The Role of Ion Channel TRPM7 in B Cell Development and Function

Mithunah Krishnamoorthy

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy (Ph.D.)

Cell and Systems Biology University of Toronto

The Role Ion Channel TRPM7 in B Cell Development and Function

Mithunah Krishnamoorthy Doctor of Philosophy (Ph.D.)

Cell and Systems Biology University of Toronto

2018

Abstract

The channel-kinase Transient Receptor Potential Subfamily M7 (TRPM7) is known to regulate magnesium homeostasis and was the first channel implicated in the survival of a B cell line. Our study is the first to show that B cells require TRPM7 for development in a murine model.

By using a mouse model where TRPM7 is specifically deleted in B cells under the control of the mb1 promotor, we show that B cells are absent in all peripheral lymphoid tissues due to apoptosis of Pre B cells. By using an in vitro stromal cell line system, we demonstrate that B cell development can be partially rescued by high levels of extracellular magnesium. Interestingly, the lack of B cells is accompanied by an expanded granulocyte population in the spleen. In addition to identifying TRPM7 as an essential factor for B cell development, we show that

TRPM7 is also an important regulator of B cell activation. DT40 B cells lacking TRPM7 fail to contract and gather antigen when activated. To investigate the role of the kinase domain of

TRPM7 we made use of B cells expressing a kinase dead point mutant. These cells were also unable to gather antigen, showing that the kinase domain is an important regulator of this process.

We also show that the kinase domain may potentially interact with another important regulator of B cell activation, PLCγ2 to mediate antigen collection and cell contraction. Importantly, primary murine B cells expressing only one allele of TRPM7 or treated with a TRPM7 inhibitor both displayed defects in antigen gathering, confirming our results in the DT40 cell line. Lastly, we show that TRPM7 is essential for antigen internalization, a process that is important for the recruitment of T cell help and ultimately, antibody production.

iii

Acknowledgements

I could not have finished this degree without the support of several individuals. While I knew that on this journey I would meet new people, I had no idea of the lasting impact that these individuals would have on my life. First and foremost, I would like to thank Dr. Bebhinn Treanor.

This work would have been entirely impossible without her. From editing what feels like a million figures, reports and presentations to providing sound advice, she is a tour de force that has always supported me. Thank you Dr. Treanor, for being an excellent mentor and for teaching me how to be a better scientist every day. I could not have asked for a better supervisor. I would also like to thank the members of my supervisory committee, Drs. Rene Harrison and Blake Richards, for their advice and guidance over the years. I would also like to thank Dr. Mauricio Terebiznik for his kindness and help.

Huge thanks goes to my Mom, who has supported me in all my endeavors, even though she did not entirely understand what I did or why I had to be in the lab all the time. Mom, I am so glad that I can always come to you for advice regarding any aspect of my life and for being the one person I can always count on no matter what. No amount of thanks can even begin to acknowledge what you have done for me. I also want to thank my Dad for being my personal chauffeur from day 1.

To my dearest lab mates, you have made coming into the lab everyday a treat. I will miss our impromptu Timmy’s runs, going out for all you can eat (until you die) sushi, Pump it up exercise sessions and random office conversations. I will always cherish those moments; I know they will put a smile on my face for years to come. Thank you, Tina Zhao, Anh Cao, Josephine

Ho, Laabiah Wasim, Trisha Mahtani, Hifza Buhari, Nouf Alluqmani and Logan Smith for your friendship and support. Special thanks go to Tina, for her unwavering emotional support and for

iv being the shoulder I can always go to cry on. She has never failed to cheer me up when I was feeling down and discouraged. For that, I will be forever grateful. Thank you, Tina, for spending almost a year troubleshooting those terrible total ERK blots. Honestly, I have no idea how you kept going. Your level of persistence is inhuman and is something I strive to achieve going forward. I will never forget how important your friendship has been to my success.

Declaration

Chapters 2 and 3 have been submitted to the journal of Science Signalling under the following titles:

M. Krishnamoorthy, F.H.M Buhari, T. Zhao, P. Brauer, K. M. Burrows, E. Cao, V.

Moxley-Paquette, A. Mortha, J. C. Zúñiga-Pflücker, and B. Treanor. The ion channel TRPM7 is required for B cell lymphopoiesis. Science Signalling, accepted.

M. Krishnamoorthy, L. Wasim, F.H.M Buhari, T. Zhao, T. Mahtani, J. Ho, S. Kang, A-

L. Perraud, C. Schmitz, and B. Treanor. The channel-kinase TRPM7 regulates antigen gathering and internalization in B cells, Science Signalling, accepted.

I performed all the experiments contained within this thesis with the exceptions of those performed by Laabiah Wasim and Josephine Ho (Figures 3.2A-C, 3.4B-D, and 3.6C-D), Hifza

Buhari (Figure 2.3, 3.5C-D), Tiantian Zhao (Figure 2.5, Figures 3.8 H-I), and Trisha Mahtani

(3.8A)

Table of Contents

Abstract ...... ii Acknowledgements ...... iv Declaration...... vi List of Figures ...... x List of Tables ...... xi List of Abbreviations ...... xii

CHAPTER 1: Introduction ...... 1 1.1 B Cell Biology ...... 1 1.1.1 Overview ...... 1 1.1.2 B cell development ...... 2 1.1.3 B cell activation ...... 5 1.1.4 Microclusters in the spreading and contraction response ...... 8 1.1.5 Antigen internalization and presentation ...... 14 1.2 Secondary Lymphoid Tissues ...... 15 1.2.1 Overview ...... 15 1.2.2 Spleen ...... 15 1.2.3 Lymph nodes ...... 17 1.2.4 Peyer’s patches...... 18 1.3 The Transient Receptor Potential Channel Superfamily ...... 19 1.3.1 Overview ...... 19 1.3.2 TRPM subfamily ...... 20 1.4 TRPM7 Structure and Function ...... 24 1.4.1 Overview ...... 24 1.4.2 Structure of TRPM7 ...... 25 1.4.3 Function of TRPM7 ...... 28 1.5 Magnesium Function ...... 31 1.5.1 Overview ...... 31 1.5.2 Importance of magnesium in the immune system ...... 33 1.6 Thesis Rational and Hypothesis ...... 34

vii

CHAPTER 2: TRPM7 is Essential for B Cell Development ...... 36 2.1 Introduction ...... 36 2.2 Results ...... 38 2.2.1 Generation of a TRPM7 conditional knockout ...... 38 2.2.2 Peripheral lymphoid tissues are devoid of B cells in TRPM7-/- mice...... 40 2.2.3 Secondary lymphoid organs have altered architecture ...... 43 2.2.4 The population of splenic myeloid cells is increased in TRPM7-/- mice...... 46 2.2.5 B cell development is abrogated at the Pro B cell stage in TRPM7-/- mice ...... 48 2.2.6 Kinase activity of TRPM7 is dispensable for B cell development ...... 51 2.2.7 B cell development can be partially rescued with extracellular magnesium ...... 53 2.3 Discussion ...... 55 2.4 Materials and Methods ...... 59 2.4.1 Animals ...... 59 2.4.2 Cell isolation and flow cytometry ...... 59 2.4.3 Apoptosis assay ...... 59 2.4.4 Antibodies ...... 60 2.4.5 Tissue sectioning and immunostaining ...... 60 2.4.6 In vitro B cell development assay ...... 60 2.4.7 Data analysis ...... 61

CHAPTER 3: TRPM7 is Important for Antigen Gathering and Presentation ...... 62 3.1 Introduction ...... 62 3.2 Results ...... 65 3.2.1 TRPM7 regulates the actin cytoskeleton in B cells...... 65 3.2.2 Centralization of antigen is altered in TRPM7 deficient B cells...... 68 3.2.3 TRPM7 kinase activity is important for B cell contraction...... 72 3.2.4 Mutation of phosphorylation sites in the C2 domain of PLCγ2 alter B cell spreading and contraction dynamics...... 76 3.2.5 Expression of human TRPM7 in TRPM7 deficient B cells restores antigen centralization...... 79 3.2.6 B cells expressing less TRPM7 accumulate more antigen upon stimulation. .... 81 3.2.7 Pharmacological inhibition of TRPM7 increases antigen accumulation B cells. 83 3.2.8 TRPM7 is important for antigen internalization and presentation...... 87 3.3 Discussion ...... 90

viii

3.4 Materials and Methods ...... 96 3.4.1 Cell preparation and culture ...... 96 3.4.2 Planar lipid bilayer ...... 96 3.4.3 Cell spreading ...... 97 3.4.4 Immunostaining ...... 97 3.4.5 Sample preparation for scanning electron microscopy ...... 97 3.4.6 Cell stimulation and immunoblotting ...... 97 3.4.7 qPCR ...... 98 3.4.8 Flow cytometric assay for BCR internalization...... 98 3.4.9 Presentation assay...... 98 3.4.10 MHC class II upregulation assay...... 98 3.4.11 Microscopy ...... 99 3.4.12 Image processing and data analysis ...... 100

CHAPTER 4: Discussion ...... 101 4.1 Thesis Summary ...... 101 4.2 B Cell Development and Pre-BCR Signalling ...... 103 4.3 TRP Channels in Lymphocyte Function ...... 105 4.4 The Role of Ions in Immune Function and Human Disease...... 107

References ...... 115

List of Figures

Figure 1.1 B cell development ...... 3 Figure 1.2 B cell signalling ...... 7 Figure 1.3 Schematic diagram illustrating the spreading and contraction response in B cells...... 9 Figure 1.4 Secondary lymphoid organs ...... 16 Figure 1.5 TRPM7 structure ...... 26 Figure 2.1 TRPM7 is essential for B cell development ...... 41 Figure 2.2 Splenic architecture is altered in TRPM7-/- mice ...... 44 Figure 2.3 Architecture of lymph nodes in TRPM7-/- mice ...... 45 Figure 2.4 Myeloid population is expanded in TRPM7 deficient mice ...... 47 Figure 2.5 B cell development is abrogated at the Pro B cell stage in TRPM7-/- mice ...... 50 Figure 2.6 Kinase activity of TRPM7 is dispensable for B cell development ...... 52 Figure 2.7 Supplementation with magnesium partially supports B cell development...... 54 Figure 3.1 TRPM7 is involved in the regulation of the actin cytoskeleton ...... 67 Figure 3.2 B cell spreading and contraction response is impaired in TRPM7 deficient cells ...... 69 Figure 3.3 TRPM7 deficient B cells acquire more antigen and have prolonged signalling ...... 71 Figure 3.4 TRPM7 kinase activity is important for cell contraction in response to membrane bound antigen ...... 73 Figure 3.5 TRPM7 kinase activity is important for actin clearing and BCR signalling ...... 75 Figure 3.6 PLCγ2 T1045A is unable to centralize antigen ...... 78 Figure 3.7 Expression of human TRPM7 in TRPM7 deficient B cells restores antigen gathering ...... 80 Figure 3.8 Reduced expression of TRPM7 leads to altered antigen accumulation and signalling in primary B cells ...... 82 Figure 3.9 Pharmacological inhibition of TRPM7 increases antigen accumulation in B cells...... 85 Figure 3.10 Pharmacological inhibitor NS8593 is specific for TRPM7 ...... 86 Figure 3.11 TRPM7 is important for antigen internalization and presentation ...... 89 Figure 4.1 TRPM7 may regulate B cell activation by phosphorylating PLCγ2 ...... 102

List of Tables

Table 1.1 Enzymes requiring magnesium as a co-factor ...... 32 Table 2.1 Conditional deletion of TRPM7 in B cells does not alter embryonic survival ...... 39 Table 2.2 Proportion of T cells is increased in peripheral lymphoid tissues of TRPM7-/- mice ...... 42

List of Abbreviations

7-AAD 7- aminoactinomycin D

ADPR Adenosine diphosphate ribose

AP-2 Adaptor Protein 2

APC Antigen Presenting Cell

ATP Adenosine Triphosphate

Ba2+ Barium

BAFF B cell Activation Factor

BCR B Cell Receptor

BCR-Ag B Cell Receptor-Antigen

BiFC Bimolecular Fluorescence Complementation

BLNK B Cell Linker btk Bruton’s Tyrosine Kinase

Ca2+ Calcium

CaM Calmodulin

CCL CC Chemokine Ligand

CCP Clathrin Coated Pits

CD Cluster of Differentiation

Cd2+ Cadmium

CFS/ME Chronic Fatigue Syndrome/ Myalgic Encephalomyletis

CLP Common Lymphoid Progenitor

Co2+ Cobalt

Con A Concanavalin A

CRAC Calcium Release Activated Channel

xii cSMAC Central Supramolecular Activation Cluster

CTL Cytotoxic T Lymphocyte

CXCL13 CXC Chemokine Ligand 13

CXCR5 CXC Chemokine Receptor 5

DAG Diacylglycerol

DGK Diacylglycerol Kinase

DMSO Dimethyl Sulfoxide

DN Double Negative

DNA Deoxyribonucleic Acid eEF2 Eukaryotic Elongation Factor 2 eEF2K Eukaryotic Elongation Factor 2 kinase

ELISA Enzyme-Linked Immunoabsorbent Assay

ER Endoplasmic Reticulum

ERK Extracellular Signal-Regulated Kinase

ERM Ezrin – Radixin – Moesin

FERM 4.1 Protein Ezrin – Radixin – Moesin

Flt3 FMS Tyrosine Kinase 3

Flt3L FMS-Like Tyrosine Kinase 3 Ligand

FRAP Fluorescence Recovery After Photobleaching

FRET Fluorescence Resonance Energy Transfer

FSC Forward Scatter

GEF Guanine Nucleotide Exchange Factor

GTP Guanine Nucleotide Triphosphate

HEK Human Embryonic Kidney

xiii

HEL Hen Egg Lysozyme

HEV High Endothelial Venules

HSC Hematopoietic Stem Cell

HSH Hypomagnesemia with Secondary Hypocalcemia

ICAM-1 Intracellular Adhesion Molecule 1

Ig Immunoglobulin

IL Interleukin

ILC Innate Lymphoid Cells

IP3 Inositol 1,4,5-triphosphate

IP3R Inositol 1, 4, 5-triphosphate Receptor

IRM Interference Reflection Microscopy

ITAM Immunoreceptor Tyrosine-based Activation Motifs

IκB Nuclear Factor κ Binding Inhibitor

K+ Potassium

KD Kinase dead

KO Knockout

LFA-1 Lymphocyte Function Associated Antigen

Li+ Lithium

LN Lymph Node

LPS Lipopolysaccharide

LT Lymphotoxin

LTi Lymphoid Tissue inducer Cells

LTo Lymphoid Tissue organizing Cells

LTβR Lymphotoxin β Receptor

xiv

M cell Microfold cell

MagNum Magnesium Nucleotide Regulated Metal Ion Current

MAPK Mitogen Activated Protein Kinase

Mg2+ Magnesium

MHC Major Histocompatibility Complex mIg Membrane Immunoglobulin

Mn2+ Manganese

MPPC Multipotent progenitor cells mTORC Mammalian Target of Rapamycin Complex

Na+ Sodium

NAD Nicotinamide Adenine Dinucleotide

NFAT Nuclear Factor of Activated T cells

NFκB Nuclear Factor κ Binding

Ni2+ Nickle

NK cell Natural Killer cell

NUDT9 Human Nucleo-side Diphosphate-Linked Moiety X-Type Motif 9

OCT Optimal Cutting Temperature

PALS Periarteriolar Lymphoid Sheath

PBS Phosphate Buffered Saline

PHA Phytohaemagglutinin

PI3K Phosphoinositide-3 Kinase

PIP2 Phosphotidyl-inositol 4,5-bisphosphate

PIP3 Phosphotidyl-inositol 3, 4, 5-triphosphate

PKC Protein Kinase C

PLC Phospholipase C pMHC Peptide Major Histocompatibility Complex pSMAC Peripheral Supramolecular Activation Cluster

PTEN Phosphatase and Tensin homologue

RAG Recombination Activation Genes

RNA Ribonucleic Acid

ROS Reactive Oxygen Species

RPMɸ Red Pulp Macrophages

RT Room Temperature

SCF Stem Cell Factor

SEM Scanning Electron Microscopy

SH2 Src Homology Domain 2 siRNA Short Interfering RNA

SK Calcium Activated Potassium Channel

SLC41A2 Solute Carrier 41 Member A2

SLO Secondary Lymphoid Organs

SR Sarcoplasmic Reticulum

Sr2+ Strontium

SSC Side Scatter

TCR T cell Receptor

TIRFM Total Internal Reflection Microscopy

TRP Transient Receptor Potential

TRPA Transient Receptor Potential Ankyrin

TRPC Transient Receptor Potential Canonical

xvi

TRPM6 Transient Receptor Potential Melastatin 6

TRPM7 Transient Receptor Potential Melastatin 7

TRPML Transient Receptor Potential Mucolipin

TRPN Transient Receptor Potential Nitric oxide Mechanopotential

TRPP Transient Receptor Potential Polycystin

TRPV Transient Receptor Potential Vanilloid

VCAM-1 Vascular Adhesion Molecule 1

VLA-4 Very Late Antigen 4

WASp Wiskott-Aldrich Syndrome protein

WT Wildtype

XMEN X-linked Immunodeficiency with Magnesium defect and Epstein–Barr virus

infection and neoplasia

Zn2+ Zinc

xvii

CHAPTER 1 Introduction

B cells are an important part of the adaptive immune system, which produce protective antibodies that can clear infection and retain an immunological memory in case of re-infection. This thesis investigates the role of the ion channel TRPM7 in development (Chapter 2) and function (Chapter 3) of B cells.

1.1 B Cell Biology

1.1.1 Overview

The adaptive immune system is essential to protect the body against a diverse range of invading pathogens. This system is responsible for producing high affinity antibodies that specifically target infectious agents for destruction and establishing immunological memory that quickly mounts an immune response upon re-encountering a pathogen. B (bursa or bone marrow derived) cells are a crucial part of this system whose major function is to recognize specific pathogens (antigen) via their B cell receptor (BCR) and differentiate into antibody producing plasma cells or long lived memory B cells1.

Up until the 1960’s, the accepted model of cell mediated immunity featured one type of lymphocyte that was thought to originate in the thymus, form plasma cells, and produce antibodies in response to antigen. However, this model could not explain why removal of the thymus in mice or chickens still resulted in antibody production2. In 1965, Max Cooper and colleagues discovered that removal of the chicken bursa of Fabricius, a hindgut lymphoid organ, prevented the proliferation of antibody secreting cells and antibody production in chickens that were immunized with Brucella abortus. However, lymphocytes were still detected in the thymus of bursectomied chickens. This suggested that lymphocytes can be delineated into B (bursa derived) lymphocytes or T (thymus derived) lymphocytes where B lymphocytes are important 1 for producing antibodies3. From then onward, the search began for the mammalian counterpart of the bursa. Initially, systematic excisions of lymphoid organs from rabbits and lamb were performed to determine the origin of B cells. These excision experiments did not affect the production of antibody. Finally, in 1974, B cells were found to develop in the bone marrow and fetal liver of mammals by using in vitro culture systems2.

1.1.2 B cell development

B cells develop in the fetal liver of embryos or the bone marrow of adults before migrating to secondary lymphoid organs (SLO)4. They are first localized in the endosteum of the bones as hematopoietic stem cells (HSC) and once fully developed, leave the bone marrow and enter into circulation5. HSCs are self-renewing cells that have the potential to become cells of the erythroid, myeloid and lymphoid lineage6. Eventually, HSCs differentiate into non-renewing multipotent progenitor cells (MPPC) which express a cell surface receptor tyrosine kinase Flt3

(Fms Tyrosine Kinase 3) that can stimulate stromal cells. Stromal cells are a network of non- lymphoid connective tissue that are always in close contact with developing lymphocytes and promote their development through the expression of cytokines and adhesion molecules (Figure

1.1)7. Flt3 ligand on stromal cells binds to Flt3 and promotes differentiation of MPPCs into common lymphoid progenitors (CLP)5. Stromal cells then produce IL-7 which differentiate CLPs into the earliest committed B-lineage cell, the Pro B cell6.

B cells all have a unique BCR, which is generated in development by the recombination of heavy and light chain genes (Figure 1.1). Each stage of rearrangement delineates a certain stage of B cell development. In the Pro B cell stage, cells do not express any surface Ig but undergo recombination of the heavy chain8. The heavy chain gene consists of several variable

(V), diversity (D) and joining (J) gene segments which can be recombined in several combinations, leading to multiple heavy chain rearrangement possibilities. The double stranded 2

Immature Large B cell Pre B cell

CLP Small Pre B cell

Stromal cells Pro B cells

Stromal cell Pre-BCR IgM

Adhesion Surrogate Molecules SCF IL-7 Light Chain Integrin c-Kit

Igα/β Stop light chain rearrangement Heavy chain rearrangement Stop heavy chain rearrangement Pro B cell Large Pre B cell Immature B cell

___ Figure 1.1 B cell development. Common lymphoid Progenitors (CLP) interact with stromal cells in the bone marrow and differentiate into Pro B cells. Pro B cells begin heavy chain rearrangements upon engagement with stem cell factor (SCF) and adhesion molecules. Once a functional heavy chain is made, a Pre-BCR is expressed on the surface. Pre-BCR signalling arrests further heavy chain rearrangement and initiates light chain rearrangement. The expression of a functional heavy and light chain (IgM) marks the cell as an immature B cell.

DNA breaks that are needed for somatic recombination are mediated by recombination activation genes (RAG) that express RAG1 and RAG2 endonucleases that are tightly regulated during development. The DH and JH genes are combined in the early Pro B cell stage and the VH gene is

8 combined with the already recombined DH-JH gene segment in the late Pro B cell stage . At this stage, some stromal cells express stem cell factor (SCF), which binds to c-kit on the developing lymphocyte and facilitates B cell proliferation and survival6.

In the large Pre B cell stage, the rearranged heavy chain is expressed with a surrogate light chain complexed with Igα/β and is termed the Pre-BCR. The expression of the Pre-BCR is an important checkpoint in B cell development where the newly formed heavy chain is tested for functionality since recombination generates many non-functional heavy chains9. At this stage approximately half of the cells produce viable re-arrangements10. Cells that do not produce functional heavy chain rearrangements die via apoptosis, whereas cells that produce a functional

Pre-BCR survive, undergo several rounds of proliferation, and then proceed with rearranging the light chain genes9. This signal also arrests further recombination of heavy chain genes by restricting its access to recombinase activity and provides survival signals to the cell.

The recombination of light chains occurs at the small Pre B cell stage, where the VL and

JL gene segments are arranged. The successful recombination of light chains leads to the expression of a complete IgM-BCR11. These cells are classified as immature B cells. Immature

B cells are then tested for their ability to bind self-antigen before leaving the bone marrow. If cells can bind to self-antigen they are deemed autoreactive, and either die via apoptosis, undergo receptor editing, where the light chain genes undergo further re-arrangement or are rendered anergic, in which B cells are unresponsive to stimuli12. If immature B cells are not auto-reactive, they can leave the bone marrow through the sinus and migrate to SLOs. There, immature B cells

4 develop into mature B cells, which express both IgM and IgD isoforms of the BCR. These cells can then be activated by antigens that enter SLOs.

1.1.3 B cell activation

B cell activation is an essential step for B cell functionality. Each B cell possesses a unique

BCR that recognizes only one feature (epitope) of a pathogen. Therefore, an entire population of

B cells can recognize millions of epitopes. B cells are found mainly in the spleen, gut and lymph nodes of the body where they come in contact with antigens which are either soluble or membrane-bound. Antigens come to be membrane-bound when they are presented on the surface of antigen presenting cells (APCs) such as follicular dendritic cells, dendritic cells and macrophages. This predominant form of antigen is the main activator of B cells in vivo13,14.

BCR activation consists of two stages. In the first stage, antigen binds to the BCR, triggering an intracellular signalling cascade and receptor mediated endocytosis of the antigen15.

The endocytosed antigen is then processed into peptides, which are then presented at the surface of the B cell on Major Histocompatibility Complex Class II (MHC class II) proteins16. The second stage of activation occurs when CD4 T cells recognize the peptide on MHC class II presented on

B cells via their T cell receptor (TCR). This binding initiates a signalling cascade within the T cell that leads to the secretion of Interlukin-4 (IL-4) and expression of CD40L which promote B cell proliferation17. With each round of proliferation, BCRs undergo somatic hypermutation which produces BCRs with slightly different binding affinities for antigen. If the BCR has higher binding affinity to antigen it can endocytose more antigen, which leads to better presentation of antigen to T cells, and consequently more T cell ‘help’13,18. Conversely, if the BCR produced by somatic hypermutation has low binding affinity, the B cell will not endocytose as much antigen, and thus will not receive the T cell help needed to proliferate. This micro-evolutionary process produces a B cell expressing a BCR with extremely high affinity to the antigen and is termed 5 affinity maturation1,19. Producing high affinity antibodies during an immune response is crucial for clearing infection. The antibody can specifically bind to pathogen and either neutralize its interactions with host cells or recruit effector cells like macrophages.

The BCR consists of a membrane-bound immunoglobulin (mIg) containing heavy and light chains linked by disulfide bonds and an Igα/β heterodimer which contains immunoreceptor tyrosine-based activation motifs (ITAMs)20. The binding of antigen to the mIg triggers the recruitment of Src family tyrosine kinase Lyn which phosphorylates the ITAMs of Igα/β21. mIg is unable to signal on its own, and requires Igα/β to transduce the signal. The tyrosine kinase Syk then docks to the phosphorylated ITAMs via Src Homology 2 (SH2) domains and becomes activated22. Activated Syk phosphorylates B cell linker (BLNK), a scaffold protein that recruits a variety of SH2 containing proteins, including Phospholipase C (PLC) γ223. PLCγ2 is activated by a B cell specific tyrosine kinase called Bruton’s Tyrosine Kinase (Btk). Once PLCγ2 is activated, it can convert phosphotidyl-inositol 4, 5-bisphosphate (PIP2) into diacylglycerol

(DAG) and inositol 1, 4, 5-trisphosphate (IP3). IP3 binds to IP3 receptors (IP3R) on the endoplasmic reticulum (ER), leading to a calcium influx24. The increase of calcium in the cytoplasm triggers the opening of calcium release activated channels (CRAC) on the plasma membrane that allows for additional influx of calcium. Calcium ions can then bind to several protein moieties, one being calmodulin (CaM), which then activates the phosphatase calcineurin.

Calcineurin dephosphorylates the transcription factor nuclear factor of activated T cells (NFAT) which then subsequently translocates to the nucleus25. Additionally, DAG can indirectly activate protein kinase C (PKC) β which leads to downstream translocation of another transcription factor nuclear factor κ B (NFκB) to the nucleus via the removal of inactivator IκB25.The translocation of extra-cellular regulated kinase (ERK) is also dependent on DAG binding to upstream GTPases

APC Legend Phosphorylation Direct interaction

Ag Indirect interaction Translocation

Igα/β BCR CRAC Lyn

Syk Ca2+ Ca2+ BLNK PIP PKCβ 2 PLCγ2 Btk Ca2+ DAG IP3 IP3R ER Gene transcription Ras CaM

Degradation

IκB NFκB ERK NFAT

__ Figure 1.2 B cell signalling. BCR engagement by membrane-bound antigen on antigen presenting cells (APC) triggers the phosphorylation of Igα/β by Lyn which recruits the binding of Syk. Syk recruits the adaptor protein B cell linker (BLNK) that forms a scaffold for the binding of phospholipase Cγ2 (PLCγ2). PLCγ2 is activated by Bruton’s tyrosine kinase (Btk) and initiates the breakdown of phosphatidyl inositol 4, 5-bisphosphate (PIP2) into diacylgycerol (DAG) and inositol triphosphate (IP3). IP3 binds to the IP3 receptor (IP3R) on the endoplasmic reticulum (ER), resulting in a release of calcium (Ca2+) into the cytoplasm. Calcium release activated channels (CRAC) allow the influx of extracellular calcium upon sensing an initial rise in intracellular calcium concentration. Calcium ions can bind to calmodulin (CaM), leading to the downstream dephosphorylation of nuclear factor of activated T cells (NFAT). Dephosphorylated NFAT translocates to the nucleus. DAG binds to protein kinase β (PKCβ) and Ras which results in the downstream translocation of nuclear factor κ light chain of activated B cells (NFκB) and extracellular regulated kinase ERK respectively. Within the nucleus, NFκB, ERK and NFAT initiate gene transcription.

like ras26. Ultimately, translocation of transcription factors into the nucleus initiate the transcription of genes that leads to proliferation and differentiation of B cells (Figure 1.2)25.

1.1.4 Microclusters in the spreading and contraction response

When engaged by membrane-bound antigen, B cells form small discrete clusters

(microclusters) of BCR-antigen (BCR-ag) on the plasma membrane27. The formation of microclusters is accompanied by a two phase spreading and contraction response (Figure 1.3)27.

