Human memory B cells during development and after treatment of pemphigus vulgaris autoimmunity Open Access
Cho, Alice (Spring 2019)
Abstract
B cells are important immune effector cells, contributing to long-term protection from pathogens through antibody secretion or differentiation into long-lived memory B cells (MBCs). Developing MBCs must be diverse in order to bind all possible pathogens, but exclude self-reactive B cells, such as those causing pemphigus vulgaris (PV). PV is an human autoimmune blistering skin disease, driven by autoantibodies primarily targeting the well-characterized antigen Desmoglein-3 (Dsg3). Consequently, PV is a powerful model to study B cell-mediated autoimmunity in an antigen-specific fashion. In order to better understand human MBCs during autoimmunity, we studied their development during PV pathogenesis, and functionality after recovery from treatment with the B cell-depletive therapy, Rituximab.
First, we addressed how MBCs contribute to PV pathogenesis. We found that the emergence of activated Dsg3-specific MBCs correlates with disease presentation. Thus, we pursued an in-depth, single-cell analysis of Dsg3-specific MBCs by generating and characterizing a large panel of monoclonal antibodies from two recently diagnosed PV patients. Additionally, a unique, paired sample collected from one of the donors 15 months priorto symptom onset allowed us to determine how autoimmunity develops. Overall, our data suggests that Dsg3-specific B cells are generated from activation of low-affinity or non-autoreactive B cell precursors, possibly via bystander activation, which undergo extensive affinity maturation to select for high affinity, pathogenic B cells. The accumulation of these selected cells appears to drive initial disease presentation.
Next, we examined the quality of B cell responses to influenza vaccination in pemphigus patients previously treated with Rituximab. We found that despite numerical recovery of total peripheral B cells, patients lacked detectable influenza-specific MBCs. However, Rituximab-treated patients still mounted robust recall responses to influenza vaccination. This suggests that MBCs likely survive Rituximab treatment, possibly by residing in tissues, and can provide potent recall responses.
Overall, this dissertation provides novel insight into how autoimmune MBCs develop and contribute to PV pathogenesis, and how MBCs may recirculate through the periphery after Rituximab treatment. We believe that these results have implications for improving clinical care of PV patients, and raise important questions regarding the antibody-mediated mechanisms underlying how Dsg3-binding antibodies cause PV symptoms.
Table of Contents
TABLE OF CONTENTS
Abstract
Acknowledgements
Chapter 1: Introduction
B CELL IMMUNE RESPONSES
B cell development
B Cell Activation
Differentiation into effector B cells
Tolerance
PEMPHIGUS VULGARIS
Autoimmune Blistering Skin Diseases
Desmoglein-3 as the major target in Pemphigus Vulgaris
B cells mediate PV disease
Contribution of other immune cells to PV pathogenesis
Characterization of Dsg3-specific monoclonal antibodies
Mechanism of action for pathogenic Dsg3-specific mAbs
RITUXIMAB THERAPY
Treatment of PV disease with Rituximab
Relapse in PV disease after Rituximab treatment
Mechanism of relapse in other B cell-mediated autoimmune diseases
Impact of Rituximab on B cell responses to influenza vaccination
SUMMARY
FIGURES AND LEGENDS
FIGURE 1. Structure of skin and desmosomes.
FIGURE 2. Pathogenesis and treatment of pemphigus vulgaris.
Chapter 2: Single-cell analysis suggests that ongoing affinity maturation drives the emergence of pemphigus vulgaris autoimmune disease
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
SUPPLEMENTARY MATERIALS AND METHODS
AUTHOR CONTRIBUTION
ACKNOWLEDGEMENTS
FIGURES AND LEGENDS
FIGURE 1. Activated Dsg3-specific memory B cells are detected exclusively in patients with active PV disease.
FIGURE 2. Dsg3-specific MBCs are clonally restricted, show signs of extensive antigenic selection, and are exquisitely specific for Dsg3.
FIGURE 3. Pathogenic mAbs bind the EC1, EC2, or EC4 domains of Dsg3.
FIGURE 4. Dsg3-specific mAbs display a restricted repertoire and bind to 1 or 2 sterically-distinct epitopes in EC1, EC2 and EC4 domains of Dsg3.
FIGURE 5. Dsg3-specific MBCs are readily detected in PV patients prior to disease onset, and undergo extensive affinity maturation during disease development.
FIGURE 6. Somatic hypermutation is necessary for antibody binding of Dsg3.
FIGURE 7. Synergistic increase of pathogenic potency by targeting multiple domains in Dsg3.
FIGURE S1. Kinetics of serum antibody titers after treatment with B cell depletive therapy.
FIGURE S2. No Dsg3-specific circulating plasmablasts are detected in symptomatic PV patients.
FIGURE S3. Dsg3-specific serum antibodies were primarily IgG1 and IgG4 isotype.
FIGURE S4. Assessment of mAbs binding to Dsg3 expressed on cell surface.
FIGURE S5. Similar sterically-distinct epitopes detected by flow cytometry-based blocking assay were representative of other PV patients at diagnosis.
FIGURE S6. Antigen selection and ongoing affinity maturation of Dsg3-specific memory B cells.
FIGURE S7. Similar sterically-distinct epitopes are detected at pre-onset and diagnosis time points.
TABLES
TABLE S1. Characteristics of subjects at time of enrollment.
TABLE S2. Repertoire analysis of Dsg3-specific mAbs isolated from two PV patients.
TABLE S3. Characteristics of antibodies selected for germline reversion.
Chapter 3: Robust memory responses against influenza vaccination in previously Rituximab-treated pemphigus patients
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
SUPPLEMENTARY MATERIALS AND METHODS
AUTHOR CONTRIBUTION
ACKNOWLEDGEMENTS
FIGURES AND LEGENDS
FIGURE 1. Reconstitution of the B cell compartment after Rituximab treatment.
FIGURE 2. Lack of memory B cells (MBCs) in peripheral blood in Rituximab-treated patients.
FIGURE 3. Robust vaccine-induced plasmablast responses likely originating from memory recall responses.
FIGURE 4. Comparable serological responses to vaccination in patients and healthy controls.
FIGURE 5. Previous Rituximab depletion has no impact on the generation of new influenza-specific memory B cells.
FIGURE 6. Vaccine-induced plasmablasts display comparable repertoire breadth in patients and healthy controls.
FIGURES S1. Anti-desmoglein autoantibody titers decrease after Rituximab treatment.
FIGURE S2. Impact of AIBD disease and treatment on plasmablast responses to influenza vaccine.
FIGURE S3. Comparable frequencies of seroprotection in patients and healthy controls.
FIGURE S4. R848+IL2 mitogen cocktail is an effective alternative to assess antigen-specific memory B cells.
TABLES
TABLE 1. Characteristics of subjects at time of enrollment.
TABLE SI. Characteristics of subjects used for plasmablast repertoire analysis
Chapter 4: Discussion
SUMMARY
FUTURE CONSIDERATIONS
B cells in PV patients who relapse after Rituximab treatment
Skin-resident B cells
Pathogenic mechanism of human-derived Dsg3-specific antibodies
CONCLUSION
FIGURES AND LEGENDS
FIGURE 1. Presence of Dsg3-specific memory B cells correlate with relapse in disease after treatment with Rituximab.
FIGURE 2. Human-derived pathogenic monoclonal antibodies can engage endocytosis and degradation of Dsg3 from the surface of keratinocytes.
References
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