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CD22 Is Indispensable for Galectin-9-Mediated Inhibition of B Cell Signaling

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CD22 Is Indispensable for Galectin-9-Mediated Inhibition of B Cell Signaling ` CD22 is indispensable for Galectin-9-mediated inhibition of B cell 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 cell signaling 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 adaptive immune system, producing antibodies and conferring long-term immunity. B-cell activation is initiated by recognition of foreign particles (antigens) by B-cell receptors (BCR). Previous research in our lab found that the lectin, 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-glycans 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. ii Fathima Hifza Mohamed Buhari Treanor Lab 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. iii Fathima Hifza Mohamed Buhari Treanor Lab 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. v Fathima Hifza Mohamed Buhari Treanor Lab 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 Lipid Raft 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 vii Fathima Hifza Mohamed Buhari Treanor Lab 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-glycan 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 viii Fathima Hifza Mohamed Buhari Treanor Lab 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 Antigen Akt Protein Kinase B APC Antigen presenting cell Asn Asparagine N Asparagine B220 B cell isoform
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