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CD22 is indispensable for Galectin-9-mediated inhibition of signaling

By Hifza Buhari

A thesis submitted in conformity with the requirements for the degree of Master of Science. Department of Cell & Systems Biology, University of Toronto © Copyright by Hifza Buhari 2019

CD22 is indispensable for Galectin-9-mediated inhibition of B Thesis

Title: CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Degree: Master of Science Year of Convocation: 2019 Name: Fathima Hifza Mohamed Buhari Graduate Department: Cell & Systems Biology University: University of Toronto

Abstract

B-cells are an important component of the , producing antibodies and conferring long-term immunity. B-cell activation is initiated by recognition of foreign particles

() by B-cell receptors (BCR). Previous research in our lab found that the , Galectin-

9 (Gal9), regulates B-cell activation; however, the mechanism remains unclear. In this study, we demonstrate using super-resolution microscopy that Gal9 regulates the organization of a lattice of inhibitory and stimulatory receptors to inhibit B-cell signaling. We found that the inhibitory receptor CD22 is crucial for this Gal9-mediated mechanism. Furthermore, we demonstrate that

CD22 N-linked glycosylation is required for Gal9-mediated inhibition of signaling. To our knowledge, our study is the first to highlight the importance of galectins in regulating CD22 organization, as well as define N- in CD22 as imperative in regulating B-cell signaling.

Our findings may have important implications for the therapeutic application of Gal9 to alleviate

B-cell mediated autoimmune diseases.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Declaration

Dual dSTORM of WT and 5Q mutant (controls) were acquired by Laabiah Waasim. Analysis of dual dSTORM was performed by Myuran Yoganathan. All other work presented in this thesis is my own.

Data in this thesis has or will be submitted for publication in peer-reviewed publications.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Acknowledgements

When I joined Dr. Treanor’s lab four years ago to do my undergraduate thesis, I didn’t know much about B cells or how to conduct experiments. I am now leaving the lab after gaining those skills but also so much more, importantly the mentorship and friendships that I will cherish forever.

First of all, I would like to thank Dr. Bebhinn for the amazing support she provided as I learned and grew, for teaching me what a supportive and productive mentor looks like, and for believing in me. I cannot thank her enough.

Thank you to my committee members, Dr. Harrison and Dr. Terebiznik, for supporting my decision to complete my Masters early and for asking insightful questions. Thank you to Bruno for the training and technical help he provided when I was down in the CNS.

Thank you to my mom who works tirelessly, and who takes care of me when I forget to take care of myself. And thank you to my dad, who lives and works more than a 1000 miles away, in order to ensure I can gain an education. Thank you to my siblings; Izaaz and Jasmin datha for bringing me dinner when I spent late-nights in the lab, and Amrin for bringing me cups of tea during the late nights while I drew up presentations. I could never be where I am now without them.

Thank you to my long-term lab mates; Anh, Tina, Trisha, and Laabiah, for being the amazing people they are and being pillars of support. Anh lit up my lab environment with his boisterous energy and sensitive nature, and I thank him for providing me with laughter during the fun times and support during the low points. Thanks to Tina, hard-working and sensitive, playfully making fun of me on every turn, but would provide me the best words of advice when I doubted myself. iv Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Thank you to Trisha, my knowledgeable and charismatic friend, and one of the sweetest humans

I have had the privilege to know. And a huge thank you to Laabiah, the delightful tree-climbing human I know; a person who reminds me to be passionate about science, and also what it means to be the truest form of a friend.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Table of Contents

Abstract…………………………………………………………………………………..…….…ii Declaration…………………………………………………………………………….……...…..iii Acknowledgements………………………………………………………………………...... iv - v Table of Contents…...….…………………..……………………………………..…...... …vi - vii List of Figures………………………………………………………………………….…....…..viii Abbreviations…………………………………………..……………………………..…...…ix - xi INTRODUCTION…………………………………………...…………………..…….….…1 – 23 1 B cells……………………………...…...…………………...…...……..………....1 – 2 1.1 B cell activation and signaling…………..………………………………...... 2 – 5 1.2 Important receptors of B cell signaling……………………………...…….....6 – 14 1.2.1 IgM-BCR……………………………...………...……...…………….6 – 7 1.2.2 CD19……………………………...…………………………...……..8 – 9 1.2.3 CD45…………………...…………………………...………..……10 – 11 1.2.4 CD22……………………...…………………………………...…..12 – 14 1.3 Spatiotemporal regulation of membrane receptors……………………...... 15 1.3.1 Lipid Domains…………………...…………………………...... 15 - 16 1.3.2 Actin Cortex..……………………...……………………………………16 1.3.3 Glycosylation…………………………………………………....…17 – 18 1.3.3.1 Novel regulators: Galectin lattice……………………...... 19 – 20 1.3.3.1.1 Galectin-9……………………….…………..20 – 22 1.4 Hypothesis and Aims…………………………………………………………….23 METHODS…………………………………………………………………………………24 – 34 2.1 Mice……………………………………………………………………...………24 2.2 Murine Cell Isolation…………………………………………………………....24 2.3 Cell Line and Culturing…………………………………..………………..24 – 25 2.4 rGal9 Treatment………………………………………………………..…..……25 2.5 Gal9 surface staining………………………………………………..……..25 – 26 2.6 Fluorescent immunostaining of murine B cells for confocal microscopy.…26 – 27 2.7 Staining …………………………………………………..………..…27 2.8 Calcium Signaling……………………………………………………..…………28 2.9 Western Blots………………………………………………………..…..…28 – 30 2.9.1 Murine B cells…………………………………………….………….28 – 29 2.9.2 Daudi B cells…………………………………………….…………...29 – 30 2.10 Statistical analysis………………………………………………………………30 2.11 Dual dSTORM…………………………………………………...………..31 – 34 2.11.1 Daudi B cell sample preparation………………………………………….31 2.11.2 dSTORM acquisition and image reconstruction……………...... 31 – 32 vi Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

2.11.3 dSTORM post-processing, analysis and quantification………………………33 2.11.4 Coordinate based co-localization analysis……………………………………34

RESULTS…………………….…………………………………….………………………35 – 45 3.1 Galectin-9 increases the molecular density of CD19………..……….…….35 – 36 3.2 Addition of rGal9 does not induce activation of B cells……….…………..37 – 38 3.3 Treatment of Daudi B cells with rGal9 induces IgM-CD22 colocalization..39 – 42 3.4 Treatment of rGal9 causes coalescence of lipid rafts enriched in CD22 and CD45 ……………………………………………………………………………..42 – 44 3.5 Gal9-KO B cells do not have significant increase in phosphorylation of CD19……..…………………………………………………………………44 – 45 3.6 Treatment of B cells with rGal9 suppresses B cell signaling.……………...46 – 47 3.7 CD22 is required for Gal9-mediated inhibition of B cell signaling………...48 – 49 3.8 CD22 glycosylation is crucial for mediating IgM-CD22 interactions….…..50 – 51 3.9 CD22 N-glycans are crucial for Gal9-mediated CD22 clustering…………52 – 53 3.10 Gal9-mediated inhibition of B cell signaling is dependent on CD22 N-glycans …………………………………...……………………………..…………54 – 55 DISCUSSION……………………………………………………...…………………….…56 – 69 References………………………………………………………..…………………………70 – 78

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

List of Figures

Figure 1. The BCR signaling cascade

Figure 2. Schematic structure of a typical LacNAc N-

Figure 3. The galectin family

Figure 4. The Gal9 lattice increases the molecular density of CD19 but not FcγRIIb

Figure 5. rGal9 treatment does not induce Ca2+ signaling in B cells

Figure 6. Exogenous Gal9 increases association of IgM and CD22

Figure 7. rGal9 induces coalescence of lipid raft domains containing CD22 and CD45

Figure 8. Phosphorylation of CD19 and Akt is not altered in Gal9-KO B cells

Figure 9. Treatment with exogenous Gal9 suppresses B cell signaling

Figure 10. CD22 is necessary for Gal9-mediated inhibition of BCR signaling

Figure 11. Gal9-mediated association of IgM and CD22 is dependent on N-glycans of CD22

Figure 12. Gal9-mediated CD22 clustering is dependent on N-glycans of CD22

Figure 13. CD22 N-glycans are crucial in Gal9-mediated regulation of B cell signaling

Figure 14. Working model depicting Galectin-9’s role in regulating receptor organization

and inhibition of B cell signaling

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Abbreviations

µg/ml Microgram per millimeter µM Micromolar µm2/s micrometer squared per second µH IgM µ heavy chain δH IgD δ heavy chain 5Q-mutant Cluster of Differentiation 22 with 5 N to Q mutations Å Angstrom (1 × 10-10 meters) Ag Akt Kinase B APC Antigen presenting cell Asn Asparagine N Asparagine B220 B cell isoform of 220kDa BCR B cell receptor BAFF B cell activating factor BSA Bovine serum albumin BLNK B-cell linker Btk Bruton’s tyrosine kinase Cµ IgM µ heavy chain constant domain Ca2+ Calcium ion CD19 Cluster of Differentiation 19 CD21 Cluster of Differentiation 21 CD22 Cluster of Differentiation 22 CD45 Cluster of Differentiation 45 CD81 Cluster of Differentiation 81 CRD Carbohydrate recognition domain Csk C-terminal Src-kinase CT-B Cholera toxin B DAG Diacylglycerol Dual dSTORM Dual direct stochastic optical reconstruction microscopy ECD Extracellular domain EDTA Ethylenediaminetetraacetic acid ER Endoplasmic reticulum ERK Extracellular signal-regulated kinase F-actin Filamentous actin FBS Fetal bovine serum Gal1 Galectin-1 Gal9 Galectin-9 Gal9-KO Galectin-9 Knockout GEM -enriched membrane GlcNAc N-Acetylglucosamine GPI Glycosylphosphatidylinositol ix Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Grb2 Growth factor receptor-bound protein 2 HIV Human immunodeficiency HRP Horseradish peroxidase IFNγ Interferon gamma Ig Immunoglobulin IgD Immunoglobulin D IgM Immunoglobulin M IgM-BCR IgM isotype B cell receptor IgG Immunoglobulin G IgE Immunoglobulin E Igα Immunoglobulin alpha Igβ Immunoglobulin beta IL Interleukin IP3 Inositol 1,4,5-triphosphate ITAMs Immunoreceptor tyrosine-based activation motifs ITIMs Immunoreceptor tyrosine-based inhibitory motifs JAK Janus kinases LatA Latrunculin A LR Lipid raft Man MAPK activated protein kinase Mgat β1,6-N-acetylglucoaminyl transferase MHC II Major histocompatibility complex class II N-glycan Asparagine-linked glycosylation NaCl Sodium chloride NaN3 Sodium azide Neu5Ac N-acetyl neuraminic acid Neu5Gc N-glycolyl neuraminic acid NF-κB Nuclear factor-κB NFAT Nuclear factor of activated T-cells PBS Phosphate buffered saline pERK Phosphorylated extracellular signal-regulated kinase PFA Paraformaldehyde PI3K Phosphatidylinositol-,4,5-bisphosphate 3-kinase PIP2 Phosphatidylinositol-4, 5-biphosphate PIP3 Phosphatidylinositol-3, 4, 5-triphosphate PKC Protein kinase C PLC-γ2 Phospholipase C gamma 2 PTPase Protein phosphatase PTPRC Protein tyrosine phosphatase, receptor type, C pY Phosphotyrosine Q Glutamine RA rGal-9 Recombinant galectin-9 RPMI Roswell Memorial Institute media x Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

SEM Standard error of means Ser Serine SFK Src family kinases SH2 domain Src homolog 2 domain Shc SHC-transforming protein SHIP-1 SH2 domain-containing inositol 5'-phosphatase 1 SHP-1 Src homology domain containing phosphatase-1 SIAE Sialic acid acetylesterase Siglec Sialic acid-binding immunoglobulin-like lectin SLE Systemic lupus erythematosus SPT Single-particle tracking SSc Systemic sclerosis STAT Signal transducer and activator of transcription Syk Spleen tyrosine kinase T1D Type 1 Diabetes TAPA-1 Target of the anti-proliferative antibody 1 TCR receptor Thr Threonine TIRFM Total internal reflection fluorescence microscopy Tyr Tyrosine Vµ IgM µ heavy chain variable domain v/v Volume/volume Vav Protein encoded by VAV gene WASP Wiskott-Aldrich Syndrome protein WT Wild-type Y Tyrosine

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

INTRODUCTION

1 B cells

B lymphocytes are an important component of the adaptive immune system, producing antibodies and conferring long-term immunity. The production of these essential effector responses relies on the ability of B cells to be activated upon encountering specific foreign particles, termed antigens. B cells recognize antigen through B cell receptors (BCRs)1. Antigen-

BCR ligation initiates an intracellular signaling cascade leading to B cell expansion, proliferation and differentiation into plasma cells or memory cells; the former secreting antigen-specific antibodies, the latter conferring long-term protection2.

B cells are derived from hematopoietic stem cells and begin their development in the bone marrow3. They undergo several developmental checkpoints to prevent recognition of self- antigens, and upon expression of the IgM-BCR isotype, leave the bone marrow and home to secondary lymphoid organs such as the spleen and/or circulate the blood, as naïve B cells3. In secondary lymphoid organs, B cells are presented antigen in membrane-bound form on the surface of subscapular macrophages or follicular dendritic cells, which leads to BCR signaling and activation4,5. The regulation of BCR signaling and resulting strength of the intracellular response determines the fate of the cell,5,6. For example, blocking the ability of positive regulators to regulate BCR signaling such as BLNK and CD19 has been found to lead to an absence of peritoneal cells, whereas blocking negative regulators, such as CD22, induced a greater production of peritoneal B cells6. Indeed, dysregulation of BCR signaling has been found to manifest in autoimmunity and B cell . For example, polymorphisms in the B cell

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis signaling kinases Lyn and Blk have been linked with the systemic lupus erythematosus (SLE)7. Furthermore, B cells from SLE patients are significantly more sensitive during antigenic challenge due to their increased susceptibility to respond to upregulated BAFF

7 (B cell activating factor), common in the serum of SLE patients . BAFF is important in promoting cell survival and differentiation, and BAFF receptors is upregulated by BCR stimulation7,143. In addition to autoimmune diseases, dysregulated BCR signaling has also been associated with the pathogenesis of B cell lymphomas. For example, the identification of

Bruton’s Tyrosine kinase (Btk) as an important component of the BCR signaling cascade led to development of selective Btk inhibitors for patients with B cell non-Hodgkin lymphoma8.