During the initial spreading phase, the B cell forms lamellipodia, a sheet-like protrusive structure that spreads over the antigen bearing surface27. As BCRs on the leading edge of the lamellipodia come in contact with more antigen, more BCR-ag microclusters are formed27. The extent of spreading is determined by BCR engagement at the leading edge of the lamellipodia, where increased antigen density and affinity result in more spreading27. However, the cell does not spread more once an upper limit of antigen affinity is reached27. This spreading is then followed by a much slower contraction step, which collects the accumulated microclusters into one large central aggregate called the central supramolecular activation cluster (cSMAC)27. The peripheral supramolecular activation cluster (pSMAC) surrounds the cSMAC, and consists of several integrin molecules such as lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) bound to their respective ligands, intracellular adhesion molecule 1 (ICAM-

1) and vascular adhesion molecule-1 (VCAM-1) respectively. Both ICAM-1 and VCAM-1 are found on antigen presenting cells and facilitate the accumulation of antigen. These molecules are important for B cells to respond to antigens that have lower affinities for the BCR or present in lower densities28,29. In primary B cells, spreading and contraction occurs over a period of approximately 10 minutes27. It is unclear whether antigen is endocytosed immediately after or

BCR I -CAM F-Actin Ag Integrin

B cell Side ViewSide

APC Bottom View Bottom

Steady state Spreading Contraction __ Figure 1.3 Schematic diagram illustrating the spreading and contraction response in B cells. Upon BCR (red dots) binding of membrane-bound antigen (Ag/stars) on antigen presenting cells (APC), B cells spread over the membrane to collect antigen in microclusters and contract to gather the antigen into the center of the contact (cSMAC) for subsequent endocytosis. Integrins (green dots) and f-actin (blue lines) surround the cSMAC.

9 during B cell contraction. Nevertheless, endocytosed antigen is processed and presented to T cells13. Since more antigen is collected when antigen is denser or has higher affinity to the BCR, the spreading and contraction response allows the B cell to discriminate between antigens of different affinities. This is important in the context of affinity maturation and the production of high affinity antibodies27. Microclusters are formed in both B cells and T cells upon lymphocyte activation by membrane-bound ligands30,31. In both B and T cells, each microcluster is an active site of signalling as evidenced by the recruitment of several signalling molecules31,32. Each microcluster in B cells contain 50 to 500 BCR-ag conjugates and has a diameter of ~300 to 600 nm even when activated by a wide range of different antigen affinities30,31,33,34. The factors that determine microcluster size are still unresolved. However, when actin was depolymerized using pharmacological agents, the formation of microclusters was abrogated and the clusters were quick to fall apart. This indicates that the size and shape of microclusters may be determined by actin which acts a fence surrounding microclusters35.

To understand how and why microclusters form, investigating the organization of surface

BCR before antigen engagement is crucial. Initially, BCRs were thought to exist as monomers on the surface of B cells which would crosslink upon engagement with antigen and form microclusters36. Via fluorescence resonance energy transfer (FRET) experiments, studies showed that FRET did not occur between individual molecules of BCR, indicating that BCRs potentially exist as monomers36. However, within the same study, FRET did not occur between Igα and Igβ which are known to exist as bona fide dimers. Therefore, this study may not be the best way to study the organization of surface BCR. Other studies have used bimolecular fluorescence complementation (BiFC) to show that BCRs exist as oligomers37. To perform this experiment,

BCRs with either the C terminal or N terminal portion of yellow fluorescent protein (YFP) were both expressed heterologously in cells. If BCRs exist as oligomers, then the C terminal and N

10 terminal fragments will join to form a complete functional YFP molecule which can be detected by flow cytometry or pull-down assays. Indeed, these experiments detected functional YFP, indicating that BCRs exist as oligomers. Additionally, these oligomers were detected on primary

B cells using super-resolution microscopy techniques38. Upon binding antigen, BiFC signal was found to decrease, indicating that the BCRs move apart from each other. The dissociation of

BCRs was thought to allow signalling molecules to access the ITAMs on Igα and initiate signalling39.

Upon formation, microclusters recruit numerous intracellular signalling molecules and adapters to the plasma membrane. This collection of proteins is termed the signalosome. Kinases such as Syk and Lyn are recruited initially, followed by PLCγ240. The signalosome also transiently recruits positive regulators like CD19 and excludes negative regulators such as the phosphatase CD4531,32. Interestingly, the formation of microclusters are not completely dependent on signalling through the BCR. This was shown in Lyn-deficient B cells which still assembled microclusters upon antigen binding, albeit fewer being formed40. Conversely, BCR signalling is essential for the spreading of the B cells. When transgenic B cells expressing signalling deficient chimeric BCR with high affinity for antigen were stimulated with membrane-bound antigen, they failed to spread and had a severe defect in antigen accumulation27.

In fact, deletion of any one of several signalling molecules downstream of BCR engagement lead to spreading defects, including Lyn, Syk, PLCγ2 or its activator Btk, and guanine exchange factors (GEF) like Vav40. Notably, PLCγ2 has been demonstrated to retain Vav in microclusters and vice versa40. Vav is a GEF for Rho GTPases like Cdc42, Rac1 and Rho, which are involved in actin remodeling41. The synergistic co-operation between PLCγ2 and Vav provides a mechanism that links microcluster formation to cell spreading40.

The reorganization of actin is critical for the formation of microclusters and lamellipodia during the spreading response. Indeed, B cells treated with actin depolymerizing agents cannot spread and collect ~75% less antigen when activated with membrane-bound antigen27. As mentioned previously, Vav is recruited to BCR microclusters and can activate the GTPase Cdc42.

Cdc42 is important for binding and changing the conformation of Wiskott-Aldrich Syndrome protein (WASp) to an active form42. Active WASp can then bind the actin nucleation protein

Arp2/3 which binds an actin filament43. Globular actin monomers are added to either side of the existing actin filament by Arp2/3 in a “Y” shaped configuration44. Thus, activation of several

Arp2/3 complexes generate a sheet of branched actin that pushes upon the plasma membrane, initiating the formation of lamellipodia44. The process for actin remodeling is important for lymphocyte function as mutations in the WASp gene leads to immunodeficiency. The resulting disease, Wiskott-Aldrich Syndrome, results in symptoms such as eczema and recurrent infections42. Deficiencies in any of the components of the above mentioned pathway (either Vav,

Cdc42, WASp or Arp2/3) results in a lack of spreading in B cells and ultimately abrogates B cell function 40,45,46.

While actin polymerization is important, the initial depolymerization of the actin cytoskeleton is equally necessary. In fact, pre-treatment of B cells with the actin stabilizing drug jasplikinolide prevented the formation of microclusters and cell spreading when activated by membrane-bound antigen47. In the steady state, BCR on the plasma membrane are constantly moving, but are restricted to an area by underlying actin48. Actin is tethered to the plasma membrane via the Ezrin-Radixin-Moesin (ERM) family of proteins49. These proteins interact with both the plasma membrane and actin using a N terminal FERM (4.1 protein, Ezrin-Radixin-

Moesin) domain and C terminal actin binding domain respectively and are regulated by conformational opening upon phosphorylation49. Upon BCR engagement, actin is

12 depolymerized, in concert with the dephosphorylation of ezrin, which releases actin from the plasma membrane35,50. BCR mobility increases, which is thought to permit BCR interaction with signalling molecules as well as form microclusters that initiate a signalling cascade51. This idea is supported by the finding that a calcium flux is elicited by B cells treated with the actin depolymerizing agent latrunculin A48. Cofilin is an actin severing protein that aids in actin depolymerization and was found to be important in B cell spreading and microcluster formation47.

When dephosphorylated by slingshot phosphatase upon B cell activation, cofilin is activated.

Expression of a phosphomimetic cofilin mutant in B cells leads to defective antigen accumulation and cell spreading. The activation of cofilin was found to be dependent on the activation of the

GTPase Rap1, which is activated downstream of PLCγ2 upon BCR engagement47. Ezrin- mediated actin remodeling is also important for microcluster formation as B cells expressing either a constitutively active or a dominant negative mutant of Ezrin resulted in larger or smaller microclusters respectively35.

The contraction response that works to gather antigen is not studied as well as lymphocyte spreading. During T cell activation, actin treadmilling is thought to coalesce microclusters into the cSMAC52. The process of actin treadmilling involves monomers of actin constantly being added to the barbed end of the actin filament while monomers are removed from the non-barbed end53. This produces a net movement of actin that pushes microclusters toward the cSMAC52.

Myosin IIA may also participate in TCR microcluster movement along actin filaments54. This is demonstrated by an arrest in microcluster movement upon treatment with the myosin inhibitor blebistatin54. B cell contraction may be mediated by dynein centered mechanism55. Dynein is a motor protein that transports cargo along tubulin microfilaments toward the nucleus of the cell56.

In B cells, the adaptor proteins Grb2, Dok3 and the ubiquitin ligase Cbl recruit dynein to BCR microclusters and assist in the movement of microclusters. Knocking out either Grb2, Dok3 or

Cbl resulted in an arrest in microcluster movement, while neither microcluster formation or spreading were interrupted55. Disruption of the microtubule network also resulted in a lack of microcluster movement55. These studies demonstrate that several mechanisms of contraction may exist.

1.1.5 Antigen internalization and presentation

The major method of antigen internalization is clathrin mediated endocytosis57. Clathrin is a large triskelion-shaped protein that is composed of three heavy and light chains58. The associated adaptor protein 2 (AP-2) binds to PIP2 in the plasma membrane which induces a conformational change in AP-2. This exposes more PIP2 and clathrin binding sites on AP-2 and allows for more clathrin to be recruited to the plasma membrane, forming clathrin-coated pits

(CCP). CCPs are localized stochastically on the membrane in the resting state59. After the formation of the cSMAC, AP-2 can also bind to Igαβ of the BCR and initiate the recruitment of

CCP60. The addition of more clathrin to the CCP curves the membrane inward. When the clathrin cage is almost completely formed, the formed vesicle still needs to be detached from the plasma membrane. This requires actin polymerization and Myosin 1E and VI to provide the mechanical force required to pinch off the vesicle while also liberating the antigen from APCs61,62.

Hydrolases are locally secreted into the IS to promote the extraction of membrane-bound antigen63. Once the vesicle is free from the plasma membrane, the clathrin coat is stripped off and it becomes an early endosome64. Early endosomes then mature into late endosomes which can fuse with lysosomes. Proteases within the lysosome then degrade the antigen into peptides that can be loaded onto MHC II that can then be shuttled to the plasma membrane for presentation to T cells65. This triggers T cells to produce cytokines that further activate B cells to proliferate and differentiate into antibody producing cells.

1.2 Secondary Lymphoid Tissues

1.2.1 Overview

Secondary lymphoid tissues efficiently trap and concentrate antigens which initiate immune responses by mature naïve T and B lymphocytes. SLOs include many tissues with the spleen, lymph nodes (LN), and Peyer’s patches being prominent ones (Figure 1.4). These organs provide an environment to support the interaction between antigen presenting cells and rare antigen-specific lymphocytes.

1.2.2 Spleen

In humans, the spleen (Figure 1.4) is a fist-sized organ located behind the stomach which can be divided into two sections; the red pulp and the white pulp. The red pulp is responsible for filtering the blood for dead or dying red blood cells or microorganisms and consists mainly of transiting erythrocytes66. Red pulp macrophages dispose of dead erythrocytes and help recycle iron67. The white pulp, which accounts for half the splenic tissue, is organized into lymphoid sheaths, which surrounds arterial vessels and contains compartmentalized regions of B cells and

T cells. T cells are found in an area immediately adjacent to blood vessels termed the periarteriolar lymphoid sheath (PALS) and B cells are distributed around the PALS in tightly packed follicles.

The compartment that immediately surrounds the follicles is the marginal zone, where blood borne microorganisms in the red pulp can be engulfed by marginal zone macrophages and dendritic cells66. These APCs can then activate T cells and initiate the adaptive immune response16. Unlike other SLOs, the spleen is not directly connected to the lymphatic system, so the blood is its only access to antigens66.

Spleen

Red Pulp B cells T cells

Peyer’s Patch

M Cell Follicle Associated Lymphatics Epithelium

B cells

T cells Lymph node T cells (paracortex) B cells Medullary Cords Efferent Lymphatic Medullary Sinus Vessel

Artery Vein

Efferent Afferent Lymphatic Lymphatic Vessel Vessel Figure 1.4 Secondary Lymphoid Organs. Cross sections of secondary lymphoid organs (Spleen, Peyer’s Patch and Lymph node)

The distinct organization of the white pulp is mediated by several cytokines secreted by stromal cells. Follicular stromal cells and adjacent follicular dendritic cells secrete CXC chemokine ligand 13 (CXCL13) which attract B cells that express CXC chemokine receptor 5

(CXCR5). As CXCL13 binds to CXCR5 on B cells, they are induced to express lymphotoxin

α1β1 (LT α1β1) on the cell surface, which induces stromal cells to produce more CXCL13 in a positive feedback loop68. T cells are recruited to the PALS by stromal cells expressing CC chemokine ligand 19 (CCL19) and CC chemokine ligand 21 (CCL21)69.

1.2.3 Lymph nodes

Lymph nodes are a set of encapsulated bean-shaped lymphoid tissues all interconnected by lymphatic vessels (Figure 1.4). Extracellular fluid from tissues (lymph) drains through lymphatic vessels, filters through lymph nodes and returns back to the blood. Similar to the white pulp of the spleen, lymph nodes are comprised of T cell zones and B cell zones. The center of the lymph node is comprised of T cells in the paracortex and B cells are found in follicles around the paracortex. The paracortex is infiltrated by high endothelial venules (HEVs), specialized blood vessels that allow the entry of lymphocytes into lymph nodes70. Macrophages and dendritic cells are also present in a thin layer surrounding B cell follicles70. During infection, dendritic cells travel through the lymphatics to the draining lymph node to present antigen collected in peripheral tissues to T cells in order to activate them. B cells can also be activated by antigen collected by dendritic cells, follicular dendritic cells, and subcapsular macrophages14,71,72. In addition, free antigen in the lymph, if small enough (~4 nm radius), can permeate through the lymph node in channels called conduits without the help of dendritic cells to activate B cells73. With T cell help, activated B cells proliferate and form germinal centers which make up the B cell follicle19. If the cognate lymphocyte is not met, the dendritic cell will exit the lymph node via the efferent lymphatics in search of another lymph node with the appropriate cognate B or T cell73.

1.2.4 Peyer’s Patches

Peyer’s patches are lymphoid tissues found underneath a layer of epithelial cells on the small intestine (Figure 1.4). Each Peyer’s patch contains numerous B cell follicles and T cells fill the spaces in between the follicles. The layer between the follicles and the epithelium is known as the subepithelial dome and is populated by many dendritic cells74. Antigens enter from the gut lumen through a specialized epithelial cell called the microfold cell (M cell) which have a folded luminal surface. M cells are constantly endocytosing molecules from the gut lumen and passing them to dendritic cells in the subepithelial dome75. Dendritic cells activate antigen-specific T cells and together, they activate B cells to produce antibodies that are class switched to IgA. IgA antibodies neutralize pathogens without initiating an inflammatory response76. If the specific T cell cannot be found, dendritic cells leave the Peyer’s patch via the efferent lymphatics to the mesenteric lymph nodes. All Peyer’s patches drain into the mesenteric lymph nodes77.

Development of Peyer’s patches begins during embryogenesis. The development of each individual patch along the small intestine are temporally separated, where proximal Peyer’s patches are formed before distal ones78. Initiator cells on the small intestine express IL-7 which induce LTi cells to produce LTαβ that activate lymphoid tissue organizing (LTo) cells through the lymphotoxin β receptor (LTβR). LTo cells express adhesion molecules like VCAM and chemokines such as CXCL13, CCL19 and CCL21. HEVs form through the mass of cells and B,

T and dendritic cells are recruited to their specific compartments in the rudimentary Peyer’s patch79.

1.3 The Transient Receptor Potential Channel Superfamily

1.3.1 Overview

The Transient Receptor Potential (TRP) channels are a large superfamily of cation channel proteins that are present on the plasma membrane of eukaryotic cells. These channels are found in yeast, indicating that they existed before the evolution of metazoans80. The superfamily consists of 28 members which are divided into two groups based on their sequence homology. The groups are then further divided into subfamilies also based on their sequence similarity. Group 1 consists of TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA

(ankyrin), and TRPN (Nitric Oxide Mechanopotential) subfamilies. Group 2 consists of TRPP

(Polycystin) and TRPML (Mucolipin) subfamilies. All of the group 1 and 2 subfamilies are expressed in humans except the TRPN channel80. Both groups have 6 transmembrane segments; however, group 2 channels possess a large loop connecting transmembrane segments one and two. The pore forming loop is found between the fifth and sixth segments of all TRP channels81.

Usually, TRP channel proteins form homo or heterotetramers to make one pore in the plasma membrane. The cation selectivity of the pore depends on the channel subunits that make up the tetramer. TRP channels are homologous to voltage gated K+, Na+ and Ca2+ channels, but lack a complete set of positively charged amino acid residues on the fourth transmembrane domain that acts as a voltage sensor82. Besides the transmembrane segments, each channel has C and N terminal tail that extend into the cytoplasm of the cell80.

Interestingly, these channels have a wide range of activation mechanisms, cation specificities and are expressed in many tissues. Many of these channels are found to have roles in sensory physiology such as taste and olfaction and often act as signal integrators83. Although members of a subfamily may have similar sequences, their function, regulation mechanisms and cation specificities can differ greatly. For example, TRPM4 and TRPM5 are selective for

19 monovalent cations, and only produce current in the presence of Ca2+ currents, while TRPM6 and

TRPM7 are selective for magnesium currents80. Some TRP channels can also be activated by pH, mechanical stimuli or G-protein coupled receptors. One such channel activated by G proteins is

TRPC1. TRPC1 was the first identified member of the TRP family80. Discovery of this channel lead to the discovery of other TRP channels.

Several TRP channels have also been implicated in human disease. Mutations in group two channels TRPP and TRPML leads to autosomal dominant polycystic kidney disease and autosomal recessive mucolipidosis IV respectively83. Mutations in TRPM6 leads to hypomagnesia and hypocalcemia (HSH), which can be rectified with a diet high in magnesium84.

Also, mutation of another group 1 channel, TRPC6, leads to autosomal dominant segmental glomerulosclerosis in the kidneys85. This shows that the TRP family of ion channels are important in regulating a wide array of cellular processes.

1.3.2 TRPM subfamily

The TRPM subfamily consists of 8 members and shares 20% sequence homology over 5

C-terminal transmembrane domains with TRPC channels83. Unlike other TRP channels, TRPM channels lack ankyrin repeats. TRPM channels can be placed into 3 groups based on sequence and functional similarity: TRPM1/3, TRPM4/5, and TRPM6/7. TRPM2 and TRPM8 are more closely related to each other than the rest of the subfamily, but do not have similar function, thus do not belong to any group. Many of these channels have been implicated in immune function82-85.

TRPM1 was the first mammalian TRPM channel identified in melanoma cells (hence the name melastatin) and had non-selective permeability to cations80. Levels of TRPM1 expression can be used as a predictor of melanoma progression, as decreased levels of TRPM1 indicate more

20 aggressive melanomas90. Furthermore, a truncated isoform of TRPM1 exists and is devoid of any transmembrane domains91. This truncated version is thought to associate with the full isoform of TRPM1 and prevent its insertion into the plasma membrane92. Only weak TRPM1 currents were detected from the plasma membrane and might be due to its higher expression in intracellular vesicles. Knockdown of TRPM1 results in lower melanin expression, suggesting that

TRPM1 has a role in regulating melanin content in cells91. TRPM1 is also highly expressed in the retina and plays a role in repolarizing retinal bipolar cells93. Although this was the first TRPM channel discovered, very little is known about its structure93.

Expressed highly in the brain and immune cells, TRPM2 is a channel protein that has a C terminal adenosine diphosphate ribose (ADPR) pyrophosphatase domain (NUDT9 homology domain) which can be activated by reactive oxygen species (ROS) and ADPR94,95. The channel is sensitive to oxidative stress and activation can lead to cell death93,96 Intracellular calcium is needed for channel activation and can potentiate the effects of ROS or ADPR97. TRPM2 also seems to play a role in T cell activation by mediating influx of calcium upon Concanavalin A

(Con A) stimulation98. Jurkat T cells produce ADPR, a metabolite of Nicotinamide Adenine

Dinucleotide (NAD) in response to Con A. ADPR was found to induce TRPM2 dependent Ca2+ currents. Inhibition of ADPR formation reduced ConA but not store operated calcium flux and prevented ConA induced cell death in T cells98. TRPM8 is most closely related to TRPM2; however, it does not contain any associated enzymes. TRPM8 is activated by cold temperatures and menthol and can flux calcium99. High expression of TRPM8 in the prostate has been used as a biomarker for prostate cancer100.

TRPM3 is most closely related to TRPM1 out of all the TRPM channels and is highly expressed in the brain80,94. It encodes the largest number of isoforms of all the TRPM channels

21 due to alternative splicing of mRNA. Different variants of TRPM3 form heterotetramers with each other to form a pore in the plasma membrane. However, the constituents of the heterodimer determine the channel specificities for certain cations101. Interestingly, TRPM3 has be implicated in Chronic Fatigue Syndrome/Myalgic Encephalomyletis (CFS/ME), a disease linked to reduced natural killer (NK) cell cytotoxicity102. NK cells in CFS/ME patients have reduced expression of

TRPM3 and also have reduced calcium flux when stimulated. This reduction in calcium flux was attributed to the decrease in TRPM3 expression in CFS/ME patients103.

First observed in rat cardiac myocytes, TRPM4 is a non-selective channel for monovalent cations and is permeable to Na+, K+, Cs+, and Li+104. It is ubiquitously expressed, but most highly expressed in the testis94. Although it is not permeable to Ca2+ ions, the channel is activated by high intracellular Ca2+ concentrations and is responsible for depolarization of the plasma membrane in response to activation105. There are two isoforms of TRPM4, TRPM4a and

TRPM4b. TRPM4a lacks the first 174 amino acid residues from the N terminus and has little activity. TRPM4b is the longer isoform and can form a working channel104. TRPM4 has been found to play an important role in mediating intracellular Ca2+ level oscillations in Jurkat T cells that were stimulated with phytohaemagglutinin (PHA). When TRPM4 levels were knocked down via siRNA, there was a lack in calcium oscillation, but instead, a prolonged and elevated level of

Ca2+. The knockdown of TRPM4 also led to increased IL-2 production, which was speculated to be due to increased Ca2+ flux89.

Closely related to TRPM4 is TRPM5. Like TRPM4, TRPM5 is a nonselective monovalent cation channel that is activated by high intercellular Ca2+ and is able to depolarize plasma membranes106. Also, the sensitivity of TRPM5 to intracellular Ca2+ is much higher than

TRPM4 despite having about 50% sequence similarity107. Although TRPM5 was thought to be

22 mostly expressed in taste receptor cells, it is also expressed in splenic B cells and to a lesser extent splenic T cells108; however, the role of TRPM5 in lymphocyte function has not been elucidated. Interestingly, TRPM5 has been implicated in mucosal immunity. In the gut, TRPM5 is exclusively expressed in tuft cells, highly specialized chemosensory intestinal epithelial cells.

Upon helminth infection, tuft cells increase in number and are important for clearing helminths from the gut. In TRPM5-/- mice infected with helminths, tuft cells do not increase in number and over time helminth number does not decrease. It was also found that TRPM5 deficient tuft cells do not produce IL-25, an important cytokine for innate lymphoid cell (ILC) activation, which are important in clearing parasitic gut infections109.

TRPM6 is a rare kinase domain possessing channel protein and is the closest known relative of TRPM780. TRPM6 is permeable to divalent cations, mainly magnesium and calcium110. It can also form heterotetramers with TRPM7 where the phosphorylation of TRPM7 by TRPM6’s kinase domain can dictate the sensitivity of the channel to cations111. It was also found that the interaction between TRPM6 and TRPM7 was important for trafficking TRPM6 to the plasma membrane112. Mainly expressed in the kidneys and colon, TRPM6 is thought to regulate the body’s level of magnesium and, to a lesser extent, calcium94,110. Mutations in TRPM6 leads to HSH, a disease resulting in seizures and muscle spasms due to magnesium deficiency84.

As magnesium deficiency is implicated in immunodeficiency (See section 1.5), TRPM6 may play a role in regulating immune function by regulating magnesium uptake113.

Of all the TRPM channels, TRPM7 is the only channel that has been implicated in survival, where the global deletion of TRPM7 in a murine model is embryonic lethal114. It is ubiquitously expressed, but expression is highest in the heart, liver, bone and adipose tissue in humans94. Like TRPM6, TRPM7 is permeable to magnesium and calcium while also having a

23 kinase domain115. However, TRPM7 is thought to be responsible for regulating magnesium homeostasis at a cellular level. Channel activity is inhibited by intracellular free magnesium and adenosine triphosphate (ATP)-Mg2+ 115. TRPM7 has also been implicated in regulating the actin cytoskeleton. Whether TRPM7 achieves this with its channel domain, kinase domain or a combination of both is debated116,117. Since the actin cytoskeleton is critical for immune cell function, TRPM7 may play a role in the regulation of immune cell activation48. Since TRPM6 is not expressed in B cells, TRPM7 may be especially important in B cell function108.

1.4 TRPM7 Structure and Function

1.4.1 Overview

TRPM7 was discovered independently by 3 different groups in 2001115,118,119. Runnels et al. used a yeast two hybrid to screen a rat brain protein library using the C2 domain containing C terminus region of Phospholipase C β1. One putative interactor of the PLCβ1 fragment strangely resembled the myosin heavy chain kinase of Dictyostelium discoideum, a species of soil-living amoeba, and eukaryotic elongation factor 2 kinase (eEF2K). Sequence alignment of the yeast two hybrid putative binding partner revealed that it was a part of a larger open reading frame. This larger protein was 1863 amino acids in length and had a molecular mass of 212 kDa. Interestingly, the larger protein had high sequence similarity to the melastatin subfamily of TRP channels, and thus this newly discovered protein was dubbed TRPM7. Sequence analysis of TRPM7 predicted

6 transmembrane domains and a zinc finger motif within the kinase domain118. Also in 2001,

Nadler et al. used a bioinformatics approach to discover TRPM7 in the process of classifying ion channels in hematopoietic cells. They then went on to generate TRPM7-deficient DT40 B cells to study the role of TRPM7 in cell physiology, and discovered that they could only successfully delete one allele of TRPM7; deleting both alleles produced no viable cells. To test whether TRPM7 was indeed important for cell survival, Nadler and colleagues cloned and

24 expressed a tamoxifen inducible Cre-mediated deletion of TRPM7 in DT40 B cells. Upon inducing deletion, cells underwent growth arrest and died within 72 hours. This was the first study to implicate an ion channel in cell survival. Also, this study was the first to identify that TRPM7’s currents are regulated by magnesium and ATP. Aptly named magnesium-nucleotide regulated metal ion currents (MagNuM), TRPM7 currents are inhibited in the presence of intracellular free magnesium or Mg-ATP115. The third group to clone TRPM7 was Ryazanova and colleagues119.

In attempt to clone unconventional α-kinases, they looked for proteins with similar homology to eEF2K and myosin heavy chain kinase. In the process they found that one alpha kinase was covalently linked to a channel and was expressed in mouse melanoma cells and kidney cells119.

TRPM7 is encoded by 39 exons and has been mapped to chromosome 15 in the q21.2 region in humans. In mice, TRPM7 is also coded by 39 exons, but located on chromosome 2120.

There is 95% sequence similarity between mouse and human TRPM7115. TRPM7 consists of a cytoplasmic N terminus, 6 transmembrane domains, a TRP box domain, a coiled-coiled domain, and a kinase domain (Figure 1.5)115,118.

1.4.2 Structure of TRPM7

TRPM7 (Figure 1.5) shares high homology regions in the N-terminus with other TRPM channels121. The highly conserved sequences, called melastatin homology regions, on the N- terminus imply a potential role in TRPM7 function however these roles remain largely undefined.

One potential role of the N terminus maybe to mediate vesicle fusion as the N-terminus of

TRPM7 was found to associate with snapin, a protein important for vesicle fusion and neurotransmitter release122. The N-terminus may also play a role in transporting TRPM7 to the plasma membrane, as has been suggested for TRPM2 since deletion of the first 110 amino acids

A B Pore

Plasma membrane

TRP Box Melastatin Coiled Coil Region Homology Region Thr/Ser Rich Region

Kinase

Figure 1.5 TRPM7 Structure. (A) Monomer of TRPM7 channel in a lipid membrane. The pore forming loop is indicated by P (B) Homotetramer of TRPM7 where each teal cylinder represents a single unit of TRPM7. Kinase domains are represented as purple cones. Pore indicated by arrow

of the N-terminus prevented its transportation to the plasma membrane121. Perhaps, the N- terminus has a similar function in transporting TRPM7.

