MULTI-FUNCTIONAL EFFECTOR RESPONSES ELICITED from Igm MEMORY STEM CELLS

MULTI-FUNCTIONAL EFFECTOR RESPONSES ELICITED FROM IgM MEMORY STEM CELLS Item Type Dissertation Authors Kenderes, Kevin Rights Attribution-NonCommercial-NoDerivatives 4.0 International Download date 29/09/2021 18:50:57 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/20.500.12648/2017 MULTI-FUNCTIONAL EFFECTOR RESPONSES ELICITED FROM IgM MEMORY STEM CELLS by Kevin Kenderes A Dissertation in Microbiology and Immunology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies of State University of New York, Upstate Medical University. Approved ___________________________ (Sponsor’s signature) Date_______________________ Table of Contents List of Figures iv List of Tables vi Abbreviations vii Acknowledgements x Abstract xi Chapter I: Introduction 1 History of Humoral Immunity 2 Development of Naive B cells 4 The Primary B cell Response 7 T cell dependent B cell responses 7 T cell independent B cell responses 10 Secondary B cell Responses 12 Ehrlichia muris 15 Immune Responses to E. muris 17 T-bet-Expressing B Cells 19 Summary 21 Chapter II: Materials and Methods 22 Chapter III: CD11c+ T-bet+ IgM memory Cells Exhibit Stem Cell-like 27 Properties Abstract 28 Introduction 29 Results 33 ii Discussion 76 Chapter IV: Summary 84 Model 85 Future directions 90 Summary 95 Appendix I: Tetanus Toxin binds to bystander B cells following immunization 97 Abstract 98 Introduction 99 Materials and Methods 102 Results 105 Discussion 124 Future Directions 128 References 130 iii List of Figures Figure 3.1. Labeling Aicda-expressing CD11c+ T-bet+ IgM memory cells in vivo 34 Figure 3.S1. Phenotype of Aidca-expressing memory B cell subsets. 39 Figure 3.2. IgM memory cells differentiate only after challenge infection. 44 Figure 3.S2. IgM memory did not differentiate following transfer into naive mice. 46 Figure 3.S3. IgM memory cells circulate to the lymph nodes. 48 Figure 3.S4. IgM memory B cells generate secondary IgM and IgG responses. 52 Figure 3.3. IgM Memory cells enhance primary B cell responses. 54 Figure 3.4. IgM memory B cells generated all effector and memory subsets 58 following challenge infection. Figure 3.5. CD11c expression did not affect IgM memory B cell differentiation. 62 Figure 3.6. IgM memory B cells retain multi-lineage potential after serial transfer. 65 Figure 3.7. IgH repertoire analyses of differentiated IgM memory cells. 69 Figure 3.S5. Clonal relationship between recipient mice and lineages. 74 Figure 4.1. Gradient model of memory B cells 88 Figure A.1. Tetanus toxin tetramer binding was specific and anatomically 107 localized. iv Figure A.2. Tetanus toxin binding cells were detected following immunization 110 with other adjuvants. Figure A.3. Tetanus toxin binding cells are quiescent. 114 Figure A.4 Tetanus toxin binding was not mediated by the binding of the toxin to 118 its ligand. Figure A.5. Tetanus toxin binding was mediated by the BCR. 122 v List of Tables Table 3.1. Surface Marker Expression on IgM Memory Cells (MFI) 38 Table3.S1. Surface Marker expression on CD11c+ and Cd11cneg IgM Memory Cells 41 vi Abbreviations ABCs Age-associated B cells AID Activation-induced cytidine deaminase APRIL A proliferation-inducing ligand ASC Antibody-secreting cell BAFF-R B cell activating factor receptor BCR B cell receptor BM Bone marrow BrdU Bromodeoxyuridine CD Cluster of differentiation CDR3 Complementarity-determining region 3 CLP Common lymphoid progenitor Cr-2 Complement receptor 2 CSR Class switch recombination CXCL12 C-X-C motif chemokine 12 CXCR3 C-X-C motif chemokine receptor 3 CXCR4 C-X-C motif chemokine receptor 3 dbpA Decorin-binding protein A DC Dendritic cell DTaP Diphtheria, tetanus, and pertussis vaccine ELISA Enzyme-linked immunosorbent assay EMLA E. muris-like agent exfPB Extrafollicular plasmablast vii eYFP enhanced yellow fluorescent protein FcR Fc receptor FcRL4/5 Fc receptor-like molecule 4/5 FcgRIIb Fc gamma receptor IIb FcmR Fc mu receptor FDC Follicular dendritic cell GABA gamma-Aminobutyric acid Gal Galactose GalNAc N-Acetylgalactosamine GC Germinal center APC Antigen-presenting cell HEL Hen egg lysozyme HME Human monocytic ehrlichiosis HSC Hematopoietic stem cell i.p. Intraperitoneal i.v. Intravenous ICOS Inducible co-stimulator ICOS-L Inducible co-stimulator ligand IFNg Interferon gamma IOE Ixodes ovatus Ehrlichia LMPP Lymphoid-primed multi-potent progenitors LPS Lipopolysaccharide MBC Memory B cell viii MHC Major histocompatibility complex MPP Multi-potent progenitor MZ Marginal zone OMP-19 Outer membrane protein 19 PC Plasma cell PD-L2 Programmed death ligand 2 RAG Recombinase activating gene SA Streptavidin SA:TTCF Streptavidin tetanus toxin C fragment tetramer SHM Somatic hypermutation STAT-1 