Testing of this inhibitor in demonstrated that its use reduced the level of circulating autoantibodies, highlighting the importance of regulation of BCR signaling and the impact of dysregulated signaling on the pathogenesis of autoimmune disease and lymphomas8. Hence, understanding B cell activation and signaling is crucial in order to identify potential targets for immunotherapies.

1.1 B cell activation and signaling

B cell activation occurs through two signals, with the first signal being engagement of BCR with antigen. The BCR consists of antigen-binding immunoglobulin heavy and light chains, and a transmembrane heterodimer of Igα and Igβ, each bearing a single immunoreceptor tyrosine- based activation motif (ITAM)9. Upon antigen-BCR ligation, antigen-BCR microclusters are formed10. Antigens that BCR recognize can either be soluble or membrane- bound, with the predominant form in vivo being membrane-bound, usually on the surface of antigen-presenting

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis cells (APC) such as dendritic cells or macrophages11,12,13,14. B cells acquire antigen by spreading over the APC surface and then contracting, aggregating antigen-BCR microclusters to the center of the B cell contact with the APC, which are subsequently internalized15. The antigen is then processed and presented on major histocompatibility complex (MHC) class II in order to activate T cells16. T cell activation causes the upregulation of CD40 ligand (CD40L), which binds to the B cell costimulatory receptor CD40, leading to T cell of soluble such as IL-4, IL-5, and IL-617. Together, these provide a second signal for B cell activation.

Signaling emanating from BCR engagement and T cell help leads to B cell proliferation and differentiation into plasma or memory B cells.

Upon antigen binding, BCRs translocate into glycosphingolipid-rich microdomains on the plasma membrane called lipid rafts18. These lipid raft domains have high levels of protein tyrosine kinases such as Lyn18. Lyn phosphorylates the ITAM motifs of Igα/Igβ19,20, which facilitates the recruitment of other signaling molecules such as Syk 18,21 (Figure 1). The recruitment of Syk leads to phosphorylation of other key proteins, such as B cell linker

(BLNK)22,23,24. BLNK acts as a scaffolding protein and is key to the initiation of several signaling cascades. Lyn also phosphorylates BCR co-receptor CD1925, which provides binding sites for the p85 adapter subunit of phosphoinositide 3-kinase (PI3K)26. Furthermore, Lyn also has an important negative regulatory role as it phosphorylates CD22 and FcγRIIB, two inhibitory coreceptors containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), that dampen

BCR signaling27,28. Shrivastava et al. discovered that the positive regulatory function of Lyn is inhibited by receptor-like protein tyrosine phosphatase family protein, CD4529.

CD19-mediated recruitment of PI3K leads to phosphorylation of phosphatidylinositol 4,5-

30,31 bisphophate (PI(4,5)P2) yielding phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) , and 3 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis recruitment of Bruton’s tyrosine kinase (Btk)32. BLNK facilitates recruitment of PLCγ2, which is

22,23,24 phosphorylated by Syk and Btk . Phosphorylated PLCγ2 catalyzes the cleavage of PIP2 into

33 second messengers inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) . IP3 binds IP3 receptors on the endoplasmic reticulum causing the influx of intracellular and extracellular Ca2+ into the cytosol34. This activates transcription factors nuclear factor-κB (NFκB) and nuclear factor of activated T cells (NFAT)33. DAG activates protein kinase C (PKC) which controls extracellular regulated kinases (ERK1/2), crucial for regulating B cell proliferation and differentiation33.

B cell activation and BCR signaling are crucial for cellular outcomes, determining whether the B cell will undergo proliferation, differentiation, or apoptosis33. Therefore, regulation of B cell activation and signaling is critical and mechanisms of regulation are an important area of active research.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Figure 1. The BCR signaling cascade. BCR binding of antigen (Ag) induces phosphorylation of ITAM motifs of Igα/β by Lyn (positive regulation indicated by green arrows). This recruits Syk, which leads to the recruitment of BLNK, creating a scaffold for the recruitment of proteins such as BTK and PLCγ2. The BCR signaling cascade is also amplified by the recruitment of CD19, which facilitates recruitment of PI3K and Akt. PI3K is involved in phosphorylating PIP2 into PIP3, allowing for the recrutiment of Btk. Akt recruitment is crucial for providing survival signals to the cell. PLCγ2 hydrolyzes PIP2 into 2+ diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 leads to release of intracellular Ca stores. DAG leads to the recruitment of protein kinase C, which phosphorylates proteins like ERK activating transcription factors that regulate proliferation and differentiation. Lyn activity is negatively regulated by CD45 (negative regulation indicated by red arrows). Lyn also phosphorylates inhibitory receptors such as CD22, which attentuates the BCR signaling response by recruiting phosphatases that dephosphorylate Syk and CD19. Purple circles indicate glycosylation.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

1.2 Important receptors of B cell signaling

Recent evidence has demonstrated that protein receptors are highly compartmentalized or laterally segregated into specific domains, and dysregulation of their spatial organization can lead to disease states. For example, Blouin et al. found that differential partitioning of IFNγ receptor into actin-rich domains instead of lipid domains through a gain-of-glycosylation mutation, caused lack of JAK/STAT signaling upon IFNγ stimulation, leading to susceptibility to mycobacterial infection in patients35. Therefore, an active area of research is focused on understanding the B cell membrane landscape and elucidating key mechanisms by which receptors are organized. This section highlights key receptors and co-receptors of B cell signaling and our current limited but crucial knowledge on the spatiotemporal organization of cell surface proteins and its influence on B cell signaling.

1.2.1 IgM-BCR

Mature naïve B cells that circulate the periphery express both IgM-BCR and IgD-BCR isotypes36, 37. Transcription for IgM µ heavy chain (µH) is seven times higher than that of IgD’s

δH likely due to the high turnover of IgM receptors38. At a given moment however, there are approximately 20,000 to 150,000 IgM molecules on the surface of B cells, which are organized into distinct nanoclusters separate from other receptors such as IgD and CD19 (with a small amount of overlap)39,40. Signal transduction via the IgM-BCR is crucial for B cell development in the bone marrow, tolerance induction, cell death, proliferation and differentiation33.

IgM-BCR is a 190-kDa membrane immunoglobulin composed of two heavy chains and two light chains41. There are five immunoglobulin domains in the heavy chain (Vµ, Cµ1-4) linked by bridges, and a cytoplasmic domain containing three amino acids (lysine, valine, 6 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis lysine)41. The light chains are attached to the heavy chain by a disulfide bridge to Cµ141. The structure of IgM-BCR is similar to serum IgM with the difference of a 41- (a.a) carboxyl terminus found in the IgM-BCR heavy chain41. Twelve of these amino acids are part of the extracellular surface of the protein, 26 form the transmembrane domain, and the last 3 form the cytoplasmic tail41.

While it is known that IgM-BCR is a glycosylated protein, the actual structure of the glycans and their ligands are currently unknown or are under investigation. Moreover, the N- glycan type and sites we describe for IgM-BCR have been extrapolated from identification of these sites from serum IgM, and therefore there could be unknown variations. There are five predicted N-linked glycosylation sites on IgM (Asn171, Asn332, Asn395, Asn402, Asn563)45.

There are variations in the type of glycans attached to these different glycosylation sites, with sites Asn171, Asn332, and Asn395 predominantly possessing complex N-type glycans, whereas

Asn402 and Asn563 possess high mannose type N-glycans (Man6-9GlcNAc2)45.

Using the super resolution technique direct stochastic optical reconstruction microscopy

(dSTORM), IgM-BCR has been shown to exist in distinct nanoclusters on the surface of B cells39. These clusters are approximately 120-160 nm in diameter and contain approximately 20-

50 molecules of IgM39. In resting B cells, these IgM-BCRs diffuse through the membrane, restricted by the actin cytoskeleton42. IgM-BCR but not IgD-BCR has been found to be

43 segregated from GM1 gangliosides in resting B cells . As GM1 ganglioside-containing regions play a crucial role as signaling domains (refer to section 1.3.1), the regulation of different BCR isotypes into different domains may have important implications on cell fate upon BCR stimulation.

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1.2.2 CD19

CD19 is a 95-kDa transmembrane B cell specific costimulatory receptor. It has been found that compared to IgM-BCR ligation alone, IgM-BCR and CD19 co-ligation during antigenic stimulation enhances activation 100-1000 times more. CD19-deficient mice have defects in Ca2+ mobilization, antibody production, germinal center formation and affinity maturation48-51, demonstrating its crucial role in B cell activation.

CD19 has two Ig-like extracellular domains (ECDs) and a cytoplasmic tail comprising nine tyrosine residues, three of which are phosphorylated to recruit several intracellular signaling molecules32. CD19 exists in a protein complex with the complement receptor CD21, the tetraspanin protein CD81, and Leu1358,59. However CD19 also exists in excess of these other proteins and can be found in a monomeric state58. There are approximately 88,000 CD19 molecules on the B cell surface40. Similar to IgM, CD19 also exists in distinct nanoscale clusters

(which can also be referred to as protein islands), but is separate from IgM-BCR as measured by super-resolution imaging39. Small proportions of IgM-BCR however, have been found to co- immunoprecipitate with CD19. Upon antigen-BCR ligation, BCR is released from the actin cytoskeleton allowing it to interact with CD19 which is held stationary by CD8151. CD19 diffusion on the B cell membrane in resting cells is very low, with a diffusion rate of 0.006 µm2/s and is not impacted by actin depolymerization as seen with IgM-BCR. However, in the absence of CD81, CD19 diffuses three times faster, demonstrating the importance of protein-protein interactions in regulating the organization and mobility of receptors and consequently signaling.

The ligand of CD19 ligand elusive, although its existence as a complex with several other proteins has provided mechanisms for its recruitment during antigen-induced B cell activation.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

CD21 functions as a ligand-binding accessory molecule for CD19 through its recognition of complement-coated antigen52. It should be noted that there are differences between CD21- deficient and CD19-deficient mice, with more pronounced impairment (reduced serum IgM and

IgG1 levels and germinal center formation) in CD19-deficient mice, suggesting that there are other mechanisms which regulate CD19 recruitment and signaling53. Indeed, CD19 can be recruited to BCR upon antigenic stimulation independent of CD21, but instead through membrane-proximal cytoplasmic residues of CD1953.

CD19 has nine tyrosine residues in its cytoplasmic tail; however mutational analysis of the human sequence has demonstrated that only three tyrosines (Y391, Y482, and Y513) are phosphorylated upon BCR stimulation54-56. These tyrosines are phosphorylated by the Src family kinase Lyn. Phosphorylated Y391 (pY391) creates a binding site for Vav, a guanine nucleotide exchange factor, while pY482 and pY513 cooperatively create a binding site for phosphatidylinositol 3-kinase (PI3K)56. The association of PI3K with CD19 increases its catalytic activity leading to phosphorylation of PIP2 to PIP3. PIP2 hydrolysis as outlined above, ultimately leads to upregulation of NFκB and NFAT. Vav is an important actin regulatory protein and has been found to play an important role in the spreading and contraction response and the accumulation of antigen140. CD19 activation is also crucial in recruiting Syk55.

Furthermore, PIP3 production following CD19 ligation leads to recruitment of Akt, a serine/threonine kinase that promotes cell survival and apoptosis57. CD19 is a crucial co-receptor of B cell signaling, with CD19-deficient B cells having reduced phosphotyrosine levels in BCR-

Ag microclusters, decreased Syk, and decreased Vav recruitment85. Furthermore, Ca2+ flux in response to membrane-bound antigen is completely abrogated in CD19-deficient B cells, demonstrating the importance of its function in B cell signaling. 9 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

1.2.3 CD45

CD45 is a receptor-like protein tyrosine phosphatase expressed on immune cells. It has multiple isoforms, with B cells expressing the largest isoform, B220. It is one of the most copious expressed, covering ~10% of the cell surface60.

CD45-B220 is composed of all three of the alternatively-spliced exons at the N-terminal which comprise a heavily glycosylated extracellular domain, a transmembrane domain, and an extensive cytoplasmic domain composed of 2 phosphatase homology domains61. As determined from the cDNA sequence, the 220 kDa form of CD45 has 17 predicted N-glycosylation sites63.

33% of the N-glycans on CD45 are tri- and tetra-antennary complex-type sugar chains composed of poly(N-acetyllactosamine) groups62. Most of the N-glycans on CD45 including the poly(N- acetyllactosamine)-containing heavily-branched sugar chains were found to be at least mono- or di-sialylated, which facilitates CD22-CD45 interactions63. CD45 has also been found to contain -type sugar chains, which may carry I antigens64.

CD45 is localized in lipid raft domains on the cell membrane, which positions it close to its Src-family kinase substrates. Depoil et al. found using Total Internal Reflection Fluorescence

Microscopy (TIRFM) that CD45 distribution is homogeneous across the B cell membrane in resting cells10. However, upon antigen-BCR ligation, CD45 is excluded from antigen-BCR microclusters, which is consistent with findings that CD45 is partitioned from lipid raft domains upon TCR stimulation10,69. CD45 also expresses the ligand for CD22, and it has been shown that

CD22 organization is grossly altered in CD45-deficient mice, highlighting that these proteins are in close association with each other in B cells, and regulated by glycan-binding40.