TRPM7 has 6 hydrophobic domains which were determined via sequence analysis. These hydrophobic domains were later determined to be transmembrane domains that span between amino acids 756 and 1095115,123. When in a homotetramer, or heterotetramer with TRPM6, the 4 pore-forming loops found between the 5th and 6th transmembrane domain forms the channel that is permeable to cations (Figure 1.5)115. In the pore forming loop, there are four negatively-charged residues that are conserved across all TRPM channels. This cluster of negative charges from all the subunits of the tetramer are thought to be important for ion selectivity124. For example, although TRPM7 is permeable to all divalent cations, it exhibits selective permeability as follows:

Zn2+> Ni2+> Ba2+> Co2+>Mg2+> Mn2+> Sr2+> Cd2+> Ca2+ 125, 115.

The coiled-coil domain in TRPM7 is located between amino acid residues 1198 and

1250123. These domains are known to be widespread protein-protein interaction structural motifs126. Each helix comprises a repeating heptamer, that when coiled form a hydrophobic strip down the helix. The hydrophobic strips of several helices can interact together to form a coiled coil127. When the putative coiled-coil domains of TRPM7 were purified, gel filtration of these proteins showed that the domains are capable of self-directed assembly128. Indeed, X-ray crystallography studies of the coiled-coil domain of TRPM7 shows that it exists as a tightly twisted, 4 stranded symmetrical coiled coil. This demonstrates that the coiled-coil domain is responsible for coordinating the quaternary structure of TRPM7128.

The TRP box is a conserved stretch of hydrophobic residues found on the C-terminal side of the coiled-coil domain (AA 1109 – 1128)83,123. It is thought to serve as a coiled-coil zipper that holds the channel in a closed conformation. Some studies have suggested that the TRP box has

129 putative PIP2 binding sites that could regulate channel activity . Whether PIP2 is a positive or negative regulator of channel activity is still debated123.

The kinase domain is the most unique feature of TRPM7. Other than TRPM6, there are no other known channel proteins with kinase domains80. This kinase is an atypical α-kinase which phosphorylates serine and threonine residues in the context of α-helices (hence its name) instead of β-turns, loops or irregular structures like conventional kinases130. In humans, only 6 other α- kinases have been identified and all bear similar homology to each other130. In order to stabilize the tertiary structure, α-kinases have a zinc finger motif that holds a single zinc atom. Disrupting the zinc finger motif results in loss of zinc binding and subsequent loss of kinase function. Despite insignificant sequence homology between conventional and α-kinases, X-ray crystallography reveals that the central catalytic core of α-kinases is structurally similar to conventional kinases.

In fact, sequence comparisons show that residues important for phosphorylation have been conserved between the two families of kinases131. Understanding the structural components of

TRPM7 can provide some insight on its function.

1.4.3 Function of TRPM7

As mentioned previously, TRPM7 is important for the survival of DT40 B cells115. Whole cell currents from B cells lacking TRPM7 demonstrated no evidence of magnesium currents, indicating that TRPM7 is the main regulator of magnesium in DT40 B cells132. Expression of other magnesium transporters like Solute Carrier Family 41 member 2 (SLC41A2) can partially rescue growth in TRPM7-KO DT40 B cells132. While this demonstrates that magnesium is important for cellular growth, the partial rescue by SLC41A2 also highlights the unique gating capabilities of the channel domain of TRPM7.

As a tetramer, the kinase domains of each TRPM7 subunit can autophosphorylate each other128,133. In vitro phosphorylation assays show that TRPM7 undergoes extensive autophosphorylation at a C-terminal serine/threonine-rich region in an incremental fashion. Once fully phosphorylated, the kinase domain could phosphorylate other substrates such as non-muscle

Myosin II134. The extensive phosphorylation of TRPM7 is thought to initiate a conformational change in the kinase domain, enabling it to better bind to other proteins. Alternatively, TRPM6 can also facilitate the phosphorylation of TRPM7134.

The kinase domain of TPRM7 has several putative binding partners. One of the first binding partners identified was PLC118. Confirmed by pulldown assays, the kinase domain of

TRPM7 can associate with several isoforms of PLC118. Originally, activation of PLC was

135 correlated with inactivation of the TRPM7 channel which was attributed to hydrolysis of PIP2 .

Other studies have shown a directly contradictory result, where PLC agonists increase TRPM7 currents and elicit TRPM7 dependent flux of calcium136. Additionally, TRPM7 was shown to phosphorylate PLCγ2 in B cells on a serine and threonine residue in the C2 domain. One of these phosphorylation sites were reported to be important in regulating PLC activity under hypomagnesic conditions. WT DT40 B cells could flux calcium in both normomagnesic and hypomagnesic conditions, while DT40 cells expressing the PLCγ2 serine mutant could only flux in normomagnesic conditions. These findings indicate that phosphorylation of PLCγ2 by

TRPM7 is important for regulating calcium flux in hypomagnesic conditions137. TRPM7 was also shown to modulate protein expression in response to extracellular magnesium by interacting with eEF2K138. eEF2K is responsible for inhibiting translational elongation by ribosomes via the phosphorylation of eEF2139. In hypomagnesic conditions, TRPM7 was found to phosphorylate eEF2K, which in turn phosphorylated eEF2 and stopped translation138. These findings show that

TRPM7 is important for sensing and responding to magnesium levels in the environment through both channel and kinase domain

The kinase domain of TRPM7 was also shown modulate cell morphology of neuroblastoma cells by phosphorylating myosin IIA heavy chain. In vitro kinase assays with purified TRPM7 and myosin IIA heavy chain show that phosphorylation of myosin is dependent on the kinase domain of TRPM7116. Indeed, when TRPM7 is over expressed in neuroblastoma cells and activated by a PLC agonist, cells spread larger and form more focal adhesions116. The association of the kinase domain of TRPM7 and myosin is perhaps not surprising, as the α-kinase of TRPM7 is very similar to the myosin heavy chain kinase found in Dictyostelium119. Myosin heavy chain kinases inhibit Myosin IIA by phosphorylating the heavy chain tail on a specific threonine residue. This leads to filament disassembly and release of cortical tension140. Through this mechanism, TRPM7 can indirectly regulate the actomyosin network in cells.

The kinase domain of TRPM7 also phosphorylates annexin-1, a calcium and phospholipid binding protein that modulates inflammatory responses and cell death141. Annexin was found to be phosphorylated at the fifth serine, located at the N-terminus of annexin-I142. Since TRPM7 is also implicated in proliferation and cell death, it could be that regulation of apoptosis involves the phosphorylation of annexin-I.

Although the channel and kinase domain affect different cellular processes, the question arises whether each domain can function independently or not. Initially, Runnels et al showed that a TRPM7 kinase deletion mutant was unable to initiate a current118. In 2003, another study showed that phosphotransferase deficient kinase mutant only affects the sensitivity of the channel to inhibition by Mg2+ and does not prevent currents from forming143. Conversely, kinase deletion mutants are more sensitive to inhibition by Mg2+143. The discrepancy between the two TRPM7

30 mutants remains unexplained. Further strengthening the notion that TRPM7 can produce a current independently of the kinase domain, studies have shown that the kinase domain of TRPM7 can be cleaved from the channel domain, resulting in a still functional kinase domain and channel144,145. The kinase is also catalytically active when expressed as a soluble cytoplasmic variant134.

TRPM7 has been demonstrated to be important in the migration of fibroblasts117, T cells146, and tumour cells147,148. In Swiss 3T3 fibroblasts, breast cancer, and nasopharngeal carcinoma cells, wound healing assays showed that TRPM7 knockdown via siRNA prevented cell migration113,147,148. Furthermore, siRNA knockdown of TRPM7 decreased the velocity of migrating activated T cells146. In both T cells and nasopharngeal carcinoma cells, the decrease in migration was attributed to the decrease in calcium flux caused by the lack of TRPM7. In fibroblasts, migration defects were inferred to be due to a lack of intracellular magnesium as expression of other magnesium channels rescued TRPM7 knockdown117. Taken together, these studies suggest that the channel domain of TRPM7 is important for regulating migration.

1.5 Magnesium Function

1.5.1 Overview

Magnesium ions act as co-factors for several enzymes and are important for the active conformation of proteins (Table 1.1)149. At the cellular level, magnesium has also been implicated in mediating cell contraction, motility and proliferation143,150. In fact, magnesium was found to regulate phospholipase activity in a specific isoform of PLC found in proliferating B cells151.

Magnesium is the most abundant divalent cation in the mammalian cell, where total concentrations range from 14 mM to 20 mM121. Most magnesium (4-5 mM) is present in the cytosol in a complex with ATP or nucleotide-phosphate moieties149. Free magnesium is harder to

Enzyme Function Diacylglycerol kinase ζ Requires magnesium to phosphorylate diacylglycerol to (DGK ζ) phosphatidic acid152. Important for the downregulation of signal in lymphocytes153. Phosphatase and tensin Requires magnesium to dephosphorylate phosphatidyl inositol homologue (PTEN) 3, 4, 5-triphosphate to phosphatidyl inositol 4, 5-bisphosphate. Important for the downregulation of signal in lymphocytes154. Dynein Cytoskeletal motor protein that moves cargo toward the minus end of tubulin155. Requires magnesium for progressive movement. Important in the movement of microclusters to the cSMAC55 Large ribosomal subunit Requires magnesium to associate with the small ribosomal subunit in order to translate mRNA into protein156. Phospholipase C γ1 (PLCγ1) Requires magnesium to hydrolyze phosphatidyl 4, 5- bisphosphate into diacylglycerol and inositol triphosphate. Important for the activation of T cells113. Rho family GTP binding Hydrolyses Guanine Triphosphate to guanine diphosphate proteins which regulates several the reorganization of actin, which (cdc42, Rac1, RhoA) contributes to several cellular processes including the formation of filopodia and lamellipodia. Magnesium was found to slow on and off rate of bound nucleotides157. DNA polymerase β Magnesium binding to catalytic site is critical for selection of correct nucleotide during DNA replication and repair by inducing subtle conformational changes158. DNA restriction enzyme Requires magnesium bound to catalytic site to cleave DNA159. EcoRV

Table 1.1 Enzymes requiring magnesium as a co-factor

32 measure, but is estimated to range from 0.5 mM to 1 mM in the cytoplasm of mammalian cells and makes up just 5% of total cellular magnesium149. In humans, the serum concentration of magnesium is around 1mM149.

The chemical properties of Mg2+ ions make it a potential antagonist for Ca2+ ions. Ca2+ has a larger atomic radius, but Mg2+ has a higher affinity for electronegative oxygen, which leads to a greater hydrated radius160. Hydrated Mg2+ can bind in place of Ca2+ and prevent the conformational changes associated with Ca2+ binding. For example, Mg2+ competes with Ca2+ for the binding site on the helix-loop-helix motif found on calmodulin160. Although the binding of

Mg2+ does not change the conformation of calmodulin, it prevents the binding of calcium and effectively slows down its function160. Taken together, these properties of magnesium demonstrate that it is important for cellular function.

1.5.2 Importance of magnesium in the immune system

Since the 1930’s a myriad of studies involving rats on magnesium deficient diets demonstrate the importance of magnesium to the immune system. Initial studies followed the progression of symptoms of rats deprived of magnesium in their diet. After 4 days of magnesium deprivation, blood vessels have dilated, resulting in reddened skin. Eventually, these rats have seizures which results in death 37 days after starting the magnesium deficient diet161. In more recent studies, magnesium deprivation resulted in splenomegaly, and accelerated thymic involution along with increased neutrophils and lymphocytes in the peripheral blood162–164.

Intestinal mucosa are also effected, as rats fed magnesium deficient diets for 3 days had abnormally flattened microvilli as well increased intestinal resident neutrophils and lymphocytes165. In the context of the B cell response, serum IgG and fecal IgA of these mice were also decreased165. This indicates that dietary magnesium is important for the function of the immune system.

The importance of magnesium in human immune function is highlighted by the X-linked disease XMEN (X-linked immunodeficiency with magnesium defect and Epstein–Barr virus infection and neoplasia) which is characterized by uncontrolled viral infections, and impaired thymic production of T cells and T cell function166. XMEN patients lack the expression of a magnesium channel called MagT1. MagT1 fluxes magnesium after T cell stimulation and was found to be important for the activation of PLCγ1, an important signalling molecule in T cell activation113. However, in B cells, the deletion of MagT1 has no effect on B cell signalling113.

This suggests that another channel must regulate magnesium in B cells.

1.6 Thesis Rational and Hypothesis

Understanding the mechanisms regulating B cell development and activation is important for expanding potential therapies for several B cell related immune disorders. Currently, there is a poor understanding of ion channels in the immune system. Knowledge regarding the role of ion channels in the immune system can be useful to developing pharmaceutical agents to manipulate the activity of these channels.

TRPM7 is of particular interest since it has been implicated in the regulation of cellular magnesium homeostasis115.The homeostasis of magnesium is especially important as magnesium is known to be a co-factor for various enzymes and is important in protein translation and thus, cell survival167. This is evidenced by growth in arrest in DT40 B cells that have been cultured in media lacking magnesium168. Cell survival is tightly regulated in B lymphopoeisis, and may require the regulation of magnesium. Thus, the aim of this thesis was to examine the role of

TRPM7 and magnesium in B cell development. Although TRPM7 is more permeable to other cations, magnesium is of special interest given its ability to regulate TRPM7 channel currents.

Since TRPM7 has been implicated in embryonic survival and survival of a B cell line114,115, we

34 hypothesized that TRPM7 is necessary for B cell development. Our study is the first to explore a

B cell specific deletion of TRPM7 in a murine model.

TRPM7 is a rare channel with a kinase domain and thus may be involved not only in the regulation of ion entry but also the phosphorylation of downstream effector molecules. B cell activation in vivo is triggered by APCs that present antigen in a membrane bound form13,169. In response to this type of antigen, B cells undergo a spreading and contraction response that requires significant remodeling of the actin cytoskeleton27. Actin remodeling has been shown to be highly dependent on the activation of PLCγ240, however the exact mechanism that couples

BCR engagement and subsequent PLCγ2 activation to actin remodeling is unclear. Since the kinase domain of TRPM7 has shown to phosphorylate residues of PLCγ2137, we hypothesized that TRPM7 may regulate actin reorganization and ultimately B cell activation through PLCγ2.

CHAPTER 2 TRPM7 is Essential for B Cell Development

Author Contributions

I would like to acknowledge the following individuals for contributing to this work. Hifza Buhari generated Figure 2.3. Tiantian Zhao generated Figure 2.5.

2.1 Introduction

Transient receptor potential (TRP) channels are a large and diverse superfamily of ion channels which have a large range of cation specificities and cellular functions. Some of these channels play a role in maintaining homeostasis of ions like calcium and magnesium80.

Magnesium plays a very important role in cellular systems, as several enzymes use cations as co- factors that are needed to function. For example, the subunits of ribosomes require magnesium to associate and begin translation156. The importance of magnesium to survival is evidenced by rats that die onset 37 days of being placed on a magnesium depleted diet161. Moreover, rats on a magnesium depleted diet show symptoms of increased inflammation, such as redness of the skin and edema162. Ion deficiencies due to mutations in magnesium channels leads to illness, highlighting the role magnesium plays in human health86,87,113.

TRPM7 is a member of the TRPM (melastatin) subfamily of TRP channels, along with its sister channel TRPM6. They both conduct magnesium and calcium currents into the cell and are the only known channel proteins to have an associated kinase domain80,115. This atypical kinase domain has the potential to integrate signals from the extracellular environment into the cell137,138. While TRPM7 is ubiquitously expressed, TRPM6 is mostly expressed in the colon and kidney94. Interestingly, a spontaneous point mutation in TRPM6 leads to a magnesium absorption defect in humans called hypomagnesaemia with secondary hypocalcaemia (HSH), and can be remedied by a high magnesium diet84. Despite having over 50% sequence similarity with 36

TRPM6, mutations in TRPM7 lead to a more drastic consequence – death114,115. It is the only ion channel that has been implicated in cell survival; deletion of TRPM7 results in embryonic lethality before embryonic day 7 in mice114. Embryonic lethality is also seen in zebrafish and toads when TRPM7 is mutated170,171.

TRPM7 was first identified in 2001 as a constitutively active magnesium channel that is inhibited by intracellular free Mg2+ or ATP-Mg. This study knocked-out TRPM7 in the DT40 chicken B cell line and found that TRPM7-KO cells underwent growth arrest and eventually apoptosis, unless supplied with high levels of extracellular magnesium115. This implies that magnesium plays an important role in cell survival. More importantly, it demonstrates that

TRPM7 is solely responsible for magnesium homeostasis within these cells. TRPM7 has also been conditionally knocked-out in murine T cells. Unlike DT40 B cells, thymocyte development is only delayed in T cells lacking TRPM7172. Notably, although there is a delay in thymopoiesis, the number of peripheral T cells is only modestly reduced, indicating that TRPM7 is not necessary for T cell development in a murine model172. These findings led us to pose the question whether

TRPM7 is important for development of B cells in vivo.

To investigate the role of TRPM7 in B cell development, we generated a conditional

TRPM7 knockout mouse by crossing floxed TRPM7 mice with mb1-cre mice. The progeny of this cross express cre-recombinase under the promotor of mb1, a B cell specific marker, and results in the knockout of TRPM7 specifically in B cells. Our findings show that TRPM7 is absolutely critical for the development of B cells. Specific deletion of TRPM7 in B cells leads to the loss of B cells in all peripheral lymphoid tissues, and lack of Peyer’s patches on the small intestine. Interestingly, in our TRPM7 knockout model, we observed a larger population of

CD11b+ granulocytes, especially neutrophils. This expanded population has never been described before in mouse models lacking B cells. The lack of B cells was attributed to apoptosis of Pre B

37 cells. By culturing hematopoietic cells (HSC) extracted from murine bone marrow isolated from

WT and TRPM7-/- mice with the stromal cell line OP9, we demonstrated in vitro, that addition of high levels of extracellular magnesium to HSCs partially rescued B cell development. This shows that the Mg2+ transport function of TRPM7 is essential for B cell survival during development.

2.2 Results

2.2.1 Generation of TRPM7 conditional knockout

To study the role in TRPM7 in murine B cell development, we created a conditional knockout in B cells by crossing TRPM7flox/flox mice with mice expressing cre recombinase under the B cell specific mb1 promotor (mb1cre/+)173. The resultant progeny consisted of

TRPM7flox/floxmb1-Cre- (WT), TRPM7flox/+mb1-Cre+ (TRPM7+/-), and TRPM7flox/floxmb1-Cre+

(TRPM7-/-) mice. To assess embryonic survival of these progeny, we compared the frequencies of each genotype to expected frequencies according to Mendelian inheritance. The frequency of genotypes was similar to expected frequencies, indicating that deletion of TRPM7 from the B cell lineage does not affect embryonic development (Table 2.1).

A TRPM7fl/fl mb1+/+ X TRPM7+/+mb1cre/+ Genotype Expected Actual TRPM7fl/+mb1+/+ 50% 46.9 % (53) TRPM7fl/+mb1cre/+ 50% 53.1 % (55)

B TRPM7fl/flmb1+/+ X TRPM7fl/fl mb1cre/+ Genotype Expected Actual TRPM7fl/flmb1+/+ 50% 54.9 (84) TRPM7fl/flmb1cre/+ 50% 45.1% (69)

C TRPM7fl/fl mb1cre/+ X TRPM7fl/+ mb1cre/+ Genotype Expected Actual TRPM7fl/+mb1+/+ 25% 26.4% (52) TRPM7fl/flmb1+/+ 25% 27.9 % (55) TRPM7fl/+ mb1cre/+ 25% 21.8 % (43) TRPM7fl/flmb1cre/+ 25% 23.9 % (47)

Table 2.1 Conditional deletion of TRPM7 in B cells does not alter embryonic survival. Mendelian inheritance tabulations for the indicated breeding schemes showing expected and actual genotypes. Number in brackets indicates number of weaned pups. Genotype effect on survival was assessed using Chi-squared test (A;X2=0.0007, B; X2=0.04, C; X2=1.72)

2.2.2 Peripheral lymphoid tissues are devoid of B cells in TRPM7-/- mice.

To examine B cells in peripheral lymphoid tissues, we stained for B cell specific markers

CD19 and B220. Using flow cytometry, we were unable to detect B cells (CD19+B220+) in the spleen, lymph nodes, or blood of TRPM7-/- mice. In contrast, TRPM7+/- mice had similar percentages of B cells to WT mice in all peripheral lymphoid organs, indicating that some expression of TRPM7 is sufficient to rescue development of B cells (Figure 2.1A). We also examined T cells (CD3+) in these peripheral organs. The percentage of T cells increased in

TRPM7-/-, however the absolute number of T cells decreased in the spleen, signifying that B cells are important for splenic T cell development (Table 2.2). A similar trend is seen in B cell deficient

µMT mice174. We also wondered whether the development of unconventional B1 B cells would be compromised like conventional B2 cells seen in spleen and lymph nodes of TRPM7-/- mice.

Cells obtained via peritoneal lavage were immunostained for CD23 and B220, then analyzed via flow cytometry. Indeed, both B2 (B220+CD23+) and B1 (B220+CD23-) B cells were absent in

TRPM7-/- mice. WT and TRPM7+/- mice had similar percentage of B1 and B2 B cells in the peritoneum (Figure 2.1B).

Peyer’s patches are also important peripheral lymphoid organs found on small intestines.

As other B cell deficient mice have small or non-existant Peyer’s patches175, we wanted to determine if Peyer’s patches were altered in TRPM7-/- mice. To investigate this, small intestines were isolated from mice and treated with 7% acetic acid to aid in visualization of Peyer’s patches.

Intestines obtained from TRPM7-/- mice lacked visually discernible Peyer’s patches (Figure

2.1C). TRPM7+/- mice had similar number of Peyer’s patches, with a similar percentage of B cells as Peyer’s patches from WT mice (Figure 2.1C, D).

Figure 2.1 TRPM7 is essential for B cell development. (A) Flow cytometry analysis of B220+CD19+cells from the spleen, lymph node, and blood of WT, TRPM7 +/-, or TRPM7-/- mice. (B) Peritoneal lavage samples were stained with antibodies against B220, CD23 and B2 cells were identified as B220+CD23+; B1 as B220+ CD23-. (C) Peyer’s patches (circles) from the small intestine (duodenum to ileum) from WT, TRPM7 +/-, or TRPM7-/- mice. (D) Peyer’s Patches isolated from WT and TRPM7+/- mice were stained with antibodies against CD19 and B220 and analyzed by flow cytometry. Quantification of percentage of the indicated population is shown to the left of the flow plots. Data are representative of at least 3 mice of each genotype. A, B were analyzed using Kruskall-Wallis test followed by Dunn’s multiple comparison test, *P<0.05, D was analyzed using Mann-Whitney test; ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001

A WT TRPM7 +/- TRPM7-/- Spleen 43.10± 2.60 50.88 ± 6.92 59.43 ± 4.503 * Lymph Node 71.25 ± 2.89 77.05 ± 3.59 89.92 ± 3.691 * Blood 52.17 ± 5.38 50.73 ± 8.35 79.67 ± 1.48* Peyer’s Patch 20.73 ± 3.634 15.14 ± 3.677 N/A

B WT TRPM7 +/- TRPM7-/- Spleen 1.83x107 ± 3.80 x106 2.76x107 ± 1.09x107 4.58x106 ± 1.64x106* Lymph Node 4.67x105 ± 7.26 x104 7.83x105 ± 5.60 x104 6.23x105 ± 2.02 x105 Peyer’s Patch 5.69x104 ± 8.49 x103 7.79x104± 1.91 x104 N/A

Table 2.2 Proportion of T cells is increased in peripheral lymphoid tissues of TRPM7-/- mice. Quantification of (A) percentage or (B) absolute number of T cells in spleen, 2 lymph nodes or 4 Peyer’s Patches as mean ± SEM. Data are representative of at least 3 mice of each genotype. Comparisons are made against WT using Kruskall-Wallis test followed by Dunn’s multiple comparison’s test *p<0.05.

2.2.3 Secondary lymphoid organs have altered architecture

As TRPM7-/- mice have no peripheral B cells, we wondered how this would change the architecture of secondary lymphoid organs such as the spleen. Without B cells, spleens from

TRPM7-/- were 50% smaller in mass than WT or TRPM7+/- spleens (Figure 2.2A, B). To determine whether B cell deficient spleens have altered architecture, spleens from WT, TRPM7+/- and TRPM7-/- were cryosectioned and stained for B cells (IgD+), T cells (CD3+) and monocyte/granulocytes (CD11b+). We could not detect any B cells in the spleen, consistent with our flow cytometry data in TRPM7-/- mice (Figure 2.2C). The follicles of B cell deficient spleens were non-existent, while T cell zones remained organized in compartments. Interestingly, there appeared to be an expanded population of CD11b+ cells in TRPM7-/- spleens.

Lymph nodes analyzed by flow cytometry also show a complete lack of B cells, which prompted us to investigate their architecture as well. Surprisingly, inguinal lymph nodes from

WT, TRPM7-/- and TRPM7+/- mice were similar in size (Figure 2.3A). When cryosectioned and visualized via immunostaining, we observed that lymph nodes are mostly filled with T cells

(CD3+) and lacked B cells (IgD+). No discernible change in CD11b+ populations were observed between WT, TRPM7-/- and TRPM7+/- lymph nodes (Figure 2.3B).

Figure 2.2 Splenic architecture is altered in TRPM7-/- mice. (A) Spleens isolated from WT, TRPM7+/-, or TRPM7-/- were weighed and quantified as a percent of total body mass in (B). (C) Epifluorescent microscopy images of splenic cryosections derived from WT, TRPM7 +/-, or TRPM7-/- mice. Cryosections were stained with antibodies against IgD (cyan), CD3 (magenta) and CD11b (yellow). Scale bar 0.5 mm. Data are representative of at least 3 mice of each genotype. Statistical significance was assessed by Kruskall-Wallis test followed by Dunn’s multiple comparison’s test **p<0.0, ***p<0.001

Figure 2.3 Architecture of lymph nodes in TRPM7-/- mice. (A) Inguinal lymph nodes extracted from WT, TRPM7+/-, or TRPM7-/- mice. (B) Representative epifluorescence microscopy images of lymph node tissue cryosections derived from WT (top), TRPM7+/- (middle), or TRPM7-/- (bottom). Tissue sections were stained with monoclonal antibodies against IgD (cyan), CD3 (magenta) and CD11b (yellow). Data are representative of at least 3 mice of each genotype. Scale bar 0.5 mm.

2.2.4 The population of splenic myeloid cells is increased in TRPM7-/- mice.

We observed an expanded population of CD11b+ cells in the spleen of TRPM7-/- mice, which is indicative of an increase in myeloid cells. Myeloid cells are important cells of the innate immune system and comprised of neutrophils, eosinophils, monocytes, dendritic cells and macrophages. To examine these populations, we stained total splenocytes from WT and

TRPM7-/- mice with CD11b, F4/80, Ly6G, and CD11c. Total splenocytes from TRPM7-/- mice had a larger population of FSChiSSChi cells (Figure 2.4A), signifying an expanded population of granulocytes, which are cells that have a granule rich cytoplasm176. Indeed, we observed a 3-fold increase in eosinophils (CD11b+SSChi), a 2.6-fold increase in monocytes (CD11b+SSChi) and a

5.6-fold increase of neutrophils (CD11b+Ly6G+) in TRPM7-/- splenocytes (Figure 2.4C, E).

Furthermore, there was no difference in the population of red pulp macrophages (RPMΦ)

(CD11b-F4/80+) and dendritic cells (CD11b+CD11c+) between WT and TRPM7-/- mice (Figure

2.4B, D).

Figure 2.4 Myeloid population is expanded in TRPM7 deficient mice. Flow cytometry analysis of the spleen of WT and TRPM7-/- mice. Gating strategies are shown on the far left. Single cell suspensions of total splenocytes were stained with CD11b, F4/80, Ly6G, and CD11c. Quantification of the data are shown in the far right column and indicate the percentage of cells in the indicated gates. (A) Cells were gated on granulocytes (FSChiSSChi) (A-E) Cells were gated sequentially as follows: (A) DAPInegSinglets; (B) Red Pulp MΦ (CD11bloF4/80hi); (C) Neutrophils (CD11bhiLy6Ghi); (D) Dendritic cells (CD11bhiCD11chi); (E) Eosinophils (CD11bhiSSChi) and monocytes (CD11bhiSSClo). Data were pooled from at least 3 independent experiments with at least 3 mice for each phenotype. Statistical significance was assessed by Mann-Whitney test *p<0.05, **p<0.01, ***p<0.001

2.2.5 B cell development is abrogated at the Pro B cell stage in TRPM7-/- mice

TRPM7 has been implicated in cell motility, we therefore wanted to determine if the loss of B cells from peripheral lymphoid tissues is due to a defect in development or cell migration147.

