IL-21 among others (218-221) that are essential for the establishment and maintenance of GCs as well as differentiation of GC B cells (217, 222). .

FDCs are radiation-resistant stromal cells specialised in displaying antigen in its intact form within the GC (60) and providing soluble factors critical for GC B cell homeostasis (223, 224). The functions of FDCs include controlling the turnover of GC B cells during selection (225, 226) and to serve as an antigen reservoir for BCR stimulation and affinity-based selection in the GC (59, 61, 66, 205, 227). In the GC, the FDC network is concentrated in the light zone (191) and are capable of retaining captured antigen-immune complexes for extended periods of time (61, 140). Non-cognate mature B cells in secondary lymphoid organs have been shown to be responsible for transporting these antigen-immune complexes to the GC light zone and transferring them to FDCs (228-230). FDCs capture antigen-immune complex via complement receptors 1 and 2 (231-234), since deletion of these receptors resulted in significantly reduced antigen presentation and impaired T-dependent responses (203). While GC B cell homeostasis greatly depended on FDCs (66, 205), FDCs are mutually dependent on B cells for their normal maturation and development (235). Maturation of functional FDCs require lymphotoxins (LTα1β1) and tumor necrosis factor (TNF) produced by mature B cells to fully develop into their mature functional state (236, 237). Similarly, studies using LTα1β1-deficient or TNF-deficient mice demonstrated significantly impaired FDCs formation that also failed to develop robust GC responses (236, 238, 239).

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BAFF is also produced by Tfh cells (75, 76) and more abundantly by FDCs (240) within the GC. Since BAFF:BAFFR signalling is imperative for the maturation and survival of peripheral B cells, absence of BAFF or BAFFR thus have a significant impact on Tfh cells and FDC development (241) and thus is likely to impair GC formation in multiple ways. Nevertheless, the provision of BAFF by Tfh cells and FDCs has also been implicated in the maintenance and affinity-based selection in the GC reaction (75, 82, 241).

1.4.3.3 Differentiation and maintenance of long-lived antibody responses

Long-lived PCs are terminally differentiated, non-dividing cells that produce antibodies and thus distinct from the short-lived extrafollicular PBs generated early in B cell responses (135). Long-lived PCs are affinity matured as a result of successive rounds of SHM and positive selection of high affinity antibody with the GC (49). The differentiation of GC B cells into PCs is regulated by the transcription factor IRF4 (242) which initiates the transcription of Blimp-1/PRDM-1 necessary for PC differentiation (243-245). Blimp-1 further induces the expression of transcription factor XBP1 that controls the unfolded protein response responsible for the production and secretion of antibodies (246, 247). The surface phenotype of PCs can be defined as B220lo, CD38lo, IgD-, CD138+, as well as with high levels of intracellular Ig (248). When PCs exit the GC, they enter the blood stream mediated by S1PR1 expression (249) and upregulate CXCR4, which directs PCs to the CXCL12-rich bone marrow niche where they reside (250, 251). The bone marrow niche provides cytokines such as APRIL (252, 253), CXCL12 (137) and CD80 (254) that act on distinct receptors BCMA, CXCR4, and CD28 expressed on PCs respectively, all of which converge to upregulate the pro-survival protein MCL-1 critical for PC survival (255).

1.4.3.4 Germinal centre maintenance, selection and their terminal differentiation into plasma cells

The maintenance of GC B cells is largely dependent on the master transcription factor BCL-6 that programs three main functions (256): i) to

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tolerate AID-induced DNA damage during SHM and CSR (257, 258), ii) to suppress anti-apoptotic molecules in GC B cells such that high turnover of somatically mutated GC B cells can be screened and deleted efficiently (259, 260), and iii) to control other factors/pathways that supplement BCR and CD40 signalling on the GC B cell (261). Thus, GC B cells are highly susceptible to apoptosis (262) and express low levels of pro-survival molecules of the Bcl2- family pro-survival molecules (263, 264) such that inhibition of apoptosis in GC B cells lead to the B cells expressing normally counter-selected antibodies to become MBCs and PCs (136).

The purpose of the GC reaction is the generation and selection of useful antibodies against specific T-dependent antigens. High affinity cells are preferentially selected to proliferate to enter the PC compartment (49) while low affinity cells are negatively selected by apoptosis (265). BCR signalling from the antigen itself is fundamental to the initiation of the GC response but the extent of its contribution to the selection and differentiation into long-lived PC fate has been controversial (183, 199, 266, 267).

The role of Tfh cells as the primary initiator of the GC B cell differentiation into PCs has been suggested both mathematically (268, 269) and experimentally (193, 206). Help from cognate Tfh cells was shown to drive GC B from the light zone towards the dark zone and their subsequent differentiation into PCs (141, 193, 202). ICOS:ICOSL signalling provided by Tfh cells is also essential for the optimal cell-cell contact and cytokine feedback that drives affinity maturation, and by further promoting T cell production of cytokine IL-21 that controls GC robustness and selection of high affinity antibodies (215). In addition, T cell-derived BAFF was also implicated in the affinity-based selection and survival of high affinity GC B cells towards PC differentiation (75) but this remains to be confirmed.

Nonetheless, antigen-binding affinity of the BCR also contributes to the selection process (139, 265). However, the extent to which active BCR signalling controls GC B cell selection and commitment to the PC fate is still not entirely clear. A recent study using Nur77 reporter mice suggested direct BCR

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signalling in light zone GC B cells correlated to induced for PC differentiation, including cMyc, Ifr4, Blimp-1, thus implicated that active BCR signalling possibly facilitates the selection of high affinity GC B cells (270, 271). Recent work from our laboratory also demonstrated that blocking of antigen access on FDCs within already established GCs but not the blocking of T cell help, completely abrogated the initiation of PC differentiation (140). These studies collectively highlighted that both the engagement of high affinity antigen displayed on FDCs and a second costimulatory signal from cognate Tfh cell work in concert to regulate affinity-based selection into the long-lived PC compartment.

1.4.4 Establishing immunological memory to T-dependent antigens

1.4.4.1 Definition of memory B cells in long-lived immunity

Memory B cells (MBCs) by definition are long-lived quiescent B cells capable of eliciting rapid recall antibody responses upon antigen-stimulation compared to their naïve B cell counterparts (272-274). Murine MBCs can persist for long periods and can be identified and distinguished from GC B cells based on their B220+, CD38hi (275), CD138- phenotype, lack of PNA-binding (276) and dynamic expression of CD73 (150, 155, 277). Human MBCs, on the other hand, are typically classified as CD27+, IgDlo/- and CD148+ (278).

Historically, MBCs were thought to solely derive from the GC, but it has recently been shown that MBCs can also be generated in the absence of GC formation (150, 153). Thus, it is now clear that two waves of distinct MBC populations arise from T-dependent responses: i) early MBCs generated prior to and independent of the GC reaction and thus lacking SHM events, and ii) somatically mutated, often affinity matured MBCs derived from the GC (155, 272, 279). These distinct MBC populations can be generated in different magnitudes depending on the T-dependent stimulus (155, 272, 279). However, the differentiation programs that govern the bifurcation of activated B cells into GC-independent and GC-dependent memory fates are still undetermined. Furthermore, no definitive phenotypic markers have yet been identified to distinguish class-switched GC-derived and GC-independent MBCs, with the

18 Chapter 1: Introduction

exception of SHM in their Ig gene and time of their appearance during T- dependent responses (150, 154, 155, 272). The existence of both GC- dependent and GC-independent MBCs highlights that the criteria to obtain “memory” are not dependent on their affinity for antigen or affinity maturation of their BCR to become highly specific for a single determining epitope. This possibly reflects an evolutionary adaptation to retain a spectrum of antigen- specificities for related T-dependent antigens that may help to counteract the issue of "original antigenic sin” (280), the concept in which immunological memory initially established for a dominant antigen is continually reinforced at the expense of effective responses to new, related antigens.

1.4.4.2 Classical GC-dependent memory B cells

GC-derived MBCs contain somatic mutations in their Ig, indicating their passage through the GC reaction, and so are typically affinity matured (281, 282). Affinity matured MBCs of both IgM+ and IgG+ isotypes are found in abundance in both mice and humans and differ in sensitivity to recall and longevity (154, 156, 283, 284). The signals determining GC B cell differentiation into MBCs are only recently becoming better understood (155, 278, 279). The evidence to date suggests that GC B cells with low to moderate BCR affinities for foreign antigens are promoted to become GC-derived MBCs (136, 285, 286). A recent study elegantly demonstrated that low affinity class-switched GC B cells in the light zone were “preferentially” selected into the memory compartment based on the strength from T cell help (282). Recent work from our laboratory also identified CCR6-expressing light zone low affinity cells in the GC to be precursors destined to become MBCs (287). In addition, the transcription factor BACH2 has recently been revealed to be responsible for GC B cell differentiation into post-GC MBCs (288, 289).

1.4.4.3 GC-independent early memory B cells

GC-independent early MBCs are generated during the initial expansion and proliferation of B cells in response to T-dependent antigens (154, 272) and characteristically do not contain somatically mutated Ig genes (150, 154, 277,

19 Chapter 1: Introduction

290, 291). The existence of early MBCs was confirmed in immunisation studies with BCL-6-deficient mice, where robust unmutated memory responses were generated and maintained in the absence of GCs and Tfh cell differentiation (151). However, the formation of these early MBCs is dependent on T cell- mediated costimulation and requires CD40-signals provided by activated CD4+ T cells other than Tfh cells (150, 151). Furthermore, class switching of undifferentiated B cell blasts occurs outside the GC (135) and both IgM+ and IgG+ early MBCs have been identified, showing distinct functionality and responsiveness to antigens (151, 153, 154, 276, 284).

1.4.4.4 Heterogeneity, Ig class and effector function of MBCs

Both IgG+ and IgM+ MBCs are found in both early and late memory responses to T-dependent antigen (154, 284). Specific isotypes have been shown to elicit specialised functions and responsiveness to antigen re-exposure in unswitched vs. class-switched MBCs (155). In vivo investigations on the function and threshold of reactivation between different memory Ig classes have been performed using adoptive transfer of IgG+ vs. IgM+ MBCs into unimmunised mice (154, 284). These studies revealed that IgG1+ MBCs were predisposed to differentiating into antibody-producing cells following reactivation (154, 284, 289). Interestingly, the history of antigenic-stimulation in IgG1+ MBCs but not signalling through the IgG receptor, was found to confer their rapid differentiation into antibody-producing PBs, for the re-stimulation of IgG+ naïve B cells engineered with nuclear transfer from an IgG1+ MBC did not respond similarly (289). On the contrary, IgM+ MBCs were shown to preferentially establish secondary GC responses that can undergo further affinity maturation and CSR (154, 284). Bona fide IgM+ MBCs generated from primary responses have also been shown to have exceptionally longer half-lives compared to IgG+ MBCs, and have been linked to SHM and the higher amount of AID expression found in IgG+ MBCs (154, 156). It is worth noting however, that many of these studies of IgG+ vs. IgM+ MBCs were primarily performed on GC-dependent MBCs and not GC-independent class-switched MBCs (154), hence it would be invaluable to further investigate how these early MBC subsets might differ to GC-dependent MBCs.

20 Chapter 1: Introduction

A major challenge in studying MBCs is the lack of definitive markers to distinguish between unswitched and class-switched MBCs generated from the GC-independent and GC-dependent pathways. Recent studies have reported further heterogeneity exists within the T-dependent MBCs compartment, with the expression of PD-L2, CD80 and CD73 appear to be associated with their maturity towards PC vs. GC differentiation (150, 154, 277, 281, 292). The expression of CD73 was shown to distinguish between class-switched GC- derived MBCs (IgG+, CD73+) and unswitched early MBCs (IgM+, CD73-) (150, 154). On the other hand, the expression of PD-L2 and CD80 surface markers were linked to the mutational content in the Ig variable genes of MBCs (155, 277, 292, 293). These studies found that “double positive” (CD80+, PD-L2+) MBCs accumulated the most SHM in their Ig variable gene, followed by “single positive” (CD80-, PD-L2+) cells with intermediate frequency of mutational load, and “double negative” (CD80-, PD-L2-) cells that were found to be predominantly IgM+ and had relatively fewer mutations (277, 292). The expression of these surface markers, however, reportedly did not correlate to specific MBC Ig isotypes (294). Since IgG+ MBCs generated in these experiments were primarily GC-derived that contain SHM (292), the usefulness of these surface markers in distinguishing GC-dependent vs. GC-independent class-switched MBC subsets have not been elucidated.

1.4.4.5 Memory B cell program, survival and maintenance

MBCs have been shown to persist in the absence of antigen-driven BCR signalling (295, 296). It has also been suggested that their maintenance are independent of CD40:CD40L signalling (297) or ICOS:ICOS-L signalling (290) from T cells. While these studies suggested that both BCR and T cell help might be dispensable for MBCs maintenance, these theories remain controversial (155) and it is possible that additional factors are involved in the maintenance of long-lived memory responses (155, 274, 278).

BAFFR expression is mostly restricted to the B cell lineage and is required in maintaining naïve B cell survival; hence BAFF:BAFFR signalling could also play a role in sustaining MBC survival. The inhibition of BAFF during

21 Chapter 1: Introduction

late T-dependent B cell responses in mice, however, revealed the survival of GC-dependent class-switched MBCs to be BAFF-independent (253, 272, 299). However, IgM+ MBCs were also reported to be sensitive to BAFF-mediated survival (298). Thus, it is still unclear whether BAFF:BAFFR signalling impacts differently on MBCs derived from the GC-dependent vs. GC-independent pathways.

In both mice and humans, the survival gene signature found in resting MBCs is strikingly similar to naïve B cells (299, 300), but MBCs show a unique BCR signalling signature that could explain their heightened and rapid responsiveness to a secondary challenge (293, 301, 302). Transcription factor BACH2 was recently shown to be important for the generation of GC-derived MBCs by repressing PC transcription factor Blimp-1 (282). T-dependent MBCs have been shown to upregulate anti-apoptotic genes such as Bcl-2, Bcl-xL (274, 299, 303), Mcl-1 (301, 305, 306) and Bcl2a1 (140, 300) and demonstrated selective sensitivity to their inhibition (307, 308). The inactivation of pro- apoptotic antagonists BIM (304) and PUMA (305) also differentially prolonged the survival of IgM+ and IgG+ memory subsets (274). Thus, BACH2-regulated transcription of survival proteins critical in hematopoietic cell survival might be linked to the maintenance of GC-dependent MBCs (300, 306-308). However, these studies were performed on GC-derived MBCs, hence the transcriptional regulation that delineates early MBCs is still to be elucidated.

1.5 BAFF:BAFFR signalling in B cell responses, health and diseases

BAFF:BAFFR signalling is imperative for the selection of transitional B cells by providing critical survival signals to maintain the naive B cell repertoire (70, 71). The expression of BAFFR is highly conserved in human and in mice, including naïve B cells, activated B cells and class-switched MBCs that continue to express this receptor at high levels (309). Therefore, the dysregulation of BAFF or BAFFR unquestionably leads to incompetent humoral responses and serious immune complications.

22 Chapter 1: Introduction

In human BAFFR deficiency, patients often manifest clinical immunodeficiency, with characteristically reduced IgG and IgM serum antibodies and are susceptible to recurrent infections (310). Furthermore, the abnormal expression of BAFFR has also been linked to some B cell malignancies and lymphoproliferative disorders (311-314). Moreover, the overproduction of BAFF is strongly associated with autoimmune diseases, in which both humans and mice presented with abnormally high levels of BAFF consequently develop autoantibodies and autoimmune conditions (81, 86, 315). The dichotomy of BAFF:BAFFR signalling in these immune disorders highlights its importance in controlling B cell responses. BAFFR-expression is restricted to specific B cell subsets in humans and in mice. BAFFR remains upregulated during GC and MBC differentiation (309, 316), with the latter also expressing TACI (145, 312, 317). However, BAFFR expression is strictly inhibited in PCs that instead express both TACI and BCMA (65, 91, 312). These observations collectively suggest BAFF:BAFFR signalling might play a role in GC and MBC homeostasis.

Investigations on BAFF:BAFFR signalling in regulating T-dependent responses have been explored using BAFFR-mutant (A/WySnJ), BAFFR- deficient (Tnfrsf13c-/-) and BAFF-deficient (Tnfsf13b-/-) murine models (82, 93, 97, 241). When challenged with T-dependent antigens NP-CCG or NP-KLH, these mice initiate GC responses that are attenuated with reduced serum antibody titres and a complete absence of MBCs (82, 97). These studies suggested BAFF:BAFFR signalling was required for the ongoing maintenance of T-dependent responses. However, the loss of peripheral B cells in BAFF- deficiency and BAFFR-deficiency greatly perturbs secondary lymphoid tissue architectures (122) and impairs optimal B cell survival, FDC maturation and Tfh cell differentiation (82, 241); hence the cell-intrinsic role of BAFF:BAFFR signalling in B cells participating in T-dependent B cell responses remains unclear. Moreover, BAFF was recently implicated to be required in facilitating affinity-based selection of high affinity GC B cells (75, 241) but had been shown to be dispensable for high affinity MBC survival (253). Altogether, these

23 Chapter 1: Introduction

observations indicated that further investigation is necessary to fully elucidate the impact of BAFF:BAFFR signalling in regulating GC and MBC responses.

24 Chapter 1: Introduction

1.6 SWHEL system to study antigen-specific B cell responses in vivo

The SWHEL Ig knock-in mouse model developed in our laboratory features a rearranged anti-HEL heavy chain variable gene derived from the

HyHEL10 monoclonal antibody (mAb) (VH10) targeted to the endogenous

C57BL/6 Ig heavy chain (IgH) (318). SWHEL B cells carrying the targeted and the HyHEL10 ĸ light chain transgene express surface IgM and IgD that bind HEL with same high specificity and affinity as the HyHEL10 (2 x 1010 M-1) (319) and are capable of switching to all classes of Ig and undergoing SHM in the GC (136, 318). Another advantage of the SWHEL transgenic model is its unbiased development into different mature (Fo and MZ) B cell subsets, which were shown previously in our laboratory to exhibit different recruitment kinetics in response to T-dependent stimuli (49), thus providing a more accurate representation of the heterogeneity in normal B cell responses.

In order to mimic physiologically relevant number of antigen-specific B cells in the normal B cell repertoire, studies of B cell responses using small 4 numbers (3 x 10 ) of SWHEL B cells are typically performed via adoptive transfer into CD45 congenic recipient mice. Thus donor SWHEL B cells are tracked in congenic recipients throughout a T-dependent challenge with HEL proteins conjugated to multivalent carrier sheep red blood cell (SRBC) (49). Naïve 3X SWHEL B cells binds HEL (a mutant variant of HEL with three distinct amino acid substitutions in the HyHEL10 epitope of HEL) at an immunogenic but 1000-fold reduced affinity (2 x 107 M-1) (136). Immunisations using HEL3X allow B cell responses to be studied at a physiologically relevant antigenic affinity, enabling high-resolution analysis of the kinetics, affinity maturation and selection in T-dependent responses (52, 135, 136); hence this in vivo system can be integrated with various gene-deficient or transgenic mice strains to investigate the B cell-intrinsic or cell-extrinsic effects of any gene of interest.

Thus, the SWHEL adoptive transfer can be used to investigate T- dependent B cell responses using the following broad approaches: i) Wild type + (WT) SWHEL B cells (CD45.1 ) are transferred into gene-deficient or transgenic congenic recipients (CD45.2+) – to examine how cell-extrinsic or environmental

25 Chapter 1: Introduction

factors associated with the genetic lesion impact on the response of WT B cells, + and conversely ii) gene-deficient or transgenic SWHEL B cells (CD45.1 ) can be transferred into WT congenic recipients (CD45.2+) – to examine B cell-intrinsic impact of the genetic modification in question in a WT, normal environment.

SWHEL B cell responses in assorted tissue samples are tracked by donor CD45 expression and phenotyped using flow cytometry and HEL-specific antibody responses can be detected in the serum. The detailed experimental procedures can be found in Chapter 2 and will be discussed throughout this thesis.

26 Chapter 1: Introduction

1.7 Project aims and hypothesis

The dichotomy of the BAFF/BAFFR axis in the onset of immune disorders highlights the importance of BAFF:BAFFR signalling in controlling B cell responses. However, the specific role(s) of BAFF:BAFFR signalling in regulating T-dependent B cell responses is still unclear, particularly its involvement in regulating GC survival and selection. Importantly, while earlier evidence suggests GC-derived MBCs do not depend on BAFF for their maintenance, it is still unclear whether BAFF:BAFFR signalling impacts differently on MBCs generated from the GC-independent pathway. Thus, given the pivotal function of BAFF:BAFFR signalling in B cell homeostasis and its significant association in health and diseases, we hypothesise that BAFF:BAFFR signalling might be important in regulating T-dependent B cell responses, particularly since the knowledge to date are unclear and are limited by the lack of appropriate murine models. Shaped by these hypotheses, this PhD thesis will address the following broad aims:

1. To establish a murine model suitable for investigating BAFF:BAFFR

signalling in T-dependent B cell responses

2. To examine the impact of BAFF-deficiency and BAFFR-deficiency in

regulating normal B cell responses

3. To investigate the role of BAFF:BAFFR signalling in regulating GC

responses and B cell memory

27 Chapter 2: Material and Methods

Chapter 2

Material and Methods

2.1 Mice and Adoptive Transfer

2.1.1 Mice

-/- -/- ΔB/ΔB SWHEL Ig Tg (49, 52), Tnfsf13b (40), Tnfrsf13c (82), Traf3 (106), Cd3e-/- (320), Bcl2l11-/- (119) mice have been previously described. WT C57BL/6 mice and CD45.1 congenic Ptprc mice were sourced and purchased from Australian BioResources (Moss Vale, Australia) or Australian Resources Centre (Perth, Australia). Tnfsf13b-/- mice were received in-kind from Fabienne Mackay (UMelb, Melbourne) and Cd3e-/- mice were received in-kind from Axel Kallies (WEHI, Melbourne). Bcl2l11-/- mice were generated using CRISPR-Cas9 technology from the MEGA facility at the Garvan Institute. All mice were bred and maintained in specific pathogen-free animal housing at Australian BioResources and the Garvan Institute of Medical Research Biological Testing Facility. Animal experiments were performed under approved guidelines of the Garvan Institute of Medical Research/ St Vincent’s Hospital Animal Ethics Committee.

2.1.2 Recombinant HEL proteins

Hen Egg Lysozyme (HEL) was purchased from Sigma-Aldrich. HEL3X and HEL4X mutants were produced in-house by Katherine Bourne using methods previously described (136). Yeast (Pichia pastoris) expressing HEL mutant proteins were cultured, supernatants collected and purified via ion- exchange chromatography using HiTRAP SP FF columns (GE Healthcare). Purified HEL mutant were stored in PBS at -80 oC.

HEL proteins were desalted into conjugation buffer in the following manner prior to conjugation to SRBC: Proteins were thawed and their concentrations confirmed by spectrophotometry at 280nm. PD10 columns (GE Healthcare) were flushed and equilibrated with 30mL of conjugation buffer.

28 Chapter 2: Material and Methods

Between 100 – 200 µg of each protein was loaded and absorbed into the column. Precisely 2.5 mL of conjugation buffer was added to elute protein through to the base of the column. The 2.5 mL of flowthrough was discarded. To elute the protein of interest, 3.5 mL of conjugation buffer was added onto the column and proteins were eluted in 5 fractions of the following volumes: 250 µL, 1000 µL, 250 µL, 250 µL and 250 µL following optimisation by the Brink laboratory. Protein concentration of each fraction was measured by spectrophotometry at 280nm and the fractions containing significant amount of proteins were pooled and stored at 4 oC for no more than 3 months.

2.1.3 Conjugation of HEL proteins to SRBC

10 µg of desalted HEL (or HEL3X) was conjugated to 10 x 109 SRBC (Institute of Medical and Veterinary Sciences, Adelaide) and was scaled up proportionally for large scale experiments. 1.5 mL of SRBC in Alsever’s solution was transferred into 50 mL falcon tube. Cells were washed at least 3 times or until lysis ceased in 30 mL of chilled DPBS (1x) (Thermo Fisher Scientific), spun at 1065 g for 6 minutes at 4 oC and finally resuspended in 10 mL of conjugation buffer. Cells were spun and buffer was removed by vacuation. SRBC pellet was gently resuspended in 800 µL of conjugation buffer followed by the addition of 10 µg of HEL protein. Cell and protein mixture was placed on ice and left to shake for 10 min on a platform rocker. 100 µL of 100 mg/mL EDCI (N-(3- Dimethylaminoapropyl)-N’-ethylcarbodiimide hydrochloride) (Sigma Aldrich) was added into the mixture and rocked for a further 30 min on ice. Cells were washed 3 times in chilled DPBS (1x) or until lysis ceased.

HEL-conjugated SRBC were resuspended at a final concentration of 1 x 109 cells/mL, such that each recipient mouse received 2 x 108 HEL-conjugated SRBC in 200 µL intravenously (i.v.). To assess conjugation efficiency, Hy9- A647 was used to detect HEL-conjugated to SRBC via flow cytometric analysis.

29 Chapter 2: Material and Methods

2.1.4 Recipe: Conjugation Buffer

D-Mannitol (Sigma) 350 mM

Sodium Chloride (NaCl) 10 mM

---

Prepared in Milli-Q Water

30 Chapter 2: Material and Methods

2.1.5 Preparation of Spleen, Bone Marrow and Blood

Donor and recipient mice were euthanised using CO2 chambers followed by cervical dislocation. Spleens were collected through keyhole surgery and placed into 2% FBC in RPMI (2FR) medium. For each spleen, a single cell suspension was prepared by pushing splenic tissue through a 70 µm cell strainer (BD Biosciences) using a 1 mL syringe plunger. Suspension was spun at 453 g for 5 min at 4 oC. Supernatant was discarded and erythrocytes were lysed by resuspending into Red Blood Cell (RBC) lysis solution with 1 mL of Fetal Bovine Serum (FBS) underlay, spun and resuspended in 5 mL of 2FR medium. Lymph nodes were isolated and mashed through a 70 µm cell strainer and resuspended into 2FR directly without RBC lysis. Live cells were diluted and counted in Trypan Blue Solution (Sigma Aldrich).

Bone marrow was typically obtained from the femur and tibia. Excess muscle tissue was removed from bones, and the end joints of each bone were cut to expose bone marrow. Bones were then loaded into open-ended PCR tubes and spun at 6000 g for 5 min into 1.5 mL eppendorf tubes containing 500 µL 2FR medium. Bone marrow clumps were separated by aspirating through a 25-gauge needle and pushed through a 70 µm cell strainer (BD Biosciences) to generate a single cell suspension.

Blood was obtained from cardiac puncture and spun down at 16100 g for 10 min. Serum was collected and stored at -20 oC until required for ELISA analysis.

2.1.6 Preparation of SWHEL splenocytes prior to adoptive transfer

Spleen from SWHEL donor mice were isolated and prepared as described in Chapter 2.1.5. To quantify the number of HEL-binding B cells prior to 6 adoptive transfer, 2 x 10 cells were stained with 200 ng/mL of HEL and Fc receptors were blocked with anti-CD16/23 antibodies. Cells were then washed with 0.1%BSA/0.1%Azide in 1x PBS (PBA) buffer and stained with anti-B220 and monoclonal HyHEL9 antibodies to determine the percentage of HEL- binding cells using flow cytometric analysis. Cell density of suspension was

31 Chapter 2: Material and Methods adjusted in 2FR medium such that each recipient mouse received 3 x 104 HEL- binding B cells in a 50 µL volume.

2.1.7 Recipe: 2% FBC in RPMI (2FR) Medium

Fetal Bovine Serum (FBS) 2% (v/v)

β-mercaptoethanol 55 µM

Penicillin 50 units/mL

Streptomycin 50 µg/mL

L-Glutamine 2 mM

---

Prepared in RPMI 1640 Medium (Gibco/Invitrogen)

2.1.8 Recipe: RBC Lysis Buffer

Ammonium Chloride (NH4Cl) 150 mM

Potassium Bicarbonate (KHCO3) 0.055 mM

EDTA (Edetate disodium) 0.1 mM

---

pH 7.2 – 7.4

Prepared in Milli-Q water

2.1.9 Adoptive Transfer

+ SWHEL mice on either SJL/Ptprca (CD45.1 ) background aged around 6 – 12 weeks were typically used as donors. Mice of 7 – 12 weeks bearing congenic CD45.2 were typically used as recipients. HEL-conjugated SRBCs

(HEL-SRBCs) were mixed with SWHEL splenocytes in vitro 5 – 10 min prior to tail vein injections. Each recipient received a 250 µL mixture of 2 x 108 HEL- SRBCs and 3 x 104 HEL-binding B cells unless specified otherwise. The day of adoptive transfer is defined as day 0; the days of tissue harvest from recipient mice are defined as the number of days post-immunisation.

32 Chapter 2: Material and Methods

2.2 Antibodies and reagents for data analyses

2.2.1 Monoclonal antibodies (mAbs)

A complete list and sources of monoclonal antibodies used for Flow Cytometry, FACS, ELISA analysis, as well as in tissue culture stimulation can be found in Table 2.2. Purified HyHEL9 was sourced from UCSF Hybridoma Core and conjugated to AlexaFluor 647 using the AlexaFluor 647 Protein Labelling Kit (Invitrogen) according to manufacturer’s instructions. Purified anti- CD38 antibody (eBiosciences) was conjugated to Pacific Orange Antibody Labelling Kit (Invitrogen) according to manufacturer’s instructions.

2.2.2 Other reagents

Mouse serum for blocking during FACS staining was purchased from Jackson ImmunoResearch and reconstituted according to manufacturer’s instructions, or obtained via cardiac puncture of unimmunised C57BL/6 mice. Blood obtained was centrifuged at 16100 g for 10 min and serum was collected.

33 Chapter 2: Material and Methods

Table 2.2. List of monoclonal Abs, clones, conjugates and sources used for tissue culture, flow cytometry, immunofluorescence histology, MACS separation and ELISA analysis. Target Clone Fluorochrome/ Source conjugate CD16/32 2.4G2 Purified UCSF Hybridoma Core IgG1 A85-1 Biotin, FITC, PE BD Biosciences

CD38 90 Purified, PE, FITC, BD Biosciences BV510 CD45R/B220 RA3-6B2 PE, BV786 BD Biosciences

CD45.1 A20 PerCp-Cy5.5, FITC, eBiosciences PE CD45.2 104 PerCp-Cy5.5, FITC, eBiosciences PECy7, UV395 CD93 (C1qR) AA4.1 APC, PE BioLegend

Fas (CD95) Jo2 PE, PE-Cy7, BV510 BD Biosciences

BAFFR 9B9 Biotin Adipogen

CD21/35 7E9 FITC, Pacific Blue BioLegend (CR2/CR1) CD23 B3B4 PE, PE-Cy7, Biotin BioLegend

CD35 8C12 Biotin BD Biosciences

CD40 FGK4.5 Purified Bio X Cell

Streptavidin PE, BV421, BD Biosciences BUV395 Streptavidin AlexaFluor555 Invitrogen

CD3e eBio500A2 Biotin eBioscience

IgD 11-26c.2a A647 BioLegend

PNA FITC Vector Laboratories

Mouse R&D Systems Recombinant IL-4

34 Chapter 2: Material and Methods

2.3 MACS Cell Separation

2.3.1 MACS depletion of mature B cells

For experiments involving adoptive transfer of immature SWHEL B cells, mature B cells of the CD35+ or CD23+ phenotypes were labelled using anti- CD35-biotin (8C12) and anti-CD23-biotin (B3B4) antibodies, followed by negative selection using Manual MACS® Cell Separation Column (Milteyi Biotec).