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While there are two phosphatase homology domains, only that which is closest to the membrane (d1) is enzymatically active61. The exact role CD45 plays in B cell signaling remains unclear, as it has been shown to play positive and negative regulatory roles, similar to its substrate Lyn. Lyn activity is upregulated by phosphorylation of its activating tyrosine (Tyr397) by C-terminal Src-kinase (Csk), and dephosphorylation of its inhibitory tyrosine (Tyr508) by

CD4565. Indeed, while Lyn is hyper-phosphorylated at the inhibitory tyrosine in CD45-deficient mice, there are developmental defects in T and B cells and these cells do not proliferate after antigenic stimulation, providing evidence for its positive regulatory role in B cell signaling66-68.

However, other studies have demonstrated an inhibitory effect of CD45, where the postulated general mechanism is through CD45’s constitutive dephosphorylation of both of Lyn’s regulatory sites in resting cells. Upon antigen-BCR ligation, CD45 is segregated from Lyn- containing domains through an unknown mechanism, allowing Lyn to become active and regulate B cell signaling. CD45 is then recruited back to these domains, where it can enforce its inhibitory effect on Lyn29,69. Hence the regulation of CD45 organization across time and space has important implications for the regulation of BCR signaling, and elucidating mechanisms of its organization and recruitment is a crucial area of research.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

1.2.4 CD22

CD22 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) receptor family70. It is a B cell-lineage-restricted inhibitory receptor bearing three immunoreceptor tyrosine-based inhibitory motifs (ITIMs) on its cytoplasmic tail. CD22 is crucial in preventing abnormal B cell activation upon BCR-antigen ligation, and is therefore vital for maintaining homeostasis in adaptive immunity. Consequently, CD22 deficiencies and genetic variants have been implicated in autoimmune disease and hyperactive B cells71.

CD22 is a 140-kD transmembrane spanning the surface of the B cell membrane.

It has seven immunoglobulin (Ig) domains (d1-7) and 12 N-linked glycosylation sites on the extracellular domain (Figure 1). Six of the N-glycosylation sites are located on the first three Ig domains: N67, N101, N112, N135, N164, and N231. Mutating glycosylation site N101 prevents protein expression72. The d1 domain is the site of recognition of sialic-acid bearing α 2,6- linkages. The structure of CD22 was recently crystallized at 2.1 Å resolution, and revealed that

CD22 is inflexible with a “tilted elongated rod” structure, with the binding site ideal for binding to bi-, tri- and tetra-antennary sialic acid terminal glycans72.

Sialic acids, the ligand of CD22, are a diverse range of monosaccharides, commonly found as terminal entities at the non-reducing end of glycans found on the surface of glycoproteins, , and glycolipids73. Sialic acid-binding is important for and signaling70. Sialic acids are expressed on hematopoietic stem cells, certain endothelial cells, and

T and B cells. Human and mice CD22 have different sialic acid ligands, with human CD22 preferring Sia N-acetyl neuraminic acid (Neu5Ac) while murine CD22 has higher specificity to non-human N-glycolyl neuraminic acid (Neu5Gc)74. CD22 itself expresses its own ligand, as

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis does CD45 and IgM-BCR. This allows CD22 to bind to itself and other receptors in a “cis” configuration, or with ligands in a “trans” configuration. The competition by cis and trans ligands has important implications for B cell signaling and adhesion75-78.

Upon antigen-BCR ligation, CD22 translocates to BCR microclusters, inducing the phosphorylation of the ITIMs by Lyn. Two of the phosphorylated domains allows for the recruitment and activation of Src homology domain containing phosphatase-1 (SHP-1), a protein tyrosine phosphatase, which inhibits Ca2+ signaling82. The human Y828 (or mouse Y807) binds

Grb2 and Shc upon phosphorylation, which recruits a protein complex containing SH2 domain- containing inositol 5-phosphatase (SHIP)79-81; however, SHIP does not appear to play a role in the inhibitory function of CD2286. Furthermore, CD22 plays an important role in regulating the

CD19 pathway, where it suppresses CD19-induced Vav phosphorylation85. SHP-1 has also been shown to dephosphorylate CD22 in vitro, and therefore may be involved in terminating the response83-84.

CD22 is organized into nanodomains of approximately 100 nm on the B cell surface. There are only approximately 65,000 CD22 molecules per B cell, and therefore is vastly outnumbered by CD45 and BCR40. While in discrete protein islands, CD22 also associates to a degree with

IgM, IgD, and CD19 islands. As mentioned above, CD22 can bind in cis to itself, forming clusters of homo-oligomers87. These cis-cis interactions can prevent binding and CD22 activation and redistribution of CD22 (for example to BCR-antigen microclusters) upon antigen-BCR ligation, creating a signaling threshold for B cell activation77,40,80. Like IgM-BCR, CD22 organization is not regulated by the actin cytoskeleton40,42. Using single-particle tracking (SPT), it was demonstrated that CD22 mobility but not organization is restricted by the actin

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis cytoskeleton, implicating another entity in regulating its organization. CD22 is also highly mobile, diffusing four to five times faster than IgD-BCR or CD19, and two times faster than

IgM-BCR40.

Due to the importance of CD22 in maintaining a threshold of B cell activation, CD22 genetic polymorphisms, dysregulation of enzymes associated in glycosylating CD22 ligands, and circulation of anti-CD22 antibodies have been linked to human autoimmune disease88-89. For example, patients with rheumatoid arthritis (RA), type 1 diabetes (T1D), and SLE have been found to have loss-of-function mutations in the enzyme sialic acid acetylesterase (SIAE), which is involved in the deacetylation of N-glycan sialic acids, increasing ligand binding to CD22.

SIAE mutant mice have a break in B cell tolerance and produce autoantibodies90-93.

Furthermore, 13 single-nucleotide polymorphisms in CD22 have been identified in RA and SLE, seven of which cause amino acid substitutions in the extracellular domain80. The Q152E substitution was found to be significantly associated with SLE. An interesting study found that

Systemic Sclerosis (SSc) and SLE patients have anti-CD22 antibodies that inhibit tyrosine phosphorylation of CD22, and alter the regulation of B cell activation94. How these changes impact CD22 function to culminate in outward disease states is unknown. These molecular and clinical findings demonstrate how CD22 dysregulation can lead to B cell hyperactivity and autoimmune disease and the importance of understanding its regulation in B cell activation and signaling.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

1.3 Spatiotemporal regulation of membrane receptors

As discussed above for BCR and its co-receptors, proteins are highly compartmentalized and segregated on the membrane. Some of the key structures that regulate spatiotemporal organization of these proteins have been elucidated, which include lipid raft domains, the actin cytoskeleton, glycosylation, and the glycan-binding family of secreted proteins, known as galectins.

1.3.1 Lipid domains

Lipid domains play a crucial role in protein compartmentalization, which in turn is important for membrane trafficking and organizing signaling platforms95. Lipid raft domains (10-

200 nm) are composed of clusters of sphingolipids (glycosphingolipids and sphingomyelin) and cholesterol95-96. These lipid raft microdomains have high concentrations of glycosylphosphatidylinositol (GPI)-anchored proteins, membrane proteins, and Src-family tyrosine kinases (important kinases in the initiation of B cell signaling). Indeed, IgM-BCRs are selectively excluded from lipid raft domains in the steady-state, but upon antigen stimulation

IgM-BCRs coalesce with lipid rafts97. Furthermore, this partitioning of BCR into lipid raft domains has been implicated in Syk recruitment and increased tyrosine phosphorylation, providing further evidence for enhanced BCR signaling upon association with membrane rafts98.

Interestingly, patients with SLE have reduced Lyn translocation into lipid rafts compared to healthy individuals, which was also associated with increased B cell expansion and anti-DNA autoantibody production99. This reduction in Lyn translocation was hypothesized to provide evidence for Lyn’s negative regulatory role, however the exact mechanism of this finding is

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis unknown. Therefore, understanding the mechanisms in which proteins are segregated into or out of lipid raft domains has important implications in autoimmunity.

1.3.2 Actin Cortex

The actin cytoskeleton is a highly dynamic cortical structure involved in maintaining the integrity of the plasma membrane, regulating internalization and transport of molecules100, and more recently signaling101. It is composed of F-actin filaments, which are ~50 nm-2 µm in thickness attached to the plasma membrane via membrane adapter proteins and lipids102. The first study that found a link between the actin cytoskeleton and signal regulation was the identification of mutations in the actin-regulatory protein, Wiskott-Aldrich syndrome protein

(WASP), after isolation via positional cloning from Wiskott-Aldrich Syndrome (WAS) patients103. WAS is an immune disorder characterized by defects in T and B cell function such as the failure of B cells to respond to carbohydrate-based antigens103. Indeed, since then several studies have elucidated several mechanisms by which the cytoskeleton plays a crucial role in regulating BCR signaling. BCR signaling was found to be regulated by actin by (1) restricting

BCR and coreceptor diffusion and (2) differentially compartmentalizing IgM-BCR and its co- receptor CD19 in resting B cells96,42,101,102. Interestingly, however, a recent paper published by

Gasparrini et al. found that while the mobility of CD22 was regulated by the actin cytoskeleton, its organization was not. Visualization of the organization of CD22 using superresolution microscopy demonstrated that CD22 nanoclusters were not affected by disruption of the intricate underlying actin meshwork. These findings have highlighted that there are other key regulators involved in membrane protein organization.

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1.3.3 Glycosylation

Glycosylation is an ancient mechanism in which saccharides are added to other carbohydrates, proteins and lipids but only recently been has its importance in cell signaling and protein organization been discovered104. Glycosylation is currently an enigmatic and complex topic due to our inability to predict post-translational modifications based on the genome.

Indeed, the complete glycome has yet to be elucidated, but the simple pentasaccharide sequences themselves have been estimated to be around 7000 in number105. The existence of glycoproteins was discovered by Pavy in 1893; however, he hypothesized that the carbohydrate portions function as precursors for proteins106. Subsequent work by Neuberger and others, demonstrated through repeated crystallization and various failed physical separation experiments that albumin had a fixed quantity of carbohydrates attached to the protein molecule106. Naturally occurring protein glycans are either defined as N-linked or O-linked. In the case of N-linked glycosylation,

N-acetylglucosamine (GlcNAc) is covalently bound to the amide side chain of asparagine

(Figure 2)107. N-linked glycosylation begins in the endoplasmic reticulum (ER) and the Golgi apparatus108. Glycosylation is modified by the differential expression and quantity of glycan- modifying enzymes (glycosidases and glycosyl transferases) depending on the cell-type. The binding of different to glycans is dependent on their affinity to different complexities of branching. Glycan branching of molecules is sequential and mediated by monoacylglycerol acyltransferase (Mgat) enzymes, which use a UDP-GlcNAc as a donor108.

The cumulative effects of anabolic and hydrolytic activities? of glycosyl transferases? produce a diverse range of glycans104. Glycans play crucial roles in promoting proper protein conformation, adherence, migration, signaling, and protein internalization104. Indeed,

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis inactivation of the glycan-modifying enzyme Lunatic Fringe glycosyltransferase manifests in defective Notch signaling and is found in the disease human spondylocostal dystosis109.

Interestingly, genetic deficiencies in the production of ST6Gal-1 glycosyltransferase reduce the expression of the α 2,6 sialic acid ligand on the B cell surface, and this was associated with altered membrane organization, increased BCR colocalization with CD22, and increased inhibition of B cell signaling110. Hence, understanding protein-glycan binding is crucial in elucidating mechanisms of protein organization and compartmentalization on the B cell surface.

Figure 2. Schematic structure of a typical LacNAc type N-glycan (adopted from Lee et al., 201872). Terminal can be branched by addition of a β1-3GlcNAc, which can receive a β1-4Gal, forming two N-acetyllactosamine (LacNAc) units. These reactions form poly-N- LacNAc. Gal9 has increased affinity for this form of branched glycans.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

1.3.3.1 Novel regulators: Galectin lattice

Galectins (formerly known as S-type lectins) are a family of β-galactoside-binding proteins evolving over 700 million years ago111. There are 15 mammalian galectins. Specifically, they bind to N-acetyllactosamine sequences (Galβ(1,4)GlcNAc) of O- and N-linked glycoproteins111.

Galectins 1,2,5,7,10,11,13,14, and 15 contain only 1 carbohydrate-recognition domain (CRD) and are known as prototype galectins (Figure 3)112. Galectin 3 contains a single CRD and an N- terminal domain that allows oligomerization. Galectins 4, 6, 8, 9, and 12 are tandem-repeat galectins that have two different CRDs connected by a flexible linker region112. Galectin affinity for glycans on glycoproteins is dependent on the number of glycosylation sites as well as the diversity of β-galactoside-binding epitopes created by glycosyltransferases113. Galectins form lattice-like structures through the binding of different glycoproteins and on the extracellular surface of cells. They are involved in a wide range of functions such as membrane protein expression and trafficking, cell trafficking and migration, cell signaling, and apoptosis114.

Recently our lab identified that galectin-9 (Gal9) plays a role in regulating antigen-BCR microcluster formation upon interacting with membrane-bound antigen and plays a crucial function in regulating B cell signaling

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Figure 3. The galectin family. Visual representation of differenone CRD that can dimerize. Tandem-repeat galectins have two different CRDs attached by a flexible linker region. Chimera- type galectins have one CRD with a proline-tyrosine-rich N-terminus that allows oligomerization.

1.3.3.1.1 Galectin-9

Gal9 is widely expressed in a variety of organs including immune-related tissues and cells115. Active research on Gal9 function has found that it plays crucial but distinct roles in different cell types. For example, galectin-binding to glucose transporter 2 was found to slow its diffusion and internalization, allowing for increased transport of glucose into pancreatic β cells116. Use of Gal9 as a therapeutic treatment in autoimmune mouse models such as MRL/lpr lupus prone mice has been shown to ameliorate a variety of different autoimmune phenotypes such as proteinuria, arthritis, anti-dsDNA production, and hematocrit117. The authors hypothesized that this may be due to regulating T cell function and plasma cell . Here we provide an alternative mechanism in which Gal9 may inhibit autoimmune disease progression through regulating B cell signaling.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Gal9 is a tandem-repeat galectin with two CRDs attached by a flexible linker. The N- terminal CRD is comprised of 148 amino acids and the C-terminal CRD has 149 amino acids118.