Cells were isolated from the bone marrow of WT, TRPM7+/-, and TRPM7-/- and examined using

B220, IgM, and IgD expression to assess Pre-Pro B cells (B220+IgD-IgM-), immature B cells

(B220+IgD-IgMlo), transitional B cells (B220+IgD-IgMhi), and mature B cells (B220+IgDhiIgMhi) by flow cytometry. TRPM7-/- bone marrow had ~80% less B220+ cells compared to WT and

TRPM7+/- bone marrow (Figure 2.5A). Of these cells, 95% were IgM- B cells in TRPM7-/- mice, whereas only ~53% were IgM- in WT or TRPM7+/-. Additionally, TRPM7-/- bone marrow was devoid of immature, transitional or mature B cells, unlike WT or TRPM7+/- (Figure 2.5A) To further define stages of B cell development, we stained bone marrow cells for B220, IgM, CD24 and CD43. Pre-Pro B cells were identified as B220+IgM-CD43+CD24-, Pro B cells as B220+IgM-

CD43+CD24+, and Pre B cells as B220+IgM-CD43-CD24+. In TRPM7-/- mice, we observe an increase in the proportion of Pre-Pro and Pro B cells in comparison to WT and TRPM7+/-.

Quantification of absolute number of cells at each stage of development shows that cell numbers at the Pre-Pro and Pro stages from WT, TRPM7-/-, and TRPM7+/- are similar. However, there are significantly more Pre B cells in WT and TRPM7+/- than TRPM7-/- mice, demonstrating that there a loss of cells at the Pre B cell stage (Figure 2.5B). To determine if the lack of Pre B cells is due to apoptosis, we further stained bone marrow cells with Annexin V and 7-Aminoactinomycin D

(7AAD) to distinguish between early and late apoptosis. Annexin V binds to phosphotidylserine which usually present on the inner leaflet of the plasma membrane. As the cell begins to die, phosphotidylserine is flipped to the outward facing leaflet. 7AAD is a dye that fluoresces upon binding DNA. This dye can only enter the cell if the plasma membrane has been compromised, which occurs later in apoptosis. Therefore, cells in early apoptosis are AnnexinV+7AAD- while

48 cells in late apoptosis are AnnexinV+7AAD-. Similar proportions of late apoptotic cells were observed in both pre-pro and Pro B cells of WT, TRPM7+/- and TRPM7-/- cells. Strikingly, Pre B cells in TRPM7-/- mice have a larger percentage of cells in late apoptosis compared to WT and

TRPM7+/-. There is also a small increase of TRPM7-/- Pro B cells that are in early apoptosis.

Although, not statistically significant, it suggests that developing B cells in TRPM7-/- mice begin to die at the Pro stage and become more apoptotic as they progress to the Pre B cell stage. This demonstrates that TRPM7 is required for the progression of B cell development to the Pre B cell stage, whereby the absence of TRPM7 leads to the death of Pre B cells.

Figure 2.5 B cell development is abrogated at the Pro B cell stage in TRPM7-/- mice. Flow cytometry analysis of bone marrow from WT, TRPM7+/-, or TRPM7-/- mice. (A) Cells were stained with antibodies against B220, IgD and IgM. Pre-Pro B cells were identified as B220+IgD- IgM-, Immature B cells as B220+IgD-IgMlo, Transitional B cells as B220+IgD-IgMhi, mature B cells as B220+ IgDhi IgMhi. (B) Bone marrow cells were stained with antibodies against CD24, CD43, IgM. Cell viability was additionally assessed with Annexin V and 7AAD. Pre B cells were identified as B220+IgM-CD24+CD43-, Pro B cells as B220+IgM-CD24+CD43+, and Pre-Pro B cells as B220+IgM-CD24-CD43+. Cells in early apoptosis (Annexin V+7AAD-) and late apoptosis (Annexin V+7AAD+) were identified for each B cell subpopulation. Data are representative of at least 4 mice of each genotype. Statistical significance was assessed by Kruskall-Wallis test followed by Dunn’s multiple comparison’s test *p<0.05, **p<0.01, ***p<0.001, ns = not significant

2.2.6 Kinase activity of TRPM7 is dispensable for B cell development

To determine if the kinase activity of TRPM7 is important for B cell development, we made use of a knock-in mouse model expressing TRPM7K1646R/K1646R (TRPM7KR/KR), which abolishes kinase activity. Peripheral lymphoid tissues were examined for the presence of B cells in both WT and TRPM7KR/KR mice by isolating cells from the spleen, inguinal lymph nodes, blood and Peyer’s patches and staining for B220 and CD19. We observed that TRPM7KR/KR peripheral lymphoid tissues had similar percentage of B cells compared to WT mice (Figure 2.6). This demonstrates that the kinase function of TRPM7 is not required for B cell development.

Figure 2.6 TRPM7 Kinase activity is dispensable in B cell development. Flow cytometry analysis of spleen, lymph node, blood, Peyer’s patches (P.P.), peritoneal lavage and bone marrow of WT and KI mice. Spleen, lymph node and blood and Peyer’s patch (P.P) derived cells were stained for CD19 and B220. B cells were identified as B220+CD19+cells. Quantifications are shown to the right of the figure. Data are representative of 3 mice of each genotype. Statistical significance was determined with Mann-Whitney statistical test. Ns = not significant.

2.2.7 B cell development can be partially rescued with extracellular magnesium

DT40 B cells lacking TRPM7 and embryonic stem cells from a TRPM7 kinase deficient mouse model are unable to survive unless supplemented with high levels of extracellular magnesium115,177. To determine whether supplying high levels of extracellular magnesium to

TRPM7 deficient hematopoietic stems cells (HSCs) would rescue B cell survival in development, we made use of a stromal cell line (OP9-R7FS) stably expressing IL-7, Flt3L, and stem cell factor

(SCF). OP9-R7FS stromal cells are established from mouse embryonic stem cells and provide an environment that promotes HSC differentiation into B cells178,179. HSCs were purified from bone marrow of WT and TRPM7-/- mice and co-cultured in vitro with OP9- R7FS cells (Figure 2.6A).

Cells were cultured with additional 0, 5 or 10 mM MgCl2. After 9 days of co-culture, cells were stained for B220 and CD19 to check for the presence of B cells (B220+CD19+) via flow cytometry

(Figure 2.7B). Regardless of magnesium concentration, 80% of WT HSCs differentiated into

B220+CD19+ cells. Interestingly, there is a dose-dependent increase in the proportion of

B220+CD19+ cells derived from TRPM7-/- HSCs in response to extracellular magnesium.

However, even 10 mM MgCl2 could not fully rescue B cell development. This finding suggests that the magnesium transport function of TRPM7 is important for B cell development.

Figure 2.7 Supplementation with Mg2+ partially supports B cell development. (A) Flow cytometry analysis of hematopoietic stem cells (HSCs) enriched from WT or TRPM7-/- bone marrow. Cells were stained with antibodies against B220 and CD19 to assess the presence of B cells post enrichment and quantified below. (B) HSCs were enriched from WT (top row) or TRPM7-/- (bottom row) bone marrow and co-cultured with OP9-R7FS murine stromal cells in vitro for 9 days. Cultures were supplemented with 0, 5, or 10 mM MgCl2. Loosely adherent cells were collected and stained with antibodies against B220 and CD19. Differentiated B cells were identified as B220+CD19+ cells and quantified as a percentage of total cells. Data are representative of at least 3 mice of each genotype. Statistical significance was assessed using Wilcoxon matched pairs ranked test in (A) and Freidman’s test using Dunn’s multiple comparisons test in (B); *p<0.05. ns = not significant

2.3 Discussion

In this study we developed a mouse model where TRPM7 is specifically deleted in B cells by expressing floxed TRPM7 under the mb1-cre promotor. We observed that TRPM7 is essential for the development of B cells, as there were no peripheral B cells in our knockout model. This lack of B cells in the periphery was due to apoptosis of cells at the Pre B cell stage of development.

The survival of B cells can be partially rescued by the addition of extracellular magnesium. Thus, our study is the first to show that TRPM7 plays an essential role in B cell development in vivo.

Our findings contrast with Jin et al.’s 2008 study, where deletion of TRPM7 under the

Lck promotor largely did not affect the growth and development of T cells. In this model, thymocytes are still present in the thymus after deletion of TRPM7, which occurs very early in

T cell development, during the double-negative-1 (DN1) stage of thymopoeisis180. These TRPM7 deficient thymocytes are partially blocked at the double negative 3 (DN3) stage. Despite the developmental delay, the absolute number of DN4 cells is nearly the same between WT and

TRPM7-/- thymocytes, which results in similar numbers of peripheral T cells172.

There are several reasons why the loss of TRPM7 effects B cell development more severely than T cell development. In the absence of TRPM7, the related channel, TRPM6, which is also permeable to Mg2+ might be upregulated in thymocytes and thus prevent the total abrogation in development. As TRPM6 is not expressed in B cells, this is unlikely to occur in our model94. Another magnesium channel, MagT1 may also be upregulated. Although MagT1 is expressed in both T cells and B cells, it was only found to have a role in T cell signalling and not

B cell signalling, and therefore may play more of a role in T cell development113. Mutations in

MagT1 in humans leads to X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection and neoplasm (X-MEN). This disease is characterized by a decreased number of circulating CD4 T cells which have defective signalling113. It may also be that TRPM7 was not

55 effectively deleted by Lck-driven expression of cre recombinase. Indeed, western blotting for

TRPM7 expression in lck-cre TRPM7-/fl thymocytes show that TRPM7 expression was only knocked down by ~90% in this study172. Our study shows that the presence of even one allele of

TRPM7 is able to completely rescue B cell development; hence the incomplete deletion of

TRPM7 may account for the presence of nearly normal levels of peripheral T cells in the lck-cre

TRPM7-/fl mouse model.

To discern the role of the kinase domain and the channel domain in development, we examined peripheral B cells in TRPM7KR/KR knock in mice that express a kinase dead TRPM7 mutant. We found that the lack of kinase activity did not affect B cell lymphopoeisis. This is consistent with DT40 B cells expressing a TRPM7 kinase dead mutant that can grow as well as

WT DT40 cells143. While the kinase activity may be dispensable for development, the kinase domain itself may be important. The viability of DT40 cells that express a kinase domain deletion mutant of TRPM7 is significantly reduced unless supplemented by extracellular magnesium143.

Moreover, mice expressing TRPM7 with a deleted kinase domain are embryonic lethal.

Embryonic stem cells isolated from the embryos of these mice cannot be cultured in vitro unless extracellular magnesium is added177. Indeed, TRPM7 kinase deletion mutants exhibit enhanced magnesium dependent suppression of channel activity compared to WT TRPM7 and was shown to support low levels of intracellular magnesium when expressed in DT40 B cells143. Therefore, the kinase domain of TRPM7 is important for determining the gating properties of the channel domain which ultimately impacts cell survival.

Culturing WT DT40 B cells in media depleted in magnesium leads to growth arrest at the

115,168 G0/G1 stage, similar to TRPM7-KO DT40 cells . While the growth arrest occurred in 52% of magnesium starved DT40 WT B cells but in 73% of TRPM7-KO DT40 B cells, it still implies that B cells require magnesium to grow168. Thus, we asked whether the addition of extracellular

56 magnesium would rescue B cell development. Using the OP9 stromal cell line that expresses

SCF, IL-7 and Flt3L (Brauer and Zúñiga-Pflücker, manuscript in preparation) in a co-culture with purified HSCs from TRPM7-/- mice, B cell development was partially rescued with extracellular magnesium supplement. This experiment shows that the channel domain of TRPM7 and its role in transporting magnesium is important for B cell development. Higher concentrations of extracellular magnesium should be tested to determine if it can wholly rescue B cell development.

Based on our in vivo findings, one would expect that TRPM7 deficient HSCs would not differentiate into B cells when co-cultured with OP9 stromal cells, however we see that 10% of the cultured cells are B220+CD19+ cells, even in the absence of additional extracellular Mg2+.

There are several reasons that might explain why some differentiation occurs. First, the OP9-

R7FS stromal system overexpresses key cytokines to support B cell development. SCF, Flt3L, and IL-7 signalling all feed into the PI3K/AKT/mTORc pathways and may partially rescue development since TRPM7 also feeds into this pathway. Growth arrest in TRPM7-/- DT40 can be completely rescued if reconstituted with a constitutively active PI3K168, therefore signalling through this pathway via other cytokines may restore growth. Additionally, this assay uses media that is not completely devoid of magnesium ions. Therefore this low concentration (0.4 mM) of magnesium in the media may contribute to the non-zero proportion of B220+CD19+ cells in the condition where no additional magnesium is added extracellularly.

The expanded population of granulocytes that we observed in our TRPM7 deficient mouse model has not been described before in other B cell deficient mouse models. We detected a slight increase in monocytes and eosinophils and a greater increase in neutrophils in the spleen of TRPM7-/- mice. Interestingly, B cell deficient µMT mice infected with microorganisms

(Chlamydophila abortus and Leishmania donovani), have elevated levels of neutrophils181,182; however studies have not shown neutrophil counts before infection, and therefore it is not clear

57 whether neutrophil counts were already high prior to infection181,182. Another study has shown that lymphocytes and granulocytes share a common developmental niche in the bone marrow and compete for resources183. In Rag-/- mice that lack both B cells and T cells, flow cytometry analysis of bone marrow demonstrated that granulocyte precursors (Gr-1+) were increased compared to

WT mice. The removal of lymphocytes specifically expanded the population of granulocytes, as erythroid populations in both WT and Rag-/- stay the same proportionally183. This study suggests that our finding of increased neutrophils in our B cell deficient model may in fact be representative of all B cell deficient models. Thus, it is imperative that future studies explore granulocyte populations in B cell deficient mouse models. These mouse models are used for studying B cell immunodeficiencies, and therefore it is important to determine if the increased number of granulocytes is a direct result of the lack of B cells and the mechanism that leads to it.

2.4 Materials and Methods

2.4.1Animals

129/SvEvTac TRPM7flox/flox mice were purchased from the Jackson Laboratory and were crossed with C57BL/6 Mb1-cre mice173, kindly provided by M. Reth. TRPM7flox/+Mb1cre/+ mice from F1 progeny were then back crossed with TRPM7flox/flox mice to generate TRPM7flox/flox (WT),

TRPM7flox/+Mb1cre/+ (TRPM7+/-) and, TRPM7flox/floxmb1cre/+ (TRPM7-/-). Mice were maintained on a mixed 129/SvEvTac and C57BL/6 background. TRPM7K1646R/K1646R (TRPM7K/KR)184 were kindly provided by V.Chubanov. All experiments were approved by the Local Animal Care

Committee at University of Toronto Scarborough.

2.4.2 Cell isolation and flow cytometry

Mice were euthanized via CO2 inhalation, and lymphoid organs were dissected and used for preparing single-cell suspensions by mincing through a 70 μm nylon mesh. For isolation of peritoneal cells, the peritoneal cavity was flushed with 5 mL ice-cold PBS. Bone marrow was obtained from femurs via centrifugation. Erythrocytes were lysed from spleen and bone marrow suspension using a hypotonic lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA).

Prior to surface staining, single cell suspensions (5 x 105 cells) were incubated with 2 µM

CD16/32 (BD Pharmagen, Clone 2.4G2) for 15 min in staining buffer (3% FBS, 0.02% NaN3).

Cells were then stained with antibodies listed below for 45 min on ice and analysis was performed using an LSRFortessa or an LSRII (BD Biosciences) flow cytometer. Data were analyzed using

FlowJo (Tree Star, Ashland, OR).

2.4.3 Apoptosis Assay

Cells were stained with the appropriate antibodies as detailed above and were washed once with staining buffer. Cells were subsequently stained for 15 min at RT with 8ug/mL Annexin V-Pacific

Blue (Biolegend) in Annexin V Binding Buffer (Biolegend). Cells were then washed with

59 staining buffer before resuspending in 4 µg/mL of 7AAD in staining buffer. Cells were analysed via flow cytometery.

2.4.4 Antibodies

B220-APC (Clone:RA3-6B2, Thermofisher), B220-PE (Clone:RA3-6B2, Thermofisher),

CD11b-Alexa Fluor 547 (Clone:M1/70, BioLegend), CD11b-FITC (Clone:M1/70, BioLegend),

CD11c-APC (Clone:N418, BioLegend), CD19-APC (Clone:6D5, Thermofisher), CD19-PE

(Clone:6D5, Thermofisher), CD23-eFluor 660 (Clone:B3B4, eBioscience), CD3-FITC

(Clone:500A2, Thermofisher), CD5-Pe-Cy7 (Clone:53-7.3, eBioscience), F4/80-APC-Cy7

(Clone:BM8, BioLegend), IgD-Pe-Cy7 (Clone: 11-26c.2a, BioLegend), Ly6G-Qdot 605 Brilliant violet (Clone:1A8, BioLegend), CD43-APC (Clone: S11- BioLegend), CD24-Brilliant violet 605

(Clone: M1/69-Biolegend).

2.4.5 Tissue sectioning and immunostaining

Spleen and inguinal lymph nodes were dissected and immediately immersed in Optimal Cutting

Temperature (OCT) compound (TissueTek). Tissues were then frozen in an isopentane bath submerged in liquid nitrogen. Frozen tissue blocks were cryosectioned into 12 µm slices and subsequently fixed in -20˚C acetone on Superfrost Plus microscope slides (VWR) for 5 min and then air dried. After rehydration with PBS for 15 min, the sections were blocked with 5% FBS for 30 minutes at room temperature (RT), then stained with various antibodies in 5% FBS for 2 hrs. Sections were mounted in Fluoro-Gel with DABCO™ Anti-Fading Mounting Medium.

Slides were imaged with Zeiss Axio Scan.Z1 at 40X magnification (Apo Plan 40X /0.95 NA).

2.4.6 In vitro B cell development assay

OP9 stromal cells178 stably expressing IL-7, Flt3L, and SCF (OP9-R7FS; Brauer and Zúñiga-

Pflücker, manuscript in preparation) were cultured as a monolayer in alpha-MEM media

60 supplemented with 5% FBS, 1% penicillin/streptomycin (all Gibco, Life Technologies) at 37 ˚C with 5% CO2. HSCs were enriched from murine bone marrow by negative selection according to manufacturer’s protocol (EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit,

StemCell Technologies). 5 x 105 OP9-R7FS cells were seeded in a 6 well plate 24hrs before co- culturing with 1.0 x 105 HSCs from WT or TRPM7-/- mice. Cultures were additionally supplemented with 0, 5, or 10 mM MgCl2. 5 days later, differentiated cells were washed off the monolayer and re-plated onto a fresh confluent monolayer of OP9-R7FS cells. 3 days later, differentiated cells were collected from the monolayer and expression of B220 and CD19 were assessed via flow cytometry.

2.4.7 Data Analysis

Statistical significance was assessed by the Mann-Whitney test, Kruskall-Wallis test with Dunn;s

Comparison, Freidman’s test with Dunn’s Comparison or Wilcoxon matches pairs ranked test.

All statistical tests were performed with Prism software (Version 6.01; Graphpad).

CHAPTER 3 TRPM7 is Important for Antigen Gathering and Presentation

Author Contributions

I would like to acknowledge the following individuals for contributing to this work. Josephine

Ho and Laabiah Wasim recorded time-lapse video and generated Figures 3.2A-C, 3.4B-D, and

3.6C-D. Fathima H. M. Buhari generated Figures 3.5C-D. Trisha Mahtani generated Figure 3.8A.

Tiantian Zhao generated Figures 3.8 H-I.

3.1 Introduction

B cells are activated upon the binding of ligand (antigen) to the B cell receptor (BCR), which leads to differentiation into antibody secreting plasma cells or memory B cells1,25. In vivo, antigen is predominantly bound to the membrane of antigen presenting cells, such as follicular dendritic cells and subcapsular macrophages that reside in lymph nodes13,14,169,185. Upon binding membrane-bound antigen through BCR, B cells undergo a rapid spreading phase which is then followed by a slower contractile step. Antigen is collected in small discrete clusters

(microclusters) that form while the cell is spreading27. The density and the affinity of the antigen to the BCR determines the degree to which the B cell spreads over the presenting membrane and the number of BCR-antigen clusters formed27. BCR-antigen microclusters are collected into a central aggregate when the cell contracts and are then internalized27. This two-step spreading and contraction response increases the amount of antigen collected by the cell while at the same time discriminating between antigens of different affinities27. The internalized antigen is then processed and presented to T cells which provide additional signals that are important for further

B cell activation16.

The spreading and contraction response is largely mediated by actin remodeling. Upon activation by membrane-bound antigen, actin rapidly depolymerizes and repolymerizes to form actin sheets which drive the spreading response27,50. When actin is depolymerized via pharmacological agents in B cells, the spreading and contraction response is abrogated27.

Although several regulators of actin reorganization have been identified, the complete mechanism that couples BCR activation to actin remodeling has yet to be elucidated40,45,47,186.

One key regulator of actin in B cell activation is phospholipase Cγ2 (PLCγ2). The deletion of PLCγ2 in B cells leads to decreased spreading and antigen accumulation40. PLCγ2 was found to work synergistically with Vav, a guanine nucleotide exchange factor (GEF) that can activate key actin regulators including the Rho-family of GTPases, Rac, Rho, and Cdc42, which modulate actin nucleating proteins such as Wiskott-Aldrich Syndrome Protein (WASp)40,187. PLCγ2 also cleaves phosphatidylinositol 4, 5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-1,

4, 5-trisphosphate (IP3). IP3 binds to IP3 receptors on the endoplasmic reticulum, leading to release of calcium from the endoplasmic reticulum25. The sudden increase in cytoplasmic calcium triggers the opening of calcium release-activated calcium channels (CRACs) on the plasma membrane, resulting in influx of extracellular calcium25. This is important for the activation of many calcium-dependent molecules, including transcription factors driving cellular proliferation and differentiation25. PLCγ2 is known to have several phosphorylation sites that influence its phospholipase activity188. Recently, it has been shown that the novel ion channel transient receptor potential cation channel, subfamily M, member 7 (TRPM7) phosphorylates two other sites within the C2 domain of PLCγ2 with its unique kinase domain137. It is unknown whether phosphorylation of these sites influences phospholipase activity.

TRPM7 is one of two known channels that have an alpha kinase domain which is able to phosphorylate itself and other targets115,189. TRPM7 is ubiquitously expressed and is specific to

63 divalent cations, mainly magnesium94,115. In chapter 2, we demonstrated that TRPM7 is essential for B cell development. When TRPM7 was specifically deleted in B cells, there was a lack of peripheral B cells in mice. Furthermore, the developmental block was partially, but not wholly, alleviated when hematopoietic stem cells were supplemented with extracellular magnesium, implicating both the channel and kinase domain of TRPM7 in B cell development.

Besides regulating magnesium, TRPM7 has also been implicated in regulation of the actin cytoskeleton117,134. Overexpression of TRPM7 resulted in increased cell spreading in neuroblastoma cells, through a kinase and calcium dependent pathway, whereby TRPM7 kinase phosphorylates the heavy chain of myosin IIA, resulting in inhibition of myosin contraction116.

The function of TRPM7 in B cell activation is unclear; yet, its potential to phosphorylate PLCγ2, a key regulator of actin reorganization, marks it as a possible mediator of the actin cytoskeleton and consequently, B cell activation.

Here, we demonstrate that DT40 B cells deficient in TRPM7 or expressing a point mutation in the kinase domain are unable to centrally aggregate antigen when activated by membrane-bound antigen. Moreover, these results are recapitulated in primary B cells expressing decreased levels of TRPM7. We also demonstrate that antigen internalization is defective in

TRPM7-deficient cells and this has important functional consequences for T cell activation.

These findings implicate TRPM7 as a novel regulator of antigen gathering and internalization in

B cells.

3.2 Results

3.2.1 TRPM7 regulates the actin cytoskeleton in B cells.

Since TRPM7 has been previously implicated in regulation of the actin cytoskeleton116,117, we initially examined the effect of TRPM7 deficiency on B cell morphology in the steady-state.

In this study, we made use of the chicken B cell line DT40. DT40 B cells are advantageous since its high rate of homologous recombination allows for rapid construction of genetic knockouts190, and has been an especially helpful tool in assessing proteins that cannot be evaluated in a murine model due to embryonic lethality190. DT40 cells have been shown to closely follow the spreading and contraction dynamics of murine primary B cells40. To investigate the morphology of TRPM7 deficient B cells, wild-type (WT) and TRPM7-Knockout (KO) DT40 B cells were adhered to glass coverslips and visualized by scanning electron microscopy (SEM). Consistent with previous reports40, WT DT40 cells have numerous short microvilli (Figure. 3.1A). In contrast, cell morphology of TRPM7-KO B cells is strikingly different (Figure 3.1A). TRPM7-KO B cells possess roughly half the number of cell surface protrusions compared to WT (Figure. 3.1B), however these protrusions are twice as long (Figure. 3.1C). As actin reorganization plays a critical role in B cell spreading in response to membrane-bound antigen27, we next investigated whether deletion of TRPM7 led to defective BCR-mediated reorganization of the actin cytoskeleton. WT and TRPM7-KO cells were settled on a surface coated with immobilized anti-IgM as surrogate antigen for 10 minutes and visualized by Scanning Election Microscopy (SEM). In contrast to

WT cells, which extend lamellipodia across the antigen-bearing surface, TRPM7-deficient cells have elongated filopodia-like surface protrusions but spread less (Figure 3.1D). The altered spreading in TRPM7-KO B cells demonstrates that BCR-induced reorganization of the cytoskeleton requires TRPM7. Staining these cells for filamentous actin (F-actin) also revealed that TRPM7-KO cells have impaired spreading and altered actin organization compared to WT

65 cells (Figure 3.1E). Quantification of these images demonstrated a significant decrease in area of cell spreading (Figure 3.1F) and circularity of contact (Figure. 3.1G) in TRPM7-deficient B cells.

Taken together, these results demonstrate a crucial role for TRPM7 in regulation of the actin cytoskeleton in the steady-state and actin reorganization upon BCR stimulation.

Figure 3.1 TRPM7 is involved in the regulation of the actin cytoskeleton. (A) SEM images of WT and TRPM7-KO DT40 B cells after settling on glass coverslips. Individual cells were quantified for (B) number of filopodia (mean ± SEM for WT 93.13 ± 4.7; TRPM7-KO 43.26 ± 2.8) and (C) length of filopodia (mean of WT 1.011 ± 0.03 μm; TRPM7-KO 2.05 ± 0.06 μm). (D) SEM images and (E) confocal microscopy images visualizing actin of WT and TRPM7-KO DT40 B cells after settling on anti-IgM coated glass coverslips for 10 minutes. Dotted line indicates cell edge. (E) Contact area (mean of WT 191.5 ± 7.14 μm2; TRPM7-KO 119.6 ± 4.81 μm2) and (F) shape factor (mean of WT 0.378±0.02 AU; TRPM7-KO 0.18 ± 0.01 AU) was measured from confocal microscopy images. Data are representative of 3 independent experiments; measurements were taken from minimum 30 cells per experiment. Statistical significance was determined via Mann-Whitney test ****P < 0.0001. Scale bar, 5µm

3.2.2 Centralization of antigen is altered in TRPM7 deficient B cells.

Given our observation of altered actin organization in TRPM7-deficient B cells, we were keen to assess how deletion of TRPM7 would affect BCR-antigen microcluster formation and dynamics. In order to examine this, we made use of an artificial planar lipid bilayer system, which permits tethering of surrogate antigen via fluorescently-labeled streptavidin and mimics the membrane-bound antigen found in vivo. We visualized the spreading and contraction response on artificial planar lipid bilayers via total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM). When activated by membrane-bound antigen, both

WT and TRPM7-KO cells spread and form BCR-antigen microclusters, reaching maximal spreading by 3 minutes (Figure. 3.2A). Surprisingly, TRPM7-KO B cells more rapidly form microclusters and their area of spreading is larger than WT cells during the first three minutes of interaction (Figure. 3.2B, C). However, TRPM7-KO cells do not contract and centrally aggregate antigen as WT cells, maintaining a mean contact area of 58.297±3.146 μm2 after 15 minutes compared to 38.584±4.800 μm2 for WT. Tracking of individual clusters revealed that in WT cells, antigen microclusters translocate from the periphery of the contact to the center whereas in

TRPM7-KO cells, microcluster tracks are more confined to the periphery and do not translocate to the center of contact (Figure 3.2D). Consistent with these observations the mean diffusion coefficient of microclusters was almost three times lower in TRPM7-KO cells compared to WT cells (Figure 3.2E).