Signal transducer and activator of transcription 1 T1 Transitional 1 T2 Transitional 2 TACI Transmembrane activator and CAML interactor TCR T cell receptor TFH T follicular helper cell TH1 T helper 1 cell Ti T-independent TLR Toll-like receptor TNFa Tumor necrosis factor alpha TTCF Tetanus toxin C fragment VSVG Vesicular stomatitis virus G ix Acknowledgements I would like to thank Dr. Gary Winslow for his mentorship and support throughout graduate school. I would like to thank current and past members of the Winslow Lab for their companionship, helpful insights and discussions: Amber Papillion, Lisa Dishaw, Berenice Cabrera-Martinez, Russell Levack and Maria Popescu. In particular, I am grateful for Russell Levack for performing the IgSeq data analysis. I would also like to thank my thesis advisory committee, Steve Taffet, Gary Chan, William Lee and Brian Rudd, for their advice and input into my project. I would like to thank Jennifer Yates and Mike Tighe for performing sectioning, staining and imaging of lymph nodes. Kai Wucherpfennig generously provided the plasmid expressing TTCF and Jean Claude Weill generously provided the Aid-Cre-ERT2 mice. I would like to thank Lisa Phelps who assisted with flow cytometric cell sorting and maintain the flow cytometry core and Alice Bernat for assistance in making the graphics. Finally, I would like to thank members Department of Laboratory Animal Resources who maintained and cared for the mouse colony. Funding was provided by NIH ROI AI114545 grant and the Nappi award. x Abstract Title: Multi-functional Effector Responses Elicited From IgM Memory Stem Cells Author: Kevin Kenderes Sponsor: Gary Winslow The response of memory B cells to challenge infection is fundamental to long- term protection against pathogens. Following challenge, memory B cells can rapidly differentiate into antibody-secreting cells (ASCs) to produce a secondary antibody response. Memory B cells have also been shown to re-enter into germinal centers and undergo additional rounds of affinity maturation. Both the isotype of the B cell and the signals that generated the B cell have been proposed to modulate how memory B cells respond. Initial studies proposed BCR-intrinsic factors are responsible for the differentiation of memory cells. IgM memory cells undergo differentiation in GCs following antigen challenge, while IgG memory cells rapidly differentiate into ASCs. Other studies found no link-between BCR isotype and differentiation. We investigated the differentiation of T-bet+ CD11c+ IgM memory B cells following challenge infection. IgM memory cells differentiated into IgM-producing plasmablasts. Other IgM memory B cells entered germinal centers, underwent class switching, and became switched memory cells. Yet other donor cells were maintained as IgM memory cells. The IgM memory cells also retained their multi-lineage potential following serial transfer. The kinetics of the IgM memory response mimicked the kinetics of the primary response. Thus, IgM memory cells can differentiate into all effector B cell lineages, and undergo self-renewal, properties that are characteristic of stem cells; however, differentiation occurs with the same kinetics of the primary response. We propose that memory B cells have varying degrees of stem cell likeness. IgM memory stem cells retain the most differentiative xi capacity but respond to challenge similarly to naïve cells, while IgG effector memory cells are primed to rapidly differentiate into IgG ASCs. xii CHAPTER I: Introduction 1 B cells are a major component of the immune system and responsible for providing protection against a wide range of pathogens. B cell responses to vaccination are essential for long-term protection against pathogens. Immunity against pathogens is critical for both individual and global health. Not only does immunity provide personal protection against disease, it also slows the spread of the disease through the population, protecting those who cannot be vaccinated. Immunity is separated into the innate and adaptive response. The innate response provides non-specific protection against a broad range of pathogens. Adaptive immunity is characterized by long-term specific protection against the infecting pathogen. B cells are a major component of the adaptive immune response and provide long-term protection through the production of infection-specific antibodies. These antibodies can neutralize infectious agents and prevent disease progression. Additionally, some B cells responsible for neutralization are retained long-term as memory B cells. Because of the prominent role of B cells in long-term immunity, investigation of the primary B cell response, how memory B cells are generated, and how memory cells respond to provide

Load more