There are three endogenous forms of human Gal9 produced through alternative-splicing mechanisms. They differ only through the length of the linker domain with large linker being 58 amino acids long, the M-type with 26 amino acids and the S-type Gal9 linker domain with 14 amino acids118. The basic activity of all three variants has been found to be the same, at least in vitro.

Frontal has demonstrated that, in general, both the N-terminal and C-terminal CRDs have affinity for branched N-glycans and glycans with poly-N- acetyllactosamine, however the N-terminal CRD shows a greater affinity to glycolipid pentasaccharides119. Gal9 affinity increases as N-glycan branching from mono to tetra-antennary branching increases. To date, specific Gal9 glycan ligands and their attachment sites on glycoproteins have only been elucidated in the context of IgE. However, a very recent paper published by Giovannone et al. found, using N-glycome mass spectrometry, that naïve B cells expressed high levels of poly-LacNAc and this directly related to increased affinity for Gal9 binding, in comparison to diminished Gal9-binding to germinal center B cells due to increased expression of I-branched poly-LacNAcs120.

Recent research has identified several ligands for Gal9 in different cell types. One of the ligands identified on epithelial cells are glycolipids that are found concentrated in lipid rafts.

Interestingly, the N-terminal CRD shows greater affinity (with low dissociation constants) to glycolipids than the C-terminal CRD, highlighting the differences in binding within the Gal9 protein119. Ligands identified on T cells are Tim-3121, CD44122, CD137123 and PD1124 via surface

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis plasmon resonance. Serum IgE has also been identified as a Gal9 ligand125. Elucidation of the specific Gal9 glycan ligand sites on IgE have been identified as Asn210 and Asn222 on the Cµ1 domain of IgE, with the authors hypothesizing that the spatial arrangement between the antigen- binding sites and the Cµ1 domain suggest that the N-glycans in the region play an important role in Gal9-induced inhibition of IgE-antigen complex formation126. However, other than its binding to IgE to mediate mast cell degranulation125, the function of Gal9 had not been elucidated in B cells.

While Gal9 has been identified to play a role in initiating apoptosis of T cells as a mechanism of preventing autoimmunity, our lab discovered that Gal9 does not play a role in initiating B cell apoptosis130. We discovered that IgM-BCR and the B220 isoform of CD45 are ligands of Gal9 via mass spectrometry analysis. We also demonstrated that Gal9-KO B cells have increased antigen-BCR microcluster formation and downstream pERK signaling, key indicators of heightened B cell activation. We also identified a potential role for the inhibitory co-receptor CD22 in Gal9-mediated regulation of B cell activation as Gal9 induced co-clustering of IgM-BCR and CD22 (Cao et al., Nat. Comms), however, the complete molecular mechanism by which Gal9 regulates B cell activation and signaling is not clear.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

1.4 Hypothesis and Aims

Given previous findings from our lab that Gal9 plays a crucial role in B cell activation and regulates downstream signaling (ERK phosphorylation)130, we hypothesized that Gal9 binds specifically to inhibitory proteins to cluster them with IgM-BCR and inhibit B cell signaling.

Furthermore, our previous finding of increased CD22 density in Gal9-enriched regions but lack of identification of CD22 using mass spectrometry led us to hypothesize that CD22 might play a crucial role in Gal9-mediated B cell signaling through direct or indirect Gal9-mediated recruitment130. Finally, given that CD22 glycosylation was simultaneously discovered to play an important role in regulating B cell signaling (Wasim et al., unpublished), we hypothesized that glycosylation of CD22 is crucial in Gal9-mediated inhibition of B cell signaling.

This study had 3 main aims:

Aim 1: Examine the role of Gal9 in the spatial organization of inhibitory and stimulatory receptors

Aim 2: Examine Gal9-regulation of early B cell signaling events to elucidate mechanism of inhibition

Aim 3: Examine the importance of CD22 glycosylation in Gal9-mediated inhibition of B cell signaling

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

METHODS

2.1 Mice

Wild type (WT) mice (strain C57BL/6), were obtained from Charles River Laboratories and

Gal9-KO mice were obtained through The Scripps Research Institute (TSRI) from Steven

Beverley (Washington University). Mice aged from 2-10 months were age and sex-matched and used for all indicated experiments. Mice were contained in pathogen-free animal housing at

University of Toronto Scarborough (UTSC), Scarborough, Canada. All procedures performed on mice were permitted by the Local Animal Care Committee (LACC) at UTSC.

2.2 Murine Cell Isolation

Splenocyte single cell suspension was isolated from C57BL/6 WT and Gal9-KO mice using 70

µm cell strainer and phosphate buffered saline (PBS, PH 7.4, Life technologies). B cell isolation was conducted using negative isolation kit (Miltenyi Biotec Inc. or Stem Cell technologies) according to the manufacturer’s protocol.

2.3 Cell Lines and Culturing

Daudi B cells were cultured in 5% CO2 in RPMI-1640 with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and streptomycin (Gibco), and 50 µM 2-mercaptoethanol

(Amresco). CD22-KO B cells was graciously provided Dr. Joan Wither from the Krembil

Research Institute. CD22 5Q-mutant B cells were provided by Laabiah Wasim. Briefly, 10µg of

WT human CD22 plasmid or 5Q human CD22 plasmid containing point mutations at N67,

N112, N135, N164, and N231, from Asparagine (N) to Glutamine (Q) were transfected into

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CD22-KO Daudi B cells, through electroporation (Gene Pulser Xcell (Bio-Rad)) at 570 V, 25

µFD. Construct-positive cells were selected for using 0.8 mg/ml Geneticin (Thermo Fisher) for

30 days. Finally, FACS sorting was conducted in order to isolate positive populations by labeling cells with humanized anti-CD22 Fab fragment (pinatuzumab [16]) at 5 µg/mL.

2.4 rGal9 treatment

B cells were treated with 1 µM recombinant galectin-9 (R&D Systems) in 2% BSA in RPMI for

30 minutes at 37ºC. Cells were washed 1X with PBS or RPMI and resuspended in PBS for immediate use in experiments.

2.5 Gal9 surface staining

For murine B cell Gal9 staining, 8-well Lab-Tek chambers were coated with 1µg/ml anti-mouse

MHC-II (M5/114, Sunnybrook) antibody in PBS for 2 hours at room temperature and washed 3X with PBS. 5 x 106 primary murine B cells from WT mice were treated with 1 µM recombinant galectin-9 in 2% BSA in RPMI for 30 minutes at 37oC. Cells were washed 1X with PBS. Cells were resuspended in PBS and allowed to adhere to chambers for 15 minutes. Cells were fixed in

2% PFA for 10 minutes and then washed 2X in FACS buffer (PBS, 1% BSA, 0.1% NaN3). Cells were incubated with 2 µg/mL purified rat anti-mouse CD16/32 (BDPharmingen; 1:250v/v) in

FACS buffer for 15 minutes at 4oC. Cells were stained for Gal9 in 2 ways: 1. 1 µg/mL goat anti- mouse Gal-9 (R&D systems) in FACS buffer for 1 hour at 4oC. Cells were washed 3X with

FACS buffer. Cells were incubated with 1 µg/mL Cy3-conjugated bovine anti-goat IgG secondary or Alexa-Fluor488 Donkey anti-goat IgG secondary (Jackson ImmunoResearch) for one hour at 4oC. Cells were washed 3X with PBS. 2. Cells were immunostained with PE-

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis conjugated rat anti-mouse Gal9 antibody (clone 108A2, Biolegend) at 1:200v/v in FACS buffer for 30 minutes at 4°C. Cells were washed 3 times with FACS buffer.

To stain for rGal9 on Daudi B cells, cells were first blocked with purified anti-human

CD16/CD32 (Fc-block, 1:250 v/v; BD Biosciences, Cat. No 564220) in PBS for 15 minutes at room temperature. Cells were washed two times and immunostained with goat anti-galectin-9 antibody (R&D Systems, Cat. No. AF3535) at 1:100 v/v for 1 hour in 2% BSA PBS. Cells were washed 3 times, followed by labeling with Cy3-conjugated AffiniPure bovine anti-goat antibody

(Jackson Immunoresearch Laboratories, Cat. No. 805-165-180) at 1:1000 v/v in 2% BSA RPMI for 30 minutes at 4ºC.

2.6 Fluorescent immunostaining of murine B cells for confocal microscopy

8-well Lab-Tek chambers were coated with 1µg/ml anti-mouse MHC-II (M5/114, Sunnybrook) antibody in PBS for 2 hours at room temperature. 5 x 106 primary murine B cells from WT mice were treated with 1 µM recombinant galectin-9 in 2% FBS in RPMI for 30 minutes at 37oC.

Cells were washed 1X with PBS. Cells were resuspended in PBS and allowed to adhere to chambers for 15 minutes. Cells were fixed in 2% PFA for 10 minutes and then washed 2X in

FACS buffer (PBS, 1% BSA, 0.1% NaN3). Cells were incubated with 2 µg/mL purified rat anti- mouse CD16/32 (BDPharmingen) in PBS for 15 minutes at 4oC. Cells were incubated with 1

µg/mL goat anti-mouse Gal-9 (R&D systems) in FACS buffer for 1 hour at 4oC. Cells were washed 3X with FACS buffer. If cells were stained to visualize CD16/32, cells were incubated with 1 µg/mL Alexa-Fluor594 Donkey Anti-Rat IgG (H+L) secondary for 1 hour at 4oC. Cells were washed 2X with PBS. Cells were incubated with 1 µg/mL Cy3-conjugated bovine anti-goat

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IgG secondary or Alexa-Fluor488 Donkey anti-goat IgG secondary (Jackson ImmunoResearch) for one hour at 4oC. Cells were washed 3X with PBS. Cells were stained with fluorochrome- conjugated antibodies specific for CD45/B220 (RA3-6B2, BD Biosciences), CD22 (OX-97,

Biolegend), CD19 (1D3, Biolegend), or IgM (Jackson ImmunoResearch) for 1 hour at 4oC. Cells were washed 3X with PBS. Cells were mounted in FluoroGel with DABCOTM (Electron

Microscopy). Chambers were imaged by spinning disk confocal microscopy (Quorum

Technologies) consisting of an inverted fluorescence microscope (DMI6000B; Leica) equipped with a 63X/1.4 NA oil immersion objective and an EMCCD camera (Image EM; Hamamatsu).

Images were acquired using Metamorph software (Molecular Devices). Images were analysed using Volocity software (Perkin Elmer). The fluorescence signal of CD45, CD22, CD19, IgM or

FcγRIIb were used to define a mask delineating the membrane region. Gal9high regions were determined by the fluorescence signal of Gal9. Gal9low regions were determined by subtracting the Gal9high regions from the membrane region. The mean florescence intensity of CD45, CD22,

FcγRIIb and IgM was calculated in Volocity.

2.7 Lipid Raft Staining

Primary murine WT B cells and 1µM rGal9-treated B cells were labeled with 1µg/mL of

Cholera toxin subunit B-Alexa-Fluor 555 (Vybrant™ Alexa Fluor™ 555 Lipid Raft Labeling

Kit, ThermoFisherScientific) for 15 minutes on ice. Cells were washed 2X with PBS for 5 minutes at 4°C. Cells were then allowed to adhere to anti-MHC II-coated Lab-Tek chambers for

10 minutes. Cells were fixed with 2% PFA for 15 minutes and washed 3X with PBS. Cells were then stained for specific proteins as described above.

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2.8 Calcium signaling

Intracellular Ca2+ flux was measured by flow cytometry. Freshly isolated splenocytes (5 x 106 cells/sample) were labelled with 1 µM Fluo-4, AM (Thermo Fisher) in 10% FBS/HBSS at 37°C for 15 minutes. Cells were incubated with anti-CD45R (B220) (clone RA3-6B2; Biolegend) for 5 minutes at 4°C at a dilution of 1:200. Cells were washed 2X with PBS and resuspended in RPMI.

After collecting a baseline reading for 30s, 20 µg/ml anti-mouse kappa immunoglobulin light chain (clone HB58) or 1 µM rGal9 was added to the FACS tube, and change in fluorescence intensity was recorded on a Fortessa cytometer (BD Biosciences) and plotted using Flowjo software (TreeStar). Fold Change was calculated by dividing the fluorescence intensity at each time point by baseline intensity at 1 second.

2.9 Western Blots

2.9.1 Murine B cells

24-well plates were coated with 5 µg/ml AffiniPure F(ab’)2 fragment goat anti-mouse IgM, µ- chain specific (Jackson ImmunoResearch Laboratories) in PBS overnight at 4°C. Primary naïve splenic B-cells from WT and Gal9-KO mice were stimulated for the indicated time, then lysed with Laemmli buffer containing 100 µM DTT followed by SDS-PAGE. For rGal9 treatment,

WT B-cells were pre-treated 1 µM recombinant mouse galectin-9 (rGal9, E.coli-derived, R&D

Systems) in RPMI containing 1% FBS (both Gibco) and incubated for 30 minutes at 37°C. Cells were then washed once with RPMI and then stimulated as described above. Proteins were transferred to PVDF and blocked with 5% non-fat skimmed milk or 5% BSA in TBST (20 nM

Tris pH 7.5, 150 mM NaCl, and 0.1% Tween-20) for 1 hour at room temperature or overnight

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(for 4G10) at 4°C with shaking. Membranes were immunoblotted with the following antibodies overnight or for 3 hours (for 4G10) at 4ºC: mouse anti-β-tubulin (Sigma, 1:5000 in 1%

BSA/TBST), rabbit anti-phospho- p44/42 MAPK (ERK1⁄2 Cell Signaling, 1:1000 in 1%

BSA/TBST), rabbit anti-ERK p44/42 MAPK (ERK1⁄2 Cell Signaling), 4G10 (Sunnybrook,

1:1000 in 1% BSA/1% NaN3/TBST), phospho-CD19 (Tyr531 Cell Signaling, 1:1000 in 1%

BSA/1% NaN3/TBST) and phospho-Akt (Tyr308 Cell Signaling, 1:1000 in 1% BSA/1%

NaN3/TBST). HRP-conjugated secondary antibodies were used at 1:10,000 v/v in 5% non-fat skimmed milk in TBST or 1% BSA in TBST and incubated for 2 hours with shaking at room temperature. Membranes were incubated with SuperSignal (Thermo Fisher Scientific) chemiluminescent substrate and then imaged by ChemiDoc (Bio-Rad). Densitometric analysis of western blots was performed using ImageJ (NIH). The amount of phosphorylated protein was normalized to loading control and then expressed as a fold change relative to the 0 min time point.