Figure 3.2 B cell spreading and contraction response is impaired in TRPM7 deficient cells. B cells were settled onto artificial planar lipid bilayers containing anti-IgM as antigen (Ag). (A) Representative images of DT40 WT and TRPM7-KO are shown, (top) DIC (right) and TIRF microscopy (left) visualizing antigen (Ag), (bottom) IRM. Quantification of (B) spreading area and (C) number of clusters over time based on TIRFM images. (D) Representative examples of antigen microcluster tracks in WT and TRPM7-KO cells. (E) Mean diffusion coefficient of microclusters from individual cells (mean of WT 3.16 x 10-3 ± 3.23 x 10-4 μm2s-1; TRPM7-KO 9.23 x 10-4 ± 1.18 x 10-4 μm2s-1). Mean ± SEM calculated from minimum 20 cells averaged from minimum 5 independent experiments. Statistical significance was determined via Mann-Whitney test ****P<0.0001. Scale bar, 5μm.

To visualize the actin cytoskeleton during this process, cells were settled on antigen- containing bilayers for either 3 or 10 minutes, then fixed and stained with phalloidin to visualize

F-actin. Consistent with our live cell imaging, TRPM7-KO cells rapidly form clusters of antigen, which by 3 minutes are larger and less discrete than the microclusters seen in WT cells (Figure

3.3A), resulting in a two-fold increase in total antigen intensity in TRPM7-KO cells (Figure

3.3B). After 10 minutes of contact with the bilayers, WT cells contract, collecting antigen into a central aggregate that is surrounded by actin (Figure 3.3C). Similar clearing of actin from the center is seen in TRPM7-KO; however, the size of the aggregation of antigen and consequently the total amount of antigen accumulated is increased compared to WT cells (Figure 3.3D).

The process of B cell spreading and formation of microclusters is essential in propagating

BCR signalling. As microclusters form, several signalling molecules are recruited, leading to formation of the signalosome where each microcluster represents a unit of signalling31. Given our observation of altered BCR microcluster formation and increased antigen accumulation in

TRPM7-KO cells, we next investigated BCR signalling using plate-bound antigen as previously described35. We found that phosphorylation of extracellular signal-regulated kinase (ERK) was sustained at 30 minutes in TRPM7-KO cells compared to WT (Figure 3.3E, F). Taken together, these results demonstrate an important role for TRPM7 in antigen accumulation and BCR signalling.

Figure 3.3 TRPM7 deficient B cells acquire more antigen and have prolonged signalling. B cells were settled onto artificial planar lipid bilayers containing anti-IgM as antigen (Ag). (A) Representative confocal microscopy images of WT and TRPM7-KO DT40 B cells after 3 minutes of interaction with Ag-containing bilayer. Relative fluorescence intensity plots indicate the distribution of antigen and actin along the dashed line. (B) Quantification of antigen accumulation in (A); (mean ± SEM of WT 6.10 x 106 ± 19.4 x 105 AU; TRPM7-KO 1.15 x 107 ± 5.42 x 105 AU). (C) Confocal images of WT and TRPM7-KO DT40 B cells after 10 minutes of interaction with Ag-containing bilayer. (D) Quantification of antigen accumulation in (C); (mean of WT 5.28 x 106 ± 1.59 x 105 AU; TRPM7-KO 7.35 x 107 ± 3.40 x 105 AU). Data are representative of 3 independent experiments, measurements taken from minimum 20 cells per experiment. (E) WT and TRPM7-KO DT40 cells were settled onto anti-IgM coated plates for the indicated period of time. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti– phospho-p44 and -p42 MAPK (Erk1 and Erk2) or anti-tubulin. (F) Comparison of the normalized intensity of pERK upon stimulation in WT or TRPM7-KO cells. Mean ± SEM calculated from 3 independent experiments. Statistical significance was determined via Mann-Whitney test *P<0.05, ****P<0.0001. Scale bars, 5 µm.

3.2.3 TRPM7 kinase activity is important for B cell contraction.

To dissect the contribution of the channel domain vs the kinase domain of TRPM7, we made use of TRPM7-deficient cells which were stably reconstituted under the control of a doxycycline-regulated promotor with human TRPM7-K1648R, a point mutation that abolishes phosphotransferase activity143. In the steady-state, B cells expressing TRPM7 kinase dead mutant

(TRPM7-KD) exhibit altered cellular morphology, with fewer yet longer filopodia much like

TRPM7 deficient cells (Figure 3.4A), indicating that the kinase domain is important for cytoskeletal integrity. To investigate the role of the kinase domain during B cell activation, we visualized B cell spreading and formation of BCR microclusters using artificial planar lipid bilayers as previously described. Consistent with TRPM7-KO cells, TRPM7-KD cells were unable to contract and form a tight central aggregate, with microclusters persisting even after 15 minutes of activation by membrane-bound antigen (Figure 3.4B, C, D). However, the phenotype observed in TRPM7-KD cells is also distinct from TRPM7-KO cells in that cells are slower to spread (Figure. 3.4C), and the impairment in centralization of antigen is exaggerated (Figure

3.4D). Tracking of individual clusters revealed that clusters are largely immobilized and there is little translocation to the center of contact (Figure. 3.4E, F). The exaggerated phenotype in

TRPM7-KD cells may be consistent with a functional coupling between the kinase and channel domains as previously reported143.

Figure 3.4 TRPM7 kinase activity is important for cell contraction in response to membrane bound antigen. (A) Representative SEM image of TRPM7-kinase dead (TRPM7-KD) expressing DT40 cells in the steady-state. (B) DT40 TRPM7-KD cells were settled onto bilayers containing anti-IgM as surrogate antigen. Representative images of (top) DIC (right) and TIRF microscopy (left) visualizing antigen (Ag); (bottom) IRM. Quantification of (C) spreading area and (D) number of clusters over time based on TIRFM images. WT and TRPM7-KO comparisons are taken from Figure. 3.2B and Figure 3.2C. (E) Representative example of antigen microcluster tracks in TRPM7-KD cells. (F) Mean diffusion coefficient of microclusters from individual cells (mean of TRPM7-KD 1.14 x 10-3 ± 1.14 x 10-4 μm2s-1). WT and TRPM7-KD plots from Figure 3.2E are presented for ease of comparison.). Data are representative of 3 independent experiments, measurements taken from minimum 20 cells per experiment. Mean ± SEM calculated from minimum 20 cells. Statistical significance was determined via Mann-Whitney test ****P<0.0001. Scale bar, 5μm.

In order to observe actin during cell spreading, TRPM7-KD cells were fixed and stained for F-actin 3 and 10 minutes after spreading on the bilayer. Consistent with our live cell imaging,

TRPM7-KD cells spread and form antigen microclusters, with a similar enhancement in antigen accumulation seen in TRPM7-KO cells (Figure 3.5A, B). Notably, we observed altered actin organization with BCR microclusters co-localizing with actin (Figure 3.5 A) to a greater degree than in either WT or TRPM7-KO cells (Figure 3.3A). After 10 minutes of interaction with the antigen-containing membrane, multiple microclusters are still visible in the cell periphery of

TRPM7-KD cells and there is only a small central accumulation of antigen (Figure 3.5C).

Strikingly, in contrast to that seen in WT cells, there is minimal clearing of F-actin (Figure 3.5C).

These data suggest that the kinase domain of TRPM7 plays an important role in actin reorganization in B cells. Surprisingly, the total antigen intensity at this timepoint is similar to that seen in WT cells (Figure 3.5D), suggesting that perhaps antigen is being internalized, despite the lack of centralization. The dramatic alteration in microcluster organization and dynamics suggests that BCR signalling would also be altered. Indeed, consistent with TRPM7-KO cells, we observe sustained phosphorylation of ERK in TRPM7-KD cells 30 minutes after stimulation.

(Figure 3.5 E, F). Surface BCR expression of WT DT40 B cells was found to be slightly higher than either TRPM7-KO or TRPM7-KD cells (data not shown), therefore the difference in signalling is not due to increased BCR expression. Taken together, these data indicate that both the channel and kinase domain of TRPM7 are important for BCR microcluster dynamics and signalling.

Figure 3.5 TRPM7 kinase activity is important for actin clearing and BCR signalling. (A- D) Representative confocal microscopy images of TRPM7-KD B cells after (A) 3 or (C) 10 minutes of interaction with bilayers. Relative fluorescence intensity plots indicate the distribution of antigen and actin along the dashed line. (B) Quantification of antigen accumulation at 3 minutes (mean of WT 8.72 x 106 ± 3.11 x 105 AU; TRPM7-KD 1.25 x 107 ± 5.17 x 105 AU) or (D) 10 min (mean of WT 8.85 x 106 ± 3.46 x 105 AU; TRPM7-KD 9.32 x 106 ± 3.84 x 105 AU). Data are representative of 3 independent experiments, measurements taken from minimum 20 cells per experiment. (E) WT, TRPM7-KO, and TRPM7-KD DT40 cells were settled onto anti- IgM coated plates for the indicated period of time. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti–phospho-p44 and -p42 MAPK (Erk1 and Erk2) or anti- tubulin. (F) Comparison of the normalized intensity of pERK upon stimulation in WT, TRPM7- KO or TRPM7-KD cells. Mean ± SEM calculated from 3 independent experiments. Statistical significance was determined via Mann-Whitney test. ns, not significant; *P<0.05; ****P<0.0001. Scale bar, 5μm.

3.2.4 Mutation of phosphorylation sites in the C2 domain of PLC2 alter B cell spreading and contraction dynamics.

Recently it was found that TRPM7 kinase phosphorylates PLCγ2 within the C2 domain

(S1164) and in the linker region preceding the C2 domain (T1045). The S1164 site was found to be important in regulating signalling when B cells are in a hypomagnesic environment137. We wanted to investigate the implication of these phosphorylation sites and how they are important to modulating spreading and contraction in the context of B cell activation by membrane-bound antigen. Using artificial planar lipid bilayers and PLCγ2 deficient B cells stably over-expressing

PLCγ2 S1164A or T1045A mutants, we examined how these mutants spread and contract upon stimulation with membrane-bound antigen by live cell time-lapse TIRF microscopy. Both PLCγ2 mutants displayed phenotypes that were very different from WT cells; however, only PLCγ2-

T1045A expressing cells produce a phenotype similar to TRPM7-KD cells (Figure 3.6A).

PLCγ2-T1045A expressing cells spread and maintain a constant contact area after 3 minutes

(Figure 3.6B) and like TRPM7-KD cells, are unable to gather microclusters into a tight central aggregate even after 15 minutes (Figure 3.6A, B). PLCγ2-S1164A mutants fail to spread and consequently microcluster formation is reduced approximately 60% compared to WT or cells expressing PLCγ2-T1045A (Figure 3.6C). Similar levels of surface BCR were detected in both

PLCγ2 S1164A and T1045A expressing cells. Therefore the difference in number of microclusters is not due to differences in BCR expression. Tracking of individual microclusters indicated that the diffusion co-efficient of clusters in both mutants is similar to microclusters in

TRPM7-KD cells, albeit clusters found in PLCγ2-S1164A expressing cells were more likely to remain in the center of the contact due to the lack of cell spreading while microclusters in PLCγ2-

T1045A expressing cells remained in the periphery (Figure. 3.6E). Taken together, these data indicate that both phosphorylation sites are important for regulating the B cell spreading and

76 contraction response. However, our data suggests that the kinase domain of TRPM7 is more likely to regulate B cell activation through phosphorylating threonine 1045 of PLCγ2.

Figure 3.6 PLCγ2 T1045A expressing cells are unable to centralize antigen. (A) PLCγ2-KO DT40 cells expressing PLCγ2-S1164A or PLCγ2-T1045A were settled onto bilayers containing α-IgM as surrogate antigen. (top) DIC (right) and TIRF microscopy (left) visualizing antigen (Ag), (bottom) IRM. Quantification of (B) spreading area and (C) number of clusters over time based on TIRFM images in (A). WT and TRPM7-KD comparisons are taken from Figure. 3.2B, 3.2C, 3.4C and 3.4D. (D) Antigen microclusters were tracked in PLCγ2-S1164A and PLCγ2- T1045A expressing cells where (E) mean diffusion coefficients of microclusters from individual cells (mean of PLCγ2-S1164A 1.32x10-3 ± 1.44x10-4 μm2/s; PLCγ2-T1045A 1.29x10-3 ± 1.89x10-4 μm2/s) were measured. WT and TRPM7-KD plots from Figure 3.2D and 3.4E are presented for ease of comparison. Mean ± SEM calculated from minimum 18 cells from minimum 5 independent experiments. Statistical significance was determined via kruskall-wallis test. ****P<0.0001. Scale bar, 5μm.

3.2.5 Expression of human TRPM7 in TRPM7 deficient B cells restores antigen centralization.

To determine if the TRPM7-KO phenotype is specifically due to deletion of TRPM7, we made use of TRPM7 deficient cells which were stably reconstituted under the control of a doxycycline-regulated promotor with human TRPM7 (TRPM7-hWT). The ability of these cells to accumulate antigen at 3 and 10 minutes after initial contact with lipid bilayers was assessed.

When induced with doxycycline, hTRPM7 expressing cells recapitulate the phenotype of WT cells at both 3 and 10 minutes (Figure 3.7A, B). Consistent with previous data, antigen centralization was compromised in uninduced cells, similar to TRPM7 deficient cells. This data demonstrates that our observed phenotype in TRPM7-KO B cells is specifically due to the lack of TRPM7 and reconstitution of TRPM7 in TRPM7-KO B cells is sufficient to rescue antigen gathering.

Figure 3.7 Expression of human TRPM7 in TRPM7 deficient B cells restores antigen gathering. Representative confocal microscopy images of WT, doxycycline induced (+Dox) and uninduced (-Dox) TRPM7-hWT DT40 B cells after (A) 3 or (B) 10 minutes of interaction with bilayers . Quantification of antigen accumulation at 3 minutes (mean of WT 2.10 x 106 ± 9.48 x 104 AU; TRPM7-hWT uninduced 3.37x 106 ± 1.82 x 105 AU; TRPM7-hWT induced 2.13 x 106 ± 1.91 x 105 AU) or 10 min (mean of WT 3.06 x 106 ± 1.59 x 105 AU; TRPM7-hWT uninduced 5.95 x 106 ± 4.46 x 105 AU; TRPM7-hWT induced 3.26 x 106 ± 3.20 x 105 AU) are displayed to the right. Data are representative of 3 independent experiments, measurements taken from minimum 40 cells per experiment. Statistical significance was determined via kruskall-wallis test ns, not significant; *P<0.05; ****P<0.0001. Scale bar, 5μm.

3.2.6 B cells expressing less TRPM7 accumulate more antigen upon stimulation.

TRPM7 is a channel that is ubiquitously expressed throughout the body and is essential for development of several organ systems94,191. Deletion of TRPM7 in mice is embryonic lethal114 and the conditional knockout of TRPM7 under mb1, a B cell specific promotor, leads to the complete loss of peripheral B cells (Figure 2.1A). However, in mice where only one allele of

TRPM7 is deleted (TRPM7+/-), B cells develop normally (Figure 2.1A), but express approximately 50% of TRPM7 mRNA compared to WT B cells (Figure 3.8A). To investigate whether this decrease in TRPM7 expression can alter B cell contraction and antigen collection, we examined TRPM7+/- primary B cells at 1.5 and 10 minutes after contact with artificial lipid bilayers by confocal microscopy. At 1.5 minutes, TRPM7+/- B cells collected 50% more antigen

(Figure 3.8B, C) and spread 10% more than WT cells (Figure 3.8B, D). 10 minutes after activation, TRPM7+/- B cells did not contract as much as WT cells and also accumulated more antigen (Figure 3.8E, F, G). This led us to investigate whether downstream signalling was also altered in these cells. When stimulated by plate bound F(ab’)2 α-IgM, ERK phosphorylation was sustained in TRPM7+/- B cells compared to WT primary B cells, consistent with that observed in

DT40 TRPM7 deficient cells (Figure 3.8H, I). Indeed, the overall phenotype of primary

TRPM7+/- was similar to DT40 B cells deficient in TRPM7, despite there only being decreased expression of TRPM7 in primary B cells. Thus, these findings demonstrate that TRPM7 plays an important role in primary B cell activation.

Figure 3.8 Reduced expression of TRPM7 leads to altered antigen accumulation and signalling in primary B cells. (A) TRPM7 mRNA levels from WT and TRPM7+/- primary B cells quantified as a fold change via qPCR. Representative confocal microscopy images of WT and TRPM7+/- primary B cells after (A) 1.5 or (B) 10 minutes of interaction with bilayers. Quantification of antigen accumulation at (C) 1.5 minutes and (F) 10 minutes. Quantification of spreading area at (D) 1.5 min or (G) 10 minutes. Data are representative of 3 independent experiments, measurements taken from minimum 50 cells per experiment. (H) WT or TRPM7+/- cells were settled onto anti-IgM coated plates for the indicated period of time. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti–phospho-p44 and -p42 MAPK (Erk1 and Erk2) or total ERK1/2. (I) Comparison of the normalized intensity of pERK upon stimulation in WT or TRPM7+/- cells. Mean ± SEM calculated from 5 independent experiments. Statistical significance was determined via Mann-Whitney test *P<0.05; ****P<0.0001. Scale bar, 5μm.

3.2.7 Pharmacological inhibition of TRPM7 increases antigen accumulation B cells.

Since complete deletion of TRPM7 is impossible via genetic means, we took an additional approach to study the role of TRPM7 in B cells through the use of NS8593, a chemical inhibitor of TRPM7. NS8593 directly supresses inward and outward currents of TRPM7 by binding the magnesium binding site192, thus allowing us to assess the contribution of the channel function of

TRPM7. To test whether this inhibitor would recapitulate the knockout phenotype found in DT40

B cells and primary TRPM7+/- B cells, DT40 WT B cells were treated with NS8593 for 10 minutes before allowing cells to interact with membrane-bound antigen. Cells were fixed after 3 minutes of contact with the lipid bilayer and imaged by confocal microscopy. Consistent with our observations in TRPM7-KO DT40 B cells, inhibitor treated cells acquired 1.5 times more antigen than DMSO treated control cells (Figure 3.9A, B). We also treated primary murine B cells with

NS8593 in order to determine if supressing TRPM7 channel activity can alter antigen accumulation in primary cells. Murine B cells were treated with 30 µM NS8593 for 10 minutes before allowing them to settle on planar lipid bilayers containing antigen. After 1.5 minutes of interaction, cells were fixed and imaged by confocal microscopy. Similar to inhibitor treated

DT40 cells and TRPM7-KO cells, NS8593 treated primary B cells accumulated 1.5 times more antigen than DMSO control treated cells (Figure 3.9C, D). Thus, these results support our findings that TRPM7 channel domain plays an important role in B cell activation.

To rule out the possibility that we are observing altered antigen accumulation due to non- specific interactions of NS8593 with other channels, we treated TRPM7-KO cells with 30 µM

NS8593 for 10 minutes before settling them on membrane-bound antigen. Cells were fixed after

3 minutes of interaction and imaged by confocal microscopy. If NS8593 acts specifically on

TRPM7, treated TRPM7-KO B cells should be identical to untreated TRPM7-KO cells. Indeed,

83 we observed no difference in antigen accumulation between NS8593 treated and DMSO control treated TRPM7 deficient B cells (Figure 3.10A, B).

NS8593 was originally screened from a panel of compounds that were developed for blocking calcium activated potassium channels (SK channels)192. Since B cells express the

193 calcium activated potassium channel KCa3.1 , we wanted to test whether blocking these channels would alter antigen accumulation. Primary B cells were treated with 1 µM of Apamin, a SK channel blocker, for 10 minutes before settling onto anti-kappa containing lipid bilayers.

Cells were fixed after 1.5 min of interaction with membrane-bound antigen and imaged by confocal microscopy. We observed no difference in antigen accumulation between Apamin treated and vehicle-control cells (Figure 3.10C, D), indicating that SK channels do not play a role in antigen gathering. The lack of a difference also demonstrates that the increased antigen accumulation in NS8593 treated cells compared to vehicle-control treated cells is not due to inhibition of SK channels on B cells.

Figure 3.9 Pharmacological inhibition of TRPM7 increases antigen accumulation in B cells. Representative images of (A) WT DT40 B cells or (C) primary murine B cells treated with DMSO or 30 μM NS8593 for 10 minutes, then settled on bilayers containing anti-kappa for 3 min or 1.5 min respectively. (B) Quantification of antigen in DMSO and NS8593 treated DT40 B cells (mean ± SEM of DMSO 1.05 x 107 ± 3.24 x 105 AU; NS8593 1.60 x 107 ± 5.30 x 105 AU) or (D) primary B cells (mean ± SEM of DMSO 2.25 x 106 ± 6.29 x 104 AU; NS8593 4.69 x 106 ± 1.58 x 105 AU). Data are representative of 3 independent experiments; minimum of 100 cells were measured per experiment. Statistical significance was determined via Mann test ****P<0.0001 Scale bar, 5µm.

Figure 3.10 Pharmacological inhibitor NS8593 is specific for TRPM7. (A)TRPM7-KO B cells were treated with DMSO or 30 µM NS8593 and then allowed to settle on bilayers containing anti-IgM for 3 min. (B) Quantification of antigen accumulation of cells in (A). (C) Primary splenocytes were treated with 1 µM Apamin for 10 min or vehicle control then allowed to settle on bilayers containing anti-kappa for 1.5 min (D) Quantification of antigen accumulation in (C). Images are representative of 3 independent experiments; minimum of 100 cells were measured per experiment. Statistical significance was determined via Mann-Whitney test ****P<0.0001 Scale bar, 5µm

3.2.8 TRPM7 is important for antigen internalization and presentation.

The inefficient centralization of antigen in TRPM7-KO cells led us to ask whether

TRPM7-KO cells would also have a defect in endocytosis of antigen. B cells uptake antigen via receptor-mediated endocytosis upon activation, which leads to trafficking of BCR-antigen microclusters into endosomal compartments for processing and presentation to T cells, an essential step for full B cell activation and differentiation into antibody producing cells16. To examine the effect of TRPM7 deficiency on antigen internalization, we employed a flow cytometry assay to measure the amount of surface BCR following BCR stimulation. DT40 WT,

TRPM7-KO, and TRPM7-KD cells were stimulated with anti-IgM and incubated for various periods of time at 37°C to allow for BCR-antigen internalization and the remaining surface BCR was detected using a fluorophore-labelled anti-IgM antibody which binds to an alternative epitope. Consistent with previous studies194, WT B cells have internalized approximately 75% of

BCR after 30 minutes (Figure 3.11A, B). In contrast, antigen internalization is significantly compromised in TRPM7-KO cells, with only 45% of surface BCR internalized at this time point

(Figure 3.11A, B). Surprisingly, we found that the kinase domain is dispensable, as TRPM7-KD cells internalize BCR similar to WT cells. This indicates that the channel domain, and not the kinase domain, of TRPM7 is important for receptor-mediated endocytosis of antigen in B cells.

The observed defect in antigen internalization in TRPM7-deficient cells suggested that antigen presentation to T cells may be compromised. To investigate this, we again made use of the channel inhibitor NS8593 to examine upregulation of MHC class II. NS8593 treated primary murine B cells were stimulated with anti-IgM-coated beads for 18 hours and then immunostained for MHC class II and examined via flow cytometry. NS8593 treated B cells expressed approximately 30% less MHC class II than DMSO control treated cells (Figure 3.11C, D). To investigate whether this decrease in MHC class II expression in TRPM7 inhibited B cells would

87 affect T cell activation and thus the amount of help received by the B cell, we performed a presentation assay. A20 B cells expressing a transgenic receptor specific for hen egg lysozyme

(HEL) were incubated with HEL-coated beads for 30 minutes in the presence of NS8593 or vehicle control before uninternalized beads were washed out. B cells were then co-incubated with the HEL-specific 2G7 T cell hybridoma overnight and IL-2 levels in the co-culture supernatant were measured via ELISA (enzyme-linked immunoabsorbant assay). Consistent with our finding of reduced expression of MHC class II in primary B cells treated with the TRPM7-specific inhibitor, we found that IL-2 production was decreased approximately 50% when B cells were treated with NS8593 compared to DMSO-treated cells (Figure 3.11E). Taken together, these findings demonstrate an important role for TRPM7 in antigen internalization and consequently T cell activation.

Figure 3.11. TRPM7 is important for antigen internalization and presentation. (A) Surface BCR levels on WT, TRPM7-KO, and TRPM7-KD DT40 cells were measured after stimulating with soluble anti-IgM via flow cytometry. (B) Quantification of BCR internalization. (C, D) Primary murine B cells treated with DMSO or 30 μM NS8593 were cultured with or without beads coated with α-IgM for 18 hours followed by staining for MHC class II and analyzed by flow cytometry. (D) Quantification of mean fluorescence intensity of MHC class II. (E) A20 B cells treated with DMSO or 30 μM NS8593 were incubated with beads coated with hen egg lysozyme (HEL) for 1 hour then co-cultured with 2G7 T cell hybridoma for 18 hours. IL-2 levels in culture media were determined via ELISA. Data are representative of 3 independent experiments. Fold change relative to WT B cells treated with PBS. Statistical significance was determined via Mann-Whitney test *P<0.05; ****P<0.0001

3.3 Discussion

In this study we report that deletion of TRPM7 in DT40 B cells leads to altered cell morphology in the steady-state and when activated through the BCR. In response to membrane- bound antigen, TRPM7 deficient DT40 cells accumulated more antigen, and failed to contract.

DT40 cells expressing a kinase dead mutant also accumulated more antigen than WT cells, yet displayed a more severe defect in cell contraction. Importantly, the increased accumulation of antigen in TRPM7-KO DT40 cells was replicated in primary TRPM7+/- B cells that express only half of WT TRPM7 mRNA, and primary B cells treated with a pharmacological inhibitor of

TRPM7. Moreover, BCR-antigen internalization is compromised in TRPM7 deficient DT40 cells. Using a pharmacological inhibitor of TRPM7 in A20 B cells, we demonstrate that antigen presentation to T cells is reduced and consequently T cell activation is compromised. Taken together, our data identified an important and novel role for TRPM7 in B cell function.

We demonstrate that B cells lacking TRPM7 have altered cell morphology in the steady and activated state. Other studies have also reported the importance of TRPM7 in regulating morphology in a variety of cell types. When TRPM7 was over-expressed in human embryonic kidney (HEK) cells, the cells rounded up, lost adhesion and underwent cell death within 96 hours of inducing over-expression115. Interestingly, over-expression of TRPM7 in neuroblastoma cells had the opposite morphology to HEK cells. TRPM7 activation via bradykinin in neuroblastoma cells over-expressing TRPM7 resulted in increased cell spreading and the formation of more focal adhesions116. More similar to our findings, siRNA-mediated knockdown of TRPM7 in fibroblasts leads to exaggerated membrane extensions117. The altered cellular morphology in TRPM7 deficient cells suggests that a critical component of the cytoskeleton, such as actin, is disrupted.

Actin regulators of the Rho family GTPases, Rac and Cdc42, promote the formation of lamellipodia and filopodia respectively195. The balance in activation between Rac and Cdc42

90 could be disrupted by the loss of TRPM7, leading to the formation of more filopodia. Indeed,

TRPM7 deficient fibroblasts are unable to form lamellipodia in a wound healing assay, and have lower levels of GTP-Rac and GTP-Cdc42117, implicating TRPM7 in the regulation of these

GTPases. Interestingly, free magnesium has been shown to be a negative regulator of GDP- dissociation from Rac and Cdc42157. The lack of magnesium influx in TRPM7 deficient cells may thus interfere with normal regulation of these GTPases by their respective GEFs, consequently altering morphology of the cell.

Whether the kinase domain of TRPM7 is important in regulating cell morphology is heavily debated116,117. Our studies demonstrate that DT40 cells expressing a kinase dead TRPM7 mutant have altered morphology in the steady-state. However, these cells do not exactly phenocopy TRPM7-KO cells, suggesting that both channel and kinase domains play a role in regulating cell morphology. Spreading of neuroblastoma cells was found to be kinase domain dependent, as over expression of kinase dead mutants did not lead to spreading of cells116. The kinase domain was found to phosphorylate myosin IIA heavy chain and this was important for localized relaxation of the actin cytoskeleton116. Contrary to this study and ours, the elongated phenotype of TRPM7 deficient fibroblasts could be rescued by expressing a TRPM7 kinase dead mutant, indicating that the kinase domain is not necessary for maintaining morphology of fibroblasts. Indeed, this phenotype could also be rescued by the expression of SLC41A2, a magnesium channel117, further implicating the channel function of TRPM7 in fibroblast morphology.