Donor spleens were harvested and processed with biotin-free 2% FBS in DMEM (2FD) medium using the method described in Chapter 2.1.5. Splenocytes were resuspended into 10 mL of 2FD medium, spun down at 453 g for 5 min at 4 oC and washed twice with 10 mL MACS buffer. Final cell pellet was resuspended into 10 mL MACS buffer and counted using Trypan Blue cell viability test. Live cells were spun down at 453 g for 5 min, supernatant removed and were resuspended in a mixture of anti-CD23 and anti-CD35 biotinylated antibodies at a density of 1 x 108 cells/mL in MACS buffer. Cell and antibody mixture was incubated at 4 oC for 30 min and washed by direct addition of 10 mL 2FD medium and spun down at 453 g for 5 min at 4 oC. Supernatant was removed and cell pellet was resuspended at 1 x 108 cells/mL into MACS buffer containing anti-biotin magnetic beads (Miltenyi Biotec) and incubated at 4 oC for 30 min. 10 mL 2FD medium was directly added for washing, cells were spun at 453 g for 5 min at 4 oC and finally resuspended at 1 x 108 cells/mL into MACS buffer.

Prior to loading of the sample, magnetic separation (MS) column containing magnetic microbeads (Miltenyi Biotec) was equilibrated with 30 mL of MACs buffer. Cell suspension was filtered through 35 µm cell strainer (BD Biosciences) to removed dead cell clumps and sample was loaded onto the MS column at 800 uL at a time. Eluent containing purified cells were collected and resuspended directly into 2FD medium. The purity of depletion and percentage of HEL-binding B cells were determined by flow cytometry. A final cell density of 1.2 x 105 immature HEL-binding B cells in 50 µL 2FD medium were adoptively transferred i.v. with 200 µL of 2 x 108 HEL-SRBC into congenic recipient mice.

35 Chapter 2: Material and Methods

On the 4th day after immunisation, recipient mice were boosted again with 200 µL of 2 x 108 HEL-SRBC.

2.3.2 Recipe: MACS Separation buffer

Bovine Serum Albumin (BSA) (Bovogen Biologicals) 0.25% (w/v)

Sodium Azide (NaN3) 0.1% (w/v)

EDTA (Edetate disodium) 0.1 mM

---

Prepared in 1x PBS

2.3.3 Recipe: 2% FBS in DMEM (2FD) Medium

Fetal Bovine Serum (FBS) 2% (v/v)

β-mercaptoethanol 0.01% (v/v)

Penicillin 50 units/mL

Streptomycin 50 µg/mL

L-Glutamine 2 mM

---

Prepared in DMEM, high glucose (Gibco/Invitrogen)

36 Chapter 2: Material and Methods

2.4 Flow cytometry

2.4.1 Surface staining procedure

Spleens and bone marrow were harvested and prepared as described in Chapter 2.1.5. RBC lysis was performed followed by 1 mL FBS underlay, cells were centrifuged at 453 g for 5 min and resuspended into 5 mL of PBA buffer, counted, and approximately 2.5 – 3 x 106 cells were aliquoted into each well of a non-binding 96-well U-bottom plate. All antibody-staining steps were performed in PBA buffer and incubated for at least 25 min, washed twice with PBA buffer and spun down at 863 g for 30 sec at 4 oC to pellet the cells. In all cases, blocking of surface Fc receptors using anti-CD16/32 purified antibodies was performed prior to surface staining with other anti-mouse antibodies.

For studies involving early T-dependent responses, surface staining was performed with 200 ng/mL of HEL (Sigma Aldrich) to identify total HEL-binding

B cells. For studies involving affinity analysis of SWHEL GC and memory responses, high affinity versus low affinity B cells were identified by incubating with 50 ng/mL HEL3X, as previously optimised in the Brink laboratory. AF647 conjugated HyHEL9 antibody was used to detect HEL-binding B cells. To detect surface IgG1, unimmunised mouse serum (5%) block was performed prior HEL detection with HyHEL9 AF647. Other antibodies targeting surface markers were stained following the detection of HEL and a typical staining procedure is depicted in Table 2.4. Following each step, cells were washed by adding 180 mL PBA buffer, spun at 863 g for 30 sec, and supernatant discarded. Washes were repeated twice before performing the next staining step.

37 Chapter 2: Material and Methods

Table 2.4. Staining procedure for surface marker analysis by flow cytometry Antibodies/Reagent Step 1 Fc-block (anti-CD16/32) HEL or HEL3X 2 Anti IgG1-FITC

3 5% unimmunised mouse serum

4 HyHEL9-AF647 anti B220-BV786 anti BAFFR-biotin 5 anti CD45.1-PerCp-Cy5.5 anti CD45.2-BUV395 anti CD38-BV510 anti Fas-PE-Cy7 Streptavidin-BV421

38 Chapter 2: Material and Methods

2.4.2 Data acquisition, processing and analysis

Surface stained cells were filtered through 35 µm filters into round bottom FACS tubes (CORNING) prior to sample acquisition on flow cytometers. Data were acquired on the flow cytometric analysers LSRFortessa or LSRII SORP (BD Biosciences) and were analysed and processed using FlowJo software (TreeStar). For the analysis of rare events, flow cytometric data of samples from the same test group were concatenated (i.e. pooled/ combined) where appropriate in each experiment.

2.4.3 Recipe: PBA (FACS) Buffer

Bovine Serum Albumin (BSA) (Bovogen Biologicals) 0.1% (w/v)

Sodium Azide (NaN3) 0.1% (w/v)

---

Prepared in 1x PBS

39 Chapter 2: Material and Methods

2.5 SHM analysis

2.5.1 Standard protocol for single cell FACS

Spleens were harvested and processed according to Chapter 2.1.5. To + + FACS sort donor-derived SWHEL IgG1 GC B cells and IgG1 MBCs, a simplified staining panel devised from the strategy detailed in Chapter 2.4.1 is depicted in Table 2.5.1. Single cell sorting was performed on FACS Aria and FACS AriaIII (BD Biosciences).

Table 2.5.1. Staining strategy for sorting donor-derived IgG1+ GC B cells and IgG1+ memory B cells Antibodies/Reagent Step 1 Fc-block (anti-CD16/32)

2 Anti IgG1-biotin

4 anti B220-BV786

5 anti CD45.1-FITC anti CD38-PE Streptavidin-BV421

40 Chapter 2: Material and Methods

2.5.2 Plate set up for single cell sort

Labelled cells were FACS sorted directly into single wells of 96-well skirted PCR plate (Thermo Fisher Scientific) containing 10 µL of the following reaction master mix in Table 2.5.2. Cells sorted into plates were sealed with Microseal “B” adhesive seal (Bio-Rad), spun at 453 g for 5 min and heat digested at 56 oC for 40 min followed by enzyme inactivation at 95 oC for 8 min as shown in Table 2.5.3. After digestion, plates were spun down and stored at -80 oC for up to a week, or at 4 oC for short term storage (3 – 4 days).

Table 2.5.2. Reaction mix for FACS sorted single cell digest

10x Taq Buffer (no MgCl2, Invitrogen) 110 µL

10 mg/mL Proteinase K (Promega) 55 µL

10 mM EDTA (Sigma) 11 µL

10% Tween-20 (Sigma) 11 µL

---

Prepared in water for irrigation (Baxter) 913 µL

Table 2.5.3. Single cell digest reaction condition

1. 56 oC for 40 min

2. 95 oC for 8 min

Digested cells were kept at 4oC for short term or -80oC for long term storage.

41 Chapter 2: Material and Methods

2.5.3 Primary PCR

To amplify the VH10 genes from single SWHEL B cells digested from single cell sort, plates were thawed and spun down at 453 g for 5 min. Taq polymerase and associated buffer solutions were purchased from Invitrogen. Conditions and reagents for single cell primary PCR is listed in Table 2.5.4. Primer oligonucleotide (Sigma-Aldrich) sequences were customised and 15 µL of the following primary PCR reaction mixture was added directly into digested cells per well as shown in Table 2.5.4. DNA was PCR amplified using the conditions shown in Table 2.5.5.

Table 2.5.4. Reagents for single cell primary PCR

Primary PCR Primer Sequences:

psc-SHH upper: gtt gta gcc taa aag atg atg gtg

psc-SHH lower: gat aat ctg tcc taa agg ctc tga g

Primary PCR reaction mixture:

DNA digest in 1x Taq Buffer (no MgCl2) 10 µL

10x Taq buffer (no MgCl2) 1.5 µL

50 mM MgCl2 1 µL

10 mM dNTPs (Sigma) 0.5 µL

psc-SHH upper (10 pmol/µL) 0.1 µL

psc-SHH lower (10 pmol/µL) 0.1 µL

Taq DNA polymerase (0.5 U) 0.15 µL

---

Prepared in water for irrigation (Baxter) 11.65 µL

42 Chapter 2: Material and Methods

Table 2.5.5. Primary PCR reaction condition

1. 94 oC for 3 min

2. 95 oC for 15 sec

3. 55 oC for 3 min

4. 72 oC for 1 min

5. 72 oC for 10 min

Steps 2 – 4 were repeated for a total of 35 cycles

43 Chapter 2: Material and Methods

2.5.4 Secondary PCR

Secondary amplification of primary PCR products were performed using the following primer sequences and PCR reaction conditions listed below. 2.5 µL of primary PCR products were added into 96-well non-skirted plate (Thermo Fisher Scientific) containing 20 µL/well of secondary PCR reaction mixture and the customised primer oligonucleotides (Sigma-Aldrich) shown in Table 2.5.6. Primary DNA products were PCR amplified using conditions depicted in Table 2.5.7.

Table 2.5.6. Reagents for single cell secondary PCR

Secondary PCR Primer Sequences:

ssc-SHH upper: tct tct gta cct gtt gac agc cc

ssc-SHH lower: caa ctt ctc tca gcc gcc tc

Secondary PCR reaction mixture: Primary PCR product in 1x Taq Buffer 2.5 µL

10x Taq buffer (no MgCl2) 2.25 µL

50 mM MgCl2 1 µL

10 mM dNTPs (Sigma) 0.5 µL

ssc-SHH upper (10 pmol/µL) 1 µL

ssc-SHH lower (10 pmol/µL) 1 µL

Taq DNA polymerase (0.5 U) 0.1 µL

---

Prepared in water for irrigation (Baxter) 16.65 µL

44 Chapter 2: Material and Methods

Table 2.5.7. Secondary PCR reaction condition

1. 94 oC for 3 min

2. 95 oC for 15 sec

3. 62 oC for 3 min

4. 72 oC for 1 min

5. 72 oC for 10 min

Steps 2 – 4 were repeated for a total of 35 cycles

45 Chapter 2: Material and Methods

2.5.5 PCR product screening

To screen for successfully amplified PCR products, SYBR safe DNA gel stain (Invitrogen) was diluted at 1 in 4000 into TBE buffer. 5 µL of undiluted secondary product was mixed with 10 µL of diluted DNA dye in a 96-well non- skirted plate (Thermo Fisher Scientific), shielded from light and incubated at room temperature for 15 – 30 min on a platform shaker. Products were visualised by UV light using the Bio-Rad Geldoc System and amplified products were verified and selected for sequencing.

2.5.6 Sequencing amplified VH10 PCR products

Sequencing of SWHEL VH10 gene was performed by 2 independent services. Samples submitted for sequencing at the Garvan Molecular Genetics facility were first subjected to PCR clean-up using illustraTMExoProStarTM 1-step (GE Healthcare) to remove unincorporated primers and nucleotides. 2 µL enzyme containing Exonuclease I and Alkaline Phosphatase was mixed with 5 µL undiluted secondary product, heated at 37 oC for 15 min followed by enzyme inactivation at 80 oC for 15 min. PCR products were diluted at 1 in 10 in

Milli-Q water and 1 µL of the product was mixed with 1 µL of 3.2 pmol ssc-SHH upper primer (sequence refer to Chapter 2.5.6). For sequencing services at GENEWIZ Boston, unpurified secondary products were diluted at 1 in 60 in Milli-Q water into 96-well skirted-plate and submitted alongside with 200 µM secondary upper primer (ssc-SHH upper) according to facility’s instructions.

46 Chapter 2: Material and Methods

2.5.7 Analysis of SHM

DNA sequencing results and chromatogram traces were manually verified using plasmid editor software ApE, DNA Strider and Serial Cloner v2.6.1. Sequencing quality of DNA sequences were provided by respective sequencing facilities and verified manually using the Biostrings Bioconductor package in the R environment incorporated into a software program “Immunity- Verifier” developed at the Garvan Institute by Dr. Tyani D. Chan in collaboration Dr. Liviu Constantinescu. The DNA sequence of each clone was aligned and verified against native HyHEL10 protein sequence using a pairwise alignment function. Alignment scores below 400 and those containing significant insertions or deletions were filtered out as failing quality control. To identify mutations in the VH10 gene, DNA sequences were translated computationally according to standard amino acid sequences using the Standard Genetic Code, and the translated sequence was aligned against the HyHEL10 mAb protein sequence to determine the position of amino acid mutation.

Biostrings Bioconductor package:

(http://www.bioconductor.org/packages/release/bioc/html/Biostrings.html)

Standard Genetic Code:

(https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi#SG1).

2.5.8 Recipe: TBE Buffer (5X)

Tris base 54 g

Boric acid 27.5 g

0.5 M EDTA (pH 8.0) 20 mL

---

Prepared in Milli-Q water

47 Chapter 2: Material and Methods

2.6 ELISA

2.6.1 Staining procedure to detect HEL-specific antibodies

Serum samples were purified from whole blood as described in Chapter 2.1.5. To detect affinity matured antibody production, HEL3X and HEL4X were used to identify high affinity antibodies and those that have acquired further affinity-enhancing mutations respectively. 384-well plates (CORNING) were coated with either HEL3X or HEL4X at 5 µg/mL in NPP buffer and incubated overnight at 4 oC. An automated plate washer (BioTek) was used to wash plates with ELISA wash buffer and all incubation steps were performed at 37 oC for 1 hr. After washing, HEL-coated wells were first blocked with 1% BSA/PBS. Serum samples were then titrated in 0.1% BSA/PBS, washed and detected with anti-IgG1-biotin (A85-1) diluted in 0.1% BSA in 1% skim milk powder/PBS. Excess biotinylated antibodies were washed, follow by addition of Streptavidin conjugated alkaline phosphatase (SA-AP, 1000 U) (Roche) diluted in PBS (1x) and washed to remove unbound SA-AP. Substrate p-nitrophenyl phosphate (NPP) (MP Biomedicals Australia) was dissolved in NPP buffer and SA-AP activity was measured via spectrophotometric analysis at 405 nm. Endpoint titres were used to determine serum antibody concentrations defined by negative control from unimmunised serum and based on 99.9% confidence levels (321).

48 Chapter 2: Material and Methods

2.6.2 Recipe: NPP Buffer

Sodium Bicarbonate (NaHCO3) 35 mM

Sodium Carbonate (Na2CO3) 19 mM

Sodium Azide (NaN3) 3 mM

Magnesium Chloride (MgCl2) 1 mM

---

Prepared in Milli-Q water

2.6.3 Recipe: ELISA Wash Buffer

Tween-20 (Polysorbate 20) 1% (v/v)

Prepared in 1x PBS

49 Chapter 2: Material and Methods

2.7 Retroviral Transduction and Protein Overexpression on primary B cells

2.7.1 Cloning and Transformation

cDNA encoding membrane-bound BAFFR, BCL-2, BCL-xL were inserted between the NotI and XhoI sites of the MCS upstream of eGFP preceded by an IRES. Plasmid DNA containing the gene of interest was transformed into One Shot® TOP10 Chemically Competent E. coli (Thermo Fisher Scientific). A 25 µL aliquot of competent E. coli was mixed with 1 µL DNA, incubated on ice for 30 min. Cells were warmed in 42 oC waterbath for precisely 30 seconds and placed on ice for 2 min. 200 µL Super Optimal Broth was added to competent cells, and tubes were left shaking at 200 rpm in a 37 oC incubator shaker for 1 hr. Successfully transformed clones were selected in LB agar containing Ampicillin (Sigma) overnight at 37 oC. LB broth was spiked with a colony from the growth plate and left shaking at 37 oC overnight, followed by plasmid extraction using AccuPrep Nano-Plus Plasmid Mini Extraction Kit (Bioneer) according to manufacturer’s instructions.

2.7.2 Generation of viruses for Retroviral Transduction of primary B cell

Retroviruses were generated using Phoenix E packaging cell line (322). Phoenix cells were cultured with 10% FBS in DMEM (10FD) medium in 10 cm culture plates until 60 – 70% confluent. Medium was then replaced with serum- free DMEM and cells were transfected with 10 µg of plasmid cDNA and 5 µg of pCL-Eco (323) using FuGENE®6 transfection reagent (Roche/Promega). Supernatant was collected at 48th hr and cell culture was replenished with fresh serum-free DMEM. Viral supernatant was harvested again at 72nd hr. Supernatants were pooled from both collection days, was spun at 1258 g for 5 min and filtered through 0.45 µM filters (CORNING). Purification was further performed with 100 kDa Amicon tubes (Millipore) and retroviruses were snap frozen in liquid nitrogen for storage at -80 oC.

50 Chapter 2: Material and Methods

2.7.3 Retroviral Spin Transduction of SWHEL B cells

Splenic B cells from SWHEL mice were first isolated and prepared as describe in Chapter 2.1.5 under sterile conditions and resuspended into B Cell Medium (BCM). Splenocytes were cultured in a 12-well culture plate at 2 x 106 cells per well and stimulated with 1 mL mixture of 10 ng/mL recombinant mouse IL-4 and 10 µg/mL anti-CD40 (FGK4.5) for 20 hr at 37 oC. Cells were spun at 1258 g for 1 min, medium was gently removed and cells were resuspended into 1 mL of 4 µg/mL Polybrene (Hexadimethrine Bromide) (Sigma Aldrich) in 10FD medium and 1 mL of retroviral supernatant. Spin transduction of cells was performed at 1258 g for 50 min at room temperature. Cells in retroviral supernatant were then incubated at 37 oC for a further 3 – 3.5 hr. Following incubation, cells were spun down for 1 min, medium removed and cells were again stimulated with 1 mL of 10 ng/mL recombinant mouse IL-4 and 10 µg/mL anti-CD40 (FGK4.5) in BCM medium for 20 hr at 37 oC. Cells were harvested the next day, counted and the frequency of HEL-binding B cells was quantified as described in Chapter 2.1.6. Efficiency of transduction was determined by flow cytometric analysis of surface BAFFR (Adipogen) and GFP expression, and dead cells were excluded by DAPI staining. Cultures were washed with BCM and adoptively transferred i.v. at the cell density of 3 x 104 live HEL- binding B cells with 2 x 108 HEL-SRBC in 500 µL for each recipient mouse.

51 Chapter 2: Material and Methods

2.7.4 Recipe: Super Optimal Broth

Tryptone 20 g/L

Yeast Extract 5 g/L

Magnesium Sulfate (MgSO4) 4.8 g/L

Dextrose 3.603 g/L

Sodium Chloride (NaCl) 0.5 g/L

Potassium Chloride (KCl) 0.186 g/L

---

Prepared in LB media

2.7.5 Recipe: 10% FBS in DMEM (10FD) Medium

Fetal Bovine Serum (FBS) 10 % (v/v)

β-mercaptoethanol 0.01 % (v/v)

Penicillin 50 units/mL

Streptomycin 50 µg/mL

L-Glutamine 2 mM

---

Prepared in DMEM, high glucose (Gibco/Invitrogen)

52 Chapter 2: Material and Methods

2.7.6 Recipe: B Cell Medium (BCM)

Fetal Bovine Serum 10 % (v/v)

β-mercaptoethanol 0.01 % (v/v)

Penicillin 50 units/mL

Streptomycin 50 µg/mL

L-Glutamine 2 mM

MEM Non-Essential Amino Acid 1 % (v/v)

Sodium Pyruvate 1 mM

HEPES 10 mM

---

pH 7.2 – 7.6

Prepared in RPMI 1640 Medium (Gibco/Invitrogen)

53 Chapter 2: Material and Methods

2.8 Immunofluorescence histology

Spleens were frozen in Tissue Tek OCT compound (Scigen) and 6 – 7 µm sections were cut using CM3050 S cryostat (Leica). Cut spleen sections on glass slides were left to air dry for 1 hour followed by fixation in ice- cold acetone and were left to air dry overnight. Cut sections were rehydrated the next day in PBS (1x) for 10 – 20 mins, blocked with 0.05% BSA in PBS (1x) for 30 mins and washed in plain PBS (1x). To prevent non-specific Fc-binding, spleen sections were blocked with 5% normal mouse serum for 30 mins and rinsed with PBS (1x). Sections were stained with biotinylated anti-CD3e (eBio500A2) for 30 mins, washed and incubated with streptavidin AlexaFluor- 555 (Invitrogen) for 1 hour. Sections were washed and then stained with anti- IgD A647 (11-26c.2a) and FITC-labelled PNA for 1 hour before final wash with PBS (1x). Labelled spleen sections were left to dry in the dark and photo- bleaching anti-fade solution (Southern Biotech) was applied prior covering with coverslip. Slides were kept cool at 4 oC and protected from light. Stained sections were visualised using immunofluorescence microscope DM5500 from Leica Microsystems and analysis was performed using Image J or Adobe Creative Suite CS5.1.

2.9 Generation of Mixed Bone Marrow Chimeras

Bone marrow recipient mice of 6 – 7 weeks were lethally irradiated with two doses of 425 rad (X-RAD 320 Biological Irradiator, PXI). Irradiated mice were placed on a gel-based chow diet and maintained on water containing antibiotic, Bactrium (Roche), for two weeks post irradiation. Bone marrow stem cells were harvested from the femur and tibia collected from 14 – 16 weeks old bone marrow donors using the method described in Chapter 2.1.5. Bone marrow cells collected from multiple donors of the same genotype were counted, pooled and i.v. transferred into irradiated recipient mice. Each recipient received 1 x 107 donor bone marrow cells, was monitored daily, and was left to recover and reconstitute for at least 6 – 8 weeks before use.

54 Chapter 2: Material and Methods

2.10 Statistical analysis

Data analysis for significance was calculated using Prism 7.0 software (Graphpad). For comparison between two normally distributed groups, a two- tailed unpaired t-test with Welch’s correction was used. For comparison of more than 2 parametric datasets, unmatched one-way ANOVA was employed, with post-hoc multiple comparison using Bonferroni’s correction. For comparison of categorical data based on nominal variable significance, the Chi-square contingency test was performed. Confidence levels were set at 95% confidence intervals, where significance was denoted by: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

55 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

Chapter 3

BAFFR dynamics and the requirement for BAFF in T-dependent B cell responses

3.1 Preamble

GC formation is a hallmark feature of T-dependent B cell response. Here B cells undergo vigorous proliferation and SHM of their Ig variable region genes to generate B cells clones with a spectrum of affinities towards the target antigen. Therefore, the survival of high affinity cells and their removal of low affinity counterparts are critical processes for selecting high quality antibody- producing cells to provide long term immune protection.

The TNF superfamily ligand BAFF (TNFSF13B) is critical for the survival and maturation of naïve peripheral B cells via engagement of its high affinity receptor BAFFR (TNFRSF13C). However, the expression of BAFFR is differentially regulated when activated B cells differentiate into GC B cells, MBCs and PCs during T-dependent responses. In addition to its key role in the development of the primary B cell repertoire, therefore, it is possible that BAFFR plays additional roles following B cell activation that may impact on the development of long term immunity. However, the precise role of BAFF:BAFFR signalling in regulating T-dependent GC responses remains unclear.

Inside the GC, the major sources of BAFF are the myeloid and stromal compartments (65, 324). Nevertheless, T cells can produce BAFF and it has been proposed that Tfh cells regulate GC B cell affinity maturation and selection via BAFF (75). In this chapter, the expression of BAFFR on B cells responding to T-dependent antigens is characterised and the impact of T cell-derived BAFF on the survival and selection of GC B cells directly assessed. To investigate the function of BAFF:BAFFR signalling on genuine antigen-experienced B cells, this thesis will focus on class-switched (IgG1+) GC B cells and MBCs, in which the induction of IgG1 switching on responding B cells is indicative of their reception

56 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses of IL-4 and CD40-signalling from T cell during a T-dependent challenge (ref. Chapter 1.4).

3.2 GC B cell differentiation leads to increased BAFFR expression

To first determine and confirm that BAFFR expression is maintained on activated murine B cells in response to T-dependent antigens, WT mice were immunised with SRBC and spleens were harvested on day 3 and day 5 post- immunisation (Figure 3.1A). Isolated splenocytes were stained with B220 and surface IgG1 to identify total IgG1+ class-switched B cells. On day 3, small numbers of class-switched GCs (B220hi, Fashi, CD38lo) and early PBs (B220lo, Faslo, CD38lo) were detectable (Figure 3.1B). By day 5, both populations had significantly expanded and their phenotypes were verified based on surface expression of B220, CD86 and Forward Light Scatter-Area (FSC-A) (cell size) (Figure 3.1B, C). Interestingly, analysis of surface BAFFR expression on day 5 showed elevated levels on IgG1+ GC B cells compared to naïve B cells (B220+, IgD+, CD38hi, CD86lo) while surface BAFFR was not detected on IgG1+ PBs (Figure 3.1C). This differential expression of BAFFR suggests a potential role for BAFFR signalling in regulating GC B cells but not PBs.

57 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

A SRBC Day 0 Day 3 Day 5 i.v

Wild type C57BL/6 Harvest Harvest

SRBC-immunised Total splenic leukocytes B

0.03 53

B220 + CD38 Total IgG1 19 Day 3

8 Wild type

(C57BL/6) 0.45 3

Day 5 90

5

IgG1 Fas

+ IgG1 Early Plasmablast C + IgG1 GC B cells Day 5

B cell subset Naive IgG1+ PB Relative Cell no. IgG1+ GC

BAFFR CD86 B220 FSC-A

Figure 3.1. BAFFR is differentially expressed on activated B cell blasts upon activation by T-dependent antigen SRBC.

(A) Wild type C57BL/6 mice challenged with SRBC were analysed on day 3 and 5 post immunisation. (B) Flow cytometric analysis of splenic IgG1+ GC B cells (B220hi, IgG1+, Fashi, CD38lo) and IgG1+ early PB (B220lo, IgG1+, Faslo, CD38lo). (C) Histogram overlaying surface expression for BAFFR, CD86, B220 and + hi hi lo + FSC-A of naïve B cells (B220 , IgD , CD38 , CD86 ), IgG1 GC B cells and IgG1+ early PBs. Profiles are representative of 4 mice and are representative of 3 independent experiments.

58 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

3.3 Gating strategy to characterise BAFFR dynamics in antigen- specific SWHEL B cell responses

Since BAFFR expression is upregulated upon GC differentiation of B cells (Chapter 3.2), the SWHEL adoptive transfer system was utilised to further characterise the dynamics of BAFFR expression and to examine its role in regulating affinity maturation and selection in the GC. SWHEL B cells were isolated and transferred into WT recipient mice, challenged with HEL3X-SRBC and boosted with the same antigen 4 days later. Spleens were harvested at various time points, splenocytes prepared and surface stained with fluorescently-labelled antibodies directed against surface markers: CD45.1, CD45.2, B220, CD38 and IgG1 (Figure 3.2). Using flow cytometry, donor- derived cells were identified (CD45.1+) and B220, CD38 expression and IgG1 expression used to identify IgG1+ GC B cells (B220hi, IgG1+, CD38lo) and IgG1+ MBCs (B220hi, IgG1+, CD38hi). For the early phase of the response (days 3-6) when extrafollicular PB responses are still detectable in this system (135), the IgG1+ PB populations were identified as FSC-Ahi, B220lo and CD38lo. GC and MBC populations were further divided into their high affinity and low affinity 3X compartments based on binding affinity to HEL -staining as described in Chapter 2.4 (Figure 3.2). Since early IgG1+ B cell blasts also exhibit a B220hi, CD38hi phenotype, cells of this phenotype were only classified as MBCs from day 6 onwards since undifferentiated B cell blasts are absent by day 5 of the response (135).

59 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses FSC-A SSC-A FSC-H

FSC-A FSC-A Unstained

+ IgG1 Early

CD38 Plasmablast

CD45.2 SWHEL FSC-A Donor Derived B220hi

CD45.1 IgG1 B220 CD38

IgG1 + IgG1 + IgG1 GC B cell Memory B cell

High Affinity High Affinity 3X GC 3X Memory HEL HEL Binding Binding

Low Affinity Low Affinity Memory GC IgG1 IgG1

60 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

Figure 3.2. Gating strategy to identify donor-derived high vs. low affinity GC B cells, MBCs and early PBs using multicolour cell surface markers.

+ SWHEL B cells (CD45.1 ) were adoptively transferred into congenic recipients (CD45.2+) and were challenged with HEL3X-SRBC. Recipient spleens were harvested for analysis at various time points. Cells were stained with surface markers for B220, CD45.1, CD45.2 and CD38 to identify donor-derived (CD45.1+, CD45.2-) and endogenous-derived (CD45.2+, CD45.1-) GC B cells and MBCs, and were further stained for IgG1 and HEL3X-binding. Following data acquisition and compensation, light scatter gate was applied to identify lymphocytes (FSC-A vs. SSC-A), followed by a doublet exclusion gate (FSC-A vs. FSC-Flow cytometric). Autofluorescent cells were excluded (Unstained vs. FSC-A) and a donor gate was applied (CD45.1 vs. CD45.2). IgG1-switched GC B cells (B220hi, CD38lo, IgG1+) and IgG1-switched MBCs (B220hi, CD38hi, IgG1+) were further divided into high affinity and low affinity subsets based on HEL3X-binding.

61 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

3.4 Mapping the BAFFR expression during GC affinity maturation

To examine if BAFFR expression is differentially regulated over the 3X course of a T-dependent response, SWHEL B cells were challenged with HEL - SRBC in WT recipient mice and IgG1-switched donor B cells were analysed by flow cytometry on days 6, 9, 12 and 15 using the gating strategy described in + + Chapter 3.3 (Figure 3.3A). On day 6, SWHEL IgG1 GC and IgG1 MBC compartments showed comparable BAFFR expression, and as expected, no BAFFR was detected on IgG1+ PBs (Figure 3.3B, C). Interestingly, as the response progressed through to day 9, 12 and 15, a steady reduction in BAFFR expression was observed specifically in MBCs. GC B cells however, maintained high levels of surface BAFFR throughout the response. (Figure 3.3B, C).