2.9.2 Daudi B cells

6 Daudi cells were suspended at 1 x 10 cells/ml in RPMI 1640 and stimulated for the indicated time with 5 µg/ml AffiniPure F(ab’)2 goat anti-human IgM, µ-chain specific (Jackson

ImmunoResearch Laboratories) at 37ºC. The reaction was stopped by adding 1 ml of ice cold PBS.

Cells were centrifuged at 1200 rpm for 30 seconds. Supernatant was removed and lysis buffer (1%

NP40, 0.15 M NaCl, 20 nM Tris pH 8.9, 100 mM NaF, 10 nM Na3VO4 and Roche completeTM protease inhibitor cocktail) was added at 10 x 107 cells/ml. Cells were incubated at 4ºC with gentle shaking for 30 minutes. Cell lysate was then centrifuged at 1200 rpm for 15 minutes to remove cellular debris and the supernatant was transferred to a clean microtube. 2X Laemmli buffer

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis containing 0.1 M DTT was added to cell lysate. Samples were boiled at 95ºC for 5 minutes followed by SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked in 5% BSA in TBS-T

(20 mM Tris pH 7.5, 150 mM NaCl and 0.1% Tween 20) overnight at 4ºC. Membranes were immunoblotted with the following antibodies in 1% BSA/TBST for 5 hours at room temperature with gentle rocking: mouse anti-β-tubulin (Sigma, 1:5000 v/v), 4G10 (Sunnybrook, 1:1000 in 1%

BSA/1% NaN3/TBST), rabbit anti-phospho CD19 Y531 (Cell Signaling Technology, 1:1000 v/v), rabbit and rabbit anti-phospho ERK p44/p42 MAPK (Cell Signaling Technology, 1:1000 v/v).

Membranes were washed 3 times with TBST, then incubated with HRP-conjugated donkey anti- rabbit IgG or donkey anti-mouse IgG antibodies (Jackson ImmunoResearch, 1:10 000 v/v) in 1%

BSA/TBST for 1 hour at room temperature. Membranes were washed 5 times with TBST, then incubated with Pierce ECL Western Blotting Substrate and imaged with ChemiDoc System (Bio-

Rad). The intensity of each protein band was analyzed by ImageJ, normalized to β-tubulin and the fold changes were calculated relative to the signal of WT B cells at 0 min time point.

2.10 Statistical Analysis

Statistical analysis was performed using GraphPad Prism. Comparisons between two groups were performed using Student’s t test for data with normal distribution and Mann-Whitney for data with non-normal distribution. Comparisons between multiple groups were performed by ordinary one-way ANOVA for data with normal distribution and Kruskal-Wallis test for data with non-normal distribution. Western blot data was analyzed by two-way ANOVA followed by

Sidak’s multiple comparisons test.

2.11 dual dSTORM

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

2.11.1 Daudi B cell sample preparation

1 x 106 WT and 5Q-mutant Daudi B cells were centrifuged and resuspended in 2% FBS/PBS with 10 µg/ml of Alexa Fluor 647 labeled pinatuzumab IgG and 2 µg/ml Alexa Fluor 488 labeled anti-human IgM Fab fragment for 20 minutes at 4ºC. Cells were then washed in PBS 2X, resuspended in PBS, and incubated at 37º for 5 minutes before allowing to settle on TIRF- specialized TIRF-M 24-well plates, and allowed to spread at 37ºC for 20 minutes. Wells were then gently washed 2 X with PBS at room temperature. Right before image acquisition, dSTORM imaging buffer (PBS containing 0.1 M β-MercaptoEthylamine (MEA, Sigma-Aldrich),

3% (v/v) OxyFlourTM (Oxyrase Inc.), 20% (v/v) of sodium DL-lactase solution (L1375, Sigma-

Aldrich) adjusted to pH ~8.3) was added to the wells. Fiducial markers (100 nm Tetraspeck

Fluorescent Microspheres, Invitrogen) were added to buffer and allowed to settle for 5 minutes prior to imaging.

2.11.2 dSTORM acquisition and image reconstruction dSTORM was executed using a TIRF microscope (Quorum Technologies) based on an inverted microscope (DMI6000C, Leica), HCX PL APO 100X/1.47 oil immersion objective and Evolve

Delta EMCCD camera (Photometrics). For Alexa® 647, photoconversion was achieved with 633 nm laser (intensity ranged from 80 to 100 mW/cm2) illumination and simultaneous illumination with the 405 nm laser (intensity range from 5 to 20mW/cm2) increased the rate of conversion from the dark state. Dual-colour dSTORM images were acquired consecutively; 647-channel imaging was conducted first after which imaging was done in the 488-channel. For Alexa® 488, photoconversion was achieved with the 488-nm laser (intensity ranged from 80 to 100mW/cm2).

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

8000 images were acquired at 30 millisecond exposure with a frame rate of 33 frames/s, and EM gain of 200. Fiducial markers, which are observable in both channels, were used to align the two channels for effective image reconstruction. Polynomial transformation function was calculated using MultiStackReg plug-in of ImageJ, which allowed the 488 nm channel to be mapped onto the 647 nm channel. The transformation matrix was applied to each frame of the 488-nm channel stack. Image reconstruction was performed using ThunderSTORM plugin for ImageJ according to the parameters previously reported144. The camera setup was as follows: pixel size 101.5 nm, photoelectron per A/D count 2.4, base level [A/D count] 414 and an EM gain of 200. Image filtering was applied to remove camera noise and enhance photoswitching events using a wavelet filter (B-Spline) with a B-Spline order of 3 and B-Spline scale of 2.0. Approximate localization of molecules was detected by local maximum method with a peak intensity threshold of std

(Wave.F1) and a connectivity of 8-neighbourhood. Sub-pixel localization of molecules was identified by fitting point spread function to an integrated Gaussian using weighted least squares method with a fitting radius of 3 pixels. Single molecules may not be adequately resolved in spatially dense organizations and thereby multiple activated molecules are detected as a single blob. Multiple emitter fitting analysis (MFA) was used to estimate the number of molecules detected as a single blob with maximum 5 molecules per fitting region. To improve the multi- emitter fitting algorithm, molecules are fitted assuming the same intensity. Super resolution images were rendered with a pixel size of 20 nm.

2.11.3 dSTORM post-processing, analysis and quantification

Reconstructed dSTORM images were post-processed with drift correction using the built-in method in the ThunderSTORM plugin. The workflow for post-processing steps is shown in

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Supplementary Fig. 8B and consisted of the following steps: 1. Remove duplicates, in which repeated localizations of single molecules in one frame, which may occur when using multiple- emitter, were removed based on uncertainty radius of localization; 2. Filter, in which localizations with an uncertainty greater than 20 nm were eliminated; 3. Density filter, in which isolated localizations were removed based on the parameter of at least 2 neighbors are required in 50 nm radius for a localization to be accepted; 4. Drift correction, in which image drift was corrected using fiducial markers; and 5. Merging, in which molecules that appeared within 20 nm in multiple frames were merged together. To reduce processing time, 4000 frames were processed for cluster analysis. For each reconstructed image of the cells, a 3 x 3 µm region was manually selected but excluded from the cell boundary. The Hopkins index and Ripley’s H function analysis were performed by SuperCluster, an analysis tool kindly provided by the

University of New Mexico’s Spatio Temporal Modeling Center (http://stmc.unm.edu/). Cluster analysis was performed by a Bayesian, a model-based approach In brief, uncertainty, x and y coordinates of each localization in post-processed reconstructed images were exported. The selected 3 x 3 µm regions were analyzed by the published R code of Bayesian cluster analysis 39 with an alpha value of 20, pbackground of 0.5, rseq of (5, 200, 10) and thseq of (0, 50, 5). The analyzed data were post-processed to extract data about percentage of molecules localizing in clusters, cluster radius, number of clusters, and number of molecules per clusters.

2.11.4 Coordinate based co-localization analysis

Colocalization of dual dSTORM data was conducted using Coordinate based colocalization

(CBC) analysis 50 using ThunderSTORM. Briefly, this method uses coordinate information of each molecule instead of an intensity based approach, which would depend on the reconstruction

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis and post-processing parameters chosen. Furthermore, this method takes into account the spatial distribution of each set of localizations to prevent false positive colocalization values that result when one of the molecular species is randomly organized. First, the spatial distribution function of the neighboring localizations from the same species in each channel are calculated. Then, from the individual distribution functions, a correlation coefficient is calculated and weighted by distance to the nearest neighbour of the localization’s respective species. As a result, each single molecule of each species is attributed an individual colocalization value, which provides information on the molecule’s local environment. CBC algorithm was applied to x-y coordinate lists of localizations in the 3 x 3 µm region of 647 and 488 channels. A search radius of 70 nm, based on the radius IgM nanoclusters, was used to calculate degree of colocalization values varying from –1 (perfectly excluded) to +1 (perfectly co-localized).

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

RESULTS

3.1 Galectin-9 increases the molecular density of CD19

Our lab previously identified that treatment of naïve murine B cells with exogenous Gal9 increases the molecular density of CD22, CD45, and IgM in Gal9high regions compared to

Gal9low regions130. Thus, we hypothesized that the Gal9-lattice increases the density of other inhibitory receptors such as FcγRIIb in order to attenuate BCR signaling, and will selectively exclude co-stimulatory molecules such as CD19. To study the effect of Gal9 on CD19 and

FcγRIIb organization, we treated primary murine B cells with 1 µM recombinant Gal9 (rGal9) and then settled them in 8-well labtek chambers, and then fixed and immunostained with anti-

CD19, anti-FcγRIIb, anti-IgM, and anti-Gal9 antibodies and visualized cells by confocal microscopy. Surprisingly, we observed increased fluorescence intensity of CD19 in Gal9-rich regions (Fig. 4A, B). To quantify this difference, we created a masking algorithm using Volocity software, defining Gal9high vs Gal9low regions (Fig 4C), and then measured the mean fluorescence intensity of CD19 in these regions. We found that the mean fluorescence intensity of both IgM and CD19 was significantly increased in Gal9high regions in comparison to low regions (Fig. 4D).

Furthermore, contrary to our hypothesis, treatment with rGal9 did not induce any changes in the distribution of the inhibitory receptor FcγRIIb between Gal9high vs Gal9low regions (Fig. 4E-G).

This demonstrated that Gal9 specifically regulates the organization of the inhibitory receptors

CD22 and CD45 as well as stimulatory receptors IgM and CD19, but not other receptors such as

FcγRIIb.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

A C

B D

E

F G

Figure 4. The Gal9 lattice increases the molecular density of CD19 but not FcγRIIb. A. Representative confocal images of primary murine B cells treated with 1 µM rGal9 and immuostained for CD19 (cyan), IgM (magenta), and galectin-9 (Gal9; yellow). B. Fluorescence intensity profile of CD19, IgM, and Gal9 along the cell membrane. C. Representative example of masking output of algorithm to detect regions of high galectin-9 (Gal9high) and low galectin-9 (Gal9low). D. Mean fluorescence intensity of CD19 (left) and IgM (right) in Gal9high and Gal9low regions. E. Representative confocal images of WT B cells treated with 1 µM rGal9 and immunostained for FcγRIIb (cyan), IgM (magenta), and Gal9 (yellow). F. Fluorescence intensity profile of FcγRIIb, IgM, and Gal9 along the cell membrane. G. Mean fluorescence intensity of FcγRIIb (left) and IgM (right) in Gal9high and Gal9low regions. Data representative of at least three independent experiments. Each dot represents one cell, at least 30 cells measured per condition per experiment. Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney; ****p < 0.0001 **p < 0.01, *p < 0.05, ns not significant. Scale bar 2 µm.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

3.2 Addition of rGal9 does not induce activation of B cells

Our lab previously found using the superresolution microscopy technique dSTORM, that treatment of B cells with rGal9 significantly increases the size of IgM-BCR clusters and immobilizes IgM130. Furthermore, findings from other labs have suggested that increased IgM-

BCR oligomerization (which requires the Cµ4 of IgM) induces a reduction in the movement

BCR and this is coincident with BCR signalling127. Given our finding that CD19 is also increased in Gal9high regions, we considered whether Gal9-mediated clustering of IgM and CD19 could induce signaling. In order to test this hypothesis, we examined whether treating B cells with exogenous Gal9 induces the release of intracellular Ca2+ stores,as an indicator of activation by flow cytometry. In comparison with treating B cells with 20 µg/mL of anti-BCR antibodies, treatment with 1 µM rGal9 (our usual treatment), did not induce Ca2+ flux (Fig 5). Cells were also treated with ionomycin as an indicator of the integrity of the cells to flux intracellular Ca2+.