TRPM7 deficient DT40 B cells spread more when activated by membrane-bound antigen, which indicates that there could be altered actin polymerization. Since the actin cytoskeleton is important in defining the organization of microclusters, an alteration in the actin cytoskeleton of

TRPM7-KO B cells could explain the increased number of microclusters35. When Latrunculin A,

91 an actin depolymerizing agent, was added to B cells that have already formed microclusters, the microclusters increased in size while becoming less defined and fewer in number35. This demonstrates that the actin cytoskeleton plays a role in defining the size and number of clusters35.

Also, cortical actin plays a role in determining the activation threshold of B cells. In the resting state, BCRs are trapped within actin “fences” until they are released by actin depolymerisation that occurs during B cell activation48,50. BCRs can then join other BCRs on the plasma membrane to form microclusters51,196. The morphology that we observe in TRPM7-KO B cells in the steady state may indicate altered cortical actin, which impacts cortical tension. If cortical tension is already reduced due to decreased cortical actin at the steady state, actin may be more readily depolymerized upon BCR engagement, leading to more rapid formation of microclusters.

Despite increased microcluster formation, the mobility and centralization of microclusters are decreased in TRPM7-KO cells and abolished in TRPM7-KD cells. The depolymerisation of actin leads to an arrest in centralization of microclusters in both B cells197 and T cells32. T cell receptor (TCR) microclusters that form at the periphery of the contact are thought to be pushed to the center via actin treadmilling52. Therefore, the lack of antigen centralization in TRPM7-KO and TRPM-KD B cells could be due to a dysregulation of actin. Future studies should investigate if rates of actin polymerization are similar between WT and TRPM7-KO B cells via fluorescence recovery after photobleaching (FRAP) experiments. Alternatively (or additionally), TRPM7 may phosphorylate other components of the actomyosin network to regulate cell contraction and antigen gathering116. Upon TCR ligation, myosin light chain is phosphorylated, which induces contraction of actin filaments54. In addition inhibiting Myosin IIA with blebbistatin arrests the centripetal movement of TCR microclusters to the center54. TRPM7 was demonstrated to phosphorylate the heavy chain of Myosin IIA in neuroblastoma cells116. Although this phosphorylation induces relaxation of myosin IIA, little is known if TRPM7 acts similarly in B

92 cells. TRPM7 may instead phosphorylate myosin light chain to regulate B cell contraction. In B cells, microcluster centralization was shown to be dependent on the adaptor proteins Grb2, Dok-

3 and Cbl. These adaptor proteins tether microclusters to the microtubule motor protein dynein which co-localizes with microclusters. Upon BCR stimulation, the microtubule network orients radially, like spokes on a wheel, allowing dynein to pull the microcluster toward the center of the contact along the microtubules55 Interestingly, cytoplasmic free magnesium can promote the activity of dynein155. Therefore, the low intracellular magnesium levels in TRPM7-KO cells may downregulate dynein activity and contribute to regulating microcluster movement155.

Our study found that B cells deficient in TRPM7 or expressing TRPM7-KD have prolonged BCR signalling. We suggest that this is due to the lack of centralization of BCR- antigen microclusters. This is consistent with studies in T cells showing that peripheral clusters are important for sustained TCR signalling32,198. Moreover, when microclusters are physically restrained from moving to the central cluster, these clusters continue to associate with signalling molecules198. Thus, the sustained peripheral microclusters in TRPM7-KO and TRPM7-KD cells might explain why these cells also had prolonged signalling. This may be due to the exclusion of inhibitory phosphatases like CD45 from peripheral microclusters 31.

The kinase domain of TRPM7 was found to have several phosphorylation targets, including myosin IIA heavy chain116 and PLCγ2137. PLCγ2 plays a key role in BCR signalling and the spreading and contraction response, as evidenced by the lack of cell spreading in PLCγ2 deficient B cells, making it an especially interesting target of TRPM7. PLCγ2 is activated by

Bruton’s tyrosine kinase (Btk), yet ablation of Btk does not abrogate spreading and contraction to the extent that occurs in PLCγ2-KO cells40. This indicates that there are other mechanisms to activate PLCγ2. Studies have shown that PLCγ2 has several other phosphorylation sites which could modulate the activity of PLCγ2137,188. In particular, TRPM7 was found to phosphorylate a

93 serine (S1164) and threonine (T1045) in the C2 domain137. The phosphorylation of these residues was postulated to aid in activating B cells under hypomagnesic conditions137. Little is known about the C2 domain of PLCγ2; however, one study has shown that the C2 domain binds to the adaptor protein BLNK and facilitates a feed forward loop, where the presence of Ca2+ increases the phospholipase activity of PLCγ2. It could be that the phosphorylation of the C2 domain by

TRPM7 negatively regulates PLCγ2 activity. In our study, activation of B cells expressing

PLCγ2–T1045A had a phenotype that is similar to cells expressing a kinase dead mutant of

TRPM7, where although more clusters form, they fail to translocate into a central aggregate.

Notably, PLCγ2–S1164A expressing B cells were unable to spread and accumulate antigen as well as WT cells. This phenotype is similar to the spreading and contraction of PLCγ2-KO DT40

B cells40, suggesting that the S1164 residue is crucial to the functioning of PLCγ2 in B cells. In contrast, a study by Deason-Towne et al. demonstrated that expression of the PLCγ2–S1164A mutant in DT40 B cells leads to defective BCR signalling only in hypomagnesic conditions137.

The disparity seen between our study and this one may be due to different methods of stimulation.

Stimulation via soluble antigen and membrane bound antigen trigger different signalling pathways. Therefore, the S1164A residue may be required for membrane bound stimulation but not soluble stimulation in normo-magnesic conditions. These findings demonstrate that both phosphorylation sites are important in mediating PLCγ2 activity and ultimately B cell activation.

Whether these phosphorylation sites affect PLCγ2 translocation to the plasma membrane or phospholipase activity is unknown, and should be further investigated. However, the lack of centralization of microclusters in TRPM7-KD cells and PLCγ2-T1045A cells suggests that

TRPM7 may regulate PLCγ2 specifically through the phosphorylation of residue T1045.

The internalization of antigen is important for B cell function, since it is required for the presentation of antigen to T cells and the subsequent acquisition of T cell help16. Importantly, T

94 cell help is required for B cells to differentiate into plasma cells or memory B cells1. Our study demonstrates that TRPM7 is required for antigen internalization. Consistent with our finding that the absence of TRPM7 leads to altered actin reorganization, previous studies have shown that actin depolymerisation prevents internalization of BCR199,200. It is also important to note that B cells cannot uptake antigen from the artificial lipid bilayer system. The assays used to measure antigen gathering versus internalization are very different as B cells are activated via membrane bound antigen in one assay and soluble antigen in the other. Although TRPM7-KD expressing B cells failed to centralize microclusters, they were still capable of internalizing antigen. This demonstrates that the channel domain is important in antigen internalization. However, it is not clear how the channel domain may regulate antigen internalization as a role for magnesium in receptor mediated endocytosis has yet to be described.

Although the downstream effectors of both the magnesium transport function and kinase domain of TRPM7 remain to be elucidated, our study identifies a novel regulator of antigen gathering and internalization in B cells. This study is the first to characterize the role of TRPM7 in B cell activation and contributes to our understanding of TRP channels in the immune system.

3.4 Materials and Methods

3.4.1 Cell preparation and culture

TRPM7-/- (TRPM7-KO)143 and wild-type (WT) DT40 B cells were used. DT40 B cells and stable transfectants were maintained at 39.5°C with 5% CO2 in RPMI 1640 containing 10% heat inactivated fetal bovine serum (FBS), 1% chicken serum, 100 U/mL penicillin and streptomycin

(all Gibco), 50 µM 2-mercaptoethanol (Amresco), and 10 mM MgCl2. 18 hours prior to performing experiments, DT40 B cells were grown in media with 2 mM MgCl2. TRPM7–KO cells transfected with TRPM7–K1648R (TRPM7–KD) or human TRPM7 (TRPM7-hWT)143 were induced with 10 µg/mL doxycycline for 18 or 6 hrs prior to performing an experiment respectively. PLCy2-KO DT40 cells were transfected with PLCy2-S1164A (PLCy2 S1164A) or

PLCy2-T1045A (PLCy2-1045A) were also cultured. A20 B cells expressing HEL-specific BCRs

(D1.3) and 2G7 T cell hybridoma were maintained at 37°C with 5% CO2 in RPMI 1640 containing 10% heat inactivated FBS, 100 U/mL penicillin and streptomycin, and 50 µM 2- mercaptoethanol. All DT40 mutants were kindly provided by C. Schmitz. Primary murine splenic

B cells were isolated from C57BL/6 wild-type (WT) mice (Charles River) by negative selection according to the manufacturer’s protocol (Miltenyi Biotec). 129/SvEvTac TRPM7flox/flox mice were purchased from the Jackson Laboratory and were crossed with C57BL/6 mb1-cre mice, kindly provided by M. Reth 173. TRPM7flox/+mb1cre/+ mice to generate TRPM7flox/flox (WT) and

TRPM7flox/+mb1cre/+ (TRPM7+/-). Mice were maintained on a mixed 129/SvEvTac and C57BL/6 background. All experiments were approved by the Local Animal Care Committee at University of Toronto Scarborough.

3.4.2 Planar lipid bilayer

Artificial planar lipid bilayers containing anti-IgM as surrogate antigen were prepared in FCS2 chambers (Bioptechs) by liposome spreading as previously described40. 1, 2-dioleoyl-sn-glycero-

3-phosphocholine (DOPC) liposomes (Avanti Polar Lipids, Inc.) were mixed with either 1.0 ×

10-3 or 2.5 ×10-4 % biotinylated liposomes. Alexa Fluor 633-streptavidin (Invitrogen) was incorporated into the lipid bilayers and used to tether specific antigen. The monobiotinylated antigens used were anti-mouse  light chain (clone HB-58) or anti-chicken IgM (clone M1).

Assays were performed in chamber buffer (PBS, 0.5% FBS, 2 mM Mg2+, 0.5 mM Ca 2+, 1 g/liter

D-glucose).

3.4.3 Cell spreading

Glass coverslips were coated with 5 µg/mL anti-chicken IgM (clone M4) in PBS overnight at

4C. Cells were allowed to settle on coverslips for 10 min at 37°C before being fixed either in preparation for immunostaining or scanning electron microscopy.

3.4.4 Immunostaining

All staining was performed in FCS2 imaging chambers or on glass coverslips. Cells were fixed in pre-warmed 4% PFA for 15 minutes at 37°C, permeabilized with PBS containing 0.01% Triton

X-100 for 5 minutes and blocked in 1% BSA for 30 min RT. F-actin was stained with Alexa Fluor

488-phalloidin (Invitrogen)

3.4.5 Sample preparation for scanning electron microscopy

Cells on glass coverslips are fixed with 2% glutaraldehyde for 1 hr. at RT followed by post- fixation in 1% OsO4 for 2 hrs. Fixed cells were then dehydrated in 50%, 70%, 95% and 100% ethanol, critical point dried with liquid CO2 and sputter-coated with gold. Images were acquired using a scanning electron microscope (Hitachi S350).

3.4.6 Cell stimulation and immunoblotting

DT40 B cells (WT, TRPM7–KO. and TRPM7–KD) or primary B cells (WT or TRPM7-/-) were equilibrated in RPMI at 37°C for 10 min and plated onto immobilized stimulatory anti-IgM (clone

M1) or F(ab’)2 Goat anti- mouse IgM (Jackson) for the indicated time at 37°C. Stimulatory plates were created by plating 0.5 mL of 5 g/mL of appropriate stimulatory antibody and incubating overnight. Cells were lysed in 2X Laemmli sample buffer and resolved by 12% SDS-

PAGE, followed by immunoblotting with 1:1000 anti–phospho-p44 and -p42 MAPK (Erk1 and Erk2) (Cell signalling Technologies) or 1:10,000 anti–tubulin (Sigma-Aldrich). Blots were reprobed with 1:1000 ERK1/2 (Cell signalling Technologies) after stripping with mild stripping buffer (0.2 M Glycine, 3.5 mmol Sodium dodecyl sulfate, 0.01% Tween20, pH 2.2) for 13 min.

3.4.7 qPCR

RNA from 5x106 primary B cells was isolated with the RNeasy mini kit (Qiagen) and subsequent synthesis of cDNA was completed with the qScript cDNA synthesis kit (Quanta Bioscience) with the T100 ThermoCycler (Bio-Rad). cDNA first strand reaction was utilized to perform qPCR with power SyBr green mastermix (Applied Biosystems). Primer sets used for the reactions were:

TRPM7 5’-TTTGGTGTTCCCAGAAAAGC-3’ (sense) and 5’-

ACCAAGTTCCAGGACCACAG-3’ (antisense) and GAPDH 5’-

TCAACAGCAACTCCCACTCTT-3’ (sense), 5’-ACCCTGTTGCTGTAGCCGTAT-3’

(antisense) (Zeng et al. 2015, Bertin et al. 2014). qPCR reactions were performed in the CFX

Connect Real-Time PCR Detection System (Bio-Rad). Fold change of mRNA expression between WT and TRPM7+/- B cells was quantified by the ΔΔ cycle threshold method after normalization with housekeeping gene, GAPDH.

3.4.8 Flow cytometric assay for BCR internalization.

DT40 cells were treated with 10 µg/mL anti–chicken IgM (clone M4) in PBS on ice for 20 min before washing unbound antibodies 3 times with 5 mL of ice-cold PBS. Cells were warmed to

37°C for the indicated time periods. The internalization process was then stopped by adding 1 mL ice-cold FACS buffer (3% FBS, 0.02% NaN3). Surface BCR was stained with primary anti–

98 chicken IgM (clone M1) antibody followed by goat anti–mouse Alexa 488 secondary antibody in FACS buffer. Analysis was performed via flow cytometry (LSR Fortessa, BD biosciences) and

FlowJo (Treestar).

3.4.9 Presentation Assay.

1 x 105 D1.3 A20 B cells were treated with 30 µm NS8593 for 10 minutes 37°C before adding

α–HEL coated polystyrene beads (110 nm, Bangs Laboratories). Cells were allowed to internalize beads for 1hr before being washed of inhibitor. B cells were then added to 2G7 T cell hybridoma at a 1:1 ratio. B and T cells were co-cultured at 37°C, 5% CO2 for 18 hrs; cells were then freeze thawed to release IL-2 before determining concentration via ELISA (BD OptEIA mouse IL-2

ELISA kit) according to the manufacturer’s protocol.

3.4.10 MHC class II upregulation assay.

4 × 105 murine splenocytes were incubated in complete media containing 50 µg/mL BAFF, with or without 30µM NS8593 (Sigma-Aldrich) or vehicle (DMSO) for 10 minutes at 37°C. Cells were then stimulated with α–IgM (HB58) – coated polystyrene beads (110 nm, Bangs

Laboratories) and incubated for 18 hours at 37°C followed by immunostaining for B220, CD19 and MHC class II. Cultures were then analysed by flow cytometry.

3.4.11 Microscopy

Total internal reflection fluorescence microscopy (TIRFM) images, interference reflection microscopy (IRM) and differential interference contrast (DIC) images were all acquired by a

TIRF microscope (Quorum Technologies) consisting of an inverted fluorescence microscope

(DMI6000B; Leica), HCX PL APO 100×/1.47 oil immersion objective and Evolve Delta

EMCCD camera (Photometrics). Confocal images were acquired using a spinning disc confocal

99 microscope (Quorum Technologies) consisting of an inverted fluorescence microscope

(DMI6000B; Leica) equipped with a PL APO 64×/1.45 oil immersion objective.

3.4.12 Image processing and data analysis

The contact area of the B cell with the artificial membrane and the number of clusters were quantified using Matlab (Version R2014a; MathWorks) as previously described51, measured in at least 30 cells of each cell type. Microclusters were defined as an enrichment of at least twice the fluorescence intensity of the background. Fluorescence intensity of antigen, shape factor, and filopodia length were quantified using Volocity (Version 5.0; Elmer-Perkin). Relative enrichment of microclusters was determined using the plot intensities along line function in Volocity.

Antigen was pseudo-colored using the rainbow lookup table provided by Volocity. Densitometric analysis of western blots was performed using ImageJ (National Institute of Health). pERK levels were normalized to loading control and then expressed as a fold change relative to WT at the 0 min time point. Antigen microclusters were tracked using Matlab as previously described48.

Statistical significance was assessed by the Mann-Whitney or one way ANOVA with Tukey’s

Post Hoc test using Prism software (Version 6.01; Graphpad software).

100

CHAPTER 4 Discussion

4.1 Thesis Summary

Here, we identify a key role for the channel kinase TRPM7 in B cell development and activation. Using a murine genetic model in which TRPM7 in specifically deleted in the B cell lineage, we demonstrate that loss of TRPM7 results in a complete lack of peripheral B cells stemming from a developmental block at the Pre B cell stage in the bone marrow. Our study is the first to demonstrate that TRPM7 is necessary for the development of B cells in vivo. We further demonstrate using an in vitro model of lymphopoiesis that B cell development can be partially rescued with a high level of extracellular Mg2+, indicating an important role for the ion transport function of TRPM7. In addition to playing a key role in B cell development, our study identifies an important role for TRPM7 in B cell activation. Using DT40 B cells deficient in

TRPM7, we demonstrate that antigen gathering and internalization is defective, resulting in prolonged BCR signalling. We interrogated the kinase vs. channel function of TRPM7 in these processes and found that the kinase domain is necessary for antigen gathering during B cell contraction but dispensable for antigen internalization. We posit that TRPM7 kinase may potentially regulate antigen gathering and B cell contraction through phosphorylation of PLCγ2

(Figure 4.1), specifically at threonine 1045 in the linker region preceding the C2 domain137.

Importantly, we demonstrate that primary B cells expressing only a single allele of TRPM7, or treated with a pharmacological inhibitor that inactivates TRPM7 channel activity also displayed a defect in antigen gathering and cell contraction, validating our findings in the DT40 cell line.

Finally, we demonstrate that blocking TRPM7 function has important functional consequences, as antigen presentation to T cells, and consequently T cell activation, is compromised.

101

APC

Igα/β TRPM7 BCR CRAC Lyn

Syk Mg2+ BLNK Ca2+ PLCγ2 Btk 2+ Ag Internalization Kinase Ca PIP2 Domain dynein activity DAG 2+ IP3 Ca ER

B cell contraction IP3R Legend Cluster formation Ras Phosphorylation Gene Transcription Direct interaction Indirect interaction Putative interaction Translocation ERK Down or up regulation

Figure 4.1 TRPM7 may regulate B cell activation by phosphorylating PLCγ2. Upon BCR activation by membrane bound antigen (Ag), PLCγ2 is activated, initiating the hydrolysis of PIP2 into DAG and IP3. The kinase domain of TRPM7 phosphorylates PLCγ2 at the T1045 residue which may lead to down regulation of phospholipase activity. Subsequently, this may decrease DAG levels within the cells which leads to decreased pERK levels within the cell. Influx of magnesium may be necessary for antigen internalization as well as B cell contraction and microcluster formation.

102

Our study adds to the growing amount of evidence that TRP channels and ion transporters are important regulators of the immune system. While several of these channels have been implicated in the regulation of T cells89,201,202, far less is known about the role of TRP channels in B cells. Our data demonstrates that TRPM7 is important for BCR signalling, which may potentially regulate other signalling cascades, such as in B cell development. Successful B cell development requires Pre-BCR signalling9, which could be regulated by TRPM7. In addition to

TRPM7, other ion transport proteins have also been implicated in lymphocyte function203.

However, the importance ions themselves is much less understood. The following discussion explores some of the discoveries made in relation to TRP channels and ions in lymphocyte development and activation and how we can build on this new framework to further our understanding of the role of ions in the immune response.

4.2 B Cell Development and Pre-BCR Signalling

An important developmental checkpoint for B cells in the bone marrow is expression of

Pre-BCR, which is similar to BCR and consists of heavy chain and surrogate light chains204.

Signalling through the Pre-BCR is important for determining viability of gene rearrangements and promoting further progression in development. Pre-BCR activation begins upon its initial expression, facilitated by auto-aggregation of the surrogate light chain that triggers a signalling cascade very similar to BCR signal transduction9. Our studies have shown that TRPM7 is important for regulating BCR signalling, and also B cell development; therefore, one may postulate whether TRPM7 impacts B cell development by altering Pre-BCR signalling. In our mouse model, TRPM7 is deleted at the pre-pro stage. At this stage of B cell development, a Pre-

BCR has yet to be expressed, as heavy chain rearrangements are still underway8. However, as the apoptosis of Pre B cells occurs in TRPM7-/- mice, TRPM7 may play a role in Pre-BCR signal transduction. We found that ERK signalling is prolonged in mature TRPM7+/- B cells upon BCR

103 engagement, and therefore it could be argued that TRPM7 plays a role in Pre-BCR signalling as well. One measure of altered Pre-BCR signalling is changes in the proportion of distinct B cell subsets defining specific developmental stages205,206. For example, Src homology 2 containing 5’ inositol phosphatase (SHIP) deficient mice have a reduced number of transitional B cells and increased immature B cells in the bone marrow while also displaying prolonged calcium flux upon BCR engagement205,207. Conversely, deficiency in signalling molecules such as Syk, which are essential for the initial propagation of Pre-BCR signalling, abrogate development206.

Lymphocyte deficient mice reconstituted with Syk deficient HSCs failed to progress past the Pre

B cell stage indicated by an expanded population of Pro B cells in these mice.206 Since TRPM7+/- mice had similar proportions of transitional and mature B cells compared to WT mice, our data suggests that B cells expressing only one allele of TRPM7 do not have a defect in Pre-BCR signalling. This indicates that either TRPM7 is not important for Pre-BCR signalling, or TRPM7 expression is sufficient for Pre-BCR signalling. Future experiments should directly examine Pre-

BCR signalling as well as examine whether TRPM7 expression changes during B cell development.

Future studies should also examine the effect of deleting TRPM7 in later stages of B cell development to determine whether these stages require TRPM7 for survival. By generating

CD19cre+ TRPM7flox/flox mice, TRPM7 can be deleted specifically from the pre-B cell stage and onward208. If B cells from these mice are still viable, this model would allow us to better interrogate a role for TRPM7 in Pre-BCR signalling. Pre-BCR signalling may be altered due

TRPM7-mediated regulation of PLCγ2 as our study suggests for BCR signalling. The activity of

PLCγ2 is important for B cell development as PLCγ2 deletion from Pre B cells results in decreased numbers of peripheral B cells209. Conversely, constitutively activated Btk, which activates PLCγ2, is a key feature of uncontrolled proliferation seen in acute lymphoblastic

104 leukemia210. As our study shows that TRPM7-KO B cells have sustained ERK activation, the absence of TRPM7 in Pre B cells may also result in prolonged signalling and potentially increased proliferation. TRPM7 can be deleted even later in development in CD21/35cre+ TRPM7flox/flox mice, where TRPM7 deletion will occur in transitional B cells and onward211. During the transitional stage, B cells undergo negative selection where self-reactive B cells are deleted from the clonal repertoire by apoptosis or undergo receptor editing to alter BCR specificity12,212.

Extensive crosslinking of BCRs induces receptor internalization, which halts the maturation process and sends pro-apoptotic signals to induce cell death213. As we show that BCR internalization is defective in DT40 TRPM7-KO B cells, it could be that in transitional B cells lacking TRPM7 self-reactive BCR cannot be internalized. The lack of internalization might result in the inability to delete self-reactive B cells, therefore leading to autoimmunity213. Thus, the interrogation of deletion of TRPM7 at different stages of development is important for understanding the role of TRPM7 in B cell signalling and development.

4.3 TRP Channels in Lymphocyte Function

Our study shows the TRPM7 plays an important role in regulating B cell activation and development. In the past decade, there is growing evidence that many TRP channels are expressed and functional in tissues of the immune system108,201,214. In murine splenic B cells, several TRP channels are expressed, including members of the TRPC and TRPV family which are permeable to calcium80,108. Store operated calcium flux was thought to be regulated mainly by CRAC channels; however, in patients with CRAC channel mutations, B cells function normally215 and calcium flux defects were only detected in T cells216. This suggests that ion channels other than CRAC channels function as store operated calcium channels in B cells.

Several TRP channels have already been implicated in B cell calcium flux217,218. TRPC1 was found to regulate store operated calcium channels as well as calcium release from the

105 endoplasmic reticulum. DT40 B cells deficient in TRPC1 have decreased calcium flux when activated by anti-IgM217. TRPC1 was also demonstrated to be important for calcium oscillations, which determine the specificity and efficacy of signalling, as TRPC1-KO B cells had fewer oscillations with smaller amplitude219. Moreover, when treated with a calcium chelator, the number of calcium oscillations after stimulation was reduced in TRPC1 deficient B cells. This indicates that calcium release from the endoplasmic reticulum is also dependent on TRPC1217.

TRPC3 was also found to be important for DAG mediated calcium flux in DT40 B cells218.

Although the physiological significance of DAG regulated calcium flux is not well known, its downstream effects include activating NFAT220. Specifically, TRPC3 recruits protein kinase C beta (PKCβ) to the plasma membrane after BCR stimulation and is important for the prolonged activation of ERK218.

Another TRP channel that may be involved in B cell activation is TRPV1. TRPV1 is a calcium permeable channel initially identified as a pain receptor in sensory neurons221. Although it has not yet been reported to be expressed in B cells, it has recently been demonstrated to mediate calcium signalling in T cells202. Since calcium is regulated by a myriad of channels, TRPV1 may be a potential regulator of T cell activation. TRPV1 is likely activated by the src-family tyrosine kinase Lck in a non-store operated fashion. In the absence of TRPV1, calcium flux is dampened and consequently there is decreased production of cytokines upon T cell activation202. TRPV1 co-localizes with TRPA1 on the plasma membrane of T cells and is negatively regulated by

TRPA1 activity201. TRPA1 is a calcium permeable channel222 expressed in the spleen223, lymph nodes223, Jurkat T cells (human T cell line)223 and murine CD4+ T cells201, in addition to sensory neurons222. In the absence of TRPA1, calcium flux is increased in CD4+ T cells and is attributed to increased TRPV1 activity. This was demonstrated by specifically activating TRPV1 using the agonist capsaicin in TRPA1 deficient T cells. In the absence of TRPA1, capsaicin induced

106

TRPV1 currents were 200% higher than with TRPA1201. TRPV1 activation by capsaicin also induced cell death or autophagic pathways in thymocytes224,225. This suggests that TRPV1 could influence development of thymocytes. Since autophagic pathways are important for removing autoreactive cells during B cell development, TRPV1 may play a role in this process.

In addition to regulating calcium signalling, some TRP channels have been implicated in

BCR internalization. TRPML and TRPP channels are two small subfamilies of TRP channels that are non-selective to cations and are predominantly expressed intracellularly226. While TRPML2 and TRPML1 have been reported to colocalize with MHC class I and MHC class II respectively in Hela cells, not much is known about their role in the immune system227,228. Interestingly, however, both TRPML1 and TRPML2 were found to localize to intracellular vesicles that also contained endocytosed BCR. This suggests that TRPML channels may be important for BCR internalization229.

The above mentioned studies collectively show that TRP channels are important for regulating lymphocyte signalling. Although few of these studies report TRP channels in lymphocyte development, their significance in signalling pathways implicates a potential role of mediating Pre BCR signalling during development and warrants further investigation.

4.4 The Role of Ions in Immune Function and Human Disease.

The intake of various ions is necessary for numerous physiological functions; however, the study of ions has predominantly focused only on calcium. The earliest report of calcium involvement in lymphocyte activation was in 1970, where radioactive Ca2+ was observed to be taken up by human T cells upon stimulation with the carbohydrate binding protein phytohaemagglutinin. Now, calcium is recognized as an important second messenger, playing a role in signalling pathways of nearly all cell types and all immune cells230. Binding of ligand to

107

BCR231, TCR178, Fc receptor232 and chemokine receptors233 or interactions between co- stimulatory molecules72 all initiate an increase in intracellular calcium. The importance of calcium signalling in the immune response is evidenced by patients that lack functional CRAC channels234, resulting in severe combined immunodeficiency disease, since calcium signalling is needed for the transcription of several cytokines that are required for proliferation, differentiation and the activation of immune cells25,234.

Our study is one of the first to show that magnesium regulation is important for the function and development of B cells, shedding light on the virtually unexplored field of magnesium regulation in B cells. As mentioned previously, magnesium can act as a calcium agonist due to its charge and atomic radius160. As we show prolonged signalling in TRPM7 deficient B cells, the reduction of magnesium in these cells may have removed a negative regulator of calcium. Indeed, rats that have been fed a magnesium depleted diet show increased inflammation, as evidenced by higher levels of IL-6 and neutrophils in the blood162, important contributors to the acute inflammatory immune response164. Since many of the signalling pathways that lead to inflammation require calcium signalling, the loss of magnesium may have resulted in more calcium signalling230. This is consistent with decreased inflammatory symptoms, such as reduced ear redness and reduced number of leukocytes in the in blood, in magnesium deficient mice that are fed a calcium deficient diet235. Magnesium may compete for calcium binding sites and thus reduce activation of calcium signalling pathways. At the cellular level, there is evidence that magnesium inhibits calcium release from the sarcoplasmic reticulum

(SR) upon IP3 binding in muscle cells. This study showed that magnesium non-competitively

236 inhibits IP3 from binding IP3 receptors and inhibiting the release of calcium from the SR .