To further characterise the expression of BAFFR throughout maturation of GC responses, high affinity and low affinity donor IgG1+ GC B cells were identified as described in Chapter 3.3. A key advantage of the SWHEL system is that progression of antigen-specific B cells through affinity maturation and selection in the GC can be visualised at high resolution (52, 136). On day 6, the + SWHEL IgG1 GC B cell compartment was comprised primarily of low affinity GC B cells (Figure 3.4A, B, C). By day 9, high affinity GC B cells had emerged and occupied 50-60% of the GC B cell compartment (Figure 3.4A, B, C). By day 12, at least 90% IgG1+ GC B cells had acquired affinity-increasing mutations to become high affinity cells (Figure 3.4A, B, C). Between day 12 and 15, the

SWHEL GC response reached its peak and was dominated by high affinity clones (>90%) with only a small fraction of surviving low affinity cells (<5%) (Figure 3.4A, C). Interestingly, no differences in BAFFR MFI were observed between high affinity versus low affinity IgG1+ GC B cells across all time points (Figure 3.4D).

62 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

A Wild type HEL-binding B cell + Day 0 Day 4 Day 6 Day 9 Day 12 Day 15 HEL3X-SRBC i.v

3X HEL -SRBC Harvest Harvest Harvest Harvest Wild type Boost Recipient

Donor B cell subset B C + IgG1 Early Plasmablast + + IgG1 Memory B cells IgG1 Donor B cells + IgG1 GC B cells

6.3 hi 11 B220 non- CD38

FSC-A Plasmablast Relative Cell no. Day 6

87 83

0.7 3.8

Day 9

94 94

0 0.5

Day 12

96 99

0 0.7

Day 15

95 98

B220 IgG1 BAFFR

63 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

Figure 3.3. BAFFR is differentially expressed on activated SWHEL B cells responding to T-dependent antigen HEL.

+ SWHEL B cells (CD45.1 ) were adoptively transferred into wild type recipients (CD45.2+) to investigate BAFFR expression during T-dependent B cell 3X activation. (A) SWHEL B cells were challenged with HEL -SRBC and antigen boosted on day 4. Donor-derived splenic responses (CD45.1+, CD45.2-) were analysed on day 6, 9, 12 and 15 based on gating strategy depicted in Figure 3.2. (B) Flow cytometric analysis of IgG1+ donor-derived B cells (B220+, IgG1+) showing early PBs (B220lo, FSC-Ahi, IgG1+) and non-PBs (B220hi, IgG1+). IgG1+ non-PBs were further categorised into GC B cells (IgG1+, CD38lo) and MBCs (IgG1+, CD38hi). Numbers on plots represent percentages (%). Plots depicted here are representative of 5 mice and are representative of 2 independent experiments. (C) Histogram overlay of surface BAFF-R levels for various donor-derived IgG1+ subsets identified in (B): early PBs (green), GC B cells (blue) and MBCs (red).

64 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

A + B + Donor IgG1 GC Donor IgG1 GC 105 s Affinity Compartment 4 10 High Affinity 0.4 Low Affinity 103

splenocyte 2 6 10 Binding Day 6 0 3X 101

0

HEL 10

99 Cells per 1 <0.1 69 6 9 12 15

C 100 0

Day 9 80 20 % Low finity f

A 60 40

31 A f finit 40 60 91 y % High

20 80 Day 12 0 100 6 9 12 15 4000 D B cell subset 9 Naive B cell 3000 I High Affinity GC

94 F Low Affinity GC M

R 2000 Day 15 BAFF 1000 6 0 IgG1 6 9 12 15 Days post Immunisation

Figure 3.4. BAFFR expression does not correlate with high and low affinity compartments in the GC.

+ 3X Donor-derived SWHEL IgG1 GC responses to HEL -SRBC challenge were analysed on day 6, 9, 12 and 15 as depicted in Figure 3.3. (A) Flow cytometric analysis showing surface IgG1 counterstained with competitive binding of 3X limited HEL (50ng/mL) to identify high affinity and low affinity GC B cells. Numbers on plot represent percentages (%). Plots depicted here are representative of 3 mice in one experiment. (B) Enumeration and (C) Frequency of high affinity vs. low affinity IgG1+ GC B cells overtime. (D) Mean fluorescent intensity (MFI) of surface BAFFR expression of high vs. low affinity GC B cells compared to naïve B cells.

65 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

3.5 T cell-derived BAFF is dispensable for GC and memory B cell survival

Since GC B cells maintained higher surface levels of BAFFR compared to naïve B cells, MBC and early PB, it was possible that BAFF-BAFFR signalling may play a role in regulating GC B cell responses. Inside the GC, BAFF is produced by stromal, myeloid and T cells (240). A recent study also suggested Tfh cells to be a local source of BAFF in the GC that contributes to the positive selection of high affinity B cell clones (75). Thus, to determine whether T cell-derived BAFF contributes to the regulation of GC B cell responses, a series of mixed bone marrow chimeras containing WT T cells versus BAFF-deficient T cells were generated (Figure 3.5).

To do this, T cell-deficient (Cd3e-/-) recipient mice were lethally irradiated in two doses (2 x 425 rad), followed by reconstitution with a bone marrow -/- mixture of one of the following compositions: i) 20% WT plus 80% Cd3e , or ii) 20% Tnfsf13b-/- plus 80% Cd3e-/-. After hematopoietic reconstitution, 100% of the T cells in each case either expressed BAFF normally (WT chimeras) or lacked BAFF expression (BaffΔT chimeras) (Figure 3.5A, B). After 8 weeks of reconstitution, SWHEL B cells were transferred into the different chimeric recipient mice and challenged with HEL3X-SRBC. Chimeras were antigen boosted again 4 days later and spleen cells were harvested to determine donor- derived B cell responses on day 14 and 21 (Figure 3.6A).

The absence of T cell-derived BAFF was found to have no apparent impact on T-dependent responses mounted by SWHEL B cells (Figure 3.6B). Firstly, comparison of the frequency and numbers of total donor-derived GC B cells (B220hi, CD38lo) on day 14 showed that GC responses were indistinguishable in WT and BaffΔT chimeras (Figure 3.6B, C). Similarly, day 21 responses also revealed no significant differences in GC B cell numbers in recipient mice lacking T cell-derived BAFF (Figure 3.6B, C). Further examination of the IgG1-switched compartments on both day 14 and 21 revealed no difference in frequency or numbers of IgG1+ GC B cells (IgG1+,

66 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

CD38lo) (Figure 3.6B, D). The absence of T cell-derived BAFF also did not alter the output of IgG1+ MBCs (IgG1+, CD38hi) (Figure 3.6B, E).

67 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

A 100% T cells can produce BAFF

Mixed Lethally irradiated T cell constitution Bone Marrow recipients in chimera 20%

Wild type (WT)

Cd3e-/- 80% WT T cells (20% WT plus 80% Cd3e-/-)

T cell-deficient (Cd3e-/-)

B 100% T cells cannot produce BAFF

Mixed Lethally irradiated T cell constitution Bone Marrow recipients in chimera

20%

BAFF-deficient (Tnfsf13b-/-)

Cd3e-/- Baff 80% (20% Tnfsf13b-/- plus 80% Cd3e-/-)

T cell-deficient (Cd3e-/-)

Figure 3.5. Generation of mixed bone marrow chimera to investigate the role of T cell-derived BAFF in regulating GC and MBC responses.

Bone marrow chimeras were generated by reconstituting lethally irradiated T cell-deficient (Cd3e-/-) mice with a mixture of (A) 80% Cd3e-/- plus 20% WT bone marrow, or (B) 80% Cd3e-/- plus 20% BAFF-deficient (Tnfsf13b-/-) bone marrow. Chimeric recipients were left to reconstitute for 6 – 8 weeks.

68 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

A

Wild type HEL-binding B cell + Day 0 Day 4 Day 14 Day 21 HEL3X-SRBC i.v

3X HEL -SRBC Harvest Harvest Baff Boost Chimeric Recipient

B Donor B cells Chimeric Baff Recipient:

0.6 1.1 CD38 Day 14

52 46

1.2 1.9

Day 21

44 49 IgG1

C D E Donor IgG1+ GC Donor IgG1+ Memory 10 5 105 103 s s s

10 4 104 102 splenocyte splenocyte splenocyte

6 3 6 6 10 3 10 0 0 0

101 2 10 2 10 Cells per 1 Cells per 1 Cells per 1 1 0 10 1 10 10 14 21 14 21 14 21 Days post Immunisation Days post Immunisation Days post Immunisation Chimeric Recipient Wild type Baff T

69 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

Figure 3.6. T cell-derived BAFF is dispensable for the survival and maintenance of donor-derived GC and MBC responses.

+ SWHEL B cells (CD45.1 ) were adoptively transferred into WT or BaffΔT bone marrow chimeras (CD45.2+) generated from the strategy depicted in Figure 3.5. 3X (A) SWHEL B cells were challenged with HEL -SRBC and boosted again with antigen on day 4. Donor-derived B cell responses (CD45.1+, CD45.2-, B220+) in WT vs. BaffΔT chimeric recipients were examined on day 14 and 21. (B) Flow cytometric analysis of donor-derived IgG1+ GC B cells (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi) in WT vs. BaffΔT recipients. Numbers on plots represent percentages (%). Data showing 5 concatenated mice from one experiment. (C) Enumeration of donor-derived GC B cells, (D) IgG1+ GC B cells and (E) IgG1+ MBCs on day 14 and 21 post immunisation. Results were analysed using unpaired Student t-test with Welch’s correction post-hoc. Data were considered not significant unless specified.

70 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

3.6 Affinity-based selection in the GC does not require T cell-derived BAFF

The unchanged size of the donor GC responses in recipients that lack T cell-derived BAFF were not entirely surprisingly as non-hematopoietic FDCs and fibroblastic reticular cells have been shown to be the major sources in the spleen (78, 240) and these would all be capable of BAFF expression in each type of chimera. Nevertheless, since Tfh cells directly conjugate with B cells in the GC, it remained possible that T cell-derived BAFF may play a more qualitative role in the GC, particularly in light of recent data indicating the involvement of BAFF in T cell-mediated selection of high affinity B cell output in the GC reaction (75).

To assess selection in the GC directly, the relative antigen affinity of + SWHEL donor IgG1 GC B cells in the spleen of the chimeras was examined on days 14 and 21 of the response to HEL3X-SRBC. Interestingly, no significant difference in the frequency of high affinity GC B cells was observed when T cell- derived BAFF was absent. (Figure 3.7A, B, C). This suggested GC B cells can affinity mature and undergo normal selection independent of BAFF derived from T cells. Moreover, the emergence of high affinity IgG1+ MBCs on day 14 and 21 were consistently observed at comparable frequencies between the two groups (Figure 3.7D, E, F).

Furthermore, serum anti-HEL3X antibody analysis on days 14 and 21 of

SWHEL responses in WT and BaffΔT chimeras revealed similar titres of high affinity antibodies (Figure 3.8A, B). Thus, within the PC and MBC compartments, there was no evidence of defective selection for high affinity cells in the absence of T cell-derived BAFF. To examine this in further detail, + 3X single IgG1 GC B cells from day 21 SWHEL responses to HEL in WT versus BaffΔT chimeras were FACS sorted and subjected to SHM analysis and will be described below in Chapter 3.7.

71 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

Affinity Compartment Low Affinity High Affinity

Donor IgG1+ GC A B 100 DAY14 0 Donor IgG1+ GC

80 20 % Low

Chimeric y Baff Recipient: finit 60 40 f

A A

f

90 89 finit

3X 40 60 y % High

HEL 20 80 Binding Day 14

0 100 Wild type Baff 7 9 C 100 DAY21 0 84 78

80 20 % Low y Day 21

finit 60 40 f

A A

f finit 40 60 14 21 y IgG1 % High 20 80

0 100 Wild type Baff Chimeric Recipient

Donor IgG1+ Memory E DAY14 D Donor IgG1+ Memory 100 0

Chimeric 80 20 % Low Recipient: Baff y

finit 60 40 f

A A

50 55.7 f finit 3X 40 60 y % High HEL Binding Day 14 20 80

0 100 Wild type Baff 48.8 44 F DAY21 82.8 76.8 100 0

80 20 % Low Day 21 y

finit 60 40 f

A A

f finit 16.7 23 40 60 y

IgG1 % High 20 80

0 100 Wild type Baff Chimeric Recipient

72 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

Figure 3.7. T cell-derived BAFF is dispensable for GC B cell affinity maturation and selection into the MBC compartment.

+ + lo + Donor-derived SWHEL IgG1 GC B cells (IgG1 , CD38 ) and MBCs (IgG1 , CD38hi) in response to HEL3X-SRBC in wild type (WT) or BaffΔT conditions were identified as in Figure 3.6. (A) Flow cytometric analysis of donor-derived IgG1+ GC B cells in WT vs. BaffΔT chimeric recipients on day 14 and 21. High and low affinity B cells were distinguished based on HEL3X-binding (50ng/mL) counterstained with surface IgG1. (B) Proportion of high vs. low affinity SWHEL IgG1+ GC B cells on day 14 and (C) day 21. (D) Flow cytometric data showing donor-derived IgG1+ MBCs in WT vs. BaffΔT chimeric recipients on day 14 and + 21. (E) Proportion of high vs. low affinity SWHEL IgG1 MBCs on day 14 and (F) day 21. Numbers on plots represent percentages (%). Flow data showing 5 concatenated mice. Results were analysed using unpaired Student t-test with Welch’s correction post-hoc. Data were considered not significant unless specified.

.

73 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

A Day 14 anti-HEL3X IgG1 4.0 Chimeric Recipient

3.0 Wild type e Baff T Unimmunised 2.0 wild type Absorbanc 1.0

0.0 102 103 104 1/serum dilution

B Day 21 anti-HEL3X IgG1 4.0

3.0

2.0

1.0

0.0 102 103 104 1 / serum dilution

Figure 3.8. T cell-derived BAFF is not required for the generation and selection of high affinity antibodies.

SWHEL B cells adoptively transferred into WT or BaffT chimeric recipients and challenged with HEL3X-SRBC and antigen boosted as depicted in Figure 3.6. Blood was harvested on day 14 and 21. (A) HEL3X-binding IgG1 serum antibodies from SWHEL responses in WT (black) and BaffT (blue) recipients were measured by ELISA and demonstrated comparable high affinity antibody titres on day 14 and (B) day 21. Serum from unimmunised WT mice (grey) was used as negative control. Serum samples were titrated in duplicates. Data represent mean absorbance at 405 nm.

74 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

3.7 Wild type GC B cell responses show similar SHM patterns in chimeras with wild type versus BAFF-deficient T cells

The gating strategy for sorting donor IgG1+ GC B cells was as depicted in Chapter 3.3. In recipients reconstituted with WT T cells, IgG1+ GC B cells showed the expected dominance of the Y53D substitution in the CDR2 region (present in 95% of clones) that confers high affinity binding of HEL3X (Figure 3.9A). Two additional affinity-enhancing substitutions towards HEL3X, Y58F (33%) and S31R (28%), were also frequently selected at this time point (Figure 3.9A). Only a very small frequency of the low affinity IgG1+ GC compartment did not carry Y53D mutations (Figure 3.9A). Donor IgG1+ GC responses generated in BaffΔT chimeras were found to show nearly identical SHM patterns as in their WT counterparts, with comparable frequencies of Y53D (95%), Y58F (43%) and + S31R (22%) mutations (Figure 3.9A). Out of all SWHEL IgG1 GC B cell clones analysed in WT vs. BaffΔT chimeric recipients, the proportion of high and low affinity GC B cells were comparable between the two groups. SWHEL responses in WT chimeras vs. BaffΔT chimeras revealed similar frequencies of Y53D+ high affinity clones without either Y58F or S31R, at 51% and 47% respectively (Figure 3.9B). The percentages of Y53D+ clones that further acquired Y58F or S31R substitutions were also comparable between WT chimeras (44%) and BaffΔT chimeras (45%) (Figure 3.9B). Collectively, these results found no evidence that T cell-derived BAFF has an impact on the positive selection of high affinity B cells in the GC.

75 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

Day 21 Donor IgG1+ GC

A Chimeric Recipient: Baff 100 100

n Y53D n = 39 n = 62 Y53D o i

t 95% SHM = 4.1 SHM = 4.1 95% u 80 80 it t s b

u 60 60 s d i

c Y58F

a 40 S31R 40 Y58F 33% S31R o 28% 43% in 22%

m 20 20 A

% 0 1 4 7 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 12 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 12 82C 1031061091 82C 1031061091 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 HyHEL10 heavy chain variable region amino acid residue number

B Chimeric Recipient: Baff

44% n = 39 45% n = 62 SHM = 4.1 SHM = 4.1 51% 47%

HyHEL10 heavy chain region Amino Acid substitution Unmutated Y53D (high affinity) Y53D + affinity mutations Other mutated

76 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

Figure 3.9. GC B cells undergo normal SHM and selection for affinity- enhancing mutations in the absence of T cell-derived BAFF.

3X SWHEL B cells were challenged with HEL -SRBC in WT or BaffΔT chimeric recipients as depicted in Figure 3.6. Donor-derived IgG1+ GC B cells (CD45.1+, CD45.2-. B220hi, IgG1+, CD38lo) were single cell sorted on day 21 for SHM analysis. (A) SHM analysis showing percentage amino acid substitutions at the Ig heavy chain variable region, with Y53D (red) substitution associated with high affinity binding to HEL3X, and additional affinity-increasing mutations at Y58F (blue) and S31R (orange). CDRs 1, 2 and 3 have been shaded in blue, green and yellow respectively. n = number of clones analysed and SHM = average number of mutations per clone. Data were pooled from 5 chimeric recipient mice. (B) Proportion of high affinity and other mutated and unmutated clones constituting the donor-derived IgG1+ GC compartment in WT vs. BaffΔT chimeric recipients. Unmutated clones are indicated in red, other mutated clones in yellow, high affinity clones with Y53D substitution in dark blue, and high affinity Y53D+ clones with additional affinity- enhancing mutations (Y58F+ and/or S31R+) are represented in light blue.

77 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

3.8 Discussion

The aim of this chapter was to evaluate the dynamics of BAFFR (TNFRSF13C) expression and its involvement following B cell activation by T- dependent antigens and, in particular, to establish a murine model to investigate the role of BAFFR signalling during T-dependent B cell responses.

Work presented here first examined the suitability of the model antigen

HEL and its complementary SWHEL in vivo system to investigate the function of BAFFR during T-dependent B cell responses. Surface detection of BAFFR by flow cytometric analysis revealed distinct BAFFR levels in class-switched (IgG1+) GC, early PBs and MBCs in both polyclonal responses to SRBC (Figure 3X 3.1C) and monoclonal SWHEL responses to HEL (Figure 3.3C). Notably, PBs do not express BAFFR (Figure 3.3C) but class-switched GC B cells showed increased BAFFR expression compared to their naïve ancestors and class- switched MBCs, consistent with observations made in a recent report (75). GC B cells from human tonsils also predominantly express BAFFR, but at mildly lower levels compared to naïve and MBCs, and notably, PCs showed no BAFFR expression, in agreement with studies reported by others (74). The upregulation of BAFFR expression on GC B cells suggested BAFFR may play a part in affinity-based selection in the GC. Furthermore, BAFF is produced by Tfh cells but in a larger part by myeloid and stromal cells such as FDCs inside the GC (41, 324). Thus considering the highly competitive environment and rapid turnover of B cells in the GC (225, 262), the potential for BAFFR-mediated survival in regulating such a process is alluring.

Taking advantage of the SWHEL in vivo system’s sensitivity to track affinity maturation, we sought to detect any differential expression of BAFFR throughout the GC response, with particular interest in its expression by high vs. low affinity GC B cells (Figure 3.4A, B, C). Interestingly, we observed a uniform increase in surface BAFFR levels in both HEL-specific high and low affinity GC B cells (i.e. day 9) but no correlation were observed between affinity and BAFFR expression (Figure 3.4D).

78 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses

Interestingly, one recent study suggested the specific source of Tfh cell- derived BAFF was essential for the positive selection of high affinity GC B cells, such that Tfh cell-derived BAFF impacted upon SHM and selection of high affinity antibodies in the GC (75). Earlier studies have shown that blocking BAFF did result in decreased high affinity antibody titres in NP-CGG immunised mice, but blocking BAFF was found to significantly dampen GC responses without affecting SHM (92, 241). Therefore the dampened GC responses, not impaired selection, might explain the poor antibody responses in these studies. In addition, PC survival through BCMA-signalling is mediated by either BAFF or APRIL (91, 253); hence the dual specificity of the inhibitors (i.e. TACI-Fc or BCMA-Fc) used was a confounding factor in determining the effects of BAFF on GC B cell selection from these studies (92, 241). Nevertheless, another study using WT and BAFF-deficient animals to generate reciprocal bone marrow chimeras clearly demonstrated that radiation-resistant stromal cells alone were sufficient to establish and sustain robust high affinity antibody responses (240). These apparently contradictory results indicated that the role of BAFF from T cells in GC selection required further investigation.

In order to address some of these discrepancies and determine the role of T cell-derived BAFF in the GC response, we generated mixed bone marrow chimeras that specifically lacked T cell-derived BAFF (Figure 3.5). To ensure that T cells could only be derived from donor marrow, lethally irradiated T cell- deficient mice were used as bone marrow recipients. Analysis of antigen- specific donor SWHEL B cells and serum antibodies revealed no differences between GC and class-switched MBC survival in the absence of T cell-derived BAFF (Figure 3.6B, C, D, E). Further investigation using affinity analysis (HEL3X-binding) also yielded no difference in the responding B cells' capacity to affinity mature or to become low affinity and high affinity MBCs (Figure 3.7). Our results are consistent with the idea that BAFF from Tfh cells can be substituted by other non-hematopoietic sources such as FDCs (240). Thus given the study addressed here (75) suggesting T cell-derived BAFF is essential for fine-tuning the selection of high affinity mutations in the CDR regions of high affinity B cells, + SWHEL IgG1 GC B cells from recipients deficient in T cell-derived BAFF were

79 Chapter 3: BAFFR dynamics and requirement for BAFF in T-dependent B cell responses single cell sorted for SHM analysis (Figure 3.9). However, SHM analysis revealed no differences in mutation patterns between GC B cells generated in chimeric mice containing WT or BAFF-deficient T cells (Figure 3.9). Our results also revealed comparable positive selection in the CDR regions corresponding to further affinity-enhancing substitutions for low affinity HEL-variant (HEL3X) that consequently lead to normal production of high affinity antibodies (Figure 3.8; 3.9).

Taken together, our results indicate BAFF produced by T cells is dispensable for regulating selection in the GC in this system. Our findings contrast those suggested in the recent study, in which BAFF from T cells apparently facilitated the selection of high affinity antibodies in an NP- immunised system (75). The discrepancy between the two sets of results is not immediately clear but may reflect differences in the two model systems used such as the employment of adjuvant in the NP-experiments, or the significantly lower affinity and scope for increasing affinity in this hapten (i.e. NP) system. Nevertheless, our data clearly demonstrate that Tfh cell-derived BAFF does not play a critical part in the selection of high-affinity specificities in the GC. Even though BAFF from Tfh cell appears redundant for GC selection, the role of BAFF:BAFFR signalling – primarily mediated by non-hematopoietic sources of BAFF – remains an important question. The precise contribution of BAFFR in regulating GC responses and B cell memory will be investigated in more detail in the subsequent chapters.

80 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Chapter 4

Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

4.1 Preamble

Attempts to investigate the in vivo functions of the BAFF:BAFFR axis have to a large extent involved experimentation on BAFF-deficient (Tnfsf13b-/-) and BAFFR-deficient (Tnfrsf13c-/-) or BAFFR-mutant (A/WySnJ) mice (93, 97, 325). BAFF-deficient and BAFFR-deficient mice showed severely impaired GC and MBC responses that can be initiated but could not be sustained (82, 93, 241). Since BAFF:BAFFR signalling is necessary for the maturation and survival of B cells during development, BAFF-deficient or BAFFR-deficient mice contain reduced numbers of peripheral B cells, significantly smaller secondary lymphoid organs and have poor survival of mature B cells (39, 40, 326). Due to these defects, it remains unclear what the specific role(s) of BAFF:BAFFR signalling are in the control and maintenance of GC and MBC responses independent of the impairments to B cell maturation and the resulting impacts on secondary lymphoid organ structure. This chapter aims to examine the extent to which the abnormal physiology in BAFF-deficiency and BAFFR- deficiency impacts on the induction, control and maintenance of normal B cell responses to T-dependent antigens.

81 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

4.2 Loss of mature B cells in BAFF-deficient and BAFFR-deficient mice leads to abnormal lymphoid organ architecture

Flow cytometric analysis was performed on spleens from unimmunised BAFF-deficient (Tnfsf13b-/-) and BAFFR-deficient (Tnfrsf13c-/-) mice to confirm the lymphoid tissue abnormalities under these conditions. Splenic B cells were categorised based on B220 and CD93 expression to identify immature transitional B cells (B220+, CD93+) and mature B cells (B220+, CD93-) (Figure 4.1A). Naïve B cell subsets were delineated based on CD21 and CD23 expression to identify Fo and MZ B cells (Figure 4.1A). Spleens obtained from Tnfsf13b-/- and Tnfrsf13c-/- animals were comparable in size and showed the expected reductions in B cell numbers compared to WT mice (Figure 4.1B, C). Tnfsf13b-/- and Tnfrsf13c-/- animals characteristically displayed very few mature hi + B cells (B220 , CD93 ) compared to WT control (Figure 4.1A). Moreover, the size of the mature B cell subsets of the Fo (CD23hi, CD21int) and MZ (CD23lo, CD21hi) phenotypes were markedly reduced in Tnfsf13b-/- and Tnfrsf13c-/- mice compared to their WT counterpart, whereas the numbers of immature transitional B cells (CD23lo, CD21lo) were only marginally affected (Figure 4.1A, D). Interestingly, the loss of Fo B cells in Tnfsf13b-/- mice was slightly greater than in Tnfrsf13c-/- mice (Figure 4.1A, D), consistent with previous reports that BAFF regulates both CD21 and CD23 expression on naïve B cells but through pathways other than BAFFR signalling alone (38, 82).

Immunofluorescence histology performed on spleens taken from WT, Tnfsf13b-/- and Tnfrsf13c-/- mice challenged with SRBC further demonstrated these BAFF-deficient and BAFFR-deficient mice had severe lymphoid tissue abnormalities (Figure 4.2). WT spleen sections showed well-defined B cell follicle formations and persistent GC formations on day 14, whereas Tnfsf13b-/- and Tnfrsf13c-/- animals showed poorly developed follicles in their secondary lymphoid organs and significantly reduced numbers of GCs in response to SRBC challenge (Figure 4.2). Meanwhile T cell numbers and T cell zone were unaffected in these deficient animals compared to WT controls (Figure 4.2).

82 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

A Unimmunised spleen Total splenic leukocytes Genotype: Wild type Tnfsf13b-/- Tnfrsf13c-/-

8.7 9.3 9.8 CD93

39 5.8 5.4 B220 + Total B220 lymphocytes

82 2.8 26 CD23

7.1 7.9 94 1.4 67 1.3 CD21/35 B Total Leukocytes C Total B cells 109 80 Genotype

n Wild type 60 **** * Tnfsf13b-/- 108 **** ** Tnfrsf13c-/- 40 % Spleen 107

Cells per splee 20

106 0

Splenic B cell subsets D 108 **** **** n 107 * **** **** 106

Cells per splee 105

104 Immature Follicular Marginal Zone B cells B cells B cells

83 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Figure 4.1. BAFF-deficient and BAFFR-deficient mice are lymphopenic and lack mature B cells in their secondary lymphoid organs.

Spleens from unimmunised wild type, BAFF-deficient (Tnfrsf13b-/-) and BAFFR-deficient (Tnfrsf3c-/-) mice were phenotyped using cell surface markers. (A) Flow cytometric analysis showing splenic B cell subsets: follicular (Fo) and transitional-2 (T2) B cells (CD21/35int, CD23hi), marginal zone (MZ) B cells (CD21/35hi, CD23lo) and immature transitional-1 B cells (CD21/35lo, CD23lo). Numbers on plots represent percentages (%) of associated windows and are representative of 5 mice. (B) Enumeration of total splenic leukocytes. (C) Frequency of splenic B cells (B220+). (D) Enumeration of mature B cell (B220+, CD93-): Fo B cells (CD21/35int, CD23hi, CD93-), MZ B cells (CD21/35hi, CD23lo, CD93-), and total immature transitional B cells (B220+, CD93+) in Tnfsf13b-/- and Tnfrsf13c-/- mice. Data are representative of 5 mice from one experiment and are representative of two replicate experiments. Results were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, *P<0.05, ****P<0.0001.

84 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Wild type

Tnfsf13b-/-

Tnfrsf13c-/-

IgD PNA CD3 6 um sections, 10x Day 14 SRBC immunised Figure 4.2. Abnormal lymphoid organ architecture in BAFF-deficient and BAFFR-deficient mice.

Three-colour immunohistology of day 14 spleen section from SRBC- immunised wild type, Tnfsf13b-/- and Tnfrsf13c-/- mice showing loss of lymphoid follicular structures, reduced Fo and MZ B cells and an absence of germinal centres (GCs) in Tnfsf13b-/- and Tnfrsf13c-/- mice but not in wild type mice. Sections were stained with IgD (blue) to identify B cell follicle, CD3 (red) to identify T cell zone and PNA (green) to identify GC as denoted by arrows. Data here are representative of 6 mice from one experiment. Data are representative of two independent replicate experiments.

85 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

4.3 Strategy to examine T-dependent responses of wild type B cells in BAFF-deficient and BAFFR-deficient mice

In order to determine how the lymphoid tissue abnormalities and compositions present in BAFF-deficiency and BAFFR-deficiency affect normal B cells responses, SWHEL B cells were adoptively transferred into WT, BAFF- deficient (Tnfsf13b-/-) and BAFFR-deficient (Tnfrsf13c-/-) recipient mice and challenged with HEL3X-SRBC i.v. (Figure 4.3). Recipients were antigen-boosted 4 days post primary immunisation and spleens were harvested on day 6 and 14 for flow cytometric analysis (Figure 4.3). The gating strategy described in Chapter 3.3 was adapted to identify and analyse transferred donor-derived B cells. Following compensation of acquired data, live lymphocytes were gated (FSC-A vs. SSC-A) and doublets were excluded (FSC-A vs. FSC-H). Autofluorescent cells were excluded based on emission into empty V610/V660 channel (Unstained vs. FSC-A). Congenic markers (CD45.1 and CD45.2) were + - stained to identify SWHEL donor-derived (CD45.1 , CD45.2 ) and endogenous host (CD45.2+, CD45.1-) B cell responses to HEL3X-SRBC. Antibodies targeting surface B220, CD38 and IgG1 were used to identify IgG1-switched GC B cells (B220+, IgG1+, CD38lo) and IgG1-switched MBCs (B220+, IgG1+, CD38hi) (ref. Chapter 3.3).