Thus, our findings indicate that Gal9-mediated clustering of IgM and CD19 does not induce B cell activation and signaling.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Figure 5. rGal9 treatment does not induce Ca2+ signaling in B cells. Intracellular Ca2+ flux in primary naive B cells stimulated with anti-kappa (black line) or rGal9 (blue line) measured by flow cytometry. Addition of stimulation (anti-kappa or rGal9) is indicated by black arrow and ionomycin by red arrow. Data are representative of two independent experiments.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

3.3 Treatment of Daudi B cells with rGal9 induces IgM-CD22 colocalization

Given our findings that Gal9 reorganizes IgM-BCR into larger clusters but does not induce B cell signaling, we hypothesized that Gal9 forms an intricate and regulated lattice of inhibitory and stimulatory receptors to coordinate BCR signaling. To test our hypothesis, we treated Daudi B cells with 1 µM rGal9, settled the treated and untreated cells on fibronectin- coated specialized TIRF imaging chambers, labeled the cells with Alexa 488-labeled anti-IgM

Fab fragment and Alexa 647-labeled anti-CD22, and then conducted dual dSTORM imaging using Total Internal Reflection Fluorescence Microscopy (TIRF). dSTORM is a super-resolution technique which allows for the visualization and separation of two points as small as 10-30 nm apart, providing resolution of single-molecules on the cell surface. Dual dSTORM allows for the examination and colocalization of two different fluorophores at high resolution. 4000 images were combined to generate reconstructed super-resolution images. Visual inspection of reconstructed dSTORM images revealed an increased association between IgM and CD22 in rGal9-treated B cells (Fig. 6A). To quantify our findings we used the Hopkin’s Index, H function, and coordinate-based colocalization (CBC). First, we examined the difference in IgM and CD22 clustering between untreated and treated cells using the Hopkin’s Index. The Hopkin’s index measures the clustering tendency compared to a random distribution (random distribution is 0.5). We found that both IgM and CD22 were significantly more clustered upon rGal9 treatment (Hopkin’s Index: 0.85 and 0.98 respectively) compared to WT B cells (Hopkin’s

Index: 0.8 and 0.97 respectively) (Fig. 6 B, D). The H function is derived from Ripley’s K function and assesses the degree of clustering; where distance of the H function peak is indicative of cluster radius and peak height emphasizing the density of molecules in clusters. We

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis found that the peak height of the H function was increased in rGal9-treated cells for both IgM and CD22, indicating that the number of molecules in clusters were increased (Fig. 6 C, E).

Consistent with our hypothesis, we found that addition of rGal9 significantly increased colocalization between IgM and CD22, as indicated by coordinate-based colocalization (CBC) analysis, with CBC values ranging from -1 (perfectly segregated) to +1 (perfectly colocalized)

(Fig 6. F,G). In rGal9 treated B cells, CBC values were right-shifted indicating higher degree of colocalization compared to untreated cells (Fig. 6F). Furthermore, the median CBC value for rGal9-treated B cells was significantly higher at 0.27 compared to 0.18 for untreated B cells indicating higher degree of colocalization (Fig. 6G). This finding highlights the role of Gal9 in mediating the interaction between inhibitory and stimulatory receptors on the B cell surface.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

A B C

D E

F G

Figure 6. Exogenous Gal9 increases association of IgM and CD22. A. Representative merged TIRFM (top), dSTORM (middle), dSTORM zoom (bottom) images showing surface CD22 (magenta), IgM BCR (green), exogenous recombinant galectin-9 (rGal9) (cyan) on WT Daudi B-cells (WT) (left) and recombinant galectin-9 treated WT Daudi B-cells (WT rGal9) (right). dSTORM ROI (3 x 3 µm) is outlined in yellow (middle) and magnified in dSTORM zoom (bottom). Colocalization between channels shown in white. Scale bars represent 5 µm and 1 µm (zoom). B-G.Quantification of at least 15 ROIs from WT and WT rGal9 Daudi B-cells pooled from 4 independent experiments. B. Hopkin’s index showing randomness of CD22 organization (1000 localizations plotted) (0.5 indicates completely random distribution). C. H function derived from Riplely’s K showing degree of clustering of CD22. D. Hopkin’s - index showing randomness of IgM organization (1000 localizations plotted). E. H function derived from Riplely’s K showing degree of clustering of IgM. F. Coordinate-based colocalization (CBC) histograms of the single-molecule distributions of colocalizations between 41 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

CD22 and IgM. G. Coordinate-based colocalization (CBC) values of data from F.. Lines/errors represent means ± SEM. **** p < 0.0001 by Mann Whitney. Non-significant differences are not indicated.

3.4 Treatment of rGal9 causes coalescence of lipid rafts enriched in CD22 and CD45

Lipid rafts have been shown to play an important role as signaling platforms in a wide range of cells. Gal9 has been previously reported to bind to Forssman pentasaccharide-containing glycosphingolipids, which are enriched in lipid rafts129. Hence, we hypothesized that Gal9 may play a role in organizing inhibitory receptors in lipid raft domains through this association. To test this hypothesis, we treated primary naïve murine B cells with rGal9, labeled lipid raft domains with fluorescently conjugated cholera toxin β-subunit (CT-B) and immunostained for

Gal9, CD45, and CD22 (Fig 7 A, E). We found that treatment of WT B cells with rGal9 coalesced lipid raft domains into a polarized cap, and that these regions almost exclusively contained Gal9 on the cell surface (Fig 7A). To quantify this observation, we generated masks as described earlier, but this time defining CT-B-high (lipid raft high; LRhigh) versus CT-B-low

(lipid raft low; LRlow) regions (Fig 7B) and examined the localization of CD45 and CD22 in these domains. We found that LRhigh regions had significantly higher mean fluorescence intensity of CD45 and CD22 compared to LRlow regions (Fig 7 C-D, F-G). The striking reorganization of lipid raft domains upon treatment of exogenous Gal9 as well as the increased density of these inhibitory receptors highlights the importance of Gal9 in organizing membrane architecture.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

A C

B D

E

F G

Figure 7. rGal9 induces coalescence of lipid raft domains containing CD22 and CD45.

A. Representative confocal images of primary B cells treated with 1 µM rGal9 and immunostained for CD45 (cyan) & Gal9 (yellow), and fluorescent cholera toxin (CT-B; gray) to label lipid rafts. B. Fluorescence intensity profile of CD45, CT-B, and Gal9 along the cell membrane. C. Representative example of masking output of algorithm to detect regions of high CT-B (lipid raft high; LRhigh) & low CT-B (LRlow). D. Mean fluorescence intensity of CD45 (left) & Gal9 (right) in LRlow & LRhigh regions. E. Representative confocal images of primaryB cells treated with 1 µM rGal9 and immunostained for CD22 (cyan), Gal9 (yellow), and fluorescent CT-B (gray). F. Fluorescence intensity profile of CD22, CT-B, and Gal9 along the cell membrane. G. Mean fluorescence intensity of CD22 (left) and Gal9 (right) in LRlow and LRhigh regions. Data are representative of at least three independent experiments. Each dot represents one cell, at least 30 cells measured per condition per experiment. Mean ± SEM

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis indicated by the red bar. Statistical significance assessed by Mann-Whitney; ****p < 0.0001, ***p < 0.001. Scale bar 2 µm

3.5 Phosphorylation of CD19 and Akt is not altered in Gal9-deficient B cells

Our findings above have elucidated that Gal9 plays a crucial role in organizing important signaling receptors on the surface of B cells. Furthermore, our lab previously found that Gal9- deficient (Gal9-KO) murine B cells have significantly increased phosphorylation of ERK at the

10 and 15 minute mark following anti-IgM stimulation130. In order to further interrogate the signaling cascade affected by Gal9 we isolated splenic B cellss from wild-type (WT) and Gal9-

KO mice and stimulated them with anti-IgM F(ab’)2 for 0, 5, 10, and 30 minutes. Cells were then lysed and separated by SDS-PAGE followed by immunoblotting by anti-phosphotyrosine antibody (pY) (Fig. 8A). We found a global increase in tyrosine phosphorylation in Gal9-KO B cells compared to WT B cells, and saw a significant increase in phosphorylated bands around the

100-kDa mark. Given that CD19 is a 95-kDa protein and a substrate for dephosphorylation by

CD22, we further examined the difference in phosphorylation of CD19 and its downstream kinase Akt (Fig 8. B-C) by western blotting. Surprisingly, we did not see any significant differences in the phosphorylation of either CD19 or Akt in Gal9-KO B cells compared to WT cells (Fig. 8B-C).

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

A B

C

Figure 8. Gal9-KO B cells do not have significant enhancement in phosphorylation of CD19 and Akt. Primary naïve B cells from WT and Gal9-KO mice were settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with A. anti-phosphotyrosine (pY) and anti-ERK1/2 B. anti-phospho-CD19 and C. anti-phospho-Akt and anti-β-tubulin. Data are representative of at least 3 independent experiments. Quantifications normalized to β-tubulin loading control and fold change calculated from WT 0 min. Quantification of the fold increase in pCD19 and pAkt with the mean ±SEM indicated by bar are shown to the right. Data were analyzed by two-way ANOVA, followed by Sidak’s multiple comparisons test.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

3.6 Treatment of B cells with rGal9 suppresses B cell signaling

Our lab previously found that the ligands for Gal9 onprimary naïve murine B cells are not saturated upon cell isolation but 10-times more Gal9 can bind to the B cell surface130. Thus, we we hypothesized that treatment of B cells with exogenous Gal9 would amplify inhibtion of B cell signaling, and thus provide a tool to investigate its mechanism of action. Hence, we isolated splenic B cells from WT mice, treated them rGal9, and stimulated them with anti-IgM F(ab’)2 for

0, 5, 10, and 30 minutes. Cells were then lysed and separated by SDS-PAGE followed by immunoblotting by anti-phosphotyrosine antibody (pY) (Fig. 9A). We found a global decrease in tyrosine phosphorylation in rGal9-treated B cells compared to untreated B cells, which is consistent with our previous finding of increased total tyrosine phosphorylation in Gal9-KO B cells. We then examined phosphorylation of CD19 and Akt upon BCR stimulation in rGal9- treated cells and found an almost complete abolishment of phosphorylation of these proteins (Fig

9. B-C). These findings identify the CD19 pathway as an important target of Gal9-mediated regulation of B cell signaling.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

A B

C

Figure 9. Treatment with exogenous Gal9 suppresses B cell signaling. Naive B cells from WT mice were treated with 1 µM rGal9 and settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with A. anti- phosphotyrosine (pY) B. anti-phospho-CD19, and C. anti-phospho-Akt and anti-β tubulin. Quantifications normalized to β-tubulin loading control and fold change calculated from WT 0 min. Quantification of the fold change in pCD19 and pAkt over time, averaged over three independent experiments, with the mean ± SEM indicated by the bar is shown in the right panel. Statistical significance measure by two-way ANOVA followed by Sidak’s multiple comparisons test; **p <0.01, *p < 0.05.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

3.7 CD22 is required for Gal9-mediated inhibition of B cell signaling

Our lab previously conducted mass spectrometry analysis and identified IgM-BCR and

CD45 as ligands of Gal9130. However, even though CD22 was not identified as a ligand, we found its distribution to be significantly altered upon treatment of exogenous Gal9 in WT B cells.

To examine the importance of this alteration and the requirement for CD22 in Gal9-mediated inhibition of B cell signaling, we treated human Daudi WT and CD22-deficient B cells with 1

µM rGal9, stimulated the cells with anti-IgM F(ab’)2 for 0 and 10 minutes, lysed the cells, separated the proteins using SDS-PAGE and immunoblotted for total phosphotyrosine (pY), pCD19, and pERK and β-tubulin (Fig. 10A-B). We found that treatment with rGal9 suppressed total tyrosine phosphorylation, as well as phosphorylation ofCD19, and ERK in WT Daudi B cells, but strikingly, rGal9 did not suppress total tyrosine phosphorylation or phosphorylation of

CD19 and ERK in CD22-KO B cells (Fig. 10 A-C). These findings identify a requirement for

CD22 in Gal9-mediated inhibition of B cell signaling.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

pY C

Figure 10. CD22 is necessary for Gal9-mediated inhibition of BCR signaling. WT and CD22 KO Daudi B cells treated with rGal9 were stimulated with 5.0 µg/ml anti-IgM F(ab’)2 for indicated times, lysed and subjected to SDS-PAGE followed by immunoblotting with A. anti- phosphotyrosine and B. anti-phospho-CD19 and anti-phospho-ERK. C. Quantifications (shown below) normalized to β-tubulin loading control and fold change calculated from WT 0 min. Blots are representative of at least three independent experiments.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

3.8 CD22 glycosylation is crucial for mediating IgM-CD22 interactions

We identified that CD22 is indispensable for Gal9-mediated inhibition of B cell signaling. Previous research has emphasized the sialic acid binding domain in regulating the role of CD22 in B cell signaling. However, the role of CD22 N-glycosylation has not been examined.

Given our finding of Gal9’s regulation of CD22 organization, we wanted to examine the importance of CD22 N-glycans in regulating B cell signaling. To do this, we utilized a CD22 construct with asparagine (N) to glutamine (Q) mutations in 5 of the 6 terminal N-glycosylation sites (5Q-mutant). N101 was not mutated due to lack of expression of CD22 upon mutation of this N-glycan site. We treated WT and 5Q-mutant B cells with rGal9 and conducted dual dSTORM imaging and analysis as described above. Visual examination of rGal9-treated WT cellsrevealed a significantly higher colocalization of IgM and CD22 (Fig. 11A). In constrast, we found that rGal9-treated 5Q-mutant B cells had left-shifted CBC values, indicating significantly lower degree of IgM and CD22 colocalization (Fig. 11B). Indeed the median CBC value for rGal9-treated WT cells was 0.27 compared to 0.19 for rGal9-treated 5Q-mutant cells (Fig. 11C).