Furthermore, an increase of extracellular magnesium increases the charge difference between the inside and outside of the cell. To equalize the electrochemical gradient, calcium flux would

108 increase237. Intracellular magnesium also increased calcium ATPase activity, moving more calcium ions back into the sarcoplasmic reticulum and out of the cell cytosol238. While these mechanisms have not been shown to occur in B cells, analogous signalling pathways suggest that magnesium may potentially regulate calcium flux in B cells in a similar fashion. To investigate this, WT B cells could be stimulated in the presence of extracellular magnesium or not, and the resulting calcium flux observed via flow-cytometry. Additionally, calcium release from intracellular stores can be delineated from total calcium flux by activating B cells in the presence of a calcium chelator. TRPM7 deficient B cells are deficient in Mg2+143, and therefore can also be used to study the effect of magnesium on calcium fluxes.

As magnesium plays an important role in many cellular processes, it is not surprising that many clinical disorders have been associated with magnesium deficiency237. In fact, 20–61% of intensive care unit sepsis patients are reported to have hypomagnesemia (low blood serum magnesium)239. Patients with hypomagnesia also have higher mortality rates in addition to longer periods of hospitalization237. Since rats with hypomagnesemia exhibit inflammatory symptoms161,164, one can speculate that hypomagnesemia in patients makes it more likely that they will develop sepsis. In a study conducted in 2015, serum magnesium levels of 2369 patients were recorded on admission to the Mayo clinic (Rochester, Minnesota). Patients were characterized into six categories based on serum magnesium concentration (<1.5, 1.5-1.7, 1.7-

1.9, 1.9-2.1, 2.1 – 2.3, >2.3 mg/dL). This study demonstrated a correlation between serum magnesium levels and incidences of septic shock. Patients with 2.1–2.3 mg/dL of magnesium had the lowest occurrence of septic shock. Interestingly, higher incidences of septic shock were associated with both high (>2.3 mg/dL) and low (<1.5 mg/dL) concentrations of magnesium240.

This suggests that magnesium plays an important role in regulating the immune response in humans. However, the molecular mechanisms that lead to such consequences are still unknown.

109

The mutation in mammalian magnesium channel MagT1 especially highlights the importance of magnesium in human health. As mentioned before, mutations in MagT1 leads to

XMEN, an immunodeficiency disease characterized by chronic viral infections and impaired T cell signalling113. MagT1 is also important for regulating cytotoxic functions of Natural Killer

(NK) cells and cytotoxic T cells (CTLs)241. NK cells are cytotoxic lymphocytes that recognize virally infected host cells, and release lytic granules in response, effectively killing the infected cell. CTLs work very similarly to NK cells, also killing virus infected host cells; however, CTLs only release cytotoxic granules upon TCR binding to MHCI that present viral peptide on the host cell. Chaignee-Delalande and colleagues demonstrate that lytic capabilities are defective in NK cells and CTLs in XMEN patients241. The decreased cytotoxic capabilities were attributed to low expression of NKG2D; a receptor needed to trigger lytic granule release in both NK cells and

CTLs. When these cells were supplemented with extracellular magnesium, NKG2D expression increased in both cell types and NK cells regained their cytotoxic capabilities; however, CTL cytotoxicity was only partially rescued. To test whether magnesium can improve XMEN symptoms, patients were administered magnesium threonate orally for 175 days. Within the first

2 days of treatment, the percentage of virally infected cells decreased by 50%. This demonstrates that magnesium is important for the function of NK cells and CTLs and the clearance of viral infection241.

As discussed previously, multiple lines of interrogation have demonstrated the importance of ions to immune function. Calcium and magnesium are the focus of many of these studies, whereas other ions such a potassium and zinc are lacking fundamental studies investigating their function in the immune system. Despite being the most abundant cation intracellularly242, little is known about potassium regulation and its importance in lymphocytes. Potassium was initially identified as important to T cell activation as pharmacological inhibition of potassium channels

110 prevented PHA induced T cell proliferation243. Two types of potassium channels exist in human

244–246 and murine T cells, Kv1.3 and Kca3.1 . These two potassium channels have similar tetrameric structure on the plasma membrane that change in conformation to let potassium ions through247. Kv3.1 channels open when the inside of the cell accumulates more positive charges

(depolarization)247. This usually occurs upon calcium flux during T cell activation. As more calcium ions enter the T cell, Kv1.3 channels open and efflux potassium ions230. Since there is a higher concentration of potassium ions intracellularly, the opening of Kv1.3 channels allows the passive movement of potassium ions down the K+ electrochemical gradient248. The loss of potassium ions maintains the charge gradient across the cell, allowing more calcium ions to

248 enter . Kca3.1 channels, on the other hand, are activated by increases in cytosolic levels of

246 calcium ions . Kca3.1 channels sense calcium via a c-terminally bound calmodulin domain and

249,250 help sustain calcium signalling . Therefore, Kca3.1 channels are activated in addition to

Kv1.3 channels upon calcium flux during T cell activation246. Upon T cell activation by APC,

Kv1.3 and Kca3.1 channels, that are initially evenly distributed on the plasma membrane, form puncta at the contact between the T cell and APC251,252. Studies have suggested that potassium channels may localize to the contact to be in close proximity to other protein kinases which can then regulate its activity253,254. Interestingly, the addition of extracellular potassium to T cells can negatively regulate T cell activity255. In a cancer microenvironment, the necrosis of tumour cells releases potassium which was found to supress cytokine expression in T cells. While extracellular potassium did not alter calcium flux or ERK signalling, it did lower Akt phosphorylation255. Akt signalling is important for cell survival and protein synthesis pathways256. The mechanism that leads to the decrease in Akt phosphorylation is unknown.

B cells also have Kv1.3 and Kca3.1 channels that are thought to modulate calcium flux similarly to T cells193. Indeed, potassium channel blockers inhibit B cell activation and

111 subsequent proliferation when stimulated by lipopolysaccharide (LPS)257. When activated through the BCR, both types of channels flux potassium ions258. Whether these channels localize to BCR-antigen microclusters during activation by membrane-bound antigen is unknown.

However, the reorganization of Kv1.3 and Kca3.1 in T cells upon T cell stimulation suggest that reorganization may occur in B cells as well. As B cells differentiate after activation, the

193 expression of Kv1.3 and Kca3.1 channels begins to change . The differences in channel expression between stages of B cell differentiation allows for the manipulation of specific B cell subsets by targeting either Kv1.3 or Kca3.1 channels with channel blockers. This is evidenced by

Kca3.1 inhibitors decreasing DNA synthesis in CD40 activated naïve B cells but not memory B cells. The reverse was true for Kv1.3 inhibitors. Since blocking potassium channels inhibits proliferation of B cells, selective inhibition of potassium channels maybe a useful therapeutic for immunological disorders such as autoimmunity193. However, the functional importance of differential expression of both types of potassium channels in B cells is unknown.

In relation to TRPM7 and our findings, another ion of interest is zinc, as TRPM7 is most permeable to zinc125. Zinc is an essential trace element that is required for the structural stability of many metalloproteins259. In humans, zinc deficiency due to a mutant zinc transporter channel results in acromatitis enteropathica, a disease that results in skin lesions and recurrent infections, and can be treated with lifelong zinc supplements260. Mice fed a zinc-deficient diet show similar symptoms in addition to a highly atrophied thymus and prolonged duration of infections261. When immunized, these mice also showed reduced production of IgM262. Zinc has the potential to be a second messenger since zinc serum concentrations are 10,000 fold more than intracellular concentrations; providing a concentration gradient that can generate a zinc influx, potentially through TRPM7125 or another zinc transporter203,263,264. Indeed, activation of T cells leads to a rise in cytoplasmic zinc concentrations dependent on the zinc transporter ZIP6265. Notably, BCR

112 signalling is increased in B cells lacking the zinc transporter ZIP10 and was attributed to decreased CD45 phosphatase activity266. However, when ZIP10 knockout mice were immunized with either T independent or T dependent antigen, they produced significantly less IgM and IgG than WT immunized mice. The presence of zinc is also important for the BCR induced proliferation of B cells, as ZIP10 deficient B cells failed to proliferate when stimulated with anti-

IgM, but not with LPS266. This shows that zinc selectively regulates BCR signalling. ZIP10 was also found to be important for B cell development. ZIP10flox/flox, mb1-cre mice have a 50% decrease in the number of splenic B cells due to an increase in caspase levels in Pro B cells, resulting in increased apoptosis267. Similarly, B lymphopoiesis was defective in mice fed a zinc deficient diet for 30 days. These mice had a 40–80% decrease in the number of pre-Pro B cells,

Pro B cells, immature B cells and mature B cells in the bone marrow, with increasing cell death seen in later stages of development261. The decrease in developing B cells was attributed to increased cellular apoptosis. Hypozinc conditions induce stress signals, which are thought to increase the production of glucocorticoids that subsequently triggers the transcription of genes needed for apoptosis. The signalling pathway is regulated by bcl2, an anti-apoptotic factor261. As

B cells earlier in development have more bcl2 expression, it might explain why these cells undergo less apoptosis261,268. Our study also shows defects in B cell development and prolonged signalling. Since TRPM7 is most permeable to zinc125, there is a possibility that the lack of

TRPM7 results in abrogated zinc influx. Therefore, the role of TRPM7 in cellular zinc regulation should be futher explored in future studies.

Ions and their respective transporter channels are underappreciated regulators of immune function. Our study is one of the first to demonstrate that magnesium and TRPM7 are important for the regulation of B cell activation and development, providing a framework for further studies on other ion channels. Already, calcium, magnesium, potassium, and zinc have been implicated

113 in regulating signalling pathways in lymphocytes, influencing development261,267, cytokine production25,162, differentiation193,234,261, and cytotoxicity234,241, however these studies have yet to define the mechanisms that underlie the aforementioned phenomena Thus, the study of ions and their transporters is important; especially for the development of specific channel blockers that can potentially treat inflammation or autoimmune diseases.

114

References 1. Rajewsky, K. Clonal Selection and Learning in the Antibody System. Nature 381, 751– 757 (1996). 2. Cooper, M. D. The early history of B cells. Nat. Rev. Immunol. 15, 191–7 (2015). 3. Cooper, M. D., Peterson, R. & Good, R. Delineation of the thymic and bursal lymphoid systems in the chicken. Nature (1965). 4. Asma, G. E. M., Van den Bergh, R. L. & Vossen, J. M. Development of pre-B and B lymphocytes in the human fetus. 407–414 (1984). 5. Nagasawa, T. Microenvironmental niches in the bone marrow required for B-cell development. Nat. Rev. Immunol. 6, 107–116 (2006). 6. Busslinger, M. Transcriptional Control of Early B Cell Development. Annu. Rev. Immunol. 22, 55–79 (2004). 7. Jacobsen, K. & Osmond, D. G. Microenvironmental organization and stromal cell associations of B lymphocyte precursor cells in mouse bone marrow. Eur. J. Immunol. 20, 2395–2404 (1990). 8. Osmond, D. G., Rolink, A. & Melchers, F. Murine B lymphopoiesis: Towards a unified model. Immunol. Today 19, 65–68 (1998). 9. Herzog, S., Reth, M. & Jumaa, H. Regulation of B-cell proliferation and differentiation by pre-B-cell receptor signalling. Nat. Rev. Immunol. 9, 195–205 (2009). 10. Rolink, A. G., Schaniel, C., Andersson, J. & Melchers, F. Selection events operating at various stages in B cell development. Curr. Opin. Immunol. 13, 202–207 (2001). 11. Yancopoulos, G. D. & Alt, F. W. Regulation of the assembly and expression of variable- region genes. Annu. Rev. Immunol. 4, 339–68 (1986). 12. Pelanda, R. & Torres, R. M. Central B-Cell Tolerance : Where Selection Begins. 1–16 (2016). doi:10.1101/cshperspect.a007146 13. Batista, F. D., Iber, D. & Neuberger, M. S. B cells acquire antigen from target cells after synapse formation. Nature 411, 489–94 (2001). 14. Junt, T. et al. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450, 110–4 (2007). 15. Harwood, N. E. & Batista, F. D. New insights into the early molecular events underlying B cell activation. Immunity 28, 609–19 (2008). 16. Lanzavecchia, A. Antigen-specific interaction between T and B cells. Nature 314, 537– 539 (1985). 17. Parker, D. C. The functions of Antigen Recognition in T cell dependent B cell activation. Semin. Immunol. 5, 413–420 (1993). 18. Batista, F. D. & Neuberger, M. S. B cells extract and present immobilized antigen: implications for affinity discrimination. EMBO J. 19, 513–520 (2000).

115

19. MacLennan, I. Germinal centers. Annu Rev Immunol 429–457 (1994). 20. Reth, M. Antigen receptor tail clue. Nature 338, 383–384 (1989). 21. Kurosaki, T. GENETIC ANALYSIS OF B CELL. (1999). 22. Rowley, B., Burkhardt, A., Chao, H.-G., Matsueda, G. & Bolen, J. Syk Protein-tyrosine Kinase is regulated by tyrosine-phosphorylated Iga/b immunoreceptor tyrosine activatio motif binding and autophosphorylation. J. Biol. Chem. 11590–11594 (1995). 23. Fu, C., Turck, C. W., Kurosaki, T. & Chan, A. C. BLNK: A central linker protein in B cell activation. Immunity 9, 93–103 (1998). 24. DeFranco, A. L. The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9, 296–308 (1997). 25. Dal Porto, J. M. et al. B cell antigen receptor signaling 101. Mol. Immunol. 41, 599–613 (2004). 26. Johnson, G. L. Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases. Science (80-. ). 298, 1911–1912 (2002). 27. Fleire, S. J. et al. B cell ligand discrimination through a spreading and contraction response. Science 312, 738–41 (2006). 28. Carrasco, Y. R., Fleire, S. J., Cameron, T., Dustin, M. L. & Batista, F. D. LFA-1/ICAM- 1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 20, 589–599 (2004). 29. Carrasco, Y. R. & Batista, F. D. B-cell activation by membrane-bound antigens is facilitated by the interaction of VLA-4 with VCAM-1. EMBO J. 25, 889–99 (2006). 30. Campi, G., Varma, R. & Dustin, M. L. Actin and agonist MHC-peptide complex- dependent T cell receptor microclusters as scaffolds for signaling. J. Exp. Med. 202, 1031–6 (2005). 31. Depoil, D. et al. CD19 is essential for B cell activation by promoting B cell receptor- antigen microcluster formation in response to membrane-bound ligand. Nat. Immunol. 9, 63–72 (2008). 32. Varma, R., Campi, G., Yokosuka, T., Saito, T. & Dustin, M. L. T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25, 117–27 (2006). 33. Bunnell, S. C. et al. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158, 1263–1275 (2002). 34. Yokosuka, T. et al. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat. Immunol. 6, 1253–62 (2005). 35. Treanor, B. L., Depoil, D., Bruckbauer, A. & Batista, F. D. Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity. J. Exp. Med. 208, 1055–68 (2011). 36. Tolar, P., Sohn, H. W. & Pierce, S. K. The initiation of antigen-induced B cell antigen

116

receptor signaling viewed in living cells by fluorescence resonance energy transfer. 6, 1168–1176 (2005). 37. Yang, J. & Reth, M. Oligomeric organization of the B-cell antigen receptor on resting cells. Nature 467, 465–469 (2010). 38. Mattila, P. K., Batista, F. D. & Treanor, B. L. Dynamics of the actin cytoskeleton mediates receptor cross talk : An emerging concept in tuning receptor signaling. 212, 267–280 (2016). 39. Yang, J. & Reth, M. The dissociation activation model of B cell antigen receptor triggering. FEBS Lett. 584, 4872–4877 (2010). 40. Weber, M. et al. Phospholipase C-gamma2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen. J. Exp. Med. 205, 853–68 (2008). 41. Jaffe, A. B. & Hall, A. RHO GTPASES: Biochemistry and Biology. Annu. Rev. Cell Dev. Biol. 21, 247–269 (2005). 42. Imai, K., Nonoyama, S. & Ochs, H. D. WASP (Wiskott-Aldrich syndrome protein) gene mutations and phenotype. Curr. Opin. Allergy Clin. Immunol. 3, 427–36 (2003). 43. Machesky, L. M. & Insall, R. H. Scar1 and the related Wiskott–Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Curr. Biol. 8, 1347–1356 (1998). 44. Goley, E. D. & Welch, M. D. The ARP2/3 complex: an actin nucleator comes of age. Nat. Rev. Mol. Cell Biol. 7, 713–726 (2006). 45. Liu, C. et al. N-WASP Is Essential for the Negative Regulation of B Cell Receptor Signaling. PLoS Biol. 11, (2013). 46. Westerberg, L., Greicius, G., Snapper, S. B., Aspenström, P. & Severinson, E. Cdc42, Rac1, and the Wiskott-Aldrich syndrome protein are involved in the cytoskeletal regulation of B lymphocytes. Blood 98, 1086–94 (2001). 47. Freeman, S. A. et al. Cofilin-mediated F-actin severing is regulated by the Rap GTPase and controls the cytoskeletal dynamics that drive lymphocyte spreading and BCR microcluster formation. J. Immunol. 187, 5887–900 (2011). 48. Treanor, B. L. et al. The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor. Immunity 32, 187–99 (2010). 49. Bretscher, A., Edwards, K. & Fehon, R. G. ERM proteins and merlin: integrators at the cell cortex. Nat. Rev. Mol. Cell Biol. 3, 586–599 (2002). 50. Hao, S. & August, A. Actin Depolymerization Transduces the Strength of B-Cell Receptor Stimulation □. Mol. Biol. Cell 16, 2275–2284 (2005). 51. Mattila, P. K. et al. The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling. Immunity 38, 461–74 (2013). 52. Kaizuka, Y., Douglass, A. D., Varma, R., Dustin, M. L. & Vale, R. D. Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse

117

formation in Jurkat T cells. (2007). 53. Bugyi, B. & Carlier, M.-F. Control of actin filament treadmilling in cell motility. Annu. Rev. Biophys. 39, 449–470 (2010). 54. Ilani, T., Vasiliver-Shamis, G., Vardhana, S., Bretscher, A. & Dustin, M. L. T cell antigen receptor signaling and immunological synapse stability require myosin IIA. Nat. Immunol. 10, 531–539 (2009). 55. Schnyder, T. et al. B cell receptor-mediated antigen gathering requires ubiquitin ligase Cbl and adaptors Grb2 and Dok-3 to recruit dynein to the signaling microcluster. Immunity 34, 905–18 (2011). 56. Kardon, J. R. & Vale, R. D. Regulators of the cytoplasmic dynein motor. Nat. Rev. Mol. Cell Biol. 10, 854–865 (2009). 57. Salisbury, J. L., Condeelis, J. S. & Satir, P. Role of coated vesicles, microfilaments, and calmodulin in receptor-mediated endocytosis by cultured B lymphoblastoid cells. J. Cell Biol. 87, 132–141 (1980). 58. Winkler, F. K. & Stanley, K. K. Clathrin heavy chain, light chain interactions. EMBO J. 2, 1393–1400 (1983). 59. Cocucci, E., Aguet, F., Boulant, S. & Kirchhausen, T. The first five seconds in the life of a clathrin-coated pit. Cell 150, 495–507 (2012). 60. Busman-Sahay, K., Drake, L., Sitaram, A., Marks, M. & Drake, J. R. Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs. PLoS One 8, (2013). 61. Boulant, S., Kural, C., Zeeh, J.-C., Ubelmann, F. & Kirchhausen, T. Actin dynamics counteract membrane tension during clathrin-mediated endocytosis. Nat. Cell Biol. 13, 1124–1131 (2011). 62. Krendel, M., Osterweil, E. K. & Mooseker, M. S. Myosin 1E interacts with synaptojanin- 1 and dynamin and is involved in endocytosis. FEBS Lett. 581, 644–650 (2007). 63. Yuseff, M. I. et al. Polarized Secretion of Lysosomes at the B Cell Synapse Couples Antigen Extraction to Processing and Presentation. Immunity 35, 361–374 (2011). 64. Clark, M., Massenburg, D., Siemasko, K., Hou, P. & Zhang, M. B-cell antigen receptor signaling requirements for targeting antigen to the MHC class II presentation pathway. Curr. Opin. Immunol. 16, 382–7 (2004). 65. Yuseff, M.-I., Pierobon, P., Reversat, A. & Lennon-Duménil, A.-M. How B cells capture, process and present antigens: a crucial role for cell polarity. Nat. Rev. Immunol. 13, 475–86 (2013). 66. Mebius, R. E. & Kraal, G. Structure and function of the spleen. Nat. Rev. Immunol. 5, 606–616 (2005). 67. Knutson, M. & Wessling-Resnick, M. Iron metabolism in the reticuloendothelial system. Crit Rev Biochem Mol Biol 38, 61–88 (2003). 68. Ansel, K. M. et al. A chemokine-driven positive feedback loop organizes lymphoid

118

follicles. Nature 406, 309–314 (2000). 69. Förster, R. et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99, 23–33 (1999). 70. Drayton, D. L., Liao, S., Mounzer, R. H. & Ruddle, N. H. Lymphoid organ development: from ontogeny to neogenesis. Nat. Immunol. 7, 344–353 (2006). 71. Mandel, T. E., Phipps, R. P., Abbot, a & Tew, J. G. The follicular dendritic cell: long term antigen retention during immunity. Immunol. Rev. 53, 29–59 (1980). 72. Tseng, S. Y., Waite, J. C., Liu, M., Vardhana, S. & Dustin, M. L. T cell-dendritic cell immunological synapses contain TCR-dependent CD28-CD80 clusters that recruit protein kinase C-θ. J Immunol 181, 4852–4863 (2008). 73. von Andrian, U. H. & Mempel, T. R. Homing and cellular traffic in lymph nodes. Nat. Rev. Immunol. 3, 867–878 (2003). 74. Ahlawat, S., De Jesus, M., Khare, K., Cole, R. A. & Mantis, N. J. Three-Dimensional Reconstruction of Murine Peyer’s Patches from Immunostained Cryosections. Microsc. Microanal. 20, 198–205 (2014). 75. Mabbott, N. A., Donaldson, D. S., Ohno, H., Williams, I. R. & Mahajan, A. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunol. 6, 666–677 (2013). 76. Coombes, J. L. & Powrie, F. Dendritic cells in intestinal immune regulation. Nat. Rev. Immunol. 8, 435–446 (2008). 77. Nagler-Anderson, C. Man the barrier! Strategic defences in the intestinal mucosa. Nat.Rev.Immunol. 1, 59–67 (2001). 78. Mebius, R. E. Organogenesis of lymphoid tissues. Nat. Rev. Immunol. 3, 292–303 (2003). 79. Eberl, G. & George, R. The development of intestinal lymphoid tissues at the interface of self and microbiota. Mucosal Immunol. 2, 478–485 (2009). 80. Venkatachalam, K. & Montell, C. TRP channels. Annu. Rev. Biochem. 76, 387–417 (2007). 81. Gaudet, R. TRP channels entering the structural era. J. Physiol. 586, 3565–3575 (2008). 82. Birnbaumer, L. et al. On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp proteins. Proc. Natl. Acad. Sci. U. S. A. 93, 15195–15202 (1996). 83. Montell, C. Physiology, Phylogeny, and Functions of the TRP Superfamily of Cation Channels. Sci. Signal. 2001, re1-re1 (2001). 84. Walder, R. Y. et al. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat. Genet. 31, 171–174 (2002). 85. Schlondorff, J., del Camino, D., Carrasquillo, R., Lacey, V. & Pollak, M. R. TRPC6 mutations associated with focal segmental glomerulosclerosis cause constitutive

119

activation of NFAT-dependent transcription. AJP Cell Physiol. 296, C558–C569 (2008). 86. Schlingmann, K. P. et al. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat. Genet. 31, 166–170 (2002). 87. Hermosura, M. C. et al. A TRPM7 variant shows altered sensitivity to magnesium that may contribute to the pathogenesis of two Guamanian neurodegenerative disorders. Proc. Natl. Acad. Sci. U. S. A. 102, 11510–5 (2005). 88. Melzer, N., Hicking, G., Göbel, K. & Wiendl, H. TRPM2 Cation Channels Modulate T Cell Effector Functions and Contribute to Autoimmune CNS Inflammation. PLoS One 7, 1–6 (2012). 89. Launay, P. TRPM4 Regulates Calcium Oscillations After T Cell Activation. Science (80- . ). 306, 1374–1377 (2004). 90. Duncan, L. M. et al. Down-Regulation of Novel Gene Melastatin Correlates with potential for melanoma metastasis. Cancer Res. 32, 2230–2238 (1998). 91. Oancea, E. et al. TRPM1 Forms Ion Channels Associated with Melanin Content in Melanocytes. Sci. Signal. 2, ra21-ra21 (2009). 92. Xu, X. S., Moebius, F., Gill, D. L. & Montell, C. Regulation of melastatin , a TRP- related protein , through interaction with a cytoplasmic isoform. 98, 10692–10697 (2001). 93. Ramsey, I. S., Delling, M. & Clapham, D. E. An Introduction to TRP Channels. Annu. Rev. Physiol. (2006). doi:10.1146/annurev.physiol.68.040204.100431 94. Kunert-Keil, C. et al. Tissue-specific expression of TRP channel genes in the mouse and its variation in three different mouse strains. BMC Genomics 7, 1–14 (2006). 95. Perraud, A. et al. ADP-ribose gating of the revealed by Nudix motif homology. 411, 595–599 (2001). 96. Nishida, M., Hara, Y., Yoshida, T., Inoue, R. & Mori, Y. TRP Channels: Molecular Diversity and Physiological Function. Microcirculation 13, 535–550 (2006). 97. Mchugh, D., Flemming, R., Xu, S., Perraud, A. & Beech, D. J. Critical Intracellular Ca2+ Dependence of Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel Activation *. J. Biol. Chem. 278, 11002–11006 (2003). 98. Gasser, A. et al. Activation of T Cell Calcium Influx by the Second Messenger ADP- Ribose. J. Biol. Chem. 281, 2489–2496 (2006). 99. Mckemy, D. D., Neuhausser, W. M. & Julius, D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. 416, (2002). 100. Zhang, L. & Barritt, G. J. TRPM8 in prostate cancer cells : a potential diagnostic and prognostic marker with a secretory function ? 27–38 (2006). doi:10.1677/erc.1.01093 101. Oberwinkler, J., Lis, A., Giehl, K. M., Flockerzi, V. & Philipp, S. E. Alternative splicing switches the divalent cation selectivity of TRPM3 channels. J. Biol. Chem. 280, 22540– 22548 (2005).

120

102. Brenu, E. W. et al. Natural killer cells in patients with severe chronic fatigue syndrome. Autoimmun. Highlights 4, 69–80 (2013). 103. Nguyen, T. et al. Impaired calcium mobilization in natural killer cells from chronic fatigue syndrome/myalgic encephalomyelitis patients is associated with transient receptor potential melastatin 3 ion channels. Clin. Exp. Immunol. 187, 284–293 (2017). 104. Launay, P. et al. TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell 109, 397–407 (2002). 105. Nilius, B. et al. Voltage dependence of the Ca2+-activated cation channel TRPM4. J. Biol. Chem. 278, 30813–30820 (2003). 106. Prawitt, D. et al. TRPM5 is a transient Ca2+-activated cation channel responding to rapid changes in [Ca2+]i. Proc. Natl. Acad. Sci. U. S. A. 100, 15166–71 (2003). 107. Ullrich, N. D. et al. Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice. Cell Calcium 37, 267–278 (2005). 108. Inada, H., Iida, T. & Tominaga, M. Different expression patterns of TRP genes in murine B and T lymphocytes. Biochem. Biophys. Res. Commun. 350, 762–767 (2006). 109. Howitt, M. R. et al. Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut. Science (80-. ). 351, 1329–1333 (2016). 110. Voets, T. et al. TRPM6 Forms the Mg 2+ Influx Channel Involved in Intestinal and Renal Mg2+ Absorption. J. Biol. Chem. 279, 19–25 (2004). 111. Zhang, Z. et al. The TRPM6 Kinase Domain Determines the Mg - ATP Sensitivity of TRPM7 / M6 Heteromeric Ion Channels *. 289, 5217–5227 (2014). 112. Chubanov, V. et al. Disruption of TRPM6 ͞ TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia. 101, (2004). 113. Li, F.-Y. et al. Second messenger role for Mg2+ revealed by human T-cell immunodeficiency. Nature 475, 471–6 (2011). 114. Jin, J. et al. The channel kinase, TRPM7, is required for early embryonic development. Proc. Natl. Acad. Sci. U. S. A. 109, E225-33 (2012). 115. Nadler, M. J. S. et al. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature 411, 590–5 (2001). 116. Clark, K. et al. TRPM7, a novel regulator of actomyosin contractility and cell adhesion. EMBO J. 25, 290–301 (2006). 117. Su, L.-T. et al. TRPM7 regulates polarized cell movements. Biochem. J. 434, 513–21 (2011). 118. Runnels, L. W., Yue, L. & Clapham, D. E. TRP-PLIK, a Bifunctional Protein with Kinase and Ion Channel Activities. Science (80-. ). 291, 1043–1047 (2001). 119. Ryazanova, L. V., Pavur, K. S., Petrov, A. N., Dorovkov, M. V & Ryazanov, A. G. Novel type of signaling molecules: Protein kinases covalently linked with ion channels. Mol. Biol. 35, 271–283 (2001).