86 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Donor Recipient

i.v Wild type (WT) 3 x 104 HEL-binding B cells

2 x 108 -/- SWHEL (WT) HEL3X-SRBC Tnfsf13b (Baff)

-/- Tnfrsf13c (Baffr) Wild type HEL-binding B cell + Day 0 Day 4 Day 6 Day 14 HEL3X-SRBC i.v

3X HEL -SRBC Harvest Harvest WT or Baff Boost or Baffr Recipient

Figure 4.3. Adoptive transfer strategy to investigate the impact of BAFF- deficient and BAFFR-deficient conditions on wild type B cell responses.

+ -/- SWHEL B cells (CD45.1 ) were adoptively transferred into WT, Tnfsf13b and Tnfrsf13c-/- congenic recipients (CD45.2+) and challenged with HEL3X-SRBC and antigen boosted again on day 4 to investigate normal B cell responses in BAFF-deficient and BAFFR-deficient environments. Endogenous responses - + + (CD45.1 , CD45.2 ) to SRBC and donor-derived SWHEL responses (CD45.1 , CD45.2-) to HEL3X were examined concurrently in WT, Tnfsf13b-/- and Tnfrsf13c-/- recipient spleens on day 6 and day 14.

87 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

4.4 Normal induction but attenuated endogenous GC and memory B cell responses in BAFF-deficient and BAFFR-deficient mice

First, to evaluate how BAFF-deficient and BAFFR-deficient animals respond to T-dependent antigen and how their endogenous responses might affect donor SWHEL responses that shared the same GC niche, the responses of endogenous B cells (CD45.2+, CD45.1-) to HEL3X-SRBC immunisation were investigated in the WT, Tnfsf13b-/- and Tnfrsf13c-/- recipients (Figure 4.4).

On day 6 of the response, Tnfsf13b-/- and Tnfrsf13c-/- mice showed similar frequencies of IgG1-switched GC B cells (B220hi, IgG1+, CD38lo) compared to WT mice (Figure 4.4A, B), indicating that BAFF:BAFFR signalling was not required for CSR to occur in activated B cells. However, due to the lower overall numbers of B cells, the size of the endogenous GC responses in Tnfsf13b-/- and Tnfrsf13c-/- mice were consistently 10 to 15-fold lower than in WT mice (Figure 4.4C, D). By day 14, the size of the GC response in Tnfsf13b-/- and Tnfrsf13c-/- mice were further reduced in relation to WT mice (Figure 4.4C, D). The generation of IgG1+ MBCs (B220hi, IgG1+, CD38hi) in response to SRBC in Tnfsf13b-/- and Tnfrsf13c-/- mice were also markedly reduced at all time points (Figure 4.4A, E). These observations confirmed that the immunisation of Tnfsf13b-/- and Tnfrsf13c-/- mice could initiate small GCs but could not sustain long-lived B cell responses to T-dependent antigens. Thus, to further investigate whether these impaired responses were due to B cell-intrinsic defect or were caused by cell-extrinsic abnormalities, donor-derived responses were examined.

88 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Endogenous SRBC Response Total splenic B cells

Genotype Wild type Tnfsf13b-/- Tnfrsf13c-/- A 0.07 0.04 0.05 CD38 Day 6

0.74 0.81 0.76

0.07 0.01 0.01

Day 14

0.89 0.32 0.36 IgG1

B Total GC C Total GC 50 107 *** *** Genotype 40 106 *** *** n Wild type

+ -/- 30 105 Tnfsf13b Tnfrsf13c-/- 4 % IgG1 20 10 Cells per splee 10 103

0 102 6 14 6 14 Days post Immunisation Days post Immunisation

D IgG1+ GC E IgG1+ Memory 106 105 ** ** *** ** ** ** *** *** 104 n 105 n

103 104 102 Cells per splee 103 Cells per splee 101

102 100 6 14 6 14 Days post Immunisation Days post Immunisation

89 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Figure 4.4. BAFF-deficient and BAFFR-deficient mice fail to sustain endogenous GC and MBC responses to SRBC.

+ -/- SWHEL B cells (CD45.1 ) adoptively transferred into WT, Tnfsf13b and Tnfrsf13c-/- congenic recipients (CD45.2+) were challenged with HEL3X-SRBC and antigen boosted on day 4. WT, Tnfsf13b-/- and Tnfrsf13c-/- spleens were harvested and recipient’s endogenous B cells (CD45.2+, CD45.1-) excluding donor B cells (CD45.1+, CD45.2-) were analysed. (A) Flow cytometric analysis showing recipient-derived (CD45.2+, CD45.1-, B220+) IgG1+ GC B cells (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi) day 6 and 14 post immunisation. (B) Frequency of IgG1-switching in recipient-derived GC B cells (CD45.2+, CD45.1-, B220hi, Fashi, CD38lo) for WT, Tnfsf13b-/- and Tnfrsf13c-/- recipients. (C) Enumeration of recipient-derived total GC (B220hi, Fashi, CD38lo), (D) IgG1+ GC and (D) IgG1+ MBCs identified in (A). Plots are representative of 4 mice from one experiment and are representative of 2 independent replicate experiments. Results were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, **P<0.01, ***P<0.001.

90 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

4.5 Cell-extrinsic factors compromise wild type B cell responses in BAFF-deficient and BAFFR-deficient mice

To examine how the abnormal lymphoid tissue architecture in BAFF deficient and BAFFR deficient animals might contribute to the compromised B -/- -/- cell responses in Tnfsf13b and Tnfrsf13c mice, donor-derived SWHEL responses in WT, Tnfsf13b-/- and Tnfrsf13c-/- recipient mice were analysed using flow cytometric analysis. On day 6, splenic donor-derived B cells + - + -/- -/- (CD45.1 , CD45.2 , B220 ) in Tnfsf13b and Tnfrsf13c mice were respectively 8-fold and 25-fold greater than the frequencies of cells found in WT recipients (Figure 4.5A). However, enumeration revealed equivalent donor cell numbers in these recipient mice comparable to WT controls (Figure 4.5B). By day 14, donor-derived B cell numbers in Tnfsf13b-/- and Tnfrsf13c-/- recipients significantly decreased compared to WT recipients (Figure 4.5A, B). These observations indicated that while normal donor B cells can initially respond to antigens and clonally expand in the absence of environmental BAFF and BAFFR, the tissue microenvironments associated with global BAFF-deficiency and BAFFR-deficiency impaired normal T-dependent B cell responses.

Flow cytometric analysis of GC and MBC responses generated by SWHEL donor B cells showed striking differences between Tnfsf13b-/- and Tnfrsf13c-/- and WT recipient mice (Figure 4.6A). On day 6, IgG1 switching in donor GC B cells (B220hi, IgG1+, CD38lo) was slightly elevated in Tnfsf13b-/- (42%) and Tnfrsf13c-/- (47%) recipients compared to WT (32%) (Figure 4.6A, B) but the numbers of total GC and IgG1+ GC B cells were comparable in all recipient groups (Figure 4.6C, D). However, by day 14 the numbers of donor-derived GC B cells were significantly reduced in Tnfsf13b-/- and Tnfrsf13c-/- recipients compared to the WT counterpart (Figure 4.6C, D). These findings implicated that the environments in BAFF-deficient and BAFFR-deficient mice facilitated the initiation but not the perpetuation of normal GC B cell responses. - Intriguingly, while SWHEL GC B cell responses were terminated in both Tnfsf13b /- -/- + + hi and Tnfrsf13c recipient mice, donor-derived IgG1 MBCs (IgG1 , CD38 ) specifically persisted in Tnfrsf13c-/- (BAFFR-deficient) but not Tnfsf13b-/- (BAFF- deficient) mice (Figure 4.6A, E). These observations suggested that BAFF,

91 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses which is still present in these recipients, might contribute to the development of the MBC responses. Thus, whilst these experiments could not clarify the importance of BAFF:BAFFR signalling intrinsic to the responding B cells in the GC response, they did suggest that it may play a B cell-intrinsic role in regulating MBC responses.

92 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

A Total splenic B cells

Recipient: Wild type Tnfsf13b-/- Tnfrsf13c-/-

0.6 4.2 8.1

CD45.2 Day 6

0.6 0.4 1.1

Day 14

CD45.1

B Total Donor B cells 106 * Recipient ** Wild type 105 Tnfsf13b-/- Tnfrsf13c-/- 104

3

Cells per spleen 10

102 6 14

Days post Immunisation

Figure 4.5. BAFF-deficient and BAFFR-deficient environments inhibit the progression of wild type B cell responses.

-/- -/- SWHEL B cells were adoptively transferred into WT, Tnfsf13b and Tnfrsf13c congenic recipients and challenged with HEL3X-SRBC, recipient spleens were harvested on day 6 and 14 as depicted in Figure 4.3. (A) Flow cytometric + - + analysis showing donor-derived SWHEL B cells (CD45.1 , CD45.2 , B220 ) in WT, Tnfsf13b-/- and Tnfrsf13c-/- recipient mice. (B) Enumeration of total donor- derived B cells on day 6 and day 14. Plots are representative of 4 mice from one experiment and data are representative of 2 independent replicate experiments. Data were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, *P<0.05, **P<0.01.

93 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

A Donor B cells Recipient: Wild type Tnfsf13b-/- Tnfrsf13c-/-

4.0 3.1 6.0 CD38 Day 6

16 29 21

0.3 0 4.2

Day 14

53 34 4.5 IgG1 B Total Donor GC C Total Donor GC 80 106 ** Recipient ** ** ** n 60 105 Wild type

+ * Tnfsf13b-/- -/- 40 104 Tnfrsf13c % IgG1 Cells per splee 20 103

0 102 6 14 6 14 Days post Immunisation Days post Immunisation D Donor IgG1+ GC E Donor IgG1+ Memory 106 105 ** * ** 4

* n 10 n 105 * ** 103 104 102 103 101 Cells per splee Cells per splee 102 100

<0.1 101 6 14 6 14 Days post Immunisation Days post Immunisation

94 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Figure 4.6. Attenuated wild type GC responses but intact MBC responses in BAFFR-deficient not BAFF-deficient recipient mice.

+ - + Donor-derived SWHEL B cells (CD45.1 , CD45.2 , B220 ) challenged with HEL3X-SRBC in WT, Tnfsf13b-/- and Tnfrsf13c-/- recipients were examined on day 6 and 14 as described in Figure 4.3. (A) Flow cytometric profiles demonstrating donor-derived (CD45.1+, CD45.2-) IgG1+ GC B cells (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi). (B) Frequency of IgG1-switching in total donor-derived GC B cells (CD45.1+, CD45.2-, Fashi, CD38lo) in WT, Tnfsf13b-/- and Tnfrsf13c-/- conditions. (C) Enumeration of total donor-derived GC (Fashi, CD38lo), (D) IgG1+ GC and (E) IgG1+ MBCs identified in (A). Results are representative of 4 mice from one experiment and are representative of 2 independent replicate experiments. Data were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, *P<0.05, **P<0.01.

95 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

4.6 GC affinity maturation is intact but cell-extrinsic absence of BAFF impairs memory B cell survival and absence of BAFFR accumulates low affinity memory B cells

In light of the differences in donor-derived responses in BAFF-deficient and BAFFR-deficient recipients, we next examined how the affinity of the B cells in the GC and MBC populations were impacted. HEL3X staining was utilised to + visualise high affinity and low affinity SWHEL IgG1 GC B cells using the method detailed in Chapter 2.4 and the gating strategy detailed in Chapter 3.3. The output of donor-derived high affinity and low affinity IgG1+ MBCs in WT, Tnfsf13b-/- and Tnfrsf13c-/- recipient mice were also visualised using this method (ref. Chapter 3.3).

At the peak of SWHEL GC maturation on day 14, at least 91% of donor- derived IgG1+ GC B cells in WT recipients have affinity matured to become high affinity (Figure 4.7A, B). Interestingly, donor-derived IgG1+ GC responses in Tnfsf13b-/- and Tnfrsf13c-/- recipient mice also showed similar frequencies of high affinity cells, at 95% and 90% respectively despite the greatly reduced overall GC responses (Figure 4.7A, B). Conversely, comparison of donor- derived IgG1+ MBC responses in WT, Tnfsf13b-/- and Tnfrsf13c-/- recipients showed differing proportions of high vs. low affinity cells (Figure 4.7A, C). The proportions of high and low affinity MBCs in Tnfsf13b-/- recipients could not be determined as only very few MBCs were present in these mice (Figure 4.6A, E; 4.7A, C). In Tnfrsf13c-/- recipients, however, donor-derived IgG1+ MBCs showed a major skewing towards low affinity cells with only ~3% exhibiting high affinity compared to 68% of IgG1+ MBCs in WT recipients (Figure 4.7A, C). This was intriguing as IgG1+ GC B cells displayed normal affinity maturation, suggesting three possibilities: i) the selection in the GC to become MBCs was impaired by loss of environmental BAFFR, or ii) the survival of GC-derived low affinity MBCs was prolonged, or iii) that these were MBCs generated in a GC-independent pathway. The experiments that follow were designed to resolve between these possibilities.

96 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

A Day 14 Donor B cells

Recipient Wild type Tnfsf13b-/- Tnfrsf13c-/-

3X 91 95 90 HEL Binding IgG1+ GC

8.9 5.3 8.4

68 N.D 2.4 IgG1+ Memory

32 N.D 98 IgG1

B C Donor IgG1+ GC Donor IgG1+ Memory 100 0 100 0 **** 80 20 80 20 % Low % Low y y finit 60 40 finit f 60 40 f A A

A A f f finit finit 40 60 40 60 y y % High % High

20 80 20 80

N.D 0 100 0 100 -/- -/- -/- -/-

ild type ild type W Tnfsf13b W Tnfrsf13c Tnfsf13b Tnfrsf13c Recipient Recipient

Affinity Compartment Low Affinity High Affinity

97 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Figure 4.7. Wild type responses accumulate low affinity MBCs despite normal GC affinity maturation in BAFFR-deficient recipient mice.

+ + Donor-derived SWHEL IgG1 GC and IgG1 MBCs described in Figure 4.6 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric profiles + showing high vs. low affinity compartments in donor-derived SWHEL IgG1 GC and IgG1+ MBC responses 14 days post immunisation. (B) Proportion of high vs. low affinity IgG1+ GC B cells and (C) IgG1+ MBCs depicted in (A). N.D denotes “not determined” due to low cell numbers. Plots are representative of 4 mice from one experiment and data are representative of 2 independent replicate experiments. Results were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, ****P<0.0001.

98 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

4.7 Absence of BAFFR extrinsic to responding wild type B cells promotes the persistence of low affinity memory B cells

To further investigate the abnormal accumulation of donor-derived low -/- affinity MBCs in Tnfrsf13c mice, SWHEL donor responses in WT and Tnfrsf13c-/- recipients were examined on day 11 and day 19 using flow cytometry and SHM analysis (Figure 4.8A). On day 11 donor-derived IgG1+ GC B cell responses were comparable between WT and Tnfrsf13c-/- recipients + -/- (Figure 4.8B, C). By day 19 however, SWHEL IgG1 GC responses in Tnfrsf13c mice were diminished significantly compared to WT mice (Figure 4.8B, C). Notably, the number of donor-derived IgG1+ MBCs in Tnfrsf13c-/- recipients was consistently similar to WT control (Figure 4.8D), confirming the results presented previously (ref. Chapter 4.5).

On day 11, affinity analysis revealed normal or even slightly elevated frequencies of high affinity GC B cells in Tnfrsf13c-/- mice compared to WT mice (Figure 4.9A, B). The frequencies of high vs. low affinity GC B cells on day 19 were also comparable between the two groups (Figure 4.9A, C). However, these results did not correlate with the proportions observed for high and low affinity IgG1+ MBCs (Figure 4.9D). Thus, although high affinity MBCs were present in similar numbers in WT and Tnfrsf13c-/- mice on day 11 (Figure 4.9D, E), the numbers and frequency of low affinity MBCs were significantly increased in Tnfrsf13c-/- mice and were not normally seen in WT mice (Figure 4.9E, G). This preferential accumulation of low affinity MBCs in Tnfrsf13c-/- mice was found to persist until day 19 of the response (Figure 4.9F, H).

99 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

A

Donor Recipient

i.v

3 x 104 Wild type HEL-binding B cells

2 x 108 3X SWHEL HEL -SRBC

Tnfrsf13c-/-

B Donor B cells

Recipient Wild type Tnfrsf13c-/-

0.8 2.5 CD38 Day 11

47 18

0.4 1.4

Day 19

57 4.8 IgG1

C Donor IgG1+ GC D Donor IgG1+ Memory 107 105 Recipient 106 n ** n 104 Wild type 105 Tnfrsf13c-/- 104 103

103 2 Cells per splee Cells per splee 10 102

101 101 11 19 11 19 Days post Immunisation Days post Immunisation

100 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Figure 4.8. BAFFR-deficient environment attenuates GC but not MBC responses of wild type B cells.

+ (A) SWHEL B cells (CD45.1 ) were adoptively transferred into WT and Tnfrsf13c-/- recipients (CD45.2+) and challenged with HEL3X-SRBC. Recipients were antigen boosted again on day 4 and spleens were harvested at day 11 and 19 for single cell sorting and SHM analysis of donor-derived responses (CD45.1+, CD45.2-). (B) Flow cytometric analysis showing donor-derived IgG1+ GC (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi) on day 11 and 19. (C) Enumeration of donor-derived IgG1+ GC and (D) IgG1+ MBCs. Numbers denote percentages (%) of associated window. Data are representative of 5 mice and are representative of 3 independent experiments. Results were analysed using unpaired Student t-test with Welch’s correction post-hoc. Data were considered not significant unless specified, **P<0.01.

101 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Affinity Compartment A Low Affinity Donor + IgG1 GC High Affinity -/- Recipient Wild type Tnfrsf13c Donor IgG1+ GC DAY 11 69 84 100 0 3X B *

HEL 80 20 Binding

Day 11 % Low y

finit 60 40 f A

A f

30 15 finit 40 60 y

96 97 % High

20 80

Day 19 C 0 100 Wild type Tnfrsf13c-/- DAY 19 100 0 4 3 80 20

IgG1 % Low y 60 40

+ finit f

D A Donor IgG1 Memory A

f finit 40 60 -/- y

Recipient Wild type Tnfrsf13c % High 42 6 20 80 3X

0 100 HEL Wild type Tnfrsf13c-/- Binding Day 11 Recipient Donor IgG1+ Memory 57 94 DAY 11 G 100 0 88 2

80 20 % Low y Day 19 *

finit 60 40 f A

A f finit 40 60 y

12 98 % High IgG1 20 80

0 100 Wild type Tnfrsf13c-/- E F DAY 19 Day 11 Day 19 H 100 0 105 105

104 104 80 20 n n * **** 3 3 ** * % Low 10 10 y

finit 60 40 2 2 f

10 10 A

A f finit 101 101 40 60 y % High Cells per splee Cells per splee 100 100 20 80 <0.1 <0.1

High Affinity Low Affinity High Affinity Low Affinity 0 100 Wild type Tnfrsf13c-/- Recipient Wild-type Tnfrsf13c -/- Recipient

102 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Figure 4.9. Wild type B cell responses accumulate low affinity MBCs but not low affinity GC B cells in BAFFR-deficient condition.

+ + Donor-derived SWHEL IgG1 GC and IgG1 MBCs described in Figure 4.8 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric analysis showing high vs. low affinity IgG1+ donor GC responses in WT and Tnfrsf13c-/- recipient mice. (B) Proportion of high vs. low affinity GC B cells on day 11 and (C) day 19. (D) Flow cytometric analysis showing high vs. low affinity IgG1+ donor MBC responses in WT and Tnfrsf13c-/- recipient mice. (E) Enumeration of high affinity and low affinity IgG1+ MBCs on day 11 and (F) day 19). (G) Proportion of high vs. low affinity memory cells on day 11 and (H) day 19. Numbers represent percentages (%) in associated windows. Data are representative of 5 mice from one experiment and are representative of 3 independent replicate experiments. Data were analysed using unpaired Student t-test with Welch’s correction post-hoc. Data were considered not significant unless specified, *P<0.05, ****P<0.0001.

103 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

4.8 Wild type B cell responses accumulate unmutated memory B cells in BAFFR-deficient recipient mice

+ To determine the nature of the low affinity SWHEL donor-derived IgG1 MBCs that accumulate in BAFFR-deficient recipient mice, single IgG1+ GC B cell and IgG1+ MBC from WT and Tnfrsf13c-/- recipients were sorted on day 11 and 19 for SHM analysis of Ig heavy chain VH10 sequence as described in Chapter 2.5. The gating strategy outlined in Chapter 3.3 was used to identify and sort donor-derived IgG1+ GC B cells (CD45.1+, B220+, IgG1+, CD38lo) and IgG1+ MBCs (CD45.1+, B220+, IgG1+, CD38hi).

+ -/- On day 11, SWHEL IgG1 GC B cells sequenced from WT and Tnfrsf13c recipient mice showed similar rates of SHM (Figure 4.10A). A significant fraction of the GC had also acquired the high affinity Y53D substitution in both WT and Tnfrsf13c-/- mice (Figure 4.10A). Comparable frequencies of Y53D+ high affinity GC clones were found in both WT (59%) and Tnfrsf13c-/- (68%) recipient mice (Figure 4.10A). At this time point, a small fraction of GC B cells with no mutations were still present in both WT (2%) and Tnfrsf13c-/- (3%) recipients (Figure 4.10A). It was also revealed that very few of the high affinity Y53D+ GC B cells in either set of recipients had also acquired the Y58F or S31R substitutions that further increase affinity for HEL3X-binding (Figure 4.10A). By -/- day 19, all SWHEL GC B cells carry somatic mutations in WT and Tnfrsf13c mice and the proportions of Y53D+ high affinity GC clones were also similar between the two recipient groups (100% and 97%, respectively) (Figure 4.10B). The proportions of Y53D+ high affinity cells that had acquired further affinity- enhancing substitutions (Y58F and/or S31R) were also comparable between WT control (53%) and Tnfrsf13c-/- mice (38%), suggesting that SHM and affinity maturation of GC B cells were not affected by BAFFR-deficiency despite the overall decreased GC B cell responses (Figure 4.10B).

SHM analysis of the donor-derived MBC compartment however, revealed significant differences between WT and Tnfrsf13c-/- recipient groups (Figure 4.10C, D). Day 11 analysis revealed 54% of donor-derived IgG1+ MBC from WT mice contained high affinity Y53D+ substitutions, with 15% also possessing the

104 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses affinity-enhancing mutations Y58F and/or S31R (Figure 4.10C). This was in stark contrast to the small proportion of Y53D+ high affinity MBCs (4%) and the MBC clones with additional affinity-enhancing substitutions (Y58F and/or S31R) (1%) sequenced from Tnfrsf13c-/- recipients (Figure 4.10C). Instead, the majority + -/- of SWHEL IgG1 MBCs sorted from Tnfrsf13c mice contained no somatic mutations (74%), suggesting that these were likely to be GC-independent, early MBCs (Figure 4.10C). Notably, unmutated donor MBCs were observed at much lower frequencies (15%) in WT recipients (Figure 4.10C). Day 19 SHM analysis of donor-derived IgG1+ MBCs further confirmed these observations (Figure 4.10D). In WT recipients, the majority of MBCs were GC-derived and possessed the high affinity Y53D substitution (93%) as well as a substantial portion had also acquired additional affinity-enhancing substitutions (Y58F and/or S31R) (17%) (Figure 4.10D). However, of all donor-derived IgG1+ MBC clones analysed from Tnfrsf13c-/- mice, all MBC clones were devoid of somatic mutations, indicating that these were GC-independent, early MBCs (Figure 4.10D). Thus, when WT B cells are challenged with T-dependent antigen in mice that lack BAFFR but do express BAFF, the production and/or survival of unmutated MBCs appears to be the only component of the response that remains unaffected.

105 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Donor IgG1+ GC

Recipient Wild type Tnfrsf13c-/-

2% 3% A

n = 44 n = 37 SHM = 2.8 SHM = 2.0 Day 11 59% 68%

B

53% 38% n = 43 n = 34 Day 19 SHM = 4.3 SHM = 3.8 47% 59%

HyHEL10 heavy chain region Amino Acid substitution Unmutated Donor IgG1+ Memory Y53D (high affinity) Y53D + affinity mutations Other mutated Recipient Wild type Tnfrsf13c-/- **** C 1% 15%

n = 46 3% n = 43 Day 11 SHM = 2.4 SHM = 0.4 74% 39% 15%

D

17% n = 41 n = 33 Day 19 SHM = 3.0 SHM = 0 100% 76%

****

106 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

Figure 4.10. Single cell SHM analysis reveals wild type B cell responses accumulate unmutated, early MBCs in BAFFR-deficient environment.

+ + Donor-derived SWHEL IgG1 GC and IgG1 MBCs identified in Figure 4.8 were single cell sorted on day 11 and 19 for SHM analysis. (A) Data showing percentages of high affinity vs. low affinity clones constituting the donor IgG1+ GC compartment in WT vs. Tnfrsf13c-/- conditions on day 11 and (B) day 19. (C) Frequency of high affinity vs. low affinity clones constituting the donor-derived IgG1+ memory cell compartment in WT vs. Tnfrsf13c-/- recipients on day 11 and (D) day 19. Unmutated clones are indicated in red, other mutated clones in yellow, high affinity clones with Y53D substitution in dark blue, and high affinity Y53D+ clones with additional affinity-enhancing mutations (Y58F+ and/or S31R+) are represented in light blue. n = number of clones analysed and SHM = average number of mutations per clone. Data are pooled from 5 recipient mice and are representative of 3 independent experiments. Significance was determined by Chi-Sq contingency test, ****P<0.0001.

107 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses

4.9 Discussion

The aim of this chapter was to examine how BAFF-deficiency and BAFFR-deficiency in recipient mice impact on the ability of WT B cells to generate robust T-dependent responses.

Using flow cytometric analysis and immunohistochemistry staining, the B cell constitution and lymphoid tissue architecture found in BAFF-deficient and BAFFR-deficient mice were first characterised. The loss of mature B cells in these mice led to poorly structured lymphoid follicles and largely absent GCs by day 14 after SRBC challenge (Figure 4.1; 4.2). These findings were confirmed by flow cytometric analysis where BAFF-deficient and BAFFR-deficient mice initiated and formed small endogenous GCs to SRBC-challenge that did not persist beyond 14 days post-immunisation (Figure 4.2; 4.4A, C). The impaired GC B cell responses in these mice correlated with a large decrease in both IgM and IgG antibody titres (data not shown) and the complete absence of IgG1+ MBCs (Figure 4.4A, E).

To determine whether these defective responses were due to lack of BAFF:BAFFR signalling in responding B cells or due to cell-extrinsic factors associated with BAFF-deficiency and BAFFR-deficiency, WT SWHEL B cells were adoptively transferred into BAFF-deficient and BAFFR-deficient recipients as described in Chapter 4.3. The results showed that SWHEL B cells initiated GC responses in both BAFF-deficient and BAFFR-deficient mice by day 6 but these responses failed to persist (Figure 4.6A, C, D). Since the donor SWHEL B cells did express BAFFR, these findings suggested that the availability of BAFF alone is insufficient for maintaining robust GC responses. Instead, factors extrinsic to the responding B cells that are common to both BAFF-deficient and BAFFR-deficient mice must explain the impaired GC responses. Since non- cognate B cells and mature FDCs in the GC microenvironment are important components in controlling and maintaining robust GC responses in vivo (66, 201, 228, 327, 328), one possibility is that the lack of endogenous mature B cells in BAFF-deficient and BAFFR-deficient mice disrupts FDC maturation sufficiently to disrupt normal GC progression. Furthermore, BAFF-deficient mice

108 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses have been shown to lack a mature FDC-reticulum, leading to poor immune complex trapping and antigen presentation (233, 241). The successive treatments with BAFF-inhibitor (BCMA-Fc) in WT mice have also been shown to result in the decline in peripheral B cell numbers and reduced FDC maturation, consequently leading to the dissolution of GCs (241). Thus, the lack of competent FDC networks may explain the attenuation of SWHEL GC responses in these BAFF-deficient and BAFFR-deficient recipient mice.

A surprising finding from this work was the significant contrast in the + development of IgG1 SWHEL MBCs in BAFF-deficient and BAFFR-deficient mice (Figure 4.6A, E). Although donor IgG1+ GC B cell responses were poor in both BAFF-deficient and BAFFR-deficient recipients, only responses in BAFF- deficient mice were completely devoid of MBCs (Figure 4.6A, E). Instead, donor IgG1+ MBC responses were maintained at comparable levels in BAFFR- deficient and WT recipients (Figure 4.6A, E). Since BAFF-deficient and BAFFR- deficient mice have very similar overall phenotypes (ref. Chapter 4.2), the most likely explanation for this difference is that the BAFFR-expressing donor B cells are being acted upon by the BAFF present in BAFFR-deficient recipients. In other words BAFF appears to act on responding B cells to specifically promote the MBC response. This result was intriguing because blockade of BAFF using BAFFR-Ig treatment has been reported to have no impact on IgG-switched high affinity MBC survival (253).

Interestingly, no differences in the affinity maturation of donor GC B cells were observed in WT, BAFF-deficient or BAFFR-deficient conditions (Figure 4.7A, B; 4.9A, B; 4.10A, B). This suggested that BAFF:BAFFR signalling has little impact on the affinity-based selection in the GC. Furthermore, high affinity antibodies were readily detected in these deficient mice (97, 241). In agreement with this, previous studies have shown that BAFF-deficient and BAFFR-deficient mice challenged with T-dependent antigens (NP-CGG or NP-KLH) generated GC responses with normal SHM frequency, albeit these responses were attenuated (93, 241). In our system, however, SWHEL donor-derived low affinity MBCs accumulated in abnormally high numbers in BAFFR-deficient mice compared to WT mice (Figure 4.7A, C). Moreover, these low affinity MBCs

109 Chapter 4: Impacts of BAFF and BAFFR deficiency on T-dependent B cell responses persisted in BAFFR-deficient recipients during all stages of the response, while high affinity MBCs were markedly reduced (Figure 4.7A; 4.9D, E). SHM analysis of these MBCs further indicated these were primarily unmutated, GC- independent MBCs (Figure 4.10C, D). Thus, these results indicated the lymphoid tissue abnormalities in BAFF-deficient and BAFFR-deficient recipient mice contribute significantly to impairing WT B cell survival and inhibiting the persistence of WT GC responses. The attenuation of GC responses in these recipients, however, did not impact on GC B cell affinity maturation; the selection of somatically mutated affinity-matured MBCs was normal. Therefore, the non-sustainable GCs may explain the reduced output of high affinity MBCs. Meanwhile, the low affinity MBCs accumulating in BAFFR-deficient but not BAFF-deficient recipient mice strongly implicated that BAFF mediated the production and/or survival of GC-independent, early MBCs. Thus, the deletion of BAFF receptor (BAFFR) on responding B cells will be examined to dissect the role of BAFF:BAFFR signalling in the control and maintenance of GC- dependent vs. GC-independent MBC responses in the next Chapter.