Furthermore, rGal9-treated 5Q-mutant B cells had left-shifted CBC values in comparison to untreated 5Q-mutant cells (Fig. 11D), where the median CBC value for rGal9-treated 5Q-mutant was 0.19 versus 0.28 for 5Q-mutant B cells (Fig. 11E). While this highlighted the importance of these CD22 N-glycans in Gal9-mediated association between IgM and CD22, interestingly, we also found that treating 5Q-mutant B cells with exogenous Gal9 actually decreased the association between IgM and CD22.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

A

B C

D E

Figure 11. Gal9-mediated association of IgM and CD22 is dependent on N-glycans of CD22. A. Representative merged TIRFM (top), dSTORM (middle), dSTORM zoom (bottom) images showing surface CD22 (cyan), IgM BCR (magenta), rGal9 (yellow) on WT Daudi B-cells (WT) (first panel), rGal9 treated WT (WT rGal9) (second panel), 5Q-mutant Daudi B-cells (5Q) (third panel), rGal9 treated 5Q (5Q rGal9) (fourth panel). dSTORM ROI (3 x 3 µm) is outlined in yellow (middle) and magnified in dSTORM zoom (bottom). Colocalization between channels shown in white. Scale bars represent 5 µm and 1 µm (zoom). B-E. Coordinate-based colocalization (CBC) between CD22 and IgM single-molecules showing quantification of at least 15 ROIs from Daudi B-cells pooled from 4 independent experiments. B. CBC histograms in WT rGal9 and 5Q rGal9 C. CBC values of data from B. D. CBC histograms in 5Q and 5Q rGal9. E. CBC values of data from D. Lines/errors represent means ± SEM. ****p<0.0001 by Mann Whitney.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

3.9 CD22 N-glycans are crucial for Gal9-mediated CD22 clustering

We further wanted to examine the influence of CD22 N-glycans on clustering of IgM and

CD22. Using the Hopkin’s Index we found that CD22 is highly clustered in the 5Q-mutant and clustering decreases upon treatment with rGal9, in contrast to treating WT B cells with rGal9 where CD22 clustering is increased (Fig. 12 A). IgM clustering is significantly increased upon rGal9-treatment in both WT and 5Q-mutant B cells (Fig 12 C). We found that treatment of 5Q- mutant with rGal9 did not induce an increase in the molecular density of CD22 within clusters

(as we saw no shift in the H function peak), as it did in WT B cells treated with rGal9 (Fig. 12B).

However, we found that, consistent with increased clustering of IgM upon treating 5Q-mutant B cells with rGal9, the peak of the H-function was higher (compared to untreated 5Q-mutant B cells) indicating increased density of IgM molecules in clusters (Fig. 12D).

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

A B

C D

Figure 12. Gal9-mediated CD22 clustering is dependent on N-glycans of CD22. Analysis of dual dSTORM data of CD22 and IgM nanoclusters in WT and 5Q CD22 mutant expressing cells treated or not with rGal9. A. Hopkin’s index (1000 localizations plotted) and B. H function of Ripley’s K of CD22 in WT and 5Q mutant cells treated or not with rGal9. C. Hopkin’s index (1000 localizations plotted) and D. H function of IgM in WT and 5Q mutant cells treated or not with rGal9. WT and WT+ rGal9 data plotted for comparison. Numerical annotations in A and C represent mean values. Quantification of at least 15 ROIs pooled from 4 independent experiments. Lines/errors represent means ± SEM. * p < 0.05, ** p <0.01, **** p < 0.0001 by Kruskal-Wallis.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

3.10 Gal9-mediated inhibition of B cell signaling is dependent on CD22 N- glycans

Finally, we wanted to examine whether these gross alterations in the organization of IgM and CD22 due to mutating these N-glycan sites on CD22 impacted Gal9-mediated inhibition of

B cell signaling. We treated human Daudi WT, 5Q-mutant, and CD22-KO B cells with 1 µM rGal9, stimulated the untreated and treated cells with anti-IgM F(ab’)2 for 0 and 10 minutes, lysed the cells, separated the proteins using SDS-PAGE and immunoblotted for total phosphotyrosine (pY), pCD19, and pERK and β-tubulin (Fig. 13A-B). We found that, in contrast to WT B cells, rGal9 treatment did not suppress total tyrosine phosphorylation , nor phosphorylation of CD19 and ERK in 5Q-mutant B cells, similar to CD22-KO B cells (Fig. 10

A-C). These findings identify the importance of CD22 N-glycans in Gal9-mediated inhibition of

B cell signaling.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

A B

C

Figure 13. CD22 N-glycans are crucial in Gal9-mediated regulation of B cell signaling. WT, 5Q-mutant and CD22-KO Daudi B cells treated with 1 µM rGal9 were stimulated with 5.0 µg/ml anti-IgM F(ab)2 for indicated times, lysed and subjected to SDS-PAGE followed by immunoblotting with A. anti-phosphotyrosine, B. anti-phospho-CD19 and anti-phospho-ERK and β-tubulin. C. Quantification of pCD19 and pERK (shown below) normalized to β-tubulin loading control and plotted as fold change relative to WT 0 min. Blots and quantification representative of at least three independent experiments.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

DISCUSSION

Here, we identify a mechanism through which Gal9 organizes membrane receptors through glycan interactions to modulate the B cell signaling response. Our current working model proposes that Gal9 binds to and organizes a coordinated network of stimulatory molecules such as IgM-BCR and CD19, with inhibitory receptors CD45 and CD22, to directly inhibit B cell signaling (Figure 14). Based on our findings, we believe that Gal9 binds to IgM-BCR and CD45 in order to regulate CD22 and CD19 function in glycosphingolipid-rich domains. Crucially, we identify that Gal9-mediated inhibition of B cell signaling is entirely dependent on CD22, specifically, one or more of the five terminal N-glycans on CD22.

56 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

A

B

C

D

Figure 14. Working model depicting Galectin-9’s role in regulating receptor organization and inhibition of B cell signaling. (A) Top view of IgM and CD22 organization on the surface of resting WT B cells (left), 5Q-mutant Daudi B cells (middle), and CD22-KO B cells (right). In resting WT B cells, galectin-9 mediates basal interaction between IgM and CD22 to allow for regulated BCR signaling. Mutation of five N-linked glycosylation sites from asparagine to glutamine on CD22 leads to increased CD22 clustering, decreased IgM clustering, decreased CD22 phosphorylation, and increased BCR signaling. (B) Side view of IgM, CD45, CD22, and CD19 organization on the surface of resting WT B cells (left), 5Q-mutant Daudi B cells (middle), and CD22-KO B cells (right), based on results and predictions. In WT B cells, Gal9 directly binds to IgM-BCR and CD45 and may inhibit B cell signaling through CD45’s constitutive dephosphorylation and inhibition of Lyn which resides in glycosphingolipid- 57 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis rich domains. We further hypothesize that Gal9 may directly (as depicted in model) or indirectly bind to the inhibitory receptor CD22 and the stimulatory receptor CD19, and together with CD45 and IgM-BCR, inhibit B cell signaling. In 5Q-mutant B cells, CD22 clustering is enhanced, which we hypothesize may be due to the sialic acid-mediated homotypic clustering which inhibits CD22 function. (C) Top view of IgM and CD22 organization on the surface of recombinant galectin-9 (rGal9) treated WT (left), 5Q- mutant (middle), and CD22-KO (right) B cells. In resting WT B cells, treatment with rGal9 increases IgM and CD22 nanoclusters and increases IgM-CD22 colocalization leading to suppression of BCR signaling upon BCR stimulation. Treatment of 5Q-mutant with rGal9 increases IgM cluster size and decreases IgM- CD22 colocalization. rGal9 treatment does not suppress BCR signaling in 5Q-mutant B cells as observed in WT B cells, demonstrating that galectin-9 mediated regulation of BCR signaling is dependent on CD22 N-glycans. Treatment of CD22-KO B cells with rGal9 does not show dampening of the BCR signaling response as seen in WT B cells upon BCR stimulation, demonstrating that CD22 is indispensable for Gal9-mediated inhibition of B cell signaling. (D) Side view of IgM, CD22, CD19, and CD45 organization on the surface of recombinant galectin-9 (rGal9) treated WT (left), 5Q-mutant (middle), and CD22-KO (right) B cells, depicted based on results and predictions. In WT B cells, rGal9-treatment increases the molecular density of CD45, CD22, CD19 and IgM-BCR in glycosphingolipid-rich domains, which in turn suppresses the BCR signaling response. In 5Q-mutant B cells, CD22 and IgM colocalization decreases, demonstrating that the N-glycans are crucial for mediating the interaction between IgM and CD22. A crucial missing link is whether CD45 organization is altered in the 5Q-mutant B cell and CD22-KO B cells, and whether it is important in mediating the association between IgM and CD22 via these N-linked glycosylations. Future studies need to be conducted in order to elucidate the molecular interactions between these key signaling receptors in the absence and presence of Gal9 and CD22 N-linked glycans.

58 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

The fluid mosaic model, proposed by Singer and Nicholson in 1972, theorized that the distribution of proteins across the membrane is random where molecules are allowed to freely diffuse uninhibited145. Recent research, however, demonstrates that organization of proteins is non-random and regulated by factors such as the actin cytoskeleton42, lipid composition97, protein-protein interactions58-59, and protein-carbohydrate interactions104. For example, numerous studies have demonstrated that the actin cytoskeleton regulates protein organization and mobility102,137. Importantly, this actin-dependent organization and restriction of mobility has functional significance as simple actin depolymerization leads to spontaneous BCR signaling42.

Furthermore, the actin-based compartmentalization of the BCR from CD19 on the nanoscale level regulates BCR signaling and prevents spontaneous B cell signaling in resting state39.

However, there are still many levels of receptor organization for which the mechanism is currently unknown. For example, Gasparrinni et al. show that CD22 organization is not regulated by the actin cytoskeleton40. Therefore, we hypothesized that the galectin lattice may mediate an additional layer of membrane protein organization.

Galectins bind to β-galactoside side chains on glycoproteins to form a network, known as the galectin-glycoprotein lattice, and have been previously implicated in regulating signaling, adhesion, and migration in a variety of cell types. Our lab identified that the galectin, Gal9, plays a crucial role in inhibiting B cell activation. We observed that treatment of B cells with rGal9 significantly suppressed BCR-antigen microcluster formation and downstream ERK signaling.

We further found that Gal9 directly binds to IgM-BCR and CD45 using mass spectrometry.

Interestingly, we found that CD22 was significantly clustered in Gal9high regions using confocal microscopy, and hence we hypothesized that Gal9 may play a role in aggregating inhibitory 59 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis receptors while excluding costimulatory receptors such as CD19. Surprisingly, contrary to our hypothesis, we found that Gal9 significantly enhances the density of CD19 in Gal9high regions while not affecting the distribution of the inhibitory receptor FcγRIIb (Fig 4). We also asked whether the increased clustering of IgM-BCR and CD19 induced by rGal9 treatment may induce immediate B cell activation and signaling, as we only assess B cell activation using anti-BCR crosslinking antibodies 30 minutes post rGal9 treatment. We used flow cytometry and calcium assays to examine whether rGal9 treatment induced calcium flux, a commonly used and robust indicator of early BCR signaling. We found that addition of rGal9 did not induce calcium flux as seen when we treated cells with anti-BCR antibodies, demonstrating that rGal9 treatment does not activate B cells (Fig 5). Furthermore, even though we did not see a significant increase in the phosphorylation of CD19 in Gal9-KO B cells upon BCR stimulation (Fig. 8B), examination of stimulated rGal9-treated WT B cells demonstrated a significant suppression of the phosphorylation of CD19 and its downstream kinase Akt (Fig. 9B-C). We hypothesize that the difference in CD19 phophorylation between Gal9-KO and rGal9-treated B cells is because Gal9- ligands are not fully saturated in endogenous conditions, and therefore the addition of rGal9 acts as an important tool to exaggerate a phenotype that might not be resolvable when observed at endogenous levels. Our results hence demonstrate that Gal9 plays a crucial role in regulating

CD19 function.

Our finding that Gal9 regulates the organization and function of CD19 raises further questions in terms of how Gal9 organizes the receptor landscape on B cells. First, how is CD19 recruited into Gal9high regions? One of our hypotheses is that CD19 may be recruited indirectly into Gal9high regions independent of direct Gal9-binding through association with other receptors. For example, CD19 co-immunoprecipitates with IgM-BCR, and this association is 60 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

135 dependent on 17 amino acids in the cytoplasmic domain of CD19 . This was demonstrated with the use of chimeric CD19 receptors which only contained the transmembrane domain and membrane proximal 17 amino acids of the cytoplasmic domain135. This suggests that CD19 may be recruited into Gal9high regions through its association to IgM-BCR via its cytoplasmic domains. However, as only approximately 1% of IgM-BCR associates with CD19 through this interaction135, it may not explain the vast difference we see in the co-localization of IgM-BCR and CD19 in rGal9-treated WT B cells. Interestingly, Carter et al. also found that CD22 and

SHP-1 also co-immunoprecipitate with CD19135. However, the CD22 band was stronger in the lysate prepared using Brij-96 compared to the Nonidet P-40 lysate. As Brij-96 is a milder detergent than Nonidet P-40, it allows detection of other membrane proteins, due to the incomplete disruption of the membrane135. Therefore, the authors hypothesized that the interaction between CD22 and CD19 may be indirect135. This interaction, similar to CD19 with

IgM-BCR, is also mediated by CD19’s cytoplasmic domain, however unlike IgM-BCR, this association was only examined after BCR stimulation135. Therefore, it is currently unknown whether there is direct interaction or association between CD22 and CD19 in the resting state. As the structure of the N-glycans that exist on CD19 have not been identified, it could be that CD22 binds directly to CD19 via its sialic acid-binding domain if CD19 N-glycans contain α-2,6, sialic acid linkages. To test this hypothesis, B cells can be treated with sialidases, which are enzymes which cleave and remove terminal sialic acids from the surface of glycoproteins or glycolipids146. CD22 and CD19 organization and colocalization on sialidase-treated cells can then be examined using dual dSTORM, in order to identify if sialic acid mediates an interaction between these two receptors. Our third hypothesis is that Gal9 may bind directly to one or more of the five N-glycans on CD19 to regulate its organization with other proteins and lipids on the

61 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis cell membrane (Figure 14). Indeed, our previous mass spectrometry results identified CD19 as a potential ligand for Gal9 in 2 out of 3 experimental repeats (Cao et al., unpublished). Therefore,

Gal9 may directly bind CD19 to IgM-BCR or CD45 in order to regulate BCR signaling. Further experimental repeats of the mass spectrometry results may provide evidence for this interaction.