121

120. Jackson, M. F., Sun, H., Tymianski, M. & Macdonald, J. F. in New Strategies for Stroke Intervention 175–188 (2009). doi:10.1007/978-1-60761-280-3 121. Perraud, A.-L., Knowles, H. M. & Schmitz, C. Novel aspects of signaling and ion- homeostasis regulation in immunocytes. The TRPM ion channels and their potential role in modulating the immune response. Mol. Immunol. 41, 657–73 (2004). 122. Krapivinsky, G., Mochida, S., Krapivinsky, L., Cibulsky, S. M. & Clapham, D. E. The TRPM7 ion channel functions in cholinergic synaptic vesicles and affects transmitter release. Neuron 52, 485–96 (2006). 123. Visser, D., Middelbeek, J., Van Leeuwen, F. N. & Jalink, K. Function and regulation of the channel-kinase TRPM7 in health and disease. Eur. J. Cell Biol. 93, 455–465 (2014). 124. Owsianik, G., Talavera, K., Voets, T. & Nilius, B. Permiation and Selectivity of TRP Channels. Annu. Rev. Physiol. 68, 685–717 (2006). 125. Monteilh-Zoller, M. K. et al. TRPM7 Provides an Ion Channel Mechanism for Cellular Entry of Trace Metal Ions. J. Gen. Physiol. 121, 49–60 (2003). 126. Lupas, a N. & Gruber, M. The structure of a-helical coiled coils. Adv. Protein. Chem. 70, 37–78 (2005). 127. Crick, F. Packing of alpha-helices: Simple Coiled-Coils. Acta Crystallogr. 6, 986–997 (1953). 128. Fujiara, Y. & Minor, D. L. X ray crystal structuure of a TRPM assemble doamin reveals an antiparallel four stranded coiled coil. 383, 854–870 (2009). 129. Xie, J. et al. Phosphatidylinositol 4,5-bisphosphate (PIP2) controls magnesium gatekeeper TRPM6 activity. Sci. Rep. 1, 146 (2011). 130. Middelbeek, J., Clark, K., Venselaar, H., Huynen, M. A. & Van Leeuwen, F. N. The alpha-kinase family: An exceptional branch on the protein kinase tree. Cell. Mol. Life Sci. 67, 875–890 (2010). 131. Yamaguchi, H., Matsushita, M., Nairn, A. C. & Kuriyan, J. Crystal structure of the atypical protein kinase domain of a TRP channel with phosphotransferase activity. Mol. Cell 7, 1047–1057 (2001). 132. Sahni, J., Nelson, B. & Scharenberg, A. M. SLC41A2 encodes a plasma-membrane Mg 2+ transporter. Biochem. J. 401, 505–513 (2007). 133. Matsushita, M. et al. Channel function is dissociated from the intrinsic kinase activity and autophosphorylation of TRPMT/ChaK1. J. Biol. Chem. 280, 20793–20803 (2005). 134. Clark, K. et al. Massive autophosphorylation of the Ser/Thr-rich domain controls protein kinase activity of TRPM6 and TRPM7. PLoS One 3, (2008). 135. Runnels, L. W., Yue, L. & Clapham, D. E. The TRPM7 channel is inactivated by PIP(2) hydrolysis. Nat. Cell Biol. 4, 329–36 (2002). 136. Langeslag, M., Clark, K., Moolenaar, W. H., Van Leeuwen, F. N. & Jalink, K. Activation of TRPM7 channels by phospholipase C-coupled receptor agonists. J. Biol. Chem. 282, 232–239 (2007).

122

137. Deason-Towne, F., Perraud, A.-L. & Schmitz, C. Identification of Ser/Thr phosphorylation sites in the C2-domain of phospholipase C γ2 (PLCγ2) using TRPM7- kinase. Cell. Signal. 24, 2070–5 (2012). 138. Perraud, A., Zhao, X., Ryazanov, A. G. & Schmitz, C. The channel-kinase TRPM7 regulates phosphorylation of the translational factor eEF2. Cell. Signal. 23, 586–593 (2011). 139. Ryazanov, A. G., Shestakova, E. & Natapov, P. Phosphorylation of elongation factor 2 by EF-2 kinase affect rate of translation. Nature 334, 170–176 (1988). 140. Egelhoff, T. T., Lee, R. J. & Spudich, J. A. Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo. Cell 75, 363–371 (1993). 141. Gerke, V. & Moss, S. E. Annexins : From Structure to Function. Physiol. Rev. 82, 331– 371 (2002). 142. Dorovkov, M. V & Ryazanov, A. G. Phosphorylation of annexin I by TRPM7 channel- kinase. J. Biol. Chem. 279, 50643–6 (2004). 143. Schmitz, C. et al. Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell 114, 191–200 (2003). 144. Krapivinsky, G., Krapivinsky, L., Manasian, Y. & Clapham, D. E. The TRPM7 chanzyme is cleaved to release a chromatin-modifying kinase. Cell 157, 1061–72 (2014). 145. Desai, B. N. et al. ScienceDirect.com - Developmental Cell - Cleavage of TRPM7 Releases the Kinase Domain from the Ion Channel and Regulates Its Participation in Fas- Induced Apoptosis. Sciencedirect.Com 22, 1149–1162 (2012). 146. Kuras, Z., Yun, Y. H., Chimote, A. A., Neumeier, L. & Conforti, L. KCa3.1 and TRPM7 channels at the Uropod regulate migration of activated human T cells. PLoS One 7, (2012). 147. Meng, X. et al. TRPM7 mediates breast cancer cell migration and invasion through the MAPK pathway. Cancer Lett. 333, 96–102 (2013). 148. Chen, J.-P. et al. TRPM7 regulates the migration of human nasopharyngeal carcinoma cell by mediating Ca 2+ influx. Cell Calcium 47, 425–432 (2010). 149. Romani, A. & Scarpa, A. Regulation of cell magnesium. Arch. Biochem. Biophys. 298, 1–12 (2000). 150. Gilbert D’Angelo, E. K., Singer, H. A. & Rembold, C. M. Magnesium relaxes arterial smooth muscle by decreasing intracellular Ca2+ without changing intracellular Mg2+. J. Clin. Invest. 89, 1988–1994 (1992). 151. Chein, M. M. & Cambier, J. Divalent Cation Regulation of Phosphoinositide metabolism. J. Biol. Chem. 265, 9201–9207 (1990). 152. Kato, M. & Takenawa, T. Purification and characterization of membrane-bound and cytosolic forms of diacylglycerol kinase from rat brain. J. Biol. Chem. 265, 794–800 (1990).

123

153. Wheeler, M. L., Dong, M. B., Brink, R., Zhong, X. P. & De Franco, A. L. Diacylglycerol kinase ζ limits B cell antigen receptor-dependent activation of ERK Signaling to inhibit early antibody responses. Sci. Signal. 6, 1–24 (2013). 154. Woscholski, R., Waterfield, M. D. & Parker, P. J. Purification and biochemical characterization of a mammalian phosphatidylinositol 3,4,5-trisphosphate 5- phosphatase2260. J. Biol. Chem. 270, 31001–31007 (1995). 155. Walter, W. J., Koonce, M. P., Brenner, B. & Steffen, W. Two independent switches regulate cytoplasmic dynein’s processivity and directionality. Proc. Natl. Acad. Sci. U. S. A. 109, 5289–93 (2012). 156. Klein, D. J., Moore, P. B. & Steitz, T. a. The contribution of metal ions to the structural stability of the large ribosomal subunit. RNA Soc. 1366–1379 (2004). doi:10.1261/rna.7390804.2 157. Zhang, B., Zhang, Y., Wang, Z. X. & Zheng, Y. The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins. J. Biol. Chem. 275, 25299–25307 (2000). 158. Yang, L., Arora, K., Beard, W. A., Wilson, S. H. & Schlick, T. Critical role of magnesium ions in DNA polymerase β’s closing and active site assembly. J. Am. Chem. Soc. 126, 8441–8453 (2004). 159. Cowan, J. Structural and catalytic chemistry of magnesium dependent enzymes. Biometals 15, 225–235 (2002). 160. Gifford, J. L., Walsh, M. P. & Vogel, H. J. Structures and metal-ion-binding properties of the Ca 2+ -binding helix–loop–helix EF-hand motifs. Biochem. J. 405, 199–221 (2007). 161. Kruse, H. ., Orent, E. & McCollum, E. . Studies on magensium deficiency in animals I. symptomatology resulting from magensium deprevation. J. Biol. Chem. 65, 519–539 (1932). 162. Mazur, A. et al. Magnesium and the inflammatory response: Potential physiopathological implications. Arch. Biochem. Biophys. 458, 48–56 (2007). 163. Malpuech-Brugère, C. et al. Accelerated thymus involution in magnesium-deficient rats is related to enhanced apoptosis and sensitivity to oxidative stress. Br. J. Nutr. 81, 405– 411 (1999). 164. Malpuech-Brugère, C. et al. Inflammatory response following acute magnesium deficiency in the rat. Mol. Basis Dis. 1501, 91–98 (2000). 165. Zimowska, W. et al. Morphological and immune response alterations in the intestinal mucosa of the mouse after short periods on a low-magnesium diet. Br. J. Nutr. 88, 515– 22 (2002). 166. Li, F. et al. XMEN disease : a new primary immunodeficiency affecting Mg 2+ regulation of immunity against Epstein-Barr virus. Blood 123, 2148–2152 (2014). 167. de Baaij, J. H., Hoenderop, J. G. J. & Bindels, R. J. M. Magnesium in man: implications for health and disease. Physiol Rev 95, 1–46 (2015).

124

168. Sahni, J. & Scharenberg, A. M. TRPM7 ion channels are required for sustained phosphoinositide 3-kinase signaling in lymphocytes. Cell Metab. 8, 84–93 (2008). 169. Carrasco, Y. R. & Batista, F. D. B cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node. Immunity 27, 160–71 (2007). 170. Elizondo, M. R. et al. Defective skeletogenesis with kidney stone formation in dwarf zebrafish mutant for trpm7. Curr. Biol. 15, 667–671 (2005). 171. Liu, W. et al. TRPM7 regulates gastrulation during vertebrate embryogenesis Wei. Dev. Biol. 350, 348–357 (2011). 172. Jin, J. et al. Deletion of TRPM7 Disrupts Embryonic Development and Thymopoiesis Without Altering Mg 2+ Homeostasis. Science 322, 756–760 (2008). 173. Hobeika, E. et al. Testing gene function early in the B cell lineage in mb1-cre mice. Proc. Natl. Acad. Sci. U. S. A. 103, 13789–13794 (2006). 174. Ngo, V. N., Cornall, R. J. & Cyster, J. G. Splenic T zone development is B cell dependent. J. Exp. Med. 194, 1649–60 (2001). 175. Gonnella, P. A., Waldner, H. P. & Weiner, H. L. B Cell-Deficient ( MT) Mice Have Alterations in the Cytokine Microenvironment of the Gut-Associated Lymphoid Tissue (GALT) and a Defect in the Low Dose Mechanism of Oral Tolerance. J. Immunol. 166, 4456–4464 (2001). 176. Manz, M. G. & Boettcher, S. Emergency granulopoiesis. Nat. Rev. Immunol. 14, 302– 314 (2014). 177. Ryazanova, L. V. et al. TRPM7 is essential for Mg2+ homeostasis in mammals. Nat. Commun. 1, 109 (2010). 178. Cho, S. K. et al. Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 96, 9797–802 (1999). 179. Nakano, T., Kodama, H. & Honjo, T. Generation of Lymphohematopoietic Cells from Embryonic Stem Cells in Culture. Science (80-. ). 265, 1098–1101 (2017). 180. Shimizu, C. et al. Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of lck proximal promoter activity. Int. Immunol. 13, 105–17 (2001). 181. Buendia, A. et al. B-Cell Deficient Mice Show an Exacerbated Inflammatory Response in a Model of Chlamydophila abortus Infection. Microbiology 70, 6911–6918 (2002). 182. Smelt, S. C., Cotterell, S. E. J., Engwerda, C. R. & Kaye, P. M. B Cell Deficient Mice Are Highly Resistant to Leishmania donovani Infection, but Develop Neutrophil- Mediated Tissue Pathology. J. Immunol. 164, 3681–3688 (2000). 183. Ueda, Y., Kondo, M. & Kelsoe, G. Inflammation and the reciprocal production of granulocytes and lymphocytes in bone marrow. J. Exp. Med. 201, 1771–1780 (2005). 184. Ryazanova, L. V. et al. Elucidating the role of the TRPM7 alpha-kinase: TRPM7 kinase inactivation leads to magnesium deprivation resistance phenotype in mice. Sci Rep 4,

125

7599 (2014). 185. Phan, T. G., Grigorova, I., Okada, T. & Cyster, J. G. Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nat. Immunol. 8, 992–1000 (2007). 186. Castello, A. et al. Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K- Akt pathway in B cells. Nat. Immunol. 14, (2013). 187. Song, W., Liu, C. & Upadhyaya, A. The pivotal position of the actin cytoskeleton in the initiation and regulation of B cell receptor activation. Biochim. Biophys. Acta (2013). doi:10.1016/j.bbamem.2013.07.016 188. Kim, Y. J., Sekiya, F., Poulin, B., Bae, Y. S. & Rhee, S. G. Mechanism of B-Cell Receptor-Induced Phosphorylation and Activation of Mechanism of B-Cell Receptor- Induced Phosphorylation and Activation of Phospholipase C- ␥ 2. 24, 9986–9999 (2004). 189. Schmitz, C., Perraud, A.-L., Fleig, A. & Scharenberg, A. M. Dual-function ion channel/protein kinases: novel components of vertebrate magnesium regulatory mechanisms. Pediatr. Res. 55, 734–7 (2004). 190. Winding, P. & Berchtold, M. W. The chicken B cell line DT40: a novel tool for gene disruption experiments. J. Immunol. Methods 249, 1–16 (2001). 191. Jin, J., Desai, B. N., Navarro, B., Donovan, A. & Andrews, N. C. Supporting Online Material for Deletion of Trpm7 Disrupts Embryonic Development and Thymopoiesis without Altering Mg 2 + homeostasis * These authors contributed equally to this work This PDF file includes : 192. Chubanov, V. et al. Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7. Br. J. Pharmacol. 166, 1357–76 (2012). 193. Wulff, H., Knaus, H.-G., Pennington, M. & Chandy, K. G. K+ channel expression during B cell differentiation: implications for immunomodulation and autoimmunity. J. Immunol. 173, 776–86 (2004). 194. Niiro, H. et al. ( Bam32 ) Regulates B Cell Antigen Receptor Internalization 1. (2017). doi:10.4049/jimmunol.173.9.5601 195. Nobes, C. D. & Hall, A. Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81, 53–62 (1995). 196. Batista, F. D., Treanor, B. L. & Harwood, N. E. Visualizing a role for the actin cytoskeleton in the regulation of B-cell activation. Immunol. Rev. 237, 191–204 (2010). 197. Treanor, B. L. & Batista, F. D. Organisation and dynamics of antigen receptors: implications for lymphocyte signalling. Curr. Opin. Immunol. 22, 299–307 (2010). 198. Mossman, K. D., Campi, G., Groves, T. J. & Dustin, M. L. Altered TCR Signaling from Geometrically Repatterned Immunological Synapses. Science (80-. ). 310, 1191–1193 (2005).

126

199. Brown, B. K. & Song, W. The actin cytoskeleton is required for the trafficking of the B cell antigen receptor to the late endosomes. Traffic 2, 414–427 (2001). 200. Barois, N., Forquet, F. & Davoust, J. Actin microfilaments control the MHC class II antigen presentation pathway in B cells. J Cell Sci 111 ( Pt 1, 1791–1800 (1998). 201. Bertin, S. et al. The TRPA1 ion channel is expressed in CD4+ T cells and restrains T- cell-mediated colitis through inhibition of TRPV1. Gut 1–13 (2016). doi:10.1136/gutjnl- 2015-310710 202. Bertin, S. et al. The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4(+) T cells. Nat Immunol 15, 1055–1063 (2014). 203. Feske, S., Skolnik, E. Y. & Prakriya, M. Ion channels and transporters in lymphocyte function and immunity. Nat Rev Immunol 12, 532–547 (2012). 204. Nishimoto, N. et al. Normal pre-B cells express a receptor complex of M heavy chains and surrogate light-chain proteins. Immunology 88, 6284–6288 (1991). 205. Brauweiler, A. et al. Differential regulation of B cell development, activation, and death by the src homology 2 domain-containing 5’ inositol phosphatase (SHIP). J Exp Med 191, 1545–1554 (2000). 206. Cheng, A. M. et al. Syk tyrosine kinase required for mouse viability and B-cell development. Nature 378, 303–306 (1995). 207. Okada, T. et al. Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. J.Biol.Chem. 274, 27359–27370 (1999). 208. Rickert, R. C., Roes, J. & Rajewsky, K. B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 25, 1317–1318 (1997). 209. Hashimoto, A. et al. Cutting Edge: Essential Role of Phospholipase C- 2 in B Cell Development and Function. J. Immunol. 165, 1738–1742 (2000). 210. Feldhahn, N. et al. Mimicry of a constitutively active pre–B cell receptor in acute lymphoblastic leukemia cells. J. Exp. Med. 201, 1837–1852 (2005). 211. Kraus, M., Alimzhanov, M. B., Rajewsky, N. & Rajewsky, K. Survival of resting mature B lymphocytes depends on BCR signaling via the Igα/β heterodimer. Cell 117, 787–800 (2004). 212. Carsetti, R., Köhler, G. & Lamers, M. C. Transitional B cells are the target of negative selection in the B cell compartment. J. Exp. Med. 181, 2129–2140 (1995). 213. Goodnow, C. C., Sprent, J., de St Groth, B. F. & Vinuesa, C. G. Cellular and genetic mechanisms of self tolerance and autoimmunity. Nature 435, 590–597 (2005). 214. Philipp, S. et al. TRPC3 mediates T-cell receptor-dependent calcium entry in human T- lymphocytes. J. Biol. Chem. 278, 26629–26638 (2003). 215. Le Deist, F. et al. A primary T-cell immunodeficiency associated with defective transmembrane calcium influx. Blood 85, 1053–62 (1995). 216. Feske, S. et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC

127

channel function. Nature 441, 179–185 (2006). 217. Mori, Y. et al. Transient receptor potential 1 regulates capacitative Ca(2+) entry and Ca(2+) release from endoplasmic reticulum in B lymphocytes. J. Exp. Med. 195, 673–81 (2002). 218. Numaga, T. et al. Ca2+ influx and protein scaffolding via TRPC3 sustain PKCbeta and ERK activation in B cells. J. Cell Sci. 123, 927–38 (2010). 219. Dolmetsch, R. E., Xu, K. & Lewis, R. S. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392, 933–936 (1998). 220. Thebault, S. et al. Differential role of transient receptor potential channels in Ca 2+ entry and proliferation of prostate cancer epithelial cells. Cancer Res. 66, 2038–2047 (2006). 221. Caterina, M. et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824 (1997). 222. Bandell, M. et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 41, 849–857 (2004). 223. Stokes, A. et al. TRPA1 is a substrate for de-ubiquitination by the tumor suppressor CYLD. Cell. Signal. 18, 1584–1594 (2006). 224. Amantini, C. et al. Distinct thymocyte subsets express the vanilloid receptor VR1 that mediates capsaicin-induced apoptotic cell death. Cell Death Differ. 11, 1342–1356 (2004). 225. Farfariello, V., Amantini, C. & Santoni, G. Transient receptor potential vanilloid 1 activation induces autophagy in thymocytes through ROS-regulated AMPK and Atg4C pathways. J. Leukoc. Biol. 92, 421–431 (2012). 226. Qian, F. & Noben-Trauth, K. Cellular and molecular function of mucolipins (TRPML) and polycystin 2 (TRPP2). Pflugers Arch. Eur. J. Physiol. 451, 277–285 (2005). 227. Karacsonyi, C., San Miguel, A. & Puertollano, R. Mucolipin-2 localizes to the Arf6- associated pathway and regulates recycling of GPI-APs. Traffic 8, 1404–1414 (2007). 228. Thompson, E. G., Schaheen, L., Dang, H. & Fares, H. Lysosomal trafficking functions of mucolipin-1 in murine macrophages. BMC Cell Biol. 8, 54 (2007). 229. Song, Y., Dayalu, R., Matthews, S. A. & Scharenberg, A. M. TRPML cation channels regulate the specialized lysosomal compartment of vertebrate B-lymphocytes. Eur. J. Cell Biol. 85, 1253–1264 (2006). 230. Feske, S. Calcium signalling in lymphocyte activation and disease. Nat. Rev. Immunol. 7, 690–702 (2007). 231. Baba, Y. et al. Coupling of STIM1 to store-operated Ca2+ entry through its constitutive and inducible movement in the endoplasmic reticulum. Proc. Natl. Acad. Sci. U. S. A. 103, 16704–9 (2006). 232. Beaven, M. A. & Baumgartner, R. A. Downstream signals initiated in mast cells by Fc RI and other receptors. Curr. Biol. 766–772 (1996).

128

233. Hsu Sf et al. Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway. J. Immunol. 166, 6126–6133 (2001). 234. Feske, S., Giltnane, J., Dolmetsch, R. E., Staudt, L. M. & Rao, A. Gene regulation mediated by calcium signals in T lymphocytes. Nat Immunol 2, 316–324 (2001). 235. Bussière, F. I., Gueux, E., Rock, E., Mazur, A. & Rayssiguier, Y. Protective effect of calcium deficiency on the inflammatory response in magnesium-deficient rats. Eur. J. Nutr. 41, 197–202 (2002). 236. Volpe, P., Alderson-Lang, B. & Nickols, A. Regulation of inositol 1,4,5-trisphosphate- induced Ca2+ release. I. Effect of Mg2+. Am. Physiol. Soc. (1990). 237. Tong, G. M. & Rude, R. K. Magnesium deficiency in critical illness. J. Intensive Care Med. 20, 3–17 (2005). 238. Laurant, P. & Touyz, R. M. Physiological and pathophysiological role of magnesium in the cardiovascular system: implications in hypertension. J. Hypertens. 18, 1177–1191 (2000). 239. Velissaris, D., Karamouzos, V., Pierrakos, C., Aretha, D. & Karanikolas, M. Hypomagnesemia in Critically Ill Sepsis Patients. J Clin Med. Res 7, 911–918 (2015). 240. Thongprayoon, C., Cheungpasitporn, W. & Erickson, S. B. Admission hypomagnesemia linked to septic shock in patients with systemic inflammatory response syndrome. Ren. Fail. 37, 1518–1521 (2015). 241. Chaigne-Delalande, B. et al. Mg2+ Regukates Cytotoxic Funtions of NK and CD8 T Cells in Chronic EBV Infection Through NKG2D. Science (80-. ). 1, 186–191 (2013). 242. Romani, A. M. P. Cellular magnesium homeostasis. Arch. Biochem. Biophys. 512, 1–23 (2011). 243. Chandy, K. G., DeCoursey, T. E., Cahalan, M. D., McLaughlin, C. & Gupta, S. Voltage- gated potassium channels are required for human T lymphocyte activation. J. Exp. Med. 160, 369–85 (1984). 244. Douglass, J. et al. Characterization and functional expression of a rat genomic DNA clone encoding a lymphocyte potassium channel. J. Immunol. Methods 144, 4841–4850 (1990). 245. Attali, B. et al. Cloning, functional expression, and regulation of two K+ channels in human T lymphocytes. J. Biol. Chem. 267, 8650–8657 (1992). 246. Grissmer, S., Nguyen, a N. & Cahalan, M. D. Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology. J. Gen. Physiol. 102, 601–30 (1993). 247. Kuang, Q., Purhonen, P. & Hebert, H. Structure of potassium channels. Cell. Mol. Life Sci. 72, 3677–3693 (2015). 248. Cahalan, M. D. & Chandy, K. G. The functional network of ion channels in T lymphocytes. Immunol. Rev. 231, 59–87 (2011). 249. Fanger, C. M. et al. Calmodulin Mediates Calcium-dependent Activation of the

129

Intermediate Calmodulin Mediates Calcium-dependent Activation of the Intermediate Conductance K Ca Channel , IKCa1 *. 274, 5746–5754 (1999). 250. Fanger, C. M. et al. Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels. J. Biol. Chem. 276, 12249–12256 (2001). 251. Nicolaou, S., Neumeier, L., Peng, Y., Devor, D. & Conforti, L. Compartmentalizes in the Immunological Synapse of Human T Lymphocytes. Am J Physiol Cell Physiol 292, 1431–1437 (2008). 252. Panyi, G. et al. Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells. Proc. Natl. Acad. Sci. U. S. A. 101, 1285–90 (2004). 253. Szabò, I. et al. Cell Biology and Metabolism : Tyrosine Phosphorylation-dependent Suppression of a Voltage-gated K + Channel in T Lymphocytes upon Fas Stimulation Alerts : • When this article is cited Tyrosine Phosphorylation-dependent Suppression of a .(Voltage- gated K ؉ . 271, 20465–20469 (1996 254. Chung, I. & Schlichter, L. C. Native Kv1.3 channels are upregulated by protein kinase C. J. Membr. Biol. 156, 73–85 (1997). 255. Eil, R. et al. Ionic immune suppression within the tumour microenvironment limits T cell effector function. Nature 537, 539–543 (2016). 256. Bhaskar, P. T. & Hay, N. The Two TORCs and Akt. Dev. Cell 12, 487–502 (2007). 257. Amigorena, S., Choquet, D., Teillaud, J. L., Korn, H. & Fridman, W. H. Ion channel blockers inhibit B cell activation at a precise stage of the G1 phase of the cell cycle. Possible involvement of K+ channels. J. Immunol. 144, 2038–45 (1990). 258. Partiseti, M., Korn, H. & Choquet, D. Pattern of potassium channel expression in proliferating B lymphocytes depends upon the mode of activation. J. Immunol. 11, 2462– 2470 (1993). 259. Hirano, T. et al. Roles of Zinc and Zinc Signaling in Immunity: Zinc as an Intracellular Signaling Molecule. Adv. Immunol. 97, 149–176 (2008). 260. Ackland, M. L. & Michalczyk, A. Zinc deficiency and its inherited disorders -a review. Genes Nutr. 1, 41–49 (2006). 261. Fraker, P. J. & King, L. E. Reprogramming of the Immune System During Zinc Deficiency. Annu. Rev. Nutr. 24, 277–298 (2004). 262. Cook-Mills, J. M. & Fraker, P. J. Functional capacity of the residual lymphocytes from zinc-deficient adult mice. Br J Nutr 69, 835–848 (1993). 263. Haase, H. & Rink, L. Functional significance of zinc-related signaling pathways in immune cells. Annu. Rev. Nutr. 29, 133–52 (2009). 264. Rükgauer, M., Klein, J. & Kruse-Jarres, J. D. Reference Values for the Trace Elements Copper, Manganese, Selenium, and Zinc in the Serum / Plasma of Children, Adolescents, and Adults. J. Trace Elem. Med. Biol. 11, 92–98 (1997).

130

265. Yu, M. et al. Regulation of T cell receptor signaling by activation-induced zinc influx. J. Exp. Med. 208, 775–785 (2011). 266. Hojyo, S. et al. Zinc transporter SLC39A10/ZIP10 controls humoral immunity by modulating B-cell receptor signal strength. Proc. Natl. Acad. Sci. U. S. A. 111, 11786–91 (2014). 267. Miyai, T. et al. Zinc transporter SLC39A10/ZIP10 facilitates antiapoptotic signaling during early B-cell development. Proc. Natl. Acad. Sci. U. S. A. 111, 11780–5 (2014). 268. Merino, R., Ding, L., Veis, D. J., Korsmeyer, S. J. & Nuñez, G. Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes. EMBO J. 13, 683–691 (1994).

131