110 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Chapter 5

The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.1 Preamble

In the previous chapter it was demonstrated that the impacts of BAFF- deficiency or BAFFR-deficiency extrinsic to the responding B cells impair the prolongation of GC B cell responses. In addition, donor-derived GC- independent IgG1+ MBCs were found to accumulate in BAFFR-deficient but not in BAFF-deficient mice. To clarify the B cell-intrinsic function of BAFF:BAFFR signalling in controlling T-dependent B cell responses, SWHEL mice were crossed onto a BAFFR-deficient mice background and the responses of these B cells were assessed in WT recipient mice. In this way, this chapter aims to dissect the role of BAFFR in regulating the GC reaction and B cell memory.

5.2 Strategy to examine B cell-intrinsic function of BAFF:BAFFR signalling

To elucidate the B cell-intrinsic function of BAFF:BAFFR signalling in regulating GC and MBC responses, SWHEL donor B cells from WT (SWHEL) and -/- + BAFFR-deficient (SWHEL.Tnfrsf13c ) mice (CD45.1 ) were adoptively transferred at an equivalent cell number (3 x 104) into WT congenic recipients (CD45.2+) and challenged with HEL3X-SRBC i.v. (Figure 5.1). As expected,

BAFFR-deficient SWHEL B cells were immature and showed impaired responsiveness in vivo compared to mature WT SWHEL B cells. This was -/- evident when the kinetics of SWHEL and SWHEL.Tnfrsf13c B cell expansion in response to a primary immune challenge were examined on day 3, 4 and 5 (Figure 5.1).

111 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Donor Recipients

i.v

3 x 104 SWHEL HEL-binding B cells (Wild type) 2 x 108 Wild type HEL3X-SRBC

-/- SWHEL.Tnfrsf13c (Baffr

WT or Baffr HEL-binding B cell + Day 0 Day 3 Day 4 Day 5 HEL3X-SRBC i.v

Harvest Harvest Harvest Wild type Recipient

Figure 5.1. Adoptive transfer strategy to elucidate B cell-intrinsic functions of BAFFR in T-dependent responses.

-/- + SWHEL and SWHEL.Tnfrsf13c B cells (CD45.1 ) were adoptively transferred into wild type recipients (CD45.2+) and challenged with HEL3X-SRBC to investigate the role of BAFFR expression on the responding B cell. Recipient spleens were harvested on day 3, 4 and 5 consecutively to examine the early kinetics of B cell activation and differentiation in the presence and absence of cell-intrinsic BAFFR expression.

112 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.3 Immature BAFFR-deficient B cells show poor expansion but normal differentiation in response to T-dependent antigens

-/- SWHEL and SWHEL.Tnfrsf13c donor-derived B cells were identified with CD45.1+ and visualised by saturated HEL-staining (200ng/mL) as described in Chapter 2.4. Early PBs and early GC B cells were identified based on surface expression for B220, CD38, Fas and BCR.

-/- Enumeration revealed SWHEL.Tnfrsf13c donors cells were consistently

10-fold less frequent than SWHEL responses across all time points, despite equivalent numbers of HEL-binding B cells being transferred in each case

(Figure 5.2A, B). On day 3, SWHEL B cell blasts expanded substantially in -/- the spleen but SWHEL.Tnfrsf13c responders were undetectable (Figure 5.2A). lo lo lo hi On day 4, early PBs (B220 , Fas , CD38 ) and early GC B cells (B220 , hi lo Fas , CD38 ) began to differentiate and increase in the SWHEL donor response (Figure 5.2C). However, only very small numbers of expanded -/- SWHEL.Tnfrsf13c B cell blasts were identifiable at this point (Figure 5.2C). It -/- was only by day 5 that SWHEL.Tnfrsf13c early GC B cells and early PBs were readily detectable by flow cytometry (Figure 5.2C). This indicated that these -/- immature, BAFFR-deficient SWHEL.Tnfrsf13c precursors were disadvantaged in mounting comparable responses to their mature SWHEL counterparts. Nevertheless, day 5 flow cytometric analysis revealed similar frequencies of -/- early GC and early PB differentiation between SWHEL and SWHEL.Tnfrsf13c B cells, albeit the latter being at significantly lower numbers (Figure 5.2.C). By way of validation, histogram overlay of surface B220, BCR and BAFFR levels on early GC, early PBs and activated B cell blasts from day 5 responses further -/- confirmed the absence of surface BAFFR expression on SWHEL.Tnfrsf13c B cells, whilst SWHEL B cell blasts and early GC B cells retained BAFFR expression and early PBs did not express BAFFR (Figure 5.2C, D).

113 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Donor B cells

SWHEL. SWHEL. A C SWHEL SWHEL Tnfrsf13c-/- Tnfrsf13c-/- 87 23

CD38 Day 3 1 1

HEL Binding HEL 1 50

51 18

Day 4 24 9

5 46

8 5

75 79 Day 5

8 6 B220 Fas

Early Plasmablasts GC B cells Early B cell blasts B D Total HEL-binding cells Day 5 104

s * e SWHEL 103 * cyt * 102 spleno

6 0 101 Relative Cell no. 100 SWHEL. -/- Tnfrsf13c Cells per 1 <0.1 3 4 5 Days post Immunisation

Donor B220 HEL BAFF-R SWHEL Binding -/- SWHEL.Tnfrsf13c

114 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Figure 5.2. Poor clonal expansion but normal differentiation of activated BAFFR-deficient B cells.

-/- SWHEL and SWHEL.Tnfrsf13c B cells adoptively transferred into WT recipients were challenged with HEL3X-SRBC and early B cell responses in the spleen were examined on day 3, 4 and 5 as depicted in Figure 5.1. (A) Flow cytometric plots showing early expansion of activated HEL-binding (200ng/mL) SWHEL B cells (CD45.1+, CD45.2-) during days 3, 4 and 5. (B) Enumeration of total donor- derived HEL-binding B cells shown in (A). (C) Flow cytometric analysis showing differentiation of activated B cell blasts (CD38hi, Fasint) into early PBs (CD38lo, Faslo) and early GC (Fashi, CD38lo) overtime. (D) Histogram overlay showing B220, HEL-binding (BCR expression) and BAFFR levels for early PBs, early GC B cells and activated B cell blasts depicted in (C). Numbers represent percentages (%) in associated windows. Data are representative of 4 mice. Results were analysed using unpaired Student t-test with Welch’s correction post-hoc. Data were considered not significant unless specified, *P<0.05.

115 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.4 Strategy to examine the impact of B cell maturity in BAFFR- deficient B cell responses

-/- Early kinetic studies of immature SWHEL.Tnfrsf13c B cell responses demonstrated severely reduced activated B cell precursor frequencies that resulted in smaller responses compared to SWHEL donor responses (Figure 5.2). Whilst this may have been due to the lack of BAFFR expression per se, it may have also been simply due to the relative immaturity of the -/- SWHEL.Tnfrsf13c versus the WT SWHEL B cells. To test this, immature SWHEL

B cells that express BAFFR were generated by crossing SWHEL mice onto a BAFF-deficient (Tnfsf13b-/-) background (Figure 5.3A). The normal composition hi, of WT SWHEL donor HEL-binding B cells consisted of mature Fo (CD23 , CD21/35lo), MZ (CD23int,, CD21/35hi) and immature transitional (CD23lo, CD21/35lo, CD93+) B cell subsets (Figure 5.3B, C). On the other hand, HEL- -/- -/- binding B cells from SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c donor mice were highly immature (CD23lo, CD21/35lo, CD93+) (Figure 5.3B, C). Importantly, -/- -/- however, immature SWHEL.Tnfsf13b but not SWHEL.Tnfrsf13c B cells retained expression of BAFFR (Figure 5.3C).

-/- -/- Next, SWHEL, SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c B cells were adoptively transferred into WT recipients and challenged with HEL3X-SRBC (Figure 5.3A). To compensate for the poor initial responsiveness that seemed likely to be associated with immature donor B cells, increased numbers of -/- -/- 5 SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c donors cells (1.2 x 10 HEL-binding 4 cells) were transferred compared to SWHEL donor cells (3 x 10 HEL-binding cells) (Figure 5.3A). Recipients were boosted with HEL3X-SRBC on day 4 and analysed on day 10 and 21 using a similar gating strategy to that described in Chapter 3.3.

116 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

A Donor Recipients

3 x 104 HEL-binding B cells

SWHEL (Wild Type) i.v 1.2 x 105 HEL-binding B cells

-/- SWHEL.Tnfsf13b Wild type (Baff) 1.2 x 105 HEL-binding B cells

SWHEL.Tnfrsf13c-/- (Baffr)

WT or Baff or Baffr HEL-binding B cell i.v Day 0 Day 4 Day 10 Day 21 +

HEL3X-SRBC

3X HEL -SRBC Harvest Harvest Wild type Boost Recipient

B HEL-binding B cell phenotype Donor SWHEL. SWHEL. SWHEL Genotype Tnfsf13b-/- Tnfrsf13c-/- 58 0.1 7.3 CD23

29 9.8 99 0.6 91 0.5 CD21/35 C Donor SWHEL -/-

Cell no. SWHEL Tnfsf13b

Relative . SWHEL.Tnfrsf13c-/-

HEL BAFFR CD93 Binding

117 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Figure 5.3. Adoptive transfer strategy to investigate B cell-intrinsic BAFFR signalling and cell maturity in regulating T-dependent responses.

-/- -/- SWHEL donor mice were crossed onto the Tnfsf13b and Tnfrsf13c backgrounds to investigate B cell-intrinsic function of BAFF:BAFFR signalling in 4 -/- 5 T-dependent responses. (A) SWHEL (3 x 10 ), SWHEL.Tnfsf13b (1.2 x 10 ) and -/- 5 + SWHEL.Tnfrsf13c (1.2 x 10 ) HEL-binding B cells (CD45.1 ) were adoptively transferred into wild type congenic recipients (CD45.2+) and were challenged with HEL3X-SRBC. Recipients were antigen boosted again 4 days later and spleens were harvested on day 10 and 21. (B) Flow cytometric plots showing immature vs. mature phenotypes of HEL-binding B cells: Immature B cells (B220+, CD93+, CD23lo, CD21/35lo), Fo B cells (B220+, CD93-, CD23hi, CD21/35lo) and MZ B cells (B220+, CD93-, CD23lo, CD21/35hi). (D) Histogram overlay showing BAFFR and immature marker CD93 expression on SWHEL, -/- -/- SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c HEL-binding B cells.

118 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.5 Immature B cells form sustainable GC responses but memory B cells are significantly reduced in BAFFR-deficient responses

-/- -/- SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c donor B cells mounted comparable GC responses to those from control SWHEL B cells (Figure 5.4A). Surface IgG1 staining also revealed similar frequencies of IgG1 switching in donor-derived GC B cells (IgG1+, CD38lo) from all three groups (Figure 5.4A, B). However, despite the 4-fold greater number of donor B cells used, the GC -/- -/- responses generated from SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c B cell responses were typically lower than those produced from WT SWHEL B cells

(Figure 5.4A, C, D). Importantly, however, using immature donor SWHEL B cells -/- that did express BAFFR (i.e. those from SWHEL.Tnfsf13b mice) did not improve the GC response compared to those that lacked BAFFR expression (ie those -/- from SWHEL.Tnfrsf13c mice) (Figure 5.4A, C, D). Thus, in gross terms, lack of

BAFFR expression by donor SWHEL B cells has no detectable impact on the initiation and perpetuation of the GC response in this system.

Unlike the GC response, the numbers of donor-derived IgG1+ MBCs -/- were found to be significantly lower on day 10 of the SWHEL.Tnfrsf13c B cell -/- response compared to both the SWHEL and SWHEL.Tnfsf13b controls (Figure + -/- 5.4A, E). The low numbers of IgG1 MBCs in SWHEL.Tnfrsf13c response was even more striking on day 21, being 5-fold and 10-fold lower than the -/- SWHEL.Tnfsf13b and SWHEL donor responses, respectively (Figure 5.4E). In contrast to the slightly reduced GC B cell responses, this reduced MBC -/- production from SWHEL.Tnfrsf13c B cells could not be ascribed to the immature phenotype of the donor B cell population, but instead is likely to reflect the absence of cell-intrinsic BAFF:BAFFR signalling over the course of the response. Thus, these results suggested that BAFFR expression on responding B cells is required for the normal production and/or maintenance of MBCs in this system (Figure 5.4A, E).

119 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

A Donor B cells

SWHEL. SWHEL. Donor: SWHEL Tnfsf13b-/- Tnfrsf13c-/-

0.3 0.6 0.2 CD38 Day 10

45 43 48 0.3 0.3 0.09

Day 21

57 66 51 IgG1

B C Total Donor GC Total Donor GC 100 106

s Donor 5 80 10 ** * *** SWHEL * 104 * SWHEL.Tnfsf13b-/- + 60 1 HEL -/- splenocyte SW .Tnfrsf13c 6 103 0 40 % IgG 102

20 101 Cells per 1 0 100 10 21 10 21 Days post Immunisation Days post Immunisation

D E Donor IgG1+ GC Donor IgG1+ Memory 105 103 s s

104 ** 102 * ** **** ** 103 1 splenocyte splenocyte 10 6 6 0 0 102 100 101 Cells per 1 Cells per 1 <0.1 100 10 21 10 21 Days post Immunisation Days post Immunisation

120 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Figure 5.4. Loss of BAFFR signalling but not cell immaturity impairs + BAFFR-deficient IgG1 MBC survival.

-/- -/- Donor-derived SWHEL, SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c B cells (CD45.1+, CD45.2-, B220+) challenged with HEL3X-SRBC in wild type recipients were analysed on day 10 and 21 as depicted in Figure 5.3. (A) Flow cytometric analysis showing IgG1+ GC B cells (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi) on day 10 and 21 post challenge. (B) Frequency of IgG1-switching in total donor-derived GC. (C) Enumeration of total donor- derived GC B cells, (D) IgG1+ GC B cells and (E) IgG1+ MBCs on days 10 and 21. Numbers on plots indicate percentages (%) of associated windows. Flow plots are representative of 5 mice from one experiment. Enumerated plots are pooled from 2 independent replicate experiments with 5 mice each. Data were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

121 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.6 Delayed affinity maturation but normal selection in immature BAFF-deficient and BAFFR-deficient GC B cell responses

To further examine whether BAFF:BAFFR signalling had a role in regulating GC B cell and antibody selection, the affinity for the antigen was + -/- assessed on IgG1 donor populations from SWHEL, SWHEL.Tnfsf13b and -/- 3X SWHEL.Tnfrsf13c responses to HEL -SRBC challenge. Binding to limited concentrations of HEL3X was used to determine to high and low affinity compartments using the gating strategy described in Chapter 3.3.

Day 10 analysis of high vs. low affinity IgG1+ GC compartments showed -/- significantly greater frequencies of low affinity B cells in both SWHEL.Tnfsf13b -/- (35%) and SWHEL.Tnfrsf13c (38%) responses compared to WT SWHEL control (18%) (Figure 5.5A, B). These observations suggested immature B cells from -/- -/- SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c donors were slower to undergo SHM and to affinity mature compared to mature SWHEL B cells. However, later analysis on day 21 revealed comparable frequencies of low affinity GC B cell -/- compartments between all donor responses: SWHEL (4%), SWHEL.Tnfsf13b -/- (6%) and SWHEL.Tnfrsf13c (3.5%) (Figure 5.5A, C). Thus, no obvious defect -/- in affinity maturation was seen in SWHEL.Tnfrsf13c GC responses compared to -/- SWHEL.Tnfsf13b and SWHEL donor GCs, indicating that B cell-intrinsic BAFFR- deficiency did not impact significantly upon the affinity-based selection in the GC (Figure 5.5A, C).

122 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

A Donor IgG1+ GC

SWHEL. SWHEL. Donor: HEL SW Tnfsf13b-/- Tnfrsf13c-/-

88 77 80 3X HEL Binding Day 10

11 21 19

95 93 94

Day 21

4.1 6.6 4.8 IgG1

B DAY 10 C DAY 21 100 0 100 0 ** ** 80 20 80 20 % Low % Low y y 60 40 60 40 finit finit f f

A A A A

f f finit finit 40 60 40 60 y y % High % High 20 80 20 80

0 . . 100 0 . . 100 -/- -/- -/- -/- HEL HEL HEL HEL HEL HEL SW SW SW SW SW SW

Tnfsf13b Tnfrsf13c Tnfsf13b Tnfrsf13c Affinity Compartment Low Affinity High Affinity

123 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Figure 5.5. Cell immaturity delays affinity maturation but has no impact on affinity-based selection in BAFF or BAFFR deficient GC responses.

-/- -/- + Donor-derived SWHEL, SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c IgG1 GC B cells identified in Figure 5.4 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric analysis showing high vs. low affinity IgG1+ GC compartments on day 10 and 21. (B) Proportion of high vs. low affinity GC B cells on day 10 and (C) day 21. Flow cytometric plots showing 5 concatenated mice from one experiment. Non-responders were excluded from the analysis and enumerated data were pooled from 2 independent replicate experiments with 5 mice each. Enumerated data were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, **P<0.01.

124 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.7 Low affinity but not high affinity memory B cells are depleted in BAFFR-deficient B cell responses

+ -/- The specific depletion of donor IgG1 MBCs in SWHEL.Tnfrsf13c responses pointed to the potential role of BAFF:BAFFR signalling in producing + and/or maintaining MBCs. Donor-derived IgG1 MBCs in SWHEL responses were predominantly low affinity (95%) on day 10. Similar frequencies of low -/- -/- affinity cells were also seen in SWHEL.Tnfsf13b (98%) and SWHEL.Tnfrsf13c (99%) donor responses (Figure 5.6A, B). By day 21, high affinity IgG1+ MBCs -/- -/- were readily detectable in SWHEL, SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c donor responses (Figure 5.6A, C). Although fewer MBCs were found in -/- + SWHEL.Tnfsf13b responses, low affinity IgG1 MBCs showed comparable -/- frequencies between SWHEL (23%) and SWHEL.Tnfsf13b (28%) donor responses (Figure 5.6A, C). However, examination of donor-derived IgG1+ -/- MBCs from SWHEL.Tnfrsf13c responses revealed a complete absence of low affinity but not high affinity MBCs compared to the responses mounted by both -/- SWHEL and immature SWHEL.Tnfsf13b donor B cells (Figure 5.6A, C). Taken together, these findings suggested low affinity MBC survival may be dependent on cell-intrinsic BAFF:BAFFR signalling.

125 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

A Donor IgG1+ Memory

SWHEL. SWHEL. Donor: SWHEL Tnfsf13b-/- Tnfrsf13c-/-

5 1 0 3X HEL Binding Day 10

95 98 100

77 71 100

Day 21

23 28 0 IgG1

B C DAY 10 DAY 21 100 0 100 0 **

80 20 80 20 % Low % Low y y 60 40

finit 60 40 finit f f

A A A A

f f finit finit 40 60 40 60 y y % High % High 20 80 20 80

0 100 0 100 -/- -/- -/- -/- HEL HEL HEL HEL HEL HEL SW SW SW SW SW SW

Tnfsf13b Tnfrsf13c Tnfsf13b Tnfrsf13c

Affinity Compartment Low Affinity

High Affinity . .

126 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Figure 5.6. BAFFR-deficient low affinity MBCs fail to persist but high affinity MBCs remain intact during T-dependent responses.

-/- -/- + Donor-derived SWHEL, SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c IgG1 MBCs identified in Figure 5.4 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric profiles showing high vs. low affinity IgG1+ MBCs on day 10 and 21. (B) Proportion of high vs. low affinity MBCs on day 10 and (C) day 21. Flow cytometric plots showing 4 concatenated mice from one experiment. Non-responders were excluded from the analysis and enumerate data were pooled from 2 independent replicate experiments with 5 mice each. Enumerated data pooled from replicate experiments were analysed using One- way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, **P<0.01.

127 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.8 Strategy to examine the cell-intrinsic function(s) of BAFFR signalling using immature wild type versus BAFFR-deficient B cells

-/- The absence of low affinity MBCs in SWHEL.Tnfrsf13c responses suggested BAFF:BAFFR signalling was important for the maintenance of low affinity MBC survival. However, to ensure our findings were not due to -/- differences in initial maturation status between SWHEL, SWHEL.Tnfsf13b and -/- SWHEL.Tnfrsf13c donor cells, immature B cells from SWHEL and -/- SWHEL.Tnfrsf13c donor mice were isolated using negative selection as described in Chapter 2.3.

Mature B cell markers CD23 and CD35 were used to deplete all CD23+, + -/- CD35 mature B cells from spleens of SWHEL and SWHEL.Tnfrsf13c donor mice

(Figure 5.7A). Greater than 99% purity was achieved for both SWHEL and -/- SWHEL.Tnfrsf13c donor splenocytes (Figure 5.7B, C). Immature B cells purified from SWHEL donor showed intact BAFFR expression compared to -/- SWHEL.Tnfrsf13c donor (Figure 5.7D). Purified immature donor cells were then adoptively transferred into WT recipient mice and were challenged with HEL3X- SRBC for flow cytometry and SHM analysis at subsequent time points (Figure 5.7A).

128 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

A Donor Recipient

i.v

5 SWHEL 1.2 x 10 Mature B cells HEL-binding cells Depletion (anti-CD23 & anti-CD35) 2 x 108 HEL3X-SRBC Wild type

SWHEL.Tnfrsf13c-/-

B C Before depletion Post depletion

Donor SWHEL. SWHEL. SWHEL SWHEL Genotype Tnfrsf13c-/- Tnfrsf13c-/- HEL Binding

B220

64 2.9 0.9 1.5 CD23

11 11 80 0 86 0.2 83 0 CD21/35

D HEL-binding B cells Donor

SWHEL (before depletion) Cell no. Relative SWHEL (post depletion) -/- SWHEL.Tnfrsf13c (before depletion) -/- SWHEL.Tnfrsf13c (post depletion)

BAFFR

129 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Figure 5.7. Adoptive transfer strategy to compare immature B cell responses using wild type and BAFFR-deficient mice.

-/- + Immature B cells from SWHEL and SWHEL.Tnfrsf13c donor mice (CD45.1 ) were enriched to investigate immature BAFFR-deficient B cell responses using -/- immature WT B cells as control. (A) SWHEL and SWHEL.Tnfrsf13c splenic B cells were negatively selected using anti-CD23 and anti-CD35 antibodies. Purified immature HEL-binding B cells (1.2 x 105) were adoptively transferred into WT congenic recipients (CD45.2+) and challenged with HEL3X-SRBC. (B) Flow cytometric analysis showing purity of depletion for mature (CD23+, CD21/35+) HEL-binding B cells before depletion and (C) post depletion (CD23+, CD21/35-). (D) Histogram overlay confirming intact BAFFR expression on purified immature SWHEL B cells before (black) and after (grey) depletion -/- compared to SWHEL.Tnfrsf13c B cells before (red) and after (orange) depletion.

130 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.9 BAFF:BAFFR signalling is dispensable for GC maintenance but is critical for survival of memory B cells

+ - - Immature B cells (CD45.1 , CD23 , CD21/35 ) purified from SWHEL and -/- SWHEL.Tnfsf13c splenocytes were i.v. injected into congenic WT recipients (CD45.2+) and simultaneously immunised with HEL3X-SRBC. Recipients were boosted 4 days post challenge and splenic responses were analysed on day 12 and 21 to further dissect role of BAFF:BAFFR signalling in regulating GC and MBC responses (Figure 5.8A).

-/- 3X Immature SWHEL and SWHEL. Tnfrsf13c B cell responses to HEL - SRBC immunisation were identified using the staining approach as previously described in Chapter 3.3. Consistent with our previous observations, no significant differences in the donor-derived GC responses were observed -/- between SWHEL and SWHEL.Tnfrsf13 responses (Figure 5.8B, C). Day 12 analysis showed similar frequencies of donor-derived GC B cells (B220+, lo -/- CD38 ) in SWHEL and SWHEL.Tnfrsf13c responses (Figure 5.8B). The frequencies of IgG1 switching in donor-derived GC (B220hi, IgG1+, CD38lo) were also comparable between both groups across all time points (Figure 5.8B). In -/- addition, immature SWHEL and SWHEL.Tnfrsf13c responses showed similar GC and IgG1+ GC B cell numbers on both day 12 and day 21 (Figure 5.8C, D). These findings confirmed that cell-intrinsic BAFFR-deficiency had no impact on GC B cell survival, in agreement with work presented earlier in this chapter (ref. Chapter 5.5). However, donor-derived IgG1+ MBCs responses were significantly -/- diminished in SWHEL.Tnfrsf13c donor responses (Figure 5.8B, E). On both day 12 and day 21, a 3-fold reduction in IgG1+ MBCs numbers was consistently -/- observed for SWHEL.Tnfrsf13c compared to immature SWHEL controls (Figure 5.8B, E). These observations again suggested that normal IgG1+ MBC production and/or survival was BAFFR-dependent.

131 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

A Immature WT or Baffr HEL-binding B cell + Day 0 Day 4 Day 12 Day 21 3X i.v HEL -SRBC

3X HEL -SRBC Harvest Harvest Wild type Boost Recipient

B Donor B cells SWHEL. Donor: SWHEL Tnfrsf13c-/-

0.4 0.09 CD38 Day 12

76 74

1.3 0.3

Day 21

73 75 IgG1

C D + E + Total Donor GC Donor IgG1 GC Donor IgG1 Memory 104 104 103 s s s

2 103 103 10 ** 1 splenocyte splenocyte splenocyte 10 * 6

6 2 6 102 10 0 0 0

100 101 101 Cells per 1 Cells per 1 Cells per 1 <0.1 100 100 12 21 12 21 12 21 Days post Immunisation Days post Immunisation Days post Immunisation

Donor

SWHEL -/- SWHEL.Tnfrsf13c

132 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Figure 5.8. BAFFR-deficient B cells form normal GC responses but diminished MBC responses compared to immature wild type B cells.

-/- - - SWHEL and SWHEL.Tnfrsf13c immature B cells (CD23 , CD21/35 ) were enriched and purified by negative selection using anti-CD23 and anti-CD35 as -/- + described in Figure 5.7. (A) SWHEL and SWHEL.Tnfrsf13c B cells (CD45.1 ) were adoptively transferred into wild type recipients (CD45.2+) and challenged with HEL3X-SRBC. Recipients were boosted with antigen again 4 days later and spleens were harvested on day 12 and 21 to examine donor-derived responses (CD45.1+, CD45.2-). (B) Flow cytometric analysis showing donor-derived IgG1+ GC B cells (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi) on day 12 and 21 post immunisation. (C) Enumeration of total donor-derived GC B cells, (D) IgG1+ GC B cells and (E) IgG1+ MBCs on day 12 and 21 post challenge. Numbers on plots represent percentages (%) of associated window. Flow plots showing 5 concatenated mice from one experiment. Data are representative of 2 independent replicate experiments. Data were analysed using unpaired Student t-test with Welch’s correction post-hoc. Data were considered not significant unless specified, *P<0.05, **P<0.01.

133 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.10 Persistence of low affinity memory B cells is dependent on BAFFR signalling but independent of cell maturity

To further investigate the impact of immaturity and BAFFR-deficiency on the selection of GC and MBCs, affinity analysis was performed on donor- derived IgG1+ populations using HEL3X-staining as described in Chapter 2.4. Consistent with our previous observations, IgG1+ GC B cells derived from -/- immature SWHEL.Tnfrsf13c donor underwent comparable affinity maturation 3X towards HEL -binding similar to those derived from immature SWHEL B cells

(Figure 5.9A). On day 12, the frequencies of high affinity GC B cells in SWHEL -/- (90%) and SWHEL.Tnfrsf13c (92%) donor responses were comparable (Figure 5.9A, B). Day 21 analysis also revealed similar frequencies of high affinity IgG1+

GC B cells in responses derived from immature SWHEL (92%) and -/- SWHEL.Tnfrsf13c (94%) donor B cells (Figure 5.9A, C). These findings indicated that upon controlling for donor B cell maturity, SWHEL and -/- SWHEL.Tnfrsf13c GC B cells can undergo SHM and affinity mature with similar kinetics.

Consistent with data presented earlier in this chapter (ref. Chapter 5.7), day 12 analysis again revealed significantly reduced frequencies of low affinity + -/- IgG1 MBCs in responses derived from immature SWHEL.Tnfrsf13c (15%) donor versus SWHEL (38%) donor B cells (Figure 5.9D, E). Even more striking + was, the complete absence of low affinity but not high affinity IgG1 MBCs -/- observed in the SWHEL.Tnfrsf13c B cell responses on day 21 compared to the small but significant presence of low affinity MBCs in SWHEL B cell responses (15%) (Figure 5.9D, F). As with the previous experiments using undepleted donor B cell populations, these data indicate that there is a cell-intrinsic requirement for BAFF:BAFFR signalling in the persistence of low affinity IgG1+ MBCs.

134 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Affinity Compartment Low Affinity High Affinity Donor IgG1+ GC

A B 100 DAY 12 0 Donor IgG1+ GC

SWHEL. 80 20 % Low Donor: SWHEL -/- y Tnfrsf13c 60 40 finit

f A A f

90 92 finit

3X 40 60 y % High

HEL 20 80 Binding Day 12 0 100 SWHEL SWHEL. Tnfrsf13c-/- 9.7 7.4 C 100 DAY 21 0 92 94

80 20 % Low y 60 40 Day 21 finit

f A A f

finit 40 60 y

6.2 % High 4.9 20 80 IgG1 0 100 SWHEL SWHEL. Tnfrsf13c-/- Donor IgG1+ Memory

D + E DAY 12 Donor IgG1 Memory 100 0 *

SWHEL. 80 20 % Low Donor: SWHEL -/- Tnfrsf13c y 60 40 finit

f A A f

64 79 finit 3X 40 60 y HEL % High Binding Day 12 20 80

0 100 SWHEL SWHEL. Tnfrsf13c-/- 34 15 F DAY 21 91 100 100 0 *

80 20 % Low y Day 21 60 40 finit

f A A f

finit 40 60 7.3 0 y % High IgG1 20 80 0 100 SWHEL SWHEL. Tnfrsf13c-/-

135 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Figure 5.9. Normal GC affinity maturation but an absence of low affinity MBCs in BAFFR-deficient donor responses.

-/- + Donor-derived immature SWHEL and SWHEL.Tnfrsf13c IgG1 GC B cells and IgG1+ MBCs identified in Figure 5.8 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric plots showing high and low affinity -/- + compartment of SWHEL and SWHEL.Tnfrsf13c IgG1 GC B cells on day 12 and 21. (B) Proportion of high vs. low affinity IgG1+ GC B cells on day 12 and (C) day 21. (D) Flow cytometric plots showing donor-derived high and low affinity compartments of IgG1+ MBCs on day 12 and 21. (E) Proportion of high vs. low affinity IgG1+ MBCs on day 12 and (F) day 21. Numbers on plots represent percentages (%) of associated window. Flow plots showing 5 concatenated mice from one experiment. Data are representative of 2 independent replicate experiments. Data were analysed using unpaired Student t-test with Welch’s correction post-hoc. Data were considered not significant unless specified *P<0.05.

136 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.11 SHM analysis indicates normal affinity-based selection in BAFFR-deficient GC B cell responses

To further interrogate the cell-intrinsic requirements for BAFFR in the GC response, SHM analysis was undertaken. Single IgG1+ GC B cells derived from -/- 3X immature SWHEL and SWHEL.Tnfrsf13c donor B cell responses to HEL -SRBC were FACS sorted on day 12 and 21 and the Ig heavy chain VH10 gene from sorted single SWHEL B cells was sequenced using the method described in

Chapter 2.5.