Alternatively, single-site mutagenesis of CD19 N-glycan sites and the corresponding effects on rGal9-mediated association of CD19 and CD22 can be examined via dual dSTORM. Another experimental approach could be conduction of a pull-down assay using rGal9 as a bait protein, and immunoblotting for CD19. Nonetheless, to our knowledge, our study is the first to demonstrate an extracellular mechanism regulating the interaction of CD19 with membrane receptors in the steady-state and the impact this has on BCR signaling upon BCR stimulation.

Another important question arising from our results is how the Gal9-mediated increase in

CD19 density actually inhibits B cell signaling upon BCR stimulation. As we have previously shown that Gal9high domains have increased molecular density of IgM-BCR, CD45, and CD22, we hypothesize that the colocalization of CD22 and CD19 may allow mutual regulation of each other in order to fine-tune BCR signaling. Indeed, CD19 is hyper-phosphorylated in CD22- deficient mice, demonstrating that CD22 may play a crucial role in the regulation of of CD19 phosphorylation138. Upon BCR ligation and CD19 phosphorylation by Lyn, Vav is recruited and binds to CD19 via the phosphorylated Tyr-39185,139. Vav has been found to play a crucial role in the antigen acquisition response upon BCR ligation140. Furthermore, Vav1/3-KO mice cannot endocytose BCR and present antigen to T cells136. Hence, CD19-mediated recruitment of Vav is crucial for the B cell response. Sato et al. found that Vav phosphorylation was reciprocally regulated by CD22 and CD19, where Vav was hyperphosphorylated in CD22-deficient B cells, but hypophosphorylated in CD19-deficient B cells85. Hence, closer association between these 62 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis proteins may allow for optimal Vav phosphorylation, regulated antigen-microcluster formation and consequent T cell help. Reciprocally, CD22 phosphorylation requires CD19 expression, where CD19 deficiency leads to decreased phosphorylation of CD22, which in turn causes its decreased association with SHP-1, thereby blocking CD22 function85. Hence, we hypothesize that Gal9 localization of CD19 and CD22 into similar domains on the B cell surface plays a crucial role in regulating BCR signaling.

Given we hypothesize that CD22 plays a crucial function in Gal9-mediated inhibition of

B cell signaling, we wanted to further characterize Gal9-mediated organization of CD22 and the impact on its function. We used dual dSTORM to study the impact rGal9 has on the organization of CD22 and its association with IgM-BCR. Dual dSTORM allows for the distinction of 2 particles separated by a distance as small as 10 nm, and therefore the single-particle resolution of molecules such as CD22 and IgM-BCR. We found that rGal9 significantly increased the size and density of IgM and CD22 clusters, and increased the association between IgM and CD22 (Fig.

6). In order to examine whether this CD22-IgM association was important in Gal9-mediated inhibition of B cell signaling, we treated WT and CD22-KO B cells with rGal9 at 10 minutes.

Contrary to rGal9-treated WT B cells, CD22-KO B cells had increased and sustained ERK and

CD19 phosphorylation (Fig. 10), demonstrating that CD22 is indispensable for Gal9-mediated inhibition. These findings ask us to hypothesize which structural feature of CD22 allows it to play a role in Gal9-mediated inhibition of B cell signaling. These are two important structural domains; CD22’s α-2,6, sialic binding domain, and the terminal N-glycans that are present on the surface of CD22. The sialic acid binding domain has been heavily studied, and has been found to bind IgM-BCR and CD45. Since Gal9 binds directly to CD45 and IgM-BCR, it could be that

CD22 is recruited by binding to these protein receptors via the sialic acid binding domain. 63 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Conversely, CD22 could be recruited independent of its sialic acid binding domain, through interactions to other protein receptors mediated by its N-terminal glycans.

In order to test the second hypothesis, we used a CD22-mutant Daudi B cell line in which the five N-terminal glycosylation sites were mutated from asparagine to glutamine (N67Q,

N112Q, N135Q, N164Q, N231Q). We then treated 5Q-mutant B cells with rGal9 and examined

CD22-IgM colocalization. We found that in the absence of the five N-terminal glycans and treatment of rGal9, IgM-CD22 association was significantly abrogated compared to either WT rGal9-treated B cells or untreated 5Q-mutant B cells (Fig 11). The decreased association between

IgM and CD22 in rGal9-treated 5Q mutant B cells may be due to the increased clustering and density of IgM, which further segregates IgM-BCR away from CD22 (Fig. 12). Indeed, this may also explain why we see a decreased Hopkin’s index for CD22 upon rGal9 treatment in 5Q- mutant B cells, as it demonstrates that CD22 organization is more random upon mutation of the

N-glycans. Furthermore, there is no difference in IgM-BCR density and clustering in WT rGal9- treated vs 5Q rGal9-treated cells (Fig. 11C), and this is consistent in demonstrating that mutation of the N-glycans of CD22 directly affects CD22’s ability to be regulated by Gal9. The increased density of CD22 in untreated 5Q mutant B cells may be attributed to homotypic CD22 binding via the N101 glycosylation, in the absence for Gal9 to mediate interactions with other glycoproteins such as IgM-BCR on the cell membrane. We also found that the 5Q-mutant B cells had enhanced BCR signaling, and that this was not suppressed upon rGal9-treatment, highlighting that CD22 N-glycans are crucial for Gal9-mediated inhibition of B cell signaling

(Fig. 13). While our study provides a crucial first step in identifying the importance of CD22 glycosylation and Gal9-mediation of B cell signaling, many questions remain to be answered, as discussed below. 64 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

One important question is the identification of the role of each CD22 N-glycan. While the overarching effect of mutating the five terminal N-glycans was abrogation of CD22 function and

Gal9-mediated inhibition of BCR signaling, it could be that the function of each glycan is masked by this cumulative effect. For example, in CD19/CD22 double-deficient mice, CD19 deletion masked the effects of the CD22 deletion, even though they have strikingly different roles in B cells138. On plasma B cells, removal of sialic acid off the single glycosylation on the membrane protein B cell maturation antigen (BCMA), leads to increased BCMA retention on the surface, and reduced risk of apoptosis when treated with BCMA ligands147. In order to determine the role of each N-glycan and whether they have redundant effects, single site mutagenesis of each asparagine could be performed, followed by studies such as dual dSTORM or co- immunoprecipitation to examine protein interactions or binding partners. Furthermore, developing a better understanding of the role of each glycan site may help, as we discover diseases in which these glycosylation sites are mutated, to develop therapeutics to alleviate disease.

While we demonstrate that Gal9 is crucial for regulating CD22 organization via its N- glycans in order to inhibit B cell signaling, we currently do not know whether this is (a) through direct Gal9-mediated ligation of CD22 to IgM-BCR or CD45 or (b) through Gal9-binding of

IgM-BCR to CD45, where either protein can then be bound to CD22 via another lectin. For the first hypothesis, there is currently no evidence to suggest that CD22 is a direct ligand for Gal9, as it was not identified in either mass spectrometry or through far-western immunoblotting130.

However, these assays may provide poor resolution of this association due to the expression of five times more IgM-BCR molecules compared to CD22 molecules on the B cell surface 40.

More sensitive approaches may be used to detect this association in the future, such as Hydrogen 65 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Deuterium Exchange Mass Spectrometry which has been suggested to have an improved sensitivity141. The second hypothesis suggests that Gal9 mediates CD22 association with IgM or

CD45 through an indirect mechanism via a currently unknown ligand. Both hypotheses suggest a crucial missing link and an area of future study, which is the examination of the organization and localization of CD45 in rGal9-treated WT and 5Q-mutant B cells. CD45 is integral for CD22 organization, as CD45-deficient B cells have increased CD22 clustering40, similar to the increased clustering we observed in the 5Q-mutants. However, in CD45-deficient cells BCR signaling was still repressed40, as opposed to the 5Q-mutant in which CD22 function is abrogated. As CD45 is known to play positive regulatory effects, as well as negative regulatory effects, which may be mediated through CD22 recruitment, it could be that the presence of CD22 is able to rescue the absence of the negative role of CD4566-68, 29, 69. In cells in which the sialic acid binding site was mutated, CD22 clusters decreased, CD22 mobility increased, and CD22 phosphorylation increased, which inhibited B cell signaling40. Based on these findings, it could be hypothesized that Gal9 may cause the aggregation of CD45 with IgM-BCR, and along the way pull CD22 with it via CD22 sialic acid binding domain, and away from its homotypic ligands. Furthermore, CD22’s preferential binding to CD45 is because of the abundance of glycan ligands and not due to affinity of individual galectin-glycan interaction. Therefore in the

5Q-mutant, Gal9-mediated compartmentalization of CD45 and IgM-BCR from CD22 may allow

CD22 to interact more with itself to form larger oligomeric clusters, masking the ability for

CD22 to engage with IgM-BCR complexes in order to inhibit BCR signaling. In order to test this hypothesis, dual dSTORM of CD22-CD45 and/or IgM-CD45 organization and colocalization can be performed in rGal9 treated 5Q-mutant cells.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

The findings that Gal9-mediated organization of several key BCR signaling receptors into a similar region on the B cell membrane suggests that there may be a specific lipid environment into which these membrane receptors were recruited. We found that treatment of

WT murine B cells with rGal9 induced the coalescence of lipid raft domains (which usually exist in puncta on the surface of B cells142), and increased the molecular density of the inhibitory receptors CD22 and CD45 in these domains (Fig. 7). As Gal9 has been previously found to bind to glycosylated lipids on the membrane of epithelial cells119, Gal9 may bind directly to glycolipids in lipid rafts and glycosylated proteins to mediate their close interaction. What is the significance of Gal9-mediated recruitment of inhibitory receptors CD45 and CD22 into lipid raft domains? Lipid raft domains are signaling platforms enriched in Src-family kinases such as Lyn.

It can be hypothesized that the increased association of CD45 and Lyn allows for the inhibition of Lyn’s positive regulatory function by continuously dephosphorylating it29,69. The increased localization of CD45 and CD22 in lipid rafts in the resting state suggests that upon antigen-BCR ligation, these inhibitory receptors will be able to terminate BCR signaling more effectively.

Importantly, it is imperative to look at the Gal9 lattice and the role of N-glycosylation in the broader context and potentially in vivo. Our study highlights the important role of the Gal9 lattice in cis-binding on the surface of B cells. However, B cells in vivo are also involved in complex cell-cell interactions; required for development, differentiation, and homeostasis.

Whether Gal9 may influence and modulate these interactions through trans-binding is still unknown. Indeed, Gal1 has been implicated in pre-B cell signaling and development, by binding to the surrogate light chain of pre-BCR and adhesion molecules on stromal cells132. Given the important role Gal9 plays in B cell activation and signaling, it may also play a role in binding receptors in trans on T cells during the secondary signal phase to modulate T cell help. 67 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

Furthermore, Gal9 may play a potential role in mediating cell-cell interactions to maintain homeostasis. Shinzaki et al. found that blocking galectin-glycan interactions during cell-cell interaction between B cells and macrophages, by treating cells with , suppressed IL-10 production through an unknown mechanism which is crucial in preventing colitis133. Colitis is a long-term inflammation found in the inner lining of the colon. Further investigation into galectin involvement in trans-binding, their ligands, and the effects of galectin binding may lead to potential therapeutic targets for diseases such as colitis.

Finally, another important area of study is the changes to receptor glycosylation and the resulting effect on galectin binding over time and space in B cells. Indeed, recently, Giovannone et al. found that compared to naïve B cells, germinal center B cells significantly upregulated I- branched glycans which led to less Gal9 binding120. This might be hypothesized to allow easier signaling during germinal center formation, and in turn allow for high affinity antibody production upon affinity maturation. Given that naïve and memory B cells are only 2 subsets of the repertoire of different B cells expressed in the body, further characterizing glycosylation and their sites on glycoproteins can improve our comprehension of the galectin lattice and B cell glycobiology. Additionally, whether there are differences in glycosylation of B cells localized in different regions of the human body is also unknown. Understanding these changes can provide insight into the fine-tuning and differential regulation of B cell signaling and how this regulation influences B cell outcomes in these specific regions. Another imperative reason for understanding changes in glycosylation is due to the increased evidence that plasma Gal9 is significantly elevated during a wide variety of disease states such as HIV infection136, dengue137, and influenza135. Indeed, as one of the hallmarks for HIV pathogenesis is HIV-induced immune cell over activation such as polyclonal B cell activation, it is imperative to examine whether the 68 Fathima Hifza Mohamed Buhari Treanor Lab

CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis upregulation of plasma Gal9 is due to increased production and secretion by cells, or whether the glycosylation states are changing on these cells, inducing decreased binding of Gal9 to B cells, followed by enhanced activation. Further research into these areas are crucial for a more comprehensive and clear understanding of the galectin-glycan network in human biology and disease.

Our study highlights the importance of the galectin lattice and glycosylation in receptor landscape organization to regulate BCR signaling. Given the importance of B cell signaling in various autoimmune diseases and such as chronic lymphocytic leukemia, our finding of galectin-9 inhibition of B cell signaling provides detailed mechanistic understanding of action, and may have important therapeutic implications for the future, such as using recombinant

Galectin-9 as a treatment for autoimmune diseases and B cell malignancies.

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

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CD22 is indispensable for Galectin-9-mediated inhibition of B cell signaling Thesis

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