Out of all donor-derived IgG1+ GC B cell clones sequenced from day 12, a great proportion had acquired the high affinity Y53D+ substitution in both -/- SWHEL (97%) and SWHEL.Tnfrsf13c (89%) responses (Figure 5.10A). The -/- average rate of SHM between SWHEL and SWHEL.Tnfrsf13c GC B cells were identical (Figure 5.10A). The proportions of high affinity Y53D+ GC clones that had acquired further affinity-enhancing mutations (Y58F and/or S31R) were low -/- and indistinguishable between SWHEL (5%) and SWHEL.Tnfrsf13c (4%) donors responses at this time point (Figure 5.10A). On day 21, SHM patterns of SWHEL -/- + and SWHEL.Tnfrsf13c IgG1 GC B cells were again similar (Figure 5.10B). + Comparable frequencies of Y53D high affinity cells were observed in SWHEL -/- (96%) and SWHEL.Tnfrsf13c (92%) (Figure 5.10B). At this time point, the + frequencies of Y53D cells that had also acquired Y58F or S31R were also -/- similar in SWHEL control (41%) and SWHEL.Tnfrsf13c (44%) responses (Figure 5.10B).

To confirm the efficiency of positive selection for these high affinity antibodies in the GC, a serum ELISA was performed as described in Chapter 2.6. Measurement of serum anti-HEL3X IgG1 revealed no significant differences in the IgG1 antibody titers derived from the immature SWHEL versus -/- SWHEL.Tnfrsf13c responses on days 12 or 21 (Figure 5.11). This was consistent with the sequencing data revealing the positive selection of high affinity GC fractions in both donor groups (Figure 5.10). These data collectively revealed no evidence of cell-intrinsic BAFFR-deficiency impacting on the

137 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses affinity-based selection of GC B cells or the production of high affinity antibodies that confers long-lived humoral immunity.

138 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Donor IgG1+ GC

SWHEL. Donor: SWHEL Tnfrsf13c-/- A 5% 4%

n = 68 n = 75 SHM = 3.0 SHM = 3.1 Day 12

92% 85%

B

55% 48% n = 95 n = 78 SHM = 4.0 SHM = 4.5 Day 21 41% 44%

HyHEL10 heavy chain region Amino Acid substitution Unmutated Y53D (high affinity) Y53D + affinity mutations Other mutated

Figure 5.10. Single cell SHM analysis reveals normal selection for high affinity somatic mutations in BAFFR-deficient GC B cells.

-/- + Donor-derived immature SWHEL and SWHEL.Tnfrsf13c IgG1 GC B cells identified in Figure 5.8 were single cell sorted and sequenced for SHM analysis on day 12 and 21. (A) Data showing percentages of single cell sorted high -/- + affinity vs. low affinity clones constituting SWHEL and SWHEL.Tnfrsf13c IgG1 GC compartment on day 12 and (B) day 21. Unmutated clones are indicated in red, other mutated clones in yellow, high affinity clones with Y53D substitution in dark blue, and high affinity Y53D+ clones with additional affinity-enhancing mutations (Y58F+ and/or S31R+) are represented in light blue. n = number of clones analysed and SHM = average number of mutations per clone. Data were pooled from 5 mice.

139 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

A Day 12 anti-HEL 3X IgG1 B Day 21 anti-HEL3X IgG1 4.0 4.0

3.0 3.0 e

2.0 2.0 orbanc s b A 1.0 1.0

0.0 0.0 101 102 103 104 105 101 102 103 104 105 1 / serum dilution 1 / serum dilution C Day 12 anti-HEL4X IgG1 D Day 21 anti-HEL4X IgG1 4.0 4.0

3.0 3.0 e

2.0 2.0 orbanc s b A 1.0 1.0

0.0 0.0 101 102 103 104 105 101 102 103 104 105 1 / serum dilution 1 / serum dilution Donor Response

SWHEL -/- Tnfrsf13c .SWHEL Unimmunised Wild type

Figure 5.11. BAFFR-deficient B cells produce high affinity antibody responses comparable to immature wild type B cells.

-/- Immature SWHEL and SWHEL.Tnfrsf13c B cells adoptively transferred in wild type recipients and challenged with HEL3X-SRBC and antigen boosted as depicted in Figure 5.8. Blood was harvested on day 12 and 21. (A) HEL3X- binding IgG1 serum antibodies from SWHEL responses (black) and -/- SWHEL.Tnfrsf13c responses (red) were measured by ELISA and demonstrated comparable high affinity antibody titres on day 12 and (B) day 21. (C) HEL4X- binding serum IgG1 ELISA was used to further assess the presence of antibodies from high affinity clones that had acquired affinity-increasing -/- mutations in SWHEL (black) and SWHEL.Tnfrsf13c (red) responses on day 12 and (D) day 21. Serum from unimmunised wild type mice (grey) was used as negative control. ELISA data here are representative 5 individual mice from one experiment and are representative of 2 independent replicate experiments.

140 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.12 SHM analysis reveals the absence of unmutated early memory B cells in BAFFR-deficient responses

SHM analysis was also performed on single IgG1+ MBC derived from -/- responses of immature SWHEL and SWHEL.Tnfrsf13c donor B cells (Figure 5.12). Day 12 SHM analysis revealed that 32% of IgG1+ MBCs were unmutated -/- in SWHEL responses compared to only 9% in SWHEL.Tnfrsf13c responses (Figure 5.12A). Of the somatically mutated IgG1+ MBCs present in each case, around 50% carried the high-affinity Y53D substitution (Figure 5.12A). Analysis of single IgG1+ MBCs on day 21 revealed that the somatically mutated MBCs -/- derived from SWHEL and SWHEL.Tnfrsf13c responses possessed very similar mutation profiles. Thus, among these GC-derived MBCs, the proportions of + + + + Y53D clones, Y53D plus Y58F and/or S31R clones and clones carrying other somatic mutations were indistinguishable (Figure 5.12B).

These together confirmed that selection of GC B cells into the long-lived MBC pool was not affected by the lack of BAFFR expression. In contrast, -/- unmutated MBCs were found to be completely absent from SWHEL.Tnfrsf13c donor B cell responses by day 21 whilst still comprising 15% of the MBCs derived from SWHEL B cells (Figure 5.12B). These findings were consistent with the flow cytometric data described earlier demonstrating low affinity IgG1+

MBCs to be present in the WT SWHEL B cell responses but were essentially -/- absent from the SWHEL.Tnfrsf13c donor B cell responses (Figure 5.9D, F). Collectively, these data indicated that somatically mutated, GC-derived MBCs are not impacted upon by the absence of BAFFR, but unmutated, GC- independent early MBCs required cell-intrinsic BAFF:BAFFR signalling to persist.

141 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

Donor IgG1+ Memory

SWHEL. Donor: SWHEL Tnfrsf13c-/- *** A 9% 32% 36% n = 31 47% n = 45 SHM = 1.1 SHM = 1.9 Day 12 42% 32% 2%

B 17% 15% 23%

n = 27 n = 22 14% SHM = 2.4 SHM = 2.9 Day 21 59% 18% 54%

** HyHEL10 heavy chain region Amino Acid substitution Unmutated Y53D (high affinity) Y53D + affinity mutations Other mutated

Figure 5.12. Single cell SHM analysis shows unmutated, early MBCs are absent in BAFFR-deficient donor responses.

-/- - - Purified immature SWHEL and SWHEL.Tnfrsf13c B cells (CD23 , CD21/35 ) were challenged with HEL3X-SRBC in wild type recipients as depicted in Figure 5.8. Recipient splenocytes were single cell sorted on day 12 and 21 for SHM analysis. (A) Data showing percentages of single cell sorted high affinity -/- + vs. low affinity clones constituting SWHEL and SWHEL.Tnfrsf13c IgG1 memory compartment on day 12 and (B) day 21. Unmutated clones are indicated in red, other mutated clones in yellow, high affinity clones with Y53D substitution in dark blue, and high affinity Y53D+ clones with additional affinity- enhancing mutations (Y58F+ and/or S31R+) are represented in light blue. n = number of clones analysed and SHM = average number of mutations per clone. Data are pooled from 5 mice. Significance was determined by Chi-Sq contingency test, **P<0.01, ***P<0.001.

142 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

5.13 Discussion

The aim of this chapter was to elucidate the precise role of B cell-intrinsic BAFF:BAFFR signalling in regulating GC and MBC responses to T-dependent antigen. Earlier work discussed in Chapter 4 demonstrated that the BAFF appeared to prolong the survival of unmutated IgG1+ MBCs. These findings pointed to the dependence of early MBCs for BAFF that were distinct from GC- derived MBCs (253). As there had been no definitive factors identified to be essential for MBC maintenance and survival, our observations shed important insights on how the longevity of MBC subsets were differentially maintained (155). To further investigate the B cell-intrinsic function of BAFF:BAFFR signalling in controlling T-dependent responses, the work presented here utilised the SWHEL adoptive transfer approach after crossing them onto a -/- BAFFR-deficient background (SWHEL.Tnfsf13b ) such that SWHEL responding B cells no longer expressed BAFFR.

BAFF:BAFFR signalling is critical for the maturation of peripheral B cells as the deficiency in BAFF or BAFFR expression both resulted in the termination of B cell development at the transitional T2 stage (39, 93). Transitional immature B cells in secondary lymphoid tissue had been shown to be highly sensitive to BCR-induced cell death compared to mature follicular B cells, as a safety feature in regulating the deletion of immature B cells that exhibited cross- reactivity to self outside the bone marrow niche (329-332). In light of this, the potential impact of B cell immaturity in establishing early T-dependent -/- responses from immature SWHEL.Tnfrsf13c donor cells was first compared -/- with mature SWHEL B cells (Figure 5.1). SWHEL.Tnfrsf13c B cell differentiation into early PBs and early GC B cells were unaffected by loss of BAFFR signalling (Figure 5.2C). However, kinetic analysis across day 3, 4 and 5 -/- revealed severely delayed clonal expansion of SWHEL.Tnfrsf13c B cell blasts compared to SWHEL donor cells that consequently led to significantly smaller HEL-specific B cell responses (Figure 5.2A, B, C). These findings confirmed the -/- propensity of immature SWHEL.Tnfrsf13c B cells to die during early phase of activation compared to mature SWHEL B cells, hence the reduced numbers of

143 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses activated precursor B cells in BAFFR-deficient responses led to overall smaller GC and antibody responses compared to WT mice.

In order to uncouple the effects of B cell immaturity from the survival signals mediated by BAFFR activation, immature HEL-specific B cells were generated from BAFF-deficient mice by crossing SWHEL donor mice onto the BAFF-deficient background (Tnfsf13b-/-). This gave rise to BAFFR-expressing -/- immature HEL-binding B cells (SWHEL.Tnfsf13b ) that were deprived of BAFF:BAFFR signalling during their development similar to BAFFR-deficient -/- (SWHEL.Tnfrsf13c ) B cells (Figure 5.3B, C). In agreement with our earlier -/- -/- findings, immature SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c donor B cells generated slightly smaller day 10 GC responses than those from their mature

WT SWHEL counterparts even when the number of immature HEL-binding B cells transferred was 4-fold higher (Figure 5.4A, C). However, examination on day 21 revealed the size of GC responses were comparable across all the groups regardless of the maturity or BAFFR expression of the responding B cells (Figure 5.4A, C, D). These observations indicated that, while immature B cells established smaller GCs compared to mature B cells, the maintenance of GC B cell responses did not require B cell-intrinsic BAFFR expression. This was somewhat surprising, as survival signals delivered through BAFFR upon B cell activation had long been speculated to be crucial for GC B cell survival and maintenance (82, 97). Thus, the work presented here demonstrated that cell- intrinsic BAFF:BAFFR signalling on responding B cells is in fact dispensable for the progression of established GC B cell responses.

Since the survival signals delivered through BAFF:BAFFR signalling is required for the positive selection of immature B cells upon entering the peripheral B cell pool (36, 65), it had been speculated that BAFF:BAFFR signalling might also be involved in regulating the affinity-based selection during GC B cell responses (60, 75). To accurately examine the contribution of BAFFR signalling in GC selection, affinity analysis was performed on donor-derived + -/- -/- IgG1 GC B cells from immature SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c responses on days 10 and 21 (Figure 5.5). Interestingly, both immature -/- -/- SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c GC B cell responses showed slightly

144 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

delayed affinity maturation compared to mature SWHEL B cells during the early GC phase (day 10) (Figure 5.5A, B). This could be explained by the delayed activation and expansion of these immature B cells as discussed earlier (ref. Chapter 5.3). However, analysis on day 21 revealed similar proportions and -/- numbers of high affinity GC B cells in SWHEL.Tnfrsf13c responses compared to -/- its BAFFR-expressing mature (SWHEL) and immature (SWHEL.Tnfsf13b ) counterparts (Figure 5.5A, C). These findings collectively suggested that BAFFR was dispensable for the affinity maturation of GC B cells and the counter-selection of low affinity cells during affinity-based selection.

A striking revelation from this work was the reduced numbers of IgG1+ -/- MBCs generated from the responses of SWHEL.Tnfrsf13c compared to SWHEL -/- + and SWHEL.Tnfsf13b donor B cells, in which a 5 to 10-fold reduction in IgG1 -/- MBC numbers was observed in SWHEL.Tnfrsf13c responses 21 days post- + immunisation (Figure 5.4A, E). Notably, low affinity IgG1 MBCs were generated -/- -/- in both SWHEL.Tnfsf13b and SWHEL.Tnfrsf13c donor responses on day 10 (Figure 5.4E; 5.6A, B), suggesting that BAFF:BAFFR signalling was not required for the formation of these low affinity, class-switched MBCs. By day 21, however, these MBCs were specifically absent from the responses of -/- SWHEL.Tnfrsf13c donor B cells (Figure 5.6A, C). Importantly, however, the absence of BAFFR did not result in the loss of high affinity IgG1+ MBCs from -/- the responses of SWHEL.Tnfrsf13c donor B cells (Figure 5.6A, C). This result was consistent with previously published observation that affinity matured MBCs derived from the GC do not require BAFF-mediated survival signals (253, 298).

A potential caveat to the approach described above was the phenotypic -/- differences of immature SWHEL.Tnfrsf13c B cells versus immature -/- SWHEL.Tnfsf13b B cells that developed in the complete absence of BAFF (ref. Chapter 4.2). Whilst the ablation of CD23 (FcεRII) expression on B cells is a hallmark consequence in BAFF-deficiency (38), BAFFR-deficiency does allow the development of a small subset of CD23lo immature B cells (93). Another adoptive transfer approach was therefore developed in which purified immature

145 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses

- -/- (CD23 ) B cells from SWHEL and SWHEL.Tnfrsf13c donors mice were utilised (Figure 5.7).

-/- Purified immature B cells from SWHEL and SWHEL.Tnfrsf13c donors were adoptively transferred and challenged in WT recipient mice (Figure 5.7; 5.8A). In agreement with our previous observations, day 12 and day 21 flow -/- cytometric analyses of immature SWHEL and SWHEL.Tnfrsf13c responses again revealed no significant differences in GC establishment and maintenance (Figure 5.8B, C, D). This reinforced the redundant role of BAFF:BAFFR signalling in sustaining GC B cell survival. In addition, affinity analysis of IgG1+ -/- GC B cells derived from immature SWHEL and SWHEL.Tnfrsf13c donor B cells revealed comparable affinity maturation kinetics and similar frequencies of high and low affinity GC B cells on days 12 and 21 (Figure 5.9A, B, C). Single cell SHM analysis of donor-derived GC B cells reaffirmed that the absence of BAFFR had no impact on SHM or affinity maturation (Figure 5.10). This was further reflected in the similar titres of high affinity HEL-specific IgG1+ antibodies -/- detected in the serum of SWHEL and SWHEL.Tnfrsf13c responses (Figure 5.11). The data presented here provided definitive evidence that cell-intrinsic BAFF:BAFFR signalling does not play a significant role in affinity-based selection within the GC to mount long-lived antibody responses.

Although GC survival and selection did not require BAFF:BAFFR signalling, this work revealed the BAFFR-dependent nature of GC-independent, class-switched MBCs that does not apply to somatically mutated, GC-derived MBCs. In agreement with our earlier findings, IgG1+ MBCs were decreased in -/- the response of SWHEL.Tnfrsf13c versus SWHEL immature B cells (days 12 and day 21) (Figure 5.8B, E). Further analysis revealed greatly reduced numbers of -/- low affinity cells in SWHEL.Tnfrsf13c MBC compartment that also corresponded to a specific depletion of unmutated early MBCs (Figure 5.9D, E, F). In contrast, somatically mutated, affinity matured MBCs persisted in the responses from -/- SWHEL.Tnfrsf13c donor B cells (Figure 5.9D, F; 5.12).

Based on these results, it seems clear that the maintenance of GC- independent versus GC-derived MBCs has a differential requirement for cell-

146 Chapter 5: The role of BAFF:BAFFR signalling in regulating GC and memory B cell responses intrinsic BAFF:BAFFR signalling. Thus, the work presented here adds to our understanding of how MBC longevity is regulated (155, 279). Previous experiments in which BAFF activity was depleted in immunised mice have indicated that the maintenance of long-lived MBCs is BAFF-independent (241, 253, 298). However, human IgG+ MBCs were reported to be responsive to BAFF:BAFFR mediated anti-apoptotic effects in vitro, suggesting that BAFF could be required in promoting their survival and reactivation to become antibody-producing cells (90). These discrepancies likely reflect the differences in experimental approach and the failure to discriminate between GC-derived and GC-independent MBC subsets in these systems. The findings from this work raise some interesting questions regarding the survival and maintenance of MBCs: Why is BAFFR expression maintained on GC B cells and GC-derived MBCs if it is not required for their survival? How is BAFFR signalling differentially regulated between the two MBC subsets? One possible explanation is the difference in transcriptional reprogramming of MBCs that have originated from the GC (281, 282, 289, 299, 301), especially given that GC B cells are seemingly insensitive to BAFF:BAFFR mediated survival signals. In addition, BAFFR-mediated NF-κB2 activation had been associated with the upregulation of multiple BCL-2 pro-survival family members (112, 333, 334), thereby exhibiting anti-apoptotic effects in naïve B cells (121, 335). A recent study further suggested that inhibition of BCL-2 and BCL-xL by BH3-mimetic inhibitors selectively terminated the persistence of MBCs (336), implicating a potential cross-talk between BAFFR signalling and the Bcl2-family proteins in regulating long-lived MBC responses. Thus, the potential cross-talk between BAFFR and BCL-2 family survival pathways in MBC maintenance will be explored further in the next chapter.

147 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Chapter 6

Distinct survival pathways regulate GC-dependent and GC- independent B cell memory

6.1 Preamble

Work discussed in Chapter 5 demonstrated that the survival of class- switched, early MBCs is dependent on BAFF:BAFFR signalling. Thus the absence of BAFFR expression by unmutated MBCs resulted in their demise, whereas GC-derived, mutated MBCs lacking BAFFR could persist. To further investigate the contribution of BAFF:BAFFR signalling in controlling MBC homeostasis, this chapter begins by exploring the impact of enhanced BAFFR signalling. This was done in two ways – first by retroviral overexpression of

BAFFR on normal SWHEL B cells and second through the removal of negative regulator of BAFFR signalling and the NF-κB2 pathway, TRAF3 (103, 106, 107) (ref. Chapter 1.3.2). BAFFR-dependent activation of NF-κB2 pathway has been implicated in the induction of multiple pro-survival BCL-2 family members such as BCL-2, BCL-xL and BCL2A1 (111, 335, 337), as well as the repression of BH3-only pro-apoptotic member BIM (113). Given the importance of these mitochondrial-targeted gatekeepers downstream of BAFFR/NF-κB2 activation in regulating immune cell death and survival, this chapter also aims to evaluate the impact of these Bcl2-family members on regulating MBC responses.

148 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.2 Strategy to examine the impact of enhanced BAFFR signalling on normal B cells during T-dependent responses

To examine the effect of increased signalling through BAFFR on T- dependent B cell responses, BAFFR was overexpressed on WT SWHEL B cells using MSCV-based retroviral overexpression system described in Chapter 2.7. A cDNA encoding for mouse BAFFR (membrane form) was inserted into the retroviral packaging plasmid (pMIG) with a 3’ IRES-linked eGFP, such that translation of the gene of interest correlated with IRES-initiated eGFP translation.

B cells isolated from spleens of SWHEL donor mice were stimulated with anti-CD40 and IL-4 in vitro, followed by spin transduction to integrate empty (Control-GFP) or BAFFR-overexpressing (BAFFR-GFP) retroviral vectors (Figure 6.1A). Successfully transduced HEL-binding B cells expressed eGFP and, in the case of the BAFFR-GFP vector, showed increased levels of surface

BAFFR expression (Figure 6.1B, C). Control-GFP and BAFFR-GFP transduced + SWHEL B cells (CD45.1 ) were adoptively transferred into congenic WT recipient mice (CD45.2+) and challenged with HEL3X-SRBC for subsequent analysis (Figure 6.1A).

149 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

A Donor Recipient

Spin transduction + Control In Vitro stimulation + anti-CD40 + IL-4 GFP B cells In Vitro stimulation anti-CD40 + IL-4 Wild type 3 x 104 HEL-binding B cells i.v 2 x 108 Day -2 Day -1 Day 0 HEL3X -SRBC SWHEL

BAFFR overexpressing GFP+ B cells Wild type

B 48hr post stimulation & spin transduction SWHEL live lymphocytes

Retrovirus Mock (Media) Control-GFP BAFFR-GFP

8.3 8.4 8.0 HEL Binding

B220 B220

0 4.37 4.43

GFP C Transduced SWHEL B cells Mock + Control-GFP

Cell no. + Reltaive BAFFR-GFP

GFP BAFFR FSC-A

150 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.1. Adoptive transfer strategy to investigate enforced BAFFR signalling via retroviral overexpression.

BAFFR was overexpressed on SWHEL B cells to investigate the impact of enforced BAFFR signalling in mediating GC and MBC responses. (A) SWHEL B cells were stimulated in vitro and retroviral transduced with retrovirus containing overexpression vector encoding BAFFR protein controlled by 5’ LTR mediated transcription. Virus containing empty vector was used as control. Transduced + SWHEL B cells (CD45.1 ) were adoptively transferred into congenic WT recipients (CD45.2+) and were challenged with HEL3X-SRBC. (B) Flow cytometric profiles of SWHEL B cell culture 48 hours post anti-CD40 and IL-4 stimulation and spin transduction with media only (left), Control-GFP virus

(centre) and BAFFR-GFP virus (right). Stimulated SWHEL B cells were stained for surface B220 and HEL-binding (200ng/mL), live cell gate was applied (FSC- A vs. SSC-A) and doublets and debris were excluded to determine the frequency of GFP expression in HEL-binding B cells (B220+, HEL-binding, GFP+). Numbers on plot indicate percentages (%) of associated windows. (C)

Histogram overlay showing GFP, BAFFR and FSC-A levels of SWHEL B cells transduced with from mock (grey), Control-GFP (blue) and BAFFR-GFP (red) retroviruses.

151 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.3 BAFFR overexpression on responding B cells increases memory B cell output

To investigate the impact of BAFFR overexpression in regulating B cell + + responses, adoptively transferred Control-GFP SWHEL and BAFFR-GFP 3X SWHEL B cells were challenged with HEL -SRBC. Recipient mice were boosted with antigen 4 days later and splenic responses were analysed on day 12 and 21 (Figure 6.2A).

+ + Since Control-GFP and BAFFR-GFP SWHEL B cells were in vitro stimulated and transduced independently prior to adoptive transfer, it was necessary to normalise the analysis of the two donor responses to take into account the differences in transduction efficiencies of Control-GFP+ versus + BAFFR-GFP SWHEL B cells that were derived from independent transduction events. Data normalisation was achieved using the following formula, in which the percentage of GFP+ cells in the donor-derived population was divided by the percentage of GFP+ cells in the donor B cells transferred on day 0:

+ + i.e. % GFP in Donor-derived IgG1 GC (or MBC) = Normalised + Frequency % GFP in Donor B cells transferred on day 0

Following data normalisation, flow cytometric analysis revealed no significant + + difference between the size of the Control-GFP and BAFFR-GFP SWHEL IgG1+ GC responses (CD45.1+, CD45.2-, eGFP+, IgG1+, CD38lo) on either day 12 or 21 (Figure 6.2B, C). However, BAFFR-GFP+ responses showed consistently greater frequencies and numbers of IgG1+ MBCs (IgG1+, CD38hi) (Figure 6.2B, C). These data suggested BAFFR overexpression had no impact on GC B cell numbers but may specifically prolong MBC survival.

152 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

A

Transduced GFP+ vs non-transduced GFP- HEL-binding B cell + Day 0 Day 4 Day 12 Day 21 HEL3X-SRBC i.v

HEL3X-SRBC Harvest Harvest Wild type Boost Recipient

B GFP+ Donor B cells Donor: Control-GFP+ BAFFR-GFP+

0.8 2.7 CD38 Day 12

75 70

1.3 3.7

Day 21

71 73 IgG1

+ C GFP Donor Compartments 20 Day 12 Day 21 Transduced Donor + Control-GFP + BAFFR-GFP 15 ****

** 10

5 Normalised Frequency (%) 0 + + + + IgG1 GC IgG1 Memory IgG1 GC IgG1 Memory

153 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.2. BAFFR-overexpression on B cells significantly increases IgG1+ MBC numbers during T-dependent responses.

SWHEL B cells were retroviral transduced with Control-GFP or BAFFR-GFP + + viruses as described in Figure 6.1. (A) Control-GFP and BAFFR-GFP SWHEL B cells (CD45.1+) were adoptively transferred into congenic WT recipients (CD45.2+) and were challenged with HEL3X-SRBC. Recipients were again boosted with antigen 4 days later and spleens were harvested on day 12 and 21 for analysis of donor-derived GFP+ responses (CD45.1+, CD45.2-, GFP+, B220+). (B) Flow cytometric plot showing GFP+ donor-derived B cells gated on IgG1+ GC B cells (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi) on day 12 and 21. (C) Normalised frequency of GFP+ IgG1+ GC B cells and GFP+ IgG1+ MBCs on day 12 and 21. Data were normalised to the percentage (%) GFP+ of HEL-binding B cells post transduction in Control-GFP+ and BAFFR-GFP+ shown in Figure 6.1B. Data are representative of 5 mice from one experiment and are representative of 2 independent replicate experiments. Results were analysed using unpaired Student t-test with Welch’s correction post-hoc. Data were considered not significant unless specified, **P<0.01, ****P<0.0001.

154 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.4 Normal GC affinity maturation but accumulation of low affinity memory B cells in responses of BAFFR-overexpressing B cells

Affinity analysis using non-saturating HEL3X-staining described in Chapter 2.4 was performed to investigate the impact of enhanced BAFFR signalling in regulating affinity-based selection. Examination of donor-derived IgG1+ GC B cells revealed no significant differences in affinity maturation for + + Control-GFP and BAFFR-GFP SWHEL responses (Figure 6.3A). Analysis on day 12 showed a minor increase in the frequency of low affinity GC B cells in BAFFR-GFP+ donor compared to Control-GFP+ donor responses (Figure 6.3A, B). However, day 21 analysis revealed comparable proportions of high vs. low affinity cells in Control-GFP+ and BAFFR-GFP+ donor responses (Figure 6.3A, C). Thus increased BAFFR expression may marginally prolong the survival of low affinity GC B cells but not sufficiently to have a major impact on the selection of high affinity GC B cells.

By contrast, the affinity profile of donor-derived IgG1+ MBCs was found + + to differ greatly between BAFFR-GFP and Control-GFP SWHEL donor responses (Figure 6.3D). On day 12, roughly equal proportions of high affinity (51%) and low affinity (49%) MBCs were generated by Control-GFP+ responses (Figure 6.3D, E). However, the frequency of low affinity IgG1+ MBCs was significantly elevated in BAFFR-GFP+ donor (81%) (Figure 6.3D, E). The preferential accumulation of low affinity MBCs in responses from BAFFR-GFP+ donor B cells was also evident on 21 days post-immunisation (Figure 6.3D, F). Thus, day 21 analysis revealed that Control-GFP+ IgG1+ MBCs comprised of only 9% low affinity cells compared to 60% among BAFFR-GFP+ IgG1+ MBCs (Figure 6.3D, F).

155 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Affinity Compartment Low Affinity High Affinity GFP+ IgG1+ GC A B 100 DAY 12 0 GFP+ Donor IgG1+ GC *

+ + 80 20 % Low Donor: Control-GFP BAFFR-GFP y 60 40 finit

92 85 f A A 3X f

finit 40 60 y HEL Binding

Day 12 % High 20 80

0 100 7.6 13 Control BAFFR 94 88 C 100 DAY 21 0

80 20 % Low

Day 21 y 60 40 finit

f A A f

finit 4.9 10 40 60 y

IgG1 % High 20 80

0 100 Control BAFFR GFP+ IgG1+ Memory

D E 100 DAY 12 0 GFP+ Donor IgG1+ Memory ***

80 20 % Low + +

Donor: Control-GFP BAFFR-GFP y 60 40 50 18 finit

f A 3X A f

finit 40 60 y HEL Binding Day 12 % High 20 80

0 100 49 81 Control BAFFR 90 DAY 21 54 F 100 0 *** 80 20 % Low

Day 21 y 60 40 finit

f A A f

finit 9.6 45 40 60 y

IgG1 % High 20 80

0 100 Control BAFFR Overexpression

156 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.3. BAFFR-overexpressing B cell responses show normal affinity maturation in the GC but accumulate low affinity MBCs.

+ + + + Control-GFP and BAFFR-GFP SWHEL IgG1 GC B cells and IgG1 MBCs identified in Figure 6.2 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric plots showing high and low affinity compartment of GFP+ IgG1+ GC B cells from Control-GFP+ and BAFFR-GFP+ + SWHEL responses on day 12 and 21. (B) Proportion of high vs. low affinity IgG1 GC B cells on day 12 and (C) day 21. (D) Flow cytometric plots showing high and low affinity compartment of GFP+ IgG1+ MBCs from Control-GFP+ and + BAFFR-GFP SWHEL responses on day 12 and 21. (E) Proportion of high vs. low affinity IgG1+ MBCs on day 12 and (F) day 21. Flow plots are representative of 5 mice from one experiment. Enumerated data are pooled from 2 independent replicate experiments with 5 mice each. Enumerated data were analysed with unpaired Student t-test and Welch’s correction post-hoc. Data were considered not significant unless specified, *P<0.05, ***P<0.001.

157 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.5 SHM analysis reveals the persistence of unmutated memory B cells in BAFFR-overexpressing B cell responses

Earlier work in Chapter 5 showed that low affinity, unmutated early MBCs were specifically depleted in BAFFR-deficient donor responses to T-dependent antigen (ref. Chapter 5.12). It was possible, therefore, that the converse expansion of low affinity IgG1+ MBCs in BAFFR-GFP+ donor responses may in turn reflect the ability of BAFFR-overexpression to extend the survival of GC- independent MBCs. To test this, single GFP+ IgG1+ GC B cell and IgG1+ MBC from Control-GFP+ and BAFFR-GFP+ responses were FACS sorted on day 21, followed by SHM analysis of Ig VH10 using method described in Chapter 2.5.

Day 21 SHM analysis showed that Control-GFP+ and BAFFR-GFP+ IgG1+ GC B cells displayed no differences in their rate of SHM (Figure 6.4A). It was also revealed that almost identical percentages of B cells with high affinity Y53D substitution were observed in Control-GFP+ (95%) and BAFFR-GFP+ (98%) donor responses, and that those carrying additional affinity-enhancing substitutions (Y58F and/or S31R) were also present at similar frequencies (44% and 49%, respectively) (Figure 6.4A).

In contrast to the GC, SHM analysis of IgG1+ MBC clones from Control- GFP+ and BAFFR-GFP+ responses demonstrated very different compositions. Thus, whilst all IgG1+ MBC clones sorted from Control-GFP+ responses carried somatic mutations, only 60% of those from the BAFFR-GFP+ MBC response were somatically mutated (Figure 6.4B). It is worth noting, however, the low number of clones sampled here (n = 14 and n = 10 from Control-GFP+ and BAFFR-GFP+ responses, respectively) was firstly due to the rarity of GFP+ donor IgG1+ MBC populations sorted; and secondly due to subsequent PCR steps further reducing the yield of successfully amplified single cell, but these factors did not invalidate our hypothesis. Although only a small number of GFP+ IgG1+ MBC clones could be recovered from these responses, these data were consistent with the flow cytometric analysis of antigen affinity (Figure 6.3; 6.4) and indicated that enhanced BAFF:BAFFR signalling specifically prolonged the survival of early, unmutated MBCs. The absence of Y53D+ clones carrying

158 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory additional affinity-enhancing mutations (Y58F and/or S31R) from the BAFFR- GFP+ MBC response (Figure 6.4B) was not expected but may be due to the low number of clones sampled in this response; moreover, high affinity MBC clones were evident from the flow cytometric analysis of BAFFR-GFP+ responses (Figure 6.3D) that complemented these SHM results. Thus overexpressing BAFFR on responding B cells evidently promoted the persistence of unmutated, GC-independent MBCs but had no impact on affinity matured, GC-derived MBC responses.

159 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Day 21 GFP+ Donor B cells

Donor: Control-GFP+ BAFFR-GFP+

A

n = 39 n = 52 + 44% 51% IgG1 SHM = 4.5 49% SHM = 4.4 49% GC

B

40% 36% + n = 14 n = 10 IgG1 SHM = 2.2 SHM = 2.1 64% Memory 50%

* HyHEL10 heavy chain region Amino Acid substitution Unmutated Y53D (high affinity) Y53D + affinity mutations Other mutated Figure 6.4. Single cell SHM analysis reveals the accumulation of unmutated, early MBCs in BAFFR-overexpressing donor responses.

+ + + + Control-GFP and BAFFR-GFP SWHEL IgG1 GC B cells and IgG1 MBCs identified in Figure 6.2 were single cell sorted and sequenced for SHM analysis on day 21. (A) Percentages of donor-derived high affinity vs. low affinity clones constituting GFP+ IgG1+ GC and (B) GFP+ IgG1+ MBC compartments in Control- + + GFP and BAFFR-GFP SWHEL responses. Unmutated clones are indicated in red, other mutated clones in yellow, high affinity clones with Y53D substitution in dark blue, and high affinity Y53D+ clones with additional affinity-enhancing mutations (Y58F+ and/or S31R+) are represented in light blue. n = number of clones analysed and SHM = average number of mutations per clone. Data are representative of 5 pooled mice and are representative of 2 independent experiments. Significance was determined by Chi-square contingency test, *P<0.05.

160 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.6 Strategy to examine constitutive activation of BAFFR signalling via TRAF3-inactivation

The molecular mechanism underlying BAFFR signalling was previously reported in our laboratory (103) (ref. Chapter 1.3.2). Activation of BAFFR leads to the degradation of TRAF3, a downstream TNF-Receptor Associated Factor responsible for regulating NIK-dependent activation of the alternative NF-κB pathway (NF-κB2), thereby facilitating primary B cell survival. TRAF3- inactivation constitutively restores BAFFR signalling in BAFFR-deficient B cells and enables BAFF-independent survival of naïve B cells by mimicking excessive BAFF:BAFFR signalling (107) (ref. Chapter 1.3.2).

To further examine the impact of hyperactivation of BAFFR signalling pathway on T-dependent B cell responses, SWHEL mice were crossed with Traf3ΔB/ΔB mice in which B cells specifically lack expression of TRAF3. Lineage- specific Traf3 gene deletion was achieved using Traf3lox/lox mice heterozygous for Cd19-Cre allele as previously described (106, 338). SWHEL and ΔB/ΔB + SWHEL.Traf3 B cells (CD45.1 ) were adoptively transferred into WT recipients (CD45.2+) and immunised with HEL3X-SRBC (Figure 6.5). Recipient mice were antigen-boosted 4 days later and spleens were harvested on days 12 and 21 to examine splenic B cell responses using gating strategies devised in Chapter 3.3 (Figure 6.5).

161 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Donor Recipients

4 i.v SWHEL 3 x 10 HEL-binding B cells

8 2 x 10 Wild type HEL3X-SRBC

/ SWHEL.Traf3

WT or Traf3 HEL-binding B cell + Day 0 Day 4 Day 12 Day 21 HEL3X-SRBC i.v

3X HEL -SRBC Harvest Harvest Wild type Boost Recipient

Figure 6.5. Adoptive transfer strategy to examine the impact of hyperactivated BAFFR signalling in T-dependent responses.

B/B + SWHEL and SWHEL.Traf3 B cells (CD45.1 ) were adoptively transferred into wild type recipients (CD45.2+) and challenged with HEL3X-SRBC. Recipients were boosted on day 4 and spleens were harvested on day 12 and 21 to investigate the impact of extended BAFFR signalling through TRAF3- inactivation in regulating GC and MBC responses.

162 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.7 TRAF3-inactivation on responding B cells increases memory B cell output but has no impact on the GC response

Mimicking maximal BAFFR signallng by removing TRAF3 expression in ΔB/ΔB SWHEL.Traf3 B cells had no major impact on their GC responses. Flow ΔB/ΔB cytometric analysis of SWHEL and SWHEL.Traf3 donor B cell responses (CD45.1+, CD45.2-, B220+) showed similar GC sizes and IgG1 switching + compared to SWHEL donors on day 12 (Figure 6.6A, B, C). Donor IgG1 GC B + hi ΔB/ΔB cell (IgG1 , CD38 ) numbers were comparable between SWHEL.Traf3 and

SWHEL donor responses (Figure 6.6D). Enumeration on day 21 also + demonstrated comparable GC and IgG1 GC B cell numbers in SWHEL and ΔB/ΔB SWHEL.Traf3 responses (Figure 6.6C, D). Conversely, assessment of MBC responses on day 12 revealed significantly elevated output of IgG1+ MBCs + hi ΔB/ΔB (IgG1 , CD38 ) from SWHEL.Traf3 donor responses compared to SWHEL ΔB/ΔB controls (Figure 6.6A, E). These SWHEL.Traf3 MBCs also persisted at 10- fold greater in cell numbers than the MBCs generated from SWHEL responses up to 21 days post-immunisation (Figure 6.6E).

163 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

A Donor B cells

Donor: SWHEL SWHEL. Traf3/

0.2 0.8 CD38

Day 12

43 36

0.2 1.3

Day 21

34.6 34 IgG1 B C Total Donor GC Total Donor GC 100 106 s

80 105 Donor

SWHEL

+ 60 104 / 1 SWHEL.Traf3 splenocyte 6 0 40 103 % IgG

20 102 Cells per 1 0 10 1 12 21 12 21 Days post Immunisation Days post Immunisation D Donor IgG1+ GC E Donor IgG1+ Memory 106 104 s s

105 103 ** 2 * 104 10 splenocyte splenocyte 6 6 0 0 101 103

100 102 Cells per 1 Cells per 1 <0.1 101 12 21 12 21 Days post Immunisation Days post Immunisation

164 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.6. TRAF3 inactivation has no impact on GC size but significantly increases MBC numbers.

ΔB/ΔB + - + Donor-derived SWHEL and SWHEL.Traf3 B cells (CD45.1 , CD45.2 , B220 ) challenged with HEL3X-SRBC in wild type recipients were analysed on day 12 and 21 as depicted in Figure 6.5. (A) Flow cytometric analysis showing donor- derived IgG1+ GC B cells (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi) on day 12 and 21. (B) Frequency of IgG1-switching in total donor-derived GC. (C) Enumeration of donor-derived GC B cells, (D) IgG1+ GC B cells and (E) IgG1+ MBCs on day 12 and 21. Data are representative of 5 mice from one experiment and are representative of 2 independent replicate experiments. Data were analysed with unpaired Student t-test with Welch’s correction post- hoc. Data were considered not significant unless specified.

165 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.8 TRAF3-deficient B cells undergo normal affinity maturation and selection in the GC but selectively perpetuate low affinity memory B cell responses

Since no discrepancies were observed in GC B cell numbers (Figure 6.6A, C, D), affinity maturation was next examined. Day 12 analysis of donor- derived IgG1+ GC responses revealed similar frequencies of high affinity B cells ΔB/ΔB in SWHEL.Traf3 responses (85%) compared to SWHEL control (91%) (Figure 6.7A, B). Comparison made on day 21 also consistently showed no discrepancies in the proportion of high vs. low affinity compartments between ΔB/ΔB the two groups: SWHEL (95% high affinity) and SWHEL.Traf3 donor (93% high affinity) (Figure 6.7A, C). Thus, no alteration to the affinity maturation of IgG1+ GC B cells was observed due to hyperactivation of the BAFFR signalling pathway by TRAF3 deletion, once again indicating a lack of impact of BAFFR signalling on affinity-based selection in the GC.

Although affinity maturation in the GC was apparently normal in ΔB/ΔB ΔB/ΔB SWHEL.Traf3 donor responses, the SWHEL.Traf3 MBC compartment was significantly enriched in low affinity B cells (Figure 6.8A). On day 12, IgG1+ ΔB/ΔB MBCs from SWHEL.Traf3 donor responses were dominated by the accumulation of low affinity cells (76%) compared to SWHEL control (26%) (Figure 6.8A, B). By day 21, the frequency of low affinity donor-derived IgG1+

MBCs in SWHEL responses had dropped to 8% whereas they remained ΔB/ΔB dominant in the SWHEL.Traf3 responses at a frequency of 58% (Figure 6.8A, C). Interestingly, the numbers of high affinity IgG1+ MBCs produced in the two responses were similar whereas the numbers of low affinity IgG1+ MBCs ΔB/ΔB were greater than 10-fold increased in SWHEL.Traf3 donor responses compared its SWHEL counterpart (Figure 6.8D, E). Overall, very similar results were obtained using retroviral overexpression of BAFFR and deletion of TRAF3 – that is the major impact in each case was to specifically expand the numbers of low affinity MBCs.

166 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Affinity Compartment Low Affinity

High Affinity DAY 12 A B 100 0 Donor IgG1+ GC

80 20 % Low y HEL SWHEL. Donor: SW / 60 40 finit

f Traf3 A A f

finit 85 91 40 60 3X y

% High 20 80 HEL Binding Day 12 0 100 SWHEL SWHEL. Traf3 14 8 DAY 21 C 100 0 95 93

80 20 % Low y 60 40 Day 21 finit

f A A f

finit 40 60 y

4 6 % High 20 80 IgG1 0 100 SWHEL SWHEL. Traf3

Figure 6.7. TRAF3-deficient GC B cells undergo normal affinity maturation and affinity-based selection.

B/B + Donor-derived SWHEL and SWHEL.Traf3 IgG1 GC B cells identified in Figure 6.6 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric plots showing high vs. low affinity IgG1+ GC cells on day 12 and 21. (B) Proportion of high vs. low affinity GC B cells on day 12 and (C) day 21. Data are representative of 5 mice from one experiment and are representative of 2 independent replicate experiments. Data were analysed using Student t-test followed by Welch’s correction. Data were considered not significant unless specified.

167 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Affinity Compartment Low Affinity High Affinity

A B DAY 12 100 0 Donor IgG1+ Memory

80 **** 20 % Low

Donor: SWHEL SWHEL. y / 60 40 finit

f Traf3 A A f

73 23 finit

3X 40 60 y

% High 20 80 HEL Binding Day 12 0 100 SWHEL SWHEL. Traf3 26 76 C DAY 21 100 0 92 42

80 **** 20 % Low

Day 21 y 60 40 finit

f A A f

finit 40 60 7 58 y % High IgG1 20 80

0 100 SWHEL SWHEL. Traf3

D E High Affinity Low Affinity 104 104 s s

103 103

2 ** 10 * splenocyte splenocyte

6 2 10 6 0 0 101

101 100 Cells per 1 Cells per 1 <0.1 100 12 21 12 21 Days post Immunisation Days post Immunisation

Donor

SWHEL / SWHEL.Traf3

168 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.8. TRAF3-deficient responses accumulate low affinity MBCs but generate comparable numbers of high affinity MBCs.

ΔB/ΔB + Donor-derived SWHEL and SWHEL.Traf3 IgG1 MBCs identified in Figure 6.6 were examined by surface staining with low concentration of HEL3X (50ng/mL) counterstained with IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric plots showing high vs. low affinity IgG1+ MBCs on day 12 and 21. (B) Proportion of high vs. low affinity memory cells on day 12 and (C) day 21. (D) Enumeration of high affinity and (E) low affinity IgG1+ MBCs from both day 12 and 21. Data are representative of 5 mice from one experiment and are representative of 2 independent replicate experiments. Data were pooled from replicate experiments and were analysed using Student t-test followed by Welch’s correction. Data were considered not significant unless specified, ****P<0.0001.

169 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.9 Strategy to examine survival regulators in T-dependent memory B cell responses through the overexpression of pro-survival Bcl2- family proteins

Data presented so far provided strong evidence that BAFFR signalling regulates cell survival unique to IgG1+ GC-independent MBCs but not GC- derived MBCs that have undergone SHM. Engagement of BAFF with BAFFR leads to activation of NF-κB2-regulated gene transcription (103, 108, 110, 337). This can upregulate multiple BCL-2 family members, thereby providing survival signals to primary B cells (110, 111, 121, 337). The induction of Bcl-2 and Bcl-xL mRNA by BAFF:BAFFR signalling has been reported in B cells, in which the deletion of NF-κB2 protein itself ablated their induction (108) (ref. Chapter 1.3.3). Therefore, we postulated that NF-κB2 activation and the balance of Bcl2- family proteins induction might account for the survival effects delivered by BAFF:BAFFR signalling in GC-independent early MBCs.

To investigate the extent to which overexpression of pro-survival members of the Bcl2-family might mimic the effects of enhanced BAFFR expression on T-dependent B cell responses, retroviral overexpression of BCL-

2 and BCL-xL in WT SWHEL B cells was performed using the MSCV retroviral expression system described in Chapter 2.7. Bcl-2 and Bcl-xL cDNAs were cloned into plasmid (pMIG) with an eGFP preceded by an IRES sequence, such that IRES-controlled eGFP expression was indicative of the overexpressed gene of interest (ref. Chapter 2.7). Splenic SWHEL B cells were stimulated with anti-CD40 and IL-4 in vitro, followed by spin transduction with retroviruses transfected with control empty vector (Control-GFP), BCL-2 overexpression vector (Bcl2-GFP) and BCL-xL overexpression vector (BclxL-GFP) (Figure 6.9A). Efficiency of transduction was measured based on percentage GFP+ cells in transduced live lymphocytes (Figure 6.9B). BAFFR expression was further confirmed to be unaffected by BCL-2 or BCL-xL overexpression (Figure + + 6.9C). Transduced SWHEL B cells from Control-GFP , Bcl2-GFP and BclxL- + GFP cultures were then adoptively transferred into WT recipient mice and challenged with HEL3X-SRBC for subsequent analysis (Figure 6.9A).

170 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

A

Donor Recipient

Spin transduction Control vector + + In Vitro stimulation GFP B cells anti-CD40 + IL-4

In Vitro stimulation Wild type anti-CD40 + IL-4 i.v 3 x 104 Bcl-2 HEL-binding B cells overexpressing + 2 x 108 GFP B cells Day -2 Day -1 Day 0 HEL3X -SRBC SWHEL Wild type

Bcl-XL overexpressing GFP+ B cells Wild type

B 48hr post stimulation & spin transduction Live lymphocytes

Retrovirus Mock (Media) Control-GFP Bcl2-GFP BclxL-GFP

5.2 5.7 5.8 6.0 HEL Binding

B220 B220

0 5.1 4.5 5.0

GFP

C Transduced Donor Mock + Control-GFP Cell no.

Reltaive + Bcl2-GFP + BclxL-GFP

GFP BAFFR FSC-A

171 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.9. Adoptive transfer strategy to examine Bcl2-family proteins in regulating B cell responses.

Bcl-2 and Bcl-xL proteins were overexpressed on SWHEL B cells to assess the downstream gene targets of BAFFR-mediated NF-κB2 activation in regulating

GC and MBC responses. (A) SWHEL B cells were stimulated in vitro and were retroviral transduced with retrovirus containing overexpression vector encoding Bcl-2 or Bcl-xL proteins controlled by 5’ LTR mediated transcription. Virus containing empty vector was used as control. Transduced SWHEL B cells (CD45.1+) were adoptively transferred into congenic wild type recipients (CD45.2+) and were challenged with HEL3X-SRBC. (B) Flow cytometric analysis of SWHEL B cell culture 48 hours post anti-CD40 and IL-4 stimulation and spin transduction with media only (left), Control-GFP virus (mid-left), Bcl2-GFP virus

(mid-right) and BclxL-GFP virus (right). Stimulated SWHEL B cells were stained for surface B220 and HEL-binding (200ng/mL), live cell gate was applied (FSC- A vs. SSC-A) and doublets and debris were excluded to determine the frequency of GFP expression in HEL-binding B cells (B220+, HEL-binding, GFP+). Numbers on plot indicate percentages (%) of associated windows. (C)

Histogram overlay showing GFP, BAFFR and FSC-A levels of SWHEL B cells transduced with from mock (grey), Control-GFP (black), Bcl2-GFP (blue) and BclxL-GFP (red) retroviruses.

172 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.10 BCL-2 or BCL-xL overexpression on responding B cells generate abnormally expanded GC and memory B cell responses

+ + + + Control-GFP , Bcl2-GFP and BclxL-GFP SWHEL B cells (CD45.1 ) were transferred into congenic WT recipients (CD45.2+) and simultaneously immunised with HEL3X-SRBC. Recipients were antigen-boosted on day 4 and splenic responses were examined on day 12 and 21 (Figure 6.10A).

Flow cytometric analysis of GFP+ donor B cells (CD45.1+, CD45.2-, B220+) on day 12 revealed large increases in cell numbers and frequencies of both IgG1+ GC (IgG1+, CD38lo) and IgG1+ MBC (IgG1+, CD38lo) populations in Bcl2-GFP+ and BclxL-GFP+ groups compared to Control-GFP+ (Figure 6.10B). Similar to the overexpression experiments described previously in Chapter 6.3, it was necessary to normalise the analysis of these transduced donor responses to take into account the differences in transduction efficiencies of + + + Control-GFP vs. Bcl2-GFP vs. BclxL-GFP SWHEL B cells that were derived from independent transduction events. To do this, the frequency of GFP+ cells in the donor-derived population was divided by the percentage of GFP+ cells in the donor B cells transferred on day 0 using the following formula:

+ + i.e. % GFP in Donor-derived IgG1 GC (or MBC) = Normalised Frequency % GFP+ in Donor B cells transferred on day 0

The pro-survival effects on IgG1+ GC B cells were found to be comparable + + between Bcl2-GFP and BclxL-GFP donor responses (Figure 6.10B). After normalisation of the data, comparison of donor-derived IgG1+ GC responses in BCL-2 or BCL-xL overexpressing groups revealed between ~5- and ~14-fold increased in GC size compare to Control-GFP+ responses on days 12 and 21

(Figure 6.10B, C). Meanwhile, SWHEL donor B cells overexpressing BCL-2 or + BCL-xL also yielded significantly expanded IgG1 MBC responses at both time points (Figure 6.10B, C). The effects of BCL-2 and BCL-xL overexpression resulted in a ~15-fold increase in IgG1+ MBC frequencies compared to Control- + + GFP responses (Figure 6.10C). Since a similar frequency of GFP donor cells were transferred and challenged between all three donor groups (Figure 6.9B), these results indicated that BCL-2 and BCL-xL overexpression expanded the

173 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

GC B cell response but seemingly had a more immediate and greater impact on the size of the MBC response (Figure 6.10B, C).

174 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

A

Transduced GFP+ vs non-transduced GFP- HEL-binding B cell + Day 0 Day 4 Day 12 Day 21 HEL3X-SRBC i.v

HEL3X-SRBC Harvest Harvest Wild type Boost Recipient

B GFP+ Donor B cells

Donor: Control-GFP+ Bcl2-GFP+ BclxL-GFP+

1 10 7 CD38

Day 12

53 35 45 1 4 3

Day 21

80 67 52 IgG1 C GFP+ Donor Compartments 30 Day 12 Day 21 Donor + Control-GFP 25 + Bcl2-GFP + BclXL-GFP 20 **** **** **** **** **** **** 15

**** 10

5 Normalised Frequency (%)

0 IgG1+ GC IgG1+ Memory IgG1+ GC IgG1+ Memory

175 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.10. Overexpression of Bcl-2 or Bcl-xL significantly expands both IgG1+ GC and IgG1+ MBC numbers.

SWHEL B cells were retroviral transduced with Control-GFP, Bcl2-GFP or BclxL- GFP viruses as depicted in Figure 6.9. (A) Control-GFP+, Bcl2-GFP+ and BclxL- + + GFP SWHEL B cells (CD45.1 ) adoptively transferred into wild type recipients (CD45.2+) and were challenged with HEL3X-SRBC. Recipients were boosted with antigen after 4 days and spleens were harvested on day 12 and 21 for analysis of donor-derived GFP+ responses (CD45.1+, CD45.2-, GFP+). (B) Flow cytometric analysis showing GFP+ donor-derived B cells gated on IgG1+ GC B cells (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi) on day 12 and 21. (C) Normalised frequency of GFP+ IgG1+ GC B cells and GFP+ IgG1+ MBCs on day 12 and 21. Data were normalised to the percentage (%) GFP+ of HEL-binding B cells post transduction shown in Figure 6.9B. Data are representative of 5 mice and are representative of 2 independent experiments. Results were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, *P<0.05, **P<0.01.

176 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.11 Excess BCL-2 or BCL-xL levels disturbs affinity-based selection in IgG1+ GC and memory B cell compartments

The increase in both IgG1+ GC B cells and IgG1+ MBCs with BCL-2 and BCL-xL overexpression demonstrated that the direct dysregulation of Bcl2- family proteins had a much broader impact on B cell survival during T- dependent responses than the overexpression of BAFFR alone as described earlier (ref. Chapter 6.2). To clarify the extent in which BCL-2 and BCL-xL overexpression impacted on GC and MBC responses, HEL3X-bindng affinity was examined for the IgG1+ compartments on day 12 and 21 using flow cytometry (Figure 6.11; 6.12).

On day 12, assessment of high versus low affinity compartments revealed significantly greater frequencies of low affinity IgG1+ GC B cells in both + + Bcl2-GFP (27%) and BclxL-GFP (25%) donor responses compared to Control- GFP+(15%) responses (Figure 6.11A, B). In addition, later analysis also demonstrated Bcl2-GFP+ and BclxL-GFP+ responses to have higher fractions of low affinity IgG1+ GC B cells compared to Control-GFP+ response up until day 21 of the response (Figure 6.11A, C). On day 21, around 5% low affinity cells were observed in Control-GFP+ donor group, consistent with the affinity maturation and counter-selection of SWHEL B cells normally expected from a day 21 HEL3X response (Figure 6.11A, C). However, the low affinity IgG1+ GC compartments in Bcl2-GFP+ and BclxL-GFP+ donor responses continued to be significantly higher, at 16% and 15% respectively. The increased proportions of low affinity GC B cells in these responses were also concomitant with elevated cell numbers (Figure 6.11B, C). It was noteworthy, however, that the proportions and numbers of high affinity GC B cells did increase in Bcl2-GFP+ and BclxL-GFP+ donor responses compared to day 12 (Figure 6.11A, B, C). These results indicated that the cell-intrinsic capabilities of GC B cells to affinity mature and increase affinity for antigen-binding were unaffected by increased BCL-2 or BCL-xL levels. However, BCL-2 and BCL-xL overexpression clearly prolonged the survival of low affinity GC B cells that were normally counter- selected during affinity-based selection in the GC.

177 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

More strikingly, day 12 analysis of donor-derived IgG1+ MBCs revealed both Bcl2-GFP+ and BclxL-GFP+ responses accumulated large numbers of low affinity MBCs, whereas very few high affinity MBCs were present at this point of the response (Figure 6.12A). Accordingly, analysis of high vs. low affinity MBC compartments also demonstrated significantly elevated proportion of low affinity MBCs in Bcl2-GFP+ (93%) and BclxL-GFP+ (93%) responses compared to the Control-GFP+ counterpart (53%) (Figure 6.12A, B). Similarly, analysis on day 21 also showed significantly elevated numbers of IgG1+ MBCs in Bcl2-GFP+ and BclxL-GFP+ donor groups compared to Control-GFP+ responses (Figure 6.12A). Further characterisation of these MBC responses revealed low affinity cells continuing to persist in significantly increased proportions in Bcl2-GFP+ (42%) and BclxL-GFP+ (48%) responses compared to the Control-GFP+ responses (4%) (Figure 6.12A, C). Although the increase in high affinity MBCs numbers in Bcl2-GFP+ and BclxL-GFP+ donor responses could be attributed to the larger GC responses, the accumulation of low affinity MBCs was evident early in these responses (i.e. day 12), and to a much greater degree than the expansion of the GC at this early time point (Figure 6.11A, B; 6.12A, B). These observations demonstrated that the overexpression of BCL-2 or BCL-xL prolongs the survival of low affinity MBCs, similar to BAFFR overexpression and TRAF3-inactivation presented earlier (ref. Chapter 6.4 and 6.8). However, there is an additional expansion of the GC response and high affinity MBCs that arises from BCL-2 or BCL-xL overexpression but not enhanced BAFFR signalling, suggesting that BAFFR-independent signals operate to promote the survival of these other responding B cell populations.

178 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

A GFP+ Donor IgG1+ GC

Donor: Control-GFP+ Bcl2-GFP+ BclxL-GFP+

85 72 75 3X HEL Binding Day 12

14 27 25

94 84 85

Day 21

5 15 14 IgG1

B Day 12 C Day 21 100 0 100 0

80 20 80 20 % Low % Low y y *** *** finit 60 40 finit 60 40 f f

A A A A

f f finit finit 40 60 40 60 y y % High % High 20 80 20 80

0 100 0 100 Control Bcl-2 Bcl-xL Control Bcl-2 Bcl-xL Overexpression Overexpression Affinity Compartment Low Affinity High Affinity

179 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.11. Bcl-2 or Bcl-xL overexpression prolongs the survival of low affinity cells during affinity-based selection in the GC.

+ + + + Control-GFP , Bcl2-GFP and BclxL-GFP SWHEL IgG1 GC B cells identified in Figure 6.10 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric analysis showing high vs. low affinity GC B cells of Control-GFP+, + + Bcl2-GFP and BclxL-GFP SWHEL donor responses on day 12 and 21. (B) Proportion of high vs. low affinity IgG1+ GC B cells on day 12 and (C) day 21. Results are representative of 5 mice from one experiment and are representative of 2 independent replicate experiments. Data were analysed using One-way ANOVA with post-hoc Bonferroni’s multiple comparison. Data were considered not significant unless specified, *P<0.05, **P<0.01, ****P<0.0001.

180 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

+ + A GFP Donor IgG1 Memory

Donor: Control-GFP+ Bcl2-GFP+ BclxL-GFP+

46 6 5 3X HEL Binding Day 12

53 93 93

94 57 50

Day 21

4 42 48 IgG1

B Day 12 C Day 21 100 0 100 0

80 20 80 20 % Low *** % Low **** y y *** **** finit finit 60 40 60 40 f f

A A A A

f f finit finit 40 60 40 60 y y % High % High 20 80 20 80

0 100 0 100 Control Bcl-2 Bcl-xL Control Bcl-2 Bcl-xL Overexpression Overexpression Affinity Compartment Low Affinity High Affinity

181 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.12. B cell responses overexpressing Bcl-2 or Bcl-xL accumulate significantly increased numbers and frequencies of low affinity MBCs.

+ + + + Control-GFP , Bcl2-GFP and BclxL-GFP SWHEL IgG1 MBCs identified in Figure 6.10 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric profiles showing high vs. low affinity IgG1+ MBCs in Control- + + + GFP , Bcl2-GFP and BclxL-GFP SWHEL responses on day 12 and 21. (B) Proportion of high vs. low affinity IgG1+ MBCs on day 12 and (C) day 21. Results are representative of 5 mice from one experiment and are representative of 2 independent replicate experiments. Data were analysed using One-way ANOVA with post-hoc Bonferroni’s multiple comparison. Data were considered not significant unless specified, ***P<0.001, ****P<0.0001.

182 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.12 Strategy to examine survival regulators in T-dependent memory B cell responses through the deletion of pro-apoptotic protein BIM

Data presented thus far showed that the overexpression of BCL-2 and BCL-xL had a major impact on prolonging the survival of early MBCs analogous to the extended BAFFR signalling in B cells using BAFFR-overexpressing or TRAF3-deficient B cells. However, BCL-2 and BCL-xL overexpression exhibited a broader and stronger impact on prolonging cell survival not restricted only to early MBCs but also for GC B cells. This suggested an overlapping but not solely dependent role of BCL-2 and BCL-xL in BAFFR mediated NF-κB2- dependent regulation of survival and apoptotic proteins during B cell responses.

To further examine this, BIM-deficient (Bcl2l11-/-) mice were crossed onto the SWHEL background and the responses of BIM-deficient donor SWHEL B cells -/- examined in adoptive transfers. Thus, SWHEL and SWHEL.Bcl2l11 B cells (CD45.1+) were adoptively transferred into congenic WT recipients (CD45.2+) and challenged with HEL3X-SRBC (Figure 6.13). Donor B cells were antigen- boosted on day 4 and were harvested on days 12, 21 and 35 post-immunisation for flow cytometric analysis (Figure 6.13).

183 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Donor Recipient

i.v

4 SWHEL 3 x 10 HEL-binding B cells

2 x 108 HEL3X -SRBC Wild type

-/- SWHEL.Bcl2l11

WT or Bim HEL-binding B cell + Day 0 Day 4 Day 12 Day 21 Day 35 i.v HEL3X-SRBC

3X HEL -SRBC Harvest Harvest Harvest Wild type Boost Recipient

Figure 6.13. Adoptive transfer strategy to examine Bcl2-family protein Bim in regulating T-dependent B cell responses.

-/- + SWHEL and SWHEL.Bcl2l11 B cells (CD45.1 ) were adoptively transferred into wild type recipients (CD45.2+) and challenged with HEL3X-SRBC. Recipients were boosted again 4 days post challenge and spleens were harvested on days 12, 21 and 35 to examine the impact of Bcl2-family protein Bim, in the context of BAFFR signalling in regulating GC and MBC responses.

184 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.13 BIM-deletion on responding B cells generates abnormally expanded GC and memory B cell responses

To examine the impact of BIM-deletion on class-switched GC and MBC -/- + - survival, SWHEL and SWHEL.Bcl2l11 donor responses (CD45.1 , CD45.2 , B220+) were analysed on day 12, 21 and 35 using flow cytometric analysis (Figure 6.14A). On day 12, the magnitude of GC responses observed in -/- SWHEL.Bcl2l11 donor was significantly larger than the SWHEL control (Figure 6.14A). Enumeration revealed significantly increased IgG1+ GC B cell numbers

(10-fold greater) than SWHEL control during both days 12 and 21 (Figure 6.14A, -/- + B, C, D). By day 35, however, SWHEL.Bcl2l11 IgG1 GC responses contracted and converged to comparable numbers as measured in SWHEL responses (Figure 6.14A, D), indicating BIM-deletion did not immortalise or prevent GC B cell death that was presumably mediated by other pro-apoptotic proteins such as PUMA (305).

Analysis of donor-derived IgG1+ MBCs (IgG1+, CD38hi) however, -/- revealed significantly increased MBCs numbers in SWHEL.Bcl2l11 responses as early as day 12 compared to the SWHEL control group, and these MBCs accumulated throughout the duration of the response (Figure 6.14A). + Enumeration further revealed the cell numbers of IgG1 MBCs derived from -/- SWHEL.Bcl2l11 donor responses were increased by 10-fold compared to

SWHEL responses (Figure 6.14E). Furthermore, the accumulated BIM-deficient MBCs persisted through to day 21 and day 35 in unchanging numbers (Figure 6.14A, E). The pronounced longevity of these IgG1+ MBCs, not IgG1+ GC B -/- cells seen in SWHEL.Bcl2l11 donor responses indicated that BIM-deletion has a distinct and significant impact on prolonging the survival of MBCs.

185 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Donor B cells A -/- Donor: SWHEL SWHEL.Bcl2l11

0.4 1.4 CD38 Day 12

56 27 0.6 1.4

Day 21

26 40

1.2 12

Day 35

37 15 IgG1

Total Donor GC B 100 C 5 Total Donor GC

s 10 ** Donor 80 4 ** 10 * SWHEL

+ ** -/- 60 SWHEL.Bcl2l11 splenocyte

6 103 40 0 % IgG1 102 20

0 Cells per 1 101 12 21 35 12 21 35 Days post Immunisation Days post Immunisation

D + E + 5 Donor IgG1 GC 5 Donor IgG1 Memory s s 10 10

4 * 10 104 * * * 103 * splenocyte splenocyte 2 6 6 103 10 0 0 101 102 100 Cells per 1 Cells per 1 <0.1 101 12 21 35 12 21 35 Days post Immunisation Days post Immunisation

186 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.14. Bim-deletion increases GC B cell and MBC numbers and prolongs their survival during T-dependent responses.

-/- + - + Donor-derived SWHEL and SWHEL.Bcl2l11 B cells (CD45.1 , CD45.2 , B220 ) challenged with HEL3X-SRBC in wild type recipients were analysed on day 12 and 21 as depicted in Figure 6.13. (A) Flow cytometric analysis showing donor- derived IgG1+ GC B cells (IgG1+, CD38lo) and IgG1+ MBCs (IgG1+, CD38hi) on day 12, 21 and 35. (B) Frequency of IgG1-switching in total donor-derived GC. (C) Enumeration of donor-derived GC, (D) IgG1+ GC B cells and (E) IgG1+ MBCs on day 12 and 21. Data are representative of 5 mice and are representative of 3 independent experiments. Results were analysed with unpaired Student t-test using Welch’s correction. Data were considered not significant unless specified, *P<0.05, **P<0.01.

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6.14 Loss of BIM disturbs affinity-based selection in IgG1+ GC and memory B cell compartments

To further investigate the impact of BIM-mediated cell death in regulating GC and MBC responses, HEL3X-binding affinity analysis was performed to -/- examine the affinity maturation of SWHEL and SWHEL.Bcl2l11 GC B cells -/- (Figure 6.15A). Despite the fact that SWHEL.Bcl2l11 donor B cells generated larger GC responses, both high affinity and low affinity GC compartments were increased proportionally at all time points during the immune response (day 12- 35) (Figure 6.15A). Affinity analysis on day 12 of donor IgG1+ GC B cells -/- showed mildly elevated frequencies of low affinity cells in SWHEL.Bcl2l11

(13%) compared to SWHEL control (6%) responses (Figure 6.15A, B). While later analysis on day 21 revealed similar proportions of high versus low affinity -/- GC B cells between SWHEL and SWHEL.Bcl2l11 donor responses (Figure 6.15A, C), further examination on day 35 showed the abnormal accumulation of -/- low affinity GC B cells in SWHEL.Bcl2l11 (18%) donor compared to SWHEL (7%) donor responses (Figure 6.15A, D). Thus these findings indicated that whilst GC B cells deficient in BIM underwent normal affinity maturation for increasing antigen-affinity, the inhibition of BIM-mediated cell death seemingly prolonged the survival of low affinity GC B cells, suggesting the induction of BIM plays a major role in controlling the death of counter-selected low affinity GC B cells, as has been concluded in previous studies (116, 119, 304, 346).

Most remarkably, flow cytometric analysis on high versus low affinity + -/- IgG1 MBC populations from SWHEL and SWHEL.Bcl2l11 donor responses revealed significant disproportions (Figure 6.16A). On day 12, SWHEL donor B cells generated many high affinity MBCs (75%), but the proportion of high -/- affinity MBCs observed in SWHEL.Bcl2l11 donor responses was significantly reduced (14%) (Figure 6.16A, B). This correlated to a striking increase in low -/- affinity MBC numbers and frequency in SWHEL.Bcl2l11 donor that accumulated and persisted throughout the later phases of the response (day 21 and 35) (Figure 6.16A, C, D). Interestingly, even by day 21 the proportion of high affinity -/- cells had not changed in SWHEL.Bcl2l11 donor responses, in spite of the relatively normal affinity maturation kinetics in the GC response of these BIM-

188 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory deficient B cells (Figure 6.16C; 6.15C). Low affinity MBCs continued to persist -/- and accumulate in SWHEL.Bcl2l11 responses up until day 35, whilst only very few low affinity MBCs remained in SWHEL control responses (Figure 6.16A, D). -/- Nevertheless, SWHEL.Bcl2l11 donor B cells did generate substantially greater numbers of high affinity MBCs compared to its SWHEL counterpart, possibly reflecting the enlarged high affinity GC compartment also found in these BIM- deficient responses (Figure 6.15A; 6.16A). Thus, the impact of removing BIM expression from responding B cells was essentially equivalent to overexpression of BCL-2 or BCL-xL – that is both the GC and high-affinity MBC compartments were expanded in addition to the low affinity MBC compartment.

189 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

A Donor IgG1+ GC

-/- Donor: SWHEL SWHEL.Bcl2l11

92 86 3X

HEL Binding Day 12

6 13

94 93

Day 21

5 6

92 81

Day 35

7 18 IgG1

B Day 12 C Day 21 D Day 35 100 100 100 100 100 100 ** * % Low % Low 80 80 80 80 % Low 80 80 y y y finit finit 60 60 finit 60 60 60 60 f f f

A A A A A A

f f f finit finit finit 40 40 40 40 40 40 y y y % High % High 20 20 % High 20 20 20 20

0 0 0 0 0 0 SWHEL SWHEL. SWHEL SWHEL. SWHEL SWHEL. Bcl2l11-/- Bcl2l11-/- Bcl2l11-/-

Affinity Compartment Low Affinity High Affinity

190 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.15. Normal affinity maturation but impaired counter-selection of low affinity GC B cells in Bim-deficient donor responses.

-/- + Donor-derived SWHEL and SWHEL.Bcl2l11 IgG1 GC B cells identified in Figure 6.14 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric plots showing high vs. low affinity compartments in donor-derived + 3X SWHEL IgG1 GC on day 12, 21 and 35 post HEL -SRBC immunisation. (B) Proportion of high vs. low affinity IgG1+ GC B cells on day 12, (C) day 21 and (D) day 35). Plots are representative of 5 mice from one experiment and are representative of 3 independent replicate experiments. Data were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, *P<0.05, **P<0.01.

191 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

A Donor IgG1+ Memory

-/- Donor: SWHEL SWHEL.Bcl2l11

75 14 3X

HEL Binding Day 12

22 85

85 18

Day 21

14 81

91 14

Day 35

8 85 IgG1

B C D Day 12 Day 21 Day 35 100 100 100 100 100 100

** % Low % Low 80 80 80 80 % Low 80 80 **** y y y **** 60 60 60 60 60 60 finit finit finit f f f

A A A A A A

f f f finit finit finit 40 40 40 40 40 40 y y y % High % High 20 20 % High 20 20 20 20

0 0 0 0 0 0 SWHEL SWHEL. SWHEL SWHEL. SWHEL SWHEL. Bcl2l11-/- Bcl2l11-/- Bcl2l11-/-

Affinity Compartment Low Affinity High Affinity

192 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Figure 6.16. Bim-deletion increases high affinity MBCs output but accumulates significantly increased numbers and frequencies of low affinity MBCs.

-/- + Donor-derived SWHEL and SWHEL.Bcl2l11 IgG1 MBCs identified in Figure 6.14 were examined by surface staining with low concentration of HEL3X (50ng/mL) and IgG1 to identify high affinity and low affinity cells. (A) Flow cytometric analysis showing high vs. low affinity compartments in donor-derived SWHEL IgG1+ MBCs on day 12, 21 and 35. (B) Proportion of high vs. low affinity IgG1+ MBCs on day 12, (C) day 21 and (D) day 35. Results are representative of 5 mice from one experiment and are representative of 3 independent replicate experiments. Data were analysed using One-way ANOVA followed by Bonferroni’s multiple comparison. Data were considered not significant unless specified, **P<0.01, ****P<0.0001.

193 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

6.15 Discussion

The first aim of this chapter was to examine the impact of aberrant BAFF:BAFFR signalling and its contribution to GC-independent and GC- dependent class-switched (IgG1+) MBC survival and maintenance, especially given the strong evidence of their differential survival requirements discussed throughout this thesis. Secondly, since BAFFR signalling activates NF-κB2- regulated transcription of genes from the pro-survival Bcl2-family (110, 111, 121, 337), the actions of BCL-2, BCL-xL and BIM – downstream targets of BAFFR signalling – were examined to review their impacts on controlling GC and MBC responses in the context of BAFFR-mediated survival (ref. Chapter 1.3.3).

Work presented in Chapter 5 demonstrated that the loss of BAFFR had no impact on regulating GC maturation and selection, but BAFFR deletion resulted in the demise of low affinity IgG1+ MBCs devoid of IgH gene somatic mutations. However, using gene-deletion of Baffr to elucidate the function of BAFFR on responding B cells had its limitations, particularly given their immaturity due to the loss of BAFF:BAFFR signalling during B cell development. Thus, to further assess the impact of BAFFR signalling on GC B cell and MBC homeostasis, BAFFR was overexpressed on WT SWHEL B cells prior their adoptive transfer into WT recipient mice (Figure 6.1). BAFFR overexpression had little impact on GC B cell survival (Figure 6.2B, C) consistent with earlier -/- observations using BAFFR-deficient (SWHEL.Tnfrsf13c ) donor B cells, where GC B cell survival was unaffected by the loss of BAFF:BAFFR signalling (ref. Chapter 5). Moreover, affinity maturation of donor-derived IgG1+ GC B cells was mostly comparable between control and BAFFR-overexpressing responses on both days 12 and 21 (Figure 6.3A, B, C). These results were further confirmed by SHM analysis on day 21, where the frequencies of Y53D+ high affinity clones + + and those with further affinity-enhancing mutations (Y53D with Y58F and/or S31R+) were comparable between control and BAFFR-overexpressing GCs (Figure 6.4A). These findings reinforced that BAFF:BAFFR signalling had no impact on regulating SHM of GC B cells or prolonging the survival of low affinity cells from affinity-based counter-selection.

194 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Intriguingly, the data showed that overexpression of BAFFR significantly accumulated and prolonged the survival of low affinity IgG1+ MBCs (Figure 6.3D, E, F). Sequencing of these BAFFR-overexpressing IgG1+ MBCs also revealed these MBCs were primarily devoid of SHM (Figure 6.4B). These results confirmed the accumulated low affinity cells were GC-independent, unmutated MBCs (Figure 6.4B). The observations here were in agreement with the earlier work presented in Chapter 5, where IgG1+ early MBCs but not GC- derived MBCs, were found to specifically require BAFFR-mediated survival signals (ref. Chapter 5.10 and 5.12). Interestingly, Y53D+ high affinity MBCs were readily detectable in similar abundance between control and BAFFR- overexpressing donor B cell responses (Figure 6.3D, E, F; 6.4B). These findings indicated that enhanced BAFFR signalling had no impact on the selection of high affinity antibodies into the MBC pool (Figure 6.4B). The work presented thus far demonstrated strong correlations between BAFFR signalling and the survival of class-switched, GC-independent MBCs but not GC- dependent affinity-matured MBCs.

The ligation of BAFFR leads to the recruitment of TRAF3 to the cytoplasmic domain of BAFFR, which triggers the degradation of TRAF3 and thus the dissociation of the TRAF2/TRAF3/cIAP1/2 complex. This leads to the degradation of NIK and promotes NF-κB2-mediated gene transcription (106, 107) (ref. Chapter 1.3.2). A study by our laboratory, Gardam et al. reported the generation of B cell specific TRAF3-inactivation in mice (Traf3ΔB/ΔB) to identify its impact on B cell survival in vivo (106). The deletion of TRAF3 resulted in increased activation of the NF-κB2 pathway in mature B cells similar to that observed in Baff Tg B cells (106). Thus, to further investigate the impact of constitutive activation of BAFFR in controlling GC and MBC responses, ΔB/ΔB SWHEL.Traf3 donor mice were generated to perform adoptive transfer studies (Figure 6.5).

Consistent with the findings discussed throughout this thesis, ΔB/ΔB hyperactivation of BAFFR signalling pathway in SWHEL.Traf3 B cells had no impact on either GC B cell survival or affinity maturation, again indicating that GC B cells operated independently of BAFFR signalling (Figure 6.6A, C, D;

195 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

ΔB/ΔB 6.7). Notably, SWHEL.Traf3 B cells generated high affinity MBC responses comparable to SWHEL control B cells (Figure 6.6E; 6.8). On the other hand, ΔB/ΔB SWHEL.Traf3 donor responses generated significantly increased low affinity IgG1+ MBCs numbers that accumulated through days 12 and 21 post- immunisation (Figure 6.8A, E). These findings were remarkably similar to observations made on BAFFR-overexpressing MBC responses (Figure 6.3D; 6.8A), further highlighting that IgG1+ early MBCs were preferentially rescued by constitutive BAFFR activation. MBCs have been reported to upregulate NF-κB- controlled survival factors such as BCL-2, BCL-xL and BCL2A1, and their survival shown to be sensitive to inhibition by Bcl2-family protein inhibitors (336). Furthermore, the induction of Bcl2-family proteins had been demonstrated to be a downstream consequence of activating the BAFF:BAFFR signalling pathway (103, 108, 110, 337). Thus, it is possible that the pro-survival effects of BAFF:BAFFR signalling observed in GC-independent early MBCs are mediated by the inhibition of apoptosis via the regulation of key molecules such as BCL-2, BCL-xL and BIM. Therefore, the impact of these Bcl2-family proteins in controlling GC-independent and GC-derived MBC responses were examined on these distinct MBC subsets in light of the specific effects delivered by BAFF:BAFFR signalling alone.

To do this, SWHEL B cells were retroviral transduced to overexpress either BCL-2 or BCL-xL and were challenged with HEL3X-SRBC in WT recipient mice (Figure 6.9; 6.10A). Interestingly, increasing the expression levels of BCL- 2 or BCL-xL proteins did not phenocopy B cell responses that either overexpressed BAFFR or lacked TRAF3. Although enhanced expression of BCL-2 or BCL-xL indeed expanded and accumulated MBCs that persisted for extended periods of time, their effects were non-specific to MBCs alone but also greatly expanded GC B cell numbers throughout the T-dependent response (Figure 6.10B, C). Our results were consistent with earlier work performed by other groups, where transgenic expression of BCL-2 or BCL-xL similarly prevented the apoptosis of GC B cells (343-345). Nevertheless, BCL-2 and BCL-xL overexpressing IgG1+ GC B cells underwent relatively normal affinity maturation, albeit the survival of low affinity GC B cells was prolonged well

196 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory beyond 21 days post-immunisation (Figure 6.11). These outcomes were in stark contrast to the effects of BAFFR-overexpression discussed earlier, where GC B cell affinity maturation, selection and survival were largely unaffected by enhanced BAFFR signalling. Thus, these findings collectively indicated that other inhibitory pathways might be involved in suppressing the induction of BCL-2 and BCL-xL expression, or in counter-acting the pro-survival effects of these Bcl2-family proteins upon BAFFR activation in GC B cells. Moreover, given that GC B cells continue to express high levels of BAFFR on the cell surface (ref. Chapter 3.4) and yet signalling through the BAFFR was shown to be dispensable for GC maintenance (ref. Chapter 5), our results strongly implicated that signalling pathways activated through other receptors such as BCR, CD40 and potentially other cytokine receptors are likely to be more potent contributors in controlling Bcl2-family proteins levels than BAFFR signalling alone (259, 339, 340, 341).

On a similar note, the aberrant expression of BCL-2 and BCL-xL in our studies also greatly expanded the sizes of both the high and low affinity IgG1+ MBC populations, but these were found to be in extreme disproportions (Figure 6.12). Most strikingly, the data presented here clearly demonstrated that increased BCL-2 or BCL-xL expression preferentially accumulated low affinity IgG1+ MBCs compared to the control counterparts (Figure 6.12). While the elevated quantities of high affinity MBCs in BCL-2 or BCL-xL overexpressing responses could be explained by the increased number of high affinity GC B cells resulting in more high affinity MBCs being generated, it is also possible that BCL-2 or BCL-xL overexpression also enhanced the survival of these affinity-matured, GC-derived MBCs (Figure 6.11; 6.12). However, the proportion of high versus low affinity MBCs from donor responses with enhanced BCL-2 or BCL-xL levels were in stark contrast to the predicted trajectory of MBCs generated from the relatively “normal” GC affinity compartments (Figure 6.11; 6.12), indicating that these enduring low affinity MBCs were likely to be generated from the GC-independent pathway. Thus, our observations were consistent with earlier studies where Bcl-2 Tg and Bcl-xL Tg mice immunised with T-dependent antigens (NP-CGG) readily accumulated low affinity class-

197 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory switched MBCs that persisted for extended periods of time (342, 343). Furthermore, these accumulated MBCs were shown to contain no somatic mutations in their IgH variable genes, indicating that BCL-2 or BCL-xL levels indeed play a major role in the maintenance of GC-independent, early MBC survival (343, 344). Thus, in agreement with our earlier hypothesis, both survival proteins BCL-2 and BCL-xL induced upon BAFFR signalling can contribute to facilitating the persistence of GC-independent early MBCs (342- 344). In addition, the pro-survival effects of BCL-2 and BCL-xL were also observed in mutated, GC-derived MBCs that were shown to be BAFFR- independent (ref. Chapter 5), indicating that the induction of these Bcl2-family pro-survival proteins might be mediated by other signalling pathways other than BAFFR.

Thus, to further extend our investigations on Bcl2-family molecules in controlling the survival of GC-independent versus GC-derived MBCs, adoptive transfer studies using BIM-deficient SWHEL mice were carried out to examine the effects of alleviating BIM-mediated apoptosis in light of the results from the earlier BCL-2 and BCL-xL overexpression studies (Figure 6.13). The BH3-only pro-apoptotic protein, BIM, plays a major role in the regulation of hematopoietic cell death and the negative selection of autoreactive B cells (119, 345). In addition, BIM was found to be downregulated in primary B cells upon BAFFR activation thereby prolonging B cell survival (108, 115), potentially mediated by the upregulation of BCL-2 and BCL-xL anti-apoptotic proteins or other factors that together sequester the pro-apoptotic effects of BIM (116, 346). Therefore, we hypothesised that BAFFR-mediated survival of GC-independent MBCs is likely to also depend on the regulation of BIM expression.

Our results showed that the loss of BIM-expression on responding B cells led to abnormal expansion of both the GC and MBC compartments, in which the sizes of both GC and MBC responses from the responding BIM- deficient B cells were significantly elevated compared to WT controls (Figure 6.14) and consistent with an earlier study (304). Interestingly, despite BIM- deficient GC B cells being found in greater numbers during the earlier phase of the response (day 12 and 21), BIM-deficient GC responses contracted to

198 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory comparable magnitudes as WT GC B cells by day 35 (Figure 6.14A, C, D). This indicated that other pro-apoptotic molecules (such as PUMA) (305) and additional pathways are likely to be also involved in regulating GC B cell death. Nevertheless, enhanced survival imposed by BIM-deletion did not impair the cell-intrinsic capabilities of GC B cells to undergo affinity maturation during a T- dependent challenge (Figure 6.15). However, similar to that seen in BCL-2 and BCL-xL overexpressing B cell responses, low affinity GC B cells persisted at greater numbers and frequency in BIM-deficient responses during all stages of the T-dependent response (day 12-35), albeit the effects were milder compared to increasing BCL-2 or BCL-xL expression levels (Figure 6.15) (ref. Chapter 6.11). Thus, these results indicated that BIM-mediated cell death did to a certain extent control the cell survival and kinetics of counter-selection of GC B cells, but other signalling pathways or survival-mediators likely play a more prominent role in regulating GC homeostasis, particularly in low affinity GC B cells (347).

Nevertheless, in agreement with our predictions the loss of BAFFR signalling’s downstream target, BIM, did lead to a significant increase in low affinity IgG1+ MBCs production during earlier phase of the T-dependent response (day 12) that accumulated up to 35 days post-immunisation (Figure 6.16). Moreover, SHM analysis on these persistent BIM-deficient IgG1+ MBCs revealed these to be GC-independent, unmutated MBCs (data not shown), consistent with an earlier study that also reported the prolonged survival of unmutated IgG+ MBCs in NP-CGG immunised BIM-deficient mice (304). However, BIM-deficient responses also generated increased numbers of high affinity MBCs compared to their WT counterparts (Figure 6.16). Thus in light of these results, our findings indicated that BAFFR-dependent survival of class- switched early MBCs is likely mediated in a BIM-dependent manner (115, 348). However, in affinity matured, GC-dependent MBCs that continue to express BAFFR on their cell surface, BAFFR signalling was dispensable, thus again suggesting that other signalling pathways control the survival of MBCs derived from the GC.

199 Chapter 6: Distinct survival pathways regulate GC-dependent and GC-independent memory

Given that the maintenance of GC B cells and GC-derived MBCs coincidentally do not depend on BAFF:BAFFR signalling, it is tempting to speculate that the passage through the GC reaction might have redefined MBC’s dependence on BAFF. This might be explained by differences in transcriptional or epigenetic signatures in GC-dependent MBCs that were imprinted during GC differentiation, or gene silencing that occurred during SHM (350) compared to GC-independent early MBCs. Such possibilities however, are yet to be proven for these distinct MBC subsets (151) and would be an important direction to further pursue this work. Thus, the work presented in this thesis provided strong evidence that distinct cell-intrinsic signalling pathways regulate the maintenance and longevity of GC-independent and GC-dependent B cell memory.

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Chapter 7

Conclusions

7.1 Research outcomes

The continuous and dynamic expression of BAFFR on responding B cell subsets during T-dependent B cell responses implicated its potential role in regulating the quality of immune protection. Driven by this motive, the major aims of this PhD project were to elucidate the function of BAFF:BAFFR signalling in the control and maintenance of GC and MBC responses against T- dependent antigens.

The results presented in this thesis showed that WT B cells adoptively transferred into BAFF-deficient versus BAFFR-deficient recipient mice can generate early, class-switched GC B cell responses but these could not be sustained, indicating that factors extrinsic to the responding B cells found in both BAFF-deficient and BAFFR-deficient mice significantly challenge the stability of established GC responses independent of isotype switching of the responding B cells. In spite of the unsustainable GC responses, the cell- extrinsic defects or the lack of provision of BAFF in these recipient mice did not prevent affinity maturation and selection of the responding GC B cells, thus indicating BAFF:BAFFR signalling on B cells is dispensable for regulating the selection process within the GC reaction. Extensive phenotypic and SHM analysis of adoptively transferred BAFFR-deficient donor B cells further demonstrated that these B cells underwent normal SHM, affinity maturation and selection in the GC. Furthermore, BAFFR-deficient donor B cells generated robust GC responses that were comparable to its immature WT counterparts, indicating the maintenance of GC B cell survival is BAFFR-independent. BAFFR-deficient B cell responses also produced high-affinity antibodies of comparable titres as immature WT B cell responses; hence it was no surprise that the deletion of T cell-derived BAFF did not affect the selection of high affinity antibodies during normal GC responses. Thus, these findings provide

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strong evidence that BAFF:BAFFR signalling is redundant in the maintenance and selection during normal GC B cell responses.

The major outcome from this work, however, was the discovery of a novel function for BAFF:BAFFR signalling in the maintenance of class-switched, unmutated early MBCs. Adoptive transfer studies of WT donor B cells into BAFF-deficient and BAFFR-deficient recipient mice showed that IgG1+ MBC responses were abolished in the absence of cell-extrinsic BAFF but not BAFFR. This indicated that B cell-intrinsic BAFF:BAFFR signalling may be critical for the maintenance of MBC responses. This was interrogated further in adoptive transfer studies using immature WT versus BAFFR-deficient donor B cells, revealing that the absence of cell-intrinsic BAFFR expression severely abrogated the survival of IgG1+ low affinity MBCs. SHM analysis identified these missing MBCs in BAFFR-deficient responses to be primarily GC- independent, unmutated MBCs. Opposite phenotypes were also observed upon overexpression of BAFFR and TRAF3-inactivation on responding B cells, in which the hyperactivation of BAFFR signalling pathway specifically extended the survival of GC-independent, unmutated IgG1+ MBCs whilst having no impact on affinity matured, mutated IgG1+ MBCs that were derived from the GC. These results highlighted that BAFF:BAFFR signalling is required for the maintenance of IgG1+ early MBCs but not GC-dependent MBCs. Although not examined in this thesis, it is likely that the dependence of BAFF:BAFFR signalling also applies to bona fide GC-independent MBCs that are of IgM or other IgG subclasses in this system. Nevertheless, some evidence has suggested that unmutated IgM+ MBCs but not mutated IgG+ MBCs were eliminated upon treatment with anti-BAFF antibodies (277, 298). Thus, it may be the passage through the GC reaction that appears to redefine the requirement for BAFFR-mediated survival in GC-independent versus GC- dependent MBCs.

In the final section of this work, the downstream gene targets of BAFFR- mediated NF-κB2 activation (i.e. Bcl-2, Bcl-xL and Bim) were examined in light of their important role in regulating the survival of hematopoietic cells (304, 305, 345, 351). Our investigations demonstrated that manipulation of these Bcl2-

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family proteins (overexpression of anti-apoptotic BCL-2 and BCL-xL, or removal of pro-apoptotic BIM) produced global effects in T-dependent B cell responses, giving rise to greater numbers of GC B cells and also to both GC-dependent and GC-independent MBCs. The impact of these Bcl2-family proteins, however, were especially prominent in the low affinity, GC-independent early MBCs, where the overexpression of BCL-2 or BCL-xL, or the deletion of BIM significantly accumulated and extended their survival beyond compare. Thus, our findings suggest that the control of these Bcl2-family proteins may be important in mediating early MBC survival upon BAFFR-activation, but that other pathways were evidently involved in regulating the survival effects of these molecules in GC and GC-dependent MBCs.

In conclusion, these findings demonstrated BAFF:BAFFR signalling is dispensable for the regulation of GC B cell responses. MBCs generated in the GC-independent pathway however, have a distinct survival requirement for BAFF:BAFFR signalling that is not shared by the BAFFR-independent MBCs derived from the GC.

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7.2 Future Directions

The revelation of BAFFR-dependent early MBCs described in this thesis highlights a novel and previously underappreciated role of BAFFR signalling in the maintenance of immunological memory beyond B cell development and maturation. This work demonstrated that BAFFR signalling is dispensable for GC B cell survival and function. The discovery of BAFFR-dependent survival in GC-independent but not GC-derived class-switched MBCs also implicated that the passaging through GC differentiation results in MBCs becoming independent of BAFFR mediated survival. This is particularly exciting as no single deterministic phenotype had been associated with their functionality as GC-independent versus GC-derived class-switched MBCs, and the signals that control the robustness, longevity and functionality of these heterogeneous MBCs are still poorly understood. The results from this work point to potentially distinct transcriptional or post-translational patterns that could be essential for distinguishing between these MBC subsets. These had not yet been fully elucidated and will be of great interest to be explored further, particularly given the increasing resolution, precision and power of single cell mRNA sequencing and DNA methylation analyses in mapping transcript and epigenetic changes.

The obvious features distinguishing the dependence of BAFF between early MBCs and GC-derived MBCs are their BCR specificity for the immunising antigen and their passage through the GC reaction. This thesis showed that the direct tampering with pro-survival and pro-apoptotic proteins of the Bcl2-family led to major differences in GC-independent and GC-derived MBC survival (155). It will be beneficial to perform long term recall studies to further examine the differential requirement for BAFF and other trophic factors in mediating the survival for GC-independent vs. GC-dependent MBCs. However, the murine model described in this thesis generates MBCs only in small frequencies. This limited further pursuit through this approach, thereby providing prospective works that could potentially be explored through the conditional deletion of BAFFR or in other antigen-specific models. Furthermore, it will be extremely useful to perform single cell RNA-Seq analysis on these GC-independent and GC-independent class-switched MBC subsets, especially since definitive

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features and markers to distinguish these subsets have yet to be fully determined. This will serve as an important method to identify gene signatures that are differentially expressed but could not be identified through surface phenotypic analysis. This could potentially provide new insights into the mechanisms regulating their maintenance during health and diseases.

Finally, it will be very interesting to apply this new understanding on MBC maintenance in a self-reactive setting. The discoveries presented here suggested BAFF:BAFFR signalling might be critical for the maintenance of not only self-reactive naïve B cells in the breakdown of tolerance but also in sustaining the survival of self-reactive IgG+ unmutated MBCs (352), hence may be an important target in understanding autoimmune conditions.

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227 Chapter 7: Conclusions

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